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"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing pr sity score matching In the two groups gastric neoplasms containing NEC components were dividedinto pure NEC and mixed tumors with less than GHMiNEN between and GHMiNEN andmore than GHMiNEN neuroendocrine carcinoma components OS was compared between thesegroups and the gastric adenocarcinoma groupResults The OS of gastric neoplasms containing neuroendocrine NEC components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or Cox multivariable regressionanalysis suggested that tumor category neoplasms containing NEC components or gastric adenocarcinoma wasan independent risk factor for prognosis Interestingly among patients receiving neoadjuvant therapy thedifference was not significantContinued on next page Correspondence buzhaodecjcrcn jijiafuhscpkueducn Jiahui Chen Anqiang Wang and Ke Ji contributed equally to this workDepartment of Gastrointestinal Surgery Key Laboratory of Carcinogenesisand Translational Research Ministry of Education Peking University CancerHospital Institute No Fucheng Road Haidian District Beijing China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of Continued from previous pageConclusions Gastric neoplasms containing any proportion of NEC components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmsKeywords Neuroendocrine carcinoma Gastric adenocarcinoma Overall survivalBackgroundGastric neoplasms containing neuroendocrine carcinomaNEC components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure NECs and mixed tumorscontaining NEC components Every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing NEC components account for approximately of thesecases [ ] Given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []According to the World Health anizationWHO digestive neuroendocrine tumor classificationneuroendocrine neoplasm NEN can be divided intothree categories based on Ki67 levels and mitotic counts HPF Grade G1 Ki67 mitoses Grade G2 Ki67 mitoses Grade G3Ki67 mitoses [] Meanwhile the AmericanJoint Committee on Cancer AJCC defines highly differentiated NEN as a neuroendocrine tumor NET and thepoorly differentiated NEN as a neuroendocrine carcinoma NEC based on the degree of tumor cell differentiation Generally G1 G2 and rare welldifferentiated G3NENs belong to the NETs while poorly differentiatedG3 NENs belong to NECs[ ] Gastric mixedneuroendocrinenonneuroendocrineneoplasm GMiNEN is a special type of gastric NEN that is definedas containing more than of both neuroendocrineand nonneuroendocrine components [] accountingfor approximately of all GNENs and of gastricneuroendocrine carcinomas GNECs [] For thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition Consideringthe heterogeneity ofMiNEN and the malignancy degree of the different components in the tumor La Rosa [ ] proposeddividing MiNEN into three categories highgradeintermediategrade and lowgrade Highgrade MiNENconsists of NEC and carcinomaadenoma intermediategrade MiMEN consists of NET and carcinoma and lowgrade MiNEN consists of NET and adenoma Thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm GHMiNEN was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandGenerally the prognosis of mixed tumors is largely determined by the most malignant component Kim et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components All of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis Therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone We sought tofind studies on the overall survival OS comparison between GHMiNEN and gastric adenocarcinoma butfailed Thus we think that a study of the comparison ofthe OS of GHMiNEN and gastric adenocarcinoma willprovide a valuable supplement to current research on GHMiNEN To overcome the bias caused by the differences between the covariates in the comparison we usedpr sity score matching PSM to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliableMethodsPatient selectionWe retrospectively collected patients diagnosed withgastric NENs and underwent radical resection at PekingUniversity Cancer Hospital Beijing from January to December The inclusion criteria were as follows pathologically confirmed pure NEC or tumorcontaining NEC components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup Patients diagnosedwith cM1 or cT4b before surgery or died from perioperative complications were excluded from the study 0cChen BMC Cancer Page of Patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for PSM analysesperformed The chisquared test and MannWhitney Utest were used to further verify the matching resultsFollowupWe followed the patients at least twice a year Serumtumor markers test gastroscope and computed tomography CT scans were used to reexamine patients aftersurgery Depending on the patients status Magneticresonance imaging MRI and Positron emission tomography computed tomography PETCT were alsoconsidered For patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationDiagnosis and classificationWe reevaluated the diagnosis and classification of GHMiNEN Mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as GHMiNEN and GHMiNENrespectively Atumor consisting of NEC is defined as pure NECAll neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the WHO classification of tumors[] Neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin Syn chromograninA CgA and neuro cell adhesion molecule CD56 orNCAM The tumor staging described in the study wasbased on the AJCC 8th Edition TNM Staging Guidelines[] All possible disagreements were discussed in ourstudy groupDefinition of variables and groupsIn this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery Patients inthe surgery group were assessed by the pTNM stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the ypTNM staging system OSrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egPr sity score matchingTo accurately compare the prognosis of GHMiNENand gastric adenocarcinoma we employed PSM to balance the differences between the two groups PSM wasperformed through the Pamatching plugin in SPSS software Logistic regression models were used toestimate pr sity scores based on gender age tumorlocation tumor size and pathological staging Given a caliper width nearest neighbor matching wasStatistical analysisAll statistical analyses were performed using SPSS statisticalsoftware IBM United States The chisquared test and MannWhitney U test were used forstatistical analysis of categorical variables and continuous variables respectively KaplanMeier method wasused for the comparison of OS The logrank test wasused to compare survival rates Multivariable Cox proportional hazards models were used to identify predictors of survival outcome P was regarded as thethreshold of significanceResultsPatient selection and PSM resultsBetween and among the patients treated atthe Gastrointestinal Cancer Center of Peking UniversityCancer Hospital a total of patients with gastric neoplasms containing NEC components met the inclusioncriteria for the study including cases of pure NECand cases of mixedtype Of these patients a total of patients received neoadjuvant therapy NEC GHMiNEN GHMiNEN GHMiNEN while the remaining patients receivedsurgery directly NEC GHMiNEN GHMiNEN GHMiNEN There were aninsufficient number of patients in group GHMiNEN group to conduct effective statistical analysisso we combined the GHMiNEN group with theNEC group for further analysis We also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery Among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly Fig Immunohistochemical specificity markers were utilizedto identify the neuroendocrine components Fig 2aSyn was expressed in almost all neoplasms containingNEC components while the positive rates ofCgA and CD56 were much lower and respectively No significant difference in the positiverate of Syn and CgA was observed between pure NEC GHMiNEN GHMiNEN and GHMiNENFig 2b c only the positive rate of CD56 was found tobe higher in the pure NEC group than that in the GHMiNEN group Fig 2dTherefore priorto OS comparison PSM wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cChen BMC Cancer Page of Fig Flow chart of patient enrolmentComparison of OS between all patients with NECcomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupBefore PSM we compared the survival curves between all patients with NEC components and patientswith gastric adenocarcinoma by the KaplanMeiermethod Fig Apparently patients with NEC components had a poorer OS than those with gastricadenocarcinoma Fig 3a p in the surgicalgroup In contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy Fig 3b p According to the proportion of NEC components patients were classifiedinto pure NEC GHMiNEN GHMiNENand GHMiNEN The OS was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison Fig 3c dFig Illustrations of immunohistochemical staining patterns in gastric neoplasms containing NEC components a An overview of the expressionof Syn CgA and CD56 in tumors containing NEC components b Syn expression in different NEC component groups c CgA expression indifferent NEC component groups d CD56 expression in different NEC component groups CD56 neuro cell adhesion molecule CgAchromogranin A NEC neuroendocrine carcinoma Syn synaptophysin Pvalue 0cChen BMC Cancer Page of Fig See legend on next page 0cChen BMC Cancer Page of See figure on previous pageFig Comparison of OS between gastric neoplasms containing NEC components and gastric adenocarcinoma a OS comparison betweengastric neoplasms containing NEC components and gastric adenocarcinoma before PSM in the surgical group b OS comparison between gastricneoplasms containing NEC components and gastric adenocarcinoma before PSM in the neoadjuvant group c OS comparison between differentNEC content groups pure NEC GHMiNEN GHMiNEN and GHMiNEN and gastric adenocarcinoma before PSM in the surgicalgroup d OS comparison between the different NEC content groups and gastric adenocarcinoma before PSM in the neoadjuvant group e OScomparison for patients in the surgical group after PSM f OS comparison for patients in the neoadjuvant group after PSM NEC neuroendocrinecarcinoma OS overall survival PSM pr sity score matchingBefore PSM significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group Table Table To balance the clinicopathological differences between the twogroups PSM was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups The detailedclinicopathological characteristics before and after PSMare shown in Table and Table As a result patients with NEC components and patients with gastric adenocarcinoma were matchedin the surgical group Table Patients with NEC components also had a poorer OS than those with gastricadenocarcinoma Fig 3e p Multivariable analysis showed that adjuvant therapy tumor category andTNM stage werefactorsTable independent prognosticTo investigate whether neoadjuvant therapy had an effect on OS patients with NEC components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group Table Interestingly KaplanMeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in OS between the two groups Fig 3f p Comparison of OS between patients with differentproportions of NEC components and patients with gastricadenocarcinomaTo investigate whether the level of NEC componentshad an effect on OS in the surgical group GHMiNEN GHMiNEN pure NEC and pure NEC plus GHMiNEN were compared with gastric adenocarcinoma after PSM The results showed that even thegroup with the lowest proportion of NEC componentsthe GHMiNEN group had a poorer OS thanadenocarcinoma Fig 4a P As expected theGHMiNEN pure NEC and pure NEC plus GHMiNEN groups each with relatively high proportionsof NEC components had worse OS than the gastricadenocarcinoma group Fig 4bd P Detailed clinical information after matching isshown in Additional file Tables S1S4PSM was also performed in the neoadjuvant group Incontrast to the results of the surgery group in the pureNEC group containing the highest proportion ofNEC componentstill no significantdifference in OS from gastric adenocarcinoma Fig5d The other three groups with lower NEC contentwere also notfrom gastricadenocarcinoma in terms of OS Fig 5ac Detailedclinicopathologicaland afterPSM are shown in Additional file Tables S5S8characteristics beforethere wassignificantly differentDiscussionAmong gastric neuroendocrine neoplasms the tumorcontaining NEC components is a special type includingpure NEC and mixed tumor containing NEC components The incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated GNENs [ ]received neoadjuvantIn previous study Kim found that in patientschemotherapywho had notprogressionfree survivalPFS of pure GNEC waspoorer than that of gastric adenocarcinoma while thePFS of mixedtype tumors was not significantly differentIn Kimsfrom that of gastric adenocarcinoma []study the mixed type was defined as NET mixed withgastric cancer rather than NEC NET is much less malignant than NEC [ ] This may be the reason whythere was no significant difference in OS between mixedtype and gastric adenocarcinomas In addition mixed tumors with less than or more than of NEC components were not included in that study which webelieve was a deficit of the study PFS is an important indicator for evaluating prognosis in many cases it can reflect the trend of OS Based on Kims research resultswe regarded tumors containing NEC components as awhole and found that the OS of these tumors was poorerthan that of adenocarcinoma in the surgical group Inthe comparison of OS between mixed tumors with different proportions of NEC components and gastricadenocarcinoma the results for pure NEC cases wassimilar to Kims While the OS of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in Kims study Cox multivariable regression analysis showed thattumor categoryneoplasm with NEC component or adenocarcinoma 0cChen BMC Cancer Page of Table Comparison of clinicopathological characteristics before and after PSM in surgical groupPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n P valueMatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomy ± ± Proximal gastrectomy Surgical procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Table Comparison of clinicopathological characteristics before and after PSM in neoadjuvant groupMatched comparisonPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedure LaparoscopicT stageT0T1T2T3T4N stageN0N1N2N3M stageM0M1ypTNM stageIIIIIIIV ± ± Gastricadenocarcinoman ± ± P valuePatients with NECcomponents n ± ± Page of P valueGastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Page of Table Univariate and multivariate analyses of survival after PSM in surgical groupPatient CharacteristicsUnivariate analysisHR CIMultivariate analysisHR CIP valueAge yearGendermale vs femaleBMIAdjuvant therapyYes vs NoTumor size¥ cm vs cmTumor categoryCarcinoma with NEC component vsGastric adenocarcinoma vsType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedureLaparoscopic vs TNM stageIIIIIIIVP value tumor size and TNM staging were independent risk factors for prognosis This suggests that the prognosis ofgastric neoplasms with NEC components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of NEC components can alsoimpair prognosis which challenges the current cutoffvalue of The proportion of each component that must theoretically be greater than was set in [] Andsince WHO has also adopted this standard to define MiNEN [] This largely avoids the overdiagnosisof MiNEN in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges In additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines [] Nevertheless it is now being questionedby an increasing number of scholars The componentsin mixed tumors are not evenly distributed For large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely []Park compared the OS between tumors with morethan NEC components and gastric adenocarcinomawith or without less than NEC and they found thattumors with an NEC composition of more than hada worse prognosis This suggests that even a small proportion of malignant components can affect prognosis[] While in Parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith NEC components less than with gastricadenocarcinomas directly nor did they compare allNECcontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyIn our study we regarded tumors containing NECcomponents as a whole and found that the OS of thesetumors was poorer than that of adenocarcinoma in thesurgical group In addition we also found that the OS ofmixed tumors with less than between and more than NEC components or pure NEC wasworse than that of gastric adenocarcinoma Analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of Syn and CgAbetween different NEC content groups only the positiverate of CD56 was found to be higher in the pure NECgroup than that in the GHMiNEN group Therole of CD56 in the diagnosis of NEC is still controversial However Syn and CgA are two wellrecognized 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC and gastric adenocarcinoma in the surgical group aOverall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparison between GHMiNEN andgastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastric adenocarcinoma d Overall survivalcomparison between pure NEC alone and gastric adenocarcinomamarkers Therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing NEC componentsStudies on the molecular mechanism of pathogenesisshow that NEC components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity LOH and mutations at multiple loci and key oncogenes such as TP53 APC and RBgenes All these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] Moreoverin the WHO definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid [] no minimumpercentage for either ingredient is established Thereforewe believe that mixed tumors containing NEC components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma We propose considering mixed tumorscontaining NEC components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiesPreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] In a retrospective study involving patientsMa et alfound that neoadjuvant chemotherapy improves the survival of patients with NEC and HMiNENof the stomach [] Van der Veen reported that 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC components and gastric adenocarcinoma in theneoadjuvant group a Overall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparisonbetween GHMiNEN and gastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastricadenocarcinoma d Overall survival comparison between pure NEC and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing NEC components [] However because only eight patients wereincluded in the neoadjuvant group Vans results arequestionable In our study among patients receivingneoadjuvanttherapy no significant difference in OSbetween mixed tumor and gastric adenocarcinoma wasobserved Even for the pure NEC group with the highestNEC contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsAlthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research In addition we proposed treatingall gastric neoplasms containing NEC components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms In the futurewe will conduct more genomics studies to confirmour ideas This study also has its limitations Due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens As a retrospective study despite our performing PSM in advance selection biascannot be completely avoided In addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cChen BMC Cancer Page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with NEC componentless than so we could only treat all mixed tumors withNEC component as a wholeConclusionsOur study demonstrated that gastric neoplasms withNEC components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies Therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients Neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12885020072817Additional file Table S1 Comparison of clinicopathologicalcharacteristics before and after PSM of 30GHMiNEN patients insurgical group Table S2 Comparison of clinicopathologicalcharacteristics before and after PSM of GHMiNEN patients in surgicalgroup Table S3 Comparison of clinicopathological characteristics beforeand after PSM of 70GHMiNEN plus pure NEC patients in surgicalgroup Table S4 Comparison of clinicopathological characteristics beforeand after PSM of pure NEC patients in surgical group Table S5 Comparison of clinicopathological characteristics before and after PSM of 30GHMiNEN patients in neoadjuvant group Table S6 Comparison ofclinicopathological characteristics before and after PSM of GHMiNEN patients in neoadjuvant group Table S7 Comparison of clinicopathologicalcharacteristics before and after PSM of 70GHMiNEN plus pure NECpatients in neoadjuvant group Table S8 Comparison of clinicopathological characteristics before and after PSM of pure NEC patients in neoadjuvant groupAbbreviationsAJCC American Joint Committee on cancer CT Computed tomography GHMiNEN Gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm GNEC Gastric neuroendocrine carcinoma HPF High power fieldMiNEN Mixed neuroendocrinenonneuroendocrine neoplasmNEC Neuroendocrine carcinoma NEN Neuroendocrine neoplasmNET Neuroendocrine tumor MRI Magnetic resonance imaging OS Overallsurvival PETCT Positron emission tomography computed tomographyPFS Progressionfree survival PSM Pr sity score matching WHO WorldHealth anizationAcknowledgmentsThanks to Dr Zhongwu Li of the Department of Pathology Peking UniversityCancer Hospital and his colleagues for their assistance in pathologicaldiagnosis and review Thanks to all colleagues in the Department ofGastrointestinal Surgery of Peking University Cancer Hospital and Dr JiangHong from the Statistics Department for their assistance in this studyAuthors contributionsAll authors contributed to the study conception and design JC performeddata collection and wrote the manuscript AW wrote and t revised hemanuscript KJ helped with statistical analysis and prepared the illustrationsZB edited the manuscript JJ conceived the study and reviewed themanuscript All authors read and approved the final manuscriptFundingThis work was supported by the National Science Foundation for YoungScientists of China Beijing Youth Talent Plan QML20191101 andScience Foundation of Peking University Cancer Hospital Thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the Ethics Committee of Peking UniversityCancer Hospital and the patients written consent was also obtained Writteninformed consent for publication was obtained and stored in PekingUniversity Cancer HospitalConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Matsubayashi H Takagaki S Otsubo T Iiri T Kobayashi Y Yokota T et alAdvanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy Gastric Cancer Park JY Ryu MH Park YS Park HJ Ryoo BY Kim MG Prognosticsignificance of neuroendocrine components in gastric carcinomas Eur JCancer La Rosa S Inzani F Vanoli A Klersy C Dainese L Rindi G Histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms Hum Pathol Ishida M Sekine S Fukagawa T Ohashi M Morita S Taniguchi H et alNeuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis Am J Surg PatholRayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc | 2 |
"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu HawaiiParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conï¬ict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR conï¬dence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conï¬ict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulï¬lling ï¬lial obligations in order to ensure equitable access to ACP AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals values goals and preferences regardingendoflife EOL care12 Contemplation of individuals EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conï¬icts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610 AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the beneï¬ts ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proï¬ciencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships uence individuals ACP engagement To ï¬ll thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawaiiwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentiï¬ed asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was afï¬liated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andï¬nancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conï¬ict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conï¬ict was measured using the 5item Family CulturalConï¬ict scale which assesses cultural and intergenerational conï¬ictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation ï¬nancial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conï¬ict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with conï¬dence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconï¬ict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not signiï¬cantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proï¬ciency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conï¬ictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and dont want tonever but want toreluctant to yes never and dont want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conï¬icte10 most cultural conï¬ict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents life which maysometimes be in opposition to their parents own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this populations lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The ï¬nding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conï¬ict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The ï¬nding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential beneï¬t and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved onesdeath and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the signiï¬cantrelationship between family conï¬ict and ACP engagement could bethat higher levels of family conï¬ict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These ï¬ndings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizeï¬ndings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conï¬ict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conï¬ictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education ï¬nancial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulï¬lling ï¬lial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Deï¬nition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Deï¬ning advance care planning for adults Aconsensus deï¬nition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneï¬ciaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conï¬ict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difï¬cult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawaii Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspeciï¬c psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratiï¬cation of older adults preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c" | 2 |
"Cardiac arrhythmias Atrial ï¬brillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular ï¬brillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are aï¬icted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial ï¬brillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular ï¬brillation VPCs ventricular premature complexes VT ventricular tachycardia Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial ï¬brillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with conï¬rmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular ï¬brillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial ï¬brillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular ï¬brillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insuï¬ciencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular ï¬bril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial ï¬brillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial ï¬brillation According to a recent survey of electrophysiology professionals atrial ï¬brillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using speciï¬c AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the ï¬rst months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insuï¬ciency and the systemic am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients aï¬icted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with conï¬rmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the ï¬rst documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is signiï¬cantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the ï¬nal weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained signiï¬cantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as deï¬nite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and ï¬uoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary ï¬ndings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpausedependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeï¬ciency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus ï¬ngolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides ï¬uoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to signiï¬cant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs niï¬cant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ¥ ms Changes in ¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ¥ ms or QTc increase of No patients manifested TdP ¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signiï¬ cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no signiï¬cant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identiï¬ed only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of signiï¬cant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this speciï¬c population HCQAZM could not be initiated or had to be interrupted in ¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the ï¬rst manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or forme fruste of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19speciï¬c risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use of various formulas among which the Bazetts correction formula is most commonly used QTc QT RRsec QTc is deï¬ned as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTcQRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F | 2 |
ovarian cancer is a silent and largely asymptomatic cancer leading to late diagnosis and worseprognosis the latestage detection and low survival rates makes the study of the spacetime evolution of ovariancancer particularly relevant in addition research of this cancer in small areas like provinces or counties is still scarcemethods the study presented here covers all ovarian cancer deaths for women over years of age in the provincesof spain during the period spatiotemporal models have been fitted to smooth ovarian cancer mortalityrates in age groups [ [ [ and [ borrowing information from spatial and temporal neighboursmodel fitting and inference has been carried out using the integrated nested laplace approximation inla techniqueresults large differences in ovarian cancer mortality among the age groups have been found with higher mortalityrates in the older age groups striking differences are observed between northern and southern spain the globaltemporal trends by age group reveal that the evolution of ovarian cancer over the whole of spain has remainednearly constant since the early 2000s differences in ovarian cancer mortality exist among the spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are essential to discoverinequalities in ovarian cancer mortality women over years of age should be the focus of followup studies as themortality rates remain constant since highmortality provinces should also be monitored to look for specific riskfactorskeywords agespacetime models disease mapping inla ovarian cancer mortality smoothing the number and scientific impact of publications on ovarian cancer are continuously increasing not in vainovarian cancer is the eighth most common cancer inwomen and the 18th most frequent overall with nearly new cases worldwide in around of cases are concentrated in developed countries whereovarian cancer is the most lethal gynecological tumorcorrespondence lolaunavarraes1department of statistics computer science and mathematics publicuniversity of navarre campus de arrosadia pamplona spain2inamat public university of navarre campus de arrosadia pamplonaspainthe highest incidences are found in northern and easterneurope austria the czech republic germany irelandlatvia lithuania the nordic countries slovakia and theuk and the united states whereas in africa andasia this tumor is virtually nonexistent this pattern isattributed mostly to the low birth rates found in developedcountries in the past years the overall number of tumors hasundergone a constant increase in spain this is due notonly to the population growth but also to the increasedlife expectancy and the use of early detection techniquesin the estimated number of new cases of ovarian the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0ctrandafir bmc public health page of cancer was representing of all female cancersbeing the fifth cause of cancer deaths in women afterlung breast colon and uterine tumors [ ] the agestandardized incidence rate calculated using the directmethod and the world standard population is per women which may be considered high as forits temporal evolution there was a slight decline between and but since then mortality rate starts toincrease slowly but constantly in addition there is a greatdeal of variability among the spanish provinces for example during the period one finds a rate of per women in the canary islands and a rate of per women in cuenca ovarian cancer is a disease affecting mostly older postmenopausal women with more than of cases beingdiagnosed in women over according to medicalexperts it is a silent and mostly asymptomatic cancer acircumstance that leads to late diagnosis and worse prognosis furthermore in the cases where symptoms doappear these may be confused with digestive problemsbloating early satiety abdominal andor pelvic pain orbenign gynecological alterations such as endometriosis orpolycystic ovary syndrome to date no method for earlydetection is available this is reflected in the fact that upto of cases are detected in the advanced stages of thedisease the etiology of ovarian cancer is poorly understoodhowever several factors associated with an increased riskof ovarian cancer have been identified age number ofovulations early menarche infertility low parity theuse of hormone replacement therapy hrt obesity physical inactivity a family history of breast andovarian cancer including brca1 and brca2 gene mutations and past and current smoking associations between exposure to asbestos in the workplace orat home and ovarian cancer have also been found further research is needed to corroborate this finding inspain as most jobs with a high exposure to asbestos arepredominantly maledominated eg mining milling orshipyard work nevertheless a study of asbestos exposure among italian women found that the main factorwas secondhand contact due to occupationally exposedrelatives for example from soiled work clothes broughthome as of this writing however there is no documented registry for asbestos exposure in the workplaceor at home in spain some epidemiological studies havedetected an increased risk of ovarian cancer in womenwith less exposure to sunlight and consequently with lowlevels of vitamin d in particular the higher the latitudethe less overall accumulated sunlight and the higher theincidence of ovarian cancer some protective factors against ovarian cancer have also been identified suchas multiparity oral contraceptives and tubal ligation orhysterectomy the 5year agestandardised relative survival in spain is estimated at less than similar tothe european average of as mentioned aboveovarian tumors are the eighth cause of cancer deathsamong women worldwide with deceases registered in overall among malign tumors in about women died yearly in spain from ovarian cancer representing of all cancer deaths and of all deaths among women the mean age of decease from ovarian cancer inspain is years cabanes analyzedthe ageadjusted mortality trends of ovarian cancer inspain for the period ovarian cancer caused deaths in this period with rates in women over showing a tenfold increase versus those in youngerwomen in women under rates increased peryear until and afterwards started to drop at a rateof per year in the age group mortalityrates significantly increased annually up to andbecame stable thereafter in women and older mortality rates increased annually up to the year anddecreased per year afterthe disproportionate impact on older women togetherwith the aforementioned concerns regarding latestagedetection and low survival rates makes the study of thespacetime evolution of ovarian cancer particularly relevant in addition it is important to mention that agegroups are not equally affected by ovarian cancer mortality and then it is necessary not just to standardize by agebut to analyze the different age groups hence the maingoal in this paper is to study the temporal evolution of thegeographical patterns of ovarian cancer mortality rates infour age groups of women aged years or moremethodsdata sourcethe study presented here covers all ovarian cancer deathscode c56 of the 10th edition of the international classification of diseases for women over years of agein the provinces of spain excluding the autonomouscities of ceuta and melilla recorded throughout theperiod by the spanish statistical officestatistical analysisa bayesian hierarchical spatiotemporal model is used toestimate rates the model is briefly described in whatfollows for better interpretation of resultsspain is divided into s provinces indexed by i s and data are available for t27 time periods corresponding to years labeled as t tfor each age group let nit represent the population at riskfor region i and time period t then conditional on themortality rates rit the number of ovarian cancer deathsoit is assumed to follow a poisson distribution with mean 0ctrandafir bmc public health page of table descriptive statistics of observed cases and crude mortality rates per women disaggregated by age groups provinceand year min minimum q1 first quartile q3 third quartile max maximumobserved casescrude ratesage group[ [ [ [ [ [ [ [ minq1medianmeanq3maxμit nitrit that isoitrit ¼ poissonμit nitritlog μit log nit log ritwhere the lograte is modelled aslog rit α ξi γt δithere α denotes the logarithm of the overall rate ξi andγt are the main spatial and temporal effects respectivelyand δit corresponds to the spacetime interaction effectssince each of these components are supposed to be gaussian markov random fields gmrf the integratednested laplace approximation inla technique hasbeen used for model fitting and inference specifically theleroux car prior distribution has been considered for the spatial random effects and a firstorderrandom walk prior distribution for the temporal randomeffects in addition the four different types of interactionintroduced by knorrheld have been considered forthe spatiotemporal random effects these interactionsallow the spacetime effects to be completely independenttype i interaction structured in time but not in spacetype ii interaction structured in space but not in timetype iii interaction or completely structured in spaceand time type iv interactionall the computations have been done using the interactive web application sstcdapp which can befound at httpsemisstcdappunavarraeslogin thisapplication provides a user interface for the analysis ofspatiotemporal areal count data allowing to fit a widevariety of spacetime models using the inla estimationtechnique in addition the application provides differentmodel selection criteria in this paper the model with the[[[[nemow rept shaed etar edurcyearfig temporal trends by age groups temporal trend of the ovarian cancer mortality crude rates by agegroups 0ctrandafir bmc public health page of lowest value of the deviance information criterion dic has been selected for further details about modelspecification prior distribution of the hyperparametersidentifiability constraints and additional model selectioncriteria see for example adin and the referencesthereinresultsa total of ovarian cancer deaths were registered inthe population of spanish women over years of age during the period since ovarian cancer is mainlyrelated to the onset of menopause the age groups we areconsidering in this paper are [ [ [ and [ a brief summary of observed cases and mortality rates per women by age groups provinceand year is shown in table clear differences areobserved in the mean and median mortality rates amongthe youngest and oldest age groups with values ranging from cases per women up to casesper women approximately respectively figure displays the global temporal trend of crude rates by agegroup here the different behaviour of the age groups iseven more evident a pronounced slope from the lastdecade of the twentieth century to the beginning of thetwentyfirst century is observed in the older age groupsmodel was fitted to smooth spatiotemporal rates ineach age group the interaction considered in the modelwas chosen on the basis of the dic values for each subgroup of ageclass the dic pointed toward a type ivinteraction for age groups [ [ and [ whereas a type ii interaction was selected for the agegroup [ to make the different terms in all themodels comparable a decomposition of the estimated logrates was computed by defining posterior spatial ξi t and spatiotemporal δtemporal γ it patterns see adini γ so that log rit α ξit note thatexpα represents the overall mortality rate for the wholeof spain during the period in order to facilitate interpretation of the results a map of spain showingits provinces is given in fig t δla coruñalugopontevedraorenseasturiasleoncantabriavizcayaguipuzcoaalavanavarrapalenciaburgosriojahuescaleridageronazamoravalladolidsoriazaragozabarcelonasegoviatarragonasalamancaguadalajaraavilamadridcacerestoledocuencabadajozciudad realalbaceteteruelcastellonvalenciaalicantebalearescordobajaenmurciahuelvasevillagranadaalmeriamalagacadizsanta cruz de tenerifepalmaslasfig administrative division of spain map with the administrative division of spain showing provinces source map was generated by the authorsusing the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of figure shows the map with the posterior meanieestimates of provincespecific mortality ratesexpα ξexceedance probabilitiesofrate being greaterthanthe overall spanish rate have also been computedsee fig i posteriorthis provincespecificthe estimated spatial pattern draws attention toasturias as a high ovarian cancer mortality rate provincefor all age groups in the age group [ the highest spatial rates are found in the northwestern provincesasturias lugo and la coruña but also in vizcaya andhuesca over cases per women in age group[ the regions with the highest estimated rates areasturias the balearic islands and valencia with an estimated rate of over cases per women in thethird age group [ the highest rates are located inthe centralnorthern areas with salamanca and asturiasleading the ranking and in the canary islands tenerifeprovince all of them with more than cases per women the oldest age group [ exhibits high rateareas in asturias barcelona gerona and guadalajarawith a rate of over cases per women all ofthese highrate provinces have a rate significantly higherthan the overall spanish rate in their respective age groupssee fig in general northern spain has greater ovarian cancermortality rates compared to the southern regions thelowest rates are found in guipúzcoa for age groups [and [ and in almerÃa for age groups [ and[ the northwestern province of la coruña showsage group [age group [ to to to to to to to to age group [age group [ to to to to to to to to fig provincespecific mortality rates estimates by agegroups posterior mean estimates of provincespecific mortality rates expα ξsource maps were generated by the authors using the library tmap from the r statistical software version no licenses are required touse or publishi 0ctrandafir bmc public health page of age group [age group [[][[[[[][[[[[][[[[age group [age group [[][[[[fig provincespecific posterior exceedance probabilities by agegroups posterior exceedance probabilities of each province in comparison withthe spanish overall rate pexpα ξstatistical software version no licenses are required to use or publish expαo source maps were generated by the authors using the library tmap from the ria surprising behaviour with high rates in the age group[ but one of the lowest rates in the age group [the estimated global temporal pattern expα γ t isvisualized in fig rates seem to have decreased duringthe last few years from to in age group [ but have remained nearly constant in the other threeage groups which experienced a sharp increase in ratesfrom up to the beginning of the twentyfirst centuryapproximatelyfigures and display maps showing the spatiotemporal evolution of ovarian cancer mortality rates foreach spanish province for the period dividedinto intervals of years for age groups [ [ [ and [ respectively specifically these mapsrepresent the posterior mean of rit expα ξi γ t δit the corresponding maps of probabilities showing theprobability that a particular rate in a given province andyear is greater than the spanish rate during that periodare not shown here to conserve space however we haveclassified a province as having a rate significantly greaterthan the spanish rate if this probability is greater than for age group [ we find that the mortality ratefor the period was highest in the northeasternregion with the province of huesca having a significantrate that lasted until in the latter years of the periodstudied only asturias showed a significant rate withinthis age group the percentage of the rates variabilityexplained by the spatiotemporal term was implying that the specific temporal evolution of each provinceis rather high in this age group 0ctrandafir bmc public health page of age group [age group [age group [age group [fig temporal trends estimates by agegroups for the whole of spain posterior mean estimates of yearspecific mortality rates expα γ and credible intervals dotted lines provide the estimated rate in each age group in the whole period in spain expαt to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publishin age group [ rates exhibit a greater variability among provinces between cases per women and about cases per women thehighest rates occurred in the late 1990s and early 2000smainly in the northern half of spain and in the balearicislands asturias lugo salamanca valladolid huescateruel gerona and the balearic islands show significantlyhigh rates at the end of the periodfor women between and years of age the lowest rates are found at the beginning of the study periodwith significantly low rates the highest significant ratesare located mainly in the northern and western parts ofspain and in the canary and balearic islands in the period the provinces with the lowest rates at the endof the period are gerona and madridwomen over years of age show significantly low ratesbetween again asturias shows the highestmortality rate at the end of the period some provinceslocated in southern spain mainly along the coastlineshow significantly low ratesthe temporal evolution of the rates for some selectedprovinces are plotted in fig the colors used in thebands are associated to the posterior exceedance probabilities of each province at year t in comparison withthe temporal pattern for the whole of spain in that yearnamely prit α γ t o in the case of barcelonathe probability that the mortality rate lies above the spanish rate reaches a maximum between for agegroups [ and [ and between forage groups [ and [ madrid behaves similar tospain in all age groups in asturias the probability is quitehigh for all age groups with a significant rising trend forage group [ from onwards la coruña showssignificantly low rates during the whole study period forthe age group [ whereas this provinces behaviourfor women over years of age is similar to the spanishratediscussionthe ovaries are one of the cancer sites where knownrisk factors are not enough to explain all the cases andthus spatiotemporal analyses provide additional important information allowing for the examination of thespatial temporal and spatiotemporal mortality patternsas the age groups are not equally affected by ovarian cancer mortality it is necessary not just to standardize by age but also to analyze the different agegroupsresults show large differences in mortality among theage groups with higher mortality rates in the older agegroups indeed the last age groups women of more than years of age double the rate of women between and more than cases in the last age groups vs casesper women on average in age group [ thiscan be explained by poor survival rates of gynaecologicalcancers in the elderly which in turn is influenced by late 0ctrandafir bmc public health page of barcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñafig temporal evolution of ovarian cancer mortality rates estimates for some selected provinces barcelona madrid asturias and la coruñatemporal evolutions of the posterior mean estimates of mortality rates rit for some selected provinces and twosided credible intervals for agegroups [ first row [ second row [ third row and [ fourth row the colors used in the bands are associated to theposterior exceedance probabilities of each province in time t in comparison with the temporal pattern of spain that is prit expα γ orepresented with a red line in the graphstdiagnoses and the failure of treatments due to comorbidity[ ]the global temporal trends by age group reveal thatthe evolution of ovarian cancer over the whole of spainhas remained nearly constant since the early 2000s particularly for women aged years or more after a sharpincrease during the period approximatelythe stabilization of the rates may be due to an increasedconcern among women regarding their personal health inparticular among older women that led them to be testedmore frequently access to better information via massmedia and the internet and the increasing effectiveness of 0ctrandafir bmc public health page of cancer treatments in the nineties access to informationvia the internet was more limited and health care was possibly less advanced which could explain in part the sharpincrease in mortality rates in the first half of the periodmortality in women between and years of age showsa slight decrease since until nearly althoughthis decrease does not seem to be significant with respectto the average mortality in this age group it seems thatin fact during the last two years of the study period ratesare starting to rise slightly but once again this trend is notsignificant as yetthe global geographical patterns show in general thatthe north has higher rates than the south a situation similar to that observed overall in europe the variabilityobserved in all age groups between northern and southernspain remains unknown although in general women inthe south are prone to marry earlier and have more children on average interestingly asturias the provincewith the highest rates in all age groups is one of thespanish provinces with the lowest average number of children the differences observed between the north and thesouth could also be explained taking into account the relationship between exposure to sunlight provitamin dand ovarian cancer although spain is in general a sunnycountry there is a great variability between the northand south in terms of average daily hours of sunlight forexample bilbao in the north receives about hoursof sunlight per year while sevilla in the south receives thus the observed increasing trend with latitude might be at least partly explained by a cumulativeexposure to sunlightheterogeneity among the provinces regarding ovariancancer mortality rates can be elucidated at least in partby a heterogeneous distribution of other risk factorsthere are differences among provinces in the age at whichwomen have their first period menarche the averageage of first childbirth and the total number of children[] the average fecundity rate in spain has beenmarkedly declining in particular the areas registering thelowest fertility rates were the basque country asturiasnavarre and aragón [ ] we should also point outthat the age of first birthing is closely related to socioeconomic development and is steadily increasing navarreand the basque country are the regions of spain wherewomen delayed childbearing the longest additionally hormonal replacement therapy has proven to have animportant influence on the appearance of ovarian cancerin postmenopausal women [] however its use inspain has been very limited another risk factor is the presence of a family history ofthe disease hereditary ovarian cancer syndrome presenting a mutation in brca genes is important between of ovarian cancer cases are linked to bcra mutation women who are carriers of the bcra1 mutationhave a chance of suffering ovarian cancer before age in spain the accumulated risk of developing ovarian cancer before age has been estimated at ci in carriers of a mutation in bcra1 and ci in carriers of a mutation in brca2 the prevalence of brca1 and brca2 mutation in spainis heterogeneous and varies according to geographical origin moreover blay showed that the brca1 andbrca2 spectrum of mutations in asturias was largely different from other areas of spain this could also explainin part the high mortality rates found in this provincediez studying a large group of spanish patientsshowed that there is only a slight difference betweenthe percentages of deleterious mutations in brca1 andbrca2 genes and respectively however somevariation due to geographic origin is present with a higherproportion of brca1 in families from the northwesternpart of spain according to vega the differencesfound in galicia could be due to founder effectsovarian cancer is also linked to lifestyle habits tobaccoand alcohol consumption the smoking habit is a riskfactor for epithelial ovarian cancer with an odds ratio of ci [ ] differences in alcoholand tobacco consumption can be found among spanishprovinces all in all to better understand the etiology of the disease and to better determine the effect of risk factors onthe spanish female population it would have been helpful to have the medical and workplace histories of all thewomen participating in this study this is the main limitation of the current work on the other hand as thereis a lack of scientific studies analyzing the associationbetween ovarian cancer mortality rates and risk factorsin the domains analyzed here age groups provinces andyears spatiotemporal analyses by age groups are essential to discover inequalities in ovarian cancer mortality todetect provinces with high risks in each age group and tokeep track of how the rates are evolving with timesdifferences in ovarian cancer mortality exist amongthe spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are very useful to look forpotential risk factors associated to the observed geographical patterns and to allocate funds among spanish regionsfor future clinical and epidemiological practice we recommend to followup women over years as the mortality rates remain constant since highmortalityprovinces should also be monitored to look more closelyfor specific risk factors some risk factors for ovarian cancer like getting old or having a family history cannot bechanged however women may slightly decrease their riskby avoiding other risk factors for example maintaining 0ctrandafir bmc public health page of a healthy weight avoiding tobacco and alcohol consumption or not receiving hormone replacement therapy aftermenopauseabbreviationsaei spanish research agency brca breast cancer dic deviance informationcriterion ue european union inla integrated nested laplaceapproximations gmrf gaussian random markov field sstcdapp spatialand spatiotemporal count data application uk united kingdomacknowledgementswe acknowledge the spanish statistical institute for providing the dataauthors contributionsstudy conception and design mdu pct acquisition of the data mdu aaanalysis of the data mdu pct aa interpretation of the data mdu aa pctwriting the pct mdu critical revision of the all authorsapproved the final manuscript and the decision to submit the manuscriptfundingthis research has been supported by the spanish ministry of science andinnovation project mtm 201782553r aeifeder ue the content of thispaper is solely the responsibility of the authors and does not represent theofficial views of the spanish ministry of science and innovationavailability of data and materialdata have been provided by the spanish statistical institute at municipalitylevel under a contract and aggregated later up on reasonable request thecorresponding author will make the datasets available mortality data fromcancer and other causes from by sex and province is available in theinteractive epidemiological information system ariadna httpariadnacneisciiiesevindexhtml of the spanish national center for epidemiologyethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesbrüggmann d pulch k klingelhöfer d pearce c groneberg d ovariancancer density equalizing mapping of the global research architectureint j health geogr bray f ferlay j soerjomataram i siegel r torre l jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin national institute for health and care excellence nice ovarian cancerthe recognition and initial management of ovarian cancer httpswwwniceukguidancecg122resourcesovariancancerrecognitionandinitialmanagementpdf35109446543557 accessed jan dos santos silva i beral v socioeconomic differences in reproductivebehaviour iarc sci publ ferlay j soerjomataram i ervik m dikshit r eser s mathers c rebelom parkin dm forman d bray f globocan v10 cancer incidenceand mortality worldwide iarc cancer base no [internet] lyoninternational agency for research on cancer available from httpglobocaniarcfr accessed jan ferlay j steliarovafoucher e lortettieulent j rosso s coebergh jcomber hea cancer incidence and mortality patterns in europeestimates for countries in eur j cancer forman d bray f brewster dh gombe mbalawa c kohler b piñerosm steliarovafoucher e swaminathan r ferlay j cancer incidence infive continents vol x iarc scientific publication no available from httppublicationsiarcfr_publicationsmediadownload3743e886b2754a75a0f70e190e9b56e5346047319c17pdfaccessed jan ledermann j raja f fotopoulou c gonzalezmartin a colombo n cs european society for medical oncology esmo guidelines workinggroup newly diagnosed and relapsed epithelial ovarian carcinomaesmo clinical practice guidelines for diagnosis treatment and followupann oncol coleman m gatta g verdecchia a esteve j sant m storm h allemanic ciccolallo l santaquilani m berrino f eurocare3 summary cancersurvival in europe at the end of the 20th century ann oncol bouchardy c rapiti e blagojevic s vlastos a vlastos g older femalecancer patients importance causes and consequences of undertreatment j clin oncol meindl a ditsch n kast k rhiem k schmutzler r hereditary breast andovarian cancer new genes new treatments new concepts deutschesärzteblatt | 0 |
Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso and Lucia Altucci Department of Precision Medicine University of Campania Luigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespeciï¬cdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneï¬cial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientiï¬c interest focusing on theidentiï¬cation of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol ï¬setin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modiï¬cations are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modiï¬cations Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT are enzymes classiï¬ed as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identiï¬ed inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespeciï¬c degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair OCallaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context speciï¬city BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deï¬ciency DAngelo Bioactive compounds in the diet can act as antioxidantand antiammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and ï¬setin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health beneï¬ts Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneï¬cial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneï¬cial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and antiammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin ï¬setin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classiï¬cation of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid proï¬le antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidproï¬le antioxidant antiammatoryAntioxidant antiammatoryAnticancer antioxidantantiammatoryAnticancercardiovascularpreventive antiammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesDAngelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonï¬avonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has antiammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger antiammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits proammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modiï¬es SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneï¬cial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneï¬cial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneï¬cial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress ï¬brosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together theseï¬ndings highlight the beneï¬cial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneï¬cialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been conï¬rmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective beneï¬t through improvement ofendothelial functionammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well deï¬ned Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientiï¬c research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efï¬cacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimers disease Parkinsonsdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood ï¬ow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneï¬cial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe ï¬avonoid polyphenol quercetin Que ²²pentahydroxyï¬avone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and antiammatory properties Nabavi Wu In recent years the scientiï¬c community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inï¬ammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inï¬ammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inï¬ammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the bodysantioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseasesammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable conï¬guration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cells antioxidant defense mechanisms moderateoxidative stress can in fact increase the cells antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These ï¬ndings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientiï¬c community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere signiï¬cantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpeciï¬cally in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats conï¬rming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these ï¬ndings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneï¬cial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ammatoryconditions is reported in Table Speciï¬cally a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and antiammatory capacities this ï¬avonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary ï¬brosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclariï¬cation Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneï¬cial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer antiammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneï¬cial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInï¬ammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efï¬cacy of Provex CV Supplement toReduce Inï¬ammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an antiammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a signiï¬cant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These ï¬ndings were also conï¬rmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Speciï¬cally BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and antiammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ammation signaling Speciï¬cally BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is | 2 |
Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group ARED PBM Group BINFRARED PBM Group CPLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring º Analyses of body contouring will be performed by non invasive methods º The waist circumference measurement will not discriminate the factors underlying the volume modifications º The habits of the participants such as diet and exercise routines may affect the results º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical TrialsReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individuals self esteem4 Studies have shown that aesthetic treatments significantly increase a patients quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6 There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10 The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18 Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions their frequency times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25 It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described below biggersmaller Ïn2 accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participants abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group ARED ± nm Group BINFRARED ± nm Group CPLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant powerone LED mWAperture diameterone LED cmPower density at aperture mWcm2Beam spot size at targetone LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContactweek for a month a total of sessionsInfrared LEDContinuousContactweek for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire The WHO Quality of Life WHOQOL BREF as well as the Body Shape Questionnaire BSQ34 Self Image Scale will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on a protected by password computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths nm and near the infrared regions nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18 Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39 If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individuals self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on a protected by password computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients Landecho MF Tuero C Valent V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts Kouris A Platsidaki E Christodoulou C et a0al Patients self esteem before and after chemical peeling procedure J Cosmet Laser Ther Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci Thornfeldt CR Thaxton PM Horn | 2 |
" the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour and patient characteristics treatment efficacy and tolerability and quality of life better patient selection might lead to improved outcomesmethods this post hoc exploratory analysis examined the effect of prognostic factors on outcomes in the randomized double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with mcrc refractory to standard chemotherapies recourse trial patients were redivided by prognosis into two subgroups those with metastatic sites at randomisation low tumour burden and ¥ months from diagnosis of metastatic disease to randomisation indolent disease were included in the good prognostic characteristics gpc subgroup the remaining patients were considered to have poor prognostic characteristics ppcresults gpc patients n386 had improved outcome versus ppc patients n414 in both the trifluridinetipiracil and placebo arms gpc patients receiving trifluridinetipiracil n261 had an improved median overall survival vs months hr ci to p00001 and progression free survival vs months hr ci to p00001 than ppc patients receiving trifluridinetipiracil n273 improvements in survival were irrespective of age eastern cooperative oncology group performance status ecog ps kras mutational status and site of metastases at randomisation in the trifluridinetipiracil arm time to deterioration of ecog ps to ¥ and proportion of patients with ps0 discontinuing treatment were longer for gpc than for ppc patients vs months and vs respectively low tumour burden and indolent disease were factors of good prognosis in late line mcrc with patients experiencing longer progression free survival and greater overall survivalintroductioninclusion of new therapeutic options into the current treatment landscape in metastatic colorectal cancer mcrc has led to an increased survival in the last couple of key questionswhat is already known about this subject º the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour characteristics and patient factors as well as treatment characteristics such as tolerability efficacy and quality of life effects trifluridinetipiracil is indicated in pretreated patients with mcrc based on results of the pivotal randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial which demonstrated significantly improved overall survival os compared with placebo with a manageable safety profilewhat does this study add º in recourse classification of patients as having good prognostic characteristics gpc defined as those with low tumour burden metastatic sites at randomisation and less aggressive disease ¥ months from diagnosis of first metastasis at randomisation identified a subgroup of patients with improved os and progression free survival with trifluridinetipiracil compared with patients with poor prognostic characteristics treated with trifluridinetipiracil and gpc patients treated with placebohow might this impact on clinical practice º low tumour burden and indolent disease were shown to be factors of good prognosis in late line mcrc with these patients experiencing longer time on treatment and greater os this suggests that these patients could be candidates to receive further lines of therapy post trifluridinetipiracildecades1 first line treatment of patients typically involves the use of vascular endothelial growth factor vegf or epidermal growth factor receptor egfr targeted agents eg bevacizumab cetuximab panitumumab to fluoropyrimidine based fluorouracil or tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesscapecitabine chemotherapy regimens depending on the presence or absence of ras mutation positive disease2 in the usa immunotherapies nivolumab±ipilimumab or pembrolizumab are also recommended for the treatment of patients with mismatch repair deficient or microsatellite instability high disease4 in the second line setting vegf targeted treatments eg aflibercept ramucirumab can also be used in combination with chemotherapy2 the optimal chemotherapeutic regimen for use beyond third line remains unclear where resistantrefractory disease and residual toxicity potentially limit the treatment options with only two possible candidates at present5the general condition and performance status of a patient are strong prognostic and predictive factors for mcrc treatment2 fitter patients are typically assigned to a more intensive treatment approach ie a combination of cytotoxic agents with a biological agent than less fit patients2 the choice of treatment in the metastatic setting is generally influenced by tumour characteristics tumour burden localisation and biology patient characteristics age eastern cooperative oncology group performance status ecog ps an function and comorbidities and treatment characteristics efficacy toxicity profile administration and quality of life qol effects2the proportion of patients with mcrc receiving active treatment decreases from line to line leaving more than half of patients who received an active treatment in the first line without treatment in the third line setting even in randomised clinical trials in folfiri plus cetuximab versus folfiri plus bevacizumab as first line treatment for patients with metastatic colorectal cancer only of patients reached third line6 data from the usa indicate that only of patients receiving a first line of treatment move into the second line move to the third line and only will receive a fourth line of treatment7 being unable to receive a subsequent line of treatment therefore appears to have a negative impact on the patients survivalis trifluridinetipiracil ftdtpi lonsurf indicated for the treatment of adult patients with mcrc who have been previously treated with or are not considered candidates for available therapies including fluoropyrimidine based oxaliplatin based and irinotecan based chemotherapies anti vegf agents and anti egfr agents for eligible patient ras wild type combination of tipiracil hydrochloride with the nucleoside metabolic inhibitor trifluridine improves its bioavailability by inhibiting its catabolism by thymidine phosphorylase8 the relatively limited non haematological toxicity of trifluridinetipiracil makes it a good option in the third line and refractory settings2 in the pivotal phase iii randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial conducted in patients with mcrc eligible for treatment in the third line and beyond treatment with trifluridinetipiracil versus placebo extended overall survival median os vs months hr p0001 and progression free survival median pfs vs months hr p000110 this effect was shown in all subgroups regardless of age ecog ps geographical region race and kras mutational status10 furthermore trifluridinetipiracil was well tolerated with few serious adverse events aes reported haematological toxicities were the most frequently observed aes10 also time to deterioration of ecog ps to ¥ was significantly improved median vs months hr p000110 with of patients treated with trifluridinetipiracil remaining at ps at discontinuation11 remaining at ecog ps is important as it could allow patients to further benefit from subsequent therapy and potentially extend their survival in recourse and of patients treated with trifluridinetipiracil remained alive at and months respectively in the refractory setting in the post hoc analysis described here we set out to explore other factors that could extend survival in the recourse population for the purposes of our exploratory analysis we defined the characteristics of good prognosis as low tumour burden metastatic sites by response evaluation criteria in solid tumors recist evaluation at randomisation and less aggressiveindolent disease ¥ months from diagnosis of first metastasis to randomisation which are known to be strong prognostic factors in patients with mcrc with good ecog ps12 our ultimate aim is to explore how clinicians can better predict individual treatment outcomes and support treatment selection through the continuum of carematerials and methodsstudy design and patientsthe study design and methodology of the recourse trial clinicaltrials gov number nct01607957 have been previously published10 in brief recourse was a phase iii randomised double blind placebo controlled study comparing the efficacy and safety of trifluridinetipiracil plus best supportive care with those of placebo plus best supportive care10 this study included patients with metastatic biopsy provendocumented adenocarcinoma of the colon or rectum who were previously treated with ¥ standard chemotherapy regimens or who had tumour progression within months of their most recent chemotherapy or who had clinically significant aes precluding readministration of standard chemotherapies patients were randomised to trifluridinetipiracil mgm2 two times a day on days and every weeks or matching placebo10 randomisation was stratified according to kras mutation status wild type vs mutant time from diagnosis of first metastasis to randomisation vs ¥ months and geographical region japan vs usa european union and australia10 all patients had adequate an function and were ecog ps tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0cof at inclusion10 the primary endpoint of the study was os and secondary endpoints included pfs objective response rate clinical benefit rate and safety10patient subgroupsin examining the effects of prognostic factors on treatment outcomes in the current analysis several subgroups of recourse patients were considered patients from recourse n800 were divided according to good prognostic characteristics gpc and poor prognostic characteristics ppc good prognosis was considered to be defined by low tumour burden metastatic sites by recist tumour evaluation at randomisation and less aggressiveindolent disease ¥ months from diagnosis of first metastasis to randomisation12 of the gpc subgroup n386 patients received trifluridinetipiracil and received placebo the remaining patients were included in the complementary ppc subgroup n414 of these received trifluridinetipiracil and received placeboanalysis outcomesos and pfs in the gpc subgroup were compared with those in the ppc subgroup these subgroups were then analysed according to other tumour and patient characteristics that is metastatic site at randomisation for those sites present in of the population liver lung lymph or peritoneum ecog ps vs kras mutation status wild type vs mutant and age vs ¥ years os and pfs with trifluridinetipiracil were compared with placebo and were analysed according to prognostic subgroups within each of the two arms finally the effect of prognostic classification of patients on ecog ps deterioration was analysed for all patients and subgroupsstatistical methodsdemographic and baseline characteristics of patients were summarised by treatment arm and subgroups using descriptive statistics n mean sd median minimum and maximum andor frequency distributions as appropriatethe differences in os pfs and time to ecog ps deterioration between trifluridinetipiracil and placebo patients or between subgroups of patients in a specific arm of treatment were assessed using the stratified log rank test stratification factors used for the randomisation from a cox proportional hazards model for each arm or each subgroup survival was summarised using kaplan meier curves and was further characterised in terms of the median with the corresponding two sided cisresultspatientsbaseline patient demographics and clinical characteristics were generally similar between gpc and ppc patients table in the trifluridinetipiracil arm slight imbalances were seen in ecog ps more gpc than ppc open accesspatients had an ecog ps of and kras status more gpc than ppc patients were kras wild type also more gpc than ppc patients had received ¥ prior regimens among the ppc group treated with trifluridinetipiracil of patients had ¥ months from diagnosis of first metastasis to randomisation but had ¥ metastatic sites and of patients had metastatic sites but months from diagnosis of first metastasis similar differences were observed in the placebo arm with the exception of kras status which was comparable in the gpc and ppc subgroupstreatmentamong trifluridinetipiracil treated patients those in the gpc group received more treatment cycles mean sd compared with patients in the ppc group mean sd online supplementary table s1 a higher proportion of gpc patients than ppc patients receiving trifluridinetipiracil had a dose delay vs respectively or dose reduction vs respectively which is consistent with a longer duration of treatment online supplementary table s1 however median dose intensity in the first four cycles was high ¥ and did not differ markedly between the groups cycle in the gpc group and the ppc group cycle and respectively cycle and respectively cycle and respectivelythe effect of good versus poor prognosis classifications on survivalsurvival curves for the gpc versus ppc subgroups are shown in figure median os was longer in the gpc subgroup than the ppc subgroup for both trifluridinetipiracil vs months hr ci to p00001 figure 1a and placebo vs months hr ci to p00001 figure 1b rates of month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively and month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively were also higher in gpc subgroups compared with ppc subgroups median pfs with trifluridinetipiracil was also longer in the gpc subgroup versus the ppc subgroup vs months hr ci to p00001 respective values for gpc versus ppc in the placebo arm were versus months hr p00699 pfs at and months in the ppc subgroup was and for trifluridinetipiracil and and for placebo respectively in the gpc subgroup these were and with trifluridinetipiracil and and with placebo respectivelyeffects of good prognostic factors on the relative efficacy of trifluridinetipiracilmedian os was prolonged with trifluridinetipiracil versus placebo in both subgroups but to a greater tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable baseline patient demographics and clinical characteristics according to prognosistrifluridinetipiracilplacebogpc subgroup n261 ppc subgroup n273 gpc subgroup n125ppc subgroup n141 females male asian other years to years ¥ yearsmedian age yearspatient age n gender n ethnicity n ecog ps n kras status n time since diagnosis of metastasis n number of prior regimens n number of metastatic sites n site of metastatic lesion n primary site of disease n liver lung lymph peritoneum ¥ ¥ mutant wild type months ¥ months colon rectum defined as metastatic sites and ¥ months since first metastasis only those in more than of the intent to treat population are included liver lung lymph and peritoneumecog ps eastern cooperative oncology group performance status gpc good prognostic characteristics ppc poor prognostic characteristicsextent in the gpc subgroup than in the ppc subgroup figure 2a similarly median pfs was prolonged with trifluridinetipiracil versus placebo in both subgroups with the greatest magnitude of benefit observed in the gpc patients figure 2banalysis of prognostic factorsthe effect of various prognostic factors on median os and pfs is shown in table their effect on month and month os and month month and month pfs is shown in online supplementary tables and for both tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessfigure overall survival os for the good prognostic characteristics gpc and poor prognostic characteristics ppc subgroups in patients receiving a trifluridinetipiracil or b placebo ap0001 one sided bp0001 two sided ftdtpi trifluridinetipiracil mos median overall survival nr not reachedtrifluridinetipiracil and placebo the gpc subgroup had better median os and pfs than the ppc subgroup irrespective of patient age ¥ vs years ecog ps vs kras mutation status mutant vs wild type and liver metastases yes vs nowhen analysing the gpc subgroup the absence of liver metastasis at randomisation n153 representing of the gpc and of the intent to treat population was found to be the best factor of prognosis further information on this group of patients is available in online tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessa overall survival os b progression free survival pfs and c time to eastern cooperative oncology group figure performance status ecog ps ¥ with trifluridinetipiracil versus placebo in the good prognostic characteristics gpc n386 and poor prognostic characteristics ppc n414 subgroups ftdtpi trifluridinetipiracil mos median overall survivalsupplementary table s4 and online supplementary figures s1 s3 among gpc patients treated with trifluridinetipiracil median os was months longer in patients with no liver metastases compared with those with liver metastases vs months table the month os rate in gpc patients treated with trifluridinetipiracil was in those without liver metastases and in those with liver metastases corresponding month os rates in these groups were and respectively online supplementary table s2 median os was also longer in patients with no liver metastases compared with those with liver metastases in the trifluridinetipiracil ppc subgroup vs months and both the gpc and ppc subgroups of the placebo arm vs months and vs months respectively table in the group of ppc patients treated with trifluridinetipiracil the month and month os rates were and respectively in those without liver metastases compared with and respectively in those with liver metastases online supplementary table s2 for the trifluridinetipiracil and placebo arms patients with baseline ecog ps had higher median os compared with ecog ps patients in both the gpc and ppc subgroups table in the trifluridinetipiracil arm age or ¥ years and kras status did not seem to affect the treatment outcome table similar results were found for pfs with an effect for all trifluridinetipiracil gpc and ppc subgroups with median pfs values ranging from to months table among gpc patients treated with trifluridinetipiracil the month pfs rate was in those with no liver metastases compared with in those with liver metastases corresponding month pfs rates in the ppc group of patients treated with trifluridinetipiracil were and respectively online supplementary table s3 no such effect was observed in the placebo arm with values ranging months whatever the prognosis at the outset for almost all subgroups median tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0ctable the effect of various prognostic factors on median overall survival os and progression free survival pfsnumber of patientsftdtpi placebomedian survival monthshr cinumber of patientsftdtpiplacebomedian survival monthshr ciopen accessosgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgrouppfsgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroup vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs vs vs to to to to to to to to to to to to to to to to to to to to to to to to to to to to liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age¥ yearsn124n53n110n65kras mutantn119n64n153n71liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age ¥ yearsn124n53n110n65kras mutantn119n64n153n71 vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to good prognostic characteristics gpc were defined as metastatic sites at randomisation and ¥ months from first metastasis to randomisationftdtpi trifluridinetipiracil ppc poor prognostic characteristics ecog ps eastern cooperative oncology group performance statustabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable effects of prognostic classification of patients on eastern cooperative oncology group performance status ecog psmedian time to deterioration to ecog ps ¥ monthsftdtpiplaceboitt population n80011good prognosis patients n386poor prognosis patients n414ftdtpi trifluridinetipiracil itt intent to treatpfs was longer and all hrs favoured treatment with trifluridinetipiracil table effects of prognostic classification of patients on ecog psdata relative to the effect of ecog ps are presented in table the proportion of gpc patients treated with trifluridinetipiracil with an ecog ps of at treatment discontinuation was among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of at treatment discontinuation similarly among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of at treatment discontinuation the median time to deterioration of ecog ps to ¥ in patients receiving trifluridinetipiracil was months in the gpc subgroup and months in the ppc subgroup figure 2ctolerability and safetythe most common aes in patients receiving trifluridinetipiracil were nausea anaemia neutropenianeutrophil count decrease diarrhoea fatigue and reduced appetite online supplementary table s5 the most common grade ¥ aes experienced by patients receiving trifluridinetipiracil were haematological anaemia neutropenianeutrophil count decrease white blood cell count decrease there was no evidence of a higher incidence of aes in patients with ppc versus gpc in the group receiving trifluridinetipiracil but there was a trend towards a higher incidence of aes in placebo recipients with ppc compared with gpc online supplementary table s5discussionthe results of our analysis show that patients in the gpc subgroup consistently performed better than those in the ppc subgroup in both the trifluridinetipiracil and placebo arms within the same subgroups patients treated with trifluridinetipiracil performed better than placebo trifluridinetipiracil has consistently been shown to provide a significant survival benefit to patients with mcrc refractory to standard therapy with a well tolerated safety profile in three large scale randomised clinical trials10 a previous subanalysis of recourse showed that trifluridinetipiracil was more effective than placebo in patients irrespective of region age racialhr ci to to to p valueecog ps at treatment discontinuation ftdtpiplaceboethnic differences or kras mutation status17 in the current analysis further categorisation of patients as having good prognosis using the criteria of metastatic sites by recist tumour evaluation at randomisation and ¥ months from diagnosis of first metastasis to randomisation12 identified a subgroup of patients with improved os and pfs with trifluridinetipiracil compared with poorer prognosis patients ie those with ¥ metastatic sites and months from first metastasis pfs and os were also improved in gpc patients treated with trifluridinetipiracil compared with gpc patients who received placebopatients with gpc received more cycles of treatment than patients with ppc because progression was delayed in this group which may have contributed to the better survival outcomes the difference cannot be explained by a difference in dose intensity since this was high and similar in both the ppc and gpc subgroups of patients receiving trifluridinetipiracil in addition there was no evidence for higher toxicity in the ppc than the gpc group in fact the haematological aes occurred at a slightly higher rate in gpc patients than in ppc patients who received trifluridinetipiracil which probably reflects a longer exposure to treatment in the gpc group more patients in the gpc than in the ppc subgroup had dose delays which suggests that grade ¥ haematological aes were appropriately managed during treatmentit is thought that the availability of more treatment options for mcrc has contributed to an improvement in os over the last years3 indeed a retrospective study in elderly patients aged ¥ years a patient population more prone to comorbidities poor performance status and the development of treatment related toxicity reported a correlation between os and the number of treatment lines received18 thus maintaining the general condition and performance status of a patient throughout the continuum of care is of great importance especially beyond the second line to ensure patients remain fit with good qol5 our analysis showed that the majority of patients in the gpc subgroup discontinued treatment with an ecog ps of at the time of disease progression suggesting that these patients could be candidates to receive further lines of therapy post trifluridinetipiracil this is important when sequencing through the continuum of care this is in line with other tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0canalyses indicating preservation of health related qol on treatment of patients with mcrc with trifluridinetipiracil19 while the post hoc nature of this analysis limits it to an exploratory analysis the relatively large number of patients analysed make these data a good tool to estimate the expected outcomes when treating patients with refractory mcrc with trifluridinetipiracil the smaller size of some of the subgroups may limit the s that can be drawn thus preventing an evaluation of other parameters that might impact on outcomes such as lactate dehydrogenase levels the exact definition of good and poor prognostic factors12 may require further validation in a prospective cohortthe current analysis shows that compared with poor prognosis patients treated with either trifluridinetipiracil or placebo and good prognosis patients treated with placebo patients with gpcs treated with trifluridinetipiracil adequate an function ecog ps metastatic sites by recist tumour evaluation at randomisation and ¥ months from diagnosis of first metastasis have an increased survival in terms of median os and month and month survival rates treatment with trifluridinetipiracil is effective and provides the majority of patients the opportunity to maintain ecog ps and the possibility to receive further treatment options through the continuum of careauthor affiliations1vall dhebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall dhebron institute of oncology uvic ucc iob quironmedical oncology vall d'hebron hospital barcelona catalunya spain3medical oncology ospedale policlinico san martino istituto di ricovero e cura a carattere scientifico per l'oncologia genova liguria italy4department of medical oncology university hospital centre besançon besancon bourgogne franche comté france5kashiwa national cancer center hospital east kashiwa chiba japan6department of medical oncology dana farber cancer institute boston massachusetts usa7centre of excellence methodology and valorization of data centex mvd institut de recherches internationales servier suresnes france8global medical affairs les laboratoires servier sas suresnes île de france france9digestive oncology ku leuven university hospitals leuven leuven flanders belgiumacknowledgements the authors would like to thank andrea bothwell who wrote the first draft of this manuscript on behalf of springer healthcare communications this medical writing assistance was funded by institut de recherches internationales servier suresnes francecontributors jt and srmv contributed to the conception and design of the study all authors were involved in the acquisition analysis and interpretation of data and in writing andor revising drafts of the manuscript all authors have read and approved the final draft of the manuscript and accept responsibility for the finished article and the decision to submit the manuscript for publicationfunding the recourse study was funded by taiho oncology and taiho pharmaceutical co this analysis was funded by servier in partnership with taihocompeting interests jt has received personal fees from array biopharma astrazeneca bayer ag beigene boehringer ingelheim chugai genentech open accessgenmab as halozyme imugene limited inflection biosciences limited ipsen kura oncology eli lilly and company merck menarini merck serono merrimack pharmaceuticals merus molecular partners novartis peptomyc pfizer pharmacyclics proteodesign sl rafael pharmaceuticals f hoffmann la roche sanofi seattle genetics servier symphogen taiho pharmaceutical vcn biosciences biocartis foundation medicine haliodx sas pharmaceuticals and roche diagnostics ga has had an advisory role or received honoraria or travel grants from hoffmann la roche merck serono amgen sanofi bayer servier and bristol myers squibb afs has had an advisory role for amgen bayer celgene roche merck serono sanofi and servier and has attended a speakers bureau for amgen astrazeneca bayer bristol myers squibb celgene lilly merck serono roche sanofi and takeda evc has received research funding from amgen bayer boehringer ingelheim celgene ipsen lilly merck merck kga novartis roche sanofi and servier and has attended advisory boards for astellas astrazeneca bayer bristol myers squibb celgene lilly merck sharp dohme merck kgaa novartis roche and servier cb has attended advisory boards for roche servier and sanofi and has received a research grant from roche ao has received honoraria from ono bms chugai taiho eisai and amgen and has received research funding from bristol myers squibb an immediate family member of ao has been employed by celgene rjm declares no conflicts of interest lv and srmv are employees of servierpatient consent for publication not requiredethics ap | 0 |
"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjonThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partners occupation and economic statusPatients occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatients marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patients hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patients family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0c" | 2 |
" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety health functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major pathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freunds adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o | 0 |
" postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in covid19 patientsmethods we evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans complete macroscopic and microscopicautopsies were performed on different ans in covid19 nonsurvivors presence of sarscov2 was evaluatedwith immunohistochemistry ihc in lung samples and with realtime reversetranscription polymerase chainreaction rtpcr test in the lung and other ansresults pulmonary findings revealed earlystage diffuse alveolar damage dad in out of patients andmicrothrombi in small lung arteries in patients latestage dad atypical pneumocytes andor acute pneumoniawere also observed four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedthere was no evidence of myocarditis hepatitis or encephalitis kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients spongiosis and vascular congestion were the mostfrequently encountered brain lesions no specific sarscov2 lesions were observed in any an ihc revealedpositive cells with a heterogeneous distribution in the lungs of of the patients rtpcr yielded a widedistribution of sarscov2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brains in autopsies revealed a great heterogeneity of covid19associated an injury and theremarkable absence of any specific viral lesions even when rtpcr identified the presence of the virus in many anskeywords covid19 sarscov2 autopsy rtpcr immunohistochemistry correspondence isabellesalmonerasmeulbacbe1department of pathology erasme hospital universit libre de bruxellesulb route de lennik brussels belgium2centre universitaire inter rgional dexpertise en anatomie pathologiquehospitali¨re curepath chirec chu tivoli ulb rue de borfilet 12a jumet belgiumfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cremmelink critical care page of severeacuteincluding coronavirusesrespiratorysyndrome coronavirus sarscov and middle eastrespiratory syndrome coronavirus merscov causesevere acute respiratory failure which is associated withhigh mortality rates the novel sarscov2 strainexhibits phylogenetic similaritiesto sarscov andcauses coronavirus disease covid19 which hasresulted in more than deaths worldwide so faras the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that sarscov2 can directly alter cellfunction by a link to the angiotensin converting enzyme ace2 receptor which is almost ubiquitous in thehuman body nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with covid19remain poorly understood controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease in this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of covid19 most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] the aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from covid19methodsstudy designin this postmortem study we included the first adultpatients years who died in our hospital either in acovid19 unit or an intensive care unit from march with confirmed sarscov2 infection ie positivertpcr assay on nasopharyngeal swab andor bronchoalveolar lavage specimen exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination the study protocol wasapproved by the local ethics committee p2020218data collectionrelevantwe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport acute respiratory distress syndrome ardsand acute kidney injury aki were defined accordingto standard definitions [ ]postmortem procedurethe belgian public health institute sciensano guidelines were integrated into our postmortem procedure the cadavers were kept in the refrigerator at °cand autopsies were performed to h after death toensure the safety of the autopsy team personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves ffp3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots in the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationall analyses were performed at normal pressurespleen bone marrow kidney bladderusing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking the material was biobanked by biobanqueh´pital erasmeulb be_bera1 cub h´pital erasmebbmrieric the banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain for the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken for safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe additional file additional material formalinfixed paraffinembedded ffpetissues underwentstandard processing to provide hematoxylin and eosinhestained sections special stains and immunohistochemistry ihc were used for lung massons trichromeperiodic acidschiff [pas] gomorigrocott anticmvihc antihsv ihc antipneumocystis j ihc and kidneypas massons trichromejones methenamine silversamplesmorphological analysismorphological analysis was performed on he stainedglass slides using the secundos digital platform tribvnhealth care chatillon francefor digital diagnosisafter the acquisition of whole slide digital scans magnification using a nanozoomer ht slide scanner hamamatsu hamamatsu city japansarscov2 detection by immunohistochemistrysince no antibody against sarscov2 has been validatedfor ihc on ffpe tissues we selected an antisarsnucleocapsid protein antibody standard ihc was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display sarsnucleocapsid protein invitrogen pa141098dilution on dako omnis agilent technologies 0cremmelink critical care page of santa clara ca usa using the envision flexdetection system according to the manufacturersprotocol the sections were counterstained withhematoxylin negative tissue controls were obtainedfrom patients who had an autopsy before the covid pandemic semiquantitative ihc evaluation wasperformed by two senior pathologists nd mr as follows negative between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one field sarscov2 detection by rrtpcrtotal nucleic acid was extracted from ffpe tissues usingthe maxwell rsc dna ffpe kit reference as1450promega corporation madison wi usa and thepromega maxwell extractor following the protocol described by the manufacturer onestep rtpcr assaysspecific for the amplification of sarscov2 e envelopeprotein gene were adapted from a published protocol briefly μl of rna ng was amplified in μl reaction mixture containing μl of taqman fastvirus 1step master mix life technologies μm ofeach forward acaggtacgttaatagttaatagcgt and reverse atattgcagcagtacgcacacaprimers and μm of probe famacactagccatccttactgcgcttcgbbq the amplification condition was °c for min for reverse transcriptionfollowed by °c for s and then cycles of °c for s and °c for s a clinical sample highly positivefor sarscov2 was diluted and used as a positive control in each analysis a clinical sample obtainedfrom a patient who was autopsied before the covid19pandemic was used as a negative control the quality ofthe rna from the samples showing negative results wasassessed by amplification of the human met rna according to a validated iso15189 accredited methodused as a routine diagnostic method in our laboratorystatistical analysisdata are reported as counts percentage or medians[interquartile ranges iqrs] all data were analyzedusing graphpad prism version graphpad software san diego ca usaresultsstudy cohortthe main characteristics of the study cohort malesout of median age [] years are given intable the time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days all except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n none ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza a and b viruseseleven of the patients were treated with mechanicalventilation eleven patients died in the icu and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n laboratory data are reported in additional file table s1macroscopic findingsone patient had had a left pneumonectomy and onepatient a right bilobectomy the lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage thrombi were found in the large pulmonary arteries in patients and lung infarction in patientspleural adhesions associated with pleural effusions were observed in cases we observed cardiomegaly in and hepatomegaly in patients the kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct the gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis in the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhagemicroscopic findingsfile as shown in figs and and additionaltable s2the main pulmonary findings includedearlystage diffuse alveolar damage dad which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryii pneumocytehyperplasia microthrombi were noted in the smallpulmonary arteries in patients ten of the patientsalso had advanced dad lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells we observed no viralinclusions or squamous metaplasiaand typeinfiltrateall the patients who survived more than weeksn had late dad lesions there was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct in of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi the other patients had longer 0cremmelink critical care page of table characteristics of the study populationidct scancomorbiditiesagesexrrtpcrpostime todeathantemorteman failureardsakiposnegnegmfmfmcadcvddiabeteshypertensioncadcrfliver cirrhosiscopdcancerhypertensioncancercvdcopdcancerggoposmaggopospostreatmentscause of deathmechanicalventilationantibioticshydroxychloroquineantibioticscorticosteroidsmechanical ventilationhydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationhydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmohydroxychloroquineremdesivircorticosteroidsantiobioticshydroxychloroquineantibioticscardiogenicshockmofrespiratory failurerespiratory failurerespiratory failuremesentericischemiamofrespiratory failurerespiratory failureantibioticsseptic shockmofmechanical ventilationrrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmorrthydroxychloroquineoseltamivirantibioticshydroxychloroquineantibioticsrespiratory failurerespiratory failuresudden deathmechanical ventilationhydroxychloroquineantibioticsmofhydroxychloroquinerespiratory failurehydroxychloroquineantibioticsrespiratory failureardsakihypoxichepatitisardsakiardsardsakihypoxichepatitisardsakiardsakihypoxichepatitisakiardsakiardsakiardsardsakihypoxichepatitisardsardsakihypoxichepatitismhypertensioncrfbcposmnonebcposmfhypertensioncadcvdcrfdiabeteshypertensiondiabetesemphysemaposggoposmhypertensiondiabetesggobcposmmmfdiabetesliver cirrhosiscancerdiabeteshypertensioncaddiabetesdiabeteshypertensiondiabetesbcposggoposggobcggobcpospos 0cremmelink critical care page of table characteristics of the study population continuedidcomorbiditiesct scanagesexmggolpposrrtpcrtime todeathfmhypertensiondiabetesliver transplanthypertensioncvdggobcposggobclpposantemorteman failureardsakipulmonaryembolismardsakipulmonaryembolismardsakipulmonaryembolismtreatmentscause of deathmechanical ventilationrrthydroxychloroquineremdesivirantibioticsmechanical ventilationrrthydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquineantibioticsseptic shockmofseptic shockmofseptic shockmoftime to death time from admission to death days cause of death was reported by the attending physician m male f female rrtpcr reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test neg negative pos positive cad coronary artery disease cvd cerebrovascular disease lp lobarpneumonia ggo groundglass opacity ma minor abnormalities bc bilateral consolidation copd chronic obstructive pulmonary disease crf chronic renal failureards acute respiratory distress syndrome aki acute kidney injury ecmo extracorporeal membrane oxygenation rrt renal replacement therapy mof multiplean failuredelays between hospitalization and death daysthey had no microthrombififteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in in the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisfig main histological findings green finding present gray finding absent black unavailable 0cremmelink critical care page of fig pulmonary histological findings a earlystage diffuse alveolar damage dad hyaline membrane he magnification with a zoom ona giant cell magnification b fibrin thrombi in a pulmonary artery he magnification c latestage dad fibroblastic proliferationhe magnification d latestage dad fibroblastic proliferation trichrome staining magnification e acute pneumonia he magnification f antisarscov immunohistochemistry ihcpositive cells magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryliver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate the histological changes in theabdominal ans including the esophagus stomachand colon are reported in additional file table s2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisbrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n we found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalsarscov2 detection in the lungs by ihcsarscov2 was identified by ihc in the lungs of ofthe patients fig howeverthere was largevariability in the distribution of sarscov2positivecells in the lung parenchymasarscov2 detection by rtpcrsarscov2 rna was detected in at least one anfrom every patient fig in the lung rtpcr waspositive in patients with threshold cycle ct valuesvarying from to viral rna was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples ct valuesfor nonpulmonary ans ranged from to eight patients had positive rtpcr in all tested ansabnormalitiesdiscussionthis postmortem study showed several histopathologicalin covid19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though sarscov2 was detected in all examined ans using rtpcr we decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe this approach enabled us to 0cremmelink critical care page of fig detection of sarscov2 by immunohistochemistry ihc in ffpe post mortem lung samples of patients semiquantitative evaluation negative result scattered positive cells between and positive cellswhole slide positive isolated cells cellswhole slide butno foci foci of positive cells more than positive cells in one field na not availabledocument the considerable heterogeneity of histologicallesions and of sarscov2 spread through the bodythe diagnosis of sarscov2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases in a previous autopsy study in covid19patients the authors reported that dad associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia no obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ards of many different causes especially inearlyphase ards the presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice as in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of sarscov2 infection finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to sarscov2in the absence of typical postmortem viral featuresour results show that rtpcr is feasible on ffpe blocksand could be used in postmortem analyses to identifythe presence of sarscov2 in multiple ans and tounderstand the spread of the virus within the humanbody the discordant rtpcr and ihc results fordetection of sarscov2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the rtpcr whereas lowlevel viral replication might not be detected by ihc moreover ihc wasbased on the only available antibodies which aretargeted against sarscov new antibodies againstsarscov2 need to be developed to improve theaccuracy of ihc in the analysis of tissue samples fromsuspected or confirmed covid19 patientsmost of the previous postmortem studies in covid19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofsarscov2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations as for previous coronavirusdiseases [ ] the lungs are the most affected ans incovid19 however dad findings werehighly 0cremmelink critical care page of fig molecular detection of sarscov2 rna in postmortem samples detection of sarscov2 by reverse transcription realtime polymerasechain reaction rtpcr in ffpe postmortem tissues of patients positive result negative result na tissue not available nc noninformative test result due to lowquality rnaheterogeneous including both earlyonset and additionallate lesions this finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase as some patients died outsidethe icu without receiving mechanical ventilation we couldnot estimate lung compliance before death the heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the dadreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections in a recent report needle postmortem biopsies suggested that covid19 is not associated withdad but rather with an acute fibrinous and anizingpneumonia afop consequently requiring corticoid treatment a diagnosis of afop is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with dad and complete lunganalysis not just biopsies are necessary to exclude theirpresence moreover afop may be a fibrinous variant ofdad the limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for sarscov2 using rtpcr when compared to almost in our series in addition we did notfind specific endothelitis as previously reported in a smallcase series considering the heterogeneity of postmortem covid19 associated lesions molecular and ihcassessments are mandatory in the histological analysis ofcovid19 tissue samplespatients with covid19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism pe in ourstudy three patients had pe already diagnosed beforedeath four patients had pulmonary infarction in a previous study acute pe was considered as the main causeof death in four patients however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of pewhen compared to our study although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofards regardless of etiology [ ] as such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in covid19 0cremmelink critical care page of patients remains to be further studied in a systematicreview of pathologicalfindings in covid19 polak identified a timeline in the histopathologicalfindings in the lung with epithelial dad denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease our resultsare consistent with those of polak except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients we did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis the cases ofacute cardiac injury reported in covid19 clinicalstudies do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia however using rtpcr we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theace2 receptor which is ubiquitous but may notdirectly cause an injury as extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatsarscov2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in covid19associated an dysfunction asrtpcr mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance just detect residual viral genomethis study has several limitations i we only includedpatients who had had a positive rtpcr on nasopharyngeal swab andor bronchoalveolar lavage to ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic ctscan findings suggestive of covid19 but had negativertpcr results this limitation in our study reflects thedifficulty of diagnosing covid19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death this delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow sarscov2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related tocovid19 finally we did not evaluate specific mechanisms involved in the pathogenesis of an injurythese results underline the heterogeneity of an injuriesduring covid19 disease and the absence of specificsarscov2 lesions using rtpcr sarscov2 couldbe detected in all ans even those without evidentmicroscopic lesionssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13054020032185additional file critical careautopsycovid additional materialprocedure to obtain brain samplesadditional file critical careautopsycovid additional table s1laboratory findings on the day of admissionadditional file critical careautopsycovid additional table s2detailed histological findings in all patientsabbreviationsace2 angiotensin converting enzyme afop acute fibrinous andanizing pneumonia aki acute kidney injury ards acute respiratorydistress syndrome covid19 coronavirus disease ct threshold cycledad diffuse alveolar damage ffpe formalinfixed paraffinembeddedhe hematoxylin and eosin ihc immunohistochemistry iqrs interquartileranges merscov middle east respiratory syndrome coronaviruspas periodic acidschiff pe pulmonary embolism rtpcr realtime reversetranscription polymerase chain reaction sarscov severe acute respiratorysyndrome coronavirusacknowledgmentsthe authors thank nathalie lijsen christophe valleys gees lacroixbarbara alexiou dominique penninck nicole haye and audrey verrellen fortechnical and logistic supports prof frdric schuind and dr djameleddineyahiacherif for neurosurgical procedure egor zindy diapath ulb forproofreading the paper and dr mariepaule van craynest for traineessupervisionauthors contributionsis had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis is ft jlv and cd drafted the paper mrcv ll pl mlr cm alt jcg lp rdm sd sr nd lp and od collected thedata mr nd and rdm did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published all authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedfundingthis study received financial support from fonds y bo«l brussels belgiumfonds erasme pour la recherche mdicale brussels belgium and appel projet spcial covid19 ulb brussels belgium the cmmi is supported bythe european regional development fund and the walloon region ofbelgium walloniabiomed grant no project cmmiulbsupport the center for microscopy and molecular imaging and its diapathdepartment cd is a senior research associate with the fnrs belgiannational fund for scientific research 0cremmelink critical care page of availability of data and materialsthe data that support the findings of this study are available from thecorresponding author on reasonable request participant data without namesand identifiers will be made available after approval from the correspondingauthor and local ethics committee the research team will provide an emailaddress for communication once the data are approved to be shared withothers the proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data additional materials may also be required during theprocess d'haene n melndez b blanchard o de n¨ve n lebrun l vancampenhout c design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas cancersbasel corman vm landt o kaiser m molenkamp r meijer a chu dk detection of novel coronavirus 2019ncov by realtime rtpcr eurosurveill de hemptinne q remmelink m brimioulle s salmon i vincent jl ards aclinicopathological confrontation chest menter t haslbauer jd nienhold r savic s hopfer h deigendesch n ethics approval and consent to participatethe study protocol was approved by the local ethics committee erasmehospital p2020218 the ethical committee has waived the need for writteninformed consentpostmortem examination of covid19 patients reveals diffuse alveolardamage with severe capillary congestion and variegated findings of lungsand other ans suggesting vascular dysfunction histopathology epub ahead of print httpsdoi101111his14134franks tj chong py chui p galvin jr lourens rm reid ah lungpathology of severe acute respiratory syndrome sars a study of autopsy cases from singapore hum pathol nicholls jm poon ll lee kc ng wf lai st leung cy lungpathology of fatal severe acute respiratory syndrome lancet hwang d chamberlain d poutanen s low de asa sl butany j pulmonarypathology of severe acute re | 0 |
continuously increasing with development of the economy and the environment [] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrnae1e2abcbasepairinge3dguriche4ecrichpretrnarna bindingproteinsrbpgnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the hydroxyl of the upstream exon reacts with the phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the hydroxyl of the intron interacts with the phosphate of the intron to form an ecircrna following an interaction between the hydroxyl of the exon and the phosphate of the exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimers disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimers disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir in gastric cancer j biochem song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelialmesenchymal transition in colon cancer cells front biosci landmark ed verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol wei j wei w xu h circular rna hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol constantin l circular rnas and neuronal development adv exp med biol van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity 701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippoyap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol idda ml munk r abdelmohsen k gorospe m noncoding rnas in 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65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int zhao j oneil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in 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circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c' | 0 |
Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [] A majority of adult patients with t816p112p133 are therapy related [] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturers instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Childrens Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patients DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturers protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patients DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent à a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilents CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of à 109L with blasts a hemoglobin count of a0 gL and a platelet count of à 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [] Among them cases showed a gain by 1q of variable sizes [ ] As an uncommon entity t816 accounts for of all cases of AML [] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB FrenchAmericanBritishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin Peoples Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr Luppi M Fabbiano F Visani G Martinelli G Venditti A Novel agents for acute myeloid leukemia Cancers Basel Cancer Research UK Acute myeloid leukaemia AML incidence statistics https wwwcance rrese archu khealt hprofe ssion alcance rstati stics stati stics bycance rtypeleuka emiaamlincid ence Accessed Aug Jung J Cho BS Kim HJ Han E Jang W Han K Lee JW Chung NG Cho B Kim M Kim Y Reclassification of acute myeloid leukemia according to the WHO classification Ann Lab Med Murphy T Yee KWL Cytarabine and daunorubicin for the treatment of acute myeloid leukemia Expert Opin Pharmacother Byrd JC Mrózek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B CALGB Blood Lindsley RC Mar BG Mazzola E et al Acute myeloid leukemia ontogeny is defined by distinct somatic mutations Blood Vardiman JW Thiele J Arber DA et al The revision of the World Health anization WHO classification of myeloid neoplasms and acute leukemia rationale and important changes Blood Saultz JN Garzon R Acute myeloid leukemia a concise review J Clin Med Döhner H Weisdorf DJ Bloomfield CD Acute myeloid leukemia N Engl J Med Strickland SA Shaver AC Byrne M et al Genotypic and clinical heterogeneity within NCCN favorablerisk acute myeloid leukemia Leuk Res Yang JJ Park TS Wan TSK Recurrent cytogenetic abnormalities in acute myeloid leukemia Methods Mol Biol Coenen EA Zwaan CM Reinhardt D et al Pediatric acute myeloid leukemia with t816p11p13 a distinct clinical and biological entity a collaborative study by the InternationalBerlinFrankfurtMunster AMLstudy group Blood Xie W Hu S Xu J Chen Z Medeiros LJ Tang G Acute myeloid leukemia with t816p112p133KAT6ACREBBP in adults Ann Hematol Haferlach T Kohlmann A Klein HU et al AML with translocation t816p11p13 demonstrates unique cytomorphological cytogenetic molecular and prognostic features Leukemia Gervais C Murati A Helias C et al Acute myeloid leukaemia with 8p11 MYST3 rearrangement An integrated cytologic cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique Leukemia Diab A Zickl L AbdelWahab O et al Acute myeloid leukemia with translocation t816 presents with features which mimic acute promyelocytic leukemia and is associated with poor prognosis Leuk Res Schouten TJ Hustinx TW Scheres JM Holland R de Vaan GA Malignant histiocytosis Clinical and cytogenetic studies in a newborn and a child Cancer Brown T Swansbury J Taj MM Prognosis of patients with t816p11p13 acute myeloid leukemia Leuk Lymphoma Barrett R Morash B Roback D et al FISH identifies a KAT6ACREBBP fusion caused by a cryptic insertional t816 in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype Pediatr Blood Cancer Schumacher J Szankasi P Kelley TW Detection and quantification of acute myeloid leukemiaassociated fusion transcripts Methods Mol Biol DÃazBeyá M Navarro A Ferrer G et al Acute myeloid leukemia with translocation 816p11p13 and MYST3CREBBP rearrangement harbors a distinctive microRNA signature targeting RET protooncogene Leukemia Veigaard C Nørgaard JM Kjeldsen E Genomic profiling in high hyperdiploid acute myeloid leukemia a retrospective study of cases Cancer Genet Yasar D Karadogan I Alanoglu G et al Array comparative genomic hybridization analysis of adult acute leukemia patients Cancer Genet Cytogenet van der Veken LT Buijs A Array CGH in human leukemia from somatics to genetics Cytogenet Genome Res Laskowska J Szczepanek J StyczyÅski J Tretyn A Array comparative genomic hybridization in pediatric acute leukemias Pediatr Hematol Oncol Mehrotra M Luthra R Ravandi F et al Identification of clinically important chromosomal aberrations in acute myeloid leukemia by arraybased comparative genomic hybridization Leuk Lymphoma Kulikowski LD Bellucco FTS Nogueira SI et al Pure duplication 1q41qter further delineation of trisomy 1q syndromes Am J Med Genet A 2008146A2663 Nowaczyk MJM Bayani J Freeman V Watts J Squire J Xu J De novo 1q32q44 duplication and distal 1q trisomy syndrome Am J Med Genet A 2003120A229 Cone EB Dalton SS Van Noord M Tracy ET Rice HE Routh JC Biomarkers for wilms tumor a systematic review J Urol Xu B Ghossein R Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma Endocr Pathol Fournier A Florin A Lefebvre C Solly F Leroux D Callanan MB Genetics and epigenetics of 1q rearrangements in hematological malignancies Cytogenet Genome Res Lancman G Tremblay D Barley K et al The effect of novel therapies in highmolecularrisk multiple myeloma Clin Adv Hematol Oncol Bacher U Schnittger S Grüneisen A Haferlach T Kern W Haferlach C Inverted duplication dup1q32q21 as sole aberration in lymphoid and myeloid malignancies Cancer Genet Cytogenet Marcellino BK Hoffman R Tripodi J et al Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53 Blood Adv Beach DF Barnoski BL Aviv H et al Duplication of chromosome [dup1q21q32] as the sole cytogenetic abnormality in a patient previously treated for AML Cancer Genet Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | 2 |
"Lung carcinoma is a prominent cause of mortality among patients with cancer Previous studies have reported the vital role of long noncoding RNAs lncRNAs in the malignant progression of lung cancer lncRNA RP11284F219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown Therefore the present study aimed to elucidate the role of lncRNA RP11284F219 in lung carcinoma progression The expression of RP11284F219 in lung cell lines and tissues was measured using reverse transcriptionquantitative PCR The endogenous expression of RP11284F219 was silenced using RNA interference and cell viabilities were measured with a Cell Counting Kit assay The invasion and apoptosis of cells were determined via Transwell assays and flow cytometry respectively The protein expression levels were measured by western blotting An increased expression of RP11284F219 was identified in both lung carcinoma tissues and cells Knockdown of RP11284F219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis The present study identified the existence of a direct interaction between RP11284F219 and microRNA miRNAmiR6273p Mechanistically it was demonstrated that RP11284F219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of miR6273p Furthermore cell division cycle and apoptosis regulator CCAR1 was identified as a target gene of miR6273p The in vivo tumor growth assay also demonstrated that the knockdown of RP11284F219 suppressed tumor growth upregulated miR6273p and downregulated Correspondence to Dr Yuan Wang Department of Medical Imaging The First Affiliated Hospital of Xi'an Jiaotong University West Yanta Road Xi'an Shaanxi PR ChinaEmail wangyuan8003126comAbbreviations CCAR1 cell division cycle and apoptosis regulator NSCLC nonsmall cell lung cancer SCLC small cell lung cancerKey words RP11284F219 lung carcinoma proliferation invasion microRNA6273p CCARCCAR1 in the xenograft model of nude mice Thus the present findings indicated the tumor promoting functions of RP11284F219 in the progression of lung carcinoma and provided a novel lncRNAmiRNA axis as a target for the management of lung cancerIntroductionPulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women Furthermore of lung cancer cases are categorized as nonsmall cell lung cancer NSCLC while the remaining are classified as SCLC Although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival Therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentialLong noncoding RNAs lncRNAs are nucleotides in length and have little or no protein coding capacity The mechanisms via which lncRNAs regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small RNAs generating different splice variants via regulating mRNA splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes In recent years previous studies have reported that various human cancer types exhibit lncRNAs dysfunction and these lncRNAs are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells In lung cancer lncRNA metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncRNA HOX transcript antisense RNA is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with NSCLC Furthermore studies have shown that the expression of lncRNA Urothelial carcinomaassociated 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONis significantly upregulated in NSCLC and may induce resistance to treatment of EGFRtyrosine kinase inhibitors by activating the AKTmTOR pathway lncRNA RP11284F219 was primarily discovered in a Pancancer transcriptomic analysis lncRNA RP11284F219 exists as a cluster of three annotated lncRNAs RP11284F219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas However the specific function and the underlying mechanism of RP11284F219 in lung carcinoma remain unknownTo the best of our knowledge the present study demonstrated for the first time that lncRNA RP11284F219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer Together with microRNA miRNAmiR6273p and cell division cycle and apoptosis regulator CCAR1 the regulatory axis of RP11284F219miR3pCCAR1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo The present study aimed to investigate RP11284F219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the RP11284F219miR3pCCAR1 axisMaterials and methodsTissue samples and cell lines Between May and Jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range years nine male patients four female patients who were diagnosed and treated in First Affiliated Hospital of Xi'an Jiaotong University The samples were dissected during the surgery and immediately flashfrozen in liquid nitrogen and transferred to ËC storage for further extraction of both RNA and protein All the tissue samples were obtained with written informed consent from the patients The protocol was approved by The First Affiliated Hospital of Xi'an Jiaotong University approval no A normal lung epithelial cell line BEAS2B and lung carcinoma cell lines NCIH460 NCIH1299 and A549 were purchased from American Type Culture Collection ATCC and cultured according to the ATCC guidelines 293T cells were purchased from Procell Life ScienceTechnology Co Ltd and cultured in DMEM supplemented with FBS cat no ATCC and 1X Penicillinstreptomycin Thermo Fisher Scientific Inc BEAS2B cells were cultured in bronchial epithelial growth medium BEGM cat no CC Clonetics Corporation according to the manufacturer's instructions NCIH460 and NCIH1299 cells were cultured in RPMI medium cat no ATCC and A549 cells in F12K medium cat no ATCC supplemented with FBS cat no ATCC and 1X Penicillinstreptomycin Thermo Fisher Scientific Inc All cells were culture at ËC with CO2RNA extraction and reverse transcriptionquantitative PCR RTqPCR Total RNA from both tissue samples and cell lines were extracted using TRIzol® reagent Invitrogen Thermo Fisher Scientific Inc For each sample ng total RNA was reverse transcribed to synthesize the firststrand cDNA using the PrimeScript RT reagent kit Takara Bio InccDNA samples were diluted times to perform the RTqPCR using SYBR Premix Ex Taq Takara Bio Inc on a CFX96 realtime PCR detection system BioRad Laboratories Inc Expression levels of mRNAs lncRNAs and miRNAs were normalized to GAPDH The primers used for RTqPCR analyses were as follows GAPDH forward 'AAC GAC CCC TTC ATT GAC C' and reverse 'TCC ACG ACA TAC TCA GCA CC' RP11284F219 forward 'AGG ATT GGC ACT CAC TTC GG' and reverse 'TCT CTC ACC ACG TCT GGT CT' and CCAR1 forward 'CTG ATG GCT AGC CCT AGT ATG GA' and reverse 'TGC CTT TCA TGC CCA CTA AAA ' The temperature protocol used to perform RT was ËC for h followed by ËC for min Thermal conditions of PCR reactions were Initial denaturation at ËC for min followed by cycles for sec at ËC and sec at ËC The mRNA expression levels were determined using the 2ÎÎCq method Oligonucleotides and cell transfection The small interfering RNA siRNA synthetic negative control siNC RP11284F219 siRNAs siRP11284F219 miRNC miR3p mimics and miR3p inhibitor were purchased from Shanghai GenePharma Co LtdAll primer sequence information is presented in Table I At a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency All of the oligonucleotides were transfected at a final concentration of nM using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instruction Cells were collected at h posttransfection for subsequent experimentsCell Counting Kit CCK assay and EdU labeling of proliferating cells A CCK was used for cell proliferation assay the cells were seeded into well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions Dojindo Molecular Technologies Inc The optical density was measured at nm using a spectrophotometer Thermo Fisher Scientific IncFor the EdU assay cells were incubated with µM EdU cat no ab219801 Abcam for h at ËC and fixed with formaldehyde at room temperature for min After a brief washing with PBS click reagent was added into each well and incubated in the dark for min at room temperature Followed by PBS washing the cells were stained with µgml DAPI at room temperature for min Images were captured using a fluorescence microscope Nikon Corporation and measured using Adobe Photoshop software Adobe Systems Inc The EdU labeled cells were analyzed with MoFlo Astrios BeckmanCoulter Inc Magnification x200Transwell assay and flow cytometry measurement of cell apoptosis Transwell assays were performed with a coating of Matrigel BD Biosciences mixed with culture medium mixed at ratio at ËC for h A total of 1x105 cells in µl serumfree medium were added to the upper layer of the Transwell chambers µm pore size Corning Inc and cultured for h The lower chamber contained the culture medium with FBS The migrated cells were fixed with 0cONCOLOGY REPORTS Table I Sequence of siRNAs and miRNA mimics and inhibitorsOligonucleotides siNC siRP11284F219 miRNC miR3p mimics miR3p inhibitor miR microRNA siRNA small interfering RNA NC negative controlSequence ''UUCUCCGAACGUGUCACGUTTUAUUGGCACCAAGGAUAGCUCGUUAAUCGGCUAUAAUACGCUCUUUUCUUUGAGACUCACUUCUUUUCUUUGAGACUCACU paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope Magnification x200Cellular apoptosis was detected using the Apoptosis Detection kit cat no KGF001 Nanjing KeyGen Biotech Co Ltd according to the manufacturer's instructions Cells were stained with fluorescein isothiocyanateconjugated annexin V and PI After incubated for min at ËC in the dark µl 1X Binding Buffer was added to each tube and stained cells were analyzed using BD FACS Canto II flow cytometry FACS Calibur BD Biosciences Data were analyzed using FlowJo software version Tree Star IncLuciferase reporter assay The RP11284F219 wildtype wt or mutant mut 'untranslated region 'UTR and CCAR1 wt or mut 'UTR sequences were cloned into the pmirGLO plasmid Youbio httpwwwyoubiocn cat no VT1439 The vectors µgml were cotransfected with miRNC or miR6273p mimic nM and Renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc Cell lysates were collected at h after transfection and the luciferase activities were detected with the DualLuciferase Reporter Assay system Promega Corporation according to the manufacturer's instructionsWestern blotting Cell were lysed using RIPA lysis buffer SigmaAldrich Merck KGaA and protein concentrations were assessed with the BCA Protein Assay kit according to the manufacturer's instructions Beyotime Institute of Biotechnology Shanghai China Equal amounts µg of cell protein lysates were loaded and separated by SDSPAGE transferred to a PVDF membrane and blocked with nonfat milk at room temperature for h The membranes were then incubated with CCAR1 primary antibody cat no ab70243 Abcam overnight at ËC followed by incubation with goat antimouse or goat antirabbit IgGhorseradish peroxidase conjugate secondary antibodies cat no ab205718 Abcam at room temperature for h GAPDH cat no ab181602 Abcam was used as loading control The signals were detected using the ECL system Protein Simple according to the manufacturer's instructionsIn vivo tumorigenicity analysis in mice Male BALBc nude mice age weeks weight g were obtained from Beijing Vital River Laboratory Animal Technology Co Ltd and housed at a room temperature of ËC with a h lightdark cycle The mice were maintained in an individually ventilated cage system under specific pathogenfree conditions temperature ËC humidity and fed with sterile food and water free access To evaluate the effect of RP11284F219 knockdown on the growth of lung carcinoma in vivo 5x106 siNC or siRP11284F219 treated NCIH1299 cells in µl serumfree medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin The animals were monitored daily and the following criteria for humane endpoint was used Severe tumor burden mm in diameter difficulty breathing significant bodyweight loss and clinical signs such as prostration hypothermia and significant abdominal distension Tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] Then weeks after inoculation the mice were euthanized by CO2 inhalation CO2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat The xenografts were imaged and weighedThe total RNA was then extracted from the xenografts as aforementioned Animal care and study were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi'an Jiaotong University approval no Target prediction Potential target miRNAs of RP11284F219 were predicted using LncBase V2 httpcarolinaimisathena innovationgrdiana_toolswebindexphprlncbasev2index The target genes of miR3p were predicted using three bioinformatics algorithms TargetScanV72 httpwwwtargetscanorgvert_72 and miRDB httpwwwmirdborgmininghtmlStatistics analysis Data were analyzed using the GraphPad Prism software GraphPad Software Inc and presented as the mean ± SD from ¥ independent experiments A twotailed unpaired Student's ttest or oneway ANOVA with Tukey's posthoc analysis were performed to evaluate the statistical significance P005 was considered to indicate a statistically significant difference\x0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11284F219 expression is upregulated in LC tissues and cell lines A Expression of RP11284F219 in LC tissues in comparison with adjacent healthy tissues was analyzed using RTqPCR P0001 vs adjacent tissues n13 B Expression of RP11284F219 in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 compared with normal human lung epithelial cell line BEAS2B was analyzed using RTqPCR P005 P0001 vs BEAS2B n3 LC lung carcinoma RTqPCR reverse transcriptionquantitative PCRResultsExpression of RP11284F219 is upregulated in lung carcinoma To investigate the potential role of RP11284F219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma The results demonstrated that the expression of RP11284F219 was significantly upregulated in tumor tissues compared with healthy tissues Fig 1A The expression of RP11284F219 was also analyzed in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 and normal human lung epithelial cell line BEAS2B Consistent with the findings in the tissue samples the expression of RP11284F219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line Fig 1B These results indicated that RP11284F219 may serve an oncogenic role in lung carcinomaKnockdown of RP11284F219 exerts antioncogenic effects in lung carcinoma cells To study the specific role of RP11284F219 in lung carcinoma cells RP11284F219 siRNA was transfected into NCIH1299 and NCIH460 cells Fig 2A After transfection the proliferation of these cells was measured using CCK and EdU assays Fig 2BD The results suggested that knocking down RP11284F219 significantly reduced the proliferation of lung carcinoma cells compared with the NC group Fig 2BD The invasiveness of siRP11284F219 transfected cells also significantly decreased as indicated by the data from the Transwell assay Fig 2F To further validate the invasive capability a RTqPCR assay was performed to detect the expression levels of invasionrelated genes and the results identified that both MMP2 and MMP9 were significantly decreased when RP11284F219 was downregulated Fig S1The results of flow cytometry measurement based apoptosis assay suggested that cells transfected with siRP11284F219 had a higher apoptotic rate compared with the siNC transfected group Fig 2E These data demonstrated the antitumor effects of RP11284F219 knockdown in lung carcinoma cells indicating an oncogenic role of RP11284F219RP11284F219 directly interacts with miR3p Based on the prediction of the online tool lncBase v2 from DIANA Prediction module httpcarolinaimisathenainnovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream miRNAs of RP11284F219 the first five miRNAs in the output list were tested Among the predicted potential targets it was found that miR6273p had the most significant upregulation in NCIH1299 cells transfected with siRP11284F219 Fig S2Using sequence alignment it was identified that miR3p was partially complementary with the 'UTR of RP11284F219 Fig 3A Subsequently 293T cells were transfected with the pmirGLORP11284F219wt or mut vector containing the wt or mut sequence of RP11284F219 'UTR with or without miR3p mimics Results from the luciferase reporter assay suggested that miR6273p mimics significantly decrease the signal of RP11284F219wt transfected cells but not the RP11284F219mut transfected cells indicating a direct interaction between the two noncoding RNAs Fig 3A Furthermore transfection of siRP11284F219 into NCIH1299 and NCIH460 cells resulted in the suppression of endogenous RP11284F219 leading to a significant increase in miR3p expression Fig 3B Thus these findings suggested an inhibitory effect of RP11284F219 on the expression of miR3p in lung carcinoma cellsThe expression of miR3p was detected in both lung carcinoma tissues and cell lines It was demonstrated that miR3p was significantly downregulated in carcinoma tissues Fig 3C and NCIH460 NCIH1299 and A549 cells Fig 3D compared with healthy tissues and cells Collectively these data suggested a direct interaction between RP11284F219 and miR6273p in which RP11284F219 suppresses the expression of miR3pRP11284F219 regulates the proliferation and invasiveness of lung carcinoma cells via miR3p To rescue the antitumor effects of siRP11284F219 in lung carcinoma cells the miR3p inhibitor which specifically downregulates the expression of miR3p was transfected into NCIH1299 and NCIH460 cells Fig 4A The results from the CCK and EdU assays demonstrated that treatment with siRP11284F219 0cONCOLOGY REPORTS Figure RP11284F219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis A RP11284F219 knockdown was achieved via RP11284F219 siRNA and the knockdown efficiency was verified using reverse transcriptionquantitative PCR n3 Cell Counting Kit assay was performed to measure the proliferation of B NCIH1299 and C NCIH460 cells after transfection with siRP11284F219 compared with the siNC group n5 D An EdU assay was performed to measure the proliferation of NCIH1299 and NCIH460 cells after transfection with siNC and siRP11284F219 Magnification x200 E Flow cytometry analysis was performed to determine the effects of RP11284F219 knockdown on apoptotic rates in NCIH1299 and NCIH460 cells n3 F Transwell assay was performed to determine the effects of RP11284F219 knockdown on NCIH1299 and NCIH460 cell invasion n3 Magnification x200 P005 P001 vs control group NC negative control siRNA small interfering RNA OD optical density and miRNC significantly decrease the proliferation of both NCIH1299 and NCIH460 cells Fig 4BD However the administration of miR3p inhibitor partially reversed the antiproliferative effect of siRP11284F219 indicating that RP11284F219 regulates the proliferation of lung carcinoma cells partially via miR6273p Fig 4BD In addition the 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11284F219 directly interacts with miR3p A Binding site between RP11284F219 and miR3p that was identified using the DIANA tools and a luciferase reporter assay was conducted in pmirGLORP11284F219wt or mut treated cells in the presence of miR6273p mimics or miRNC n3 P005 vs miRNC B Expression of miR3p in NCIH1299 and NCIH460 cells transfected with siRP11284F219 was analyzed using RTqPCR P001 vs siNC n3 miR3p expression in C LC tissues and D NCIH460 NCIH1299 and A549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using RTqPCR n3 P005 P001 vs adjacent tissue or BEAS2B cells NC negative control siRNA small interfering RNA wt wildtype mut mutant miR microRNA LC lung carcinoma miR3p inhibitor restored the reduction in the number of NCIH1299 and NCIH460 cells that migrated through the Transwell membrane induced by siRP11284F219 treatment Fig 4F These data indicated the participation of miR6273p in the RP11284F219mediated invasive effectThe qPCR assay results identified that both MMP2 and MMP9 expression levels were restored in RP11284F219downregulated cells when miR6273p was inhibited compared with the miRNC group Fig S3 In addition transfection with miR3p inhibitor also diminished the proapoptosis effect of siRP11284F219 in both NCIH1299 and NCIH460 cells Fig 4E Therefore it was suggested that RP11284F219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of miR3pRP11284F219 regulates CCAR1 via targeting miR3p To further evaluate how RP11284F219 exerts an oncogenic role via miR3p the publicly available algorithms of TargetScan httpwwwtargetscanorg and miRDB were used which identified CCAR1 as a potential target for miR6273p Fig 5A In order to validate this prediction miR6273p mimic was transfected into cells and the transfection efficiency was assessed The results demonstrated that transfection of miR6273p mimic increased the expression of miR3p by times compared with cells transfected with miRNC Fig S4After validating the upregulation of miR6273p mimic a CCAR1wt vector was constructed which contained the wt binding site between miR3p and the CCAR1 'UTR and CCAR1mut vector containing the mut sequence Fig 5A The results from luciferase reporter assays indicated that compared with the miRNC group the miR6273p mimic significantly decreased the luciferase activity of CCAR1wt treated cells but not the CCAR1mut treated cells suggesting a direct binding of miR3p to the 'UTR of CCAR1 Fig 5B Increased expression levels of CCAR1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues Fig 5C Moreover a significant decrease in both mRNA and protein expression levels of CCAR1 was detected upon transfecting NCIH1299 and NCIH460 cells with miR6273p mimics Fig 5D and E Thus CCAR1 may be a direct target of miR3p in lung carcinoma cells and tissuesRP11284F219 knockdown inhibits tumor growth and the expression of CCAR1 in vivo In order to investigate the effect of RP11284F219 on in vivo tumorigenicity NCIH1299 cells were transfected with siNC or siRP11284F219 and injected into the nude mice After weeks a significantly 0cONCOLOGY REPORTS Figure RP11284F219 regulates proliferation and invasiveness in lung carcinoma cells via miR3p A Expression of miR3p in NCIH1299 and NCIH460 cells transfected with miRNC or miR3p inhibitor was detected using RTqPCR analysis n3 P005 vs miRNC Cell Counting Kit assay was performed in B NCIH1299 and C NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor n5 D EdU assay was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor Magnification x200 E Flow cytometry analysis was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor n3 F Transwell assay was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor Magnification x200 n3 P005 vs siNC NC negative control siRNA small interfering RNA OD optical density miR microRNA 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure miR3p directly targets CCAR1 A Bioinformatic analysis of the predicted binding sites between the CCAR1 'untranslated region and miR3p B Luciferase reporter assay in CCAR1wt or CCAR1mut treated cells in the presence of miRNC or miR3p mimic n3 P005 vs miRNC C CCAR1 expression in LC tissues compared with adjacent healthy tissues was analyzed using RTqPCR n13 P001 vs adjacent tissue Expression of CCAR1 in NCIH1299 and NCIH460 cells transfected with miRNC or miR3p mimics was detected using D RTqPCR and E western blotting n3 P005 vs miRNC miR microRNA NC negative control wt wildtype mut mutant RTqPCR reverse transcriptionquantitative PCR CCAR1 cell division cycle and apoptosis regulator LC lung carcinoma slower proliferative rate of the tumors was observed in the siRP11284F219 group compared with the siNC group Fig 6A and B Furthermore the tumor volume and weight were significantly decreased in the siRP11284F219 group compared with the control group Fig 6A and B RTqPCR analysis also demonstrated that compared with the siNC group the tumors in the siRP11284F219 group expressed higher levels of miR6273p Fig 6C and lower levels of CCAR1 Fig 6D providing further evidence to the existence of the RP11284F219miR3pCCAR1 regulatory axis in lung carcinoma tumor tissuesDiscussionThe present study investigated the function of RP11284F219 in lung carcinoma It was initially found that RP11284F219 was significantly upregulated in both lung cancer tissues and cell lines Following the deduction of a potential oncogenic role of this lncRNA siRP11284F219 was transfected into NCIH460 and NCIH1299 cells and it was demonstrated that knockdown of RP11284F219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells In the mechanistic studies using online prediction tools and in vitro assays the results indicated that miR3p directly interacts with RP11284F219 by binding to its 'UTRThe function of miR627 was initially reported in colorectal cancer CRC Padi found that when upregulated by calcitriol miR627 targets the histone demethylase Jumonji domain containing 1A to increase methylation of histone H3K9 and suppresses the proliferative factors of CRC cells thus inhibiting the proliferation of CRC both in vitro and in vivo Moreover in CRC Sun discovered the role of miR in vitamin Denhanced efficacy of irinotecan via inhibition of the cytochrome P450 enzymemediated intratumoral drug metabolism miR is also reported to be a potential noninvasive diagnostic marker in gastric and breast cancer types In pulmonary diseases miR627 is downregulated in patients with chronic obstructive pulmonary disease and targets the highmobility group box protein to inhibit its expression thus improving transforming growth factorβ1induced pulmonary fibrosis The present results demonstrated the inhibitory effect of RP11284F219 on the expression of miR3p In addition it was identified that the miR3p inhibitor can neutralize the antitumor effects of RP11284F219 knockdown indicating that RP11284F219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating miR3p This antitumor role of miR6273p under the regulation of RP11284F219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human CRC gastric and breast cancer types\x0cONCOLOGY REPORTS Figure RP11284F219 knockdown inhibits tumor growth in vivo A Macroscopic image of xenografted tumors B Tumor volume in nude mice injected with NCIH1299 cells transfected with siNC or siRP11284F219 measured over weeks n5 C Weight of tumors in nude mice at weeks after injection of NCIH1299 cells transfected with siNC or siRP11284F219 n5 Expression levels of D miR3p and E CCAR1 in the tumor tissues from nude mice injected with NCIH1299 cells transfected with siNC or siRP11284F219 for weeks were detected using reverse transcriptionquantitative PCR n5 P005 P001 P0001 vs siNC miR microRNA NC negative control sh short hairpin RNA CCAR1 cell division cycle and apoptosis regulator Using the publicly available RNA interaction prediction algorithms the current study identified that CCAR1 which was initially shown as the target gene of miR6273p is also regulated by RP11284F219 Furthermore the regulatory axis of RP11284F219miR3pCCAR1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model The interaction between RP11284F219 and miR3p and the interaction between miR3p and CCAR1 were demonstrated by the dualluciferase assay Although this method has been used to validate RNARNA interactions in previous studies other assays such as RNA pulldown and RNA binding protein immunoprecipitation that would provide more direct evidence for the RNARNA and RNAprotein interactions should be performedCCAR1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as Adriamycin and etoposide Subsequently Kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors In human breast cancer cells as CCAR1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells In CRC cells Ou reported that CCAR1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor CCAR1 is essential for the expression of Wnt target genes as well as the neoplastic transformation of CRC cells In gastric cancer cells researchers have revealed the cooperation between CCAR1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells In lung cancer CCAR1 was reported to be an effector of Doxorubicininduced apoptosis Moreover Muthu demonstrated that certain chemical compounds that bind with CCAR1 can increase the expression of CCAR1 and induce apoptosis However a contradictory conclusion was reported in a recent study which observed that CCAR1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate Corroborating this finding in the current study via the targeting of miR3p the expression of CCA | 2 |
the european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food this risk assessment coversedible parts of potato plants and other food plants containing gastomato andaubergine in humans acute toxic effects of potato gas asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea for these effects the contampanel identiï¬ed a lowestobservedadverseeffect level of mg total potato gaskg body weight bwper day as a reference point for the risk characterisation following acute exposure in humans noevidence of health problems associated with repeated or longterm intake of gas via potatoes hasbeen identiï¬ed no reference point for chronic exposure could be identiï¬ed from the experimentalanimal studies occurrence data were available only for asolanine and achaconine mostly forpotatoes the acute dietary exposure to potato gas was estimated using a probabilistic approach andapplying processing factors for food due to the limited data available a margin of exposure moeapproach was applied the moes for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the p95 exposure in all surveysfor adult age groups the moes indicate a health concern only for the food consumption surveys withthe highest p95 exposures for tomato and aubergine gas the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data for horses farm andcompanion animals no risk characterisation for potato gas could be performed due to insufï¬cient dataon occurrence in feed and on potential adverse effects of gas in these species european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritykeywords glycoalkaloids gas solanine chaconine potato margin of exposure moe food feedrequestor european commissionquestion number efsaq201600811correspondence contamefsaeuropaeu leon brimer was a member of the working group on glycoalkaloids in food and feed until august wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodpanel members margherita bignami laurent bodin james kevin chipman jes 13us del mazo bettinagraslkraupp christer hogstrand laurentius ron hoogenboom jeancharles leblanc carlo stefanonebbia elsa nielsen evangelia ntzani annette petersen salomon sand dieter schrenk tanjaschwerdtle christiane vleminckx and heather wallaceacknowledgements the panel wishes to thank the following for the support provided to thisscientiï¬c output kelly niermans the panel wishes to acknowledge all european competentinstitutions member state bodies and other anisations that provided consumption and occurrencedata for this scientiï¬c outputsuggested citation efsa contam panel efsa panel on contaminants in the food chain schrenk dbignami m bodin l chipman jk del mazo j hogstrand c hoogenboom lr leblanc jc nebbia csnielsen e ntzani e petersen a sand s schwerdtle t vleminckx c wallace h brimer l cottrill bdusemund b mulder p vollmer g binaglia m ramos bordajandi l riolo f rold 13antorres r and graslkraupp b scientiï¬c opinion risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products efsa pp 102903jefsa20206222issn european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritythis is an open access under the terms of the creative commons attributionnoderivs licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodiï¬cations or adaptations are madereproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderfigure elsevier figure springer figure american chemical society springerthe efsa is a publication of the european foodsafety authority an agency of the european unionwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodsummarythe european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food in particular in potatoes andpotatoderived products this risk assessment covers edible parts of potato plants and other foodplants containing gas in particular tomato and aubergine nonedible parts of ga containing plantshave not been considered with the exception of potato sprouts the panel developed the draftscientiï¬c opinion which underwent a public consultation from february to april thecomments received and how they were taken into account when ï¬nalising the scientiï¬c opinion werepublished in an efsa technical report efsa gas are present in many plants of the family of solanaceae and contribute to plant resistanceagainst pests and pathogens gas are composed of a steroidal aglycone and an oligosaccharide sidechain in commercial potato cultivars s tuberosum the main gas are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively the aubergine fruit s melongena contains primarily the gas asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively inlycopersicum atomatine and adehydrotomatine are the major gas withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelyshuman risk assessmentin experimental animals the potato gas asolanine and achaconine show a relatively low oralbioavailability with differences between species hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats due to the limited information themetabolic proï¬les of potato gas in experimental animals could not be characterisedin humans asolanine and achaconine are systemically absorbed following ingestion for bothsubstances relatively long serum halflives were reported suggesting a possible accumulation the bloodclearance of the respective aglycone solanidine appears to be slow accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of gas nofurther information is available on metabolism and excretion of potato gas in humansthere are no toxicokinetic data on tomato and aubergine gas and their aglycones in experimentalanimals and humansin acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice reliable data on other potatogas or tomato and aubergine gas and their aglycones are missingin repeated oral dose studies on potato gas rodents showed nonspeciï¬c effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the gasthe tomato ga atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day at higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats in mice a to 2weektreatment with the aubergine ga asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per daydevelopmental studies have been performed mainly in hamsters treated with potato gas and theiraglycones for only one day or for a short very restricted time period during gestation outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia these malformations occurred at dosesof mgkg bw per day and above for gas and of mgkg bw per day and above for theaglycones no noobservedadverseeffectlevelloael could be identiï¬ed from these studies reduced postnatal survival of pups due to insufï¬cientmilk production was reported when pregnant holtzman rats had been exposed to mg of asolaninekg bw per day studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectnoael orlevelwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodfor month similar effects were observed in rhesus monkeys exposed to mgkg bw per day for monthsfrom the limited number of studies available there was no evidence for genotoxicity of the potatogas asolanine and achaconine and the aglycone solanidine as well as for the aubergine ga asolamargine however there is not sufï¬cient information to conclude on the genotoxic potential ofthese gasno longterm chronic toxicitycarcinogencity study for potato tomato or aubergine gas or for therespective aglycones could be identiï¬edin humans acute toxic effects following ingestion of potato gas include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato gas potato tga intake of mgkg bw or more further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeain severe cases paralysis respiratory insufï¬ciency cardiac failure coma and death have beenreported doses in the range of mg potato tgaskg bw are considered to be potentially lethal forhumans results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato gasregarding the mode of action adverse effects of gas may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesafter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeagas inhibit acetylcholinesterase ache and serum butyrylcholinesterase buche by a reversiblecompetitive mode of action the relative potency of inhibition of asolanine and achaconine appearsto be similar the aglycones exert weak or no inhibitory effects the excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with gasat high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol as a consequence blood cholesterollevels were lowered in rodentsthe contam panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato gas in humans the contam panel selectedthe loael of mg potato tgakg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers the available data onacute toxicity were considered insufï¬cient to establish a healthbased guidance value instead thepanel used the margin of exposure moe approach to assess a possible health concern from acuteexposure to potato tgas via foodassuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of ache activity the panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics accordingly anmoe higher than indicates that there is no health concern this moe of takes into account theextrapolation from a loael to a noael a factor of and the interindividual variability intoxicodynamics a factor of the experimental data available for repeated dose toxicity are not sufï¬cient to identify a referencepoint for chronic exposure to potato gas in humans no evidence of health problems associated withrepeated or longterm intake of gas via potatoes has been identiï¬edregarding gas or aglycones occurring in edible parts of food plants other than s tuberosum nosuitable study for determining a reference point for tomato or aubergine gas or aglycones wasidentiï¬edoccurrence data were only available for asolanine and achaconine and mostly for maincroppotatoes and new potatoes few data were available for processed food no data on the occurrenceof tomato and aubergine gas and their aglycones were submitted to efsasince the occurrence data on potato gas did not cover all the food categories containing potatoesin the consumption database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities rpc maincrop potatoes andnew potatoes and the rpc consumption database the panel decided to combine the occurrence ofnew potatoes with that of maincrop potatoes and the mean upper bound ub occurrence sum ofwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the p95 occurrence was mgkg the minimum and maximum reported concentrations were and mgkg respectivelythe acute dietary exposure to potato tgas was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes as no occurrence data wereavailable for gas in tomato and aubergine these foods were not included in the exposure assessmentprocessing of potatoes has been reported to reduce the content of gas in the ï¬nal processedproduct in general and according to the literature the peeling of potatoes reduced the ga contentby boiling in water and blanching of peeled potatoes by and frying in oil of peeledpotatoes by microwave and oven baking of unpeeled potatoes may cause a reduction in thega content by and by respectively no information has been found about thechemical nature of the ga degradation products for the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsinformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedthe mean ub exposure to potato tgas across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers the 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the conï¬dence intervalcomparing the loael for potato tgas of mgkg bw per day with the acute exposure estimatesthe moes for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the p95 exposure in all surveys for adult age groups themoes indicate a health concern only for the food consumption surveys with the highest p95exposuresthe contam panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato tga intake may be below the moe of the highestnumber of survey days with intake of potatoes below the moe of was estimated for toddlers followed by children for the other age groups the estimated tga intake was below the moeof in up to of the survey daysfor tomato and aubergine gas the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansthe contam panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato gas in food is moderate and that overall the identiï¬ed uncertainties may eithercause an over or underestimation of the riskfarm animals horses and companion animals risk assessmentinformation on the toxicokinetics of gas was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows milkno data on the potential adverse effects of potato gas in horses companion animals cats anddogs or fur animals were identiï¬ed due to an insufï¬cient database on the adverse effects of gas inruminants pigs poultry rabbits and ï¬sh an acute reference dose could not be derivedpotatoes are not grown speciï¬cally as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsdata on potato gas in feed were insufï¬cient to perform an exposure assessmentthus no risk characterisation could be performed due to insufï¬cient occurrence data of gas forfeed and the lack of or limited data on the adverse effects of gas in farm animals horses orcompanion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodrecommendationsthe following needs have been identiï¬ed to improve the risk assessment for humans and reducethe uncertaintiescid129research on the occurrence of gas and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 occurrence data on gas and their aglycones in potato processed products including foods forinfantscid129 occurrence data on gas and their aglycones in tomato and aubergine and products thereofcid129 data on the toxicokinetics of potato tomato and aubergine gas and aglycones in experimentalanimals and humanscid129 data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine gas and aglycones in experimental animalsstudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129the following needs have been identiï¬ed to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 occurrence data on potato gas and their aglycones in feedcid129studies on the kinetics and the potential adverse effects from feed material containing gas ofpotato gas in farm animals horses and companion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodtable of contentsabstractsummaryintroduction background and terms of reference as provided by the requestor interpretation of the terms of reference supporting information for the assessment chemistry analytical methods sources potatoes tomatoes aubergine previous risk assessments legislation and other standards data and methodologies methodology for data collection selection of evidence and study appraisal food and feed occurrence data submitted to efsa data collection and validation data analysis food and feed consumption data food consumption data feed consumption data food classiï¬cation methodology for exposure assessment methodology for risk characterisation assessment hazard identiï¬cation and characterisation toxicokinetics experimental animals asolanine achaconine humans mixtures of asolanine and achaconine solanidine biomarkers of exposure farm animals horses and companion animals summary on toxicokinetics toxicity in experimental animals acute toxicity studies gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum summary on acute toxicity studies repeated dose toxicity studies gas and aglycones from edible parts of s tuberosum gas and aglycones from edible parts of food plants other than s tuberosum developmental and reproductive toxicity studies developmental effects reproductive effects immunotoxicity studies studies on cardiovascular effects neurotoxicity studies genotoxicity gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum carcinogenicity studies studies on metabolic effects gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum observations in humans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and food gas from s tuberosum reports on intoxications studies in human volunteers epidemiological studies summary gas from food plants other than s tuberosum case reports adverse effects in farm animals horses and companion animals ruminants pigs poultry rabbits fish horses companion animals cats and dogs fur animals reports on intoxications mode of action membrane effects with implications for the gastrointestinal tract inhibition of cholinesterases ches comparative determination of inhibition of ches in vitro determination of inhibitory constants ki for gas on inhibition of ches in vitro inhibition of ches in vivo developmental and reproductive effects of gas and their aglycones inhibition of cholinesterases and effects in the immune system interference with metabolism considerations of critical effects and doseresponse analysis for the human risk assessment gas from edible parts of s tuberosum considerations of critical effects and doseresponse analysis derivation of a healthbased guidance value hbgv or margin of exposure moe approach gas from edible parts of food plants other than s tuberosum considerations of critical effects and doseresponse analysis consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment occurrence data occurrence data submitted to efsa previously reported occurrence data in the open literature literature on occurrence data on food occurrence data on gas in potatoes occurrence data on gas in tomatoes occurrence data on gas in aubergines occurrence data on gas in other food products literature occurrence data in feed inï¬uence of storage and processing on the content of gas gas from s tuberosum storage of potatoes processing of potatoes for food consumption processing of potatoes for feed gas from food plants other than s tuberosum summary on the inï¬uence of storage and processing on the levels of gas exposure assessment current acute dietary exposure assessment for humans previously reported dietary exposure assessments current dietary exposure assessment for farm animals horses and companion animals risk characterisation human health risk characterisation ga from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum farm animals horses and companion animal risk characterisation uncertainty analysis assessment objectives exposure scenarioexposure model wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodhazard identiï¬cation and characterisation summary of uncertainties conclusions hazard identiï¬cation and characterisation toxicokinetics toxicity in experimental animals observations in humans adverse effects in farm animals horses and companion animals mode of action margin of exposure moe approach occurrence and exposure food feed risk characterisation human health risk characterisation farm animals horses and companion animal health risk characterisation recommendations documentation provided to efsa references abbreviations appendix a major glycoalkaloids and their aglycones present in solanum species appendix b identiï¬cation and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food appendix c details of the study design of the toxicokinetic studies appendix d comparison of developmental toxicity of single dose studies appendix e inhibition of cholinesterases by gas appendix f rapid alert system for food and feed rasff reports on the presence of solanum nigrum infood products appendix g studies on the toxicity of glycoalkaloids not considered in the risk assessment appendix h additional scenario for the human risk characterisation annex a occurrence data in food and feed submitted to efsa and dietary exposure assessment forhumans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodintroductionbackground and terms of reference as provided by the requestorbackgroundmany plants in the family solanaceae contain glycoalkaloids and they are considered to be naturaltoxins the plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcamongst the most widely cultivated food crops aubergines tomatoes and potatoes are in thesolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowthe glycoalkaloids of most relevance to food safety are those occurring in the potato thepredominant toxic steroidal glycosides in potato are asolanine and achaconine they occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors concentrations ofglycoalkaloids are times greater in the peel than in the ï¬esh there is considerable variation inglycoalkaloid content among potato cultivars storage conditions especially light and temperature aremainly responsible for increases in solanine although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsbitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include ï¬ulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea more severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision there are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriespotatoes and potatoderived products are listed in the catalogue of feed materials1terms of referencein accordance with art of regulation ec no the european commission asks theeuropean food safety authority for a scientiï¬c opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsinterpretation of the terms of referencethe contam panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids gas eg tomato and aubergine nonedible parts ofga containing plants have not been considered with the exception of potato sprouts in particular thecontam panel concluded this opinion should comprise thea evaluation of the toxicity of gas in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid proï¬le ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to efsac estimation of the dietary exposure of the european population to gas in food in particular inpotatoes and potatoderived products including the consumption patterns of speciï¬c groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to gas in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the european population including speciï¬c groupsof the population if appropriate as the consequence of the estimated dietary exposure commission regulation eu no of january on the catalogue of feed materials ojl p wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in europe as the consequence ofthe estimated dietary exposure exposure to gas from weeds containing ga is only addressedin this opinion in the context of accidental intake by farm animalswhen referring to gas in potatoes the term total gas tga refers to a material comprising asolanineand achaconine as major fraction with no speciï¬cation on the occurrence of minor gas as well as band cforms of solanine and chaconine similarly when referring to tomato and aubergine the termtga refers to the gas from the corresponding species and forms thereofsupporting information for the assessment chemistrysolanine is one of the ï¬rst alkaloids that has been isolated from nature by desfosses in friedman et al in zwenger and kind reported that solanine contains a glycoside sidechain zwenger and kind only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids gas asolanine and achaconine that share the same solanidineaglycone kuhn and l¬ow since then at least different gas have been isolated and fullystructurally elucidated from over species of the solanaceae family s 13anchezmata et al alsinani and eltayeb the chemical structures and some physical properties of the most importantones are listed in appendix agas are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see figure friedman et al friedman milner et al the gas of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone gas often contain a double bond between c5 and c6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents the stereochemistry at carbons c22 and c25 is well deï¬nedtheconï¬guration is 22r 25stheitconï¬guration is 22s 25s friedman et al in solanidineis 22r 25r and in tomatidenoltomatidinein solasodinefurther diversiï¬cation is generated by the composition of the glycoside sidechain most gascontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate in commercial potato cultivars solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentfigure wild s tuberosum varieties may contain a much wider range of gas friedman et al distl and wink the aubergine fruit derived from s melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose friedman derived from sin tomato fruitthe preï¬x alpha a refers to the intact glycoside while the preï¬xes beta b gamma c anddelta d refer to the corresponding gas with progressively truncated carbohydrate sidechains due tothe action of enzymatic or acidic hydrolysis friedman milner et al wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodohoooohohoohohooohohohsolatrioseohohooohohohsolatrioseohoohoohhh22rnhsolanidine25shhhohsolanine22r 25rnhohsolasodinehhhhooohsolasonineohohoohohohoooohoohoohohoohchacotrioseohohohchacotrioseoohoohohohoooohohohoooohohohohlycotetraoseoohohhhhoohoohoohohhohohoooohoh25sohoh22snhohtomatidinelycotetraoseoohohoohoohooohhhhhhnhsolanidinechaconinehnhohsolasodinehhhsolamarginehhhhnhohtomatidenoltomatinedehydrotomatinefigure s | 0 |
Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A casecontrolstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction HardyWeinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 oddsratio OR confidence interval CI however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [] Graves ophthalmopathyGO is one common anspecific complication affecting of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO []Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acasecontrol study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbeccos modified Eagles medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at ¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturers protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3CMycNR3C1 pCMV3CMycFOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturers protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturers protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNAIP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM TrisHCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydIdC and glycerol] for min at ¦C in a final volume of μl FOXP3 or NR3C1nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive [] [] Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbSigniï¬cance of age were evaluated by t testP005P00010039a 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the HardyWeinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 OR CI which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG 0929bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with signiï¬canceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAG2495cGG b0154b2467bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with signiï¬canceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearsons correlation Spearmans correlation Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearsons correlation Spearmans correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3CE The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 BE Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GOatherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via Drug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE HardyWeinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration 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et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves disease in Chinese patients Endocrine 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Gravesophthalmopathy patients Ophthalmology 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves ophthalmopathy Hum Immunol 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol Siegmund T Usadel KH Donner H Braun J Walï¬sh PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves disease Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with GravesOphthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi | 2 |
Scars Burns HealingVolume 1011772059513120940499 reuse guidelinessagepubcomjournalspermissions The Authors journalssagepubcomhomesbhKeloids are pathological scars that grow over time and extend beyond the initial site of injury after impaired wound healing These scars frequently recur and rarely regress They are aesthetically disfiguring can cause pain itching discomfort as well as psychological stress often affecting quality of life Many treatment modalities including surgical and nonsurgical have been explored and have been reported to be beneficial however none have been absolutely satisfactory or optimal for the treatment of all keloid subtypes to date This poses a major challenge to clinicians Often a combinational therapeutic approach appears to offer the best results with higher patient satisfaction compared to monotherapy The aetiopathogenesis of keloids is not fully elucidated however with recent advances in molecular biology and genetics insight is being gained on the complex process of scar formation and hence new therapeutic and management options for keloids In this paper we explore the literature and summarise the general concepts surrounding keloid development and review both current corticosteroids surgical excision siliconebased products pressure therapy radiotherapy cryotherapy laser therapy imiquimod and 5fluorouracil and emerging stem cell therapy mitomycin C verapamil interferons bleomycin botulinum toxin type A and angiotensinconverting enzyme inhibitors treatments Increased knowledge and understanding in this area may potentially lead to the discovery and development of novel therapeutic options that are more efficacious for all keloid typesKeywordsKeloids scar recurrence wound healing treatment managementLay SummaryKeloids are problematic scars that are difficult to treat and manage The aetiopathogenesis of keloids is not clear however recent advances in molecular biology and genetics are beginning to shed light on the underlying mechanisms implicated in keloid scar formation which will hopefully lead to the development of treatment options for all keloid types This review summarises current and emerging therapiesIntroductionWound healing is an intricate and complex series of processes comprising overlapping phases of inflammation granulation tissue formation and tissue remodelling and results in tissue structure integrity and damage being restored1 Abnormal wound healing can give rise to keloids which are benign dermal fibroproliferative nodular lesions that tend to recur after excision Keloid scars Faculty of Medical Sciences The University of the West Indies Cave Hill Campus Bridgetown Barbados West Indies Pine Medical Centre 3rd Avenue Belleville St Michael Barbados West IndiesCorresponding authorNkemcho Ojeh Faculty of Medical Sciences The University of the West Indies Cave Hill Campus PO Box St Michael Bridgetown BB Barbados West Indies Email nkemchoojehcavehilluwieduCreative Commons Non Commercial CC BYNC This is distributed under the terms of the Creative Commons AttributionNonCommercial License creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0c Scars Burns Healinginfluence in keloid aetiology9 Although no one specific gene has been associated with the development of keloids a number of genes and gene loci have been identified610 Genomewide association studies and admixture mapping studies have identified singlenucleotide polymorphisms across certain loci genetically linked to keloid development including the NEDD4 gene which encodes E3 ubiquitin ligase enzyme and the myosin genes MY01E and MYO7A10 Studies have also reported the involvement of several human leucocyte antigen HLA alleles p53 bcl2 and fas genes1014 Furthermore rare genetic disorders have been reported to present with spontaneous keloids including Dubowitz syndrome Bethlem myopathy RubinsteinTaybi syndrome Noonan syndrome Geominne syndrome and others1017 These lines of evidence suggest that genetic factors play a role on keloid predispositionPathophysiology of keloidsKeloid pathology is complex involving both genetic and environmental factors Keloids form as a result of abnormal wound healing and excessive dermal fibrosis Development of keloids has been linked to overproliferation and reduced apoptosis of dermal fibroblasts overproduction of collagen fibres and other extracellular matrix ECM components as well as abnormal ECM production and remodelling1 Various cytokines growth factors and proteolytic enzymes have been implicated in the formation of keloids including transforming growth factor TGF epidermal growth factor EGF vascular endothelial growth factor VEGF plateletderived growth factor PDGF connective tissue growth factor CTGF tumour necrosis factorα TNFα insulinlike growth factor1 IGF1 fibroblast growth factor FGF interleukin6 IL6 and matrix metalloproteinases MMPs31418 Furthermore signalling pathways such as Tolllike receptor signalling SMAD signalling and fibronectin have been reported to be associated with keloid development1819Histopathology of keloidsHistologically keloids comprise an abundance of unordered dermal collagen and vasculature with high inflammatorycell infiltrate and overactive mesenchymal cells1415 In addition to collagen elastin fibronectin and proteoglycans are deposited in excess amounts in keloid scars9 Collagen creates frequent crosslinks in ordinary wounds whereas collagen is irregularly anised in keloids forming nodules in the dermis20 During normal Figure Earlobe keloids as a consequence of ear piercingarise from skin trauma or inflammation and may develop years after the initial insult and rarely regress2 The scar tissue extends beyond the original wound site and can be disfiguring and cause psychosocial issues impairing quality of life3 In addition patients may present with symptoms such as burning pain pruritus movement limitation and hyperaesthesia2AetiologyThe aetiology of keloids is still poorly understood The most common regions of the skin for keloids include upper arms skin overlying joints chest shoulders and headneck regions particularly the ear lobes Figure The anatomical location of a keloid appears to alter its morphological characteristics Some keloids can develop spontaneously however most occur years after local trauma and other events including inflammation surgery burns elective cosmesis foreign body reactions acne insect bites vaccinations or mechanical force45Epidemiology and keloid geneticsThe incidence of keloids is highest among darkerpigmented persons of African Asian and Hispanic descent and is estimated to be in the range of Males and females have an equal risk of developing keloids6 although incidence is slightly increased in females likely attributable to them having more cosmetic procedures like ear piercing7 Persons aged years are also at a higher risk of developing keloids Additional risk factors include having blood type A hyperIgE and hormonal peaks during pregnancy or puberty8Familial keloid case studies and twin studies support the notion that genetic factors have an 0cOjeh for the treatment of keloids2627 Intralesional TAC injections have been shown to reduce scar volume and height improve scar pliability and diminish associated scar pain and itching8 as well as prevent recurrence3 Corticosteroids have antiinflammatory and antimitotic properties1 Several other mechanisms have been reported by which corticosteroids reduce keloid scar including inhibition of fibroblast growth attenuation of procollagen and glycosaminoglycan synthesis reduction of endothelial budding and enhancement of collagen and fibroblast degeneration2829 Corticos teroids inhibit TGF1 expression and induce apoptosis in fibroblasts inhibit VEGF and alphaglobulins which are involved in the wound healing process28 VEGF which promotes angiogenesis was reported to be highly expressed in keloid fibroblasts compared to controls but exogenous addition of the glucocorticoid dexamethasone suppressed its expression in vitro32 Furthermore VEGF expression was overexpressed in keloid tissue which later reduced following intralesional TAC injections in vivo33TAC is typically administered at intervals of weeks until pruritic and painassociated symptoms diminish and the scar flattens34 The dose of TAC is in the range of mgmL depending on the size and anatomical location of the lesion and the age of the patient34 TAC is used either alone as a monotherapy or in combination with other treatment modalities13 The response rates to corticosteroid injections vary clinically with regression rates in the range of reported after one year and recurrence rates in the range of reported after five years3 Combined therapy comprising surgical excision followed by TAC treatment also varied with reported recurrence rates in the range of Previous clinical studies where TAC was used alone reported efficacy and good clinical outcome with this treatment including reduced keloid height length width related pruritus and erythema and improved pliability3637 A recent randomised parallelgroup study that compared the role of intralesional TAC fractional CO2 laser or intralesional verapamil in the treatment of keloid in patients showed reduction in scar height vascularity and pliability in all three groups using the Vancouver Scar Scale score however pigmentation was not completely resolved with any of the treatments The response was fastest with TAC followed by verapamil and laser and this was statistically significant38 However intralesional TAC in combination with other treatment modalities such as 5fluorouracil 5FU pulsed dye laser PDL surgery interferon IFNα2b verapamil and Figure Current and emerging treatment strategieswound healing the early wound immature collagen type III can be modified into mature collagen type I In keloid tissue it mostly comprises disanised collagen types I and III made up of palestaining hypocellular collagen clusters lacking nodules or surplus myofibroblasts21 Furthermore recent research has provided four distinct findings only present in keloid specimens presence of keloidal hyalinised collagen presence of a tonguelike advancing edge underneath normalappearing epidermis and papillary dermis a horizontal cellular fibrous band in the upper reticular dermis and a prominent fascialike band22Treatment of keloidsCurrently various forms of treatment for keloids exist however no single treatment has proven to be the most effective This review will explore and discuss current and emerging treatment modalities Figure Some of the ongoing or completed clinical trials of keloid therapy registered on clinicaltrialsgov and accessed on May are summarised in Table Studies with the status terminated suspended or withdrawn were excludedCurrent treatmentsCorticosteroidsSeveral corticosteroids can be used for the treatment of scars including triamcinolone acetonide TAC hydrocortisone acetate dexamethasone and methyl prednisolone24 However since TAC has been the most widely used corticosteroid 0c Scars Burns HealingTCNVI esahPTCNlebacilppa toN vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoC yaMnodessecca vogslairTacinlil Cnoypareht doek fo slairt lliacinilC elbaTeltit ydutSreifitnediTCNlebacilppa toN ybdewoll iednotecaenoonicmairt llanoiseartnliilsdoeK fo tnemtaerTeht n iof resal OC lanoitcarFidoeKli sdoretS lanoiseartnl iI svyparehTdoretSdetsissA resaL lanoitcarFTCNlebacilppa toNTCNlebacilppa toNTCNVI esahP TRS ypareht noitadar liaicifrepuS iEnmativ enocilis enositrocordyh yparehtyhcarbetaresod ihgh tnavudahtij wnoisicxe lacigruS cihportrepyh idoeKlidoeKlidoeKl TRS yparehTnoitadaRi l laicifrepuS fonoitauavEevitcepsorteRA ilsdoeK fo tnemtaerT rof yparehtyhcarBetaResoDhgHi isracSdoeKdnal dnaytili llbareoTeht gnitauavEydutSevitarapmoCdezimodnaRA TCNlebacilppa toN llamsaphcir teetap suogootuAllidoeKl llllamsaPhciR teetaP suogootuAyb racSdoeK fo tnemtaerTli TCB®tiKnegeRhti wdenatboiTCBtiKnegeRhti wdenatbOi l ieg tcartxenono ro draugracS noitolamredeM xirtaciCracScihportrepyh ildna sdoeK fo tnemtaerTeht rof separehTi l acipoTowT foycaciffE sracScihportrepyHTCNI esahP muiclac fonoitcenj i laruomutartnIidoeKlrefsnartortceeybdewol llof edirohclTCNIII esahP dnaenoonicmairt llanoiseartnlIidoeKl ienodnefriP l ilsdoeK fo tnemtaerTeht rof noitaroportceEmuiclaCl acipoTdnaenoonicmairTl lanoiseartnl I foycaciffEienodnefrip lacipotnoitcenji UFidoeKllygooisyhpohtaPdna tnemtaerT gnirracSdoeKli iIOALSDAL sracSdoeK fo tnemtaerT roflTCNII esahP I esahP inks gnviltneavuqeiil dereyalib fargilpAsresalidoeKl iil tenragmunmuamuirttymumydoenideslupgno l dna OC lanoitcarFidoeKl fo tnemtaerTn ii sresaL tenraGmunmuAmuirttymumydoeNli fonoitneverPdna tnemtaerTeht rof farg ilpA foydutS toli PAisdoeKl ilsdoeKdesicxE foecnerruceR desluPgnoLdnaedixoDnobraCi l anoitcarFgnisUydutSA deunitnoC cihportrepyhTCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl eht rof eciveDdesaB saGnogrAnahti WyparehtoyrC lanoiseartnlI sracScihportrepyHdnadoeK fo tnemtaerTli TCNIII esahPxirtaciC fonoitacil ppa lacipoT sracs cihportrepyHcihportrepyhidoekl ilsracS sdoeKdna sracScihportrepyH fo tnemtaerTeht n i IXRTACCI sracScihportrepyHdnadoeK fo tnemtaerTli TCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl rof yparehtoyrC lanoiseartnl I foesUeht fonoitauavEevitcepsorPl 0cOjeh TCNVI esahPresal PTKmneht foycaciffE idoekl cihportrepyheahcun siliadoekl xirtacic xirtacic racs lacigrus racS reifitnedi vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoCdeunitnoC elbaTeltit ydutSTCNlebacilppa toNresal eyddesluP cihportrepyh idoeKl desluPmnagnisU tnemtaerT racSnohtdWesluP fo tceffEi TCNlebacilppa toN thgil AVUderutcafunamnamreG idoeklsracs amredorelcSsnoitidnoC rali imSdnaamredorelcS fo tnemtaerT rof thgLAVUi resaLeyD ecivedgnitti me gnisorbif rehtosnoitidnocTCNlebacilppa toN ecivederusserPTCNVI esahPl egenocilis lacipoT cihportrepyh idoeKlilERUSSERP sdoeK raE fo tnemtaerTeht n i eciveDerusserPidoeKl fo tnemtaerTeht no l eGenocili iSgnyrdfleS tnerapsnarT fo tceffEsracssracS l ianmodbAcihportrepyHTCNlebacilppa toNypareht noitadaRiidoeKl yrtsigeRnoitadaRdoeKlii TCNIII esahPnosahtemateB fonoitacil ppa lacipoT sracs cihportrepyHsretehtaC suoneV lartneC retfA sracSTCNI esahPII esahPTCN I esahPII esahP dna sll dicacidisuf dnaetareavl isdoeklidocitrococuglnoitadarri iBVU amredorelcs idoeKl inkS fo tnemtaerTeht n i yparehT thgL BVUil B teovartlUi leraunnaamounargl sracs siliadoekl enca desilacolxirtaM l amreDderetlAhti w snoitidnoC lecFVS suogootua fo snoitcenjI axal situc racs inkSCSDAxirtacic idoekl idevireDesopdA suogootuAhtil waxaL situCdna sracS foyparehT sll eCmetS lamyhcneseMTCNlebacilppa toN gnitti meAVUderutcafunamnamreG amredorelcs idoeKlsnoitidnoC ralimetsys thgil lamounarg sracs enca leraunna imSdnaamredorelcS rof thgLAVUi ilteovartlu VU FVS sll ecnoitcarf raucsavl lamorts FVS etahpsohp lynatit muissatop PTK sll ecmets l amyhcnesemdeviredesopdai hserF fo tnemtaerTeht rof resaLPTKmn l evoNa foydutS to liP CSDA licaruoroulf UFsracS lacigruS 0c Scars Burns HealingA variety of methods can be used for the surgical removal of keloids depending on the size of the keloid anatomical location skin type and age of patient52 These include linear closure and flap coverage excision with grafting Wplasty and Zplasty53 To reduce risk of keloid recurrence the surgeon performing the excision should establish tensionfree wound closure As a general rule closure of the wound should be accomplished with minimal tension and sutures leaving everted wound borders Zplasties threelayered sutures subcutaneousfascial tensile reduction sutures or local ï¬ap surgery can be employed on a casebycase basis5455 The final outcome of the scar is often positively correlated with the experience of the operating surgeon and technique utilised as well as the patients active participation in their wound care52Siliconebased productsSince the 1980s siliconebased products have been used in the treatment of keloids and hypertrophic scars with silicone gel or silicone gel sheeting considered as firstline therapy for minor keloids and hypertrophic scars2635 There are various other forms of silicone including creams sprays gel cushion and liquid24 The precise mechanism of action of silicone products is not fully understood but it is proposed that they enhance hydration and create an occlusive environment53 which influences fibroblast regulation and decreases collagen synthesis56 Silicone gel sheeting has been shown to have minimal side effects including local irritation which can be resolved quickly24 Studies have shown that the beneficial effects of silicone gel sheets include pain reduction tenderness and pruritus and flattening the keloid57 The silicone gel sheeting is recommended to be worn from two weeks after primary wound treatment for h for months58Studies have demonstrated an improvement of up to in keloid scars after using silicone gel sheeting35 and a decrease in the incidence rates of keloids and hypertrophic scars after surgery59 In addition controlled studies have reported the clinical effectiveness of silicone gel and silicone gel sheeting in the prevention and treatment of keloids3558 However a recent metaanalysis review60 and Cochrane review61 found that even though studies published data in support of the efficacy of silicone gel sheeting in the treatment and prevention of keloid and hypertrophic scarring they provided weak evidence and were of poor quality60 Therefore given Figure Auricular keloid surgical excision used as monotherapy Auricular keloid before a and after b surgical excisonsurgery all yielded significant improvements compared to treatment with TAC monotherapy339Intralesional steroid injections can cause several adverse side effects such as telangiectasis atrophy steroid acne pigmentary changes necrosis ulcerations and systemic side effects3 There is also significant pain associated with intradermal corticosteroid injections that can be reduced when local anaesthetic lidocaine is administered to control the pain44 Furthermore the side effects have been reported to diminish when intralesional TAC is used in combination with 5FU45Surgical excisionSurgical excision is a traditional method of removing keloids Figure However excision creates a new wound and can result in a similar or larger keloid47 Therefore surgical excision is not recommended as a monotherapy as it results in high recurrence rates in the range of For better postoperative surgical outcomes surgical excision is often combined with other forms of treatment including radiotherapy intralesional corticosteroid injections IFN injection bleomycin cryotherapy pressure therapy and silicone gel or sheeting82649 Successful use of dermal substitutes and epidermal skin grafting with keloid excision has also been reported50 A recent case series study in which patients with anterior chest wall keloids were given a treatment protocol consisting of complete excision Zplasty postoperative adjuvant radiotherapy and postsurgical wound selfmanagement reported excellent outcomes with a recurrence rate of only The use of steroid tape and injections helped to resolve the recurrence of keloids51 0cOjeh the lack of substantial evidence welldesigned clinical trials and studies are required to gain a better understanding of the effectiveness of siliconebased products in preventing and treating keloidsPressure therapyPressure therapy has been used to treat and manage keloids and hypertrophic scars for decades3563 It has been routinely employed as firstline treatment in the treatment of hypertrophic scarring resulting from burns64 The underlying mechanisms of action of compression techniques remain unclear however several hypotheses exist some of which include increased pressure to the scar surface reduces perfusion and decreased oxygen to the location of injury reduces collagen synthesis It is also thought that pressure increases apoptosis reduces scar hydration stabilising mast cells and decreases angiogenesis165 The application of pressure can be achieved using a variety of materials such as adhesive plaster moulds pressure earrings and customfitted splints16 which have improved scar cosmesis and rates of keloid recurrence66 A continuous pressure of mmHg preferably at the lower range soon after wound reepithelialisation for h per day for months is recommended166869 The efficacy of pressure therapy depends mainly on the anatomical location of the scar with trunk and limb areas being more appropriate sites for pressure therapy In addition a pressure garment is predominantly used for auricular keloids where pressure clips are commonly utilised after surgery6670 As an adjuvant therapy this form of pressure garment has also been successfully used to prevent the recurrence of keloids6667 In contrast a metaanalysis review that analysed the effectiveness of pressure garment therapy for the prevention of abnormal scarring after burn injury was unable to demonstrate any beneficial effects of pressure garment therapy on prevention or treatment of abnormal scarring59 Notwithstanding success rates of pressure therapy are contingent upon patient compliance69 which can sometimes be low due to discomfort Overall pressure therapy is tolerated better and is devoid of the pain often associated with intralesional therapies and hence can be considered as a good adjuvant therapy for keloid scarsRadiotherapyIn the treatment of keloids using superficial Xray irradiation was first described71 Since then it has been used less frequently as a monotherapy and more widely as an effective adjunct treatment after surgical excision72 with success rates in the range of and recurrence rates of about Radioactive skin patches have also been used in combination with other treatment modalities for keloids7879 Radiotherapy is most commonly used h after surgical excision7480 and acts by suppressing angiogenesis and inhibiting ï¬broblast activity1 Decreased fibroblast proliferation induced cell senescence and apoptosis leading to reduction in collagen production and suppression of keloid development have also been reported81Different radiotherapy modalities have been used after surgical excision including electron beam radiotherapy brachytherapy superï¬cial and orthovoltage radiotherapy with varying degrees of success84 Mankowski et a0 al conducted a literature review of studies to compare the clinical outcome of different forms of radiation treatment used for the management of keloids77 The metaanalysis demonstrated that radiation used as monotherapy yielded higher rates of recurrence compared to combinational therapy with postsurgery excision Comparison between the different radiationbased treatments revealed that the lowest rate of recurrence was observed with brachytherapy followed jointly by Xray and electron beam The authors also reported that the rate of recurrence was dependent on anatomical site of the keloid with chest keloids having the highest recurrence rate73The adverse effects of radiotherapy often linked with dose of radiation used can be grouped into acute skin reactions and late complications Acute reactions arise as early as seven days after keloid treatment and include oedema necrosis ulceration desquamation erythema and pigmentary changes with the latter two being the most common Late complications which include changes in pigment atrophy telangiectasis and alopecia may present several weeks after radiotherapy Emollient and steroid ointment used after radiotherapy can help alleviate the side effects19 A recommended radiation dose Gy over several sessions can also minimise adverse effects1980 Radiotherapy carries a risk of malignancy8586 Therefore caution should be used in radiationvulnerable sites such as the head neck thyroid and breast and in patients aged years2 Protecting fragile ans and selecting the most appropriate sitedependent dose protocol can help minimise further complications of radiotherapy87 0c Scars Burns HealingCryotherapyCryotherapy is a lowtemperature treatment that causes vascular damage resulting in tissue necrosis88 It has been used to treat keloids as a monotherapy or in combination with other treatment methods such as intralesional steroid injections89 Various delivery methods used for cryotherapy include spray and contact probes or the intralesionalneedle cryoprobe method Compared to contact and spray methods intralesionalneedle cryoprobe was found to be the most effective method in treating keloid scars90 Positive outcomes were observed in a number of studies that used liquid nitrogen and cryotherapy to treat keloids with success rates in the range of External cryotherapy has been associated with several side effects including hypopigmentation blistering pain delayed healing and infection9093 Moreover larger keloids have been shown to need multiple cryotherapy sessions9093 To minimise side effects intralesional cryotherapy was introduced and there are now a number of nitrogenbased cryodevices that have been described for the treatment of keloid scars with two commercially available a liquid nitrogenbased device88 and an argon gasbased device94 The intralesional cryotherapy was designed to overcome the hypopigmentation seen mostly in darkskinned individuals with external cryotherapy It works by destroying the core of the keloid sparing the surface epithelial cells including melanocytes9596 As a result it enhances volume decrease while minimising the risk of hypopigmentation and other surface reactions90 A recent comprehensive review based on the preferred reporting items for systematic reviews and metaanalysis was performed to investigate the efficacy of intralesional cryotherapy on keloid scars90 The review of eight studies that met the inclusion criteria revealed that average scar volume decreased in the range of but complete eradication of the scar on average was lacking Recurrence of keloid scars was in the range of The authors also reported that patients complaints of pain and pruritus was considerably reduced however hypopigmentation was seen mostly in Fitzpatrick skin type patients after treatment90Laser therapyLaser therapy for keloid treatment was introduced in the 1980s97 Since then different systems have been used for the treatment of keloid and hypertrophic scars4898 These lasers target skin chromophores like haemoglobin and melanin based on the principle of selective photothermolysis99 Lasers can be classified as ablative and nonablative The most common ablative lasers include the 2940nm erbiumdoped yttrium aluminium garnet ErYAG laser and the 10600nm carbon dioxide CO2 laser These emit a laser beam that is absorbed by water in the skin leading to local tissue destruction and reduction of lesion volume3 Common examples of nonablative lasers include 585nm or 595nm PDLs 1064nm neodymiumdopedyttriumaluminiumgarnet NdYAG neodymiumdopedvanadate NdVan laser and nm Qswitched NdYAG laser with low fluence100 These lasers induce thermal injury to the scars microvasculature leading to thrombosis and ischaemia which result in collagen denaturation and collagen fibre realignment101532nm laser Laser therapy requires several treatments at intervals of weeks depending on scar type and type of laser used98104 with possible side effects including itching pigmentary changes blister formation and postoperative purpura98 The use of the nonfractional vascular nm PDL in the treatment of keloid and hypertrophic scars has been welldocumented105 and has response rates in the range of PDL monotherapy has been shown to be effective108110 as well as CO2 laser monotherapy38111112 In a clinical study where patients with moderate to severe keloids were treated with highenergy pulsed CO2 laser the treatment was efficacious and welltolerated with minimal side effects112 In other studies where CO2 laser ablation was compared with other forms of treatment CO2 laser was as efficient as the other forms38111 It must be noted however that these studies are small and randomised controlled studies are lacking98Laser therapy such as PDL CO2 and NdYAG have been associated with a high rate of recurrence at months111113 However optimal results can be achieved with combination treatment especially with intralesional TAC injections116 Kumar and coworkers conducted a cohort study on patients with keloids previously treated with an NdYAG laser and reported complete scar resolution and flattening in seven patients only when intralesional TAC was used after laser therapy41 Moreover combined therapy with PDL and TAC119 and PDL TAC and 5FU36 were shown to produce better clinical results In a recent study that evaluated and compared the efficacy of combination therapy of 0cOjeh scars statistically fractional CO2 laser and intralesional TAC injection or TAC injection alone in keloid and hypertrophic significant improvements were reported in overall scar quality with the combined treatment options comto TAC monotherapy120 Moreover pared combined CO2 laser and IFNα2b injections given to patients with auricular keloids resulted in no recurrence in of patients three years after treatment121 Laser therapy can also be combined with other laser treatment topical corticosteroids and cyanoacrylate glue98 and have shown promising results however larger controlled clinical studies are needed to further evaluate their efficacy and safetyRecently lasers are also being explored as tools for assisted drug delivery Kraeva et a0al proposed an alternative technique of corticosteroid administration of laserassisted drug delivery of topical TAC This was shown to be effective when used on a keloid on the posterior scalp of a patient after each CO2 laser session122 More efficient intraepidermal drug delivery options are also being investigated Singhal et a0al developed TACcontaining polymeric microps that were prepared using a cryomilling technique for freezing fracture After ablation with a fractional ErYAG laser these microps can be deposited in cutaneous micropores and provide highdose intraepidermal reservoir systems with minimal transdermal permeation leading to sustained and targeted local drug delivery123It must be noted that one of the biggest limitations in studies available at present is the lack of histological definition between keloid and hypertrophic scars so conclusions are not valid on efficacyImiquimod creamImiquimod cream is approved for the treatment of basal cell carcinoma actinic keratoses and genital warts21 As an immuneresponse modifier it stimulates the production of proinflammatory cytokines such as TNFα interleukins and IFNs by activated Tcells124 thereby changing the expression of genes associated with apoptosis125 and reducing collagen production16 Studies have reported conflicting findings regarding efficacy of imiquimod cream postoperatively following keloid excision likely due to keloid location Many studie | 2 |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | 0 |
" colorectal cancer crc is the third leading cause of cancerassociated mortality the present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for crc the present study compared mrna expression levels of patients with crc with short and longterm prognosis and of individuals with and without tumors in the cancer genome atlas tcga database differentially expressed genes degs were identified via volcano plot and venn diagram analysis gene ontology go analysis and gene set enrichment analysis gsea were performed to identify the functions of the degs and the degs were further verified using clinical crc samples a total of degs were identified as candidate genes using the tcga database and four degs [defensin 4a defb4a hyaluronan binding protein habp2 oleoylacp hydrolase and tbc1 domain family member 3g] were associated with poor prognosis of patients with crc two degs defb4a and habp2 were upregulated in tumor tissues of patients with crc in the tcga database go and gsea analyses revealed that defb4a was highly associated with immunosuppression participates in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity and was positively correlated with cd11b cd14 cd45 cd163 and il17a furthermore defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis survival analysis revealed that defb4a upregulation was associated with poor prognosis defb4a correspondence to dr yi zhang biotherapy center the first affiliated hospital of zhengzhou university jianshe east road zhengzhou henan pr chinaemail yizhangzzueducncontributed equallykey words defensin 4a colorectal cancer prognosis immunity biomarkergene knockdown experiments demonstrated that def4ba promotes cell migration these results indicated that defb4a potentially promotes tumor growth by regulating immunosuppressive activity and provided novel insights into the diagnosis and treatment of crcintroductionaccording to the global cancer statistic of colorectal cancer crc is the third most common cancer and the second leading cause of cancerassociated mortality worldwide crc was reported as the fourth most common and fatal cancer in china in patients with crc usually have a low survival rate and poor therapeutic responses and are susceptible to progression and recurrence early diagnosis and effective treatment are critical to improve the survival of patients with crc crc studies have focused on innovative ideas to identify molecular markers used to develop highprecision noninvasive screening tests for crc to increase population compliance and reduce the potentially harmful side effects associated with more invasive techniques diagnostic markers will give an indication of the likely progression of the disease targeting specific molecules in certain patients has facilitated more personalized treatments that help prevent or decelerate cancer progression the present study aimed to determine prognostic factors and novel therapeutic targets to improve the survival of patients with crcprevious studies have focused on the identification of molecules associated with tumor progression through genetic or mrna profiling and screening of patients with colon cancer for example the expression profiles of long noncoding rnas lncrnas were compared at specific tumor stages t0 t1 t2 and t3 in an azoxymethanedextran sodium sulfateinduced primary colon cancer model and upregulation of the lncrna h19 predicted a poor prognosis other studies analyzed microrna mir expression profiles between tumor tissues and matched nontumor tissues obtained from patients with crc for example mir is significantly downregulated in tumor tissues and associated with poor survival of patients with crc and may thus be considered to be a poor prognostic marker of crc furthermore high expression levels of mir 0cwu prognostic role of defb4a in colorectal cancerand mir in serum are associated with poor survival of patients with crc analysis of the cancer genome atlas tcga database revealed that mmp19 is upregulated in patients with crc and is associated with tumor progression however to the best of our knowledge no study has directly screened mrna profiles based on prognosis the present study divided patients with crc into different groups based on prognosis and screened the mrna profiles of the respective groups defensin 4a defb4a also known as bd sap1 defb2 defb4 hbd defb and defb102 belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils which serve important roles in innate immune defense against microbial infections defb4a is upregulated in cutaneous squamous cell carcinoma and basal cell carcinoma it serves an important role in esophageal carcinogenesis both in vivo and in vitro the genomic copy number of defb4a has been analyzed in patients with crohn's disease and controls and an elevated defb4a copy number has been identified as a risk factor for crohn's disease regardless of disease origin however it remains unclear whether defb4a expression is associated with the prognosis of crc furthermore the role of defb4a in the immune system remains unclear the tumor microenvironment serves a significant role in tumor progression various immune elements comprise the tumor microenvironment including bone marrowderived cells such as macrophages cd4 t cells cd8 t cells b cells natural killer cells and dendritic cells myeloid cells can differentiate into macrophages or myeloidderived suppressor cells mdscs which serve a tumorigenic role in the tumor microenvironment mdscs contribute to tumor vascular development by promoting angiogenesis and tumor growth tumorassociated macrophages tams are important regulators of tumorigenesis by inhibiting the antitumor effects of other cells thus promoting tumor growth however it remains unclear whether defb4a has a regulatory effect on the tumor microenvironment or whether it promotes crc progressionto identify candidate target genes that potentially prolong patient survival mrna expression profiles of tissues from crc samples were compared in the tcga database venn analysis was performed to determine candidate genes upregulated in tumor tissues among patients with poor prognosis subsequently immuneassociated pathway enrichment was analyzed using gene ontology go and gene set enrichment analysis gsea and the correlations between candidate target genes and certain immune cells were determined finally clinical samples and crc cell lines were obtained to verify the clinical significance of the identified genes the present results may provide insights into targeted therapy for crcmaterials and methodsacquisition of microarray data microarray data were obtained from tcga httpcancergenomenihgov rnaseq data for samples were included in the dataset project id tcgacoadread including tumor samples from patients with crc and normal tissues from healthy donorsidentification of differentially expressed genes degs tcga data were divided into two groups based on different categories patient prognosis and gene expression in tumor and normal tissues venn analysis of the two groups was performed and genes associated with crc prognosis were identifiedvenn analysis to identify candidate genes associated with patient survival the gene expression profiles in the two groups were analyzed using the venn diagram web tool httpbioinformaticspsbugentbewebtoolsvenngo analysis functional analysis of the degs was performed using go httpwwwgeneontology based on biological processes gsea gsea was conducted using gsea v403 software wwwgseamsigdbgseaindexjsp and the gene used in the present study was downloaded from the molecular signatures database msigdb oftware broadinstitutegseamsigdbindexjsp v40 msigdb curates various gene sets including canonical signaling pathways from biocarta cgapncinihgovcgap_mitelman_retire_noticehtml kyoto encyclopedia of genes and genomes wwwkeggjp pid httppidncinihgov reactome reactome and other pathway databases tcga data were analyzed via gsea and pathways with a false discovery rate fdr were considered significantpatient characteristics tissue samples were obtained from patients with crc at the first affiliated hospital of zhengzhou university zhengzhou china between april and april patients underwent surgical resection or colonoscopy and the samples were verified via pathological analysis the clinical characteristics of the patients are shown in table i a total of men and women were included in the present study the median age was years age range years crc was diagnosed by two pathologists on the basis of pathological assessment the collection of specimens was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university zhengzhou china approval no sciencelw and informed consent was obtained from each patient with available followup informationreverse transcriptionquantitative pcr total rna was extracted from pairs of tumor and normal tissue samples from patients with crc using trizol reagent invitrogen thermo fisher scientific inc total rna samples µg were incubated at Ëc for min followed by incubation at Ëc for min and Ëc for sec according to the reverse transcription reaction protocol takara biotechnology co ltd the conditions of pcr were as follows Ëc10 min Ëc10 sec Ëc10 sec Ëc10 sec cycles premix ex taq ii roche target gene expression was simultaneously assessed relative to that of gapdh a housekeeping gene and internal control the following primers were used defb4a forward 'ctc ctc ttc tcg ttc ctc ttc a' and reverse 'gca ggt aac agg atc gcc tat' and gapdh forward 'gga gcg aga tcc ctc caa aat' and reverse 'ggc tgtt 0concology letters no of cases table i characteristics of patients with colorectal carcinomacharacteristic percentagesex male female age years ¥ treatment surgery others tumor size mm ¥ pathological type adenocarcinoma others lymph node metastasis yes no tnm stage i ii iii iv liver metastasis negative positive differentiation poor mediumwell others include chemotherapy and radiotherapy gtc ata ctt ctc atg g' the present study compared the expression levels of the target genes in clinical samples using the δδcq method expression levels of defb4a and gapdh were examined for each sample and the relative expression levels of defb4a were determined using the δcq value of defb4a divided by that of gapdh cell transfection sw480 and hct116 cells were seeded into a well plate sw480 and hct116 cells were purchased from chinese academy of sciences cell bank and cultivated with dmemhigh glucose containing fbs hyclone ge healthcare life sciences and penicillinstreptomycin at Ëc in co2 the growth status of the cells was closely observed until they reached a fusion rate of and then cells were transfected with ncsmall interfering rna negative control sinc sense 'uuc ucc gaa cgu guc acg utt' and antisense 'acg uga cac guu cgg aga att' and small interfering rna targeting defb4a sidefb4a sidefb4a sense 'ucc ucu uca uau ucc uga utt' and antisense 'auc agg aau aug aag agg att' purchased from shanghai genepharma co ltd with jetprime polyplus transfection reagent polyplustransfection sa after h the medium was changed to fresh medium and cells were further incubated in co2 for h subsequently cells were collected for the subsequent experimentswound healing assay for the wound healing assay sw480 and hct116 cells were cultured in µl medium with fbs hyclone and the percentage of serum was in line with previous papers subconfluent tumor cells were scraped using a sterile micropipette tip and then serumfree medium was added next cells were imaged at and h using an inverted fluorescence microscope magnification x200 olympus corporation transwell assay in the migration test the transfected cells 1x105 were inoculated into the top chamber microns with µl serumfree medium complete medium µl containing fbs was added to the lower chamber corning inc following incubation at Ëc for h the migratory cells located under the insert were fixed and stained with crystal violet staining solution beyotime institute of biotechnology at room temperature for min and observed using an inverted fluorescence microscope magnification x200 olympus corporationstatistical analysis the Ï2 test was used to compare clinicopathological factors and continuous variables were analyzed via unpaired student's ttest or oneway anova kaplanmeier analysis and the log rank test were performed for survival analysis univariate and multivariate logistic regression models confirmed the associations between defb4a expression and clinical features prism graphpad software inc was used for statistical analysis of all clinical samples anova was followed by tukey's posthoc test and performed using spss for windows spss inc r software version r foundation for statistical computing was used for bioinformatics analysis p005 was considered to indicate a statistically significant difference all experiments were performed in triplicate and data are presented as the mean ± standard deviation resultsgenes associated with poor prognosis to identify genes associated with poor survival in the crc cohort patients were divided into two groups based on os short ¤ days os patients with a survival time of days would be included in short os and long days os fig 1a in total degs fold change were identified using a volcano plot including upregulated and downregulated genes fig 1b and c hierarchical cluster analysis revealed the expression profiles of the degs fig 1d subsequently gene expression profiles in tumor and normal tissues fold change were analyzed and it was observed that genes were upregulated and were downregulated in tumor tissues compared with in normal tissues p005 fdr fold change fig 1eg the venn diagram revealed that 0cwu prognostic role of defb4a in colorectal cancerfigure screening of degs based on tcga a patients with crc were divided into two groups based on whether or not they survived for days in accordance with the rnaseq data from tcga b volcano plot of rnaseq data from tcga the red dots and blue dots represent upregulated and downregulated degs based on a fold change of the volcano plot displays different genes when comparing patients with a prolonged os and those with a short os c a total of upregulated and downregulated genes were identified d hierarchical clustering analysis of the rnaseq data of different genes in short os and long os samples e hierarchical clustering analysis of the rnaseq data of different genes in ca and n samples f using a threshold of p005 false discovery rate and fold change degs were selected using a volcano plot when comparing ca samples with normal colon mucosa samples from tcga g a total of upregulated and downregulated genes were identified h venn diagram representing the distribution of degs in different groups a total of degs were expressed in both patients with ca and patients with a prolonged os crc colorectal carcinoma degs differentially expressed genes os overall survival tcga the cancer genome atlas ca cancer n normal degs were identified in both screening methods fig 1h details are shown in table iivalidated degs are associated with poor prognosis in tcga to determine the prognostic significance of the identified degs their expression levels were determined in cases included in tcga kaplanmeier survival analysis revealed that defb4a hyaluronan binding protein habp2 oleoylacp hydrolase olah and tbc1 domain family member 3g tbc1d3g upregulation was significantly associated with poor survival in patients with crc fig 2a prognostic significance was not observed for kiss1r or5m11 chrnb3 otx2 s100a7a flj43860 and frmd7 in the patients with crc data not shown furthermore defb4a and habp2 were upregulated in tumor tissues fig 2b a previous study have reported low serum expression levels of habp2 in patients with crc therefore defb4a was selected as a candidate marker of poor prognosis in patients with crcgo and gsea to evaluate the biological role of defb4a in crc progression go enrichment and gsea analyses were 0concology letters descriptiontable ii upregulated genes n10 associated with a poor colorectal carcinoma prognosis in the cancer genome atlas databasegene symbol defb4a habp2 olah tbc1d3g kiss1r frmd7 s100a7a otx2 or5m11 chrnb3 defensin beta 4ahyaluronan binding protein oleoylacp hydrolasetbc1 domain family member 3gkiss1 receptorferm domain containing s100 calcium binding protein a7aorthodenticlehomeobox olfactory receptor family subfamily m member cholinergic receptor nicotinic beta subunitgene id performed defb4a was demonstrated to be involved in various biological processes associated functional pathways are shown in fig 3a and b and closely associated with myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity implying that defb4a potentially regulates the immune system finally the database was searched for expression profiles of defb4a and immunerelated genes and a positive correlation between defb4a expression and the expression of immune markers such as cd11b cd14 cd45 cd163 and il17a was observed fig 3c these results suggest that defb4a is associated with poor prognosis in patients with crc potentially in an immunosuppressive myeloid leukocyte and cytokinedependent mannervalidation in patient samples and clinical relevance of defb4a to further clarify the clinical significance of defb4a expression the present study analyzed tissue samples from patients with crc the associations between their mrna expression levels and clinicopathological variables were observed detailed information of the patients is provided in table iii defb4a expression was significantly upregulated in the crc tumor tissues fig 4a additionally an association between defb4a upregulation and advanced crc stage stage i cases stage ii cases stage iii cases stage iv cases and metastasis m0 cases m1 cases was observed fig 4b and c furthermore defb4a upregulation in the tumor tissues was associated with poor prognosis p00313 fig 4d additionally defb4a upregulation was significantly associated with advanced liver metastasis p0039 stage p0005 high ca72 value p0003 tumor size p0009 and lymph node metastasis p0044 table iii therefore defb4a was considered to be a prognostic marker associated with tumor progression in patients with crc logistic regression analysis was performed to determine whether defb4a can help predict the prognosis of crc univariate analyses revealed that advanced tnm stage [odds ratio or p001] liver metastasis or p003 lymph node metastasis or p004 high ca199 level or1324 p002 a high ca level or p001 and high defb4a level or p002 were associated with the survival of patients with crc furthermore multivariate analyses revealed that advanced tnm stage or p004 histological differentiation or p001 liver metastasis or p001 ca199 level or p001 high ca level or p005 and high defb4a level or p001 were independent prognostic predictors table iv overall these results suggest that defb4a serves an important role in predicting the prognosis of patients with crcdefb4a promotes proliferation and metastasis in crc to explore the biological roles of defb4a in crc defb4a expression was knocked down in hct116 and sw480 cells fig 5a and b a wound healing assay revealed that defb4a knockdown inhibited the migration of hct116 and sw480 cells fig 5c transwell assays demonstrated that the migration of cells was decreased following knockdown of defb4a in sw480 cells compared with that in the nc group fig 5d the number of migratory cells decreased following knockdown of defb4a in sw480 cells fig 5e overall these results suggested that defb4a serves an important role in crc development discussionwith the increasing availability of highthroughput technologies numerous novel biomarkers and therapeutic targets have been identified through transcriptomic analysis of various types of tumor however such studies on biomarkers in crc have not been extensively performed the identification of crc biomarkers may help predict and prolong the survival of patients with crc in the present study mrna profiling of microarray analysis data from the tcga database was performed to identify numerous novel genes associated with poor prognosis in crc a critical role of defb4a in patients with crc was identified the mrna profiles of patients were first compared between the long os and short os groups and between the tumor and normal tissue groups in the tcga database subsequently the present study investigated the association between mrna expression and prognosis defb4a habp2 olah and tbc1d3g were identified as potential predicators of poor prognosis defb4a and habp2 0cwu prognostic role of defb4a in colorectal cancerfigure defb4a is upregulated based on data from tcga and predicts poor prognosis a kaplanmeier curve of four genes defb4a habp2 olah and tbc1d3g derived from data of patients included in the tcga dataset b mrna expression levels of defb4a habp2 olah and tbc1d3g in cancer vs control samples from patients in tcga p005 ns not significant tcga the cancer genome atlas defb4a defensin 4a habp2 hyaluronan binding protein olah oleoylacp hydrolase tbc1d3g tbc1 domain family member 3g ca cancer n normalwere upregulated in crc tissues of patients in the database however habp2 has been reported to be downregulated in the sera of patients with crc p00137 therefore defb4a was considered as a candidate gene for further analysis go and gsea were used to assess the function of defb4a in promoting disease progression and to highlight the role of defb4a in the tumor microenvironment defb4a was involved in myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity correlation analysis revealed that defb4a expression was positively correlated with immune markers including cd11b cd14 cd45 cd163 and il17a cd11b is expressed on the surface of a number of leukocytes including monocytes granulocytes and macrophages cd14 is expressed on both monocytes 0concology letters figure defb4a is positively correlated with inhibitory immune cells a gene ontology analysis revealed that defb4a is involved in leukocyte proliferation lymphocyte differentiation leukocyte mediated immunity myeloid cell differentiation negative regulation of type i interferon production and interleukin production b gene set enrichment analysis verified the results c correlation between defb4a and cd11b cd14 cd45 cd163 and il17a r and pvalues are indicated defb4a defensin 4a r pearson's correlation coefficientfigure defb4a predicts poor prognosis in colorectal cancer a defb4a mrna expression in groups of cancer tissues and normal tissues are displayed b defb4a mrna levels were compared with respect to tnm stage c defb4a mrna expression levels of patients with different cancer stages d effect of defb4a expression on overall survival in patients with colorectal carcinoma n52 p005 p001 p00001 defb4a defensin 4aand macrophages and cd45 is expressed on leukocytes m2 macrophages may be marked with cd163 and m2 macrophages serve a role in promoting tumor growth the os of patients with nonsmallcell lung cancer and those with esophageal cancer with high m2 macrophage infiltration rates is shorter than those with low m2 macrophage infiltration rates patients with high expression levels of il17a had a poor prognosis in a crc cohort previous studies have suggested that increased il17a promotes crc in various animal models analysis of clinical specimens of patients with crc demonstrated that defb4a expression was associated with 0cwu prognostic role of defb4a in colorectal cancer 0330a total n Ï2 pvaluedefb4a expressiontable iii association between defb4a expression and clinicopathological characteristics of patients with colorectal carcinoma characteristic sex male female age years ¥ site of lesion colon rectum differentiation poor well tumor size cm ¥ pathological type adenocarcinoma others lymph node metastasis no yes liver metastasis no yes stage iii iiiiv cea normal high ca normal high ca normal high afisher's exact test all others were assessed using a Ï2 test defb4a defensin 4ahigh n low n 0009a0003a 0575a0039a poor survival furthermore defb4a expression was upregulated in patients with crc with advanced and metastatic cancer patients with crc with high defb4a expression had poor survival in addition knockdown of defb4a affected the migration ability of crc cells tcga data of patients with crc were used to identify the degs between the long os days and short os days groups in addition mrna expression was compared between tumor tissues and normal tissues in the same database defb4a was highly expressed in tumors and associated with a poor prognosis defb4a upregulation was associated with poor prognosis and defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis another study used the gene expression omnibus database to screen genes that are increased in patients with recurrence hierarchical clustering and pathway analyses revealed that thrombospondin thbs2 and cartilage 0concology letters table iv logistic regression model analysis of liver metastasis predictors in patients with colorectal carcinoma characteristics sex male vs female age vs ¥ years tumor size vs ¥ mm pathological type adenocarcinoma vs others tnm stage iii vs iiiiv differentiation medium vs poor liver metastasis no vs yes lymph node metastasis no vs yes cea vs ¥ ca199 vs ¥ ca724 vs ¥ defb4a high vs low or odds ratio or pvalue univariate ci or pvalue multivariate ci figure defb4a promotes colorectal cancer cell migration a expression levels of defb4a were detected using rtqpcr following transfection of hct116 cells with sinc and sidefb4a b expression levels of defb4a were detected using rtqpcr following transfection of sw480 cells with sinc and sidefb4a c migration ability of cells was examined using a wounding healing assay d representative images were obtained for the transwell assay magnification x200 e proportions of migrated cells after h were quantified defb4a defensin 4a nc negative control rtqpcr reverse transcriptionquantitative pcr si small interfering rna p001 p0001 0cwu prognostic role of defb4a in colorectal canceroligomeric matrix protein comp are associated with the ecmreceptor interaction focal adhesion and tgf signaling pathways the hypergeometric distribution test demonstrated that the association between thbs2 and crc is stronger than that of comp pearson test results indicated that thbs2 might be considered to be a prognostic biomarker for crc to the best of our knowledge this screening method and the hypothesis that defb4a may serve a protumor role through immunosuppression have not been seen in other studies defb4a stimulates keratinocytes to release il and il proinflammatory cytokines serving as deciding factors in the pathogenesis of psoriasis furthermore defb4a induction is required for tolllike receptor tlr activation in monocytes through the convergence of il and vitamin d receptor signaling and exerts direct bactericidal effects against m tuberculosiss the antimicrobial peptides defb4a and camp are inhibited by hsamir leading to suppression of the tlr21induced vitamin d antimicrobial signaling pathway defb4a has been suggested as a biomarker for psoriasis because the clinical efficacy of targeted antibody therapy in psoriasis is associated with the inhibition of defb4a expression defb4a expression can directly be inhibited by anthralin in vitro and in vivo thus benefiting patients with psoriasis however it has remained unclear whether defb4a is involved in the immunoregulation in crc go analysis revealed that defb4a is involved in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity therefore defb4a may be associated with immunity in crc to the best of our knowledge the present study was the first to report defb4a as a prognostic marker for crc and as an immunoregulatory factor in the tumor microenvironment in patients with crc however a limitation of the present study was that the research cohort was not large enough which may affect the statistical results in addition the specific role of defb4a and immune factors in colon cancer and the underlying molecular mechanism need to be further exploredin conclusion to the best of our knowledge defb4a is upregulated in patients with crc and is closely associated with poor prognosis defb4a regulates immune function and potentially promotes immunosuppression therefore defb4a may be considered as a prognostic marker and immunotherapeutic target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos u1804281 and and funding from state's key project of research and development plan grant no 2016yfc1303500availability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author on reasonable requestauthors' contributionsyz qw and dw participated in the design and conception of the present study yz qw dw zs jl and wty were involved in data acquisition and analysis of certain clinical data qw dw zz and yw performed the clinical experiments and analysis of the data the manuscript was written by qw and critically reviewed by yz dw zz yw wny and nrm wny ks and nrm were involved in performing and analyzing the cell experiments all authors read and approved the final manuscriptethics approval and consent to participatethe present study was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university approval no sciencelw and informed consent was obtained from each patient with available followup informationpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences siegel rl miller kd and jemal a cancer statistics ca cancer j clin cidon eu the challenge of metastatic colorectal cancer clin med insights oncol vreeland tj clifton gt herbert gs hale df jackson do berry js and peoples ge gaining ground on a cure through synergy combining checkpoint inhibitors with cancer vaccines expert rev clin immuno siegel rl miller kd fedewa sa ahnen dj meester rgs barzi a and jemal a colorectal cancer statistics ca cancer j clin dickinson bt kisiel j ahlquist da and grady wm molecular markers for colorectal cancer screening gut nikolouzakis tk vassilopoulou l fragkiadaki p mari | 0 |
" colorectal cancer crc is the third leading cause of cancerassociated mortality the present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for crc the present study compared mrna expression levels of patients with crc with short and longterm prognosis and of individuals with and without tumors in the cancer genome atlas tcga database differentially expressed genes degs were identified via volcano plot and venn diagram analysis gene ontology go analysis and gene set enrichment analysis gsea were performed to identify the functions of the degs and the degs were further verified using clinical crc samples a total of degs were identified as candidate genes using the tcga database and four degs [defensin 4a defb4a hyaluronan binding protein habp2 oleoylacp hydrolase and tbc1 domain family member 3g] were associated with poor prognosis of patients with crc two degs defb4a and habp2 were upregulated in tumor tissues of patients with crc in the tcga database go and gsea analyses revealed that defb4a was highly associated with immunosuppression participates in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity and was positively correlated with cd11b cd14 cd45 cd163 and il17a furthermore defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis survival analysis revealed that defb4a upregulation was associated with poor prognosis defb4a correspondence to dr yi zhang biotherapy center the first affiliated hospital of zhengzhou university jianshe east road zhengzhou henan pr chinaemail yizhangzzueducncontributed equallykey words defensin 4a colorectal cancer prognosis immunity biomarkergene knockdown experiments demonstrated that def4ba promotes cell migration these results indicated that defb4a potentially promotes tumor growth by regulating immunosuppressive activity and provided novel insights into the diagnosis and treatment of crcintroductionaccording to the global cancer statistic of colorectal cancer crc is the third most common cancer and the second leading cause of cancerassociated mortality worldwide crc was reported as the fourth most common and fatal cancer in china in patients with crc usually have a low survival rate and poor therapeutic responses and are susceptible to progression and recurrence early diagnosis and effective treatment are critical to improve the survival of patients with crc crc studies have focused on innovative ideas to identify molecular markers used to develop highprecision noninvasive screening tests for crc to increase population compliance and reduce the potentially harmful side effects associated with more invasive techniques diagnostic markers will give an indication of the likely progression of the disease targeting specific molecules in certain patients has facilitated more personalized treatments that help prevent or decelerate cancer progression the present study aimed to determine prognostic factors and novel therapeutic targets to improve the survival of patients with crcprevious studies have focused on the identification of molecules associated with tumor progression through genetic or mrna profiling and screening of patients with colon cancer for example the expression profiles of long noncoding rnas lncrnas were compared at specific tumor stages t0 t1 t2 and t3 in an azoxymethanedextran sodium sulfateinduced primary colon cancer model and upregulation of the lncrna h19 predicted a poor prognosis other studies analyzed microrna mir expression profiles between tumor tissues and matched nontumor tissues obtained from patients with crc for example mir is significantly downregulated in tumor tissues and associated with poor survival of patients with crc and may thus be considered to be a poor prognostic marker of crc furthermore high expression levels of mir 0cwu prognostic role of defb4a in colorectal cancerand mir in serum are associated with poor survival of patients with crc analysis of the cancer genome atlas tcga database revealed that mmp19 is upregulated in patients with crc and is associated with tumor progression however to the best of our knowledge no study has directly screened mrna profiles based on prognosis the present study divided patients with crc into different groups based on prognosis and screened the mrna profiles of the respective groups defensin 4a defb4a also known as bd sap1 defb2 defb4 hbd defb and defb102 belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils which serve important roles in innate immune defense against microbial infections defb4a is upregulated in cutaneous squamous cell carcinoma and basal cell carcinoma it serves an important role in esophageal carcinogenesis both in vivo and in vitro the genomic copy number of defb4a has been analyzed in patients with crohn's disease and controls and an elevated defb4a copy number has been identified as a risk factor for crohn's disease regardless of disease origin however it remains unclear whether defb4a expression is associated with the prognosis of crc furthermore the role of defb4a in the immune system remains unclear the tumor microenvironment serves a significant role in tumor progression various immune elements comprise the tumor microenvironment including bone marrowderived cells such as macrophages cd4 t cells cd8 t cells b cells natural killer cells and dendritic cells myeloid cells can differentiate into macrophages or myeloidderived suppressor cells mdscs which serve a tumorigenic role in the tumor microenvironment mdscs contribute to tumor vascular development by promoting angiogenesis and tumor growth tumorassociated macrophages tams are important regulators of tumorigenesis by inhibiting the antitumor effects of other cells thus promoting tumor growth however it remains unclear whether defb4a has a regulatory effect on the tumor microenvironment or whether it promotes crc progressionto identify candidate target genes that potentially prolong patient survival mrna expression profiles of tissues from crc samples were compared in the tcga database venn analysis was performed to determine candidate genes upregulated in tumor tissues among patients with poor prognosis subsequently immuneassociated pathway enrichment was analyzed using gene ontology go and gene set enrichment analysis gsea and the correlations between candidate target genes and certain immune cells were determined finally clinical samples and crc cell lines were obtained to verify the clinical significance of the identified genes the present results may provide insights into targeted therapy for crcmaterials and methodsacquisition of microarray data microarray data were obtained from tcga httpcancergenomenihgov rnaseq data for samples were included in the dataset project id tcgacoadread including tumor samples from patients with crc and normal tissues from healthy donorsidentification of differentially expressed genes degs tcga data were divided into two groups based on different categories patient prognosis and gene expression in tumor and normal tissues venn analysis of the two groups was performed and genes associated with crc prognosis were identifiedvenn analysis to identify candidate genes associated with patient survival the gene expression profiles in the two groups were analyzed using the venn diagram web tool httpbioinformaticspsbugentbewebtoolsvenngo analysis functional analysis of the degs was performed using go httpwwwgeneontology based on biological processes gsea gsea was conducted using gsea v403 software wwwgseamsigdbgseaindexjsp and the gene used in the present study was downloaded from the molecular signatures database msigdb oftware broadinstitutegseamsigdbindexjsp v40 msigdb curates various gene sets including canonical signaling pathways from biocarta cgapncinihgovcgap_mitelman_retire_noticehtml kyoto encyclopedia of genes and genomes wwwkeggjp pid httppidncinihgov reactome reactome and other pathway databases tcga data were analyzed via gsea and pathways with a false discovery rate fdr were considered significantpatient characteristics tissue samples were obtained from patients with crc at the first affiliated hospital of zhengzhou university zhengzhou china between april and april patients underwent surgical resection or colonoscopy and the samples were verified via pathological analysis the clinical characteristics of the patients are shown in table i a total of men and women were included in the present study the median age was years age range years crc was diagnosed by two pathologists on the basis of pathological assessment the collection of specimens was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university zhengzhou china approval no sciencelw and informed consent was obtained from each patient with available followup informationreverse transcriptionquantitative pcr total rna was extracted from pairs of tumor and normal tissue samples from patients with crc using trizol reagent invitrogen thermo fisher scientific inc total rna samples µg were incubated at Ëc for min followed by incubation at Ëc for min and Ëc for sec according to the reverse transcription reaction protocol takara biotechnology co ltd the conditions of pcr were as follows Ëc10 min Ëc10 sec Ëc10 sec Ëc10 sec cycles premix ex taq ii roche target gene expression was simultaneously assessed relative to that of gapdh a housekeeping gene and internal control the following primers were used defb4a forward 'ctc ctc ttc tcg ttc ctc ttc a' and reverse 'gca ggt aac agg atc gcc tat' and gapdh forward 'gga gcg aga tcc ctc caa aat' and reverse 'ggc tgtt 0concology letters no of cases table i characteristics of patients with colorectal carcinomacharacteristic percentagesex male female age years ¥ treatment surgery others tumor size mm ¥ pathological type adenocarcinoma others lymph node metastasis yes no tnm stage i ii iii iv liver metastasis negative positive differentiation poor mediumwell others include chemotherapy and radiotherapy gtc ata ctt ctc atg g' the present study compared the expression levels of the target genes in clinical samples using the δδcq method expression levels of defb4a and gapdh were examined for each sample and the relative expression levels of defb4a were determined using the δcq value of defb4a divided by that of gapdh cell transfection sw480 and hct116 cells were seeded into a well plate sw480 and hct116 cells were purchased from chinese academy of sciences cell bank and cultivated with dmemhigh glucose containing fbs hyclone ge healthcare life sciences and penicillinstreptomycin at Ëc in co2 the growth status of the cells was closely observed until they reached a fusion rate of and then cells were transfected with ncsmall interfering rna negative control sinc sense 'uuc ucc gaa cgu guc acg utt' and antisense 'acg uga cac guu cgg aga att' and small interfering rna targeting defb4a sidefb4a sidefb4a sense 'ucc ucu uca uau ucc uga utt' and antisense 'auc agg aau aug aag agg att' purchased from shanghai genepharma co ltd with jetprime polyplus transfection reagent polyplustransfection sa after h the medium was changed to fresh medium and cells were further incubated in co2 for h subsequently cells were collected for the subsequent experimentswound healing assay for the wound healing assay sw480 and hct116 cells were cultured in µl medium with fbs hyclone and the percentage of serum was in line with previous papers subconfluent tumor cells were scraped using a sterile micropipette tip and then serumfree medium was added next cells were imaged at and h using an inverted fluorescence microscope magnification x200 olympus corporation transwell assay in the migration test the transfected cells 1x105 were inoculated into the top chamber microns with µl serumfree medium complete medium µl containing fbs was added to the lower chamber corning inc following incubation at Ëc for h the migratory cells located under the insert were fixed and stained with crystal violet staining solution beyotime institute of biotechnology at room temperature for min and observed using an inverted fluorescence microscope magnification x200 olympus corporationstatistical analysis the Ï2 test was used to compare clinicopathological factors and continuous variables were analyzed via unpaired student's ttest or oneway anova kaplanmeier analysis and the log rank test were performed for survival analysis univariate and multivariate logistic regression models confirmed the associations between defb4a expression and clinical features prism graphpad software inc was used for statistical analysis of all clinical samples anova was followed by tukey's posthoc test and performed using spss for windows spss inc r software version r foundation for statistical computing was used for bioinformatics analysis p005 was considered to indicate a statistically significant difference all experiments were performed in triplicate and data are presented as the mean ± standard deviation resultsgenes associated with poor prognosis to identify genes associated with poor survival in the crc cohort patients were divided into two groups based on os short ¤ days os patients with a survival time of days would be included in short os and long days os fig 1a in total degs fold change were identified using a volcano plot including upregulated and downregulated genes fig 1b and c hierarchical cluster analysis revealed the expression profiles of the degs fig 1d subsequently gene expression profiles in tumor and normal tissues fold change were analyzed and it was observed that genes were upregulated and were downregulated in tumor tissues compared with in normal tissues p005 fdr fold change fig 1eg the venn diagram revealed that 0cwu prognostic role of defb4a in colorectal cancerfigure screening of degs based on tcga a patients with crc were divided into two groups based on whether or not they survived for days in accordance with the rnaseq data from tcga b volcano plot of rnaseq data from tcga the red dots and blue dots represent upregulated and downregulated degs based on a fold change of the volcano plot displays different genes when comparing patients with a prolonged os and those with a short os c a total of upregulated and downregulated genes were identified d hierarchical clustering analysis of the rnaseq data of different genes in short os and long os samples e hierarchical clustering analysis of the rnaseq data of different genes in ca and n samples f using a threshold of p005 false discovery rate and fold change degs were selected using a volcano plot when comparing ca samples with normal colon mucosa samples from tcga g a total of upregulated and downregulated genes were identified h venn diagram representing the distribution of degs in different groups a total of degs were expressed in both patients with ca and patients with a prolonged os crc colorectal carcinoma degs differentially expressed genes os overall survival tcga the cancer genome atlas ca cancer n normal degs were identified in both screening methods fig 1h details are shown in table iivalidated degs are associated with poor prognosis in tcga to determine the prognostic significance of the identified degs their expression levels were determined in cases included in tcga kaplanmeier survival analysis revealed that defb4a hyaluronan binding protein habp2 oleoylacp hydrolase olah and tbc1 domain family member 3g tbc1d3g upregulation was significantly associated with poor survival in patients with crc fig 2a prognostic significance was not observed for kiss1r or5m11 chrnb3 otx2 s100a7a flj43860 and frmd7 in the patients with crc data not shown furthermore defb4a and habp2 were upregulated in tumor tissues fig 2b a previous study have reported low serum expression levels of habp2 in patients with crc therefore defb4a was selected as a candidate marker of poor prognosis in patients with crcgo and gsea to evaluate the biological role of defb4a in crc progression go enrichment and gsea analyses were 0concology letters descriptiontable ii upregulated genes n10 associated with a poor colorectal carcinoma prognosis in the cancer genome atlas databasegene symbol defb4a habp2 olah tbc1d3g kiss1r frmd7 s100a7a otx2 or5m11 chrnb3 defensin beta 4ahyaluronan binding protein oleoylacp hydrolasetbc1 domain family member 3gkiss1 receptorferm domain containing s100 calcium binding protein a7aorthodenticlehomeobox olfactory receptor family subfamily m member cholinergic receptor nicotinic beta subunitgene id performed defb4a was demonstrated to be involved in various biological processes associated functional pathways are shown in fig 3a and b and closely associated with myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity implying that defb4a potentially regulates the immune system finally the database was searched for expression profiles of defb4a and immunerelated genes and a positive correlation between defb4a expression and the expression of immune markers such as cd11b cd14 cd45 cd163 and il17a was observed fig 3c these results suggest that defb4a is associated with poor prognosis in patients with crc potentially in an immunosuppressive myeloid leukocyte and cytokinedependent mannervalidation in patient samples and clinical relevance of defb4a to further clarify the clinical significance of defb4a expression the present study analyzed tissue samples from patients with crc the associations between their mrna expression levels and clinicopathological variables were observed detailed information of the patients is provided in table iii defb4a expression was significantly upregulated in the crc tumor tissues fig 4a additionally an association between defb4a upregulation and advanced crc stage stage i cases stage ii cases stage iii cases stage iv cases and metastasis m0 cases m1 cases was observed fig 4b and c furthermore defb4a upregulation in the tumor tissues was associated with poor prognosis p00313 fig 4d additionally defb4a upregulation was significantly associated with advanced liver metastasis p0039 stage p0005 high ca72 value p0003 tumor size p0009 and lymph node metastasis p0044 table iii therefore defb4a was considered to be a prognostic marker associated with tumor progression in patients with crc logistic regression analysis was performed to determine whether defb4a can help predict the prognosis of crc univariate analyses revealed that advanced tnm stage [odds ratio or p001] liver metastasis or p003 lymph node metastasis or p004 high ca199 level or1324 p002 a high ca level or p001 and high defb4a level or p002 were associated with the survival of patients with crc furthermore multivariate analyses revealed that advanced tnm stage or p004 histological differentiation or p001 liver metastasis or p001 ca199 level or p001 high ca level or p005 and high defb4a level or p001 were independent prognostic predictors table iv overall these results suggest that defb4a serves an important role in predicting the prognosis of patients with crcdefb4a promotes proliferation and metastasis in crc to explore the biological roles of defb4a in crc defb4a expression was knocked down in hct116 and sw480 cells fig 5a and b a wound healing assay revealed that defb4a knockdown inhibited the migration of hct116 and sw480 cells fig 5c transwell assays demonstrated that the migration of cells was decreased following knockdown of defb4a in sw480 cells compared with that in the nc group fig 5d the number of migratory cells decreased following knockdown of defb4a in sw480 cells fig 5e overall these results suggested that defb4a serves an important role in crc development discussionwith the increasing availability of highthroughput technologies numerous novel biomarkers and therapeutic targets have been identified through transcriptomic analysis of various types of tumor however such studies on biomarkers in crc have not been extensively performed the identification of crc biomarkers may help predict and prolong the survival of patients with crc in the present study mrna profiling of microarray analysis data from the tcga database was performed to identify numerous novel genes associated with poor prognosis in crc a critical role of defb4a in patients with crc was identified the mrna profiles of patients were first compared between the long os and short os groups and between the tumor and normal tissue groups in the tcga database subsequently the present study investigated the association between mrna expression and prognosis defb4a habp2 olah and tbc1d3g were identified as potential predicators of poor prognosis defb4a and habp2 0cwu prognostic role of defb4a in colorectal cancerfigure defb4a is upregulated based on data from tcga and predicts poor prognosis a kaplanmeier curve of four genes defb4a habp2 olah and tbc1d3g derived from data of patients included in the tcga dataset b mrna expression levels of defb4a habp2 olah and tbc1d3g in cancer vs control samples from patients in tcga p005 ns not significant tcga the cancer genome atlas defb4a defensin 4a habp2 hyaluronan binding protein olah oleoylacp hydrolase tbc1d3g tbc1 domain family member 3g ca cancer n normalwere upregulated in crc tissues of patients in the database however habp2 has been reported to be downregulated in the sera of patients with crc p00137 therefore defb4a was considered as a candidate gene for further analysis go and gsea were used to assess the function of defb4a in promoting disease progression and to highlight the role of defb4a in the tumor microenvironment defb4a was involved in myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity correlation analysis revealed that defb4a expression was positively correlated with immune markers including cd11b cd14 cd45 cd163 and il17a cd11b is expressed on the surface of a number of leukocytes including monocytes granulocytes and macrophages cd14 is expressed on both monocytes 0concology letters figure defb4a is positively correlated with inhibitory immune cells a gene ontology analysis revealed that defb4a is involved in leukocyte proliferation lymphocyte differentiation leukocyte mediated immunity myeloid cell differentiation negative regulation of type i interferon production and interleukin production b gene set enrichment analysis verified the results c correlation between defb4a and cd11b cd14 cd45 cd163 and il17a r and pvalues are indicated defb4a defensin 4a r pearson's correlation coefficientfigure defb4a predicts poor prognosis in colorectal cancer a defb4a mrna expression in groups of cancer tissues and normal tissues are displayed b defb4a mrna levels were compared with respect to tnm stage c defb4a mrna expression levels of patients with different cancer stages d effect of defb4a expression on overall survival in patients with colorectal carcinoma n52 p005 p001 p00001 defb4a defensin 4aand macrophages and cd45 is expressed on leukocytes m2 macrophages may be marked with cd163 and m2 macrophages serve a role in promoting tumor growth the os of patients with nonsmallcell lung cancer and those with esophageal cancer with high m2 macrophage infiltration rates is shorter than those with low m2 macrophage infiltration rates patients with high expression levels of il17a had a poor prognosis in a crc cohort previous studies have suggested that increased il17a promotes crc in various animal models analysis of clinical specimens of patients with crc demonstrated that defb4a expression was associated with 0cwu prognostic role of defb4a in colorectal cancer 0330a total n Ï2 pvaluedefb4a expressiontable iii association between defb4a expression and clinicopathological characteristics of patients with colorectal carcinoma characteristic sex male female age years ¥ site of lesion colon rectum differentiation poor well tumor size cm ¥ pathological type adenocarcinoma others lymph node metastasis no yes liver metastasis no yes stage iii iiiiv cea normal high ca normal high ca normal high afisher's exact test all others were assessed using a Ï2 test defb4a defensin 4ahigh n low n 0009a0003a 0575a0039a poor survival furthermore defb4a expression was upregulated in patients with crc with advanced and metastatic cancer patients with crc with high defb4a expression had poor survival in addition knockdown of defb4a affected the migration ability of crc cells tcga data of patients with crc were used to identify the degs between the long os days and short os days groups in addition mrna expression was compared between tumor tissues and normal tissues in the same database defb4a was highly expressed in tumors and associated with a poor prognosis defb4a upregulation was associated with poor prognosis and defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis another study used the gene expression omnibus database to screen genes that are increased in patients with recurrence hierarchical clustering and pathway analyses revealed that thrombospondin thbs2 and cartilage 0concology letters table iv logistic regression model analysis of liver metastasis predictors in patients with colorectal carcinoma characteristics sex male vs female age vs ¥ years tumor size vs ¥ mm pathological type adenocarcinoma vs others tnm stage iii vs iiiiv differentiation medium vs poor liver metastasis no vs yes lymph node metastasis no vs yes cea vs ¥ ca199 vs ¥ ca724 vs ¥ defb4a high vs low or odds ratio or pvalue univariate ci or pvalue multivariate ci figure defb4a promotes colorectal cancer cell migration a expression levels of defb4a were detected using rtqpcr following transfection of hct116 cells with sinc and sidefb4a b expression levels of defb4a were detected using rtqpcr following transfection of sw480 cells with sinc and sidefb4a c migration ability of cells was examined using a wounding healing assay d representative images were obtained for the transwell assay magnification x200 e proportions of migrated cells after h were quantified defb4a defensin 4a nc negative control rtqpcr reverse transcriptionquantitative pcr si small interfering rna p001 p0001 0cwu prognostic role of defb4a in colorectal canceroligomeric matrix protein comp are associated with the ecmreceptor interaction focal adhesion and tgf signaling pathways the hypergeometric distribution test demonstrated that the association between thbs2 and crc is stronger than that of comp pearson test results indicated that thbs2 might be considered to be a prognostic biomarker for crc to the best of our knowledge this screening method and the hypothesis that defb4a may serve a protumor role through immunosuppression have not been seen in other studies defb4a stimulates keratinocytes to release il and il proinflammatory cytokines serving as deciding factors in the pathogenesis of psoriasis furthermore defb4a induction is required for tolllike receptor tlr activation in monocytes through the convergence of il and vitamin d receptor signaling and exerts direct bactericidal effects against m tuberculosiss the antimicrobial peptides defb4a and camp are inhibited by hsamir leading to suppression of the tlr21induced vitamin d antimicrobial signaling pathway defb4a has been suggested as a biomarker for psoriasis because the clinical efficacy of targeted antibody therapy in psoriasis is associated with the inhibition of defb4a expression defb4a expression can directly be inhibited by anthralin in vitro and in vivo thus benefiting patients with psoriasis however it has remained unclear whether defb4a is involved in the immunoregulation in crc go analysis revealed that defb4a is involved in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity therefore defb4a may be associated with immunity in crc to the best of our knowledge the present study was the first to report defb4a as a prognostic marker for crc and as an immunoregulatory factor in the tumor microenvironment in patients with crc however a limitation of the present study was that the research cohort was not large enough which may affect the statistical results in addition the specific role of defb4a and immune factors in colon cancer and the underlying molecular mechanism need to be further exploredin conclusion to the best of our knowledge defb4a is upregulated in patients with crc and is closely associated with poor prognosis defb4a regulates immune function and potentially promotes immunosuppression therefore defb4a may be considered as a prognostic marker and immunotherapeutic target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos u1804281 and and funding from state's key project of research and development plan grant no 2016yfc1303500availability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author on reasonable requestauthors' contributionsyz qw and dw participated in the design and conception of the present study yz qw dw zs jl and wty were involved in data acquisition and analysis of certain clinical data qw dw zz and yw performed the clinical experiments and analysis of the data the manuscript was written by qw and critically reviewed by yz dw zz yw wny and nrm wny ks and nrm were involved in performing and analyzing the cell experiments all authors read and approved the final manuscriptethics approval and consent to participatethe present study was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university approval no sciencelw and informed consent was obtained from each patient with available followup informationpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences siegel rl miller kd and jemal a cancer statistics ca cancer j clin cidon eu the challenge of metastatic colorectal cancer clin med insights oncol vreeland tj clifton gt herbert gs hale df jackson do berry js and peoples ge gaining ground on a cure through synergy combining checkpoint inhibitors with cancer vaccines expert rev clin immuno siegel rl miller kd fedewa sa ahnen dj meester rgs barzi a and jemal a colorectal cancer statistics ca cancer j clin dickinson bt kisiel j ahlquist da and grady wm molecular markers for colorectal cancer screening gut nikolouzakis tk vassilopoulou l fragkiadaki p mari | 0 |
Despite the proven benefits of iron chelation therapy ICT in the management of chronic iron overload and related complications compliance to longterm ICT is challenging Results from the ECLIPSE study an label randomized multicenter 2arm phase study evaluated the safety of deferasirox dispersible tablet and filmcoated tablet FCT formulations in patients with transfusiondependent thalassemia TDT or very low low or intermediate risk myelodysplastic syndrome MDS treated over weeksMethods The aim of the current study a 2year label multicenter singlearm phase study is to evaluate the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or lowerintermediaterisk MDS treated for years after the completion of weeks of treatment with deferasirox in the ECLIPSE phase studyResults Of patients enrolled completed treatment and study Adverse events AEs reported in most patients were of mild to moderate severity Headache and diarrhea were the most frequently reported AEs None of the serious AEs including death were considered treatment related No new safety signal was identified and longterm safety of deferasirox FCT was consistent with the known safety profile of deferasirox No major concerns associated with gastrointestinal tolerability renal safety or hematological abnormalities thrombocyt ianeutr ia were reported during the years Patients receiving deferasirox FCT had a treatment compliance by pill count of and persistence continuous use for ¥ days of Reduction in serum ferritin level was almost consistent starting from week across all postbaseline time points relative reduction month month Correspondence giovanbattistaruffoarnascivicoit UOC Ematologia e Talassemia AO CivicoDi CristinaBenfratelli Piazza Nicola Leotta Palermo ItalyFull list of author information is available at the end of the The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTartaglione a0et a0al Exp Hematol Oncol Page of Conclusions The results from this 2year interventional study suggest that the recommended dosing of deferasirox FCT with better tolerability palatability and compliance offers a favorable option of ICT for longterm management of iron overload and associated complications in TDTTrial registration ClinicalTrialsgov NCT02720536 Registered March wwwclini caltr ialsgovct2showNCT02 Keywords Deferasirox Chelation Filmcoated tablet Iron overload Longterm Safety Transfusiondependent thalassemia Myelodysplastic syndromeIntroductionIron overload in chronic anemias represents a serious consequence of impaired hematopoiesis and repeated blood transfusions leading to endan damage reduced quality of life and decreased survival Iron chelation therapy ICT can be a lifelong requirement in chronic transfusiondependent refractory anemias including thalassemia sicklecell disease and myelodysplastic syndrome MDS [ ] Despite the proven benefit of ICT patient compliance to longterm ICT is challenging [] Compliance with ICT is reported to influence the frequency and severity of iron overloadrelated complications [] and a substantial increase in morbidity mortality and treatment cost has been seen among patients who are noncompliant to ICT []Currently main iron chelators are available for clinical use deferoxamine deferiprone and deferasirox Parenterally administered deferoxamine was the mainstay of chelation therapy until the availability of oral chelators in [] Oncedaily oral administration of deferasirox dispersible tablet DT formulation presented a better option with greater compliance and quality of life over parenteral deferoxamine [] Currently there are no direct comparison studies for the oral chelators however oncedaily simpler use of deferasirox has been projected to be a more costeffective option than the thricedaily administration of oral deferiprone in the management of longterm complications [ ] The deferasirox filmcoated tablet FCT formulation with a simpler oral administration and improved palatability and gastrointestinal GI tolerability compared with deferasirox DT offers a better option for optimal patient acceptance and improved compliance to longterm therapy [ ] Thus an appropriate choice of ICT plays an important role in patient compliance to chelation therapyThe deferasirox FCT used in this study contained the same active substance of the iron chelator deferasirox and was strengthadjusted to achieve comparable exposure to the currently approved deferasirox DT Deferasirox FCT is available in dose strengths a0mg a0mg and a0mg and is dosed based on body weight Unlike the DT deferasirox FCT can be administered once daily orally without any need for dispersion either on an empty stomach or with a light meal [ ]Results from the ECLIPSE study an label randomized multicenter 2arm phase study that evaluated the safety of deferasirox DT and FCT formulations in patients with transfusiondependent thalassemia TDT or MDS very low low or intermediate risk treated over a0weeks have been published previously [] The purpose of the current study was to collect data on the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or very low low or intermediaterisk MDS who had the possibility to continue treatment with deferasirox FCT after completion of a0 weeks of treatment in the ECLIPSE study The study also aimed to collect efficacy data for deferasirox FCT in the reduction or maintenance of iron burden as measured by the serum ferritin levelMethodsKey inclusion and a0exclusion criteriaPatients recruited at European sites [Austria n Greece n Italy n ] who had completed the 24week treatment in the ECLIPSE study with tolerance to deferasirox and fulfilled all eligibility criteria were included in this study Patients were male or female aged ¥ a0 years with TDT or very low low or intermediaterisk MDS who had received deferasirox DT at doses ¥ a0mgkgday or ¥ a0mgkgday respectively as per clinical judgement Patients had a transfusion history of ¥ packed red blood cell units were anticipated to be transfused with ¥ a0unitsyear during the study and had serum ferritin a0ngmL at screening Key exclusion criteria in the study included patients with creatinine clearance CrCl below contraindication limit as per local label serum creatinine SCr upper limit of normal ULN alanine aminotransferase ALT ULN unless liver iron concentration was confirmed as a0mg Fedry weight within a0months prior to screening urine protein to creatinine ratio UPCR a0mgmg impaired GI function that may significantly alter the absorption of oral deferasirox or clinicallaboratory evidence of active hepatitis Bhepatitis C infection 0cTartaglione a0et a0al Exp Hematol Oncol Page of Study designThis was a 2year label multicenter singlearm phase study NCT02720536 aimed to provide additional efficacy safety and drug exposure data following the ECLIPSE study []Patients who were assigned to either the deferasirox DT or the deferasirox FCT arm and had completed the study period of a0weeks with tolerance to deferasirox in the ECLIPSE study were allowed to participate in this study It was planned that any patient continuing directly from the ECLIPSE study would receive the same dose of deferasirox FCT or an equivalent FCT dose at the start of this study corresponding to their DT dose at the end of the ECLIPSE study As all patients had a lag period between the completion of the ECLIPSE study and the start of this study ie patients who completed the ECLIPSE study switched to commercially available deferasirox DT or another ICT they entered the current study following a washout period with a starting dose that was based on clinical judgment For each patient the daily dose was calculated based on the patients actual body weight and then rounded up or down to the nearest whole tablet according to available strengths of deferasirox FCT tablets a0mg a0mg and a0mg Dose adjustments were allowed every a0months based on serum ferritin levels and clinical judgment with ± to a0mgkgday up to a maximum dose of a0mgkgdayThe study was conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki and was approved by independent ethics committees at participating sites The study is registered at wwwclini caltr ialsgovct2showNCT02 Patients or parentsguardians provided written informed consent or assent prior to enrollmentOutcomesThe primary objective of the study was to evaluate the overall safety of the deferasirox FCT formulation measured by the frequency and severity of adverse events AEs and changes in laboratory values of interest such as SCr and CrCl The secondary objective was to evaluate the efficacy of deferasirox FCT with respect to serum ferritin levels decreased or maintained according to individual therapeutic goal measured by the absolute and relative changes in serum ferritin levels over timeStatistical evaluationsNo formal sample size was calculated A maximum of patients aged ¥ a0years were originally planned to be enrolled from the ECLIPSE study patients were enrolled into this study There were no screening failuresData from all centers participating in this study were for analyses The statistical software SAS® version collected using electronic case record forms and pooled was used for analysis The analyses were descriptive in nature no hypothesis was tested Data were summarized with respect to demographic and baseline characteristics primary and secondary assessments along with safety observations All analyses were based on data collected as per protocolscheduled assessments according to or including clinical judgment of the investigatorPatient compliance to deferasirox FCT was evaluated using the count of deferasirox FCT by the relative consumed tablet count Descriptive statistics including confidence intervals CIs for the mean relative consumed tablet counts were provided Persistence defined as continuous use of deferasirox FCT without a gap for ¥ or ¥ a0days over a fixed time interval of interest was summarized at month a0 month month and month The incidence of any treatmentemergent AEs ie AEs from the start of study treatment to a0days after the last intake of study drug overall and by maximum grade severity mild moderate or severe as reported by the clinician were summarized using frequency counts and percentages of patients For each of the laboratory parameters of interest SCr CrCl ALT and aspartate aminotransferase [AST] the worst postbaseline values were summarized as shift tables based on notableextended ranges For serum ferritin and hematological parameters red blood cells [RBCs] platelets total white blood cells hemoglobin and hematocrit the absolute mean and the relative mean changes from baseline were summarized at each postbaseline visitResultsOverall patients were enrolled from countries across Europe of whom discontinued early from treatment Fig a0Of the patients enrolled the majority patients [] including patients aged a0 years had TDT and patients had MDS all aged ¥ a0years Most patients were Caucasians and females comprised of the study population All patients had a history of prior ICT Demographics and other baseline characteristics are described in Table a0In most patients deferasirox monotherapy was the last ICT before study treatment The majority of patients had received prior medication or therapy The most common prior medications by the Anatomical Therapeutic Chemical classification system class included vitamin D and vitamin D analogues patients [] proton pump inhibitors PPIs patients [] sequential preparations of progestogens 0cTartaglione a0et a0al Exp Hematol Oncol Page of Patients enrolled N53Countries Austria Greece Italy Disease TDT MDS Completed treatment and studyaDiscontinued from treatment Withdrewconsent Pregnancy Unsatisfactorytherapeuticeffect Death Adverseevents Abnormallaboratoryvalues Unwillingnessto comply withprocedures Fig Patient disposition MDS myelodysplastic syndrome TDT transfusiondependent thalassemia aThe patients who completed the study had received treatment for at least monthsand estrogens patients [] fixed combinations of progestogens and estrogens patients [] and thyroid hormones patients []Almost all patients [] received concomitant medication or significant nondrug therapies on or after the start of study treatment The most common concomitant medications by ATC class included vitamin D and vitamin D analogues patients [] anilides patients [] antibiotics patients [] other agents for local oral treatment patients [] PPIs patients [ and other antibiotics for topical use patients []All patients received at least transfusions during the study with a maximum of transfusions received by patient The majority of patients [] including the MDS patients received to transfusions Additional file a0 Table a0S1Exposure to a0treatment and a0complianceThe mean standard deviation [SD] duration of exposure to treatment was days with most patients [] being treated for at least a0weeks The majority of patients receiving deferasirox FCT were in the longest exposure category ¥ a0weeks The sum of each patients treatment exposure to deferasirox FCT was patientyears Almost all patients [] received deferasirox FCT at an average dose of ¥ a0 mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose received Table a0 MDS patients n received an average dose of ¥ a0mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose receivedPatients had a mean relative consumed tablet count of CI The proportions of patients with continuous use of deferasirox FCT with no interruption for ¥ a0days and ¥ a0days were n and n respectively at a0monthsEfficacyA decrease in mean serum ferritin levels was observed from week a0 onward except for month a0 though the median serum ferritin levels showed a consistent decrease across all postbaseline time points The mean SD actual decrease in serum ferritin level was µgL from baseline to month a0 µgL from baseline to month a0 and µgL from baseline to month a0 Fig a0 The decrease relative change in [SD] was higher at month a0 than at month a0 and month a0 [] vs [] vs [] The mean SD actual decrease in serum ferritin level from baseline to month a0 was reported as a0µgL relative change in percentage in patient at month a0 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Demographics and a0other baseline characteristicsDemographic variableAge years mean SDAge category years n ¥ to ¥ to ¥ 65aSex n Male FemaleBMI kgm2 mean SDPredominant race n Caucasian Asian OtherbMain underlying disease n MDS IPSSR risk stratification Very low risk Low risk Intermediate risk TDTTime since diagnosis years mean SD MDS TDTPrior ICT received during the ECLIPSE study n Deferasirox DT Deferasirox FCTLast ICT received before deferasirox FCT in the current study n Deferiprone Deferoxamine and deferiprone Deferasirox monotherapyTransfusion history Total number of transfusions received over the past months mean SD Time since last blood transfusion days mean SDBaseline serum ferritin µgL mean SDDeferasirox FCT N BMI body mass index DT dispersible tablet FCT filmcoated tablet ICT iron chelation therapy IPSSR Revised International Prognostic Scoring System MDS myelodysplastic syndrome SD standard deviation TDT transfusiondependent thalassemiaa The oldest patient was years oldb Other included two patients who selfidentified as whiteSafetyAdverse eventsOf the patients almost all [] reported at least AE regardless of study drug relationship Additional file a0 Table a0S2 provides an overview of all AEsThe most common AEs by preferred term were headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain The most common AEs reported in of patients by system an class SOC during the treatment with deferasirox FCT were related to infections and infestations [] influenza rhinitis gastroenteritis pharyngitis and urinary tract infection occurred in of patients by preferred term followed by GI disorders [] diarrhea nausea vomiting upper abdominal pain and abdominal pain occurred in of patients by preferred term Table a0 None of the AEs in the infections and infestations SOC were treatment related Of the AEs that were suspected to be treatment related in patients increased UPCR 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Exposure to a0treatment and a0complianceDeferasirox FCT N Duration of exposure days mean SDDuration of exposure categories weeks n to to to ¥ Patient treatment yearsAverage actual dose mgkgday mean SDAverage actual dose category mgkgday n to to ¥ Compliance Relative consumed tablet count mean SD Persistence Continuous use of deferasirox FCT with no interruption for ¥ days n Up to months n Up to months n Up to months n Up to months n Continuous use of deferasirox FCT with no interruption for ¥ days n Up to months n Up to months n Up to months n Up to months n FCT filmcoated tablet SD standard deviation diarrhea increased blood creatinine gastritis and proteinuria were the most common reported in of patients AEs by preferred term Of these suspected treatmentrelated AEs increased blood creatinine and diarrhea both of mild severity were reported in MDS patientsThe maximum grade of severity of AEs was reported as mild moderate and severe in and of patients respectively Table a0 Severegrade AEs reported by preferred term irrespective of study drug relationship included increased UPCR splenomegaly atrial fibrillation cardiac failure goiter cholestasis hepatic failure gastroenteritis lower respiratory tract infection lymph gland infection urosepsis femur fracture spinal fracture lumbar vertebral fracture rib fracture ulna fracture transfusion reaction arthralgia back pain malignant melanoma papillary thyroid cancer headache device failure renal colic calculus urinary hydronephrosis and ureterolithiasis each None of the patients had severe GI AEs Moderate and severegrade AEs by maximum severity grade were reported in patients and patients with deferasirox as prior chelation therapy and patients and patients with other ICT as prior chelation therapy respectively Of the patients moderategrade AEs were reported in patients in the to a0mgkgday dailydose group and patients in the ¥ a0 mgkgday dailydose group while severegrade AEs were reported in patients each in the to a0 mgkgday and ¥ a0 mgkgday dailydose groups respectivelySerious AEs SAEs regardless of study drug relationship were reported in patients MDS n TDT n and none of these were considered treatment related SAEs reported in patients with MDS included cardiac failure device failure femur fracture cholestasis hepatic failure and malignant melanoma reported in patient who died lumbar vertebral fracture in patient and urosepsis in patient Other SAEs reported in patients with TDT included spontaneous abortion atrial fibrillation biliary colic urinary calculus cholecystitis diverticulitis fracture goiter hydronephrosis lower respiratory tract infection lymph gland infection ovarian adenoma panic attack papillary thyroid cancer renal colic rib fracture ulna fracture and ureterolithiasis None of these SAEs were reported in more than patient each Death not suspected to be treatment related occurred in a 72yearold male patient with very lowrisk MDS as per the Revised International Prognostic Scoring System [IPSSR] The cause of this ontreatment death according to clinical judgment was malignant melanoma with multiple metastasis in the liver and spleen with unknown primary origin Another contributing factor for the death was liver failure and intrahepatic cholestasisAEs leading to discontinuation of study treatment were reported in patients of which drug ineffective AE in patient and serum ferritin abnormal AE in patient were considered treatment related Treatment was discontinued due to an AE moderate in severity not suspected to be treatment related in MDS patient AEs that led to dose adjustmentinterruption occurred in patients with the most frequently reported being increased UPCR patients [] and vomiting patients [] Treatmentrelated AEs that led to dose adjustmenttemporary interruption were reported in patients and included increased UPCR patients [] increased blood creatinine patients [] gastritis glycosuria proteinuria upper abdominal pain patients [] each and diarrhea gastric ulcer increased serum 0cTartaglione a0et a0al Exp Hematol Oncol Page of morfnitirrefmuresni egnahcLgµenilesabDSnaeMW2n44 M1n51M2n48M3n50 M4n43M5n46 M6n44 M7n40M8n37M9n42M10n36M11n41M12n36M13n43M14n39 M15n28M16n39 M17n32M18n31M19n31M20n33M21n26M23n28 M24n25M22n33M25n21M27n1M26n22Fig Change in serum ferritin from baseline µgL by time point M month SD standard deviation W week Error bars represent the ± SD values for the respective mean valuesNumber of observations at each timepointTime pointTable Common adverse events reported by a0maximum grade of a0severityAEs N a0System an class a0 a0Preferred termOverall n Mild n Moderate n Severe n Number of patients with at least one event Infections and infestations Rhinitis Gastroenteritis Pharyngitis Urinary tract infection Gastrointestinal disorders Diarrhea Nausea Vomiting Upper abdominal pain Abdominal pain General disorders and administration site conditions Asthenia Influenza Pyrexia Respiratory thoracic and mediastinal disorders Cough Oropharyngeal pain Investigationsa Urine proteincreatinine ratio increased Musculoskeletal and connective tissue disorders Musculoskeletal pain Nervous system disorders Headache Proportion of patients with AEs reported by preferred term and grouped by system an classAE adverse eventa Abnormal laboratory values reported as AEs 0cTartaglione a0et a0al Exp Hematol Oncol Page of ferritin hypertransaminasemia and increased transaminases patient [] each AEs required additional treatment in patients with TDT macular edema and skin ulcer in patient [] diarrhea radius fracture and breast discomfort in patient [] each and none were suspected to be treatment relatedAdverse events of a0special interestOverall patients reported AEs of special interest of which AEs suspected to be treatment related were reported in patients Common AEs of special interest incidence included increased UPCR patients [] severe and suspected to be treatment related patients [] proteinuria patients [] increased blood creatinine patients [] MDS n and hypertransaminasemia patients [] Hepatic failure due to metastatic liver disease MDS n and transfusion reaction TDT n of severe grade but not suspected to be treatment related were reported in patient each One patient with MDS discontinued the treatment due to decreased creatinine renal clearance moderate in severity but not suspected to be treatment related Additional file a0 Table a0S3Laboratory parametersWorst postbaseline elevations in SCr of ULN at consecutive measurements at least a0days apart occurred in patients MDS n TDT n One patient with MDS had worst postbaseline UPCR a0 mgmmol at consecutive measurements at least a0 days apart notable range Two patients with MDS had worst postbaseline CrCl value within the notable range a0 mLmin at consecutive measurements at least a0days apart and patients had worst postbaseline CrCl a0mLmin value Two patients with TDT had a worst postbaseline ALT level in the notable range ULN and baseline value Elevations of transaminases AST or ALT ULN were Table Shift tables of a0laboratory values based on a0notableextended rangesALT UL¤ ULN ULN to ¤ ULNTotalAST UL¤ ULN ULN to ¤ ULNTotalSerum creatinine¤ ULNTotalCreatinine clearance¥ ¥ to MissingTotalUrinary proteinurinary creatinine ratio mgmmol¤ MissingTotalBaselinen n n n n ¤ ULN n ¤ ULN n ¥ n ¤ n Worst postbaseline value¤ ULN n ULN to a0¤ ULN n ULN to a0¤ ULN n ULN n ULN n Notable range n Extended range n Notable range n Extended range n Two consecutive ULN n Notable range n One value ¥ to a0 n Notable range n Missing n One value n Extended range n One value n Two values n Notable range n Missing n ALT alanine aminotransferase AST aspartate aminotransferase ULN upper limit of normal 0cTartaglione a0et a0al Exp Hematol Oncol Page of uncommon only patient with TDT had a postbaseline increase in AST and ALT values ULN and baseline value Table a0Hematological parametersThe majority of patients had low RBC of hematocrit of and hemoglobin of values at baseline Among patients with a baseline platelet count ¥ 109L only patients had worst postbaseline values in the notable range ¥ to 109L Similarly among patients with a baseline absolute neutrophil count ¥ 109L the worst postbaseline values were found to be in the notable range ¥ to 109L in patient and extended range 109L in patient Mean ± SD change mean relative percentage change from baseline to month and month in key hematological parameters is represented in Additional file a0 Table a0S4DiscussionThis 2year phase interventional study evaluated the efficacy and safety of deferasirox FCT in adult and pediatric patients mean age a0 years with MDS n and TDT n who had previously completed a0weeks of deferasirox treatment in the ECLIPSE study over a mean duration of further a0days Almost of patients received an average actual deferasirox FCT dose of ¥ a0mgkgday during the study The average actual dose in MDS patients was comparatively less than that in TDT patients reflecting the notion of physicians using lower doses to treat MDS patients [] None of the patients were administered with higher than the maximum recommended doses of deferasirox FCT a0mgkgday []Various studies have demonstrated that compliance with ICT significantly improves morbidity and mortality [ ] Compliance mean relative consumed tablet count and persistence rates with deferasirox FCT during this study were found to be on the higher side Persistence rates proportion of patients with continuous use of deferasirox FCT with no interruption for ¥ a0days or ¥ a0days in this study are comparable to the realworld data on persistence in patients who switched from deferasirox DT to deferasirox FCT []Common AEs reported with deferasirox FCT in this study headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain were consistent with the known profile of deferasirox [ ] AEs reported in of the patients were of mild to moderate in severity none of the GI AEs were of severe grade nor led to discontinuation of treatment These results were in line with those of previous studies which reported that GI tolerability profile may be improved with the FCT compared with the DT Lack of excipients that cause GI irritation and ease of administration along with a light meal could have contributed to the better GI tolerability of FCT [ ] It is noted that the concomitant use of PPIs during the study was higher than reported prior use This study was not designed to investigate the reasons behind the increased use of PPIs but we assume that they might have been prescribed as a general prophylaxis to avoid GI complications from other concomitant therapies analgesics antibiotics etc prescribed in comorbid conditions infections or proceduresSAEs regardless of study drug relationship were reported in of patients and none including the death due to malignant melanoma with multiple metastasis in the liver and spleen were considered treatment related Notably of SAEs reported in patients were reported in MDS patients aged ¥ a0 years reflecting the considerable burden of the underlying hematological disorder together with the comorbidities of the affected elderly patients [] Discontinuation of treatment due to treatmentrelated AEs drug ineffective and abnormal serum ferritin during the a0years was observed in only TDT patients The low rate of SAEs and absence of treatmentrelated deaths in this study could have been a result of the greater compliance and persistence rates with deferasirox FCTThe ECLIPSE study reported that abnormal renal parametersrenal AEs were more common in patients receiving deferasirox FCT than in those receiving deferasirox DT due to an intake of higher than the recommended dose [] Most of the renal abnormalitiesAEs reported during this 2year study were mild and reversible with dosage adjustment or temporary interruption of deferasirox FCT Only patient discontinued the treatment due to decreased creatinine renal clearance which was moderate in severity and not suspected to be treatment related Increases in SCr and liver function tests in the notableextended ranges were observed in some patients and those increases were consistent with the known safety profile of deferasirox FCT [] No substantial difference in severity of all AEs was noted based on dosing groups to a0mgkgday and ¥ a0mgkgday as well Administration of the recommended doses of deferasirox FCT with constant dosage adjustment as per serum ferritin levels might have resulted in fewer renal and liver abnormaliti | 2 |
bladder cancer is the tenth most common cancer globally but existing biomarkers and prognostic models are limitedmethod in this study we used four bladder cancer cohorts from the cancer genome atlas and gene expression omnibus databases to perform univariate cox regression analysis to identify common prognostic genes we used the least absolute shrinkage and selection operator regression to construct a prognostic cox model kaplanmeier analysis receiver operating characteristic curve and univariatemultivariate cox analysis were used to evaluate the prognostic model finally a coexpression network cibersort and estimate algorithm were used to explore the mechanism related to the modelresults a total of genes were identified from the four cohorts to construct the prognostic model including eight risk genes serpine2 prr11 dsel dnm1 comp elovl4 rtkn and mapk12 and three protective genes fabp6 c16orf74 and tnk1 the 11genes model could stratify the risk of patients in all five cohorts and the prognosis was worse in the group with a highrisk score the area under the curve values of the five cohorts in the first year are all greater than furthermore this models predictive ability is stronger than that of age gender grade and t stage through the weighted coexpression network analysis the gene module related to the model was found and the key genes in this module were mainly enriched in the tumor microenvironment b cell memory showed low infiltration in highrisk patients furthermore in the case of low b cell memory infiltration and highrisk score the prognosis of the patients was the worst the proposed 11genes model is a promising biomarker for estimating overall survival in bladder cancer this model can be used to stratify the risk of bladder cancer patients which is beneficial to the realization of individualized treatmentkeywords bladder cancer cox regression prognostic model overall survivalcorrespondence yyzhucmueducn yumengcmueducn jiaxing lin and jieping yang contributed equally to this work department of urology the first hospital of china medical university shenyang liaoning china department of reproductive biology and transgenic animal china medical university shenyang liaoning chinafull list of author information is available at the end of the bladder cancer is the tenth most common cancer in the world it is more common in men than in women and the morbidity and mortality rate in men is four times higher than that in women a significant risk factor for bladder cancer is smoking with half of all cases are linked to smoking [ ] about of patients with nonmuscular invasive bladder cancer are treated by radical tumor resection followed by intravesical instillation of the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0clin a0et a0al cancer cell int page of bacille calmettegurin vaccine approximately of patients have muscular invasive or metastatic bladder cancer and are treated with radical cystectomy and neoadjuvant chemotherapy bladder cancer is a complex disease although many clinical factors and molecular markers have been identified that can predict prognosis these have low accuracy and it does not have universal applicabilitywith the continued development of gene sequencing technology and expansion of public databases it is possible to take advantage of biological information to mine sequencing data and identify biomarkers this method can utilize large sample sizes with less investment making it an important new direction to screen disease biomarkers of available databases the cancer genome atlas tcga cance rgeno menihgov database is an authoritative oncology database and the gene expression omnibus geo httpwwwncbinlmnihgovgeo database stores curated gene expression datasets many studies have constructed a multiqueue verification model based on these two databases such as nonsmall cell lung cancer [ ] and ovarian cancer prognostic models provide effective guidance for doctors and patients to make optimal treatment decisions however in the study of the bladder cancer model many models can only be verified in two or three cohorts [ ] and do not have clinical extensibilityin this study gene expression and clinical data related to bladder cancer were obtained from tcga and geo databases and common prognostic genes were screened by univariate cox proportional hazard regression this prognostic model of bladder cancer was constructed by least absolute shrinkage and selection operator lasso regression and then verified using five cohorts this robust model can help patients with bladder cancer to achieve individualized treatmentmaterials and a0methodsdata obtaining and a0processingto reduce the error of the data we searched the tcga and geo databases for bladder cancer cohorts with a sample size of more than and these cohorts need to include survival status and survival time we found a total of five cohorts the raw rna sequencing and clinical data of bladder urothelial carcinoma blca n were obtained from tcga database and the raw rna sequencing and clinical data of gse13507 n gse32548 n gse32894 n and gse48075 n from the geo database these five cohorts were analyzed on the illumina sequencing platform in r programming language software the r package edger was used to standardize the raw rna expression matrix and obtain the corresponding log valuesconstruction of a0prognostic modelthe cox proportional hazard regression model was applied to perform univariate cox proportional hazard analysis of all genes in tcgablca gse13507 gse32548 and gse32894 cohorts the hazard ratio hr from univariate cox regression analysis was used to select the genes that were positively or negatively related to prognosis a gene with hr was considered a risk gene and a gene with hr was considered a protective gene statistical significance was defined as p the genes with hr and p were selected from the four cohorts and then risk genes were obtained by overlapping four groups of genes similarly genes with hr and p were selected for the four cohorts and combined to obtain the set of protective genes a venn diagram was constructed using the online tool bioinformatics and evolutionary genomics httpbioin forma ticspsbugent bewebto olsvenn the identified risk and protective genes make up a set of prognostic genesthe data from tcgablca as a training set was used to construct a prognostic model to simplify the model the genes were selected by univariate cox regression analysis with a p value less than the r package glmnet and survival were used to do lasso regression to further screen genes and construct a cox module first the function glmnet was randomly simulated times to construct the model and establish the relationship between lambda punishment coefficient and regression coefficients coef a higher value of lambda corresponds to greater punishment with the increase of lambda some gene coef become zero indicating that the expression of the gene will not affect the model so this gene can be removed from the model then the function cvglmnet was randomly simulated times for crossvalidation cv cv is usually divided into holdout kfold and leaveoneout cv the function used kfold cv and k took the default parameter in tenfold cross validation the data set is divided into equal parts and then nine part are tested as training sets and one is used as the validation set the deviance of the tests were used to estimate the accuracy of the model when the deviance is minimum the model is the best and the coef of the model can then be obtained by using the corresponding lambda value finally we obtained the genes and the corresponding coef to build the model the prognostic model was defined as risk score ni expi · coefi where n is the number of genes expi is the expression of the ith gene and coefi is the regression coefficient of the ith gene the algorithm can prevent overfitting of the model remove highly coexpressed 0clin a0et a0al cancer cell int page of genes and finally construct a simplified model using the obtained model we calculated the risk score of each patient in the four cohortsbetween genes modulemembership and the correlation between genes and clinical traits genesignificance kaplanmeier analysisr packages survival and survminer were used for kaplanmeier analysis and the function rescat was used to find the best cutoff value of factors the cutoff was used to divide the sample into a highrisk group and a lowrisk group to construct the kaplanmeier curve with the smallest p value the risk score distribution gene expression and patient survival status data were plotted using the r package pheatmapreceiver operating characteristic curvereceiver operating characteristic roc curves of a0 years were plotted and the area under the curve auc values were calculated using the r package survivalrocunivariate and a0multivariate cox regression analysisthe risk scores and clinicopathological factors were analyzed by univariate and multivariate cox regression analysis using the r package survival the multivariate cox analysis included age sex primary tumor range t stage grade and risk score tcgablca and gse13507 also include stage lymph node and metastasisexploration of a0gene methylationcbioportal for cancer genomics is an openaccess opensource resource wwwcbiop ortal for interactive exploration of multiple cancer genomics data sets [ ] use this tool to query the relationship between gene expression and dna methylation in the bladder cancer tcga cell dataset the tool can also download gene methylation data which can be combined with clinical data for kaplanmeier analysisweighted coexpression network analysisthe set of mrna genes in the tcgablca cohort with univariate cox analysis values less than were selected to construct a bladder cancer coexpression network by weighted gene coexpression network analysis wgcna the r package wgcna was used to construct the coexpression network this method takes advantage of similarities of gene expression and groups the genes with similar expression patterns into the same module with the idea that genes in the same module may share physiological function we then explored the relationship between the clinicalfactorriskscore and module and applied pearson correlation to determine the module that was most related to the risk score the key genes were selected by the calculated correlation pathway and a0process enrichment analysisidentified genes were entered into the metascape database httpmetas cape for pathway and process enrichment analysis the enrichment analysis included kegg pathway go biological processes reactome gene sets canonical pathways and corum to evaluate the potential biological functions and pathways of the selected genescibersort and a0estimate algorithmcibersort celltype identification by estimating relative subsets of rna transcripts is a bioinformatics algorithm to calculate cell composition from gene expression profiles of complex tissues the combination of cibersort and lm22 leukocyte signature matrix can be used to calculate the content of kinds of human leukocyte subsets we used the r package cibersort to calculate the number of immune cells in each sample of the tcgablca cohort estimate estimation of sttromal and immune cells in malignant tumours using expression data is a tool that uses gene expression trends to infer the fraction of stromal and immune cells in tumor samples the immune score of each patient in tcgablca was calculated by the r package estimate immune score represents the content of immune cells and the higher the score the higher the cell contentstatistical analysisall the statistical analyses were carried out by using r programming language software rx64 all r packages were obtained from cran cranrproje ct or bioconductor httpwwwbioco nduct or the two groups were compared by the wilcoxon test and comparison between multiple groups was performed by kruskalwallis test statistical significance was defined as p difference scatter plots were constructed using the r package beeswarm we used the r package vioplot to draw violin pictures and the r package corrplot to draw correlation heat mapresultsdata processing and a0research processwe obtained the raw rna sequencing and clinical data of tcgablca n gse13507 n gse32548 n and gse32894 n we utilized data only from patients associated with rna sequencing data survival time survival status and primary tumor for further analysis the basic clinical information of the remaining patients is summarized in table a0 the sample 0clin a0et a0al cancer cell int page of table basic clinical information for a0the a0four cohortsclinical factorstcga_blcan age ¦ gender male femalet stage t2 § t2grade who2004 low highgrade who1999 g1 g2 g3vital status alive deadfollowup mean ± sd year ± gse13507n gse32548n gse32894n ± ± ± sd standard deviationsizes of these four cohorts are all greater than the grade of bladder cancer is closely related to recurrence and invasive behavior two grading methods were used in these four cohorts tcgablca and gse13507 used the who grading standard of which was divided into punlmp papillary urothelial neoplasms of low malignant potential low grade and high grade gse32548 and gse32894 used the who grading standard of which was divided into grade g1 grade g2 and grade g3 the research process is shown in fig a0construction of a0prognostic modelunivariate cox proportional hazard analysis was carried out in tcgablca gse13507 gse32548 and gse32894 cohorts there were genes in tcgablca genes in gse13507 genes in gse32548 and genes in gse32894 that met the criteria hr and p there were genes in tcgablca genes in gse13507 genes in gse32548 and genes in gse32894 that met the criteria hr and p combining the four datasets allowed identification of risk genes and protective genes fig a0 because of the large sample size of tcgablca we used this cohort to build the prognostic model first genes with univariate cox pvalues less than in tcgablca were selected then the genes were analyzed by lasso regression analysis fig a0 2a when the number of genes in the model was the deviance was the smallest fig a02b according to the lambda value the corresponding coef of the selected genes could be determined the prognostic model could then be constructed by using the corresponding coef of the genes to see more intuitively whether these genes are collinear we analyze the coexpression of these genes as shown in the fig a02c the coexpression index of none of these two genes is greater than finally we successfully constructed a prognostic module risk score serpine2 prr11 fab p6 \u200b c 16orf74 \u200b ds el d nm1 \u200b comp t nk1 \u200b elov l4 rtkn mapk12 the basic information and coef values of the genes are listed in additional file a0 table a0 s1 the average expression values transcripts per million of genes in the four cohorts are greater than which is of practical significance for detection additional file a0 table a0s2 the results of univariate regression analysis of these genes in cohorts are shown in additional file a0 table a0s3 0clin a0et a0al cancer cell int page of fig flow chart of analysiskaplanmeier analysis of a0 geneseleven genes were taken kaplanmeier analysis in cohorts using the heat map to show the results of the study fig a0 2d except for dsel in gse32894 and c16orf74 in gse32548 the other analyses were statistically significant p serpine2 rtkn prr11 mapk12 elovl4 dsel dnm1 and comp showed that the prognosis of patients with high expression was worse and the analysis of elovl4 in tcgablca was taken as an example p fig a0 2e tnk1 fabp6 and c16orf74 showed that the prognosis of the low expression group was worse and the analysis of fabp6 in tcgablca was taken as an example p fig a02fthe degree of a0dna methylation of a0tnk1 and a0c16orf74 was a0negatively correlated with a0gene expressiondna methylation can regulate gene expression we explored the relationship between expression and methylation of these genes additional file a0 figure s1ak the results showed that there was a negative correlation between tnk1 gene methylation and gene expression spearmen cor p 144e so did as c16orf74 spearmen cor p 897e then we took tnk1 and c16orf74 methylation data combined with clinical data for kaplanmeier analysis we found that the degree of methylation of these two genes can predict the prognosis of bladder cancer p additional file a0 figure s1i m and the prognosis is worse in the case of hypermethylation the expression of tnk1 and c16orf74 is inhibited by hypermethylation which leads to a worse prognosis of bladder cancerverification of a0the a0prognostic modelthe prognostic model was used to calculate the risk scores of each patient in the training set tcgablca and three test sets gse13507 gse32548 and gse32894 we identified the best cutoff value with a risk score of for tcgablca using this method the cutoff values of gse13507gse32548gse32894 were the kaplanmeier curves showed that the prognosis of patients with highrisk was significantly worse than that of patients with lowrisk in the four cohorts p fig a03ad the receiver operating characteristic roc curves of the four cohorts were drawn the a0year area under the curve auc values for the tcgablca cohort were and respectively fig a0 3e those for the gse13507 cohort were and respectively fig a03f those for the gse32548 cohort were and respectively fig a03g and those for the gse32894 group were and fig a03h additional file a0 figure s2 shows the risk score distribution gene expression values and survival status of patients in both the highrisk group and the lowrisk groupthe clinical factors and risk scores of the four cohorts were analyzed by univariate cox and multivariate cox regression analysis table a0 the results of univariate analysis showed that t stage was more effective in predicting prognosis among the clinical factors 0clin a0et a0al cancer cell int page of fig lasso cox and kaplanmeier analysis a lines of different colors represent different genes with the increase of lambda value the coef of some genes become zero indicating that they do not affect the model b the deviance of the cross validation when the partial likelihood deviance is minimum the corresponding model is the best c the coexpression heat map of genes red indicates a positive correlation blue indicates a negative correlation and the cross indicates no statistical significance d the heatmap of kaplanmeier analysis results of genes red means high expression of the gene lead to worse prognosis blue means low expression of the gene lead to worse prognosis grey means there are no significance of the analysis p means it is statistically significant the darker of the color shows the smaller of the pvalue e kaplanmeier analysis of elovl4 in tcgablca f kaplanmeier analysis of fabp6 in tcgablcaand three cohorts had statistical significance the risk scores were statistically significant in all four cohorts and the p value of three cohorts was lower than that of the t stage in multivariate cox analysis risk scores were statistically significant in three cohorts indicating that the three cohorts were independent of other clinical factors in predicting prognosis in this analysis only two cohorts of t stage had statistical significance so it is obvious that t stage is not as strong as risk score to predict the prognosis finally we compared the risk scores for different grades and t stage in the four cohorts and found that the risk scores increased 0clin a0et a0al cancer cell int page of fig the kaplanmeier analysis and roc curves of the risk score kaplanmeier curves of tcgablca a gse13507 b gse32548 c and gse32894 d red indicates highrisk group and blue indicates lowrisk group p means it is statistically significant ci confidence interval roc curves of tcgablca e gse13507 f gse32548 g and gse32894 h in years and their corresponding auc valueswith the increase of grade and t stage p additional file a0 figure s3a b in the gse32548 cohort we compared the risk scores of fgfr3 and tp53 or with the mdm2 alteration for wild type and mutant type additional file a0 figure s3c shows that a lower risk score of mutant type than that of wild type for the fgfr3 groups p in tp53 or with the mdm2 alteration the score of mutant type was higher than that of wild type p additional file a0 figure s3cseven genes and a0model were successfully verified in a0gse48705we evaluated the prognostic ability of genes and models in gse48075 n the results showed that serpine2 rtkn prr11 mapk12 elovl4 dsel and comp were statistically significant p additional file a0 figure s4 and the prognosis was worse in the high expression group which was consistent with the analysis result of the previous four cohorts the risk score of patients was calculated according to the model 0clin a0et a0al cancer cell int page of table univariate and a0 multivariate cox regression analysis of a0 clinicalfactorsriskscore with a0 overall survival rate in a0patientsvariablesunivariate analysismultivariate analysishr ciphr ciptcgablca age gender grade stage t stage node metastasis risk scoregse13507 age gender grade stage t stage node metastasis risk scoregse32548 age gender grade t stage risk scoregse32894 age gender grade t stage risk score0inf 120e 556e 991e 262e 657e 806e 544e 246e0inf 269e 214e 996e 763e 223e 164e 824e 367e 453e 129e 246e 400e 506e 388e 364e 114e 934e 613e 326e 742e 637e 871e 181e 997e 870e 183e 271e 221e 737e 200e 427e 183e 341e446e 179e 445e 445e 942e 154e 163e 561e 408e 986e 136eitalic font means statistically significanthr hazard ratio ci confidence interval inf infinityand the risk score was analyzed by kaplanmeier analysis the prognosis of the high riskscore group was worse and the difference was statistically significant p fig a04a we drew the roc curves of the risk score and the auc value of a0 year was fig a0 4b figure a0 4c showed the risk score distribution gene expression values and survival status of patients between high and lowrisk groupsweighted coexpression network analysis and a0enrichment analysisthe coexpression network was constructed with coding genes and samples in tcgablca cohort first the expression matrix was transformed into a topological overlap matrix according to β then the genes were divided into different modules fig a05a using the dynamic pruning tree method next the association analysis of clinical traits and modules fig a05b showed a high correlation between the turquoise module and risk score cor p 2e there was also a high correlation between the turquoise module and survival status cor p 9e07grade cor p 1e09stage cor p 1e we selected key genes fig a0 5c in the turquoise module according to the standard to explore the potential function of these key genes pathway and process enrichment analysis of these key genes were performed as shown in fig a05d the three most highly significantly enriched terms were extracellular matrix anization collagen fibril anization and ecm proteoglycans all related to the tumor microenvironment tmeimmune cells can be combined with a0risk scores for a0prognostic analysiswe used cibersort to calculate the infiltration ratio of immune cells in tcgablca samples and used a bar chart to show the infiltration of high and lowrisk groups fig a06a then the wilcoxon test was used to compare the difference between high and lowrisk groups the results showed that b cells naive macrophages m0 and macrophages m1 showed high infiltration in the highrisk group b cells memory dendritic cells resting and dendritic cells activated showed high infiltration in the lowrisk group p fig a06b furthermore we took the risk score and the infiltration degree of these six kinds of immune cells for joint prognostic analysis the samples were divided into four clusters for kaplanmeier analysis according to the median value of the risk score and immune cell infiltration degree the results showed that these groups could also be used for prognostic analysis p fig a06ch among them the prognostic ability of b cells memory is the best when the degree of b cells memory infiltration is low and the risk score is high the prognosis of this cluster is significantly worse than that of other clusters we used estimate to calculate the tcgablca cohorts immune score and then combined with the risk score for kaplanmeier analysis the results showed that the cluster with low immunescore and high riskscore had the worst prognosis additional file a0 figure s5 0clin a0et a0al cancer cell int page of fig validation of the model in gse48075 a kaplanmeier analysis of the riskscore b roc curves of riskscore in years c the risk score analysis from top to bottom patients risk distribution gene expression profile and survival status mapdiscussionbladder cancer is a heterogeneous disease with a high incidence and recurrence rate but there is no robust predictive tool to guide clinical treatment some recent studies have also constructed a new model for bladder cancer such as dna methylationdriven genes related model and immune genes related model these models prefer to take a kind of gene set to build the model rather than the whole genome into the screening in this study prognostic genes were screened from four cohorts with the whole transcriptome and the common prognostic genes were selected to construct the model the model successfully predicted the overall survival of five cohorts about bladder cancer patients and it is the research with the largest cohort size in the same type of researcha variety of regional source cohorts are used to jointly develop the model which makes the model have higher credibility and broader applicability in our study all genes in all cohorts were then analyzed by univariate cox proportional hazard analysis to screen common prognostic genes in four cohorts after further screening a prognostic model was constructed using the data from the tcgablca cohort instead of using the genes obtained by analysis of a single cohort to construct a prognostic model the prognostic genes common to multiple cohorts were used to make the model more stable and reliable the patients in the tcgablca cohort were from north america gse13507 was from asia and gse32548 and gse32894 were from europe it is concluded that this model has a wide range of applicabilitythe main finding of this study is that the 11gene model we developed has a robust prognostic ability and successfully predicted the prognosis of five cohorts kaplanmeier analysis showed that the prognosis of the highrisk group was worse in all the four cohorts p the 1year auc values of the tcgablca gse13507 gse32548 and gse32894 cohorts were and respectively indicating that the risk score has the ability to predict prognosis univariate and multivariate cox analysis of clinical factors and risk scores showed that the ability of risk scores to 0clin a0et a0al cancer cell int page of fig weighted coexpression network and enrichment analysis a genes were divided into different modules according to the dynamic cutting tree method and different colors represent different modules b the heatmap of the correlation between the gene module and clinical traits p indicates statistical significance c gene significance and module membership scatter diagrams of the turquoise module the dots in the red box are represented as key genes d the left side of the picture shows the interaction network map of enriched proteins and the same color indicates the same enrichment the right side of picture shows the enriched terms decreasing from top to bottom by the significance of enrichmentpredict prognosis was better than age gender grade and t stage we also analyzed the relationship between risk score and different clinical status and found increased risk score with the increase of bladder cancer t stage and grade p there are also significant differences in risk scores between wild type and mutant types of different genes we analyzed the gse32548 mutation data and found lower risk score in the group of fgfr3 mutation in contrast in the presence of a tp53 mutation or with mdm2 alteration the risk score was higher according to previous reports mutations in fgfr3 is associated with better prognosis but tp53 mutation or with mdm2 alteration is associated with worse prognosis [ ] these s indirectly verify the prognostic ability of the risk model finally the 11gene model was successfully verified in independent cohort gse48075 the model is verified by four internal cohorts and one external cohort which shows that the model has the potential to be used in the cliniceleven genes are potential prognostic markers and therapeutic targets for bladder cancer these genes have a stable prognostic ability in tcgablca gse13507 gse32548 and gse32894 cohorts and kaplanmeier analysis showed that serpine2 rtkn prr11 mapk12 elovl4 dsel and comp was successfully verified in gse48075 besides the methylation level of tnk1 and c16orf74 can also predict the prognosis of bladder cancer among them only c16orf74 and 0clin a0et a0al cancer cell int page of fig combined analysis of risk score and immune infiltrating cells a the bar chart of the infiltration of kinds of immune cells in the samples of high and lowrisk groups b the difference of kinds of immune cells between the lowrisk group and the highrisk group was analyzed an | 0 |
gastrointestinal nematodes could release excretorysecretory es proteins into the host environment to ensure their survival these es proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions especially in chronic infections in our preliminary study haemonchus contortus adhesionregulating molecule hcadrm1 was identified from h contortus es proteins hcesps that interacted with host t cells via liquid chromatographytandem mass spectrometry analysis however little is known about hcadrm1 as an es protein which may play a pivotal role at the parasitehost interfacemethods based on bioinformatics approaches multiple amino acid sequence alignment was conducted and the evolutionary relationship of hcadrm1 with adrm1 orthologues was extrapolated employing rtqpcr and immunohistochemistry assays temporal transcriptional and spatial expression profiles of hcadrm1 were investigated using immunostaining approaches integrated with immunological bioassays the immunomodulatory potentials of hcadrm1 on goat t cells were assessedresults we hereby demonstrated that hcadrm1 with immunodiagnostic utility was a mammalian adrm1 orthologue abundantly expressed at all developmental stages of h contortus given the implications of adrm1 proteins in cell growth survival and development we further investigated the immunomodulatory property of hcadrm1 as an individual es protein acting at the parasitehost interface the rhcadrm1 stimuli notably suppressed t cell viability promoted intrinsic and extrinsic t cell apoptosis inhibited t cell proliferation and induced cell cycle arrest at g1 phase simultaneously rhcadrm1 stimuli exerted critical controls on t cell cytokine secretion profiles predominantly by restraining the secretions of interleukin il4 il10 and interferongammas importantly hcadrm1 protein may have prophylactic potential for antih contortus vaccine development together these findings may contribute to the clarification of molecular and immunomodulatory traits of es proteins as well as improvement of our understanding of parasite immune evasion mechanism in h contortushost biologykeywords h contortus excretorysecretory protein adhesionregulating molecule adrm1 immunomodulation immune evasioncorrespondence lixiangruinjaueducn moe joint international research laboratory of animal health and food safety college of veterinary medicine nanjing agricultural university nanjing jiangsu peoples republic of chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0clu a0et a0al parasites vectors page of the highly conserved and regulated ubiquitin ub proteasome pathway is the primary mechanism for targeted elimination of most shortlived proteins including misfolded or damaged proteins in eukaryotic cells ub can covalently attach to cellular proteins by ub modification which is an atpdependent process mediated via different classes of ub enzymes alongside three families of shuttling factors rad23 dsk2 and ddi1 three proteasome subunits located in the subcomplex of 26s proteasome rpn1 rpn10 and rpn13 are demonstrated to be ub receptors as well as the proteasomeassociated polyubiquitin receptor rpn13 also termed as adhesionregulating molecule adrm1 is recruited by rpn2 to be assembled into the 19s regulatory p and target protein substrates linked to the small protein ub via its pleckstrinlike receptor [ ] simultaneously the cterminal adaptor domain of adrm1 serves to bind and activate the deubiquitylase uchl5uch37 and enhance its isopeptidase activity revealing a mechanism to accelerate ub chain disassembly []with engagement in the ub proteasome pathway that regulates a broad range of physiological functions adrm1 is implicated in multitudinous cellular processes such as cell growth migration survival and development particularly in cancer cells recent publications reveal that adrm1 transcription is consistently elevated in ovarian colorectal and gastric cancer tissues and knockdown of adrm1 expression in both human colon carcinoma and gastric cancer cell lines suppress cell migration and proliferation and induces cell apoptosis [] meanwhile fejzo et a0 al demonstrated that overexpression of adrm1 in ovarian cancer promoted cell growth and migration whereas blocking its expression caused cell death given the association of amounting adrm1 expression with the onset and progression of cancers adrm1 has been defined as a potential predictive and therapeutic target for clinical therapy additionally comparable expressions of adrm1 have also been observed in several lymphocyte cell lines as well as endothelial cell lines and similar physiological roles of adrm1 are described through its excessive expression in skin endothelial cells that facilitates t lymphocyte adhesion in a previous study we identified haemonchus contortus excretorysecretory es proteins hcesps that interacted with host t cells via liquid chromatography mass spectrometry lcmsms analysis haemonchus contortus adrm1 hcadrm1 protein a mammalian adrm1 homologue was ascertained among these interacting proteins additionally recombinant hcadrm1 rhcadrm1 was recognized by serum samples obtained at day and postinfection derived from experimentally h contortusinfected goats as a result of these observations hcadrm1 with immunodiagnostic utility was fostered as a hallmark of h contortus infection and a serological diagnosis assay with high sensitivity and specificity was developed using hcadrm1 antigen furthermore our preliminary analysis showed that hcesps stimuli notably induced intrinsic and extrinsic apoptosis suppressed t cell proliferation and caused cell cycle arrested hcesps consisted of multitudinous modulatory molecules such as kinases phosphatases hydrolases and proteases where the pleiotropic effects were initiated by a cascade of individual es components importantly the exact molecules that modulated t cell immune response in the parasitehost interaction warrant further investigation given the functional diversity of adrm1 and especially its engagement in cell proliferation and apoptosis hcadrm1 might be one of these dominated proteins that exert critical controls on cellular survival and death of host key effector cells therefore herein we aimed to further investigate the molecular traits of hcadrm1 and address its immunomodulatory roles at the parasitehost interfacemethodsparasite animals and a0cellsthe h contortus strain was propagated via serial passages in nematodefree goats in the animal experimental center faculty of veterinary medicine nanjing china the collection of eggs l3 xl3 male and female adults was performed as previously described [ ] sprague dawley sd rats scxk with a standard packing weight a0 g were obtained from jiangsu experimental animal center nanjing china they were maintained in a microbefree room with access to sterilized food and water ad libitumlocal crossbred and healthy goats monthsold were reared in individually ventilated cages to prevent accidental infection with nematodes alongside ad libitum access to water in pens these goats were given hay and whole shelled corn daily peripheral venous blood samples were obtained by venipuncture as described elsewhere as well as the isolation of goat peripheral blood mononuclear cells pbmcs total t cells in goat pbmcs were sorted using a magneticactivated cell sorting system macs miltenyi biotech inc auburn ca usa as described elsewhere briefly every million pbmcs in a0µl staining buffer were incubated with µl mouse antibovine cd2 primary antibody biorad kidlington uk which crossreact with goat cd2 t cells for a0min after two washes in pbs à of total pbmcs resuspended in µl staining buffer were labeled 0clu a0et a0al parasites vectors page of with µl antifitc microbeads miltenyi biotech at room temperature for min subsequently pbmcs were loaded onto the macs magnetic system miltenyi biotech for positive sorting based on the manufacturers specifications t cells were then adjusted to a density of à cellsml in rpmi gibco grand island ny usa containing heatinactivated fetal calf serum fcs gibco and a0uml penicillin a0mgml streptomycin gibco the viability of t cells was as assessed by the trypan blue exclusion test the purity of separated t cells was validated via flow cytometric determination additional file a0 figure s1 three goats biological replicates were used in every experimentsequence alignment and a0phylogenetic analysisthe cloning and amplification of the complete coding sequence of the hcadrm1 gene were performed as previously described the amplified hcadrm1 fragment was cloned into pet28a vector invitrogen carlsbad ca usa and validated by sequence analysis using blast multiple amino acid sequences of adrm1 orthologs were aligned for comparison by the clustal omega software the evolutionary analysis was conducted and extrapolated by mega x software using jtt matrixbased model and the maximum likelihood method with partial deletion option positions with less than site coverage were excluded prior to phylogenetic analysis based on the optimized evaluation of replicates for bootstrap support the evolutionary tree of adrm1 orthologues was generated with several designated and collapsed branchesexpression and a0purification of a0the a0rhcadrm1 proteinthe expression and purification of rhcadrm1 proteins were conducted as elsewhere described briefly escherichia coli bl21 de3 cells containing the reconstructed pet28ahcadrm1 plasmid were incubated with luriabertini medium containing kanamycin a0 µgml sigmaaldrich st louis mo usa and then stimulated by isopropylβdthiogalactopyranoside sigmaaldrich for the induction of rhcadrm1 expression the rhcadrm1 protein fused with a histidinetag was obtained from the supernatant of cell lysates via histrap hp purification columns ge healthcare piscataway nj usa rhcadrm1 proteins were resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis sdspage gels for size and purity validation and the concentration was determined by a bicinchoninic acid bca assay thermo fisher scientific rockford il usa employing the detoxigel affinity pak prepacked columns thermo fisher scientific rhcadrm1 proteins were exempt of lipopolysaccharide contamination as rhcadrm1 protein was dissolved in pbs pbstreated t cells served as the control group a0 µgml in functional assays the purified rhcadrm1 was stored at a0°c until further analysispreparation of a0polyclonal antibody pabto obtain antigenspecific pab rhcadrm1 proteins a0 µg blended with freunds complete adjuvant was administrated subcutaneously into sd rats with a 2week interval booster immunizations with a0 µg of rhcadrm1 proteins emulsified in freunds incomplete adjuvant were administered four times seven days after the final boost rat sera containing antirhcadrm1 pab were harvested and kept at a0 °c for further analysis the sera harvested from h contortusinfected goats antih contortus serum were stored at the veterinary parasitology teaching and research center of nanjing agricultural university nanjing chinaimmunoblot analysisrhcadrm1 and hcesps were resolved on protein gels respectively and transferred onto nitrocellulose membranes the blots were blocked using bsa in trisbuffered saline01 tween tbst for a0 h at room temperature the blots with the rhcadrm1 samples were probed with primary goat antih contortus serum in tbst or normal goat serum control at a0°c overnight while the blots with the hcesps samples were probed with primary rat antirhcadrm1 igg in tbst or normal rat igg control after five washes in tbst the blots were incubated with horseradish peroxidasecoupled rabbit antigoat or antirat igg hl secondary antibody sigmaaldrich in tbst for h at a0 °c the blots were then developed with ²diaminobenzidine dab sigmaaldrich for min and visualized by using a chemidoc imaging system biorad hercules ca usahcadrm1 transcription in a0h contortus lifecycle stagesto detect mrna expression of hcadrm1 in h contortus lifecycle stages total rna of eggs l3 xl3 female and male adults were extracted using trizol invitrogen and the resulting cdnas were synthesized in accordance with the manufacturers specifications employing specific primers for the βtubulin gene endogenous reference and target gene hcadrm1 additional file a0 table a0s1 transcriptional analysis of the hcadrm1 gene was conducted by realtime pcr using the quantstudio system applied biosystems carlsbad ca usa with a standard protocol the specificity of the primers was 0clu a0et a0al parasites vectors page of validated to ensure product purity via generation of a melt curve and the absence of primer dimers the amplification efficiencies and correlation coefficients were verified to be stable and similar based on the δδcq method the relative transcription levels of hcadrm1 were normalized on βtubulin transcription each experiment was run in triplicateimmunohistochemistry assaysfreshly collected female and male adults were washed dehydrated fixed embedded and cut into cryostat sections as previous described to minimize nonspecific binding cryosections were treated with normal goat serum in pbs containing tween pbst for a0h subsequently cryosections were served with primary antirhcadrm1 igg or sham control igg overnight at a0 °c prior to dna staining with 24amidinophenyl6indolecarbamidine dihydrochloride dapi sigmaaldrich cryosections were then incubated with cy3labeled goat antirat igg beyotime biotechnology shanghai china at a0 °c for a0 h subsequently the samples were immersed in antifade medium sigmaaldrich to prevent fluorescence fading for microscopic examination finally the sections were imaged at à magnification using a lsm710 fluorescence microscope zeiss jena germany and zen software zeiss was used for the analysis of digital imagesthe interaction of a0hcadrm1 protein with a0t cells in vitrothe interaction of hcadrm1 with goat t cells was investigated as previously described in brief freshly sorted t cells were cultured with or without a0 µgml rhcadrm1 proteins for a0h at a0°c after three washes paraformaldehydefixed t cells were permeabilized by triton x100 in pbst and blocked with bsa in pbst for a0min subsequently prior to the staining with the cy3coupled secondary antibody t cells were treated by primary antihcadrm1 pab or normal rat igg control in a humidified chamber at a0°c for a0h followed by five pbst washes t cells were subjected to gold antifade mounting solution containing dapi life technologies eugene or usa for nuclear staining immunofluorescencelabeled cells were visualized at à magnification using a lsm780 confocal microscope zeiss jena germany zen software zeiss was employed for the interpretation of digital imagescell viabilitythe modulatory effects of rhcadrm1 on goat t cell viability were determined using the cell counting kit8 assay cck8 dojindo kumamoto japan as previously described fresh sorted goat t cells activated with concanavalin a cona a0µgml were incubated in the presence of various doses of rhcadrm1 proteins and a0µgml at a0°c following a0hstimulation cell culture medium was incorporated with a0µl cck8 solution and incubated at a0 °c in the dark for a0h following incubation optical density was measured at a0nm od450 using a microplate reader biorad hercules california usa three independent tests each in triplicate were performedcell apoptosis assayflow cytometry assays were performed for t cell apoptosis determination using the annexin vpe kit bd biosciences san jose ca usa as previously described in brief freshly sorted t cells were cultured in the presence of tested doses of rhcadrm1 proteins and a0µgml followed by annexin v and 7aminoactinomycin d 7aad staining based on the kits specification the pbsstimulated t cells served as negative controls three individual tests each in triplicate were conductedcell proliferation assaycell proliferation analysis was determined using the alexa fluor clickit plus edu flow cytometry kit thermo fisher scientific via the measurement of dna synthesis directly based on the manufacturers instructions after a0h coincubation the cell culture was incorporated with 5ethynyl2²deoxyuridine edu a0μm for another a0h incubation subsequently t cells were harvested fixed with paraformaldehyde in pbs and permeabilized using the clickit saponinbased reagent followed by clickit reaction to coupled edu with alexa fluor dye after three washes with a0ml of bsa in pbs t cells were treated with 7aad staining solution bd biosciences flow cytometry was used for the determination of edu cells in the population each experiment consisting of three replicates was run in triplicatecell cycle assayflow cytometry assays were conducted for cell cycle determination using pirnase staining buffer bd biosciences according to the manufacturers dna staining protocol following coincubation with rhcadrm1 stimuli a0 µgml for a0 h t cells were harvested washed and fixed with icecold ethanol every a0 h after being frozen at a0°c for more than a0h treatedt cells were washed twice with pbs to remove remaining ethanol and resuspended in pirnase staining buffer for flow cytometry analysis each experiment consisting of three replicates was run in triplicate 0clu a0et a0al parasites vectors page of transcription analysist cells treated with different concentrations of rhcadrm1 and a0µgml for h were harvested for the transcription analysis of the cell apoptosis pathway and t cells treated with a0µgml of rhcadrm1 for a0h were collected for transcription analysis of the cell cycle pathway cells were harvested for total rna extraction and cdna obtained by reversetranscription pcr relative quantification of candidate gene expression was conducted using previously published primers [] of endogenous reference and candidate genes additional file a0 table a0s2 based on the δδcq method the relative levels of target gene transcription were normalized to reference gene expression each experiment consisting of three replicates was run in triplicatedetection of a0cytokine secretionsfor the determination of cytokine secretion levels freshly isolated t cells activated by cona a0µgml were treated with or without rhcadrm1 and a0µgml for a0h cell culture medium was harvested and determined for cytokine secretion detection using goat enzymelinked immunosorbent assay elisa kits mlbio shanghai china according to the manufacturers specifications the limit of quantification dependent upon each analytic kit ranged from between and a0pgml each experiment was run in triplicatestatistical analysisoneway and twoway analysis of variance anova with dunnetts multiple comparison test alongside the students ttest were performed for statistical analysis using graphpad premier software graphpad prism san diego ca usa differences were regarded as statistically significant when pvalues were data were denoted as minimum to maximum all points or mean ± standard deviation sdresultssequence alignment and a0phylogenetic analysisthe entire coding region of the hcadrm1 gene a0bp was amplified from the cdna of adult worms encoding a 361amino acid protein with an estimated molecular mass around a0 kda we then performed a sequence alignment of adrm1 orthologues derived from genbank on zebrafish human mouse caenorhabditis elegans and h contortus using clustal omega software hcadrm1 protein showed a moderate degree of identity to zebrafish human mouse and c elegans orthologs fig a0 1a in addition we conducted an evolutionary analysis of hcadrm1 using the maximum likelihood method involving amino acid sequences phylogenetic analysis clearly showed an evolutionary relationship of hcadrm1 with other adrm1 orthologues revealing that hcadrm1 was closely related to the teladorsagia circumcincta homologue but divergent from vertebrate sequences fig a01bprotein expression and a0immunoblot analysisthe rhcadrm1 protein fused with the histidinetag was successfully obtained in the supernatant of cell lysates after purification rhcadrm1 was visualized by coomassie blue staining as a single band with a molecular weight of a0kda fig a01c lane the specificity of the rhcadrm1 protein was determined by western blot probing with antih contortus serum or normal goat serum a single band a0 kda was observed through the specific recognition of rhcadrm1 protein by antih contortus serum fig a01c lane while no band was identified via healthy goat sera fig a01c lane meanwhile native hcadrm1 protein derived from hcesps was identified by rat antirhcadrm1 igg as a single band of a0kda fig a01c lane while no positive band was observed in the control groups fig a01c lane differential mrna expression in a0h contortus lifecycle stages and a0immunolocalizationtranscription analysis by realtime rtpcr revealed that mrna expression of hcadrm1 were detectable at all of the tested h contortus lifecycle stages the data demonstrated rising expression levels from the freeliving stages eggs and l3 to parasitic stages xl3 female and male adults simultaneously the highest level of hcadrm1 transcription was observed in male adults but not in female adults fig a01d given that the highest mrna expression was detected in adult worms we next investigated the localization of native hcadrm1 proteins within h contortus by checking the cryosections of the adult worms specific red fluorescence resulting from tagging hcadrm1 proteins by rat antirhcadrm1 igg was ubiquitously observed from the intracellular and cytoplasmic localization of somatic cells particularly in the intestinal regions and the internal membrane of cuticle for both male and female adults fig a01e however no cy3fluorescence was observed in the sections treated with normal rat igg fig a01ebinding of a0rhcadrm1 protein to a0goat t cellsbased on our preliminary lcmsms analysis we next conducted immunocytochemistry assays to verify the in vitro interaction of hcadrm1 proteins with goat t cells immunocytochemistry assay showed that intense red 0clu a0et a0al parasites vectors page of fig molecular characterization of hcadrm1 derived from hcesps a alignment of hcadrm1 amino acid sequences with other orthologues the positions of adrm1 family motifs are indicated above the multiple sequence alignment containing zebrafish xp_0213255291 human np_0012683661 mouse np_0627962 c elegans np_4983872 and h contortus w6nb91 adrm1 ortholog sequences retrieved from genbank an asterisk indicates the position with one completely conserved amino acid while period denotes weakly conserved similarity within different groups and colon represents strongly similar conservation between groups b phylogenetic analysis of hcadrm1 with vertebrate and parasite orthologues evolutionary relationships of taxa were inferred using the maximum likelihood method with protein sequences including mus musculus np_0627962 homo sapiens xp_0115268051 danio rerio xp_0213255291 toxocara canis khn872021 c elegans np_4983872 dictyocaulus viviparus kjh490541 t circumcincta pio739301 h contortus w6nb91 oesophagostomum dentatum khj973301 and ancylostoma caninum rcn481761 bootstrap support values are shown for each node the scalebar denotes the number of substitutions per site c acquisition of rhcadrm1 proteins and western blot analysis lanes m protein standard ladder lane rhcadrm1 expressed in the supernatant of cell lysates lane sdspage analysis of purified rhcadrm1 protein lane immunoblot analysis of rhcadrm1 using antih contortus serum as primary antibody lane immunoblot analysis of rhcadrm1 using normal goat serum control as primary antibody lane immunoblot analysis of hcesps using rat antirhcadrm1 igg as primary antibody lane immunoblot analysis of hcesps using normal rat igg control as primary antibody d hcadrm1 expression in h contortus lifecycle stages data are presented as the mean ± sd e immunolocalization of native hcadrm1 protein in male and female adults the immunohistochemistry assays were performed using normal rat igg control or rat antirhcadrm1 igg as primary antibody cy3coupled fluorescence red along with dapi blue was identified for the investigation of hcadrm1 distribution scalebars e µm 0clu a0et a0al parasites vectors page of cy3fluorescence resulting from tagging rhcadrm1 was observed in rhcadrm1treated t cells revealing the cytomembrane and cytoplasmic localization of rhcadrm1 fig a0 2a a1 no red fluorescence was detected in both blank and negative control groups fig a0 2a a2 and a3 the results presented here further validated the positive interactions between hcadrm1 protein and host t cellsrhcadrm1 suppressed cell viability and a0induced cell apoptosisgiven the modulatory potential of adrm proteins on cellular development and survival we next investigated the impact of rhcadrm1 proteins on t cell viability the results of cck8 determination showed that t cell viability was dramatically inhibited by the stimulation of a0 µgml anova f4 p a0 µgml anova f4 p a0µgml anova f4 p and a0 µgml anova f4 p of rhcadrm1 proteins fig a02b based on this finding an annexin vpe7aad double staining kit was employed to evaluate the proapoptotic potential of rhcadrm1 proteins flow cytometry results demonstrated that rhcadrm1 stimuli at the tested concentrations of a0 µgml anova f4 p a0 µgml anova f4 p and a0 µgml anova f4 p remarkably caused t cell apoptosis in comparison to the unstimulated group fig a0 2c d additionally transcriptional analysis of key genes involved in apoptosis signaling pathways further validated the proapoptotic effects of rhcadrm1 proteins on host t cells the treatments with and a0µgml of rhcadrm1 dramatically upregulated mrna transcripts of caspase8 anova f4 p p and p respectively and caspase3 anova f4 p p and p respectively fig a0 2e simultaneously a0 µgml anova f4 p and µgml anova f4 p of rhcadrm1 proteins significantly promoted caspase9 transcription fig a02erhcadrm1 protein restrained the a0proliferation of a0t cells and a0caused cell cycle stallingas apoptosis proliferation and cell cycle were interconnected cellular movements we next explored the modulatory potentials of rhcadrm1 stimuli on t cell proliferation and cell cycle at the tested doses of and a0 µgml flow cytometry data showed that rhcadrm1 stimuli significantly inhibited t cell proliferation in vitro fig a03a as indicated by the decreasing proportion of edu cells compared with control cells anova f4 p p and p respectively fig a0 3b given that the treatments with a0µgml rhcadrm1 had significant biological effects on cell viability apoptosis and proliferation as well as the transcription of certain key genes we next treated t cells with a0µgml of rhcadrm1 for cell cycle determination here flow cytometry analysis with pi staining demonstrated that rhcadrm1 stimuli induced cell cycle arrest in a timedependent manner fig a0 3c as indicated by the increased proportion of t cells in g1 phase at a0h anova f8 p a0h anova f8 p and a0h anova f8 p as well as the decreased proportion of t cells in s phase at a0 h anova f8 p a0 h anova f8 p and a0h anova f8 p fig a0 3d consistent with these findings transcriptional analysis of key genes in g1s checkpoints showed that mrna transcripts of ccne1 ttest t16 p and cdk2 ttest t16 p were significantly downregulated by rhcadrm1 stimuli while no significant transcriptional changes of ccnd1 ttest t16 p cdk4 ttest t16 p and cdk6 ttest t16 p were observed fig a03e given the inhibitory effects of p21 and p27 on cdks in ubmediated cell cycle progression the transcription analysis of p21 and p27 was performed in this study in addition the mrna transcripts of iκbα as the physiological substrate of adrm1 and its downstream inhibitor nfκb were determined importantly transcription of p21 ttest t16 p p27 ttest t16 p and iκbα ttest t16 p was notably enhanced by rhcadrm1 stimuli fig a0 3e whereas the mrna transcript of nfκb ttest t16 p was significantly suppressed fig a03edetermination of a0cytokine secretionsto investigate the modulatory effects of rhcadrm1 on t cell cytokine productions il2 il4 il10 il17a ifnγ and tgfβ1 secretions in the cell culture supernatant were determined via elisa assays the data showed that the exposure of goat t cells to rhcadrm1 proteins led to the alteration of their cytokine production profiles intriguingly at the tested doses of and a0µgml rhcadrm1 stimuli predominantly inhibited secretions of il4 anova f4 p p and p respectively anova f4 p p and p respectively and ifnγ anova f4 p p and p respectively fig a0 4b c e however all the tested doses of rhcadrm1 had no notable effects on secretions of il2 anova f4 p p p and p il10 0clu a0et a0al parasites vectors page of fig rhcadrm1 proteins suppressed cell viability and induced apoptosis via the interaction with goat t cells a determination of the interaction of hcadrm1 protein with goat t cells in vitro t cells treated with a1 or without a2 rhcadrm1 protein were incubated with rat antirhcadrm1 igg as the primary antibody t cells stimulated by rhcadrm1 were incubated with normal rat igg as the primary antibody a3 b rhcadrm1 significantly inhibited t cell viability cell viability was determined via the incorporation with cck8 whereas cell viability index was determined by calculating the od values of the control group as results are presented as the mean ± sd asterisks denote statistically significant differences p p p compared with the control group c flow cytometry analysis of t cell apoptosis in responses to rhcadrm1 stimuli apoptosis determination was performed via 7aad and annexin vpe staining d statis | 0 |
"Cardiac arrhythmias Atrial ï¬brillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular ï¬brillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are aï¬icted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial ï¬brillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular ï¬brillation VPCs ventricular premature complexes VT ventricular tachycardia Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial ï¬brillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with conï¬rmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular ï¬brillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial ï¬brillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular ï¬brillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insuï¬ciencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular ï¬bril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial ï¬brillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial ï¬brillation According to a recent survey of electrophysiology professionals atrial ï¬brillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using speciï¬c AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the ï¬rst months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insuï¬ciency and the systemic am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients aï¬icted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with conï¬rmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the ï¬rst documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is signiï¬cantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the ï¬nal weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained signiï¬cantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as deï¬nite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and ï¬uoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary ï¬ndings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpausedependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeï¬ciency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus ï¬ngolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides ï¬uoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to signiï¬cant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs niï¬cant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ¥ ms Changes in ¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ¥ ms or QTc increase of No patients manifested TdP ¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signiï¬ cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no signiï¬cant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identiï¬ed only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of signiï¬cant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this speciï¬c population HCQAZM could not be initiated or had to be interrupted in ¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the ï¬rst manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or forme fruste of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19speciï¬c risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use of various formulas among which the Bazetts correction formula is most commonly used QTc QT RRsec QTc is deï¬ned as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTcQRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F | 2 |
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c | 0 |
"Luciferase activity driven by the miR-182 promoter increased in H1299 cells overexpressing GFP-Sp1 (C) whereas luciferase activity decrease in cells treated with an Sp1 inhibitor mithramycin A (D). These results suggested that Sp1 is involved in miR-182 transcriptional activation. Using the TFSEARCH software we analyzed the miR-182 promoter and identified two putative Sp1 binding elements. Consequently recruitment of Sp1 to the miR-182 promoter was examined (E and 1F). Acetyl-histone3 was recruited to the Sp1 binding elements indicating that the region could recruit TFs (E panel b). Sp1 was also recruited to the miR-182 promoter (E panel c and panel d and 1F). When the Sp1 binding element at site 1 was mutated luciferase activity driven by the miR-182 promoter was abolished but no change was observed when the other Sp1 binding site was mutated indicating that the Sp1 binding element at site 1 is important for the Sp1-mediated expression of miR-182 (G). Sp1 regulates miR-182 expression (A) Scramble (shScr) and different doses of Sp1 shRNAs (shSp1) were transfected into A549 for 48 h. The miR-182 level was determined by stem-loop RT-PCR. U6 served as the internal control (panel a). Data were quantified after three independent experiments (panel b). (B) Different titer of adeno-GFP-Sp1 virus was infected IMR-90 cells for 48 h. The miR-182 level was determined by stem-loop RT-PCR (panel a). Data were quantified after three independent experiments (panel b). (C) Plasmids pGL2 or pGL2-miR-182 (-1000/+50) and GFP or GFP-Sp1 were co-transfected into H1299 cells for 24h. Cells were harvested to study the luciferase activity. Data were quantified after three independent experiments. (D) The plasmids pGL2 or pGL2-miR-182 were transfected into H1299 cells with mithramycin A treatment for 24 h. Cells were harvested for luciferase activity assays. (E) Schematic diagram indicates the location of putative Sp1 binding sites on miR-182 promoter region (panel a). ChIP assays were performed with anti-acetyl-H3 (panel b) and anti-Sp1 antibodies (panel c). DNA was extracted for PCR with miR-182 and p21 primers. Data were quantified after three independent experiments (panel d). (F) A549 cells were harvested for DAPA with a biotin-conjugated p21 and miR-182 promoter probes and samples were analyzed by Western blotting using anti-Sp1 antibodies (panel a). Data were quantified after three independent experiments (panel b). (G) Plasmids GFP or GFP-Sp1 were co-transfected with pGL2 pGL2-miR-182 WT or mutation plasmids into H1299 cells for 24 h and then cells were harvested for luciferase activity assays. Data are representative of three independent experiments each of which was performed in triplicate and presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01). Because Sp1 is highly expressed in lung cancer we studied the expression of Sp1 and miR-182 in various lung cancer cell lines and patient samples (). Compared with normal human lung cells (BEAS-2B) lung cancer cell lines expressed higher levels of miR-182 (A). We also assessed the correlation between the miR-182 and Sp1 expression patterns. Sp1 levels in clinical lung tissue samples were highly elevated in the tumorous sections of the lung accompanied by increased expression of miR-182 (B). To confirm this result Sp1 and miR-182 levels were measured in 32 lung cancer patients. Sp1 and miR-182 were upregulated by more than 1.3-fold in 59.4% of the lung adenocarcinoma specimens when compared to expression in normal tissue (C). These results indicate that Sp1 expression positively correlates with miR-182 expression (D). The miR-182 level correlates to Sp1 level Total RNA and cell lysates were prepared from indicated cell lines (A) or from clinical lung tissues of lung cancer patients (B). The miR-182 level was determined by stem-loop RT-PCR and Sp1 level was studied by Western blotting with anti-Sp1 antibodies. U6 and tubulin served as the internal control. (C) Total RNA and cell lysates were prepared from 32 paired normal lung tissues and lung adenocarcinoma samples. The miR-182 level was studied by stem-loop RT-PCR and Sp1 levels were studied by RT-PCR. (D) The relationship between Sp1 and the miR-182 level in the 32 lung cancer samples was statistically analyzed using Fisher's exact test. miR-182 increases lung tumor growth The data shown in indicated that Sp1 regulated miR-182 expression during lung tumorigenesis. To identify the specific gene targets of miR-182 we searched public miRNA target prediction databases (miRDB miRWalk and TargetScanHuman) for candidate target genes. By combining the data from these three databases we identified 161 genes potentially regulated by miR-182 (Supplementary Figure S1A). Moreover pathway analysis using Ingenuity software indicated that the cellular growth and proliferation pathway had the highest score when the association of these 161 genes with biological pathways was examined. This suggests that miR-182 may play a functional role in cancer-associated processes (Supplementary Figure S1B). Indeed when miR-182 was knocked down with miRZip-182 shRNA the percentage of cells in G2/M and sub-G1 phases increased suggesting that miR-182 positively regulated cell cycle progression in the lung cancer cells (A). To further elucidate miR-182's effect on the cell cycle cells were synchronized at prometaphase using nocodazole treatment. After removing nocodazole more miRZip-182 than miRZip cells remained in G2/M phase providing further evidence that miR-182 positively regulates cell cycle progression (B). Consistently knockdown of miR-182 expression inhibited cell growth (C). miR-182 increases cancer cell proliferation (A) The miRZip and miRZip-182 stably expressed H1299 cells were fixed with 70% ethanol and stained with propidium iodide for cell cycle analysis by FACS. (B) Mitotic cells were released into growth by removing nocodazole then fixed at indicated time points for cell cycle progression assay by FACS. (C) The growth rates of miRZip and miRZip-182 stably expressed H1299 cells were calculated by cell counting within 5 days. Data are representative of six independent experiments and presented as the mean ± SEM. (D) Bioluminescent imaging was performed on 10 severe combined immunodeficient (SCID) mice implanted with miRZip and miRZip-182 stable expression H1299 cells (106 cells/mouse) at day 14 (panel a) then image signal was analyzed using Living Image software and presented as total flux measurements in photons/second (panel b). (E) Tumors from SCID mice implanted with miRZip and miRZip-182 stable expression H1299 cells for 4 weeks are shown (panel a) and tumor weights were analyzed (panel b). Data are representative of ten independent experiments and are presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01; *** p<0.001). To confirm the effect of miR-182 on tumor formation cells stably expressing with miRZip or miRZip-182 were implanted into SCID mice and tumor growth was monitored in vivo (D). The miRZip lentivector contains a copGFP gene and the GFP signal in miRZip-182-expressing cells was lower than that in miRZip control cells (D). Furthermore tumor volume and tumor weight were also lower in miRZip-182-implanted mice than in miRZip-implanted mice (N = 10 per group) (E). These results suggest that miR-182 overexpression facilitates lung tumor growth in vivo. Sp1 inhibits FOXO3 expression by inducing miR-182 expression To investigate the molecular mechanism underlying miR-182-mediated cancer cell proliferation we studied an important miR-182 target gene FOXO3. FOXO3 expression was higher in cells stably expressing miRZip-182 than in control cells (A). Knockdown of miR-182 expression enhanced the luciferase activity of a pGL3 vector containing the 3?-UTR of FOXO3 (B) indicating that miR-182 downregulated FOXO3 expression. Further to determine whether Sp1 downregulated FOXO3 expression through miR-182 GFP-Sp1 was expressed in cells stably expressing miRZip-182 (C). Overexpression of GFP-Sp1 reduced FOXO3 protein expression in miRZip stable cells but increased FOXO3 levels in miRZip-182-expressing cells implying that different effect of Sp1 is existed on the regulation of FOXO3 expression. Regulation of FOXO3 by miR-182 and Sp1 (A) Lenti-miRZip and lenti-miRZip-182 viruses were infected into H1299 for 96 h individually. FOXO3 level was studied by Western blotting with anti-FOXO3 antibodies and miR-182 level was studied by stem-loop RT-PCR. (B) Plasmids pGL3 and pGL3-FOXO3-3'UTR were transfected into miRZip and miRZip-182 stably expressed H1299 cells for 24 h and then cells were harvested for luciferase activity assays. (C) Different doses of GFP-Sp1 adenovirus were infected into the miRZip and miRZip-182 stably expressed H1299 cells for 48 h. FOXO3 level was studied by Western blotting using anti-FOXO3 antibodies (panel a). Quantitative results from three independent experiments are shown (panel b). The level of statistical significance was determined by t-test (* p<0.05; *** p<0.001). Therefore we further investigated the relationship between Sp1 and miR-182 in the context of FOXO3 regulation. The expression of Sp1 and FOXO3 in patients with lung cancer was examined (Figure 5A). In normal tissue samples Sp1 levels were low and FOXO3 levels were high. In tumor tissue samples two Sp1 expression patterns i.e. high and low Sp1 expression were identified. Samples with higher Sp1 levels exhibited lower FOXO3 levels whereas samples with lower Sp1 levels exhibited higher FOXO3 levels suggesting that there is an inverse correlation between Sp1 and FOXO3 levels in lung specimen (Figure 5A). The levels of FOXO3 and Sp1 in the lung cancer cell lines A549 H1299 CL 1-0 and CL 1-5 were studied (Figure 5B). Higher levels of Sp1 expression were accompanied by lower levels of FOXO3 expression in A549 and CL 1-0 cells and lower levels of Sp1 expression were accompanied by higher levels of FOXO3 expression in H1299 and CL 1-5 cells suggesting that there is an inverse correlation between Sp1 and FOXO3 expression in lung tumorigenesis. Overexpression of GFP-Sp1 decreased FOXO3 mRNA and protein levels in a dose-dependent manner (Figure 5C panel a) whereas knockdown of Sp1 expression increased FOXO3 mRNA and protein levels (Figure 5C panel b). These results indicate that Sp1 negatively regulates FOXO3 expression. Figure 5 Sp1 negatively regulates FOXO3 expression through regulating miR-182 (A) The Sp1 and FOXO3 levels in clinical lung tissue samples were studied by IHC staining using antibodies against Sp1 and FOXO3 respectively. (B) Cell lysates were harvested from various cell lines for Western blotting using antibodies against FOXO3 and Sp1 and tubulin as an internal control. (C) Adeno-GFP-Sp1 viruses were infected into IMR-90 cells for 48 h and FOXO3 mRNA and protein were studied by RT-PCR and Western blotting respectively. GAPDH served as the internal control (panel a). Scramble and Sp1 shRNAs were transfected into H1299 for 48 h then FOXO3 mRNA and protein levels were studied by RT-PCR and Western blotting (panel b). (D) Scramble and Sp1 shRNAs were transfected into H1299 for 48 h and then cells were harvested at indicated time points following cycloheximide treatment for studying the Sp1 and FOXO3 levels with Western blotting. The levels of FOXO3 protein from three independent experiments were quantified using tubulin as an internal control. (E) Plasmids pGL2 or pGL2-FOXO3 (-1000/+50) were cotransfected with GFP or GFP-Sp1 into H1299 cells for 24 h then cell lysates were harvested for luciferase activity assays. (F) Adeno-GFP-Sp1 viruses were infected into H1299 cells for 24 h and cells were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Cells lysates were harvested for luciferase activity assays. (G) H1299 cells which were infected with GFP-Sp1 adenovirus for 24 h were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Total RNA was extracted at various time points following actinomycin D treatment. The mRNA levels of luciferase were determined by using quantitative RT-PCR and quantified using GAPDH as an internal control. Data are representative of three independent experiments each of which was performed in triplicate and presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01). Next we investigated the mechanism by which Sp1 regulates FOXO3 expression. FOXO3 protein half-life was studied after Sp1 knockdown. Knockdown of Sp1 expression did not affect FOXO3 protein stability (Figure 5D). We then constructed a luciferase reporter construct containing the FOXO3 promoter (-1000/+50) to study the effect of Sp1 on the promoter-mediated transcription of FOXO3 (Figure 5E). GFP-Sp1 overexpression significantly enhanced the luciferase activity indicating that Sp1 positively regulated FOXO3 transcription (Figure 5E). However FOXO3 mRNA and protein levels decreased as shown in Figure 5C. The data shown in indicated that Sp1 increased miR-182 expression which suggests that post-transcriptional processing contributes to the regulation of FOXO3 expression. Thus the 3'-UTR of FOXO3 might play an important role in stabilizing FOXO3 mRNA and in FOXO3 translation. Consequently a luciferase reporter construct containing the 3?-UTR of FOXO3 was generated. GFP-Sp1 overexpression reduced the luciferase activity (Figure 5F). Furthermore the stability of the luciferase mRNA containing the 3?-UTR sequence of FOXO3 decreased dramatically upon GFP-Sp1 overexpression (Figure 5G). These results indicate that Sp1 regulates FOXO3 expression through transcriptional and post-transcriptional regulation with a net negative effect on FOXO3 expression. miR-182 inhibits lung cancer metastasis activity The data shown in indicated that miR-182 positively regulated lung cancer cell growth. Therefore the role of miR-182 in lung cancer metastasis was studied (Figure 6). The morphology of miRZip-182 cells was markedly altered: circular structures of actin filaments were absence and pseudopodia were enriched suggesting that miR-182 decreased the cells' migratory ability (Figure 6A). Indeed knockdown of miR-182 expression increased the migration ability of lung cancer cells suggesting that miR-182 inhibits lung cancer migration (Figure 6B). Moreover transwell migration assays showed that knockdown of miR-182 expression enhanced cell's invasive capacity (Figure 6C). In mice injected with miRZip-182-treated cells the knockdown of miR-182 expression also increased the number of nodules in the lung suggesting that miR-182 represses metastatic ability in vivo (Figure 6D). The effects of miR-182 knockdown were partially reversed by knockdown of FOXO3 suggesting that miR-182 functions as a suppressor of lung cancer metastasis by repressing FOXO3 expression (Figure 6E panel a). The endothelial-mesenchymal transition (EMT) marker N-cadherin increased after miR-182 knockdown but this effect was abolished by FOXO3 knockdown. Thus miR-182 might repress lung cancer metastasis by decreasing the expression of N-cadherin (Figure 6E panel b). However the expression of other genes regulated by miR-182 might also play a role in metastasis (Figure 6F and Supplementary Figure S3). Therefore we generated gene expression profiles using microarray analysis. Functional grouping analysis using DAVID bioinformatics resources showed that 19 of the genes differentially regulated by miR-182 knockdown were related to cell migration. The expression of these genes was increased in miR-182-knockdown cells indicating that they are potential targets of miR-182 (Figure 6F). Many metastasis-related genes such as CD44 CDH9 and ADAM9 were upregulated after the knockdown of miR-182 expression (Figure 6F). Figure 6 miR-182 attenuates lung cancer cell metastasis (A) Immunofluorescent staining of Alexa Fluor 568-conjugated phalloidin that is a high-affinity probe for F-actin (red) in miRZip and miRZip-182 stably expressed H1299 cells. DNA was stained with DAPI (blue). Stained cells were photographed under a fluorescence microscope at x 600 magnification. (B) Confluent monolayers of miRZip or miRZip-182 stably expressed H1299 cells were wounded and incubated for an additional 16 h (panel a). Migratory area was calculated for quantification (panel b). (C) The migration activities of H1299 cells (2 x 104) expressing miRZip or miRZip-182 were studied by Transwell chambers. (D) The miRZip or miRZip-182 stably expressed H1299 cells (4 x 106) were suspended in 100 ?l of PBS and injected into the lateral tail vein of SCID mice. After 8 weeks all mice were killed and the number of pulmonary tumor nodules was calculated after fixation of lungs with 4% formaldehyde for 48 h (panel a) and the number of pulmonary metastatic tumor nodules was counted (panel b). (E) FOXO3 and miR-182 in H1299 cells were knockdown by shFOXO3 and miRZip-182 respectively and then migration of cells (3 x 104) was studies by Transwell chambers (panel a). In addition cell lysates were harvested from FOXO3 and miR-182 knockdown cells for Western blotting using antibodies against N-cadherin ?-catenin vimentin FOXO3 and tubulin (panel b) respectively. (F) Heat map of the 19 of genes from miRZip and miRZip-182 microarray data the red color represents genes that are upregulated and the green color represents genes that are downregulated. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01; *** p<0.001). DISCUSSION Our recent studies showed that Sp1 increased the growth of lung cancer cells but inhibits metastatic activity [23 32]. In the present study we found that Sp1 which accumulated in the early stages of cancer positively regulated miR-182 gene expression to silence FOXO3 expression and thereby promote cancer cell growth. In addition decreased levels of Sp1 in the late stages of cancer increased the expression of FOXO3 and N-cadherin leading to cancer metastasis (Figure 7). Figure 7 (A) Clinical samples from lung cancer patients of stage I and IV were used to study the Sp1 level by IHC staining with anti-Sp1 antibodies (B) Schematic diagram illustrates Sp1 regulates miR-182 to silence FOXO3 expression in early and late stages of lung cancer progression. Sp1 functions as a transcriptional activator by recruiting p300 to its target genes and as a repressor by the recruiting HDACs. Because Sp1 accumulates in several types of cancer including lung cancer [33] understanding the Sp1 transcriptional regulatory network may provide novel insights into the molecular origins and treatment of lung cancer. In our previous studies of lung cancer we found that Sp1 was highly upregulated in the early stages of cancer progression but partially down regulated in the late stages. Our previous studies also showed that regulation of Sp1 protein stability by phosphorylation and sumoylation contributed to its expression in the early and late stages of cancer respectively [32]. Kras activation and the Notch pathway might activate ERK1/2 to phosphorylate Sp1 thus stabilizing Sp1 in the early stages of cancer [32 34]. In the late stages Sp1 could be sumoylated leading to recruitment of its E3-ligase RNF4 followed by polyubiquitination and degradation [32]. To clarify the molecular mechanism underlying gene regulation by Sp1 we used microarray analysis to assess gene expression in KrasG12D-induced lung tumor transgenic mice and identified thousands of genes potentially regulated by Sp1 [23]. However some of the genes do not harbor a conserved Sp1 binding motif within their promoter region suggesting that another regulatory mechanism is involved in Sp1-mediated gene regulation. In this study we identified a novel pathway for Sp1-mediated activation wherein miR-182 expression downregulated the expression of FOXO3 a known miR-182 target gene [35]. Sp1 activated miR-182 and FOXO3 at the transcriptional level; however FOXO3 protein expression decreased. These results suggest that post-transcriptional regulation by miRNAs is a powerful mechanism by which to control the final level of protein expression. Many coding genes with Sp1 binding element(s) in their promoters harbor conserved miRNA target sequences in their 3'-UTR. To our knowledge this is first study to demonstrate that Sp1 regulates the expression of a target gene by regulating promoter activity and post-transcriptional processing in parallel. Few studies have characterized the regulation of miRNA by Sp1. Herein using a bioinformatics approach we identified several miRNAs potentially regulated by Sp1 including miR-182. We then showed that Sp1 specifically targets the miR-182 promoter region and activates miR-182 expression. miR-182 reportedly forms a gene cluster with two adjacent miRNAs (miR-96 and miR-183) [35]. The expression of miR-96 and miR-183 also decreased following Sp1 knockdown (Supplementary Figure S2A). Moreover we also investigated the binding of Sp1 to the miR-212 promoter because the latter contains 13 putative Sp1 binding sites (Supplementary Table S1). We found that Sp1 bound to the miR-212 promoter sequence (Supplementary Figure S2B and S2C). Interestingly a recent study showed that FOXO3 is a direct target of miR-212 in the neurons of patients with Alzheimer's disease [36]. miR-182 and miR-212 might cooperate to downregulate FOXO3 expression upon Sp1 overexpression. We cannot rule out this possibility. However depletion of miR-182 was sufficient to impair the Sp1-mediated reduction of FOXO3 expression in our experiments (C) suggesting that miR-182 is the major regulator of FOXO3 in lung cancer cells. Several studies have shown that miR-182 is upregulated in lung cancer. This suggests that miR-182 plays a positive role in lung tumorigenesis. However in two studies of miR-182 function in lung cancer miR-182 inhibited the proliferation of human lung adenocarcinoma cells [37 38]. Our results in this study provide several pieces of evidence to support the notion that miRNA-182 is a positive regulator of lung cancer cell proliferation. Firstly miR-182 was upregulated in the majority of lung cancer clinical samples and lung cancer cell lines examined. Secondly miR-182 knockdown inhibited cell cycle progression and cell growth. Finally miR-182 knockdown reduced lung tumor growth in vivo. Discrepancies in the role of miRNA-182 in lung cancer cell proliferation might derive from the different experimental designs of the studies. For example because miR-182 expression is upregulated in lung cancer we knocked down its expression and examined the effects on cancer cell proliferation. However other studies that described a negative role of miR-182 in lung cancer used miR-182 overexpression to study miR-182's role in cancer cell proliferation. Overexpression conditions can alter the function of many genes [39]. For example Sp1 accumulates in most of cancers; knockdown of Sp1 expression decreases cell proliferation but Sp1 overexpression also attenuates cancer cell growth [40]. Because post-translational modifications affect protein function overexpressed proteins might not be completely processed which could affect their function. Previous studies in melanoma and hepatocellular carcinoma indicated that miR-182 enhanced tumor metastasis [35 41]. However our data as shown in Figure 6 indicated that miR-182 knockdown altered cell morphology and increased migration and invasion activities. In addition miR-182 knockdown increased N-cadherin levels suggesting that miR-182 promotes the mesenchymal to epithelial transition (MET) [42]. Previous studies have shown that TIMP-2 enhances the E-cadherin/?-catenin complex in A549 lung cancer cells [43]. Whether Sp1 or miR-182 regulates TIMP-1 in lung cancer needs to be addressed in future studies. Finally miR-182 levels were lower in CL1-5 cells then in CL1-0 cells resulting in increased metastatic activity in CL1-5. Collectively our data suggest that miR-182 inhibits lung cancer metastasis. Our previous study indicated that Sp1 is down regulated in the late stages of lung cancer progression [32]. Therefore in the late stages of lung tumorigenesis miR-182 expression was down regulated compared with expression in the early stages which led to tumor metastasis through at least in part an increase in FOXO3 expression. It is still not clear why miR-182 has different roles in different types of cancer; this awaits further study. Although we found that FOXO3 is involved in miR-182-mediated lung cancer progression FOXO3 knockdown did not completely abolish the effects of miR-182 knockdown suggesting that other genes regulated by miR-182 contribute to the inhibition of metastasis by miR-182. With this in mind we determined the expression profile of miR-182-regulated genes. Many metastasis-related genes were induced in miR-182-knockdown cells including CD44 ADAM9 and CDH9. CD44 which localizes to the cell membrane is reportedly involved in cell migration in various cancer types [44]. Recent studies also showed that tumor initiating cells with high CD44 expression maintained lung cancer tumorigenicity and drug resistance [45]. Another metastasis-related gene induced by miR-182 knockdown ADAM9 cleaves membrane proteins such as E-cadherin [46]. A previous study showed that combined Kras and Wnt pathway activation increased the incidence of lung cancer formation [47]. Given that ADAM9 is also involved in the activation of the Wnt pathway Sp1 and miR-182 might connect the Kras and the Wnt pathway. In addition CDH9 also involves in the cancer metastasis [48]. In conclusion we showed that miR-182 is an Sp1-activated miRNA whose expression increased in lung cancer. miR-182 functioned not only as an oncomiR for lung cancer growth but also as a suppressor of lung cancer metastasis. MATERIALS AND METHODS Cell culture and transfection Human lung cancer cell lines A549 H1299 CL 1-0 and 1-5 were cultured in Dulbecco's modified Eagle's medium (Invitrogen Carlsbad CA) human diploid fibroblasts IMR were cultured in Minimum Essential Media (Invitrogen) and human bronchial epithelial cells BEAS-2B was cultured in RPMI 1640 Medium (Thermo Scientific Rockford IL). All of culture mediums contained 10% fetal bovine serum 100 U/ml penicillin G sodium and 100 ?g/ml streptomycin sulfate (Invitrogen). Cells were cultured at 37? and 5% CO2. Transfection of all cells with expression vectors was done using Lipofectamine 2000 (Invitrogen) according to the manufacturer's directions. Reverse transcription-polymerase chain reaction (RT-PCR) and stem-loop RT-PCR Total RNA was isolated using the Trizol reagent (Invitrogen) and 3 ?g of RNA were reverse-transcribed using the Superscript III enzyme (Invitrogen). PCR was then performed on cDNA with gene-specific primers: Sp1 F 5'-TGC AGC AGA ATT GAG TCA CC-3' and R 5'-CAC AAC ATA CTG CCC ACC AG-3'; FOXO3 F 5'-GCA AGC ACA GAG TTG GAT GA-3' and R 5'-CAG GTC GTC CAT GAG GTT TT-3'; GAPDH F 5'-GAG TCA ACG GAT TTG GTC GT-3' and R 5'-TTG ATT TTG GAG GGA TCT CG-3'; and U6 F 5'-CGC TTC GGC AGC ACA TAT AC-3' and R 5'-AGG GGC CAT GCT AAT CTT CT-3'. The protocol for the detection of mature miRNAs using a stem-loop gene-specific reverse transcription primer was performed as described previously [49]. Stem-loop primers (miR-182 5'-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA GTG TG-3'; miR-96 5'-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA GCA AA-3'; and miR-183 5'-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA GTG AA-3') were designed to specifically reverse transcribe the mature miRNA of interest. The primers for PCR were as follows: miR-182 F 5'-CGG CGG TTT GGC AAT GGT AGA ACT-3'; miR-96 F 5'-CGG CGG TTT GGC ACT AGC ACA TTT-3'; miR-183 F 5'-CGG CGG TAT GGC ACT GGT AGA ATT-3'; and R 5'-CCA GTG CAG GGT CCG AGG TAT-3'. PCR products were analyzed by ethidium bromide-containing agarose gel electrophoresis. Western blotting Cell lysates were prepared from the indicated cell lines for SDS-polyacrylamide gel electrophoresis (SDS-PAGE)" | 1 |
structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These ï¬ndings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to ï¬nd newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodiï¬ed leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant antiammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] antiammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was ï¬rst measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their speciï¬ed regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the speciï¬c enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose ï¬fty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the ï¬rst group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationï¬ve mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a signiï¬cant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was conï¬rmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a ï¬avoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosigniï¬cant change in MDA level as compared to normal nonirradiated mice Fig A signiï¬cant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modiï¬es ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were signiï¬cantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a signiï¬cant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisï¬nding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were signiï¬cantly lower ascompared to vehicletreated irradiated ones but still signiï¬cantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different from control group signiï¬cantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB ï¬le 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have ï¬ve subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferronis post test signiï¬cantly different from control group signiï¬cantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test There were no signiï¬cant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different fromcontrol group signiï¬cantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 conï¬rmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientiï¬c GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSOd6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was reï¬uxed for h The solid product formed wascollected by ï¬ltration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr Ê cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSOd6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSOd6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h ï¬ltered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this ï¬gure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr Ê cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSOd6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidiï¬edatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent ï¬atbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The ï¬nal DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespeciï¬c activity of NQO1 was determined using menadione as asubstrate as described [] Brieï¬y the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t | 2 |
New diseaseCase reportAtypical manifestations of COVID19 in general practice a case of gastrointestinal a0symptomsSardam Faraidon Wahab1 Brian Bridal L¸gstrup2 SUMMARYDuring the previous months we have seen the rapid pandemic spread of SARS CoV2 Despite being considered a respiratory virus it has become clear that other clinical presentations are possible and some of these are quite frequent In this paper a case of a man in his late 70s showing atypical symptoms in general practice is presented Apart from fever the patient complained of diarrhoea borborygmus loss of appetite and nausea He developed no respiratory symptoms during his disease Due to his symptoms malignant disease was suspected and he was referred for further testing which revealed typical COVID19 findings on a chest CT scan The occurrence of atypical symptoms is discussed including the importance of recognising these in an ongoing pandemicBACKGROUNDIn December a cumulation of patients with pneumonia of unidentified cause was registered in Wuhan Hubei Province China Prior to this six strains of human pathogenic coronaviruses had been identified In February the WHO and the International Committee on Taxonomy of Viruses established the classification of the seventh human pathogenic virus SARS CoV2 as the disease causative agent of COVID191 While the name of the virus indicates respiratory disease the ongoing pandemic has shown that COVID19 is capable of causing symptoms from several an systems either concomitant with respiratory illness or as the only manifestation Recognising the clinical characteristics of COVID19 typical as well as atypical is of high importance in the prevention of further spread of the virus In this report an atypical case of COVID19 in general practice is presented Furthermore the significance of alternative manifestations is discussedCASE PRESENTATIONThe case of a man in his late 70s is presented The patient had a medical history of herpes zoster and stroke without sequelae He first contacted his general practitioner in mid March due to days of fever which was gradually decreasing Rectal temperature was °C The patients complaints were musculoskeletal body aches influenza like symptoms and headache These symptoms had subsided at the time of initial contact He also experienced rumbling of the stomach diarrhoea a general feeling of malaise and unease weight loss and night sweats He denied shortness of breath cough or other airway related symptomsAt the objective examination the patient appeared tired and exhausted but with normal awareness contact and cerebral condition There were no signs of respiratory distress and respiratory frequency was per minute Examination of the eyes and oral cavity as well as auscultation of the heart and lungs were normal There was no palpable lymphadenopathy in the head neck and periclavicular regions Abdominal inspection palpation percussion and auscultation were with normal findings Laboratory results are presented in table Blood pressure was mm Hg and pulse was estimated to be approximately beats per minute Urine dipstick showed trace of protein The condition was diagnosed as a viral infection and blood samples were collected for further testing He was given a control appointment days laterAt his control appointment the patient reported poor appetite and nausea and food intake was sparse Otherwise symptoms had remained the same with a continuous feeling of malaise There was no stomach pain but he had abdominal discomfort His headache had diminishedThe objective examination was with unchanged general condition The patient still appeared exhausted but otherwise his respiratory condition and appearance were natural Digital rectal examination revealed an enlarged and hard prostate with normal lateral boundaries No faeces or blood was seen on the examination glove and there was no pain on examination There was an increase in C reactive protein CRP to with a normal white and red blood cell count Due to continuous diffuse symptoms primarily abdominal discomfort and weight loss the patient was sent for further investigations This included occult cancer screening with thorough blood work and a CT scan of the throat chest abdomen and pelvisTwo weeks after the onset of symptoms the CT scan showed bilateral ground glass opacities GGO in the lungs raising the suspicion of infectious or postinfectious foci As a result of these findings combined with the short history fever and an increase in CRP COVID19 was thought of as a possible diagnosis The patient was tested for SARS CoV2 with materials obtained from tracheal suctioning The test came out positive for SARS CoV2 and negative for influenza virus and respiratory syncytial virus In the mean time the patients condition had deteriorated and he 1General Medicine Hospitalsenhed Midt Vib Denmark2Cardiology Aarhus University Hospital Aarhus N DenmarkCorrespondence toSardam Faraidon Wahab sardam w hotmail comAccepted August BMJ Publishing Group Limited No commercial re use See rights and permissions Published by BMJTo cite Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseaseTable Biochemical profileTestCRP mgLAlanine aminotransferase ULMonocytes 109LLeucocytes 109LNeutrophils 109LEosinophils 109LBasophils 109LLymphocytes 109LThrombocytes 109LHaemoglobin gLErythrocyte sedimentation rate mmhourReticulocytes 1012LHaptoglobin gLFerritin µgLTransferrin µmolLPlasma iron µmolLBlood glucose mmolLPlasma creatinine µmolLeGFR mLminPlasma kappa chain Ig free mgLPlasma lambda chain Ig free mgLPlasma kappa chain to lambda chain ratio Ig freeIgM gLHigh density lipoprotein mmolLValue day day day Reference rangeDay indicates first contact to general practice CRP development is denoted and the rest are results from day The following tests were normal HbA1c INR potassium sodium alkaline phosphatase total bilirubin TSH antinuclear antibodies IgA urate alpha fetoprotein HBsAg anti HCV HAV IgM folate amylase M component IgG cobalamin B12 PSA albumin and human choriogonadotropinCRP C reactive protein eGFR estimated glomerular filtration rate HAV hepatitis A virus HbA1c Haemoglobin A1c HBsAg hepatitis B surface antigen HCV hepatitis C virus INR international normalized ratio PSA prostate specific antigen TSH thyroid stimulating hormonefelt unwell Therefore the doctor admitted the patient to the hospital at the COVID19 isolation floorAt admission oxygen saturation was without oxygen therapy Furthermore he had a respiratory frequency of per minute temperature of °C blood pressure of mm Hg and otherwise stable vital parameters During the first days of admission he developed an oxygen demand of up to Lmin to uphold an oxygen saturation of The patient was hospitalised for a total of days Throughout the course of the disease he did not complain of regular sore throat stuffy nose sneezing cough dyspnoea or other airway related symptomsINVESTIGATIONSThe suspicion of COVID19 in our patient was raised by the radiology department The chest CT scan was performed with arterial phase contrast It mainly showed peripheral but also central peribronchovascular GGO Streaky consolidations were seen in the right upper lobe Multiple swollen lymph nodes were detected in the mediastinum and both lung hila The key findings are presented in figures There were insignificant laminar subsegmental atelectasis in the posterobasal areas of both lower Figure CT scan in axial plane showing streaky consolidations in the right upper lobe arrowslobes No pericardial or pleural effusions were seen Apart from minimal mucosal thickening and polyp like changes in the right maxillary sinus there was no other pathology detected in the airwaysOUTCOME AND FOLLOWUPThe patient was seen for a follow up appointment months after discharge He reported complete recovery without sequelae On discharge he felt fatigue which gradually subsided in the following weeks There were no complications during Figure CT scan in axial plane showing ground glass opacities arrowsWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseaseTable Biochemical profile at follow up weeks after dischargeTestReference rangeValueFigure CT scan in axial plane showing ground glass opacities extending to the pleura arrowshospitalisation He particularly mentioned a pronounced positive effect of oxygen therapy which increased his appetite and general condition within hours He was slowly regaining the kg of weight that he had lost CRP alanine aminotransferase and blood monocytes had all normalised weeks after initial contact Follow up blood tests are shown in table DISCUSSIONSARS CoV and the Middle East respiratory syndrome coronavirus are among the previously identified human pathogenic coronaviruses Both are known to cause respiratory and enteric illness Gastrointestinal GI symptoms appear to be less common C reactive protein mgLAlanine aminotransferase ULMonocytes 109LLeucocytes 109LNeutrophils 109LLymphocytes 109LThrombocytes 109LHaemoglobin gLTotal cholesterol mmolLTriglycerides mmolLCreatine kinase ULLow density lipoprotein mmolLHigh density lipoprotein mmolLin SARS CoV2 compared with SARS CoV3 but studies are continually published Since COVID19 is still a novel disease it might be too early to conclude which symptoms are typical and which are not A recent comprehensive systematic review and meta analysis reported a pooled prevalence of digestive symptoms of The three most common GI manifestations were loss of appetite diarrhoea and nausea or vomiting While fever was the most frequent manifestation and relatively constant across the studies shown in table diarrhoea ranged from to In the meta analysis by Mao et al4 the frequency of diarrhoea ranged from to It is also important to note that symptoms from different an systems may present at different times in the course of the disease For instance diarrhoea and cough may occur after fever has abated5 These circumstances which may complicate the diagnostic process are important to recogniseThe cause of these different presentations is an interesting matter Factors such as genetics age immunological response and viral properties could be relevant explanations As a result of a synonymous mutation variations in viral components responsible for antigenicity and immunogenicity were found in a recent study This indicates the possible existence of strains with either increased or decreased virulence6 The founder effect must be taken into consideration though Further studies are needed to validate the significance of these findings and contribute to the understanding of the evolutionary trends of SARS CoV2 Studies of the already known coronaviruses have shown that the virions resemble a wreath or a crown due to the conformation of the surface glycoproteins This explains the Latin name corona In contrast to most enveloped viruses these glycoproteins enable the virus to withstand the conditions of the GI tract and thus spread faeco orally7 Xiao et al8 found that of patients had SARS CoV2 RNA in stool Among these still tested positive in faecal samples after converting to negative in respiratory samples Similar to SARS CoV virological studies Table Prevalence of symptoms Sore throatDyspnoeaFeverCough Diarrhoea MyalgiaFigure CT scan in coronal plane showing mediastinal lymph node enlargement arrowZhang et al21Liu et al22Xu et al23Chen et al24Myalgia or fatigueWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseasehave pointed towards the ACE2 receptor as the site of cell entry of SARS CoV29 Previous studies have shown that ACE2 is an enzyme which physiologically counters the activity of the renin angiotensin aldosterone system and thus decreases blood pressure10 It is a membrane bound receptor which is expressed in the vascular endothelia cardiovascular and renal tissue epithelium of the small GI tract and testes ACE2 is abundantly present in the alveolar epithelium of the lungs and the small intestinal tract including the duodenum jejunum and ileum11 The mechanism by which GI manifestations occur is presumably multifactorial Positive immunofluorescent staining of ACE2 and intracellular viral capsid protein in GI tissue has been reported8 Combined with the presence of viral RNA in faecal samples this suggests secretion of infectious virions in faecal matter These findings indicate that direct virus mediated tissue damage may be a possible pathophysiological explanation The endothelial expression of ACE2 facilitates the involvement of vascular beds across several ans Histopathological examination of the small intestine has shown endotheliitis of the submucosal vessels with apoptotic bodies and accumulation of inflammatory cells This may cause endothelial dysfunction vasoconstriction and mesenteric ischaemia13 Evidence of interstitial oedema with infiltration of plasma cells and lymphocytes in GI tissue has also been reported8 These findings contribute to the understanding of GI manifestations of COVID19It is of high importance that healthcare workers are familiar with typical as well as atypical presentations and the course of this novel disease Due to the focus on respiratory symptoms in the early stages of the pandemic our patient was not initially suspected for COVID19 and thus he was sent for further testing without isolation In our case the main suspicion was influenza disease or occult cancer Among the different blood tests in table the elevated ferritin level particularly stands out It is known that ferritin levels may increase during inflammatory infectious and malignant disease among others Zhou et al14 found that ferritin levels were clearly elevated in cases of COVID19 with fatal outcome compared with survivors Another recent study found significantly elevated ferritin levels in patients with severe COVID19 and it was the last blood parameter to normalise A decrease in ferritin levels was not seen along with patient improvement Therefore it was suggested that it is a sensitive marker of severe COVID19 but it cannot be used for disease assessment15 Moreover it has been found that elevated ferritin erythrocyte sedimentation rate CRP fibrinogen and procalcitonin were higher in patients with thrombotic complications16 The present knowledge thus suggests that elevated ferritin level is a marker of severe COVID19 and is associated with thrombotic complicationsAfter the CT scan the main differential diagnoses were pneumonia eosinophilic pneumonia and COVID19 A systematic review of chest CT results in patients showed that GGO was the most frequent finding and was present in of the patients while had bilateral lung involvement17 Radiological findings of GGO have also been reported in asymptomatic patients18 This demonstrates the importance of guidelines being continually updated and communicated to clinicians As already mentioned a significant number of patients are tested positive for virus RNA in stool samples Although it is still not clear to what extent SARS CoV2 spreads faeco orally testing of stool should be further investigated in order to optimise disease controlAs an additional note related to atypical symptoms a recent study found that patients might present with conjunctivitis as the only symptom of COVID1919 Finally some patients experience Patients perspectiveIt all began when I came home from tennis and felt slightly cold with a mild fever In the following days the fever continued and fluctuated between °C and °C and my appetite was decreasing At first I could not eat anything sweet Later this came to include ordinary food as well I developed a feeling of unease in my body and I was prescribed paracetamol for my fever and general condition I experienced severe sweating every time I took paracetamol My wife is a former nurse and we were both convinced that I was having influenza The Danish Health Authorities had encouraged citizens to stay home in the presence of cough fever shortness of breath or musculoskeletal aches Approximately a week after onset I went to my physician and my blood tests showed signs of mild inflammation After three days I came back for a check up appointment which showed a further increase in the inflammatory count I had no appetite and felt very ill tired and disheartened I experienced severe unease and slept very poorly After a thorough examination the doctor said At this time I dont know the cause of your condition I would like to refer you for further examination in order to rule out severe illness and malignant disease and to find the underlying cause I was given an appointment the following day for further blood tests and imaging The imaging of my lungs showed signs of possible coronavirus infection and I was admitted to hospital in isolation I was tested with a throat swab and some fluid was obtained from my airways through a tube in my nasal cavity I was then discharged and told that we would be informed about the test results Late in the evening close to midnight we were contacted with the results I had coronavirus Half an hour later an ambulance arrived and I was admitted to an isolated COVID19 unit at another hospital nearby Further tests were conducted and I was given nasal oxygen therapy because of a low oxygen level in my blood Shortly after I felt significantly better The only therapy I received during my eight days at the hospital was oxygen therapy For every day that passed I felt better and my appetite returned When I was discharged I was told that I had to be symptom free for at least hours before I could consider myself recovered Did I have any remnants of the disease I was very tired but this receded in the following three to four weeks While I am writing this it has been seven weeks since I was discharged I have recovered completely and my condition is now as good as it was before I fell sick with coronavirus I now play tennis three times a week again and I am looking forward to the reopening of the gym Throughout my illness I have received fantastic treatment by all parts of the health care service and I am deeply gratefulLearning points º Our understanding of typical and atypical symptoms of COVID19 is still under development º The existing evidence suggests that gastrointestinal manifestations are present º This calls for increased focus on atypical non respiratory symptoms of COVID19 for optimal disease control º It is important to recognise typical as well as atypical presentations of COVID19 in general practice and other places with initial contactWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0colfactory and gustatory disturbances such as hyposmia anosmia and dysgeusia in the absence of other symptoms20Acknowledgements Maria Tudb was actively involved in clinical decision making as senior consultant in general practiceContributors SFW was the lead author of the manuscript and was involved in initial care and management case identification manuscript write up and drafting editing and literature review BBL was senior supervisor and was involved in acquisition of data and imaging and performed critical revision and editing of the manuscript for important intellectual content SFW and BBL were equally involved in the design and interpretation of dataFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewedThis article is made freely available for use in accordance with BMJs website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedREFERENCES Has¶ks¼z M Kili§ S Sara§ F Coronaviruses and SARS COV2 Turk J Med Sci Jin Y Yang H Ji W et a0al Virology epidemiology pathogenesis and control of COVID19 Viruses Cipriano M Ruberti E Giacalone A Gastrointestinal infection could be new focus for coronavirus diagnosis Cureus 202012e7422 Mao R Qiu Y He J S et a0al Manifestations and prognosis of gastrointestinal and liver involvement in patients with COVID19 a systematic review and meta analysis Lancet Gastroenterol Hepatol Kobayashi K I Kaki T Mizuno S et a0al Clinical characteristics of patients with coronavirus disease in Japan a single center case series J Infect Dis Kim S J Nguyen V G Park Y H et a0al A novel synonymous mutation of SARS CoV2 is this possible to affect their antigenicity and immunogenicity Vaccines 103390vaccines8020220 [Epub ahead of print May ]New disease Patrick R Murray KSR Michael A Pfaller medical microbiology ELSEVIER Xiao F Tang M Zheng X et a0al 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Li Y Hu Y Yu J et a0al Retrospective analysis of laboratory testing in patients with severe or critical type novel coronavirus pneumonia Lab Invest Al Samkari H Karp Leaf RS Dzik WH et a0al COVID19 and coagulation bleeding and thrombotic manifestations of SARS CoV2 infection Blood Salehi S Abedi A Balakrishnan S et a0al Coronavirus disease COVID19 a systematic review of imaging findings in patients AJR Am J Roentgenol Pan Y Yu X Du X et a0al Epidemiological and clinical characteristics of asymptomatic SARS CoV2 carriers J Infect Dis Scalinci SZ Trovato Battagliola E Conjunctivitis can be the only presenting sign and symptom of COVID19 IDCases 202020e00774 Pellegrino R Cooper KW Di Pizio A et a0al Corona viruses and the chemical senses past present and future Chem Senses 2020101093chemsebjaa031 [Epub ahead of print May ] Zhang H Shang W Liu Q et a0al Clinical characteristics of cases of COVID19 in Huanggang and Taian China Infection 101007s15010020014405 [Epub ahead of print May ] Liu K Fang Y Y Deng Y et a0al Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province Chin Med J Xu X Yu C Qu J et a0al Imaging and clinical features of patients with novel coronavirus SARS CoV2 Eur J Nucl Med Mol Imaging Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Copyright BMJ Publishing Group All rights reserved For permission to reuse any of this content visithttpswwwbmjcomcompanyproductsservicesrightsandlicensingpermissionsBMJ Case Report Fellows may reuse this article for personal use and teaching without any further permissionBecome a Fellow of BMJ Case Reports today and you can º Submit as many cases as you like º Enjoy fast sympathetic peer review and rapid publication of accepted articles º Access all the published articles º Reuse any of the published material for personal use and teaching without further permissionCustomer ServiceIf you have any further queries about your subscription please contact our customer services team on or via email at supportbmjcomVisit casereportsbmjcom for more articles like this and to become a FellowWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0c' | 2 |
increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no ï¬delity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizens involvement as volunteers in giving healthand disease data to assess the critical situation for diseases spread and prevention in neighbourhood effect scientiï¬canalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sectors increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe ï¬rst integral deï¬nition of public health was given byWinslow as science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individualsThe American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian uenza HPAI H5N1 in withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difï¬culties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman Hanaï¬Bojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconï¬rmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML ï¬les from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to beneï¬t public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no ï¬delityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in ï¬ghting cholera After years the maps wereidentiï¬ed as a communication tool in understanding andtracking of infectious diseases such as the uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classiï¬ed into four generaAlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenusSevere Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoVhadearlier demonstrated that coronaviruses can cause signiï¬cant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a spatial healthinformation infrastructure In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participants number and oftheir spread local and mass and thoroughnesstime and resource investment or King described for the people with the people or by the people about Citizen Science activities Wiggins andCrownston classiï¬ed Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientiï¬c questions and education providingknowledge to citizens Haklay classiï¬ed CitizenScience into four levels based on participants engagement level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in deï¬ning researchstrategies data collection data interpretation and performing scientiï¬c analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is popularepidemiology in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or street science as a process in which general public communities actively engagedin deï¬ning problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is ï¬rst coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using ï¬ve senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undeï¬ned crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term crowdsourcing geographic information Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuites performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe ï¬rst approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents signiï¬cant concerns regarding privacy The digital contact tracing works on the principle ofcrowdsource data by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom TrackerThis mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and Kings College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare workersexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya SetuThis mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using apps crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identiï¬ed potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of worldTrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apples recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as SARSCoV Boulos and the MERSCoVGikonyo and other seasonal ï¬us online realtime or nearrealtime mapping of diseases occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identiï¬cationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identiï¬cation of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versionsprospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHOs ofï¬cial dataDashboard Dong httpscovid19whointGlobal Visualization of ofï¬cial daily counts ofconï¬rmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation conï¬rmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconï¬rmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISROs BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of conï¬rmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric compositionPM10 and NO2Source CAMS EAC4Meteorological datahumidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and speciï¬c humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to peoplesdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically signiï¬cant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare ofï¬cials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The signiï¬cant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is deï¬ned by Hawkins as an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism In other words when data generation processstarts behaving abnormal and reï¬ects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to ï¬nd clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity scienceecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Theï¬rst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically deï¬ned as follows¼ l N þ Sðà 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Ãð2Ãð3à 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem | 2 |
lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor vegf receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha ret proto oncogene and kit proto oncogene receptor tyrosine kinase we assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiespatients and methods this was an open label single centre single arm phase study eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti vegf therapy and anti epidermal growth factor receptor therapy for tumours with wild type ras patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity the primary endpoint was centrally assessed disease control rate secondary endpoints included safety response rate progression free survival and overall survival the planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of results between october and january patients were enrolled and had received or ¥ lines of prior chemotherapy for metastatic disease respectively the median number of lenvatinib cycles was range the centrally assessed disease control rate was ci to one sided p00001 patients had a partial response and had a stable disease median progression free survival was months ci to median overall survival was months ci to the most common grade ¥ adverse events were hypertension thrombocytopenia increased alanine aminotransferase and anorexia eachs lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiestrial registration number umin ctr umin000023446 and jamcct ctr jma iia00261introductionthe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly key questionswhat is already known about this subject º no studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapieswhat does this study add º lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies º no unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationshow might this impact on clinical practice º further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1 from results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapieslenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor vegfr fibroblast growth factor receptors platelet derived growth factor receptor alpha ret and kit8 preclinical studies have shown that iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 several phase trials of patients with solid tumours in the usa11 europe12 and japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cyclea total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer disease control rate dcr was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a japanese population of the select trial than that of reported in a japanese population of correct trial using regorafenib for metastatic colorectal cancer15 these results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinwe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiespatients and methodsstudy design and patientsthis study was a single arm phase study conducted at national cancer center hospital tokyo japan the inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an eastern cooperative oncology group performance status of or an age of years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the response evaluation criteria in solid tumors recist version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type ras and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment the exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti vegf all patients provided written informed consentprocedurespatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity the dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event treatment was discontinued if a dose interruption was required for more than consecutive daystumour response was assessed by the independent radiological review committee based on the ct or mri performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles urinalysis thyroid function prothrombin time international normalized ratio pt inr and tumour markers both carcinoembryonic antigen and carbohydrate antigen were measured at screening and on day of each treatment cycle adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the national cancer institute common terminology criteria for adverse events version blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment plasma levels of angiopoietin2 were measured by the human angiopoietin2 quantikine elisa kit rd systems minneapolis usaoutcomesthe primary endpoint was centrally assessed dcr which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to recist version a complete response and partial response were needed to be confirmedthe secondary endpoints were the objective response rate orr proportion of patients who had a complete response or partial response progression free survival pfs time from the enrolment until investigator assessed disease progression or death overall survival os time from the enrolment until death due to any cause and adverse events the incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisfor this single arm study the required sample size of patients provided power to reject the null hypothesis of dcr ¤ with expectation that of patients would have a disease control one sided α of considering the possibility of a few ineligible patients we planned to recruit patientsthe final analysis was planned approximately months after enrolment of the last patient we included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses for the primary analysis binomial test was performed and the centrally assessed dcr was estimated with ci using the clopper and pearson method which corresponds to one sided α of we also estimated the investigator assessed dcr a supplementary analysis of the primary iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0ctable baseline patient characteristicscharacteristicstable continuedoverall n characteristicsoverall n open access median range continued intolerant wild type mutant ras mutational status braf mutational status wild type mutant unknownmsi status mss unkown there is an overlapping this number includes patients with the ras wild type and patient with mutant rasecog eastern cooperative oncology group egfr epidermal growth factor receptor msi microsatellite instability mss microsatellite stableendpoint and orr with cis using the same method we estimated the median time and month and year probability of os and pfs with the kaplan meier method the cis for the median time were calculated using brookmeyer and crowley method the cis of month and year survival probabilities were calculated based on the greenwoods formula hrs and cis were estimated by cox regression we did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including ras status for dcr pfs and os we also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples we did all analyses with sas v94resultspatient characteristicsbetween october and january patients with unresectable metastatic colorectal cancer were enrolled all patients were eligible and received the study medication table summarises the baseline characteristics of all enrolled patients the median number of previous lines of palliative chemotherapy was range and patients had received or ¥ prior lines of chemotherapy for metastatic disease respectively the data cut off date was january with median follow up of months iqr efficacythe centrally assessed dcr was ci to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed pr table figure a total of patients had a reduction in target lesion size from baseline figure time on treatment for all patients is ¥ ¥ male female months ¥ months right sided colon left sided colorectum lung liver lymph node peritoneumage years sex ecog performance status primary site number of metastatic site metastatic an time from start of first line chemotherapy number of previous palliative chemotherapy previous chemotherapy and reason for discontinuation fluoropyrimidine refractory intolerantoxaliplatin irinotecan tas102 trifluridinetipiracil angiogenesis inhibitor anti egfr inhibitor refractory intolerant refractory intolerant refractory refractory intolerant refractory intolerantiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesstable best response to treatmentcomplete responsepartial responsestable diseaseprogressive diseasenot evaluabledisease control rate ciresponse rate cicentral assessmentn30 to to investigator assessmentn30 to to shown in online supplementary figures and events for pfs were recorded in all patients and median pfs was months ci to figure all deaths were recorded median os was months ci to with a month and year os of ci to and ci to figure safetypatients received the study treatment for four cycles at median range the median relative dose intensity was iqr dose interruptions and reductions were required in and patients respectively the major treatment related adverse events ¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocytopenia patients the reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each after treatment with lenvatinib patients received a subsequent treatment online supplementary table most patients only had mild grades adverse events table the most common grade ¥ adverse events were hypertension patients thrombocytopenia patients increased alanine aminotransferase and anorexia patients each no clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table serious adverse events occurred in four patients including figure waterfall plot analysis of maximum percentage change from baseline in measurable target lesions response evaluation criteria in solid tumors version central reviewiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accessfigure kaplan meier curves of a progression free survival pfs by investigator assessment and b overall survival os in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisin patients with wild type ras the median pfs was months ci to and that was months ci to in patients with mutant ras online supplementary figure in patients with wild type ras the median os was months ci ci to and months ci to in patients with mutant ras online supplementary figure plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776time of treatment discontinuation online supplementary table with a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median os of months ci to months compared with months ci to in the patients with higher than a first quartile level of angiopoietin2 hr ci to online supplementary figure patients with a first quartile or less level of angiopoietin2 had a median pfs of months ci to compared with months ci to in the patients with more than a first quartile level of angiopoietin2 hr ci to online supplementary figure 0copen accesstable treatment related adverse events occurring in ¥ patients n30any gradegrade ¥treatment related adverse eventhypertensionproteinuriathrombocytopeniafatiguehypothyroidismweight losshoarsenesspalmar plantar erythrodysesthesia syndromeanorexiadiarrhoeamucositis oralserum ast increasedserum creatinine increasedast aspartate transaminase discussionpatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options in this open label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity a total of out of patients had disease control including with partial responses moreover patients experienced reduction in measurable tumour size the overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmstwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in dcr pfs and os compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies dcr median pfs months median os months in the correct study and dcr median pfs months median os months in the recourse study6 interestingly the present single arm phase study of lenvatinib revealed favourable dcr and median pfs values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study moreover about half of the patients received post study treatment which led to a favourable osthe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the japanese population18 moreover we found no unexpected or off target safety signals the most common adverse events were hypertension proteinuria thrombocytopenia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction while the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti vegfvegfr treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the correct concur and consign trials7 it was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients grade ¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the select japanese population15 respectively while in patients treated with regorafenib in the correct japanese population16 to date the clear mechanism of palmar plantar erythrodysesthesia by vegf receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesseveral preclinical studies demonstrated that vegf targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory t cells and myeloid derived suppressor cells24 several clinical studies suggested that modulation of vegf mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death pd1 antibody26 regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28angiopoietin2 a relatively novel regulator of angiogenesis that acts through the tek tyrosine kinase endothelial tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer although the baseline ang2 level was a predictive biomarker in patients with thyroid cancer in the select trial17 it did not become a reliable biomarker of lenvatinib response in this study prior treatment with anti vegfvegfr antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study the decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responsethe limitations of our study include its small size which could limit the interpretation of the subgroup analyses iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0cand the absence of a comparison group however the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursin lenvatinib provided promising activity with prolonged survival relative to the anticipated median pfs in heavily pretreated patients with metastatic colorectal cancer the safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded based on these findings further investigation of lenvatinib with anti pd1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place nct03797326 and nct04008797acknowledgements the authors thank the patients and their families the members of the clinical research support office for their support with data collection and running the study and nai incorporated for editing a draft of this manuscriptcontributors all authors conceived and designed the study and drafted and revised the manuscript for publication si no hs yh at kk th nb and yy collected data ak go mk and kn analysed the data and managed data and study progress all authors interpreted the data and approved the final version of the manuscriptfunding the study was supported by the project promoting clinical trials for development of new drugs and medical devices japan medical association from the japan agency for medical research and development grant number jp18lk0201037 and by eisai cocompeting interests si has received research grants from eisai and merck biopharma th has received research grants from eisai and honoraria from merck serono yy has received honoraria from eisaipatient consent for publication not requiredethics approval the study was conducted in accordance with the declaration of helsinki and good clinical practice guidelines the study protocol was approved by the national cancer center institutional review board t4329provenance and peer review not commissioned externally peer revieweddata availability statement data are available upon reasonable request proposals should be directed to siwasa ncc go jp the data will be available for achieving aims in the approved proposalopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idssatoru a0iwasa http orcid yasuhide a0yamada http orcid references saltz lb clarke s dÃaz rubio e et a0al bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase iii study j clin oncol tabernero j yoshino t cohn al et a0al ramucirumab versus placebo in combination with second line folfiri in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study lancet oncol open access van cutsem e tabernero j lakomy r et a0al addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen j clin oncol yamazaki k nagase m tamagawa h et a0al randomized phase iii study of bevacizumab plus folfiri and bevacizumab plus mfolfox6 as first line treatment for patients with metastatic colorectal cancer wjog4407g ann oncol price tj peeters m kim tw et a0al panitumumab versus cetuximab in patients with chemotherapy refractory wild type kras exon metastatic colorectal cancer aspecct a randomised multicentre open label non inferiority phase study lancet oncol mayer rj van cutsem e falcone a et a0al randomized trial of tas102 for refractory metastatic colorectal cancer n engl j med grothey a van cutsem e sobrero a et a0al regorafenib monotherapy for previously treated metastatic colorectal cancer correct an international multicentre randomised placebo controlled phase trial lancet matsui j yamamoto y funahashi y et a0al e7080 a novel inhibitor that targets multiple kinases has potent antitumor activities against stem cell factor producing human small cell lung cancer h146 based on angiogenesis inhibition int j cancer matsui j funahashi y uenaka t et a0al multi kinase inhibitor e7080 suppresses lymph node and lung metastases of human mammary breast tumor mda mb231 via inhibition of vascular endothelial growth factor receptor vegf r and vegf r3 kinase clin cancer res wiegering a korb d thalheimer a et a0al e7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts neoplasia hong ds kurzrock r wheler jj et a0al phase i dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma clin cancer res boss ds glen h beijnen jh et a0al a phase i study of e7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours br j cancer yamada k yamamoto n yamada y et a0al phase i dose escalation study and biomarker analysis of e7080 in patients with advanced solid tumors clin cancer res nakamichi s nokihara h yamamoto n et a0al a phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in japanese patients with advanced solid tumors cancer 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versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial lancet schlumberger m tahara m wirth lj et a0al lenvatinib versus placebo in radioiodine refractory thyroid cancer n engl j med li j qin s xu r et a0al regorafenib plus best supportive care versus placebo plus best supportive care in asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial lancet oncol van cutsem e martinelli e cascinu s et a0al regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm open label phase iiib consign study oncologist iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen access ott pa hodi fs buchbinder ei inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data front oncol kato y tabata k kimura t et a0al lenvatinib plus anti pd1 antibody combination treatment activates cd8 t cells through reduction of tumor associated macrophage and activation of the interferon pathway plos one 201914e0212513 taylor mh lee c h makker v et a0al phase ibii trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors j clin oncol makker v rasco d vogelzang nj et a0al lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre open label single arm phase trial lancet oncol fukuoka s hara h takahashi n et a0al regorafenib plus nivolumab in patients with advanced gastric gc or colorectal cancer crc an open label dose finding and dose expansion phase 1b trial regonivo epoc1603 jco iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0c' | 0 |
lipases are very versatile enzymes and produced the attention of the several industrial processes lipase can be achieved from several sources animal vegetable and microbiological the uses of microbial lipase market is estimated to be usd million in and it is projected to reach usd million by growing at a cagr of from microbial lipases ec catalyze the hydrolysis of long chain triglycerides the microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules the unique lipase triacylglycerol acyl hydrolase enzymes catalyzed the hydrolysis esterification and alcoholysis reactions immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilization processes immobilized enzymes depend on the immobilization technique and the carrier type the choice of the carrier concerns usually the biocompatibility chemical and thermal stability and insolubility under reaction conditions capability of easy rejuvenation and reusability as well as cost proficiency bacillus spp achromobacter spp alcaligenes spp arthrobacter spp pseudomonos spp of bacteria and penicillium spp fusarium spp aspergillus spp of fungi are screened large scale for lipase production lipases as multipurpose biological catalyst has given a favorable vision in meeting the needs for several industries such as biodiesel foods and drinks leather textile detergents pharmaceuticals and medicals this review represents a discussion on microbial sources of lipases immobilization methods increased productivity at market profitability and reduce logistical liability on the environment and userkeywords microbial lipase fatty acids triglycerides protein engineering biosensor food industry candida antarctica lipase b calbintroductionthe serine hydrolases are present in abundantly and known as lipase enzyme which belong to triacylglycerol ester hydrolase family ec they can catalyze the hydrolysis and synthesis of longchain triglycerides to fatty acids diacylglycerol monoacylglycerol and glycerol known as carboxylesterases [ ] besides hydrolysis activity they display interesterification esterification aminolysis and alcoholysis activity which are contributed correspondence pchandrabbaugmailcom food microbiology toxicology department of microbiology school for biomedical and pharmaceutical sciences babasaheb bhimrao ambedkar university a central university lucknow uttar pradesh indiafull list of author information is available at the end of the in wide range industries [ ] lipase synthesizes esters from glycerol and longchain fatty acids in nonaqueous medium the microbial lipases are more valuable comparison to derive from plants or animals due to their variety of catalytic activities available high yield production and simplicity of genetic manipulation absence of seasonal fluctuations regular supply more stability safer and more convenient and the growth rate of microanisms very high in economically media [ ] the bacterial isolates offer higher activities such as neutral or alkaline ph optima and the thermostability associated to yeasts bacterial strains such as pseudomonas alcaligenes p aeruginosa p fragi p fluorescens bj10 bacillus subtilis b nealsonii s2mt and some species of fungi are penicillium expansum trichoderma penicillium chrysogenum the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchandra a0et a0al microb cell fact page of aspergillus niger produces lipases in higher quantities [] the increasing awareness about animal health and quality of animal produce and increasing consumption of enzymemodified cheese emc and enzymemodified dairy ingredients emdi the lipase market has been extensively increased [ ] due to the more benefits of microbial lipases over animal and plant lipases are also motivating the market growth the request for microbial sources is projected to witness significant growth in the near future due to their wide range of food processing applications [ ] the microbial lipase market is projected to dominate due to cleaning agent segment through the forecast period the growth of industrial microbial lipases in the detergents industry is the innovative key factor to replacing harsh chlorine bleach with lipase and reduced the industrial as well as sewage pollution from fresh water [ ] the microbial lipases in the form of powder is projected to dominate the microbial lipase markets due to its stability easy to handle and easier for packaging and its transportation preferred by the consumers [ ] a0these are extensively applicable in several another industries such as dairy food and beverage animal feed cleaning biofuel pharmaceuticals textile cosmetic perfumery flavour industry biocatalytic resolution esters and amino acid derivatives fine chemicals production agrochemicals biosensor and bioremediation [] additionally altering in the dietary patterns have led to augmented the consumption of dairy products in the region increasing in trepidations about superior hygiene in consciousness of personal hygiene contagious diseases and bleaching household industrial surfaces the manufacturers who operate on a global level and the rising in implementation of lipase enzymes drive the demand for microbial lipases in the region [ ]between the and the market scope of lipase is expected to reach million by globally at a cagr of the asiapacific was the largest market for lipase consumption in [ ] and during the forecast period the asiapacific market is estimated to grow at the highest cagr moreover the rising prospects in the developing markets such as india china and brazil are expected to enhance the market scope of lipases over the forecast period novozymes as denmark e i du pont de nemours and company genencor us koninklijke dsm nv netherlands and chr hansen holdings as denmark are the key industries reported for the consumption of lipases at worldwide httpwwwmarke tsand marke tscom due to the specific properties such as enantioselectivity regioselectivity and broad substrate specificity properties the lipase showing more interest between all the enzymes [ ] this present review focused on discussing the sources of microanisms immobilization methods and their potential applications of lipases including commercially availablehistorical inside or outside the cells enzymes are proteins and have ability of catalyzing the various chemical and biochemical reactions they are highly specific natural catalysts to the various types of substrates and operate under insignificant conditions of environmental factor such as temperature pressure ph with high conversion rates [ ] lipase was first discovered in pancreatic juice as an enzyme by claude bernard in which hydrolysed unsolvable oil droplets and transformed them to soluble products after that the productions of lipase have been observed in the bacteria bacillus prodigiosus b pyocyaneus and b fluorescens in and in the current scenario serratia marcescens pseudomonas aeruginosa and pseudomonas fluorescens species of bacteria have been detected for the production of lipases on large scale lipolase was the first commercial recombinant lipase industrialized from the fungus thermomycesl anugiwnosus and expressed in aspergillus oryzae in traditionally lipase has been achieved from the animal pancreas and was made applicable as digestive supplements in the form of crude or in purified grade it has been extensively used as biocatalytic procedures for the synthesis of several novel chemical compounds []definition of a0lipaseslipases ec are known as triacylglycerol acylhydrolase which acts on carboxylic ester bonds is the part of hydrolases family [ ] they do not require any cofactor and belongs to the class of serine hydrolases triglycerides hydrolyzed into diglycerides monoglycerides fatty acids and glycerol by using the lipases naturally fig a01a the carboxylic esters bonds can be hydrolyzed by esterases in addition to lipases [ ]the hydrolysis of ester bonds at the interface catalyzes by lipases between an unsolvable phase of substrate and aqueous phase where the enzymes keep on liquefied under natural conditions fig a0 1b however pseudomonas aeruginosa candida anatarctica b and burkholderia glumae possessed a lid but did not show interfacial activation [ ] esterification transesterification interesterification acidolysis alcoholysis and aminolysis conversion reaction takes place by lipases [ ]the presence of a lid and the interfacial activation are not the suitable criteria for to categorize a true lipase carboxylesterase simply defined that catalyzes the hydrolysis and synthesis of longchain acylglycerols 0cchandra a0et a0al microb cell fact page of fig a hydrolysis of triglyceride converts into glycerol and fatty acid b representation of a molecule of lipase with its featuresproperties and a0characteristics of a0lipasesthe molecular weight of lipases is in the range of a0kda and reported to be monomeric protein the position of the fatty acid in the glycerol backbone chain length of the fatty acid and its degree of unsaturation are the factors and the physical properties of lipases depend on it [ ] the sensory and nutritive values of given triglyceride also affected by these features several lipases catalyze a number of useful reactions such as esterification due to their activeness in anic solvents [ ] lipases displayed ph dependent activities generally at neutral ph or up to ph and lipases are stable chromobacterium viscosum a niger and rhizophus sp produced extracellular lipases are active at acidic ph and p nitroaeducens produced alkaline lipase and active at ph under certain experimental conditions lipases have capability to reversing the reactions which leads to esterification and interesterification in the absence of water [ ] for the expression of lipase activities the cofactors are not necessary but calcium is the divalent cation stimulates the activity [ ] co ni2 hg2 and sn2 inhibited the lipase activities drastically and zn2 mg2 edta and sds inhibited slightly the halflife values determined temperature stability profiles of lipases and lower temperature shows more stability [ ] according to the regionspecificity lipases divided into two groups and revealed with acyl glycerol substrate without display of regiospecificity only fatty acids are discharged from all three positions of glycerols in the first group of lipases [] the fatty acids regiospecifically discharged from the positions of acylglycerols in the second group of lipase triacylglycerol hydrolysed by lipases and constructed 2monoacylglycerol and free fatty acids 3diacylglycerols in a arrhizus r delemar c cylindracea and p aeruginosa the partial stereospecificity have been detected in the hydrolysis of triacylglycerols [] these enzymes may be used to extract optically pure esters and alcohols due to these properties at low water activity using the anic media offers an exceptional prospect over variation of the solvent so varying the properties of the solvents an enzymes specificity may be transformed any solvent may utilize a substantial influence on the catalytic properties of an enzyme due to the possession of soft structures and delicate [ ]kinetic model of a0lipolysisat the substratewater interface lipolysis arises so the michaelismenten model cannot be described it in a homogeneous phase which is effective only for biocatalysis in which enzyme and substrate are soluble [ ] at an interface to describe the kinetics of lipolysis simple models has been proposed and be made up of two consecutive equilibrium [ ] the alterable adsorption of enzyme to the interface ee happens in the first equilibrium phase a single substrate molecule s 0cchandra a0et a0al microb cell fact page of binds by the adsorbed enzyme e in the formation of es complex as a result in the second phase of equilibrium [ ] for the enzymesubstrate complex to the michaelis menten equilibrium this latter equilibrium is equivalent ending with the discharge of the products and renovation of the enzyme in the e form the subsequent catalytic steps take place once the es complex is formed [ ] the adsorbed lipase in the vicinity of substrate concentration at the interface is at the surface concentration instead of volumetric concentration conventional in the atmosphere [ ] the rejuvenated lipase remnant adsorbed to the interface and is only unrestricted after a number of catalytic cycles in this model fig a0the activity of lipase is a utility of interfacial conformation the enzyme can be denatured as well as triggered or neutralized and the interface is a suitable spot for restraining lipolysis the directly interaction of lipase inhibitor with the enzyme and obstructs the activity of lipase on the other hand via the adsorption to the interphase or to the substrate molecules few compounds can postpone the lipolytic reaction []lipase inhibitors are grouped into two categoriesa synthetic lipase inhibitors including phosphonates boronic acids and fats analogues andb natural compounds βlactones and several botanical foodstuffsplant extracts and metabolites chiefly polyphenols saponins as well as peptides and particular nutritive fibers lipases are essential enzymes for lipid absorption so the absorption of fat or obesity controlled by the lipase inhibition β lactones including orlistat are the natural compounds have the ability to inhibit the lipase activity [ ] over of total dietary fats the pancreatic lipase is responsible for the hydrolysis in several countries for the treatment of obesity orlistat is the registered drug fig lipase catalyzed different reactionslipase inhibitors from a0microbial sourcesfrom microanisms several metabolic products have potent pancreatic lipase pl inhibitory activity the several bacterial fungal and other marine species continued search of effective antiobesity agent screened to find new compounds with pl inhibitory activity [ ]lipstatinthe digestive activity of pancreatic lipases controls by the lipstatin is a βlactone molecule which also controls the absorption of fat in the small intestine lipstatin was first isolated from streptomyces toxytricini is a precursor for tetrahydrolipstatin also known as orlistat xenical and alli the only fdaapproved antiobesity medication for longterm use is a very potent inhibitor of pl [ ] lipase inhibitory activity was lost on opening of βlactone ring the catalytic hydrogenation product of lipstatin is crystalline tetrahydrolipstatin and generally known as orlistat is currently on the market as an antiobesity agent [ ]panclicinsstreptomyces sp nr produced panclicins is another class of potent pl inhibitors nformylalanyloxy or nformylglycyloxy substituent are two alkyl chains are found in panclicins too contains blactone structures panclicins a and b are alanine type while panclicins c d and e are glycine type of compounds the inhibitory activity was recognized to the amino acid moiety alaninecontaining compounds being two to three folds weaker than glycinecontaining compounds valilactonevalilactone first isolated from streptomyces albolongus mg147cf2 strain from shaken culture and jar fermentation valilactone potently inhibited hog pl with an ic50 of a0ngml it also influenced inhibitory activity of esterase from hog liver with an ic50 value of a0mgml ebelactonesebelactone a and b are two ebelactones were isolated from the fermentation broth of actinomycetes strain g7gl closely related to streptomyces aburaviensis both a and b revealed pl inhibitory activity with ic50 values of against hog pl are a0 ngml and a0 ngml respectively esterastinesterastin was isolated from actinomycetes streptomyces lavendulae md4c1 strain from the fermentation 0cchandra a0et a0al microb cell fact page of broth competitively esterastin introverted the hog pancreas lipase with ic50 value of a0ngml caulerpenynecaulerpenyne extracted and purified from an extract of caulerpa taxifolia competitively introverted the activity of lipase with ic50 values of a0mm and a0mm using creamed triolein and disseminated 4methylumbelliferyl oleate as substrates individually [ ] the inhibitory activity of caulerpenyne was independent of substrate concentration suggesting direct interaction but dependent on the lipase concentration with the lipase protein slightly than interacting with the substrate oral supervision of corn oil with caulerpenyne to rats demonstrated a reduced and hindered peak plasma triacylglycerol concentration signifying its potential as a lipid absorption inhibitor [ ]vibralactonevibralactone secreted from boreostereum virens microfungi is a scarce fused βlactonetype metabolite covalently but reversibly transforms the active site serine of the enzyme via acylation by the blactone the ic50 of the vibralactone was resolute to be a0mgml [ ]percyquininpercyquinin obtained from the cultures of basidiomycetes stereum complicatum st inhibited pl with an ic50 of a0mm is another βlactone metabolite in one study on βlactone class of compounds the stereochemistry 2s 3s of the βlactone ring was found to impart specificity for the pl while 2r 3r stereochemistry was accountable for inhibition of hmgcoa synthase sources for a0microbial lipasesmicrobial lipases found universal in nature and are commercially substantial due to the low manufacturing cost superior stability and more availability than animal and plant lipases naturally or recombinant microbial lipases are generally used in diverse bioengineering applications a wide diversity of microbial resources provides by nature microbes have more adaptation abilities and inhospitable atmospheres like dead sea antarctica alkaline lakes hot springs volcanic vents and contaminated soils which provides extraordinary potential for the lipases production with specific features [ ] an enormous spinoff with esteem to the enantioselectivity hydrolysis and the formation of carboxyl esters has produced ready availability the marine microfloras have more capabilities for the formation of enzymes and proteins active compounds mostly lipase fashioned extracellularly secretion from fungi and bacteria [ ]in numerous biocatalytic procedures candida antarctica lipase b calb is the most habitually used enzyme and have a more amount of patents candida rugosa lipase crl is another scientifically significant lipase from the yeast which is a mixture of different isoforms and is commercially accessible and this grounding is known as generally recognized as safe gras and used in the food industry pla1s and pla2s from fusarium oxysporum t lanuginosus a niger and trichoderma reesei between the yeast and fungal phospholipases are used in the degumming of vegetable oils and commercialized while mostly used in the food industry are pla1s pla2s and plbs extracted from a oryzae and a niger [ ] due to their high transphosphatidylation and hydrolytic activities plds isolated from actinomycete strains are commercially available and used in several industrialized procedures mostly the bacterial genera for the production of lipases and phospholipases have been reconnoitered are pseudomonas bacillus and streptomyces followed by burkholderia chromobacterium achromobacter alcaligenes and arthrobacter some lipases producing microanisms reveal new sources and applications of industrial enzymes as shown in table a0bacterial lipaseslipase has been detected initially in b prodigiosus and b fluorescens presently serratia marcescens and p fluorescens observed todays best lipase producing bacteria subsequently [] the glycoproteins and lipoproteins are bacterial lipases in most of the bacteria the enzyme production is affected by the certain polysaccharides have been observed [] some bacterial lipases are thermostable and most of the bacterial lipases are reported as constitutive and nonspecific in their substrate specificity [ ] achromobacter sp alcaligenes sp arthrobacter sp pseudomonas sp staphylococcus sp and chromobacterium sp have been exploited for the manufacturing of lipases between the bacteria fungal lipasessince ²s fungal lipases have been studied due to their affluence in thermal and ph stability substrate specificity and activity in anic solvents and downstream processing these lipases have been exploited the contemporary period machinery favors the procedure of batch fermentation and low cost extraction methods so the fungal lipases have assistances over bacteria major filamentous genera of fungi included are rhizopus aspergillus penicillium mucor ashbya geotrichum beauveria humicola rhizomucor fusarium acremonium alternaria eurotrium and ophiostoma for the production of 0cchandra a0et a0al microb cell fact page of table microbial source of a0lipase and a0their industrial applicationmicrobial sourcesfungal species fusarium solani nfccl yarrowia lipolytica aspergillus oryzae rhizomucor javanicus meih rhizomucor miehei geotrichum candidum and c antarctica candida antarctica candida rugosa candida lipolytica penicillium camembertii trichoderma lanuginosus penicillium roquefortii aspergillus niger meyerozyma guilliermondii a niger gzuf36 aspergillus flavus candida antarctica rhizomucor meihei rhizomucor meihei rhizomucor miehei candida tropicalis aspergillus oryzae penicillium abeanum rhizopus nodosus candida rugosa p chrysogenum rhizomucor meihei p chrysogenum thermomyces lanuginose m miehei c parapsilosis m miehei c antarctica m miehei rhizopus arrhizusbacterial species achromobacter sp hegn virgibacillus pantothenticus hegn pseudomonas mendocina acinetobacter radioresistens bacillus sp fh5 staphylococcus pasteuri p fluorescensapplicationsreferenceshalophilic lipase for biodiesel productiondegrades very efficiently hydrophobic and unusual substrates such as nalkanes oils fats and fatty acids as lowcost carbon sourcessaturated fatty acids synthesized faster cheese ripening flavour customized cheesenonhydrogenated solid fatscocoabutter equivalentsthrough biocatalytic processes preparation of chiral intermediates which synthesized the pharmaceutical compounds related to the elimination of bad cholesterol for the treatment of the alzheimers disease oils and fats enriched removal of size lubricants denim finishinghuman milk fat substitutecheese ripening fatty acid productionproduction of glycerolglycolipidssynthesis of saturated triacyl glyceridesproduced a lipase containing detergent lipoprime®production of characteristic flavor of blue cheese in dairy productsfaster cheese ripening flavor customized cheese dough stability and conditioningpromising feed lipase using cheese wheypotential of the enzyme in the synthesis of functional oilsfat stain elimination synthesis of pharmaceuticals polymers biodiesels biosurfactantspitch control in paper and pulp industry polycondensation ring opening polymerization of lactones carbonates in polymeras a biocatalyst in personal care products such as skin and suntan creams bath oils etcsurfactants for baking industry dairy products noodlesoils and fats enriched cocoa butter substitutes synthesis of bioactive moleculesdegradation of crude oil hydrocarbonsuse for docosahexaenoic acid enrichment of tuna oilleather processing and dehairing and fat removalactivated sludge treatment aerobic waste treatmentfood industry waste treatmentsurfactants for baking industry dairy products noodlesfood industry waste treatmentnonhydrogenated solid fatsused as aroma and fragrance in the food beverage and pharmaceutical industrieshydroxamic acids food additivesynthesis of short chain flavour thioester in solvent free mediumproduction of flavour esterstreatment of oily wastewaterdishwashinglaundry removal of fat strainused in detergent industryusing in oil degradationenantioselective transesterification of a racemate rs4methyl1heptyn4en3ol a component of the insecticide s2852 [ ] 0cchandra a0et a0al microb cell fact page of applicationsreferencesthe production of flavour estersused in leather processinginvolved in enantioselective degradation of aopp herbicidescommonly used detergents enhance the removal of oily stains from various types of fabricswaste water treatmentanic solventtolerant lipolytic enzymeanic solventtolerant lipase for biodiesel productionbaking industry for bread makingenhanced stability in methanolbiodegradation of oil and anics determination as chemical oxygen demand cod biodegradation of food wastewater from restaurantsfood processing and oil manufactureapplication in biocatalysisalkaline lipases able to removing fatty stains when used in a washing machinesolvay enzyme products applicable for is a nonionic andor anionic detergent formulationdetergent formulations containing alkaline lipase used in laundry detergent topdegrading of the fatty material in the waste water management table continuedmicrobial sources staphylococcus warneri and s xylosus bacillus sp brevundimonas sp qpt2 micrococcus sp bacillus cereus hss marinobacter lipolyticus haloarcula sp g41 bacillus subtilis geobacillus stearothermophilus pseudomonas aeruginosa hfe733 pseudomonas sp natronococcus sp p alcaligenes m1 pseudomonas plantarii chromobacterium viscosum acinetobacter sp bacillus thermocatenulatus lactobacillus casei lactobacillus paracasei lactobacillus rhamnosus and lactobacillus plantarumused in medical industrycheese industry for improvement of flavor penicillium roquefortii staphylococcus warneri s xylosus pseudomonas cepacia pseudomonas spcheese industry for cheese ripeningproduction of flavour estersbiodiesel fuel productionformation of 15deoxyspergualin in drug industry as antitumor antibiotic and immunosuppressive agentlipases [ ] other species such as candida rugosa candida antarctica t lanuginosus rhizomucor miehei pseudomonas mucor and geotrichum colletotrichum gloesporioides produced ul of lipase are the most productive strain identified from the brazilian savanna soil by using enrichment culture techniques [ ] a niger c rugosa h lanuginosa m miehei r arrhizus r delemar r japonicus r niveus and r oryzae are the principal manufacturers of these commercial lipases []purification of a0lipasesto get consistency of lipase from a large number of bacteria and fungi various novel purification technologies are available generally several steps are contains for the purification of lipases contingent upon the purity estimated for food application the extracellular microbial lipases from the culture broth eliminated by the centrifugation or filtration in the fermentation process and cells are became freed [ ] the ammonium sulphate precipitation ultrafiltration or extraction with anic solvents is concentrated the cellfree culture broth the gel filtration and affinity chromatography like several combination of numerous chromatographic approaches purified about of the using precipitation steps and then ammonium sulphate and ethanol a homogenous product produces is the final step of gel filtration the novel purification machineries such as the i membrane separation procedures ii immuno purification iii hydrophobic interaction chromatography using epoxyactivated spacer arm as a ligand and polyethylene glycol restrained on sepharose iv polyvinyl alcohol polymers as column chromatography stationary phases and v aqueous two phase systems are frequently engaged after these prepurification steps [ ] the enzyme recovery and fold purification outcomes are found acceptable using of hydrophobic interaction chromatography [ ] an 0cchandra a0et a0al microb cell fact page of acid resilient lipase has been filtered from crude profitable arrangements by size exclusion on biogelp100 and ion exchange on monoq from a niger fungi using the chromatography on hydroxyapatite octylsepharose and sephacryl s200 the lipase was purified to homogeneity from r japonicus nr400 substrates for a0lipasea chiral alcohol moiety possesses by the glycerides which is the natural substrate for lipases the lipases were mostly valuable for the resolution or asymmetrization of esters bearing a chiral alcohol moiety was assumed []methods for a0lipase assaydue to the wide substrate specificity of lipases a number of assay protocols are engaged for lipase assay at the lipid water interface the determination of lipase activity is the analytical of free lipase using various physiochemical approaches the determination activities can be carried as with all reactions catalyzed by enzymes and observing the vanishing of the substrate or by the product release for the determining of the hydrolytic activity several methods are presented such as titrimetry spectroscopy photometry fluorimetry and infrared chromatography radio activity interfacial tensiometry turbidimetry conductimetry immunochemistry and microscopy [ ] the triacylglycerol hydrolysis reaction catalyzed by lipases generally can be written asmultifold benefits such as increase in thermal and ionic stability are applicable using immobilized lipases which upturns its proficiency when the enzyme is immobilized it is easier to control reaction parameters like flow rate and substrates convenience [ ] for immobilization include large surface area low cost reusability good chemical mechanical and thermal stability and insolubility the desirable characteristics of solid supports used according to the interface among the enzyme and support the enzyme immobilization approaches can be classified like physical and chemical procedures the interactions among the enzymes and support are by weaker bonds like hydrogen bonds van derwalls exchanges which create these interactions adjustable in the physical method for the interface among the enzyme and support are stronger by covalent bonds the procedure created irrecoverable in chemical methods [ ]physical methodsadsorptionin the physical approaches of immobilization adsorption procedure the enzymes immobilized by van der waals bonds hydrophobic interactions hydrogen bonds and ionic bonds on the surface of the support the enzyme becomes adsorbed bound and the substrates used mostly for this procedure are cation and anion exchange resins activated carbon silica gel alumina triacylglycerols diacylglycerols free fatty acids monoacylglycerols free fatty acids glycerols free fatty acidsthe activity of lipases can be | 0 |
"patients with differentiated thyroid cancer DTC tumor burden of persistent disease PD is avariable that could affect therapy efficiency Our aim was to assess its correlation with the American ThyroidAssociation ATA riskstratification system and its impact on response to initial therapy and outcomeMethods This retrospective cohort study included consecutive DTC patients referred for postoperativeradioiodine RAI treatment Patients were riskstratified using the ATA guidelines according to postoperativedata before RAI treatment Tumor burden of PD was classified into three categories ie very small small andlargevolume PD Very smallvolume PD was defined by the presence of abnormal foci on postRAI scintigraphywith SPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging Small and largevolume PDwere defined by lesions with a largest size or ¥ mm respectivelyResults PD was evidenced in patients Mean followup for patients with PD was ± years Thepercentage of largevolume PD increased with the ATA risk and in low intermediate and highriskpatients respectively p There was a significant trend for a decrease in excellent response rate from thevery small small to largevolume PD groups at months after initial therapy and respectively p and at last followup visit and respectively p On multivariate analysis age ¥ yearsdistant andor thyroid bed disease smallvolume or largevolume tumor burden and 18FDGpositive PD wereindependent risk factors for indeterminate or incomplete response at last followup visitConclusions The tumor burden of PD correlates with the ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATA riskstratification to refine outcome prognostication afterinitial treatmentKeywords Differentiated thyroid cancer Tumor burden Riskstratification Radioiodine 18FDG PETCT Correspondence rciappuccinibaclesseunicancerfr1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France2INSERM ANTICIPE Caen University Caen FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cCiappuccini BMC Cancer Page of BackgroundIn patients with differentiated thyroid cancer DTCthe riskstratification system described in the American Thyroid Association ATA guidelines is auseful tool to predict the likelihood of postoperativepersistent disease PD the response to initial therapyie surgery ± radioiodine [RAI] treatment and thelongterm outcome [] Several features related to PDare likely to influence the response to treatment andthe longterm prognosis This includes the location ofPD neck lymphnodes [LN] or distant metastases the18FFluorodeoxyglucose 18FDGRAIavidity [] oravidity [] of PD the aggressiveness of pathological variants [] and the degree of celldifferentiation [] thepresence of molecular mutations BRAF TERTp []and the tumor doublingtime [] Alone or in combination with previous characteristics notably RAIaviditythe tumor burden of PD is another variable that canaffect treatment efficiency and prognosis This has beenshown in studiessometimes old and using lowresolution imaging methods focusing on patients withdistant metastases [ ] In the daily practice it is wellknown that microscopic RAIavid lesions are morelikely cured than macroscopic ones eg lung miliary vslung macronodules However no studies have specifiedthe prognostic role of tumor burden estimated usinghighresolution imaging techniques both in the settingof distant metastases and lymphnode diseaseThe aim of the study was to assess the correlationof PD tumor burden with the ATA riskstratification system and its impact on response toinitialtherapy and outcome We hypothesized thatpatients presenting postoperatively a low tumor burden of PD would have better response to initial therapy and better clinical outcomes than patients havinghigh tumor burdenMethodsPatientsThe records of consecutive patients with DTC referred to our institution for postoperative RAI treatment between January and February werereviewed For the purpose of the study patients wereriskstratified according to the ATA guidelinesbased on pathological and surgical data available aftertotalthyroidectomy and before postoperative RAItreatment postoperative risk stratification [] Dataavailable in the preoperative period such as imagingstudies showing distant metastases were also used toinform ATA risk stratification In contrast postoperative serum thyroglobulin Tg level was not used todrive RAI treatment in these patients managed before and no diagnostic RAI scintigraphy was performed before RAI treatmentrhTSHPostoperative RAI treatmentAll patients were administered an RAI regimen ± weeks after total thyroidectomy Patients were prepared after either thyroid hormone withdrawal THWinjections of recombinant humanor after two imthyrotropinThyrogen Genzyme CorpCambridge MA USA as previously described [] TSHlevel was measured the day of RAI treatment and was mUIl in all patients The RAI activity or GBq and the preparation modalities were decided byour multidisciplinary committee All patients underwenta postRAI scintigraphy combining wholebody scanWBS and neck and thorax single photon emissioncomputed tomography with computed tomographySPECTCT A complementary SPECTCT such as abdomen andor pelvis acquisition was performed in caseof equivocal or abnormal RAI foci on WBS Patientswere scanned two or file days following or GBqrespectively Initial therapy was defined as surgery iethyroidectomy ± LN dissection plus first RAI treatmentie postoperative RAI treatmentSerum Tg and antiTg antibodies TgAb assayBlood samples for stimulated serum Tg and TgAb measurements were collected immediately before the RAItreatment Serum Tg measurements were obtained withthe Roche Cobas Tg kit Roche Diagnostics Mannheim Germany with a lower detection limit of ngml and a functional sensitivity of ngml until October and with the Roche Elecsys Tg II kit Roche Diagnostics Mannheim Germany with a lower detectionlimit of ngml and a functional sensitivity of ngml thereafter TgAb was measured using quantitativeimmunoassay methods Roche Diagnostics MannheimGermany TgAb positivity was defined by the cutoffsprovided by the manufacturerPathologyPathological variants were defined according to the WorldHealth anization classification [] Poorly differentiated carcinoma widely invasive follicular carcinomaH¼rthle cell carcinoma and among PTC variants tall cellcolumnar cell diffuse sclerosing and solid variants wereconsidered as aggressive pathological subtypes [] Tumorextent was specified according to the TNM []Tumor burden of persistent diseaseAs previously described [] PD was defined as evidenceof tumor in the thyroid bed LN or distant metastasesafter completion ofinitial therapy Confirmation wasachieved either by pathology or by complementary imaging modalities neck ultrasound examination [US]postRAI scintigraphy 18FDG positron emission tomography [PETCT] CT scan or MRI and followup 0cCiappuccini BMC Cancer Page of The tumor burden of PD was classified into three categoriesie very small small and largevolume PDVery smallvolume PD was defined by the presence ofabnormal foci on posttherapeutic RAI scintigraphy withSPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging neck ultrasound CT scan orMRI Small or largevolume PD were defined by thepresence of metastatic lesions with a largest size or ¥ mm respectively regardless of RAI or 18FDGuptake Examples of patients with very small small orlargevolume PD are presented in Fig RAI and 18FDG uptake in persistent diseaseThe RAI or 18FDG uptake profile was defined at time ofPD diagnosis PD was considered RAIpositive RAI ifat least one metastatic lesion showed RAI uptake andRAInegative RAI otherwise Similarly PD was defined18FDGpositive 18FDG if at least one metastatic lesionpresented significant 18FDG uptake and 18FDGnegative18FDG otherwiseClinical outcome assessmentAs previously described [] clinical assessment ofpatients with a negative postRAI scintigraphy wasscheduled at three months with serum TSH Tg andTgAb measurements while on levothyroxine LT4treatment When the Tg level at three months was ngml in the absence of TgAb the disease status wasassessed at months by serum rhTSHstimulated Tgassay and neck US and in recent years by Tg II assayon LT4 and neck US If there was an excellent responselevel ngmlat months according to the ATA criteria iestimulatedTgor nonstimulatedTglevel ngml without TgAb and negative neck USpatients were followed up on an annual basis For anything other than an excellent response imaging modalities such as CT scan of the neck and thorax 18FDGPETCT or MRI were performed In case of a secondRAI regimen given months after the first RAI therapy for RAIavid PD postRAI scintigraphy with SPECTCT was also used to assess initial treatment responseResponses to initial therapy as assessed at monthsand status at lastvisit were categorized as excellent response indeterminate response biochemical incompleteresponse or structural incomplete response according tothe ATA guidelines []Data analysisQuantitative data are presented in mean ± standard deviation SD except for Tg levels which are presented inmedian range Patients characteristics were comparedusing Chisquare or Fishers exact test the Wilcoxontest or the KruskalWallis test as appropriate TheCochranArmitage trend test was used to examineproportions of excellent response over the differentsubgroups in the following order verysmall small andlargevolume PD The analysis of diseasespecific survival and progressionfree survival was performed usingthe Cox regression model The analysis of prognosticfactors was performed using logistic regression Statistical significance was defined as p All tests wereFig Examples of very small small and large tumor burden in patients with persistent disease PD On the left side a 43yearold female patientwith a 40mm PTC at lowrisk after initial surgery T2NxMx and very smallvolume PD ac posttherapeutic 131I WBS showed a solitary bonyfocus on the right hip a arrow Fused transaxial image of 131I SPECTCT b arrow confirmed the bony uptake and hybrid CT c arrow did notdisplay any bone abnormality On the middle part a 74yearold female patient with a 40mm PTC at lowrisk after initial surgery T2N0Mx andsmallvolume PD df posttherapeutic 131I WBS showed pulmonary metastases d red and black arrows Fused transaxial image e red arrowand hybrid CT scan f red arrow depicted RAIavid lung micronodules ef mm On the right side an 88yearold female patient with a 40mmPTC tall cell variant at highrisk after initial surgery T2N1bM1 and largevolume PD gi no abnormal RAI uptake on posttherapeutic 131I WBSwith SPECTCT whereas 18FDG PETCT showed pulmonary and mediastinal metastases g Maximum intensity image arrows Fused transaxialimage h arrow and hybrid CT scan i arrow showed high 18FDG uptake SUVmax by an 18mm lung nodule 0cCiappuccini BMC Cancer Page of twosided SAS statistical software SAS InstituteInc Cary NC USA was used for data analysisstratification Patients characteristics are reported inTable ResultsCharacteristics of patientsThe study group included papillary thyroid cancers PTC follicular thyroid cancers FTC and poorlydifferentiated thyroid cancers PDTCThere were women and men The mean agewas ± years Three hundred and seventytwo patients were prepared with rhTSH stimulation Eightytwopatients presented positive TgAb at the time of postoperative RAI treatment In the postoperative setting priorto RAI administration patients were at lowriskLR at intermediaterisk IR and athighrisk HRaccording to the ATA riskPersistent disease and tumor burdenOverall PD was detected in patientsTheir characteristics in terms of ATA risk RAI preparation modality PD sites and RAI or 18FDG uptake arepresented in Table Of patients had very smallvolumelargevolume smallvolume and PDFigure shows two points First the rate of PDincreased from in LR patients and in IR to in HR patients p Second the percentage of patients with largevolume PD increased with risk stratification from LRIR to HR patients and respectively p Table Characteristics of patients according to the ATA riskstratification system in the postoperative settingMean age ± SD yrsSex ratio FemaleMean tumor size ± SD mmHistologyPTCFTCPDTCAggressive pathological subtypesNoYesExtrathyroidal extensionMinimalGrossT status TNM T1a T1bT2T3a T3bT4a T4bN status TNM NxN0N1a N1bM status TNM M0M1Positive TgAb levelStimulated Tg level at RAI treatment rangeaaIn patients without positive TgAb levelLRn ± ± IRn ± ± HRn ± ± p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent disease according to the tumor burdenVery smallvolumePD n SmallvolumePD n LargevolumePD n Postoperative ATA riskLRIRHRPreparation modalityTHWrhTSHPD siteLNLN DMDMTB diseaseTB disease DMRAI and 18FDG statusRAI18FDG or NPRAI18FDGRAI18FDGRAI18FDGRAI18FDG NPa21 RAI18FDG NP and one RAI18FDGb15 RAI18FDG NP and two RAI18FDGc10 RAI18FDG NP and six RAI18FDG 22a 17b 16c pFig Tumor burden in patients with persistent disease correlation to the ATA riskstratification system The figure first shows that the rateof PD increased from in LR patients in IR to in HR patients p Second the percentage of patients with largevolume PDincreased with risk stratification from LR IR to HR patients and respectively p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent diseaseaccording to the ATA riskstratification systemLRn IRn HRn pPD tumor burdenVery smallvolume SmallvolumeLargevolume The distribution of very small small andlargevolume PD in LR IR and HR patients is presented in Table Outcome of patients with persistent diseaseTreatment modalities within the first year of management and during the remaining followup are detailed inTable Mean followup for patients with PD was ± years and was similar between the three groups of tumorburden p Of the patients with PD at months after initial therapy had excellent response indeterminate response biochemical incomplete response and structuralincomplete response At last followup visit the figureswere and respectively The outcome in each of the tumor burden groupsis presented in Table There was a significant trend fora decrease in excellent response rate from the verysmall small to the largevolume PD groups at months after initial therapy and respectivelyp and at last followup visit and respectively p Fig Among the patients died related to DTCduring followup Seven were in the largevolume PDgroup and one in the smallvolume PD group All hadstructural incomplete response at months after initial therapy with 18FDGpositive diseaseFigures and show diseasespecific survival DSSand progressionfree survivalPFS according to theATA riskstratification 18FDG status and tumor burdenSignificant differences in DSS were observed for bothATA riskstratification and 18FDG status but not fortumor burden Patients with 18FDGpositive disease hadshorter PFS Hazard Ratio 95CI thanthose with 18FDGnegative disease Also IR HazardRatio 95CI and HR patients HazardRatio 95CI had shorter PFS than LRpatients Finally patients with small Hazard Ratio 95CI and largevolume PD Hazard Ratio 95CI had shorter PFS than those withverysmall volume PDPrognostic factor analysis in patients with persistentdiseaseMultivariate analysis controlling for age sex postoperative ATA riskstratificationaggressive pathologicalTable Treatment modalities and outcome of patients with PD at months after initial therapy and at last followup visitaccording to tumor burdenVery smallvolumePD n months after initial therapySmallvolume PDn Largevolume PDn pVery smallvolumePD n At last followup visitSmallLargevolume PDvolume PDn n p a Treatment modalities at months after initial therapy treatments given within the first year of followup treatment modalities at last followup visittreatments given after the first year during followupb Local treatment of DM external radiation beam therapy surgery or radiofrequencyAbbreviations PD Persistent disease RAI Radioiodine DM Distant metastasesTreatment modalitiesaRAINeck surgeryNeck external radiationbeam therapyLocal treatment of DMbTyrosinekinase inhibitorsChemotherapyOutcomeExcellent response Indeterminate responseBiochemical incompleteresponseStructural incompleteresponse 0cCiappuccini BMC Cancer Page of Fig Excellent response rate according to tumor burden months after initial therapy a and at last followup visit b in patients withpersistent disease There is a significant trend for a decrease in excellent response rate from the very small small to the largevolume PD groupsat months after initial therapy and respectively p and at last followup visit and respectively p subtypes site of PD tumor burden of PD and RAI or18FDG uptake showed age ¥ years Odds ratio [OR] p distant andor thyroid bed disease OR p smallvolume OR p andlargevolume tumor burden OR p and18FDGpositive disease OR p to be independent risk factors for indeterminate biochemical orstructuralincomplete response at last followup visitTable DiscussionThis study confirms that the incidence of PD aftertotal thyroidectomy and postoperative RAI treatmentis limited in LR patients as compared to IR or HR patients Moreover it demonstrates thatthe tumor burden of PD is correlated to postoperativeriskstratification with very smallvolume lesions preferentially observed in LR patients and small and largevolume in IR or HR patients Most importantly tumorburden of PD is shown as an independent predictor ofresponse to initial therapy and to outcome These findings confirm that tumor burden of PD is a variablewhich might be taken into account to refine outcomeprognosticationTumor burden covers a large range of locoregionalandor distant metastases from a unique microscopic lesion to multiple macroscopic ones sometimes clinicallyevident Also tumor burden encompasses structural egvisible on conventionalfunctionalradiology andorlesions eg visible on RAI scintigraphy or 18FDG PETCT The diagnostic performances of imaging methodsand consequently the concept of tumor burden havedramatically evolved in the last decades The detectionof small LN disease has been improved by the combination of highresolution neck US postRAI SPECTCTand 18FDG PETCT imaging Regarding distant metastases although postRAI WBS still remains the referencefor detecting lung miliary disease the routine use ofdiagnostic CT scan and MRI now enables the detectionof infracentimetric lung bone or brain lesionsIn the past tumor burden of PD as a potential indicator of successful treatment and prognosis was assessedusing different approaches In a study on DTC patients with lung metastases diagnosed from to multivariate analysis showed that lung nodules visible on XRay vs those not visible RAIrefractory lunglesions and multiple metastatic sites were associatedwith poor survival [] In Gustave Roussys experienceoverall survival was reported in DTC patients withdistant metastases lung bone or other sites diagnosedbetween and [] Tumor extent was classifiedinto three categories according to both postRAI planarscintigraphy and Xrays Category consisted in lesionsvisible on postRAIscan but with normal Xraycategory in metastatic lesions cm on Xrays andcategory in lesions cm regardless of RAI avidityOverall metastases were RAIavid in of patientsmore frequently in patients years than 0cCiappuccini BMC Cancer Page of Fig Diseasespecific survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden cyears Multivariate analysis demonstrated that female sex young age years well differentiatedtumor RAI avidity and limited extent category wereindependent predictors ofrecentlyRobenshtok reported the outcome of patientssurvival Morewith RAIavid bone metastasis without structural correlate on CT scan or MRI among DTC patients withbone metastases between and [] After afollowup period of years all patients were alive nonehad evidence of structural bone metastases and none 0cCiappuccini BMC Cancer Page of Fig Progressionfree survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden chad experienced skeletalrelated events confirming theexcellent prognosis after RAI treatmentIn DTC patients with persistent nodal disease there isalso indirect evidence supporting that tumor burden affects treatment response and outcome In a recent retrospective study Lamartina reported the outcome of patients without distant metastases who underwenta first neck reoperation for nodal persistentrecurrentdisease [] Male sex aggressive histology and the presence of more than LN metastases at reoperation wereshown to be independent risk factors of secondary relapse following complete response achieved with first 0cCiappuccini BMC Cancer Page of Table Risk factors for indeterminate biochemical or structural incomplete response at last followup visitVariableAge years ¥ SexFemaleMaleInitial ATA riskstratificationLRIRHRAggressive histological subtypesNoYesSite of PDLN onlyDM andor TB disease with or without LNTumor burden of PDVery smallvolumeSmallvolume mmLargevolume ¥ mmRAI and 18FDG status of PDRAI18FDG or NPRAI18FDG or NPRAI or RAI18FDGPatients at risk nInitial modelOR CIp valueFinal modelOR CIp valuereoperation Conversely the excellent outcome of microscopic nodal involvement detected on SPECTCT at RAIablation was demonstrated by a study from Schmidt [] Of patients with RAIavid LN metastasesat ablation only three still showed nodes with significantuptake on a diagnostic RAI scintigraphy at monthsThe LN successfully treated by RAI were less than cmexcept in one patient whereas those still visible at months were above cm confirming that RAI is highlymore efficientin microscopic than in macroscopiclesionsIn the present study multivariate analysis showed thatage over years distant andor thyroid bed diseasesmall or largevolume tumor burden and 18FDGpositive disease were independent risk factors for indeterminate or incomplete response at last followup visit Incontrast ATA risk stratification and aggressive pathological subtypes did not emerge from multivariate analysis possibly because of the number of patients thenumber of variables tested and confounding variablesHoweverand progressionfreethe diseasespecificsurvival curves confirmed the high prognostic value ofthe ATA riskstratification In practice data supportsthat LR patients have a better outcome than the IR andHR groups not only because PD is uncommon in thosepatients but also because the excellent response rate ishigher in very smallvolume than in small or largevolume lesions We suggest that tumor burden usingthis threeclass discrimination could be implemented inthe assessment of patients with structural incomplete response to help refining the risk prediction This variablecould also be incorporated with the other risk predictorssuch as RAI or 18FDG uptake molecular profile tumorhistology degree of cell differentiation and Tg level andtumor volume doubling time to further improve riskestimatesAlthough retrospective the present study presents several strengths including the large cohort of consecutivepatients and the significant followup Patients diagnosedbetween and were uniformly evaluated usingmodern imaging studiesincluding postRAI scintigraphy with neck and thorax SPECTCT [] and 18FDG 0cCiappuccini BMC Cancer Page of PETCT with a dedicated headandneck acquisition [] Tumor burden was assessed combining functionaland anatomic imaging as adapted from previous papersof our group [ ] One can argue that it would havebeen even more pertinent to assess tumor burden withquantitative values rather than with a threeclass discrimination ie very small small and largevolumeActually a quantitative volumetric assessment is notfeasible because of the RAIavid nodal or metastatic lesions without structural correlate Also a quantitativeassessment based on RAI or 18FDG uptake is notpossible either because of RAIrefractory or nonhypermetabolic lesions Nevertheless we believe that ourdefinition is simple to use in routine practice and easilyreproducibleConclusionsThe tumor burden of PD correlates with the postoperative ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATAriskstratification to refine outcome prognostication afterinitial treatmentAbbreviationsATA American thyroid association DM Distant metastasesDTC Differentiated thyroid cancer 18FDG 18FfluorodeoxyglucoseFTC Follicular thyroid cancers HR Highrisk IR Intermediaterisk LN Lymphnodes LR Lowrisk MRI Magnetic resonance imaging NP Not performedOR Odds ratio PD Persistent disease PDTC Poorlydifferentiated thyroidcancers PETCT Positron emission tomography with computed tomographyPTC Papillary thyroid cancers RAI Radioiodine rhTSH Recombinant humanthyrotropin SPECTCT Single photon emission computed tomography withcomputed tomography Tg Thyroglobulin TgAb AntiTg antibodiesTHW Thyroid hormone withdrawal TB Thyroid bed US Ultrasoundexamination WBS Wholebody scanAcknowledgmentsWe are indebted to Gee Knight for the reviewing of the manuscriptAuthors contributionsRC and SB conceived the study and its design RC ALC VSR CL VLH DV EBand SB performed data acquisition and analysis NH performed the statisticalanalysis RC and SB drafted the manuscript All authors read and approvedthe final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used and analysed during the current study are available fromthe corresponding author on reasonable requestEthics approval and consent to participateAll procedures were in accordance with the ethical standards of theinstitutional committee and with the Helsinki declaration and its lateramendments Baclesse Cancer Centre has licensed from the FrenchCommission for Data Protection and Liberties CNIL MR004 ref v0This study was approved by the institutional review board of Baclesse hospital and all subjects gave written informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France 2INSERM ANTICIPE Caen University Caen France 3CETAPS EA Rouen UniversityRouen France 4Department of Head and Neck Surgery Fran§ois BaclesseCancer Centre Caen France 5Department of Pathology Fran§ois BaclesseCancer Centre Caen France 6Department of Oncology Fran§ois BaclesseCancer Centre Caen France 7Department of Cancer Biology and GeneticsFran§ois Baclesse Cancer Centre Caen France 8Department of Head andNeck Surgery University Hospital Caen FranceReceived February Accepted August ReferencesHaugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEPacini F Randolph GW Sawka AM Schlumberger M Schuff KG Sherman SISosa JA Steward DL Tuttle RM Wartofsky L American ThyroidAssociation management guidelines for adult patients with thyroid nodulesand differentiated thyroid Cancer the American Thyroid Associationguidelines task force on thyroid nodules and differentiated thyroid CancerThyroid Durante C Haddy N Baudin E Leboulleux S Hartl D Travagli JP Caillou BRicard M Lumbroso JD De Vathaire F Schlumberger M Longtermoutcome of patients with distant metastases from papillary andfollicular thyroid carcinoma benefits and limits of radioiodine therapy J ClinEndocrinol Metab Robbins RJ Wan Q Grewal RK Reibke R Gonen M Strauss HW Tuttle RMDrucker W Larson SM Realtime prognosis for metastatic thyroid carcinomabased on [18F]fluoro2deoxyDglucosepositron emission tomographyscanning J Clin Endocrinol Metab Michels JJ Jacques M HenryAmar M Bardet S Prevalence and prognosticsignificance of tall cell variant of papillary thyroid carcinoma Hum Patholde la Fouchardiere C DecaussinPetrucci M Berthiller J Descotes F Lopez JLifante JC Peix JL Giraud Delahaye A Masson S BournaudSalinas CBorson CF Predictive factors of outcome in poorly differentiated thyroidcarcinomas Eur J Cancer Melo M Gaspar da Rocha A Batista R Vinagre J Martins MJ Costa G RibeiroC Carrilho F Leite V Lobo C CameselleTeijeiro JM Cavadas B Pereira LSobrinhoSimoes M Soares P Gaspar da Rocha A Batista R Vinagre JMartins MJ Costa G Ribeiro C Carrilho F Leite V Lobo C CameselleTeijeiroJM Cavadas B Pereira L SobrinhoSimoes M Soares P TERT BRAF andNRAS in primary thyroid Cancer and metastatic disease J Clin EndocrinolMetab Sabra MM Sherman EJ Tuttle RM Tumor volume doubling time ofpulmonary metastases predicts overall survival and can guide the initiationof multikinase inhibitor therapy in patients with metastatic follicular cellderived thyroid carcinoma Cancer Casara D Rubello D Saladini G Masarotto G Favero A Girelli ME BusnardoB Different features of pulmonary metastases in differentiated thyroidcancer natural history and multivariate statistical analysis of prognosticvariables J Nucl Med Ciappuccini R Hardouin J Heutte N Vaur D Quak E Rame JP Blanchard Dde Raucourt D Bardet S Stimulated thyroglobulin level at ablation indifferentiated thyroid cancer the impact of treatment preparationmodalities and tumor burden Eur J Endocrinol Lloyd RV Osamura RY Kl¶ppel G Rosai J editors WHO classification oftumours of endocrine ans 4th edition Lyon International Agency forResearch on Cancer Brierley JD Gospodarowicz MK Wittekind C TNM classification of malignanttumours 8th edition Oxford Wiley Blackwell Ciappuccini R Heutte N Trzepla G Rame JP Vaur D Aide N BardetS Postablation I scintigraphy with neck and thorax SPECTCTand stimulated serum thyroglobulin level predict the outcome ofpatients with differentiated thyroid cancer Eur J Endocrinol 0cCiappuccini BMC Cancer Page of Robenshtok E Farooki A Grewal RK Tuttle RM Natural history of smallradioiodineavid bone metastases that have no structural correlate onimaging studies Endocrine Lamartina L Bet I Mirghani H Al Ghuzlan A Berdelou A Bidault FDeandreis D Baudin E Travagli JP Schlumberger M Hartl DM Leboulleux SSurgery for neck recurrence of differentiated thyroid Cancer outcomes andrisk factors J Clin Endocrinol Metab Schmidt D Linke R Uder M Kuwert T Five months' followup of patientswith and without iodinepositive lymph node metastases of thyroidcarcinoma as disclosed by 131ISPECTCT at the first radioablation Eur JNucl Med Mol Imaging | 2 |
" natural orifice specimen extraction surgery is a novel technique of minimally invasive surgery thepurpose of this study was to compare the safety of laparoscopic anterior resection with natural orifice specimenextraction noselar and abdominal incision specimen extraction aiselar for sigmoid or rectum tumorsmethods medline pubmed embase central cochrane central register of controlled trials scopus andclinicaltrials databases were systematically searched for related s up to august the primary outcomesincluded postoperative complications overall postoperative complication incisionrelated complicationanastomotic fistula and severe complication and pathologic results lymph nodes harvested proximal resectionmargin and distal resection edge the statistical analysis was performed on stata softwareresults ten studies comprising patients were used for metaanalysis compared with aiselar noselar hadmore advantages in terms of overall postoperative complication odds ratio or ci to p incisionrelated complication or ci to p distal resection edge weighted meandifference wmd cm ci to cm p recovery of gastrointestinal function wmd day ci to day p pain scores in postoperative day wmd ci to p additional analgesics usage or ci to p and hospital stay wmd day ci to day p while the operation time of noselar was prolonged wmd min ci to min p the anastomotic fistula severe complication lymph nodes harvested proximalresection margin intraoperative blood loss and longterm outcomes in noselar were comparable with aiselars the safety of noselar was demonstrated and it could be an alternative to conventional surgery inlaparoscopic anterior resection for sigmoid and rectal tumors however further randomized and multicenter trials are requiredkeywords natural orifice specimen extraction nose laparoscopic anterior resection rectal tumor sigmoid tumor metaanalysis correspondence guangenyang163com1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0che world of surgical oncology page of introductionover the past three decades laparoscopic surgery hasevolved incessantly especially in the field of colorectalsurgery it has been widely accepted by surgeons and patients in light of the better perioperative outcomes andanalogical longterm effectiveness compared with opensurgery for colorectal cancers []for conventional laparoscopic colorectal operation asmall laparotomy in the abdomen is required for specimen harvested and colorectal anastomosis because ofthe miniincision it causes many undesirable outcomessuch as incision pain wound infection scar and even incision hernia and the advantages of laparoscopic surgeryare reduced [] to minimize those drawbacks anovel surgical variant known as natural orifice specimenextraction nose surgery with the features of naturalorifice specimen extraction and totalintraperitonealanastomosis has been introduced and is becoming ahotspot [] some studies have reported the oncology and safety outcomes between nose surgery andconventional laparoscopic surgery are comparable [] and the nose surgery therefore is supposed tohave a progress of minimally invasive surgeryrecently some metaanalysis studies had comparednatural orifice specimen extraction nose with abdominal incision specimen extraction aise in laparoscopiccolorectum resection for the colorectal disease [ ]however colorectum resection comprises right hemicolectomyleft hemicolectomy and anterior resection procedures among those surgeries were largely different and the surgical procedures and the excision extension between sigmoid and rectum resection aresimilar in addition several studies compared laparoscopic anterior resection with natural orifice specimenextraction noselar with abdominal incision specimen extraction aiselar for sigmoid or rectum tureleased [] hence wemors wereconducted this metaanalysis to evaluate the safety ofnoselar in sigmoid and rectal tumorsrecentlymethodsstudy design and inclusion criteriathis metaanalysis followed the preferred reportingitems for systematic reviews and metaanalysis prisma statements the inclusion and selection criteria were determined before starting a literature searchonly when studies with fulltext on sigmoid or rectaltumors that compared noselar and aiselar andreported at least one of the endpoints of focus were retrieved and analyzed the most comprehensive researchwas recruited when overlapping researches was conducted by the same team no language restriction wasapplied conference s case reportsreviewsrobotic surgery and singleportwere not consideredlaparoscopic surgeryselection criteria conformed to the framework ofpico participant intervention comparison and outcome patients diagnosed with sigmoid or rectal tumorsbenign and malignant tumors requiring surgery wereincludedinterventions consisted of noselar andaiselar noselar was compared with aiselarin all eligible studies primary endpoint outcomes werepostoperative complications overall postoperative complication abdominal incisionrelated complication anastomotic leak and severe complication claviendindoclassification ¥ iii and pathologic results retrievedlymph nodes proximal resection edge distal resectionedge secondary outcomes included operation timeblood loss pain score numeric rating scale score additional analgesics gastrointestinalfunction recoveryhospitalization duration 5year diseasefree survivaldfs and 5year overall survival os search strategythe following databases had been searched up to august medline pubmed central cochrane central register of controlled trials embase scopus andclinicaltrials for a more accurate search the followingkeywords andor mesh terms were used sigmoid neoplasms rectal neoplasms colorectal neoplasmslaparoscopynatural orifice specimen extractiontransvaginal specimen extraction transanal specimenextraction and transrectal specimen extraction thespecific search strategies among databases existed differences the search strategy of pubmed was presented inadditional text reference s of the eligible studies were reviewed to find the potentially relevant studiesstudy selection and quality assessmentretrieved studies were independently assessed for relevance by reviewers changjian wang and jinmingchen by screening the title and in order to enhance sensitivity studies were removed only when bothreviewers excluded the study subsequently a fulltextassessment was performed on the initial screening included studies the risk of bias was assessed by thenewcastleottawa scale nos for observational studies and studies achieving five or more stars were eligible cochrane collaborations tool for assessing risk forbias was used for randomized controlled trials [ ]all discrepancies were discussed before a final decisionwas madedata collection and statistical analysisdata from the recruited studies were extracted by tworeviewers changjian wang and jinming chen andused formeananalysis outcome valuesfurther 0che world of surgical oncology page of standard deviation and median interquartile rangewere extracted from each study considering potentialheterogeneity among studies we pooled the results byusing a randomeffects model the weighted mean difference wmd and confidence intervals cis wereapplied for continuous variables and the odds ratioor and cis were used for dichotomous variablesthe continuous outcomes were adopted the inverse variance method and dichotomous outcomes were adoptedthe mantelhaenszel statistical method when a studymerely offered the outcomes with median and interquartile range an estimation based on formulas designed byhozo was performed if a study did not provide the hazard ratio hr and cis of 5year dfsorand os the methods presented by tierney wereused for data extraction from survival curves thechisquare test and isquared value were used for measuring heterogeneity and i2 p was definedas significant heterogeneity sensitivity analyses basedon nos score ¥ and the sample size of noselargroup ¥ were conducted to assess the potentialsource of heterogeneity and the robustness of the resultspublication bias was examined with a funnel plot andharbord test p was considered statistically significant the statistical analysis was performed onstata softwareresultsliterature selection and characteristicsthe initial database search identified s ofwhich were removed based on the title and assessment the rest of the literature were evaluated byfulltext assessment and studies were excludedcharacteristics of the excluded studies were presented inadditional table ten studies were finally included forfurther qualitative and quantitative synthesis [ ] all of these were retrospective studies the process of the search and selection wasshown in fig a total of patients were recruitedin those studies with patients in the noselargroup and patients in the aiselar group themain characteristics of studies and patients were presented in table and details were shown in additionalfig flow chart of studies included in the metaanalysis 0che world of surgical oncology page of sunasunasunasunasunasunarosunaiangavsunasunasunarorororomutcermutceridomgsimutceridomgsimutceridomgsimutceridomgsiralesaipurnsonerocsetisinemcepsnoitcartxenoitacolromutleamefeamlrednegmutceridomgsimutcermutcermutcerralesonpuralesaipuraeyaegaralesonpuralesaipuralesonpugnitnuocisetapcitrapngisedydutsinogerraeyydutsisedutsdeducnliehtfoscitsiretcarahcinamelbatevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorternapajlateadasihianhclateuhianhclategnianhclategnahzianhclateuohzianhclategnxiianhclateuliydutsaissurydutsevitcepsorternawatihbaruaslateevitcepsorterecnarflatetsonedevitcepsorterydutsydutsianhclategnawrnleacsawattoeltsacwensonnoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaegnarinademronoitavedidradnats±naemsadrocertondetropera 0che world of surgical oncology page of euavlpytienegoretehieuapvldmwstluserldeoopicrororalesairalesonstneitapfoonfoonisedutsnoitacilpmocsemoctuollafostluserldeoopehtelbatsemoctuoyramirpsemoctuoevitarepotsopllarevonoitacilpmocnoitacilpmocdetaerlnoisicnilautsifcitomotsananoitacilpmocerevesdetsevrahsedonhpmylsemoctuoilcgoohtapingramnoitceserliamxorpegdenoitceserlatsidnoitcnufsemoctuoyradnocesemitnoitarepossoldooblevitarepoartnilanitsetnortsagifoyrevocerdopinapevitarepotsopegasusciseganallanoitiddayatslatipsohsoraeyevfisfdraeyevfiecnereffidnaemdethgewdmwinoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaliavvruseerfesaesidsfdliavvrusllarevosoyadevitarepotsopdopoitarsddoro 0che world of surgical oncology page of table the results of the pooled outcomes were summarized in table primary outcomesall included studies reported the overall postoperativecomplication the pooled data revealed that the postoperative complication in of patients whotreated with noselar and of patientswho treated with aiselar the or was ci to p with low heterogeneity i2 fig 2a among the studies eight studies reportedthe incisionrelated complication in of patients who underwent noselar and of patients who underwent aiselar the or was ci to p with no heterogeneity i2 fig 2b nine studies reported the anastomoticfistula of which ng reported zero events in bothgroups the remaining eight studies recorded anastomotic fistula in of patients sufferednoselar and of patients suffered aiselar or was ci to p withno heterogeneity i2 fig 2c a severe complication was defined based on the claviendindo classification two of the included studies recorded severecomplication claviendindo classification ¥ iii and thesevere complication in of patients withnoselar and of patients with aiselar or was ci to p withsignificant heterogeneity i2 fig 2da total of nine studies reported lymph node harvestthere was no significant difference in lymph node harvestbetween the two groups wmd ci to p no significant heterogeneity was observed i2 fig 3a the mean number of dissectedlymph nodes in the noselar group was and theaiselar group was the proximal resection marginwas reported in studies and the wmd in the upper resection edge was cm ci to cm p with no heterogeneity i2 fig 3b the distalresection margin was reported in studies and the wmdin the inferior resection edge was cm ci to cm p with no heterogeneity i2 fig3c the length of the distal resection margin in the twogroups was cm noselar group and cmaiselar groupsecondary outcomesa total of nine studies recorded operation time and intraoperative blood loss the pooled data revealed thatthe wmd of operative duration was min ci to min p heterogeneity i2 fig 4a the wmd of blood loss was ml ci to ml p heterogeneity i2 fig 4bfig forest plot comparing postoperative complications in thenoselar group and aiselar group a overall postoperativecomplication b incisionrelated complication c anastomotic fistulaand d severe complication 0che world of surgical oncology page of fig forest plot comparing pathologic outcomes in the noselar group and aiselar group a lymph nodes harvested b proximal resectionmargin and c distal resection edgesix studies provided data about the recovery of gastrointestinal function the wmd of bowel movement was day ci to day p heterogeneity i2 fig 5a the postoperative painwas recorded in studies and studies hisada and wang recorded the postoperative pain periodand the remaining reported the pain scores in postoperative day pod the wmd of pain score in pod 0che world of surgical oncology page of fig forest plot comparing intraoperative outcomes in the noselar group and aiselar group a operation time and b blood loss was ci to p heterogeneity i2 fig 5b the additional analgesicusage rate was also reported in those studies and theor of additional analgesics usage was ci to p heterogeneity i2 fig 5c theduration of hospital stay was reported in nine studiesthe wmd of hospital stay was day ci to day p heterogeneity i2 fig 5dfiveyear diseasefree survival dfs and 5year overallsurvival os were available in two studies the hazardratio hr in the 5year dfs was ci to p heterogeneity i2 fig 6a and thehr in the 5year os was ci to p heterogeneity i2 fig 6bsensitivity analysissensitivity analysis results based on the nos score ¥ and the sample size of noselar group ¥ were presented in additional table it showed a slight inconsistency in distal resection edge operation time andrecovery of gastrointestinal function and all the otheroutcomes showed a similar trend of results between thetwo groupspublication biasa funnel plot of overall postoperative complication wasperformed to detect publication bias it showed that allthe inclusive studies were within the confidenceinterval and no publication bias was found fig inaddition a harbord test confirmed there was no publication bias p discussionas a technique used for clinical treatment the safety ofnoselar should be efficiently proved morbidity isone of the most efficient indicators for assessing thesafety of an emerging technique postoperative complications may not only lead to failures of surgery but alsothreaten lives the overall postoperative complicationrate in noselar was lower than that in aiselar severe morbidity claviendindo ¥ iiiamong the two techniques was not a significant difference the operation involving digestive tract reconstruction anastomotic leakage is a potential risk once itoccurs reoperation is usually inevitable the incidence of anastomotic leakage in noselar wassimilar with that in aiselar in addition theincidence ofincisionrelated complications in noselar was significantly lower than that in theaiselar group obviously the reduction ofcomplications in noselar has largely attributed tothe decrease of incisionrelated complications althoughthe complications in noselar were reduced the riskof bacteria contamination in the peritoneal cavity shouldnot be neglected costantino had reported the peritoneal contamination in the nose group was higherthan that of the conventional group hence measures such as bowel preparation prophylactic antibioticsperitonealtransluminalwound retractor and abdominal drains are recommended to avoid the contamination of the peritonealcavity irrigationtransanallavagethe postoperative pathology results to some extentalso reflect the safety of a surgery this metaanalysisshowed lymph nodes harvested between the two groupswas comparable and it also conformed to the minimumrequirement of the guideline retrieved more than nodes in our metaanalysis the proximal marginin the noselar group was similar with the conventional group however the distal margin in the nosegroup was longer than that of the aiselar group the 0che world of surgical oncology page of potential cause of this difference was the use of transanalspecimen eversion and extraabdominal resection technique in the nose group [ ] because of thisprocedure the distal rectal resection is performed extraabdominally under direct vision moreover circumferential resection margin crm between the two groupshave no difference [ ] in addition accordingto our metaanalysis the longterm outcomes 5yeardfs and 5year os were comparable all of those indicated the nose technique was a safety procedure in thetreatment of sigmoid and rectal cancers nevertheless aconcern about tumor seeding was raised during the procedure of enterotomy and specimen extraction it is necessary to apply several measures such as the use ofprotection devices sterile specimen bags and avoidingoverpulling and compression during specimen extraction as a minimally invasive surgery noselar had moreadvantages in alleviating patients distress the reductionof pain scores in postoperative day pod was observed and this reduction could be attributed to thetrauma in noselar being further reduced owingto less pain the need for additional analgesics was alsoreduced in addition accelerating postoperative recoverywas also observed the recovery of gastrointestinal function and hospital duration in patients who sufferednoselar was much shorter besides some scholarsmay doubt if there have alterations in sexual urinary ordefecation function in the groups according to the included studies there were no differences in functionaloutcomes such as sexual urinary or defecation betweentwo groups [ ] even though a small part of patientsexperienced function alteration and the alternation wasreversible [ ] those all demonstrated thatnoselar was a safety surgery and to some extent ithad advantages in postoperative recoverynevertheless our study has several limitations firstlyintersphincteric resections were mixed with coloanalanastomoses with sigmoid cancer in our studies although there exist some differences we mixed the twotechniques and mainly considered there existing common procedures between sigmoid and rectum resectionin laparoscopic anterior resection and some studies didnot record the methods of outcome evaluation such asblood loss evaluation to some extent it reduces thecomparability of outcomes secondly the present metaanalysis relied solely on retrospective studies and someoriginal studies not presented how patients were selectedto be candidates for one technique or another the quality of all included studies was regarded as fair or good however this type of study cannot be comparedwith a randomized controlled trial and potential biascannot be ruled out thirdly this study only recruitedone multicenter research and some outcomes includedfig forest plot comparing postoperative recovery in the noselar group and aiselar group a recovery of gastrointestinalfunction b postoperative pain pod c additional analgesicsusage and d hospital stay 0che world of surgical oncology page of fig forest plot comparing longterm outcomes in the noselar group and aiselar group a 5year dfs and b 5year oslimited studies so further multicenter randomized controlled and more comprehensive studies containing adequate outcomes are needed fourthly the results ofsome pooled results among studies existed heterogeneity the sensitivity analysis could not be detected as thecause of heterogeneity although some results existedheterogeneity the major results were homogeneity andthe heterogeneity of outcomes such as operation duration blood loss and hospital stay can be explained byclinical heterogeneity such as the difference of patientssurgeons patient management and differences in surgical proficiency in nose technology in addition the results of the major parameters were robust all in all theresults of this analysis are convincedaccording to our metaanalysisthe advantages ofnose are reduced overall complications especiallyincisionrelated complications increased distal resectionedge enhanced recovery of gastrointestinal function reduced postoperative pain reduced additional analgesicsusage and shortened hospital stay and without an auxiliary patients operated by the nose technique achievebetter aesthetics however the operative time is prolonged although the nose technique has many advantagesthere are many requirements that should befollowed before the application of this technique in colorectal surgery firstly the nose should be operated byexperienced surgeons with conventionallaparoscopiccolorectal surgery secondly the indication of noseshould follow the indication of conventionallaparoscopic colorectal resection the depth of tumor invasionshould be within t3 and body mass index bmi shouldbe less than kgm2 for transanalnose and less than kgm2 for transvaginalnose transanal nose suitsfor male or female patients and the tumor diameter isfig funnel plot of the overall postoperative complications 0che world of surgical oncology page of recommended less than cm while transvaginal noseis only applied for female patients and the tumor diameter is limited within cm and the emergent conditionssuch as bowel obstruction perforation and massivebleeding are excluded all in all as surgeons follow appropriate indicationsthe noselar for sigmoid or rectal tumors is a safesurgery and the longterm outcomes between twooperations have no difference and the benefits of thenoselar in shortterm outcomes are noticeablethese findings promote enthusiasm in support ofnose surgery as an alternative approach forthetreatment of sigmoid and rectal tumorssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1295702001982wadditional file additional file text the search strategy of pubmedadditional file additional table characteristics of the excludedstudiesadditional file additional table summary of the included studiesadditional file additional table the results of sensitivity analysisadditional file additional table quality assessment based on thenos for retrospective studiesabbreviationsnoselar laparoscopic anterior resection with natural orifice specimenextraction aiselar laparoscopic anterior resection with abdominal incisionspecimen extraction wmd weighted mean difference or odds rationos newcastleottawa scale dfs diseasefree survival os overall survivalcis confidence intervals hr hazard ratio pod postoperative day acknowledgementswe wish to thank the timely help given by junfeng hu in statistic analysisand mengdan zhou in language editingauthors contributionsguangen yang and zhong shen contributed to the conception and designof the study jun he performed the literature search and the writing of themanuscript changjian wang and jinming chen performed the data extraction haibo yao performed the data analysis qinyan yang and jianmingqiu participated in the writing of the manuscript guangen yang and haibo yao helped to revise the intellectual content the authors read and approved the final version of the manuscriptfundingthis work was funded by the zhejiang natural science foundation grantno lq19h160013 and the zhejiang medical health science and technologyproject grant no and the hangzhou health science andtechnology project grant no 2017a26availability of data and materialsall the data analyzed in this study was obtained from the included originals or related authorsethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of colorectal surgery hangzhou third hospital hangzhou zhejiang peoples republic of china 2departments ofgastroenterology pancreatic surgery zhejiang provincial peoples hospitalhangzhou zhejiang peoples republic of chinareceived february accepted july references wang cl qu g xu hw the short and longterm outcomes oflaparoscopic versus open surgery for colorectal cancer a metaanalysisint j color dis ishibe a ota m fujii s suwa y suzuki s suwa h momiyama m watanabej watanabe k taguri m midterm followup of a randomized trial ofopen surgery versus laparoscopic surgery in elderly patients withcolorectal cancer surg endosc 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prisma statement ann internmed w64 hawker ga mian s kendzerska t french m measures of adult pain visualanalog scale for pain vas pain numeric rating scale for pain nrs painmcgill pain questionnaire mpq shortform mcgill pain questionnaire sfmpq chronic pain grade scale cpgs short form36 bodily pain scalesf36 bps and measure of intermittent and constant osteoarthritis painicoap arthritis care res 201163suppl 11s240 higgins jp altman dg gotzsche pc juni p moher d oxman ad savovic jschulz kf weeks l sterne ja the cochrane collaborations tool forassessing risk of bias in randomised trials bmj 2011343d5928 ga wells bsdo the newcastleottawa scale nos for assessing the qualityof nonrandomised studies in metaanalyses hozo sp djulbegovic b hozo i estimating the mean and variance from themedian range and the size of a sample bmc med res methodol tierney jf stewart la ghersi d burdett s sydes mr practical methods forincorporating summary timetoevent data into metaanalysis trials denost q adam jp pontallier a celerier b laurent c rullier elaparoscopic total mesorectal excision with coloanal anastomosis for rectalcancer ann surg saurabh b chang sc ke tw huang yc kato t wang hm tzuliang cwfingerhut a natural orifice specimen extraction with single stapling colorectalanastomosis for laparoscopic anterior resection feasibility outcomes andtechnical considerations dis colon rectum dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg almazrou am suradkar k mauro cm kiran rp characterization ofreadmission by day of rehospitalization after colorectal surgery dis colonrectum costantino fa diana m wall j leroy j mutter d marescaux j prospectiveevaluation of peritoneal fluid contamination | 0 |
"a biomarker we validated in BRAF inhibitor-resistant NSCLC clinical specimens. These data reveal the multifaceted molecular mechanisms by which NSCLCs establish and regulate BRAF oncogene dependence provide insights into BRAFEGFR signaling crosstalk and uncover mechanism-based strategies to optimize clinical responses to BRAF oncogene inhibition. targeted therapy combination therapy 101274235 33311 J Thorac Oncol J Thorac Oncol Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 1556-0864 1556-1380 24662455 4084898 10.1097/JTO.0000000000000148 NIHMS560664 Membrane carbonic anhydrase IX (CAIX) expression and relapse risk in resected stage I-II non-small cell lung cancer Stewart D.J. MD Nunez M.I. MD Behrens C. MD Liu D. MS Lin Y. H. PhD Lee J.J. PhD Roth J. MD Heymach J. MD PhD Swisher S. MD Hong W.K. MD Wistuba I.I. MD University of Ottawa (DJS) and University of Texas MD Anderson Cancer Center (MIN CB DL YHL JJL JR JH SS WKH IIW) Correspondence: David J. Stewart MD FRCPC Head Division of Medical Oncology The Ottawa Hospital/University of Ottawa Ottawa ON Canada K1H 8L6 Telephone: 613-737-7700 70166 dstewarttoh.on.ca 25 5 2014 5 2014 01 5 2015 9 5 675 684 Background Adjuvant chemotherapy reduces recurrences of non-small cell lung cancer (NSCLC). To determine which patients need adjuvant chemotherapy we assessed factors associated with time to relapse (TTR). Methods In 230 resected stages I-II NSCLCs we correlated immunohistochemistry (IHC) scores for factors associated with cell growth rate growth regulation hypoxia cell survival and cell death with TTR. Results With a median follow-up of 82 (1-158) months for those alive and relapse-free at last follow-up median time to recurrence was not reached. The 2- and 5-year probabilities of maintaining freedom from recurrence were 80.7% (95% confidence interval [CI]:(75.3% 86.4%)) and 74.6% (95% CI:(68.6% 81.2%)) respectively. TTR curves flattened at an apparent cure rate of 70%. In multicovariate Cox models factors correlating with shorter TTR were membranous carbonic anhydrase IX (mCAIX) staining (any vs none hazard ratio [HR]=2.083 p=0.023) and node stage (N1 vs N0 HR=2.591 p=0.002). mCAIX scores correlated positively with tumor size grade squamous histology necrosis mitoses Ki67 p53 nuclear DNMT1 and cytoplasmic SHARP2 and correlated inversely with papillary histology EGFR mutation (trend) CTR1 and cytoplasmic HIF-1? VEGF DNMT1 and ERCC1. Conclusion Nodal stage and mCAIX IHC were the strongest independent predictors of shorter TTR in resected NSCLCs. mCAIX correlated with tumor size markers of tumor proliferation and necrosis and tumor genetic characteristics and paradoxically correlated inversely with the hypoxia markers HIF-1? and VEGF. Presence of mCAIX could help determine patients with high-risk of recurrence who might require adjuvant chemotherapy. carbonic anhydrase IX non-small cell lung cancer PLoS One one 1932-6203 Public Library of Science San Francisco USA 24595062 3942483 PONE-D-13-41012 .0090818 Research Biology Biochemistry Proteins Developmental Biology Morphogenesis Cell Migration Evolutionary Biology Evolutionary Systematics Phylogenetics Genetics Cancer Genetics Gene Expression Gene Function Model anisms Animal Models Medicine Oncology Basic Cancer Research FTSJ2 a Heat Shock-Inducible Mitochondrial Protein Suppresses Cell Invasion and Migration FTSJ2 Characteristics and Functions in Cancer Cells Lai Cheng-Wei 1 Chen Hsiao-Ling 2 Lin Ken-Yo 1 Liu Fang-Chueh 1 3 Chong Kowit-Yu 4 Cheng Winston T. K. 5 Chen Chuan-Mu 1 * 1 Department of Life Sciences Agricultural Biotechnology Center iEGG center National Chung Hsing University Taichung Taiwan 2 Department of Bioresources Da-Yeh University Changhwa Taiwan 3 Department of Animal Nutrition Livestock Research Institute Council of Agriculture Tainan Taiwan 4 Department of Medical Biotechnology and Laboratory Science Chang Gung University Tao-Yuan Taiwan 5 Department of Animal Science and Biotechnology Tunghai University Taichung Taiwan Ahmad Aamir Editor Wayne State University School of Medicine United States of America * E-mail: chchen1dragon.nchu.edu.tw Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: HLC CMC. Performed the experiments: CWL KYL FCL. Analyzed the data: KYC CMC WTC. Contributed reagents/materials/analysis tools: FCL WTC. Wrote the paper: CWL CMC. 2014 4 3 2014 9 3 e90818 8 10 2013 5 2 2014 2014 Lai et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Ribosomal RNA large subunit methyltransferase J (RrmJ) an Escherichia coli heat shock protein is responsible for 2?-O-ribose methylation in 23S rRNA. In mammals three close homologs of RrmJ have been identified and have been designated as FTSJ1 FTSJ2 and FTSJ3; however little is known about these genes. In this study we characterized the mammalian FTSJ2 which was the most related protein to RrmJ in a phylogenetic analysis that had similar amino acid sequence features and tertiary protein structures of RrmJ. FTSJ2 was first identified in this study as a nucleus encoded mitochondrial protein that preserves the heat shock protein character in mammals in which the mRNA expressions was increased in porcine lung tissues and A549 cells after heat shock treatment. In addition a recent study in non-small cell lung cancer (NSCLC) suggested that the FTSJ2 gene is located in a novel oncogenic locus. However our results demonstrate that the expression of FTSJ2 mRNA was decreased in the more invasive subline (CL1-5) of the lung adenocarcinoma cells (CL1) compared with the less invasive subline (CL1-0) and overexpression of FTSJ2 resulted in the inhibition of cell invasion and migration in the rhabdomyosarcoma cell (TE671). In our findings indicate that mammalian FTSJ2 is a mitochondrial ortholog of E. coli RrmJ and conserves the heat shock protein properties. Moreover FTSJ2 possesses suppressive effects on the invasion and migration of cancer cells. This research was supported by grant NSC-98-2313-b-005-012 from the National Science Council and was partly supported by the Ministry of Education Taiwan Republic of China under the aiming top university plan (ATU-101-s0508). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Heat shock proteins (HSPs) and their biological functions are highly conserved from Escherichia coli to mammals [1]. The following five major classes of HSPs have been defined: HSP70s HSP60s HSP90s HSP110s and small HSPs. Protein folding refolding and disaggregation are well-known functions of HSPs [1] [2]. However there are still a number of proteins involved in the heat shock response that remain uncharacterized upon heat shock stress [3]. The E. coli enzyme RrmJ a 23S rRNA 2?-O-ribose methyltransferase (MTase) that was identified as a novel heat shock protein is the first gene of the rrmJ-ftsH heat shock operon and was first discovered in 1991 [3] [4]. RrmJ is required for the 2?-O-ribose methylation of U2552 in 23S rRNA [5]. Um2552 is one of the four 2?-O-ribose methylated nucleotides in E. coli rRNA and is located in the A-loop of the peptidyl transferase center of the ribosome [5] [6]. In a previous study the E. coli rrmJ deletion strain lost its adaptive ability upon heat shock stress [7]. This loss may have resulted from a change in the A-loop conformation and ribosome dissociation [7][9]. The heat shock protein RrmJ is conserved in both Prokaryota and Eukaryota [10]. In Saccharomyces cerevisiae there are three homologs of RrmJ including Trm7p Mrm2p and Spb1p. Trm7p is a tRNA 2?-O-ribose MTase that catalyzes the methylation of the nucleotides at positions 32 and 34 in the anticodon loop of tRNAPheTrp [11]. Mrm2p is a mitochondrial rRNA 2?-O-ribose MTase but is encoded by the nuclear genome. Similar to RrmJ Mrm2p catalyzes the formation of Um2791 which is located in the peptidyl transferase center in mitochondrial 21S rRNA in a position equivalent to that of Um2552 in E. coli. The mrm2 deletion strain shows growth defects in a glycerol-containing medium at 37°C indicating that this protein is important for mitochondrial function and is crucial for heat shock adaptation in yeast [12]. Spb1p is a nuclear protein composed of 841 amino acids and contains two functional domains: a small N-terminal RrmJ-like domain and a large C-terminal domain that is involved in rRNA maturation. Spb1p catalyzes the methylation of U2921 and G2922 in the A-loop of 25S rRNA which are equivalent to U2552 and G2553 in E. coli 23S rRNA respectively [13][16]. E. coli RrmJ and its three homologs in yeast all contain the four-residue sequence K-D-K-E which composes the catalytic center of RrmJ [10] [17]. Moreover the structures of the substrates of these three homologs and the positions of the methylated rRNA residues are very similar to those of E. coli RrmJ [6] especially Mrm2p because it is located in the mitochondria which are believed to be degenerated bacteria [18]. In mammals there are also three RrmJ homologs designated as FTSJ1 FTSJ2 and FTSJ3 that correspond to Trm7p Mrm2p and Spb1p respectively. In previous studies the mutation of human FTSJ1 has been shown to cause nonsyndromic X-linked mental retardation (NSXLMR) " | 1 |
Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen response as defined by ¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigators discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA level by ¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the ϲ test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics years years ¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had exceptional responses defined as normalisation or ¥ decline of CA patients or normalisation PET SUVmax in In Arm B had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an exceptional CA response and had an exceptional PET response as defined by either ¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA response ¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection statusAAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA decrease FDG PET SUVmax decrease ¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigators choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a softening of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options | 2 |
Intervertebral disc degeneration IDD is the most common degenerative disease all over the word Our previous studyconï¬rmed that the downregulated circGRB10 directly interacts with miR3285p which modulate ERBB2 and leads tothe degeneration of intervertebral disc however the underpinning mechanism of circGRB10 dysregulation remainsunclear We identiï¬ed that FUS and demonstrated that circGBR10 biosynthesis in nucleus pulposus NP cells waspromoted by FUS whose expression was controlled by miR1413p In addition ERBB2 downregulation led todecreased Erk12 phosphorylation which enhanced miR1413p production in NP cells In vivo data indicated that circGRB10 inhibited IDD in rat model The present study revealed that miR1413p and FUS are key factors that regulatecircGRB10 synthesis in NP cells In addition circGBR10 participates in the molecular circuitry that controls human IDDdevelopment These ï¬ndings provide a basis for further functional diagnostic and therapeutic studies of circGRB10in IDDIntroductionThe Global Burden of Disease Study stated that lowback pain LBP represents an important cause of disability worldwide1 LBP is tightly associated with intervertebral disc degeneration IDD which involves ofall LBP cases causing significant economic and socialburdens worldwide23 According to previous reports of the world population have low back pain during theirlifetime with being chronically disabled4Currently IDD pathogenesis is largely unclear howeverit could be due to microenvironmental alterations in theintervertebral discs caused by various factors such asgenetic features aging sex a predisposing injury and theCorrespondence Wei Guo guow0319163com1Department of Orthopaedics Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R China2Department of Breast Surgery Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R ChinaFull list of author information is available at the end of the Edited by G Calinenvironment56 The main feature of IDD pathology iselevated biosynthesis of catabolic enzymes combined withreduced extracellular matrix ECM accumulation causedby imbalanced anabolism and catabolism7 Intervertebraldiscs comprise a central nucleus pulposus NP a peripheral annulus ï¬brosus AF and cartilaginous endplates which connect overlying capillary beds craniallyand caudally The NP maintains homeostasis by producing an ECM mostly comprising type II collagen andproteoglycans the main functional components of intervertebral discs which are indispensable to maintain thedisc height and absorb various mechanical loads8 It iswell known that loss of collagenII and aggrecan is anearly critical event in the degenerative cascade in Intervertebral disc tissue9 MMP13 is the most importantenzymes that hydrolyze collagens10 ADAMTS5 is classiï¬ed as the major aggrecanases due to their high efï¬ciency in cleaving aggrecan11 A large body of evidencesupporting the involvement of MMP13 and ADAMTS5in IDD pathogenesis12 During IDD the main histologicalalteration involves the centrally located NP cells which The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of after a phenotypic transformation are substituted bysmaller ï¬brochondrocytelike cells with reduced proteoglycan production and a global shift towards synthesizing ï¬brotic materials and compromising the structuralintegrity of discs1314 Therefore unveiling the mechanisms underpinning such imbalance is urgently needed forthe development of new therapeutic targets in IDDMounting evidence supports roles for circular RNAscircRNAs in IDD15 Previous research demonstratedthat circRNAs are closed RNAs produced by backsplicingof single premRNAs18 It is not completely known howcircRNAs are biosynthesized although complementaritybetween inverted sequences in ï¬anking introns and theactivity of RNAbinding proteins RBPs increase thecontiguity of splice sites contributing to backsplicing inmammalian cells19The RBP FUS affects splicing regulation23 with manysplicing factors termed FUS interactors24 FUS mutations could lead to protein mislocalization to the cytosolwith decreased nuclear FUS amounts and occurrence ofabnormal cytosolic aggregates2728 The FUS protein isinvolved in regulating intracellular RNA transport mRNAsynthesis alternative splicing and polyadenylation siteselection29 He demonstrated that FUS combinedwith circ_002136 and promoted the generation ofcirc_002136 in Glioma30 It was recently shown that FUScontrols the expression of circRNAs by binding tointrons that ï¬ank the splicing junction31 Moreover FUSwas reported to be regulated by many miRNAs includingmiR1413p3233 Studies revealed miR1413p is upregulated in NP tissue specimens from IDD cases anddemonstrated that miR1413p is associated with discdegeneration34 However the function and mechanism ofFUS as well as the interaction between FUS and miR1413p in IDD have not been reportedOur previous research conï¬rmed that circGRB10amounts are markedly reduced in NP cells from IDDpatients which accelerates IDD development by enhancing miR3285p mediated ERBB2 suppression in NPcells15 However the mechanism of circGRB10 downregulation in degenerative NP cells remains unclear Inthis study we demonstrated that the miR1413p whichis significantly increased in degenerative NP cells34 regulate expression of the FUS which is responsible for thegeneration of circGRB10 in NP cells Furthermore weshowed that ERBB2 downregulation led to decreasedErk12 phosphorylation and the decreased levels of Erk1 phosphorylation enhanced miR1413p biogenesis indegenerative NP cells promoting IDD developmentTaken togetherthese ï¬ndings suggested circGBR10contributes to the molecular circuitry controlling IDDdevelopment in humansOfï¬cial journal of the Cell Death Differentiation AssociationResultsCircGRB10 regulates NP cell functions through the ERBB2Erk signaling pathwayOur previous study revealed circGRB10 promotes NPcell survival by increasing ERBB2 amounts via suppression of miR3285p However the effect of circGRB10expression on NP cell anabolism or catabolism remainsobscure To further assess circGBR10s functions in IDDpathogenesis circGRB10 or circGRB10 small interferingRNA siRNA was transiently transfected into culturedprimary human NP cells As shown in Supplementary FigS1 overexpression and knockdown of circGRB10 haveno effect on linear GRB10 but only affect circular GRB10The immunoï¬uorescence results demonstrated that afteroverexpressing circGRB10 in NP cellssignificantlyupregulation of collagen II and aggrecan and decreasedamounts of MMP13 and ADAMTS5 were found Conversely circGRB10 knockdown resulted in oppositeeffects Fig 1a b These ï¬ndings were conï¬rmed by qRTPCR Fig 1csignificantlyincreased while MMP13Our previous research demonstrated circGRB10 inhibits IDD development by regulating ERBB2 expression inNP cells Increasing evidence supports an important rolefor the ERBB2 gene and Erk signaling pathways in theprogression of many human diseases35 Meanwhile theErk pathway is altered during IDD38 and plays a significant role in extracellular metabolism39 These resultsprompted us to assess the plausible association of circGRB10 with ERBB2 Erk signaling In this study primaryhuman NP cells underwent transfection with circGBR10circGRB10 siRNA and respective negative controlsrespectively As shown in Fig 1d e western blot assayshowed that pErk12 collagen II and aggrecan amountswereandAMADT5 levels were reduced in NP cells overexpressingandcircGRB10 Conversely pErk12aggrecan were downregulatedandAMADT5 amounts were increased in NP cells transfected with circGRB10 siRNA Fig 1d e FurthermoreERBB2 affected pErk12 in a similar way as circGRB10Fig 1d e suggesting cricGRB10 modulates IDD progression via ERBB2Erk signaling Therefore in order tofurther validated whether ERBB2 was the downstreammediator of circGRB10 in the NP cells We cotransfectedcircGRB10 and ERBB2 siRNA into NP cells andobserved that the positive effects of circGRB10 on NPcells functions were attenuated in the absence of ERBB2Fig 1f g Moreover upregulation of ERBB2 counteracted the inhibitory effect of circGRB10 knockdown onNP cells function Fig 1hi Collectively the aboveï¬ndings indicated that circGRB10 associated protectionin IDD may involve ERBB2Erk signalingcollagen IIand MMP13 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig circGRB10 regulates NP cell functions through ERBB2Erk signal pathway a CollagenII aggrecan MMP13 ADAMT5 expression wereanalyzed in circGRB10 or circGRB10 siRNA transfected cultured primary human NP cells using Immunoï¬uorescence staining analysis b Thecorresponding bar graphs show quantitative analysis of the relative ï¬uorescent value of each group n replicates per group p p Scale bar μm c qRTPCR showing the expression levels of collagen II aggrecan MMP13 ADAMT in human NP cells after circGRB10overexpression or knockdown Three independent experiments are presented as mean ± SEM error bars P P d The expressionlevels of CollagenII aggrecan MMP13 ADAMT5 and pErk12 were detected by western blot Quantitative analysis was shown in e and threeindependent repeats were performed in each experiment p f NP cell were cotransfected with circGRB10 and ERBB2 siRNA Western blotassay showed that ERBB2 siRNA blocked the effect of circGRB10 on CollagenII aggrecan MMP13 and ADAMT5 expression Quantitative analysis wasshown in g and three independent repeats were performed in each experiment p h NP cell were cotransfected with circGRB10 siRNAand ERBB2 Western blot assay showed that ERBB2 attenuated the effect of circGRB10 siRNA on CollagenII aggrecan MMP13 and ADAMT5expression Quantitative analysis was shown in i and three independent repeats were performed in each experiment p Key factors regulating circGRB10 formationIn this study we found a highly reverse complementarysequence nt upstream the ² splice site in intron andone nt downstream the ² splice site in intron which were named 2RC reversecomplementarysequence in intron and 6RC reverse complementarysequence in intron respectively Then wildtypesequence spanning from intron to intron of theGRB10 gene and multiple deletion constructs for circGRB10 were introduced into pcDNA31respectively Fig 2a Upon transfection the wildtypevector unlike the 2RC andor 6RC deletion constructs overexpressed circGRB10 indicating 2RCand 6RC may contain the binding sites that regulate circGRB10 biogenesis Fig 2bcould beregulated by RBPs postAs circRNAs are derived from premRNAs and circRNAstranscriptionally18212240 we hypothesized that circGBR10 ismodulated by RBPs posttranscriptionally in IDD development To identify the RBPs which might regulateGRB10 premRNA splicing to generate circGRB10 weincubated biotin labeled sequences cloned from circGRB10 back splicing site nt upstream P1 or ntdownstream P2 with nuclear protein extracts fromnormal human NP cells Fig 2c Nuclear proteins boundto RNA underwent separation by SDSPAGE and silverstaining Fig 2dfollowed by mass spectrometry foridentiï¬cation A total of proteins SupplementaryTable S1 were retrieved and mapped to the STRINGdatabase screening significant interactions with scoresabove Fig 2e Enrichment analysis demonstratedthat these proteins were mainly involved in thepathways of gene expression processing of capped introncontaining premRNA mRNA splicing mRNA splicingmajor pathway mRNA processing and formation andmaturation of mRNA transcript related signaling pathways Fig 2f Among these proteins were involved inthe mRNA splicing and mRNA splicingmajor pathwaySupplementary Table S2 In addition the web tool CircInteractome predicted RBPs which can potentiallybind circGRB10 premRNA Fig 2g Notably FUS wasOfï¬cial journal of the Cell Death Differentiation Associationthe only RBP that was involved in mRNA splicing andcould potentially bind to circGRB10 premRNA suggesting circGRB10 generation may be associated withFUS expression in NP cellsFUS promotes the generation of circGRB10 in NP cellsRecently FUS was reported to have a role in regulatingcircRNA biosynthesis via binding of introns surroundingthe backsplicing junctions31 As shown in Fig 3A FUSamounts in IDD NP tissues were remarkably lower thanthose of controls In addition Western blot further conï¬rmed this result Figs 3b and S2 To assess whetherFUS contributes to circGRB10 production in NP cells weoverexpressed or suppressed FUS and determined circGRB10 amounts qRTPCR demonstrated that FUSoverexpression led to significantly increased circGRB10amounts in NP cells while FUS knockdown reduced theexpression of circGRB10 Fig 3c Moreover FUS had noeffects on linear GRB10 expression Fig 3c Overexpression of FUS resulted in increased collagenII andaggrecan amounts and decreased MMP13 and ADAMT levels in NP cells while the circGRB10 siRNA attenuated these changes Fig 3d FUS knockdown resultedin downregulated collagenII and aggrecan and upregulated MMP13 and ADAMT5 in NP cells while circGRB10 markedly counteracted the effects of FUS knockdown indicating that FUS exerted its functions throughcircGRB10 Fig 3eTo assess whether FUSbinding sequences are important in circGRB10 biosynthesis FUSbinding sequenceswere searched in circGRB10 and surrounding intronsand two putative FUSbinding sites were detected Fig3f Next two short circGRB10 minigenes were engineeredincluding circGRB10s and circGRB10sEmPrecisely circGRB10s comprises presumed FUSbingingsites on both ï¬anking introns preserved with the inverselyinserted ²intron in circGRB10 removed to preventcomplementary sequences from reacting Fig 3f circGRB10sEm resembles circGRB10s but with FUS sitesdeleted from the surrounding introns Fig 3f RIPrevealed an overt interaction of FUS with circGRB10s 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig RBPs interact with GRB10 premRNA a A schematic drawing of four types of circGRB10 overexpressing vectors to The genomicregion for circGBR10 green bars with its wildtype ï¬anking introns black lines was inserted into the pcDNA31 expression vector 2RC and 6RCare indicated by red bars A series of deletions are indicated by black crosses to b qRTPCR showed the expression of circGBR10 aftertransfection with the four types of circGRB10 overexpressing vectors to Three independent repeats were performed in each experimentp c Schematic diagram of RNAs corresponding to different fragments of GRB10 premRNA P1 P2 produced by in vitro transcription inthe presence of biotin for RNA pulldown experiments d Silver stain acrylamide gel of total nuclear proteins before Input and after pulldown withbiotinlabeled RNA probe P1 P2 M molecular weight marker kDa e Proteins identiï¬ed from mass spectrometry were integrated to STRINGdatabase and constructed Proteinprotein interaction PPI network A densely connected module which contains proteins including FUS wasscreened from the PPI network and these proteins were participate in biological process of mRNA splicing via spliceosome f Pathways enrichmentanalysis of proteins in PPI network g RBPs which can potentially bind circGRB10 premRNAunlike circBsEm Fig 3g indicating FUS required theputative sites in surrounding introns for binding We nextexpressed circGBR10sin FUS overexpressing orknocked down NP cells and circGRB10s yielded elevated amounts of circGRB10 transcript after FUS overexpression and reduced amounts upon FUS knockdownconï¬rming FUS is important in circGRB10 biosynthesisin NP cellsFig 3h Next circGRB10sEm wasexpressed in NP cells and it yielded markedly reducedcircGRB10 amounts in comparison with circGRB10sFig 3h This indicated that the putative FUSbindingsequences in the surrounding introns were important incircGRB10 biosynthesis Taken togetherthe aboveï¬ndings demonstrated that FUS had a critical regulatoryfunction in circGRB10 biosynthesis in NP cells viabinding to recognition sites in the introns surrounding thecircGRB10forming exonsFUS in NP cells is regulated by miR1413pThe mechanism of FUS downregulation in NP cells ofIDD patients remains unclear Previous studies havedemonstrated that FUS is regulated by miRNAs in manydeseases3233 Therefore we hypothesized that FUS maybe regulated by miRNAs in NP cells Using the Targetscan Microt4 miRanda PITA and RNAhybird databasesall predicted miRNAs were retrieved and submitted toVenn analysis Fig 4a The results showed that FUS waspredicted to be regulated by miRNAs SupplementaryTable S3 including miR1413p Svetoni conï¬rmedthat miR1413p regulates FUS expression during neuraldifferentiation and Ji revealed miR1413p is associated with disc degeneration3334 Furthermore qRTPCRshowed that miR1413p was markedly upregulated in NPtissue samples from IDD cases in comparison with controlvalues Fig 4b Therefore we supposed that FUSexpression was regulated by miR1413p in NP cells Tofurther assess miR1413p interaction with FUS luciferasereporter assays were carried out Cotransfection of FUSWT wild type and miR1413p mimic in primary humanNP cells resulted in markedly decreased luciferase activityin comparison with the FUSmut mutantmiR1413pmimic cotransfection group Fig 4c d These ï¬ndingsOfï¬cial journal of the Cell Death Differentiation Associationwere further conï¬rmed at the gene and protein levels inhuman NP cells in vitro Fig 4e f pointing to FUS as amiR1413p target Then primary human NP cellsunderwent transfection with miR1413p mimic and miR1413p inhibitor and the corresponding negative controls respectively The results showed that circGBR10was significantly downregulated in cells overexpressingmiR1413p Fig 4g Conversely circGRB10 was upregulated in the miR1413p inhibitor group Fig 4gMoreover upregulation of FUS alleviated the suppressiveeffects of miR1413p on circGRB10 expression Fig 4hwhile FUS knockdown attenuated the effects of miR1413p inhibitor on circGRB10 upregulation Fig 4i Theabove results indicated that miR1413p regulates circGRB10 expression in NP cells primarily through targetingof FUSERBB2 regulates miR1413p in NP cells byphosphorylating Erk12Induced Erk12 signaling causes widespread miRNArepression via suppression of the main steps of miRNAbiogenesis4142 In this study we found decreased levels ofErk12 phosphorylation in circGRB10 or ERBB2 knockeddown NP cells Fig 1d e Previous studies demonstratedthat phosphorylated Erk12 can cause widespreadmiRNA repression through suppressing the major stepsof miRNA biogenesis41 As ERBB2 amounts werereduced in degenerative NP cells we hypothesized thatmiR1413p may be regulated by Erk12 phosphorylationin NP cells To explore this possibility we overexpressedor knocked down ERBB2 in NP cells qRTPCR resultsdemonstrated that overexpression of ERBB2 significantlydownregulated miR1413p while ERBB2 knockdownincreased miR1413p amounts Fig 5a In addition pretreatment of NP cells with the Erk12 phosphorylationinhibitor U0126 downregulated ERBB2 and attenuatedERBB2 induced phosphorylation of Erk12 decreasing theexpression of FUS Fig 5b Moreover blocking Erk12phosphorylation in NP cellssignificantly attenuatedERBB2s effects on miR1413p biogenesis Fig 5c anddecrease the expression of circGRB10 Fig 5d Collectivelythe above ï¬ndings demonstrated that ERBB2 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig FUS regulates the generation of circGRB10 in NP cells a qRTPCR showing FUS mRNA levels in normal and IDD NP tissues Threeindependent repeats were performed in each experiment p b Western blot showing FUS protein amounts were decreased in IDD NPtissues c qRTPCR analysis of circGRB10 expression level after FUS overexpression or knockdown in NP cells FUS overexpression led to significantlyincreased circGRB10 amounts in NP cells while its knockdown reduced circGRB10 levels Moreover FUS had no linear effects on GRB10 expressionThree independent repeats were performed in each experiment p d e qRTPCR analysis of the expression of CollagenII aggrecan MMP and ADAMT5 in NP cells f Schematic illustrating the putative FUSbinding sites on the ï¬anking introns in the circGRB10s minigene The ²terminus of the circular exons of circGRB10 was deï¬ned as position Putative FUSbinding sites A and B are located in the intron at the ² terminusof the circGRB10 exon position to and on the intron at the ² terminus of the circGRB10 exon position g RIP analysis ofFUSbinding to circGRB10s and circGRB10sEm minigenes in NP cells Bound complexes were pulleddown using an antibody against FUS qRTPCRwas then used to measure circGRB10s binding to FUS Values were normalized to the level of background RIP as detected by an IgG isotype controlh qRTPCR analysis of the expression of circGRB10 relative to GAPDH in NP cells Cells were cotransfected with FUS or FUS siRNA and a circGRB10minigene circGRB10s or circGRB10 minigene containing deleted FUSbinding sites circGRB10Em Quantitative data from three independentexperiments is presented as mean ± SEM error bars P P regulated miR1413p expression in NP cells by phosphorylating Erk12Next we found that decreased ERBB2 amounts indegenerative NP cells could promote miR1413p generation which suppressed the expression of FUS resultingin circGRB10 downregulation our previousstudydemonstrated that circGRB10 downregulation leads toERBB2 reduction by enhancing miR3285p mediatedsuppression of ERBB2 in NP cells15 These ï¬ndings suggested circGBR10 contributed to the molecular circuitrycontrolling IDD development in humans Fig 5ephosphorylation collagenII and aggrecan upregulationand inhibited the expression of MMP13 ADAMT5 inthe rat model of IDD Fig 6g Moreover Immunoï¬uorescence staining also conï¬rmed that the increasedexpression of collagenII aggrecan and the downregulation of MMP13 ADAMT5 expression in the circGRB10 group compared with noninjection group at weeks Fig 6h Jointly the above ï¬ndings suggested atherapeutic role for circGRB10 in protecting the discsrevealing circGRB10 as a candidate therapeutic target inIDDIntradiscal injection of circGRB10 alleviates IDD in a ratmodelNeedle puncture has been one of the most commonmethods to establish animal models of IDD4445 We havesuccessfully established a rat model of IDD by needlepuncture according to the above methods in this studySupplementary Fig S3 At and days upon modelingadenoviral human circGRB10 was injected into thepunctured intervertebral discs with 33G needles In vivoRNA FISH indicated circGRB10 in the NP region at weeks after surgery Fig 6b CT scan at and weeks revealed progressive disc space narrowing in allpunctured animals and a significant increase in DHI wasnoted at and weeks post surgery in rats treated bycircGRB10 Fig 6c CT scan revealed that overexpression of circGRB10 markedly preserved the progressive disc space narrowing in rat IDD modelFig 6dAnd safranin O staining results demonstrated that overexpression of circGRB10 can inhibit the degradation ofextracellular matrix of NP cells Fig 6e These resultssuggesting circGRB10 exerted protective effects in surgically induced IDD After injection of adenoviral circGBR10 FUS and ERBB2 amounts in degenerative NPtissues were remarkably elevated Fig 6f while miR1413p amounts were decreased Fig 6f In addition injectionof adenoviral circGBR10 alleviated the degenerativealterations ofincluding enhanced Erk12the NPDiscussionNumerous circRNAs are found in the human transcriptome playing critical roles in the regulation of cellfunctions174647 Our previous study showed that circGRB10 downregulation is associated with human NP cellapoptosis15 Howeverthe mechanism of circGRB10dysregulation in IDD has not been previously describedHere we found that FUS regulated and promoted circGRB10 biosynthesis by interacting with its ï¬ankingintrons In addition FUS expression in NP cell wasregulated by miR1413p Our ï¬ndings suggest a regulatory network in NP cells FUS bound to GRB10 premRNA to regulate circGRB10 synthesis while circGRB10 acted as a molecular sponge for miR3285p withsuppressive effects on ERBB2 production and modulatedIDD development downregulation of ERBB2 decreasedErk12 phosphorylation and promoted the generation ofmiR1413p which bound to the ²UTR region of FUS toinhibit its expression constituting a positive feedbackloop promoting intervertebral disc degenerationThe differential expression of circGBR10 betweennormal and degenerative NP tissues indicates that circGRB10 biosynthesis is controlled differently in variouscells with NP cells possessing speciï¬c factors required forcircRNA biogenesis Indeed introns and of the GRB10premRNA had binding sites to regulate circGRB10biogenesis Furthermore multiple RBPs were highlyOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see ï¬gure on previous pageFig miR1413p inhibits FUS expression in NP cells a The Venn diagram indicates the predicted miRNAs regulate FUS expression miR1413p was intersected predicted by different databases b Expression of miR1413p in IDD NP tissues showing that miR1413p expression wassignificantly higher than that of controls Quantitative data from three independent experiments is presented as mean ± SEM error bars P c Sequence alignment of a putative miR1413pbinding site within the ²UTR of FUS mRNA Bottom mutations in the ²UTR of FUS mRNAsequence to create the mutant luciferase reporter constructs d Luciferase reporter assay in NP cells after transfected with scramble oligo or miR1413p mimics Renilla luciferase vector and the reporter constructs Both ï¬reï¬y and Renilla luciferase activities are measured in the same sample Fireï¬yluciferase signals were normalized with Renilla luciferase signals Quantitative data from three independent experiments is presented as mean ± SEMerror bars P e f FUS expression level was detected by qRTPCR western blot in primary human NP cells Three independent experimentsis presented as mean ± SEM error bars P g NP cells from control tissues were transfected with miR1413p mimic or miR1413p inhibitorqRTPCR was used to detect the relative expression level of circGRB10 compared with controls Three independent experiments is presented asmean ± SEM error bars P h NP cells from control tissues were transfected with miR1413p with or without FUS overexpress plasmidqRTPCR was used to detect the relative expression level of circGRB10 compared with controls i miR1413p inhibitor with or without FUS siRNA wastransfected into NP cells from control tissues and the expression level of FUS Three independent experiments is presented as mean ± SEM errorbars P Fig ERBB2 regulate miR1413p expression in NP cells a miR1413p expression level in NP cells with ERBB2 overexpression or ERBB2knockdown Three independent experiments is presented as mean ± SEM error bars P b NP cells overexpressing ERBB2 were treated withU0126 or not for one hour Western blot was used to detect the phosphorylated level of Erk12 c d NP cells overexpressing ERBB2 were treated withU0126 or not qRTPCR was used to detect the expression level of miR1413p and circGRB10 Three independent experiments are presented asmean ± SEM error bars P e Schematic representation of mechanisms by which circGRB10 mediates IDD development On the basis ofï¬ndings described in the manuscript miR1413p downregulates FUS level in NP cells leading to circGRB10 decreased This downregulated circGRB10 in turn enhanced miR3285p mediated suppression of ERBB2 in NP cells leads to decreased Erk12 phosphorylation level And the decreasedErk12 phosphorylation level enhances the generation of miR1413p in NP cellsOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig cricGRB10 attenuates IDD development in vivo a Overview of the experimental setup with injections of circGRB10 or their negativecontrol at and days after surgery b Six weeks after surgery in vivo RNA FISH found circGRB10 located in the NP region Blue ï¬uorescence diamidino2phenylindole DAPI indicating cell nucleus Red ï¬uorescence indicating circGRB10 Scale bar μm c Changes in disc height indexDHI of the indicated groups after needle puncture The DHI was measured at and weeks A significant decrease of the DHI was observedin all puncture groups at week after surgery P A significant increase in DHI was noted at and weeks post surgery in rat treated withcircGBR10 P d CT scan of the indicated groups were obtained weeks after needle puncture e The intervertebral disc degenerationevaluated by Safranin O staining Scale bar µm f qRTPCR showed that the increased levels of FUS ERBB2 and the decreased level of miR1413p in the punctured discs treated with circGRB10 Three independent experiments are presented as mean ± SEM error bars P g Westernblot showing the expression levels of collagen II aggrecan pErk12 MMP13 ADAMT5 in rat NP tissues h Immunostaining for collagenII andaggrecan in IDD model treated by circGRB10 at and weeks Scale bar µmOfï¬cial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of enriched in circGRB10s ï¬anking introns and FUS contributed to circGRB10 biogenesis as shown aboveAlthough FUS induced circGRB10 biosynthesisitssilencing only decreased circGRB10 levels by asshown above It is known that two or more RBPs couldthe synthesis of circRNAs synergistically4048controlwhich might explain the above incomplete suppressionTherefore circGRB10 modulation in NP cells deservesfurther examinationAs shown above altered FUS expression might profoundly affect circGRB10 biogenesis Further deletion ofFUSbinding sequences in the introns ï¬anking of circGBR10 dramatically reduced circGRB10 amounts Takentogether the above ï¬ndings indicate FUS may directlycontrol backsplicing to upregulate circGRB10 in NPcells by interacting with putative binding sequences onboth ï¬anking introns of circGRB10Recently miR141 has been detected in NP tissuesamples from IDD cases and its levels correlate with discdegeneration Therefore miR141 NPs have been used inIDD treatment with commendable efï¬cacy34 As shownabove miR1413p which is a key regulator of IDDdirectly regulated FUS further revealing the FUScircGRB10 axis as an IDDrelated regulatory pathwayAccumulating evidence indicates that Erk signaling hasan important role in IDD394950 In the present study wefound that circGRB10 significantly upregulated collagenII and aggrecan in NP cells and mediated the protectiveeffects in IDD likely via ERBB2Erk signaling There mightalso be cellular pathways that compensate for ERBB2expression after its knockdown For example the longintergenic noncoding RNA lincRNA BCLIN25 upregulates ERBB2 by inducing promoter CpG methylation ofmiR125b resulting in miR125b repression44 A previousstudy indicated the Erk pathway regulates the miRNAgenerating complex43 In additi | 2 |
RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomens Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the seed and soil theory was ï¬rst proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells seeds leave their primary site of origin and spreador metastasize the microenvironment soil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve assoil for tumor development and might also serve as a seed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on theï¬eld of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identiï¬ed receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieï¬yintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and eï¬cienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentiï¬ed as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong TDC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deï¬ciency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKLRANK interactionsand blocking bone resorption The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identiï¬edas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a speciï¬c fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The eï¬cacy of Denosumab has been conï¬rmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspeciï¬c Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeï¬cient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyers plaque structures Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeï¬cient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inï¬ammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL speciï¬cally in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinï¬ammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a signiï¬cant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespeciï¬c deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspeciï¬c rankldeï¬cient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deï¬ciency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a speciï¬c antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to speciï¬cally eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the ï¬rst to reportthat during pregnancy RANKL deï¬ciency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκBcyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and ampliï¬edproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also signiï¬cantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identiï¬ed SNPs which were signiï¬cantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in signiï¬cantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Ampliï¬cation of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also signiï¬cantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which speciï¬cally inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will beneï¬t notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was signiï¬cantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no signiï¬cant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinï¬ltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand signiï¬cantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports ï¬ndings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thismicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas ï¬broblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a vicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs ï¬broblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer prostate cancer and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANKs cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinï¬ltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are signiï¬cantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a signiï¬cantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassigniï¬cantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs Moreover the beneï¬t of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the vicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a signiï¬cantimprovement in overall survival In these patients nostatistically signiï¬cant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab signiï¬cantly improved progressionfree survivalPFS Whether this survival beneï¬t is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the ï¬rst clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a signiï¬cant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ··] p A median followup of months showeda signiï¬cant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI Cox p These data suggest that adjuvant Denosumabcan signiï¬cantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratiï¬cations eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial conï¬rmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso signiï¬cantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment signiï¬cantly reduced the time to bonemetastasis at the site of ï¬rst occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe ï¬eld of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the bodys own immunesystem to seek out speciï¬cally target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as | 2 |
"increasing number of studies have focused on the extragastrointestinal effects ofHelicobacter pylori H pylori including metabolic syndrome fatty liver and rheumatic and skin diseasesOsteoporosis is an asymptomatic disease that can eventually lead to fractures and has a significant impact on thequality of life of elderly individuals Sex is an influential factor that plays a crucial role in the development ofosteoporosis The aim of this study was to investigate the relationship between H pylori infection and osteoporosisand to identify potential influencing factorsMethods We conducted a crosssectional study of individuals older than years old who had undergone regularphysical examinations at the Beijing Shijitan Hospital Health Examination Center from July to October Weevaluated the associations of oste ia and osteoporosis with H pylori infection and related serum markers byusing multiple linear regression and logistic regression Then we analysed the correlation between sex andpotential serum biomarkersResults There were significant relationships between H pylori infection status and bone density in premenopausalfemales but not in males P according to Fishers exact test In females H pylori positivity OR P Body Mass Index BMI OR P and homocysteine HCY OR P wereassociated with osteoporosis Calcium had a trend but no statistically significant OR P relationshipwith osteoporosis Furthermore the waisttohip ratio OR P BMI OR P andtriglyceride levels OR P were significantly different by sex after adjusting for age as a confounderConclusion H pylori positivity BMI and HCY are associated with osteoporosis in premenopausal females Chronicinflammation may be involved in the relationship between H pylori and osteoporosisKeywords Female H Pylori infection Osteoporosis Premenopausal Chronic inflammationBackgroundHelicobacter pylori H pylori a gramnegative spiralshaped microaerophilic bacterium has been shown to bean important pathogen in gastrointestinal diseases []Approximately of the world population has been Correspondence maggie19950426163com3Department of Gastroenterology Beijing Shijitan Hospital Capital MedicalUniversity Beijing Beijing ChinaFull list of author information is available at the end of the affected by H pylori and approximately millionChinese individuals are affected by this disease It maycause chronic inflammation of the gastric mucosa whichmay lead to chronic atrophic gastritis peptic ulcer diseases and gastric cancer [ ] Moreover the latest reports have described the investigation ofthe extragastrointestinal effects of H pylori including metabolicsyndrome [] fatty liver [] and rheumatic and skin diseases [] These parenteral diseases associated with H The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cWang BMC Musculoskeletal Disorders Page of pylori infection seriously affect the patients general condition and cause a series of complicationsOsteoporosis is an asymptomatic disease characterizedby a decreased density of normally mineralized bone thatusually occurs in elderly persons It has been reportedthat approximately million men and million womenover the age of in the United States have been diagnosed with osteoporosis with an estimated millionsuffering from oste ia [] In the development ofosteoporosis there is often a long latent period beforethe appearance of the main clinical manifestation pathologic fractures Moreover the most prevalent sequelae ofosteoporosis are compression fractures of the vertebralbodies and fractures of the ribs proximal femurs humeri and distal radiuses which could have a significantimpact on the quality oflife of elderly individualsTherefore the prevention and early detection of osteoporosiselderlypopulationis particularlytheimportantforA majority of studies support the idea that at anygiven age women have a higher risk of fracture thanmen [] However men tend to have worse outcomesafter fracture than women they are twice as likely to dieafter a hip fracture than women [] Moreover sexrelated factors remain unclear Therefore exploring thedifferences in factors influencing osteoporosis in malesand females will be helpful to explore the pathogenesisof osteoporosis and early prevention of its occurrenceand developmentThere are some wellestablished risk factors for theemergence of osteoporosis such as age sex body massindex and alcohol consumption [] Recent shave focused on H pylori and osteoporosis Different researchers have proposed different theories including butnot limited to inflammation induced by H pylori infection [] and malabsorption of nutrients [ ] However there is still a lack of systematic data analyses onthe relationship between H pylori and osteoporosis Theassociation between osteoporosis and H pylori has beenstudied by many Japanese scientists and remains controversial In addition there are still some deficiencies inexisting research Few studies have focused on the correlation between H pylori and osteoporosis in Chinesepremenopausal females The sample sizes have been insufficient there is a lack of investigations on the influence of sex and there is a lack of sufficient serummarkers The aim of this study was to investigate the relationship between H pylori and osteoporosis and toidentify potential influencing factorsMethodsStudy populationBriefly men and women older than years old ienot including those aged years old who had regularphysical examinations at the Beijing Shijitan HospitalHealth Examination Center from July to October were included Our research used the following exclusion criteria for the data collection periods patientsusing the following drugs and having comorbidities thatmay cause secondary osteosis glucocorticoids thyroidparathyroid drugs psychotropic drugs anticonvulsantsselective estrogen receptor modulators SERMs vitaminD calcium and bisphosphonate patients who had ahistory of gastrectomy inflammatory bowel disease malignant diseases chronic kidney disease diabetes mellitushypohyperparathyroiddisorder acromegaly and rheumatoid arthritis includingcollagen disease Study participants who had been diagnosed with H pylori infection before or had potentiallyantiH pylori drugs in the past month were recruitedas wellhypohyperthyroidismWe also excluded postmenopausal women in thepresent study The criteria for determining menopausebased on the latest guidelines included any of the following prior bilateral oophorectomy age ¥ years old age years and amenorrhoeic for ormore months in the absence of chemotherapy tamoxifen or toremifene [] The female study participantswere not pregnant or lactatingData collectionAmong all the eligible individuals eligible study participants gave informed verbal consent and providedtheir basic informationincluding demographics agesex race smoking status and medicine use All ethicsapprovals were given by the Ethics Committee of BeijingShijitan Hospital affiliated with Capital Medical University and the study was performed in accordance withthe Declaration of Helsinki Blood measurements wereperformed with fresh serum obtained after a 12h fast tominimize the confounding effects of diurnal variation onhormone concentrations and included tests for glucosemetabolism liver function renal function lipid metabolism ions calcium iron tumour markers pepsinogenPG and progastrinreleasing peptide proGRP Anthropometric measurements including waist circumference cm blood pressure mmHg body weight kgand height cm were measured by trained nurses usinga standardized protocol Diastolic and systolic bloodpressure were measured in the morning Body massindex BMI was calculated by taking a persons weightin kilograms divided by their height in metres squaredThe waisttohip ratio WHR is measured as waist circumference divided by hip circumference H pylori infection status was measured by a []C breathing test onthe same day with an empty stomach Tumour markerswere measured using enzymelinked immunosorbentassay methods 0cWang BMC Musculoskeletal Disorders Page of Diagnosis of osteoporosisThe bone mineral density BMD of lumbar vertebrae L2 was measured by DXA using a DiscoveryDXA system Hologic Bedford Massachusetts The results provided BMD gcm2 and young adult meanbone mineral density The diagnosis of osteoporosis wasperformed in accordance with the World Healthanization diagnostic criteria from the World Healthanization WHO Collaborating Center for Metabolic Bone Diseases [] A value for BMD within onestandard deviation SD of the average BMD of normaladults was regarded as normal Oste ia is defined asa BMD that lies between and standard deviationsbelow the young adult mean value BMD more than SD below the young adult mean value was classified asosteoporosis []Statistical analysesWe used SPSS statistical software version for dataanalyses Continuous variables were reported as means ±standard deviations whereas categorical variables werepresented as percentages Study subjects were first classified into three groups according to BMD classificationnormal oste ia and osteoporosis The KolmogorovSmirnov test was used to verify whether the data fit anormal distribution and all continuous variables thatdid not conform to a normal distribution underwenttransformation for analysis Summary and grouping datafor baseline characteristics the laboratory examinationwere compared using a t test for continuous variablesand Fishers exact test for categorical variables in the Hpylori and H pylori groups Moreover we divided thestudy population by sex and used Fishers exact test toverify whether there were sex differences in the relationship between H pylori and osteoporosisMultivariateadjusted odds ratios ORs and confidence intervals CIs were calculated using logistic regression among the three subgroups To further analysethe relationship between H pylori infection status andosteoporosis we created a model using total cholesterolTC triglycerides TG uric acid UA BMI WHRlowdensity lipoprotein cholesterol LDLC Creactiveprotein CRP homocysteine HCY H pylori infectionstatus calcium Ca vitamin B12 and the BMD groupsthat analysed the different sexes separately The studypopulations highdensity lipoprotein cholesterol HDLC and glucose levels were all normal so we did not include them in the modelMeanwhile we analysed the differences in markers between males and females We separated the study population according to sex and further analysed the patientsbasic data in the same way as we analysed the baselinecharacteristics We further analysed the relationship between sex and markers to find sex differences in therelationship between H pylori infection and osteoporosis The markers included TC LDLC UA TG glucoseGLU CA724 CEA BMI SP systolic blood pressureBP diastolic blood pressure WHR HCY CRP vitaminB12 Ca and Ghb Other serum biomarkers were entered into the model as factors using their normal valueas the grouping criterion All the models were adjustedfor age as a confounder A twotailed Pvalue wasconsidered to be statistically significantResultthe participants areThe baseline characteristics ofshown in Table The mean age was ± years were male and were femaleAmong the study population had osteoporosis had oste ia and hadnormal BMDs The mean ages ofthe osteoporosisoste ia and normal groups were ± ± and ± respectively There wereno significant differences in age among the participantsin the osteoporosis oste ia and normal BMD groupsFifty percent of the males had normal BMDs ofthe males had oste ia and of the males hadosteoporosis In addition of the females had normal BMDs of the females had oste ia and of the females had osteoporosis There was no significant difference in sex distribution among these threegroupsAmong all the study participants had H pyloriinfections and did not have H pylori infectionsTable shows that CA724 t P was significantly different between the study participants withH pylori infection and those without H pylori infectionand there were no significant differences between the Hpylori infection status and the BMD status groupsIn Table we separate the study population accordingto sex We found that there was a significant relationshipbetween H pylori infection status and bone density infemales P but not in males P As shown in Table there was a significant association of H pylori positivity OR CI P BMIOR CI P and HCY OR CI P with osteoporosis inpremenopausalOR CI P and TC OR CI P had a trend but neither wassignificantly associated with osteoporosisfemales CaMeanwhile we analysed the differences in markers between males and females which are shown in Table We found that age P SP P BP P P WHR P Hb P BMI platelets P UA P TC P TG P HDLC P GLU P 0cWang BMC Musculoskeletal Disorders Page of Table Baseline Characteristics of the patients according to the H pylori infection statueFemaleMalenormaloste iaosteoporosisAgeSexBMDSP mmHgBP mmHgBMI kgm2WHRHbgLPlatelet109LCa mmolLUA umolLTC mmolLTG mmolLHDLC mmolLLDLC mmolLGLU mmolLCEA ngmlCA724UmlGhbIron umolLCRP mgLHCY umolLTotal ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± H pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± H pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PvalueB12pmolLWHR Waisttohip ratio TC Total cholesterol Ghb Glycosylated hemoglobin TG Triglyceride UA Uric acid AST Aspartate aminotransferase ALT Alanineaminotransferase Ca Calcium Cre Creatinine DP Diastolic blood pressure Hb Hemoglobin HDLC Highdensity lipoprotein cholesterol LDLC Lowdensitylipoprotein cholesterol BMD Bone mineral density OR Odds ratio proGRP Progastrinreleasing peptide WHR WaisttoHip Ratio SP Systolic blood pressure PGPepsinogen GLU Glucose CRP Creactive protein HCY HOMOCYSTEINEBold indicates statistically significant values ± ± ± Table the relationship between the H pylori infection andthe BMD in different genderFemaleH pylori infection H pylori infection Normal BMDoste iaosteoporosisMaleH pylori infection H pylori infection Normal BMDoste iaosteoporosisBMD Bone mineral densityBold indicates statistically significant valuesPvaluePvalue CEA P Ghb P iron P HCY P and B12 P demonstrated significant differences between males and femalesThe relationship between sex and markers is shown inTable We found that WHR OR CI P TG OR CI P and BMI OR CI P were significantly different bysex adjusting for age as a confounderDiscussionOsteoporosis is an important health and societal burdenin elderly people not only in females but also in malesThere are numerous osteoporosisrelated fracture riskfactors including age sex race lifestyle and concomitant medical conditions [] In men osteoporosis isunderrecognized and undertreated Only a few men are 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and BMDOR95CIPvalueTC mmolLQ1365Q2521BMI kgm2Q1179Q2240UA umolLQ1187Q2358TG mmolLQ10Q2171LDLC mmolLQ10Q2313C13Q1without H pylorQ2with H pylorWHRQ1079Q2085HCYQ10 μmolLQ2 μmolLCRPQ1 mgLQ2 mgLCaQ1 mgLQ1 mgLB12Q10516pmolLQ2 pmolLAbbreviations as in Table OR Odds ratio C13 13C breathing test positiveBold indicates statistically significant valuesscreened for osteoporosis even after a fracture [] Thetreatment rate is much lower in males than in females[] Meanwhile more men than women die every yeardue to hip fractures [] Hence we also included menin the study population to determine the risk factors forosteoporosisSome studies about the influence of sex on osteoporosis remain controversial In our study there was a significant relationship between H pylori and osteoporosisin premenopausal females but not in males The reasonsfor the difference between males and females are asfollows First differences in clinical outcomes of osteoporosis in men and women may be rooted in the biologic properties of bone BarrettConnor E holds theview that there are sexspecific differences in the number of osteoprogenitor cells and in hormone responsesand regulation [ ] Second men have a greater bonesize trabecular BMD and bone area at the radius andtibia than women even after adjusting for weight andheight which may lead to a decrease in osteoporosis andfracture [] Third men undergo a slow decrease inBMD with increasing age while women experience aprofound period of rapid bone resorption especiallyafter entering menopause [] Last but not least somestudies support the idea that men are more likely to suffer from secondary diseasefor example rheumatoidarthritis alcoholism excessive smoking gonadal deficiencies and others [] which may lead to sustainablebone lossUnfortunately the relationship between osteoporosisand H pylori infection is still controversial Some studieshold the view that there is no difference between menand women in the relationship between H pylori andosteoporosis [ ] Some studies hold the view that Hpylori is related to osteoporosis only in women ShihChun Lin conducted a retrospective study including women and showed that H pylori is related to osteoporosis in females [] while others think that there is nocorrelation between them in females Daisuke Chindaconducted a study of healthy women and found thatH pylori is not a significant risk factor for oste ia[] In our study we analysed the relationship betweenH pylori infection and osteoporosis We found a significant relationship between H pylori infection status andbone density in premenopausal females but not in malesWe suspect this may be due to the difference in the aetiology of osteoporosis between males and females However we did not find any other studies on this and itrequires more systematic research for analysisAfter analysing the differences between males and females we found that there were significant differencesin BMI WHR and TG in the study population Thisstudy provides evidence for followup research on sexdifferences in the relationship between H pylori andosteoporosisMost studies hold the view that obesity is related toosteoporosis [] It is generally believed that obesitymay be a protective factor against bone loss and osteoporosis [] However the effect of obesity remains unclear On the one hand obesity has traditionally beenconsidered positive for bone because of the beneficial effect of mechanicalloading [] On the other handpeople hold the view that BMI may harm BMD Osteoblasts and adipocytes both stem from marrow mesenchymal stromal cells Osteoblasts and adipocytes are in a 0cWang BMC Musculoskeletal Disorders Page of Table Baseline Characteristics of the patients in different genderAgeSP mmHgBP mmHgBMI kgm2WHRHbgLPlatelet109LCa mmolLUA umolLTC mmolLTG mmolLHDLC mmolLLDLC mmolLGLU mmolLCEA ngmlCA724UmlGhbIron umolLCRP mgLHCY umolLTotal ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± B12pmolLAbbreviations as in Table OR Odds ratioBold indicates statistically significant values ± Female ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Pvaluecompetitive relationship and an increase in adipocyteswill inhibit osteoblasts [] In our study there was asignificant relationship between BMI and osteoporosisIncreased BMD levels in obese people may be associatedwith increased mechanical loading and strain this is acomplicated problem that cannot be generalizedIn our study we found that H pylori infection is associated with a decrease in bone density The possible reasons are as follows First H pylori infection may causesystemic inflammation and increase the production oftumour necrosis factorα interleukin1 and interleukin [] These cytokines are directly involved in the reduction of BMD We found that HCY is related toosteoporosis which supports this hypothesis Secondosteoporosis may be related to a decrease in vitamin B12levels [] Serin et als study examined patientswithout atrophy erosions or ulcers and they found thatthe histopathological scores for both antral and corpusH pylori density and inflammation were significantly inversely associated with serum vitamin B12 levels [] Inour study although we did not find a significant relationship between B12 and osteoporosis we still supportthe relevant theory The absence of our results may bedue to a lack of sufficient data and the influence of confounding factors Last but notleast most patientschronically infected with H pylori manifest pangastritiswith reduced acid secretion due to bacterial virulencefactors inflammatory cytokines and various degrees ofgastric atrophy [] Calcium is ionized in acidic conditions and absorbed in the small bowel Therefore in either hypochlorhydria or achlorhydric stomachs calciumabsorption is impaired [] Moreover the longtermuse of acid suppressants for example proton pump inhibitors may lead to osteoporosis or a decrease in BMDLimited experimental evidence indicates that PPI mayinfluence calcium absorption leading to compensatoryphysiologic responsesincluding secondary hyperparathyroidism which may cause an increase in the rate ofosteoclastic bone resorption [] The results showedthat calcium had a trend though it was not statisticallysignificant P with osteoporosis Our results donot support the theory that there is a correlation between Ca and osteoporosis but it may be that Helicobacter pyloriinfection may cause calcium absorptiondamage and affect BMD We did not analyse vitamin Dlevels which could affect both bone homeostasis and theinflammatory state [] Although H pyloriinfectioncausing a decrease in bone density is supported by mostresearchers the effect of early eradication therapy is stillinsufficient Replogle ML holds the view that early 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and genderWHRQ1079Q2085TC mmolLQ10Q2 LDLC mmolLQ10Q2313UA umolLQ1187Q2358TG mmolLQ10Q2171GLU mmolLQ10Q2611CA724UmlQ10Q2690CEA ngmlQ10Q2500BMI kgm2Q1179Q2240SP mmHgQ190Q2 BP mmHgQ160Q2 HCY μmolLQ10Q2 CRP mgLQ1 Q2 B12pmolLQ10Q2 Ca mgLQ1 OR95CI Pvalue 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and gender ContinuedQ1 GhbQ10Q261OR95CIPvalueAbbreviations as in Table OR Odds ratioBold indicates statistically significant valuesMale and female have different normal value in UA and WHR UA 149416umolL in male89357umolL in female WHR in male in femaleeradication therapy may eliminate chronic inflammationfrom H pylori [] Some s have also reported animprovement in B12 levels after complete eradication[ ] which requires further investigationDespite its relevant findings our study had several limitations First because most patients cannot rememberthe time of HP infection accurately we were not able toobtain the time of HP infection so different infectiontimes may have had an impact on the results Secondwe did not collect vitamin D data the sample size of ourdata was not large enough and the study populationonly included participants from Beijing Shijitan Hospitalmeaning that there might have confounding factors because of differences in the distribution of hospital studypopulations Further largescale studies in the generalpopulation are needed to validate our results Third thestudy participants were all Chinese and the findingsmight not be generalizable to other ethnic populationsIn addition we only found some differences betweenmen and women but failed to further explore themConclusionsH pyloriis associated with osteoporosis in premenopausal females BMI and HCY are related to osteoporosis in premenopausal females Chronic inflammationmay be involved in the relationship between H pyloriand osteoporosisSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12891020035867Additional file AbbreviationsDXA Lumbar dualenergy xray absorptiometry ORs Odds ratiosCIs Confidence intervals H pylori Helicobacter pylori SERMs Selectiveestrogen receptor modulators proGRP Progastrinreleasing peptideBMI Body mass index WHR Waisttohip ratio BMD Bone mineral densityWHO World health anization SD Standard deviation TC Totalcholesterol TG Triglycerides UA Uric acid LDLC Lowdensity lipoproteincholesterol Ghb Glycosylated haemoglobin HDLC Highdensity lipoproteincholesterol GLU Glucose PG Pepsinogen CA Calcium DP Diastolic bloodpressure Hb Haemoglobin SP Systolic blood pressure C13 13C breathingtest positiveAcknowledgementsNot applicableAuthors contributionsLH has made substantial contributions to the design of the work SSJ hasprovided the data obtained the consent of participants and analyzed thedata preliminarily ZLC has made contributions to the collection of data andparticipated in the drafting of the DFX has analyzed and interpretedof the data WJW has drafted the manuscriptSH has helped to revise themanuscript All authors have read and approved the manuscriptFundingThe study was supported by a program from the Beijing City Health System High Levels of Health Technical Personnel Training Aid and the CapitalClinical Characteristic Applied Research Project NoZ181100001718120Funds are used for the data collection portionAvailability of data and materialsThe datasets analyzed during the current study are not publicly availablebecause it includes the study population personal information which isillegal to but are available from the corresponding author onreasonable requestEthics approval and consent to participateAll the ethics approval has been given by the ethics committee of Beijingshijitan hospital affiliated to capital medical university and have beenperformed in accordance with the Declaration of Helsinki We used theparticipants data by anonymous All involved study populations were fromthe previous physical examination group and part of the population are notin Beijing All the participants received the informed consent by email Weread informed consent to patients or their immediate family members bytelephone and inform them of the purpose and significance of the studyand obtain their oral consent which is approved by the ethics committeeConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests in this sectionAuthor details1Department of Gastroenterology Beijing Shijitan Hospital Capital MedicalUniversity No Tieyi Road Beijing China 2Department of Physicalexamination center Beijing Shijitan Hospital Capital Medical University No Tieyi Road Beijing China 3Department of Gastroenterology BeijingShijitan Hospital Capital Medical University Beijing Beijing ChinaReceived March Accepted August ReferencesGlaser DL Kaplan FS Osteoporosis Definition and clinical presentationSpine Phila Pa Suppl12s6s Malfertheiner P Megraud F O'Morain CA Atherton J Axon AT Bazzoli F Management of Helicobacter pylori infectionthe Maastricht IVFlorence consensus report Gut 0cWang BMC Musculoskeletal Disorders Page of Matsuhisa T Aftab H Observation of gastric mucosa in Bangladesh the Pan BL Huang CF Chuah SK Chiang JC Loke SS Relationship betweenHelicobacter pylori infection and bone mineral density a retrospectivecrosssectional study BMC Gastroenterol GÅogowskaSzelÄ
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" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | 0 |
inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure oï¬ce of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reï¬ect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing ï¬nancialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some ï¬gures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentiï¬ed the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspeciï¬c splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to diï¬cultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the ï¬nalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center childrens hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteï¬anked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of speciï¬c assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by ï¬rst replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was ampliï¬ed from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr ampliï¬cation ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest ï¬anking genes b mouse borcs7as3mt locus with ï¬anking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the ï¬nal borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by ï¬ushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage ï¬uid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo ï¬broblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout¢ ko medium cat no supplementedwith esqualiï¬ed fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after d in culture when the inner cell mass of the embryosreached ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualiï¬ed fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin¢ ï¬nal concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells from¼ colonies were pooled and used for rna isolation to conï¬rmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to conï¬rm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modiï¬ed bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by ampliï¬cation of the pfloxxerx plasmid previously assembled in kollers laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel puriï¬ed on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpuriï¬ed qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturers directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modiï¬ed bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene ï¬anked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasï¬anked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the ï¬rst step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene ï¬anked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psï¬ijt15neojtz17sï¬i previously assembled in kollerslaboratory see table s4 by digestion with sï¬i neb cat nor0123s the blackred homology arm was ampliï¬ed primers and using the bmq455l15 bac as a templateand the purple homology arm was ampliï¬ed primers and using the rp11753c18 bac as a template all fragments were gel puriï¬ed as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column puriï¬ed as described above theloxpï¬anked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel puriï¬edand then combined in an overlap pcr primers and the resulting pcr product was gel puriï¬ed and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modiï¬ed rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturers instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturersrecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft¢ softwareversion a portion of each of the es colonies ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientiï¬c cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientiï¬c cat no volvol triton¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturers suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a ï¬nal 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentiï¬ed and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieï¬ycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturers instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identiï¬edby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a ï¬nal extension of min the f2generationconsisting of hshs homozygotes hswt heterozygotes and wtwt homozygoteswas produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghankss balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbeccos modiï¬ed eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aï¬amedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a ï¬amedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were ï¬rst homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the speciï¬cmethod is indicated in each ï¬gure legend ddpcr is minimallyinï¬uenced by diï¬erences in the eï¬ciency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantiï¬cation of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the ï¬rst h buthad free access to puriï¬ed laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the puriï¬ed dietand diw for wk to allow for clearance of as from the bodywhich was conï¬rmed by measuring total as tas levels inurine see the results section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacriï¬ced by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were ï¬ashfrozen in dryice and stored at c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacriï¬ce ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjemstyle tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the eï¬ciencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukeys multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice students ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was | 0 |
"human FTSJ2 and porcine FTSJ2. (A) The protein structure of E. coli RrmJ which was resolved by B¼gl et al. (2000) (PDB ID: 1EIZ) [7]. (B) The protein structure of human FTSJ2 which was resolved by Wu et al. (2009) (PDB ID: 2NYU) [36]. (C) The protein structure of porcine FTSJ2 which was predicted using the SWISS-MODEL website with human FTSJ2 as a template. The ?-helices and ?-strands are shown in green and yellow respectively. The SAM residues and the K-D-K-E catalytic center are shown in the ball and stick representations respectively. (TIF) Click here for additional data file. Figure S2 Porcine Ftsj2 mRNA expression in porcine tissues. (A) Expression of porcine Ftsj2 mRNA as measured by semi-quantitative RT-PCR. Porcine ?-actin mRNA was used as a loading control. (B) Quantification of the porcine Ftsj2 mRNA expression which normalized to the ?-actin mRNA expression. The values are equal to?=?the means of duplicate experiments. (TIF) Click here for additional data file. The authors would like to thank Dr. Jeremy J.W. Chen for providing the CL1-0 and CL1-5 cell lines. We also like to thank our colleagues (Drs. Tung-Chou Tsai Yu-Tang Tung and Zi-Lun Lai) in the Molecular Embryology and DNA Methylation Laboratory for their help with discussions and technical issues. References 1 AngM LiberekK SkowyraD ZyliczM GeopoulosC (1991) Biological role and regulation of the universally conserved heat shock proteins. J Biol Chem266: 24233242361761528 2 BenjaminIJ McMillanDR (1998) Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res83: 1171329686751 3 RichmondCS GlasnerJD MauR JinH BlattnerFR (1999) Genome-wide expression profiling in Escherichia coli K-12. Nucleic Acids Res27: 3821383510481021 4 OguraT TomoyasuT YukiT MorimuraS BeggKJ et al (1991) Structure and function of the ftsH gene in Escherichia coli. Res Microbiol142: 2792821925026 5 CaldasT BinetE BoulocP CostaA DesgresJ et al (2000) The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase. J Biol Chem275: 164141641910748051 6 LapeyreB (2004) Conserved ribosomal RNA modification and their putative roles in ribosome biogenesis and translation. Curr Genet12: 263284 7 B¼glH FaumanEB StakerBL ZhengF KushnerSR et al (2000) RNA methylation under heat shock control. Mol Cell6: 34936010983982 8 BlanchardSC PuglisiJD (2001) Solution structure of the A loop of 23S ribosomal RNA. Proc Natl Acad Sci USA98: 3720372511259644 9 CaldasT BinetE BoulocP RicharmeG (2000) Translational defects of Escherichia coli mutants deficient in the Um(2552) 23S ribosomal RNA methyltransferase RrmJ/FTSJ. Biochem Biophys Res Commun271: 71471810814528 10 FederM PasJ WyrwiczLS BujnickiJM (2003) Molecular phylogenetics of the RrmJ/fibrillarin superfamily of ribose 2?-O-methyltransferases. Gene302: 12913812527203 11 PintardL LecointeF BujnickiJM BonnerotC GrosjeanH et al (2002) Trm7p catalyses the formation of two 2?-O-methylriboses in yeast tRNA anticodon loop. EMBO J21: 1811182011927565 12 PintardL BujnickiJM LapeyreB BonnerotC (2002) MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase. EMBO J21: 1139114711867542 13 BonnerotC PintardL LutfallaG (2003) Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2?-O-ribose methylation of a conserved rRNA position in yeast. Mol Cell12: 1309131514636587 14 KresslerD RojoM LinderP CruzJ (1999) Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae. Nucleic Acids Res27: 4598460810556316 15 LapeyreB PurushothamanSK (2004) Spb1p-directed formation of Gm2922 in the ribosome catalytic center occurs at a late processing stage. Mol Cell16: 66366915546625 16 PintardL KresslerD LapeyreB (2000) Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro. Mol Cell Biol20: 1370138110648622 17 HagerJ StakerBL B¼glH JakobU (2002) Active site in RrmJ a heat shock-induced methyltransferase. J Biol Chem277: 419784198612181314 18 YangD OyaizuY OyaizuH OlsenGJ WoeseCR (1985) Mitochondrial origins. Proc Natl Acad Sci USA82: 444344473892535 19 FreudeK HoffmannK JensenLR DelatyckiMB des PortesV et al (2004) Mutations in the FTSJ1 gene coding for a novel S-adenosylmethionine-binding protein cause nonsyndromic X-linked mental retardation. Am J Hum Genet75: 30530915162322 20 CampbellJM LockwoodWW BuysTP ChariR CoeBP (2008) Integrative genomic and gene expression analysis of chromosome 7 identified novel oncogene loci in non-small cell lung cancer. Genome51: 1032103919088816 21 MorelloLG ColtriPP QuaresmaAJ SimabucoFM SilvaTC (2011) The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing. PLoS One6: e2917422195017 22 SimabucoFM MorelloLG Arag£oAZ Paes LemeAF ZanchinNI (2012) Proteomic characterization of the human FTSJ3 preribosomal complexes. J Proteome Res11: 3112312622540864 23 WangH BoisvertD KimKK KimR KimSH (2000) Crystal structure of a fibrillarin homologue from Methanococcus jannaschii a hyperthermophile at 1.6 A resolution. EMBO J19: 31732310654930 24 HodelAE GershonPD QuiochoFA (1998) Structural basis for sequence-nonspecific recognition of 5?-capped mRNA by a cap-modifying enzyme. Mol Cell1: 4434479660928 25 VidgrenJ SvenssonLA LiljasA (1994) Crystal structure of catechol O- methyltransferase. 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Wntcateninmediated signaling is a key pathway regulating tissue growth and development and tumorigenesis and has received increasing attention in recent years In addition to participating in healthy tissue and an development ectopic activation of the pathway can cause a variety of tumors and other pathologies The pathway plays a critical role in many processes such as proliferation differentiation apoptosis migration invasion epithelialmesenchymal transition and cancer cell stemness The importance of the Wnt signal is selfevident This review describes the underlying mechanism of Wnt signaling pathway and highlights the latest findings on the relationship between Wnt signaling pathway and tumorigenesis In addition the potential relationship between miRNAs and Wnt signaling is presented Furthermore we discuss the intrinsic link between Wnt signaling and cancer cell stemness which shed light on the malignant progression of tumor cells Finally cancer treatment strategies based on the canonical Wnt signaling pathway are summarized hoping to help clinical development Keywords Wntcatenin tumor miRNAs cancer stem cell target therapeutic strategyIntroductionWnt signaling pathway plays a crucial role in embryonic development adult tissue homeostasis and cancer1 Due to the role in cell fate and tissue development increasing attention has been paid to Wnt signaling pathway in regenerative medicine2 Moreover the pathway is also involved in many pathological processes such as proliferation differentiation apoptosis migration invasion epithelial mesenchymal transition EMT and cancer cell stemness Notably the aberrant activation of Wntcatenin signaling results in the expression of several modulators that antagonize the antitumor activity of T cells which might lead to the failure of cancer immunotherapy3 Since Wnt signaling plays a pivotal role in tumorigenesis it is a promising target for the development of cancer therapeuticsSince the discovery about years ago many reports on the relationship between Wnt signaling and malignant tumor progression have been published The Wnt family consists of cysteinerich protein species which deliver canonical Wnt signaling through the interaction with Frizzled FZD and coreceptor low density lipoproteinrelated receptors and LRP56 thereby regulating cell fate growth and tissue repair4 After a series of posttranslational modifications such as porcupine palmitoylation lipid modification and glycosylation in the endoplasmic reticulum the Wnt precursor gradually maturates into the functional Wnt ligand Then the transmembrane protein Wntless Wls binds Wnt ligand and reaches to Cancer Management and Research Wen This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphp and incorporate the Creative Commons Attribution Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the work you hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWen Dovepressthe plasma membrane via the Golgi apparatus Finally Wnt is detached from the cell membrane as components of exosomes or lipid protein ps15 As shown in Figure Wnt ligandbinding membrane receptor complex induces constitutive activation of Wntcatenin signaling which delivers downstream signaling LRP receptor phosphorylation recruits Axin to degrade Dishevelled DVL protein activation leads to inactivation of destruction complex which leads to the stability and accumulation of catenin6 Subsequently catenin is translocated into nucleus and interacts with the transcription coactivators like Tcell factor TCF and lymphoid enhancer factor LEF proteins to regulate the expression of Wnt target genes such as AXIN2 cycling and cMYC7 When Wnt signal is turned off the scaffold protein Axin adenomatous polyposis coli APC glycogen synthase kinase 3 GSK3 and casein kinase 1α CK1α form a multimeric destruction complex which induces catenin phosphorylation and ubiquitination degradation8Other mechanisms have been identified downstream of the canonical Wnt signaling pathway including the Hippo signaling cascade downstream nuclear effectors YAPTAZ Under Wnt OFF conditions YAPTAZ is an essential component of the destruction complex It recruits E3 ubiquitin ligase transducin repeatcontaining protein TrCP to promote catenin degradation in which YAP TAZ is a negative regulator of Wnt signaling However when Wnt is ON LRP56 induces the dissociation of YAP TAZ from Axin resulting in nuclear accumulation of YAP TAZ and enhancement of the expression of proproliferative genes in which YAPTAZ is a positive regulator of Wnt signaling9 Furthermore members of the Rspondin Figure An overview of Wnt signaling pathway As illustrated on the upper left side of Figure in the absence of Wnt ligand destruction complex induces the phosphorylation of catenin and the phosphorylated catenin is recognized and ubiquitinated by YAPTAZrecruited TrCP and subsequently subjected to proteasomal degradation In addition the E3 ubiquitin ligases Cullin 3KLHL12 recognize DVL and induce its degradation On the membrane ZNRF3RNF43 targets Frizzled receptor for ubiquitination and lysosomal degradation In the nucleus the inhibitory complex Groucho and histone deacetylases HDAC bind to TCFLEF transcription factors resulting in the inhibition of the Wnt signaling gene expression as shown in the lower left side of Figure On the upper right side of Figure the phosphorylated LRP receptor recruits Axin and DVL protein to the plasma membrane Activated DVL inactivates the destruction complex In this process the combination of DVL and ASPM inhibits the recognition of Cullin 3KLHL12 catenin with the assistance of histone modifying coactivators such as Pygopus Pygo and CREB binding protein CBPp300 interacts with the transcription factors TCFLEF to regulate target gene expression Frizzled ubiquitination is inhibited through the interaction of RSPO with LGR46 and ZNRF3 RNF43 Ub stands for ubiquitination and ps stands for phosphorylation Abbreviations LRP56 lowdensity lipoproteinrelated receptors and DVL dishevelled TCF Tcell factor LEF lymphoid enhancer factor APC the adenomatous polyposis coli GSK3 the glycogen synthase kinase 3 CK1α casein kinase 1α TrCP transducin repeatcontaining protein RSPO Rspondin LGR5 leucinerich repeat containing Gproteincoupled receptors ASPM abnormal spindlelike microcephaly associated HDAC histone deacetylases Pygo Pygopus CBP CREB binding proteinsubmit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alleucinerich RSPO ligand family were identified as positive effectors of Wnt signaling10 In the absence of RSPO the two homologues E3 ubiquitin ligases ZNRF3RNF43 ubiquitinated Frizzled inducing Fzd endocytosis When RSPO forms complex with repeatcontaining Gproteincoupled receptors LGR and ZNRF3 RNF43 however the Frizzled ubiquitination process is inhibited thereby inducing the activation of the Wnt signaling cascade Recently the abnormal spindlelike microcephaly associated ASPMDishevelled signal axis was proposed to participate in the generation of canonical Wnt signaling ASPM interacts with Wnt regulator DVL blocking the recognition for DVL by the ubiquitin ligase complex Cullin 3KLHL12 inhibiting the ubiquitination and subsequent degradation of DVL and maintaining a high level of Wnt activity11Besides the canonical pathway the Wnt signal also has an additional noncanonical activation pathway which is independent of catenin The noncanonical Wnt signaling pathway targets different receptors and activates multiple intracellular targets among which the most widely studied are Wntplanar cell polarity PCP and WntCalcium Ca2 pathways As shown in Figure in the WntPCP pathway Wnt ligand binds to receptor proteins to recruit and activate DVL which subsequently activates the small GTPases Rho or Rac triggering downstream Rhoassociated kinase ROCK and cJun Nterminal kinase JNK recruitment allowing cytoskeleton reanization12 In the WntCa2 pathway Fzd receptor is activated which induces the activation of DVL Then DVL recruits phospholipase C PLC which converts phosphatidylinositol 45bisphosphate PIP2 to diacylglycerol DAG and inositol 145triphosphate Figure An overview of noncanonical Wnt signaling pathway Noncanonical Wnt signaling is divided into WntPCP and WntCa2 pathways In WntPCP Wnt ligand binding receptor proteins trigger the activation of the ROCK and JNK downstream kinases allowing cytoskeletal reanization In WntCa2 noncanonical Wnt signaling increases the intracellular levels of DAG and IP3 by recruiting DVL stimulates the intracellular Ca2 release and activates downstream PKC CaN and CaMKII thereby regulating intracellular calcium fluxes and downstream calciumdependent cytoskeletal andor transcriptional responses Abbreviations ROCK Rhoassociated kinase JNK cJun Nterminal kinase PLC phospholipase C PIP2 phosphatidylinositol 45bisphosphate DAG diacylglycerol IP3 inositol 145triphosphate PKC protein kinase C CAN calcineurin CaMKII Ca2calmodulindependent protein kinase II DAAM1 the formin protein dishevelled associated activator of morphogenesis RHOA Ras homolog family member A RAC1 Rasrelated C3 botulinum toxin substrate AP1 activator protein1 CDC42 Cell division cycle protein NFAT nuclear factor of activated T cellsCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cWen DovepressIP3 IP3 stimulates intracellular Ca2 release and DAG and Ca2 together activate downstream protein kinase C PKC calcineurin CNA and Ca2calmodulindependent protein kinase II CaMKII thereby regulating intracellular calcium fluxes and downstream calciumdependent cytoskeletal and or transcriptional responses1314 Although noncanonical Wnt signaling is also an indispensable factor in the development of cancer this review mainly focuses on canonical Wnt signalingThere is growing evidence that Wntcatenin signaling is one of the main driving factors of some malignant tumors such as colorectal cancer liver cancer and breast cancer This review therefore focuses on the latest research progress on the correlation between the Wnt signal and tumors and the potential relationship between the Wnt signal and miRNAs In addition the possible relationship between the Wnt signal and CSC is discussed Finally cancer treatment strategies based on the canonical Wnt signal are summarizedWnt Signaling in Colorectal CancerColorectal cancer CRC is the third most common cancer in the world and the fourth most deadly cancer after lung liver and stomach cancer with the highest incidence in developed countries Constitutive activation of Wnt catenin signaling is a functional marker of colorectal cancer in which APC mutations occur in approximately of CRCs APCbased therapy may be therefore a promising strategy for the treatment of colorectal cancer15 It was reported that APC silencing drives hyperproliferation in the intestine and eventually forms colon adenomas Restoring APC gene led to continuous regression without recurrence whereas reestablished crypt homeostasis was found in tumor tissues carrying Kras and p53 mutations8 Wnt signaling is thus a promising target for the development of cancer therapy It was recently found that metastasisassociated in colon cancer MACC1 served as a transcriptional target of Wntcatenin signaling The DBC1 Deleted in breast cancer1 coactivator promotes selfrenewal capacity and drug resistance in colon cancer by regulating LEF1catenindependent enhancer in the MACC1 intron16 Furthermore Deptor The DEP domain containing mTOR interacting an mTOR inhibitor promotes cancer cell proliferation and survival in CRC and is a potential target for novel cancer therapies Deptor is a direct target gene for WntcatenincMyc signaling17 Silencing Deptor induces differentiation and inhibition of CRC cells by increasing ketone production and decreasing the expression of B lymphoma MoMLV insertion region Bmi1 while increasing mTOR activation A combination of AktmTOR and Wntcatenin inhibitors thus can exert a potent antitumor effect In addition to inhibition of positive regulators for Wnt signaling a negative regulator was also found as a possible target for Wnt signaling18 Vset and transmembrane domain containing 2A VSTM2A is a secreted protein that inhibits Wntcatenin signaling in colon cancer by directly inhibiting LRP6 activity and inducing LRP6 endocytosis and degradationCancer metastasis is the main cause of death in CRC patients Tocopherol alpha transfer proteinlike TTPAL and chromatin anizer special ATrich binding protein SATB1 are possibly involved in the invasion and metastasis of CRC Gou found19 that the upregulation of TTPAL is associated with a poor prognosis of colon cancer in which TTPAL directly interacts with thyroid receptorinteracting protein TRIP6 and inhibits the ubiquitination and degradation of TRIP6 TRIP6 competitively binds to the membraneassociated guanylate kinase with inverted domain structure1 MAGI1 tumor suppressor and dissociates catenin from MAGI1 Free catenin enters the nucleus to activate the oncogenic Wntcatenin signal increasing the ability of cancer cells to migrate and invade In addition overactivation of Wnt signaling promotes the interaction between TCF7L2catenin complex and SATB1 leading to SATB1 expression20 SATB1 regulates the expression of tumor growth and metastasisassociated genes regulating the interconversion of colon cancer in the invasion phenotypeWntcatenin signaling affects the malignant progression of colon cancer by regulating tumor suppressoractivator It is therefore pivotal to further explore Wnt signal related targets with a view to revealing a network of Wnt signaling in cancer and providing more options for cancer treatments The roles of Wnt signaling in the development of colon cancer were summarized in this section hoping to understand Wnt signaling from a new perspectiveWnt Signaling in Liver CancerAccording to the survey by World Health anizations International Cancer Research Center in liver cancer is one of the worlds highmortal cancers and hepatocellular carcinoma HCC is the second leading cause of cancer death worldwide catenin is expressed in the adult liver which mediates Wnt signaling and regulates the transcription and translation of genes around liver cells affecting various aspects of liver biology Here we mainly describe the roles submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alto regulate of Wnt signaling in hepatic metabolic partitioning and liver regeneration RSPOLGR45ZNRF3RNF43 module is the main signaling complex in the Wntcatenin signaling pathway liver metabolism development and regeneration21 Knocking down LGR45 in the mouse liver resulted in a loss of catenin signaling and a defect in hepatic metabolic partitioning Supplementing RSPO1 or knocking down Znrf3Rnf43 extends Wntcatenin signal gradient in the liver in a reversible and LGR45dependent manner resuming the liver defect phenotype In addition liver regeneration is possibly regulated by type I transmembrane protein TMEM9 In the model of liver injury induced by CCl4 TMEM9 enhances the rapid assembly of the vATPase lysosomal proton pump activates cathepsin activity in lysosomes induces APC degradation and activates Wntcatenin pathway leading to the promotion of liver regeneration22 Wntcatenin signaling is therefore essential for liver development whereas the abnormal activation of the catenin signal is also a sign of promoting the development of hepatocellular carcinoma and other liver diseases As shown in Figure approximately of HCC exhibited Wntcatenin signaling activity among them about of hepatocellular carcinomas had Catenin 1 CTNNB1 mutations23 which highlighted the importance of CTNNB1 in the progression of liver cancer Together with the activation levels of other signals in HCC we might hypothesize that the crosstalk between Wnt signaling and multiple signaling pathways promotes the development of liver cancer24 In addition in a mouse hepatocyte model catenin activation per se is not sufficient to induce liver cancer and other mutation events must be involved in the development of liver cancer such as mutations in a telomerase reverse transcriptase gene TERT25 or those in the gene mesenchymal epithelial transition factor MET26 This phenomenon may be related to the weak catenin activity in the mouse hepatocyte model We found that there are weak mutations in the CTNNB1 in HCC and HCA hepatocellular adenoma for example the mutation takes place at K335N387 S45 or T41 of the CTNNB127Wnt signaling plays a crucial role in the growth and development of liver However its aberrant activation may promote the occurrence of hepatocellular carcinoma and other liver lesions It is noteworthy that a liverspecific Wntcatenin signaling pathway exists in liver cells Glypican3 GPC3 is a heparan sulfate proteoglycan specifically expressed on the surface of liver cancer cells Li reported28 that GPC3 regulates the activation of Wnt signaling by dual mechanisms and promotes proliferation of hepatocellular carcinoma When FZD is not abundant GPC3 acts as an alternative Wnt receptor and recruits Wnt at the cell surface through the Nterminal CRD domain leading to the promotion of Wnt signaling When FZD is abundant GPC3 and FZD interact through their heparin sulfate HS chains and the two molecules form a triple complex with Wnt to synergistically stimulate Wnt signaling GPC3 might function as a bridge to stabilize the interaction between Wnt and FZD Further studies promote the elucidation of the roles of Wnt signaling in the development of liver cancer and provide a theoretical basis for GPC3 targeted therapyWnt Signaling in Breast CancerWnt signaling is involved in the malignant progression of breast cancer such as proliferation invasion metastasis and drug resistance In healthy breast tissue inhibition of Figure Status of Wntcatenin signal activation in HCC A Activation ratio of Wntcatenin signaling in HCC B Mutation frequency of common highmutation genes in HCCCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cWen DovepressWntcatenin activity leads to developmental disorders reduced cell proliferation during pregnancy29 and However there is increasing evidence that aberrant activation of the Wnt signal promotes the development of breast cancer It was demonstrated30 that bone marrow tyrosine kinase on chromosome X BMX is upregulated in breast cancer It inhibits the degradation of catenin by promoting GSK3 phosphorylation thereby disrupting normal Wnt signaling and promoting the malignant progression of breast cancer Rational regulation of Wntcatenin signaling is therefore a key to the treatment of breast cancer although it is difficult to specifically control the signaling In theory inhibition of Wntcatenin signaling can prevent tumor progression Surprisingly it is likely that silencing canonical Wnt signaling drives cancer cells into dormancy resulting in the hindrance of tumor cells from cancer immunoediting by the host immune system31 It was reported that Dickkopf1 DKK1 inhibits lung metastasis by antagonizing noncanonical Wnt signal of breast cancer DKK1 however could promote breastto bone metastasis by regulating canonical Wnt signal in osteoblasts Taken together it is not straightforward to manipulate canonical Wnt signaling for the treatment of metastatic cancerWnt Signaling and MicroRNAsIn recent years several studies have revealed the potential connection between noncoding RNA and Wnt signaling Table summarizes the effect of miRNAs on Wntcatenin signaling in cancerIt has been reported that miRNA expression is also controlled by Wnt signaling In HCC CTNNB1 binds to the miR18396182 promoter region and increases the transcription of the miRNAs thereby enhancing tumor cell invasion It is noteworthy that there is a mutual feedback loop between some miRNAs and Wntcatenin signaling In colon cancer miR452 induces Wnt signaling by targeting GSK3 Since T cell factorlymphatic enhancement factor is an effective transcription factor in the miR452 promoter in turn miR452 transcription is promoted by Wnt signaling forming a positive feedback loop46 In addition a negative feedback loop was found between the miRNA and Wnt signaling pathway miR145 a tumor suppressor miRNA can interact with TCF4catenin complex recruit polycomb repressive complex PRC2 histone trimethylase and suppress Wnt signal transduction Recruitment of PRC2 however results in a high level of methylation at the miR145 promoter region which downregulates the expression of miR14547 It was further demonstrated that abnormally downregulated miR145 was inversely correlated with its inhibitory regulators TCF4 and SUZ12 Suppressor of Zeste Protein Homolog indicating that miR145 forms a double negative regulatory loop with the negative regulator of colorectal cancer It is therefore imperative to further analyze the network and regulation of miRNA and Wnt catenin signaling pathway which would provide insights into the development of miRNAbased drugsWnt Signaling in Cancer StemnessIt is hypothesized that malignant tumors grow in a layered manner and cancer stem cells CSCs having selfrenewal characteristics and multidirectional differentiation ability play important roles in the tumor development and progression CSCs continuously maintain the replacement of new CSC tumorigenic subpopulations and differentiate into nonCSC progenies with high proliferative ability48 It is assumed that cancer originates in part from CSCs de Sousa e Melo demonstrated that in colorectal cancer Lgr5GFP tumor cells had higher tumorigenicity than Lgr5GFP tumor cells showing that Lgr5 cells had tumorinitiating ability49 In addition depletion of Lgr5 cells triggers differentiated cancer cells to continuously reverse to cells in a tumorpropagating state and replenish the Lgr5 CSC pool In another study a similar conclusion was provided Mature leukemia cells could reacquire clonogenic and leukemogenic properties through dedifferentiation It is worthy of note that CSC plasticity contributes to the interconversion of leukemia status through interfering with endogenous PU1 an ETS family pioneer transcription factor required for myelopoiesis50 However CSCs that transformed from differentiated cells require additional factors mutations inflammation or changes in the microenvironment for their stemness features51 For example after hepatotoxin induction hepatocytes produce tumor nodules and express progenitor cell markers52 Targeting CSCs and preventing the generation of CSCs from nonCSCs are thus a key for the development of cancer therapyThere is a wealth of evidence that CSCs are closely related to tumor invasion metastasis and drug resistance The differentiation gradient between CSCs and their non CSC progenies results in tumor heterogeneity which indicates that cells of different genotypes can exist in the same tumor including subgroups with transferability Owing to the unique nature of tumors CSCs have become the leading cause of drug resistance in cancer treatment Wnt submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alTable Effect of miRNAs on WntCatenin Signaling in CancermiRNAmiR100miR125bmiR181a5pmiR3773pmiR504miR1301miR31943pmiR186miR4543pmiR27amiR221222miR1945pmiR31miR5905pTargetsUpTMEM170BEcadherinDownDKK1 ZNRF3ZNRF3 RNF43 DKK3 APC2catenin TCF4XIAP ZEB2FZD7BCL9BCL9catenin MCRS1RPRD1A AXIN2 DKK3 SFRP1cateninWIF1 SFRP2 DKK2 AXIN2SOX17Dkk1 AXIN1 GSK3Wnt1 catenin NcadherinEffectCell TypeActivationActivationInhibitionInhibitionInhibitionInhibitionInhibitionInhibitionActivationInhibitionActivationactivationActivationInhibitionColorectal cancerColorectal cancerColorectal cancerColorectal cancerHCCHCCHCCHCCBreast cancerBreast cancerBreast cancerBreast cancerBreast cancerBreast cancerRef[][][][][][][][][][][][][][]Abbreviations XIAP Xlinked inhibitor of Apoptosis ZEB2 zinc finger Ebox binding homeobox BCL9 Bcell CLLlymphoma MCRS1 microspherule protein RPRD1A regulation of nuclear premRNA domaincontaining 1A SFRP1 secreted frizzledrelated protein TMEM170B transmembrane protein 170B WIF1 Wnt inhibitory factor Ecadherin epithelial cadherin Ncadherin neuralcadherincatenin signaling plays an essential role in the reprogramming and cancer cell stemness and is an indispensable cytokine network for promoting the progression of CSCs Reilein found that the cell types of CSCderivatives depend in part on the magnitude of spatially graded Wnt pathway activity53 The latest report54 proves the remarkable heterogeneity in Wnt activity in HCC cells among which WntactivityhighALDH1EpCAM triplepositive cells are the most stemlike and highly tumorigenic cells in all CSC populations This type of cells is called superpotent CSCs spCSC The ASPMDishevelled signal axis is highly activated in spCSCs and is heterogeneous in HCC tissues Future studies would reveal the underlying mechanism of Wnt and stem cell heterogeneity in liver cancer and provide new insights into CSCbased tumor treatment strategyWnt signaling contributes to reprogramming and maintenance of the CSC status such as the epithelialmesenchymal transition Chang found that in epithelial cells the SRYrelated highmobilitygroup box gene Sox15 tumor suppressor interacted with the cateninEcadherin complex and then interacted with the proximal promoter region of caspase3 CASP3 Caspase3 inactivates the Twist family bHLH transcription factor Twist which inhibits Wnt signaling for CSC phenotype acquisition In interstitial cells Twist1 forms a complex with cateninTCF4 and binds to the proximal promoter region of ATPbinding cassette G2 ABCG2 which induces the expression of stem cellrelated genes and promotes Wnt signaling to induce the CSC phenotype In the presence of Wnt signaling EMT regulates the CSC phenotype by inducing the conversion of two types of complexes55 In addition to activated Wnt signaling inactivated Wnt signaling can also promote malignant progression of CSCs especially tumor recurrence Autocrine inhibition of WNT signaling induces the resting state of CSCs and tumor dormancy After the primary tumor undergoes periodic proliferation and cancer immunoediting a small fraction of the progenies are transformed into latency competent cancer LCC cells and these cells exhibit Soxdependent stem phenotype LCC cells actively silence WNT signaling to enter into a quiescent state by expressing Sox2 target gene DKK1 The cancer cells then gain longterm viability through immune escape When the function of cancer immunoediting is somehow hampered LCC cells randomly enter cell cycle triggering the next metastatic exp | 2 |
purposeconjunctival squamous cell carcinoma scc is primarily treated with surgical resectionscc has various stages and local recurrence is common the purpose of this study was todetermine molecular localization of epidermal growth factor receptor egfr and the possibility of egfr as a biomarker for the management of conjunctival sccmethodsin this retrospective study we performed immunohistochemistry to evaluate egfr expression and localization in tumor cells egfr mutationspecific expression e746a750del andl858r and human papillomavirus expression in a series of conjunctival sccsresultsall tumors in our cohort were egfr positive twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining in additionwe calculated the percentages of the two most important mutations in egfr exon a750del exon l858r mutant in conjunctival sccs weobserved that the translocation of egfr from the membrane into the cytoplasm was relatedto clinical prognosis as we detected correlations between egfr cytoplasmic staining andfinal orbital exenteration and between decreased egfr membrane staining and progressionfree survivala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation sakai a tagami m kakehashi akatsuyamayoshikawa a misawa n wanibuchi h expression intracellular localizationand mutation of egfr in conjunctival squamouscell carcinoma and the association with prognosisand treatment one e0238120 101371 pone0238120editor sanjoy bhattacharya bascom palmer eyeinstitute united statesreceived april accepted august published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0238120copyright sakai this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and supporting informationfiles one 101371 pone0238120 august one 0cfunding none of the authors have any proprietaryor financial interests to declarecompeting interests none of the authors haveany proprietary or financial interests to declaresegfr is important in the pathology of ocular surface squamous neoplasia including sccand is a prognostic factor increased understanding of egfr mutations may have importantimplications for future treatment optionsegfr in conjunctival squamous cell carcinomaintroductionocular surface squamous neoplasia ossn includes several diseases such as conjunctival premalignant dysplasia carcinoma in situ and invasive conjunctival squamous cell carcinomascc the annual incidence of ossn was casesmillionyear conjunctival intraepithelial neoplasia casesmillionyear scc casesmillionyear in the united kingdom [ ] in the united states the rate of scc is 5fold higher among males and whites other previous research revealed that the risk increases with exposure to direct daylightand in outdoor workers metaanalysis demonstrated an association with human immunodeficiency virus odds ratio and human papillomavirus hpv odds ratio howeverno large epidemiological studies have been performed on people living in the far eastscholz examined clinicopathological factors and biomarkers and identified promotermutations in telomerase reverse transcriptase in of samples of conjunctival ossn associated with ultraviolet light induction recent research demonstrated that pdl1 isexpressed in almost half of conjunctival scc cases and noted the potential application ofimmune checkpoint blockade as a treatment strategy for conjunctival scc molecular targeted therapy is now used to treat most carcinomas and its use is continuingto increase uveal melanoma also has recently been reported in the ocular oncology area gefitinib is a relatively old tyrosine kinase inhibiter tki that is used as a molecular targeted therapy and its effects have been reported in various carcinomas on the other hand nobasic clinical studies on ocular tumors have been reported [] in our current study weinvestigated epidermal growth factor receptor egfr expression in our cases to assess thepossible effect of gefitinib we also examined the molecular expression and intracellular localization of egfr in conjunctival scc in east asian patientsmaterials and methodsselection of cases and collation of clinicopathologic datathis study was approved by the institutional review boards of osaka city university andkobe kaisei hospital and adhered to the tenets of the declaration of helsinki writteninformed consent was obtained from all patients before enrollment we identified patientstreated by ophthalmologists aa mt between november and july from whom wewere able to procure tissue blocks with residual tumor for each patient we collected demographic features age at initial diagnosis and at presentation to our institution and sex andprimary tumor features disease status at presentation [primary or recurrent] and in situ versusinvasive disease the american joint committee on cancer ajcc stage local recurrenceanatomic site and date metastases regional or distant and date vital status at last followup cause of death types of surgery and adjuvant therapy were also recordedimmunohistochemistry ihcimmunohistochemical studies for egfr and hpv were performed on 6μmthick tissue sections using the following antibodies antihuman egfr rabbit monoclonal antibody clone one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomasp84 414r14 cell marque rocklin ca usa antihpv mouse monoclonal antibodyclone k1h8 ab75574 abcam cambridge uk horseradish peroxidaseconjugated antirabbit igg hl goat polyclonal antibody histofine nichirei corporation tokyojapan and horseradish peroxidaseconjugated antimouse igg hl goat polyclonal antibody histofine nichirei corporationegfr mutationspecific immunohistochemical staining was performed on cases as primary antibodies we used egfr e746a750del cell signaling technologies danversma usa and egfr l858r cell signaling technologies which were manuallyapplied to the slides stained sections were viewed with an olympus bx53dp74as controls for staining benign conjunctival lesions were also stained for egfr and coloncancer samples were stained as a positive controlimage analysisslides immunostained for egfr egfr mutations and hpv were evaluated in a blinded manner by two specialists mt and ak egfr expression was visually estimated as the percentageof tumor cells with complete or partial membranous staining tumors with egfr staining in� of tumor cells were considered the diffuse staining type diffuse type and those with of tumor cells were considered the focal staining type focal type the presence orabsence and intensity of cell membrane staining were semiquantitatively divided into groupswith a score of none weak strong very strong the presence or absence andintensity of cell cytoplasmic staining were also divided into groups with a score of andsemiquantitatively analyzed none weak strong very strong egfr mutationspecific immunostaining was divided into two groups those with immunostaining that wasclearly present and those without immunostainingslides immunostained for hpv were assessed with visual evaluation for the presence ofpunctate nuclear signals within tumor nuclei at magnification and were scored as positive or negativeegfr expression in tumorsegfr expression in the tumor was analyzed with nanostring analysis archival formalinfixedparaffinembedded tumor tissue was retrieved and manually macrodissected total mrnawas isolated from the macrodissected tumor tissues using a qiagen mirneasy kit qiagenvalencia ca usa according to the manufacturers instructions the rna sample was quantified with nanodrop thermo scientific wilmington de usa and regarded as adequate ifit contained ng at minimum the sample was subsequently analyzed with the ncounterpancancer progression panel nanostring seattle wa usa according to the manufacturers instructions nanostring data processing was done with the r statistical programmingenvironment v342 considering the counts obtained for positive control probe sets rawnanostring counts for each gene were subjected to technical factorial normalization whichwas carried out by subtracting the mean counts plus two times the standard deviation from thecodeset inherent negative controls subsequently biological normalization using the includedmrna reference genes was performed additionally all counts with p after a onesidedttest versus negative controls plus two times the standard deviation were interpreted as notexpressed over basal noisestatistical analysisthe clinical and histopathologic characteristics were summarized using descriptive statisticscorrelations between immunohistochemical demographic and clinicopathologic factors were one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomaassessed using the wilcoxon rank sum and fishers exact tests progressionfree survival pfswas defined as the time from surgery to disease recurrence or death from any cause coxregression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes statistical analyses were performed usingspss statistics version software ibm japan tokyo japan values of p were considered statistically significantresultsclinicopathologic findings of our cohort are summarized in table all patients in ourcohort were east asian and included men and women with a mean age at presentation of years fourteen patients had invasive scc and had an in situtumor primary orbital exenteration was necessary for local disease control in two patients and two patients underwent additional orbital exenteration nine patients table clinicopathologic findings of cases of conjunctival squamous cell carcinomaage years mean rangesexmalefemalefollowup after primary surgery months rangetstage ajccall n n tist1t2t3t4primary surgery typelocal excisionorbital exenterationadjuvant therapynoyesadditional excisiontopical chemotherapyradiation therapyimmunohistochemical markershpv status in tumor cellsnegativepositiveegfr expression in tumorsdiffuse stainingfocal stainingnegativecell membrane egfr expression in tumorsvery strongstrongweak one 101371 pone0238120 august continued one 0ctable continuednegativecell cytoplasm egfr expression in tumorsvery strongstrongweaknegativeoutcomeorbital exenterationyesnolocal recurrence after curative therapyyesnometastasisdistantregional distantregionalnonevital status at last followupdeadalivecause of deathconjunctival scc metastasisother101371 pone0238120t001egfr in conjunctival squamous cell carcinomaall n n underwent adjuvant therapy most commonly additional local surgery topical chemotherapyand radiation therapy were performed in one patient in the adjuvant therapy group of thisgroup one patient died with disease months after diagnosis of regional and lung metastasesthe other patient was alive without disease at months after diagnosis of regional metastasestwo patients died one of which was due to conjunctival scc described above ninepatients experienced local recurrence after curative surgeryall tumors were egfr positive in our cohort twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining fig analysisof egfr intracellular staining patterns showed scores of for membrane staining and for cytoplasmic staining no significant difference was found between carcinoma in situ tisand invasive carcinoma tadv table no significant difference was found in the scoredepending on the stage egfr expression in colon cancer was used as a positive control fig2aon the other hand seven benign conjunctival lesions three pinguecula three pterygiumone dermoid cyst showed partial weak positive staining in conjunctival squamous epithelialcells especially on the cell membrane fig 2b in addition cytoplasmic staining was seen inonly one case benign cases showed scores of for membrane staining and for cytoplasmic staining cytoplasmic staining patterns were significantly different in benign compared to scc cases p table the correlation between egfr staining focal ordiffuse and egfr localization cytoplasmic staining group was not significantly different one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr expression in conjunctival scc focal egfr staining a and diffuse egfr staining b scale bar μm inset corresponding field in ahematoxylineosinstained section membrane staining very strong c and cytoplasm staining very strong d scale bar μm101371 pone0238120g001but the diffuse egfr group tended to have a higher score p and respectivelytable egfr e746a750 del and egfr l858r expression were assessed with immunohistochemistry in all patients fig the mutation at exon egfr e7446a750 del was confirmedin cases and that at exon egfr l858r point mutation was confirmed in cases with ihc table the relationship between egfr mutation and egfr stainingtable staining patterns of egfrcell membranetis in situtadv invasiven n benign tumorn cell cytoplasmtis in situtadv invasiven n benign tumorn �p value based on the nonpaired ttest101371 pone0238120t002staining patterns n totaltotalaverageaveragepp� one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig a egfr expression in colon cancer as a positive control scale bar μm b egfr expression in a control benign lesion pinguecula scale bar μminset corresponding field in a hematoxylineosinstained section101371 pone0238120g002focal or diffuse was determined using univariate linear regression analysis with correctionfor age p regarding egfr expression in tumors we compared the tis and tadv groups according toajcc t grading n4 no significant difference was found p fig the majority of patients in our cohort were hpv negative n table the positive rate of hpv immunoreactivity increased with increases in ajcc t grading but the correlation was not statistically significantthe cox regression model was used to examine and analyze the relationship between longterm prognosis including orbital exenteration and pfs and the clinicopathological statusegfr staining pattern and egfr mutation univariate cox regression analyses revealed significant correlations between egfr cytoplasmic staining and final orbital exenteration hazard ratio hr p table additionally a significant correlation was seenbetween the t stage ajcc and pfs and between egfr membrane staining and pfs hr p p respectively table local recurrence distant metastasisrate and overall survival rate were not statistically significant in addition the egfr mutationwas not significantly correlated with final orbital exenteration or pfs tables and discussionto the best of our knowledge this is one of the first studies to survey the prevalence of egfrmutations and intracellular localization in conjunctival scc and to evaluate the prognostic significance of tumor cells that express egfr in conjunctival sccin this study we found that the tumor tissue of all conjunctival sccs expressedegfr in addition we determined the percentages of the two most important mutations intable egfr staining and localizationcell membranecell cytoplasmicegfr focaln ±±egfr diffusen ±±p101371 pone0238120t003 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr mutationspecific expression in conjunctival scc a basement membrane staining in a tumor with egfr e746a750 del bwhole tumor staining in an egfr e746a750 del mutant c conjunctival scc layer cells with strong staining in an egfr l858r mutant scalebar μm101371 pone0238120g003egfr exon 746a750del exon l858r mutant in conjunctival sccs we also showed that the translocation of egfr from the membrane into the cytoplasm was related to clinical activation of cancer as correlations between egfr cytoplasmicstaining and final orbital exenteration and between decreased egfr membrane staining andpfs were noted although the number of cases examined was small the expression of cytoplasmic staining of egfr was weak but significantly different from membrane staining in thebenign disease group our hypothesis is that as egfr transitions from the membrane into thecytoplasm malignant changes progress in addition a correlation between egfr stainingfocal or diffuse and egfr cytoplasmic staining was seen and a higher score tended to bepresent in the diffuse egfr staining grouptable summary of egfr e746a750 del and egfr l858r point mutationsmutationn age y sex mt stage egfr staining patterns diffuseegfr localization score membraneexon egfr e746a750 del n fexon egfr l858r point mutationn t3 t2 tis t3 tis focalcytoplasmicm male f female101371 pone0238120t004 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig for egfr expression in tumors we compared carcinoma in situ tis and invasive carcinoma tadv groups according toajcc t grading n4 ns not significant101371 pone0238120g004intracellular transfer of egfr in the group with diffuse staining may indicate progressionand although no statistical differences were observed in this study significant findings mayemerge by increasing the number of cases in the futurein the past especially in african countries several studies on conjunctival sccs and egfrexpression have been reported they suggested a potential association with hpv [ ]other previous studies reported that posttranslational modification can promote egfrtable relationship between orbital exenteration and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750 del 8exon l858r point mutation positive 7negative hr ci p�ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t005 one 101371 pone0238120 august one 0ctable relationship between pfs and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale 15female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750del 8exon l858r point mutation positive 7negative ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t006egfr in conjunctival squamous cell carcinomahr ci p��endocytosis and lysosomal degradation of egfr thereby ensuring termination of receptor signaling [ ]in our cohort expression and localization of egfr and its association with prognosis werefirst reported in the asian race additionally intracellular translocation of egfr from membrane staining to cytoplasm staining likely by endocytosis was associated with the percent offinal orbital exenteration cytoplasmic staining hr p and pfs membranestaining hr p in our cohort regarding the difference in local changes inegfr immunoreactivity in patients without egfr expression in the tumor we compared thetis and tadv groups according to ajcc t grading a recent study showed that feedback regulatory loops can modulate growth factors and receptor tyrosine kinases such as egfr to regulate cellular functions including abnormal states such as cancer our study examined thisphenomenon clinically and confirmed a pathological difference without changes in geneexpressionegfr mutations in ossn including invasive sccs have not been examined in asianpatients since approximately egfr mutations in lung cancer had been registeredin the cosmic the catalog of somatic mutations in cancer database most are concentrated in the exon region of the intracellular tyrosine kinase domain the most frequentone is at codon of exon a deletion mutation is present at a site centered on five aminoacids elrea near amino acid and a point mutation changes leucine to argininel858r at codon of exon shigematsu in and mitsudomi in reported that egfr mutations are common in asians females nonsmokers and adenocarcinomas in lung cancer [ ] generally when egfr mutation occurs the tyrosine kinaseactivity of egfr at the atp binding site is constantly active even without growth factor cancer cell growth and survival depend on this pathway oncogene addiction egfr tkis competitively inhibit atp binding in the kinase domain and suppress autophosphorylation ofegfr blockade of signal transmission has antitumor effects previous reports of egfractivating mutations common mutations described the frequency of exon deletion mutations as and for l858r mutations in lung cancer [ ]egfr mutations were examined to verify the effect of gefitinib on positive nonsmall celllung carcinoma in two phase iii clinical trials from japan in the nej002 trial and thewjtog3405 trial gefitinib was the test treatment group the standard treatment in the former one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig schematic of movement of egfr into the cytoplasm by endocytosis to avoid excessive signaling and for recycling101371 pone0238120g005was carboplatin paclitaxel and in the latter was cisplatin in all studies the gefitinib groupshowed superior pfs [ ] in view of these findings in lung cancer in asians our findingsregarding egfr expression and mutations will provide further options for potential treatmentof ossn for pre and postsurgical treatmentthe association of scc with hpv was not confirmed because the number of cases wassmall in addition our results may not be accurate because we did not use multiplex pcrwhich is currently the most suitable genotyping method ours is the first report to show that differences in the expression form and mutations inegfr in ossn are associated with prognosis and treatmentin an animal model egfr inhibition affected epithelial cell proliferation and stratificationduring corneal epithelial wound healing and may play a role in maintaining normal cornealepithelial thickness gefitinib is an egfr inhibitor and is the first approved molecular targeted therapy for cancer treatment in japan thus understanding the pathological role of egfr in ossn andapplying it to treatment are of great significance for seeking new treatment indications inossn including conjunctival sccs in this study egfr may translocate from the cell membrane into the cytoplasm tumor cells may transfer egfr into the cytoplasm by endocytosisto avoid excessive signaling by the feedback system fig furthermore in this study theegfr mutation was present in many patients with ossn this finding may suggest a courseof treatment in the future in addition the method we used for identification of egfr mutations was not general genotyping but was a judgment of immunohistochemically stained sections although the sensitivity and specificity were high in a previous report this is still alimitation one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomathis study has important limitations first regarding egfr expression on the ocular surface changes in benign diseases and agerelated changes in normal tissues may not have beensufficiently investigated our study found that egfr mutations were also present in conjunctival scc in east asians however we did not obtain results that correlated with the final prognosis further studies including further multiinstitutional studies and an increase in thenumber of cases will be needed in the future another limitation is that double testing of formalinfixed paraffinembedded specimens and plasma with realtime pcr for detection ofegfr mutations is more common than ihc in actual clinical practice according to the literature both the sensitivity and specificity were satisfactory for these two types of mutations in addition the size of our study cohort was small n and the length of followup lessthan year in some patients may not have been sufficient for longterm outcome analysestherefore additional studies will be needed to corroborate our findingsin the results of this study indicate that egfr is an active molecular target inthe pathology of ossn including scc and is a prognostic factor the finding also suggests thatdiscovery of mutations may have important implications for future treatment optionssupporting informations1 filexlsxacknowledgmentswe gratefully acknowledge the technical assistance of the research support platform osakacity university graduate school of medicine and the clinical laboratory department of kobekaisei hospitalauthor contributionsconceptualization mizuki tagami atsushi azumidata curation atsushi sakai mizuki tagami atsuko katsuyamayoshikawa norihiko misawa atsushi azumiformal analysis mizuki tagami anna kakehashi norihiko misawafunding acquisition mizuki tagamiinvestigation mizuki tagami atsuko katsuyamayoshikawa atsushi azumimethodology mizuki tagami anna kakehashi atsuko katsuyamayoshikawa atsushiazumiproject administration mizuki tagamisupervision anna kakehashi hideki wanibuchi atsushi azumi shigeru hondavisualization atsushi sakai mizuki tagami anna kakehashiwriting original draft atsushi sakai mizuki tagamiwriting review editing mizuki tagami shigeru hondareferenceslee ga hirst lw ocular surface squamous neoplasia surv ophthalmol 101016s0039625705800542 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kiire ca stewart rmk srinivasan s heimann h kaye sb dhillon b a prospective study of the incidence associations and outcomes of ocular surface squamous neoplasia in the united kingdom eyelond mcclellan aj mcclellan al pezon cf karp cl feuer w galor a epidemiology of ocular surfacesquamous neoplasia in a veterans affairs population cornea 101097ico0b013e31829e3c80 pmid sun ec fears tr goedert jj epidemiology of squamous cell conjunctival cancer cancer epidemiolbiomarkers prev pmid scholz sl thomasen h reis h frequent tert promoter mutations in ocular surface squamousneoplasia invest ophthalmol vis sci 101167iovs1517469pmid nagarajan p elhadad c gruschkus sk ning j hudgens cw sagiv o pdl1pd1 expressioncomposition of tumorassociated immune infiltrate and hpv status in conjunctival squamous cellcarcinoma invest ophthalmol vis sci 101167iovs1926894pmid le tourneau c delord jp gonc¸alves a gavoille c dubot c isambert n molecularly targetedtherapy based on tumour molecular profiling versus conventional therapy for advanced cancershiva a multicentre openlabel proofofconcept randomised controlled phase trial lancetoncol 101016s1470204515001886 pmid el zaoui i bucher m rimoldi d nicolas m kaya g pescini gobert r conjunctival melanomatargeted therapy mapk and pi3kmtor pathways inhibition invest ophthalmol vis sci 101167iovs1826508 pmid ciardiello f tortora g a novel approach in the treatment of cancer targeting the epidermal growth factor receptor clin cancer res pmid lynch tj bell dw sordella r gurubhagavatula s okimoto ra brannigan bw activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmallcell lung cancer togefitinib n engl j med 101056nejmoa040938 pmid paez jg ja¨nne pa lee jc tracy s greulich h gabriel s egfr mutations in lung cancer correlation with clinical response to gefitinib therapy science 101126science1099314 pmid cesano a ncounter® pancancer immune profiling panel nanostring technologies inc seattlewa j immunother cancer 101186s4042501500887 pmid yu jj fu p pink jj dawson d wasman j orem j hpv infection and egfr activationalteration in hivinfected east african patients with conjunctival carcinoma one e10477101371 pone0010477 pmid mwololo a nyagol j rogena e ochuk w kimani m onyango n correlation of egfr pegfrand p16ink4 expressions and high risk hpv infection in hivaidsrelated squamous cell carcinoma ofconjunctiva infect agent cancer 1011861750937897 pmid haglund k dikic i the role of ubiquitylation in receptor endocytosis and endosomal sorting j cell sci 101242jcs091280 pmid zhang x gureasko j shen k cole pa kuriyan j an allosteric mechanism for activation of the kinasedomain of epidermal growth factor receptor cell 101016jcell pmid avraham r yarden y feedback regulation of egfr signalling decision making by early and delayedloops nat rev mol cell biol 101038nrm3048 pmid kobayashi y mitsudomi t not all epidermal growth factor receptor mutations in lung cancer are created equal perspectives for individualized treatment strategy cancer sci 101111cas12996 pmid shigematsu h lin l takahashi t nomura m suzuki m wistuba ii clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers j natl cancerinst 101093jncidji055 pmid mitsudomi t yatabe y mutations of the epidermal growth factor receptor gene and related genes asdeterminants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancercancer sci 101111j13497006200700607x pmid yun ch mengwasser ke toms av woo ms greulich h wong kk the t790m mutation inegfr kinase causes drug resistance by increasing the affinity for atp proc natl acad sci u s a 101073pnas0709662105 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kobayashi y togashi y yatabe y mizuuchi h jangchul p kondo c egfr exon mutationsin lung cancer molecular predictors of augmented sensitivity to afatinib or neratinib as comparedwith first or thirdgeneration tkis clin cancer res 10115810780432ccr151046 pmid wu jy yu cj chang yc yang ch shih jy yang pc effectiveness of tyrosine kinase inhibitors onuncommon epidermal growth factor receptor mutations of unknown clinical significance in nonsmallcell lung cancer clin cancer res 10115810780432ccr10 pmid maemondo m inoue a kobayashi k sugawara s oizumi s isobe h gefitinib or chemotherapyfor nonsmallcell lung cancer with mutated egfr n engl j med 101056nejmoa0909530 pmid mitsudomi t morita s yatabe y negoro s okamoto i tsurutani j gefitinib versus cisplatin plusdocetaxel in patients with nonsmallcell lung cancer harbouring mutations of the epidermal growth factor receptor wjtog3405 an open label randomised phase trial lancet oncol 101016s147020450970364x pmid nishiwaki m yamamoto t tone s murai t ohkawara t matsunami t genotyping of humanpapillomaviruses by a novel onestep typing method with multiplex pcr and clinical applications j clinmicrobiol 101128jcm0079307 pmid nakamura y sotozono c kinoshita s the epidermal growth factor receptor egfr role in cornealwound healing and 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" variability in the health effects of dietary fiber might arise from interindividual differences in the gutmicrobiotas ability to ferment these substrates into beneficial metabolites our understanding of what drives thisindividuality is vastly incomplete and will require an ecological perspective as microbiomes function as complexinterconnected communities here we performed a parallel twoarm exploratory randomized controlled trial in adults with overweight and classi obesity to characterize the effects of longchain complex arabinoxylan n at high supplementation doses female gday male gday on gut microbiota composition and shortchainfatty acid production as compared to microcrystalline cellulose n nonfermentable control and integratedthe findings using an ecological frameworkresults arabinoxylan resulted in a global shift in fecal bacterial community composition reduced αdiversity andthe promotion of specific taxa including operational taxonomic units related to bifidobacterium longum blautiaobeum and prevotella copri arabinoxylan further increased fecal propionate concentrations p friedmanstest an effect that showed two distinct groupings of temporal responses in participants the two groups showeddifferences in compositional shifts of the microbiota p permanova and multiple linear regression mlranalyses revealed that the propionate response was predictable through shifts and to a lesser degree baselinecomposition of the microbiota principal components pcs derived from community data were better predictors incontinued on next page correspondence jenswalteruccie nguyen k nguyen and edward c deehan contributed equally to this work1department of agricultural food nutritional science university of albertaedmonton ab t6g 2e1 canada13department of biological sciences university of alberta edmonton abt6g 2e1 canadafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cnguyen microbiome page of continued from previous pagemlr models as compared to single taxa indicating that arabinoxylan fermentation is the result of multispeciesinteractions within microbiomes this study showed that longchain arabinoxylan modulates both microbiota composition and theoutput of healthrelevant scfas providing information for a more targeted application of this fiber variation inpropionate production was linked to both compositional shifts and baseline composition with pcs derived fromshifts of the global microbial community showing the strongest associations these findings constitute a proofofconcept for the merit of an ecological framework that considers features of the wider gut microbial community forthe prediction of metabolic outcomes of dietary fiber fermentation this provides a basis to personalize the use ofdietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features toaccount for the inconsistent health effects in human intervention studiestrial registration clinicaltrialsgov nct02322112 registered on july keywords arabinoxylan dietary fiber gut microbiota interindividual variability overweight adults shortchainfatty acids epidemiologic studies consistently associate dietary fiberconsumption with a reduced incidence of obesityassociated pathologies [ ] in largescale observationalstudies whole grains and cerealderived fibers eg arabinoxylan and glucan showed stronger associationswith reduced risk of developing cardiovascular diseasetype ii diabetes gastrointestinal cancers and of allcausemortality when compared to other fiber sources [ ] asubstantial body of animal research further consolidatedthe mechanisms by which fiber reduces metabolic pathologies despite these convincing associations findings obtained from human dietary intervention trialsaimed to improve metabolic risk markers by supplementing isolated dietary fibers remain inconsistent possibly due to an individualized clinical response [ ]owing to their chemical structure dietary fibers resistdigestion in the smallintestine and reach the colonwhere they become substrates for the gut microbiotathe microbial fermentation of fiber to shortchain fattyacids scfas has been implicated in the prevention ofobesityassociated pathologies propionate and butyrate are two scfas that are especially relevant as theyhave been linked to beneficial immunological and metabolic effects intervention studies with arabinoxylanisolated from wheat endosperm for instance have demonstrated increased fecal concentrations of both butyrateand propionate dietary fibers can further modulategut microbiota composition in a structuredependentway through the enrichment of bacterial taxa that utilizethe substrate and tolerate or benefit from the environmental changes caused by fiber fermentation [ ] forexample dietary interventions with shortchain fractionsof arabinoxylan resulted in an enriched abundance ofbacterial species that can either utilize arabinoxylan oliaxos directly eg bifidobacteriumgosaccharidesadolescentis and bifidobacterium longum or benefitfrom metabolic byproducts released during axos degradation eg anaerobutyricum hallii and faecalibacterium prausnitzii although fiberinduced alterationsto the gut microbiota are significant the effects are alsohighly individualized and this variability might haveclinical ramifications that could explain the individualized clinical responses to understand the individualized response of the gutmicrobiota to dietary fiber an ecological perspective isrequired as fiber fermentation is determined by complexinterspecies interactions between members of the gutmicrobiota the process is often based on a crossfeeding cascade where primary degraders that access thefiber provide breakdown products oligosaccharides disaccharides and monosaccharides to other microbesand metabolites that result from the fermentation ofthese products also serve as substrates interindividual variation in gut microbiota composition mayresult from the absence of keystone species that initiatethe degradation of recalcitrant fibers differences inunrelated species with similar ecological functions thatcompete for the same substrate or variation instrains of the same species that differ in their capacity tometabolize the substrate these compositional variations likely determine both the competitive and cooperative relationships between community membersthat form trophic networks some of which anize intoecological guilds that collaborate to degrade complexfibers although interindividual variation in the response of the gut microbiota to fiber can influence metabolite outputs relevant to health ie propionate orbutyrate this topic and the underlying ecologicalprinciples have received little attentionthe objective of this study was to apply an ecologicalframework to characterize the compositional and metabolic responses of the human gut microbiota to a longchain arabinoxylan isolated from corn bran compared to 0cnguyen microbiome page of a fiber that is not fermented by the gut microbiotamicrocrystalline cellulose mcc we further assessedwhether nutritional and microbiotarelated factors couldexplainamongindividualsresponsesobservedthevariableresultssubject characteristics and protocol adherenceto compare the effects of arabinoxylan and mcc weconducted a 6week parallel twoarm exploratory randomized controlled trial in individuals with overweightand classi obesity where females received gday andmales gday of either fiber fig of the subjectsenrolled and randomized to an intervention arm sevenwithdrew from the study in the arabinoxylan groupthree experienced challenges consuming the supplementand one reported constipation in the mcc group twowithdrew due to personal reasons and one due to constipation and weretherefore excluded from analysesadditional file fig s1 subjects that completed thestudy protocol n included females and males aged ± years with a body mass indexbmi of ± kgm2 no differences in age sex orbmi were detected between the intervention groups atbaseline additional file table s1 overall protocoladherence assessed by the amount weight of returnedsupplement was ± and ± in the arabinoxylan and mcc arms respectivelyeffect on the composition of the fecal microbiotafecal microbiota diversitynonmetric multidimensional scaling analysis of euclidean distances between subjects based on centered logratio clrtransformed operationaltaxonomic unitotu data showed that the two treatment groups harbored bacterialcould not becommunitiesthatdifferentiated at baseline p permutational multivariate analysis of variance [permanova] fig 2aoneweek supplementation with arabinoxylan alteredthe global fecal bacterial community which became significantly different from the fecal microbiota of subjectsreceiving mcc p this effect was maintaineduntil the end of the fiber intervention p thesechanges occurred by arabinoxylan inducing temporalshifts in fecal microbiota composition determined as theaverage diversity between the individuals treatmentand baseline samples which were significantly largerwhen compared to the mcc group p mannwhitney test fig 2b in addition while mcc increasedinterindividual differences diversity between subjectsp generalized estimated equation [gee] modelarabinoxylan reduced it p fig 2canalysis of αdiversity showed that arabinoxylan reduced fecal bacterial diversity shannons index p gee model fig 2d but not richness total otusafter weeks of supplementation overall these findingsshowed that while the nonfermentable mcc had no detectable effects on measures of bacterial diversity arabinoxylan altered the global bacterial community within week inducing temporal shifts in composition and a reduction of both interindividual variation and αdiversityeffect on the relative abundance of bacterial taxa and coabundance response groupsneither arabinoxylan nor mcc altered microbiota composition at the phylum level at lower taxonomic levelschanges in the relative abundance of two bacterial families were detected at weeks of arabinoxylan relative tobaseline and mcc namely an increase in bifidobacteriaceae q wilcoxon test fig 2e additional file table s2 and a decrease in erysipelotrichaceae q fig study design shaded study week blocks indicate a scheduled clinic visit the x indicates the task was completed during the study weekcdhq ii canadian diet history questionnaire ii stool characteristics selfreported stool consistency and bowel movement frequency 0cnguyen microbiome page of fig arabinoxylan alters the global composition of fecal bacterial communities and induces distinct shifts in taxa a nonmetricmultidimensional scaling nmds plot based on euclidean distance metrics of arabinoxylan and microcrystalline cellulose groups at each timepoint intersubject diversity showing changes in the distance between subjects over time euclidean distances b between fecal microbiotas ofsubjects at each study time point intersubject and c between each subjects fecal microbiota at baseline and during w1 and w6 of treatmentintrasubject d αdiversity displayed as shannon index and total otus of the fecal microbiotas of subjects at each time point e absolutechange δw6bl in relative abundance of bacterial taxa affected by the dietary intervention data analyzed using permanova for a gee modelswith bonferroni correction for b and d and mannwhitney tests for c for e data were analyzed using either wilcoxon tests to assess withingroup changes relative to baseline or mannwhitney tests to assess betweengroup changes ie ax vs mcc with fdr correction diversityand compositional data were reported as mean ± sd and centered logratio transformed prior to the statistical analyses bl baseline otuoperational taxonomic unit w1 week w6 week at the genus level arabinoxylan increased thegenera bifidobacterium and prevotella when comparedto both baseline and mcc and enriched blautia whencompared to mcc otu level analysis revealed that otus changed during arabinoxylan treatment relative tobaseline henceforth referred to as significant otusin particular otus related to bifidobacterium longumotu4 prevotella copri otu6 bacteroides plebeiusotu53 bacteroides sp otu56 bacteroides ovatussuccinatutensotu26otu38 blautia obeum otu85 subdoligranulum spotu11 clostridium leptum otu46 mollicutesotu32 and muribaculaceae otu79q phascolarctobacteriumbecame enriched while otus related to ruminococcusbromii otu5 eubacterium oxidoreducens otu41bacteroides uniformis otu7 and faecalibacillus sppotu21 declined in relative abundance supplementation with mcc only increased the family lachnospiraceaegenus parasutterella q numerically the dominant compositional effects of arabinoxylan were to a large degree specific to b longumotu4 and p copri otu6 as these taxa increased inrelative abundance by an average of 46fold and 4fold while other otus increased by and thein an attempt to identify groups of cooperating species that could function as ecological guilds in the 0cnguyen microbiome page of degradation of arabinoxylan we adapted a clustering approach conceptually similar to that described by tong instead of absolute proportions of taxa weused arabinoxylaninduced shifts to identify clusters ofspecies whose responses were intercorrelated this analysis revealed a total of seven coabundance responsegroups cargs fig 3a five of which showed statistically significant responses to arabinoxylan while noneresponded to mcc additional file table s2 thecarg that showed the largest increase in relative abundance was carg1 p wilcoxon test whichconsisted of six out of the eleven otus that increasedthrough arabinoxylan fig 3b among those six otusbshift andshowed significant connections to all but one member ofcarg1 rs q spearmans correlations usingpermutation tests suggesting arabinoxylan may be degraded through cooperative interactions between theselongum otu4 exhibited the largesttaxa in carg6 p copri otu6 exhibited the largestresponse but only showed one strong connection withanother member of the carg bacteroides massiliensisotu98 rs q which suggests that pcopri might act to a larger degree independently to degrade arabinoxylan fig 3b the majority of taxa thatdecreased during arabinoxylan consumption particularlyb uniformisotu7 clustered within carg7 andshowed negative correlations with taxa of carg1carg2 and carg6 suggesting competitive or antagonistic interactionstemporal responses of otus and cargsto determine if short and longterm treatment witharabinoxylan and mcc differed in their effects on thefecal microbiota we compared shifts from baseline toweek w1 with those from baseline to week w6however there were no detectable differences betweenfig identification of coabundance response groups cargs during arabinoxylan supplementation a heatmap shows the change δw6bl inrelative abundance of otus affected by arabinoxylan p wilcoxon test the hierarchical dendrogram shows clustering of centered logratio clr transformed otus rows based on spearmans correlation distances by the completelinkage clustering algorithm and then groupedon the dendrogram into seven cargs by permanova p subjects columns clustered based on euclidean distances colors from blue tored indicate the direction and magnitude of change b coresponse network analysis each node represents an otu where the size isproportional to the change δw6bl in relative abundance the shape indicates direction of change positive circle negative square and thecolor references the respective carg to which it was clustered lines between nodes represent significant positive red line or negative blueline spearmans correlations rs values ¥ or and q values bl baseline otu operational taxonomic unit w6 week 0cnguyen microbiome page of the two time frames q wilcoxon test data notshown in addition comparison of baseline w1 andw6 values by friedmans test indicated that the effectsof arabinoxylan occur rapidly within week with nofurther detectable changes at weeks fig 4a considering these findings analyses on compositional changeswere performed with w6 data unless otherwise statedinterindividual variation in responses to arabinoxylanbacterial shifts in response to arabinoxylan and theirmagnitude were highly individualized fig 4b for instance absolute increases in relative abundance rangingfrom to to 429fold change were detected inseven subjects for the otu classified as blongumotu4 while other subjects showed either a muchsmaller increase a decrease or the species was undetectable otus related to b obeum otu85 subdoligranulum sp otu11 b ovatus otu26 and c leptumotu46 were enriched by arabinoxylan in around twothirds of the subjects less frequently enriched wereotus classified as potu6 b plebeiusotu53 and bacteroides sp otu56 p copri otu6responded in only four subjects but effects were largewith the species expanding beyond to 7foldchange ofthe total bacterial community in threesubjectscoprito determine drivers of these individualized responseswe used multiple linear regression mlr analyses totest if responses in otus that showed numerically thelargest shifts p copri b longum b obeum and subdolifig temporal and individualized responses of the otus and cargs affected by arabinoxylan and microcrystalline cellulose a plots show thetemporal response of the ten most abundant otus detected in of subjects and the seven cargs centered logratio transformed datawere analyzed by friedmans test with dunns correction to assess withingroup changes between time points ie δw1bl and δw6w1 bbubble plot shows individualized differences δw6bl in relative proportions of the ten most abundant otus percentage of total microbiotacomposition and cargs sum of otus detected after weeks of arabinoxylan and microcrystalline cellulose supplementation the size of thebubble is proportional to the change in abundance relative to baseline while the color of the bubble represents the direction of the change redincrease black decrease the x indicates that the otu was either undetected or the change was relative abundance bl baseline cargcoabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of granulum sp and in cargs with significant responsescargs and could be predicted by baselinediet or microbiota composition baseline microbiota allotus and significant otus and diet variables werefirst reduced in their dimensionality by principal component analysis pca and then treated as predictors thisanalysis revealed that individualized responses of bacterial taxa and cargs to arabinoxylan and mcc could notbe predicted by baseline diet or microbiota compositionq additional file fig s2effect on stool characteristics and bowel movementswhile fecal moisture content was not changed by eitherfiber q wilcoxon test additional file table s3subjects consuming arabinoxylan reported softer stoolconsistencies when compared to subjects consumingmcc treatment effect p gee model additional file fig s3a both arabinoxylan and mcc ledto an increase in bowel movements relative to baselinep gee model additional file fig s3b withno difference detected between treatment groups treatment effect p effect on fecal ph and scfasfecal ph and scfa concentrations did not change after weeks of either fiber treatment q wilcoxon testadditional file table s3 considering that absoluteconcentrations of fecal scfas are affected by their absorption in the gut we additionally assessedchanges in the percentages of acetate propionate andbutyrate relative to total scfa concentrations at w6which has been previously shown to vary little across colonic regions this analysis revealed an increase inthe percentage of propionate produced through arabinoxylan when compared to mcc q mannwhitney test and a reduction in the percentage ofbutyrate relative to baseline q wilcoxon test although differences in butyrate were not detected whencompared to mcc q further investigation ofthe ratio between propionate and butyrate showed an increase in propionate relative to butyrate when comparedto baseline q and mcc q suggestingthat arabinoxylan supplementation directed the outputof scfas in favor of propionatecharacterization of the temporal response in the threeprimary scfas also showed an increase in fecal propionate concentrations by arabinoxylan at w1 p friedmans test fig 5a although propionate concentrations remained elevated at w6 this increase was notstatistically significant when compared to baseline p this loss of significance was caused by an increasein the interindividual variation at w6 fig 5b visualevaluation of the individualized temporal response ofpropionate to arabinoxylan revealed clear separation offig temporal and individualized output of fecal scfas in response to arabinoxylan and microcrystalline cellulose supplementation a line plotsshow the temporal response of acetate propionate and butyrate reported as mean ± sd b individualized temporal propionate response of w6responders red and w6nonresponder black grouped based on δw6w1 data analyzed for a and b using friedman test with dunnscorrection to assess withingroup changes between time points and for b using mannwhitney tests to assess differences betweengroup ateach time point bl baseline carg coabundance response group otu operational taxonomic unit scfa shortchain fatty acid w1 week w6week 0cnguyen microbiome page of subjects into two distinct patterns fig 5b based on thedirection of change from w1 to w6 ie positive ornegative subjects were grouped into w6respondersδ w6w1 and w6nonresponders δ w6w1 in general w6responders showed a higher outputof propionate at w6 p friedmans test butnot at w1 while the opposite isseen in w6nonresponders p the two groups differed bypropionate concentrations at w6 p mannwhitney testw6propionate responders and nonresponders differ intheir microbiota response to arabinoxylanmicrobiota compositionalbaseline and shifts anddiet data were ordinated using pca and then differencesandbetween w6propionaterespondersnonresponders were tested using permanova thisthe bacterial communities ofanalysis revealed thatw6responders wereindistinguishablefrom w6nonresponders at baseline but differed in their response to arabinoxylan δw6baseline fig thiswas detected if analysis was based on all otus p the significant dietresponsive otus p or the seven cargs p in contrastneither baseline microbiota composition fig nordietary factors additional file fig s4a separatedaccording to w6 response p in addition comparing w6respondersinterms of their baseline total grain whole grain andtotal fiber consumption or their stool consistency andbowel movement frequency during treatment did notreveal any differences either p mannwhitneyand w6nonrespondersfig the individualized temporal propionate response to arabinoxylan associates with compositional responses in the fecal microbiota principalcomponent analysis plots based on euclidean distance comparing the relative abundance of fecal microbiota both at baseline and arabinoxylaninduced shifts δw6baseline between w6responders red and w6nonresponders black microbiota variables ie otu or carg thatcontributed the most to intersubject variation were shown as vectors on the plot when statistical significances were determined by permanovap carg coabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of test additional files and fig s3c and fig s4btogether these findings indicate that the temporal response in fecal propionate concentrations is primarilyassociated with the shifts in the microbiota and notbaseline microbiota composition or dietindividualized scfa responses can be predicted by gutmicrobiota featuresas with compositional responses gut microbiota functional responses to fiber interventions have been shownto be individualized [ ] but what drives this variation is poorly understood we applied mlr to determine whether fecal scfa responses could be explainedby stool consistency and bowel movement frequencydiet or microbiotarelated factors and then comparedthe quality ofthe models using corrected akaikeinformation criterion aicc values where lower valuesmean higher quality these analyses revealed that thew6 scfa response to arabinoxylan could be predictedby the fecal microbiota fig additional file fig s5but not by baseline diet stool consistency or bowelmovement frequency reported during treatment additional file fig s6a and fig s6b the best modelswere achieved for propionate especially when principalcomponents pcs generated from w6 shifts of all otuswere used as predictors fig 7a additional file tables4 models were of lower quality when w6 shifts of significant otus cargs pcs of cargs or single otuswere used suggesting that global community measuresexhibited stronger linear relationships with the propionate response than single or groups of taxa although themodels that used baseline and w1 shifts of otus asfig individualized arabinoxylaninduced propionate responses could be explained by baseline gut microbiota composition and microbiotashifts a heatmap shows the linear associations between the individualized propionate response δw6bl dependent variable columns andmicrobiota profiles bl δw1bl δw6bl predictors rows cells represent individual multiple linear regression models with fdr correction thatassess whether the predictors explain the individualized propionate response multivariate microbiota data were simplified into principalcomponent pc variables pc1 pc2 and pc3 prior to analysis each model contained the best one or two predictors of pcs individual cargs orsignificant otus selected by stepwise regression all models were adjusted by fiber dosesex colors from white to red indicate relative aicchighest aicc value x lower aicc values red indicate higher quality models b scattercorrected akaike information criterion values calculated byplots show the linear relationship between propionate responses δw6bl and either the baseline contribution of all otus to pc1 or the shiftsof carg1 color and size of each point indicate propionate response magnitude and the shaded area specifies the confident interval thetop six otus that contributed the most to either pc1 of all otus or carg1 are further provided ax arabinoxylan bl baseline carg coabundance response group mcc microcrystalline cellulose otu operational taxonomic unit w1 week w6 week aicc value 0cnguyen microbiome page of predictors were of lower quality than those based on w6shifts they are still valid showing q values less than after benjaminihochbergs false discovery rate fdrcorrection linear relationships between propionate responses and significant predictors using baseline pc1 ofall otus and shifts carg1 were further visualizedusing scatter plots fig 7b reaffirming the quality ofthe analysis as a majority of subjects fall within the confidence regionssignificant models could also be designed for acetateand butyrate responses to arabinoxylan additional file fig s5 interestingly in contrast to propionate the bestmodels to predict butyrate responses were achieved usingshifts of a single otu e oxidoreducens otu41 aknown butyrate producer however overallthemodels for acetate and butyrate were of much lower quality than those for propionate in summary while individualized responses in scfas showed no association withdiet they could be predicted by microbiota shifts andbaseline composition in contrast to the analysis of the effects of arabinoxylan not one single mlr model wasfound to be significant for mcc indicating that the statistical approach based on mlr models did not detect anyassociations independent of fiber fermentationdetermining the role of bacterial taxa in propionateresponsemlr analyses were applied to determine connectionsbetween arabinoxylan responding otus within cargs and and fecal propionate concentrations fig 8athis analysis revealed that shifts in p copri otu6 didnot predict propionate responses while blongumotu4 and correlated taxa in carg1 showed strongerlinear relationships the highest quality models were obtained with b obeum otu85 b plebeius otu53and p succinatutens otu38 all of which encodemetabolic pathways for propionate production such analysis provides a potential explanation for themetabolic interactions between proposed primary degraders secondary fermenters and metabolite utilizersthat result in the promotion of propionate in responseto arabinoxylan fig 8bdiscussionin the present study we characterized the impact of a6week highdose corn bran arabinoxylan supplementation on the composition and function of the fecalbacterial community in healthy adults with overweightand classi obesity arabinoxylan treatment changedfig relationship between propionate responses to arabinoxylan and proposed primary degraders secondary fermenters and metaboliteutilizers a individual multiple linear regression models determine otu responses δw6bl that predict the fecal propionate response δw6blyaxis shows the coefficient for each predictor as in the average propionate response when otu relative abundance increases xaxis showsthe p value for each predictor all models were adjusted by fiber dosesex where bubble size represents the adjustedr2 b proposed model ofbacterial crossfeeding in the gut during degradation of complex soluble arabinoxylans otu operational taxonomic unit 0cnguyen microbiome page of showing two distinctcommunity structure and induced specific shifts inthe composition of the gut microbiota that manifestedthemselves after week of treatment without furtherchanges at w6 arabinoxylan induced increases inpropionate output both compositional and functionalresponses were highly individualized with propionateresponsestemporal patternscompositional responses to arabinoxylan could not bepredicted and functional responses were independentof stool consistency bowel movement frequency andbaseline diet however baseline microbiota composition and especially the compositional shifts correlatedwith propionateresponses the nonfermentablemcc showed virtually no effect on gut microbiotacomposition or functionan understanding of compositional and functional responses of the gut microbiota to changes in diet requiresan ecological framework arabinoxylan supplementation provides resources that can be used by microbesthat possess the traits to either access the chemicalstructures directly or utilize public goods released duringarabinoxylan degradation in our study the dominant effects of arabinoxylan were directed toward twobacterial species b longum and p copri while nine additional otus showed smaller increases including threebacteroides species eg b ovatus b plebeius and bacteroides sp this high degree of specificity toward blongum over other bifidobacterium species is in agreement with other studies testing longchain arabinoxylans[] and genomic analyses that showed that genesencoding arabinoxylandegrading glycosidase eg xylosidase and αarabinofuranosidase are conservedonly among b longum strains [ ] in contrast to thespeciesspecific enrichment of b longum arabinoxylanenriched several species within the phylum bacteroidetesthat possess the genetic and functional traits necessaryfor accessing arabinoxylan [] although arabinoxylan utilization is not universally conse | 0 |
"differentiation of human stromal mesenchymal stem cells hMSCs is a critical procedure for thedevelopment of osteoblast SNHG14 is a newly discovered lncRNA that has been barely studied Our preliminaryexperiments showed that SNHG14 may be dysregulated in the differentiation of hMSCs In this study we focusedon elucidating the relationships among SNGH14 miR2861 and osteoblastic differentiation of hMSCsMethod To investigate the roles of SNHG14 and miR2861 in hMSCs differentiation qRTPCR luciferase activity celltransfections the detections of ALP activity and Alizarin Red staining were performedResult We found that the expression of SNHG14 was enhanced while the expression of miR2861 was suppressedin serum and hMSCs from patients with osteoporosis SNHG14 could target miR2861 and shSNHG14 suppressedosteoblast differentiation of hMSC MiR2861 suppressed osteoblast differentiation of hMSC In addition the effectsof SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861Conclusion In our experimental data showed that the induction effects of SNHG14 on osteoblastdifferentiation of hMSC were attenuated by miR2861 SNHG14 could induce osteogenic differentiation of hMSCin vitro by targeting miR2861Keywords SNHG14 Osteogenic differentiation Human stromal mesenchymal stem cells miR2861BackgroundMesenchymal stem cells have the capabilities of selfrenewal and multilineage differentiation which are critical factors in the regeneration or repairment of bone tissues [ ] Human bone marrow mesenchymal stem cellhMSCs could fully differentiate to many cell types including osteoblasts chondrocytes and adipocytes [ ]The differentiation of hMSCs is thus critical for the development of osteoblast Studies have modulated the cell signaling pathways to control the differentiation of hMSCs to Correspondence vs4190163com Mingchang Du and Bo Wu contributed equally to this workThe Orthopedic Hospital of Shenyang No Dong bei da ma lu road Dadong district of Shenyang Shenyang City Liaoning Province PRChinaosteoblasts [ ] However the underlying mechanismsremain to be elusiveNoncoding RNAs have become the hotspot in severalresearch fields including long noncoding RNAs lncRNAs nt [] and microRNAs miRNAs nt [] Various lncRNAs have been reported to be involved in theosteoblastic differentiation of hMSCs For instance downregulation of lncRNAANCR promoted osteoblast differentiation by targeting EZH2 and regulating the expression ofRunx2 [] LncRNA H19 was reported to mediate BMP9induced osteogenic differentiation of MSCs through theNotch signaling [] LncRNA SNHG14 is a newly discovered lncRNA that has been barely demonstrated regardingits biological roles in human diseases It was reported thatSNHG14 promoted microglia activation by regulating miR The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cDu BMC Musculoskeletal Disorders Page of 1455pPLA2G4A in cerebral infarction [] Very limitedinformation has been revealed for its functions in hMSCsMiRNAs are another group of noncoding RNAs thathave been widely reported in human diseases ManymiRNAs exert essential roles in the differentiation ofhMSCs to osteoblast For example microRNA138 wasrevealed to regulate the osteogenic differentiation of human stromal mesenchymal stem cells in vivo [] Another study also reported thatthe microRNA320RUNX2 axis regulates adipocytic differentiation of human mesenchymal skeletal stem cells [] MoreovermiR2861 has been demonstrated to participate in theregulatory feedback loop during differentiation of mouseosteoblast []From our preliminary experiment we noticed thatSNHG14 may be dysregulated in hMSCs differentiationand miR2861 may share the common binding sequenceswith lncRNA SNHG14 In this study we aimed to clarifythe role of lncRNA SNHG14 in the formation of osteoblast from hMSCs focusing on elucidating the relationshipsand osteoblasticdifferentiation of hMSCsamong SNGH14 miR2861MethodsHuman samplesIn this study patients with hip fracture were recruited atThe Orthopedic Hospital of Shenyang Patient sampleswere divided into two groups patients in each groupincluding the treatment group osteoporosis patientswith a fracture and the control group nonosteoporosispatients with a fracture Serum and bone tissues werecollected during endoprosthesis and gamma nail wasimplanted into the proximal femur All patients enrolledin this study signed the informed consent This studywas approved by the Research Ethics Committee of TheOrthopedic Hospital of ShenyanghMSC preparationshMSCs were obtained from the bone marrow from femurs of patients during total hip or knee arthroplastydue to osteoarthritis or hip fracture The Ethics ReviewBoard of Orthopedic Hospital of Shenyang ShenyangCity Liaoning Province approved our study All hMSCswere obtained from postmenopausal women with anaverage age of years old age range years oldDensitometric examinations were performed using aLunar iDXA apparatus GE Lunar Madison WI USADiagnosis of oste ia or osteoporosis were made usingthe WHO Tscore criteria Tscore or Tscore respectively All the subjects in the osteoporosis group had vertebral fracturesCell separationThe RosetteSep Isolation kit STEMCELL Canada wasused to isolate hMSCs Cells were cultured at °C in awet environment with CO2 The culture mediumwas refreshed every week When cells reached confluence they were trypsinized and used immediatelyCell cultureWe cultured hMSCs in αminimum essential mediumαMEM containing fetal bovine serum FBS Invitrogen antibiotics and glutamax IGIBCO USAOsteogenesis was induced by fresh osteoblast inductionmedium OIM with M dexamethasone SigmaAldrich D4902 mM lascorbic acid SigmaAldrichA8960 mM glycerophosphateSigmaAldrichG9422 and mM 125vitaminD3 Alkaline phosphatase ALP was used to assess osteoblast phenotypeAlizarin Red staining was used to test matrixmineralization The medium was changed every dthroughout the experiments and cells were harvested atindicated time pointsqRTPCRTotal RNAs were extracted from serum bone tissues orhMSCs by Trizol Invitrogen USA The Reverse Transcription Kit Applied Bio USA was used to synthesizecDNAs The qRTPCR reactions were prepared usingSYBR Select Master Mix Applied Bio USA and PCRwas carried out on an ABI 7900fast thermocycler Applied Bio USA The relative expression was calculatedby 2ÎÎCT method The sequences of the primers arelisted belowSNHG14F ²GGGTGTTTACGTAGACCAGAACC3²SNHG14R ²CTTCCAAAAGCCTTCTGCCTTAG3²GAPDHF ²GAAGGTGAAGGTCGGAGTC3²GAPDHR ²GAAGATGGTGATGGGA TTTC3²OCF F ²GGCGCTACCTGTATCAATGG3²OCR ²GTGGTCAGCCAACTCGTCA3²Runx2F ²CGAATAACAGCACGCTATTAA3²Runx2R ²GTCGCCAAACAGATTCATCCA3²OSXF ²GCCAGAAGCTGTGAAACCTC3²OSXR ²GCTGCAAGCTCTCCATAACC3²ALPF ²TAGTGAAGAGACCCAGGCGCT3²ALPR ²ATAGGCCTCCTGAAAGCCGA3²miR2861F ²AACGAGACGACGACAGAC3²miR2861R ²GGGGCCUGGCGGUGGGCGG3²U6 ²GCCCCCGCCTCCGCCGCCGCC3² and ²ATATGGAACGCTTCACGAATT3²Cell transfectionsVectors with shSNHG14 miR2861 mimic and miR inhibitor all from Genepharma were transfectedto hMSCs via Lipofectamine Sigma USA At dposttransfection qRTPCR was conducted to detect 0cDu BMC Musculoskeletal Disorders Page of gene expressions The miR2861 mimic sequence was²GGGGCCUGGCGGCGGGCGG3² Mimic controlsequence was ²UUCUCCGAACGUGUCACGUTT3²The antagomir sequence was ²CCGCCCGCCGCCAGGCCCC3² The antagomir control sequence was ²CAGUACUUUUGUGUAGUACAA3²ALP activityhMSCs were collected and washed The cells were lysedby Triton X100 for min and centrifuged at g for min The supernatant was used for ALP analysis by ALP Assay Kit Abcam USAAlizarin red stainingThe osteoblasts were cultured by OIM for weeks andthen fixed by ethanol Next the cells were incubated by Alizarin Red solution for an hour at CThe results were recorded for analysisLuciferase assayPrimers were designed for the potential miR2861 binding sequence of AKT2 ²UTR SNHG14 ²UTR andthen cloned into the Sac IXba I sites of pmirGLODualLuciferase reporter vector The reconstructed plasmidswere confirmed by sequencing and named pmirGLOSNHG14WT and pmirGLOAKT2wt1 We also commercially synthesized mutant reporter constructs by mutating three nucleotides of each potential miR2861binding site and designated as pmirGLOSNHG14MUT pmirGLOAKT2mut1 Cells of confluencewere seeded in triplicate in 96well plates The wildtypeWT or mutant reporter constructs Mut were cotransfected into SiHa cells in the 96well plates with nmolL miR2861 or nmolL miRNC by using lipofectamine Invitrogen CA USA respectively Reporterposttransfection using the DualLuciferase Reporter AssayKit Promega following the manufacturers instructionsFirefly luciferase activity values were normalized fortransfection efficiency using the corresponding Renillaluciferase activity Three independent experiments wereperformedassays weregeneperformed hWestern blot analysisCell protein lysates were separated in or SDSPAGE gel h posttransfection followed by transferring to polyvinylidene difluoride membrane PVDFWestern blot analysis was performed with monoclonalantip53 Santa Cruz antiAKT2 Abcam primary antibodies AntiGAPDH antibody Santa Cruz was used asan internal control The membrane was washed and incubated with horseradish peroxidase HRPconjugatedsecondary antibody Cell Signaling Technology USAComplexes were visualized with SuperSignal West PicoChemiluminescent Substrate Pierce and the expressionlevels of these proteins were evaluated by Quantity OnesoftwareStatistical analysisData were shown as mean ± stand deviation SD Comparisons were performed by ttest between groups oroneway ANOVA among multiple groups P wasconsidered statistical significant differencesResultsSNHG14 was upregulated but miR2861 was downregulatedin serum and hMSCs from patients with osteoporosisThe expression of SNHG14 and miR2861 in serum andhMSCs of osteoporosis patients were analyzed Compared to participants without osteoporosis n theexpression levels of SNHG14 in serum and hMSCs ofn were greatly elevatedosteoporosis patiensFig 1a and c In addition the expression of miR2861was dramatically downregulated in hMSCs of osteoporosis group Fig 1d In addition a negative relationshipbetween the expression of SNHG14 and miR2861 in theserum of the osteoporosis group was observed Fig 1bfurtherinvestigated theSNHG14 was targeted by miR2861Werelationship betweenSNHG14 and miR2861 As shown in Fig 2a the common binding site between SNHG14 and miR2861 wasobserved After successfully transfecting miR2861 intohMSCs Fig 2b the cotransfection of SNHG14 ²UTR with miR2861 led to the suppression of luciferaseactivities compared with that of SNHG14 MUT Figue2C Moreover the transfection of shSNHG14 elevatedthe expression levels of miR2861 Fig 2d The expression levels of SNHG14 were also reduced in cells transfected with miR2861 Fig 2e Thees data indicated thatSNHG14 was targeted by miR2861reduced in cellsshSNHG14 suppressed osteoblast differentiation of hMSCTo investigate the effects of SNHG14 on hMSC osteoblast differentiation we induced hMSCs differentiationto osteoblasts after transfection with shSNHG14 orshNC As shown in Fig 3a the expression levels ofSNHG14 weretransfected withshSNHG14 The suppression of SNHG14 markedly lowered osteoblastic differentiation which was indicated bylower expression levels of the osteoblastspecific genesRUNX2 Osterix OSX ALP OC and decreased ALP activity Figs 3bd We observed matrix mineralizationin vitro by Alizarin red staining in shSNHG14transfected hMSCs compared with cells transfected withshNC It was obvious that shSNHG14 could suppresshMSCs differentiation to osteoblasts weeks posttransfection 0cDu BMC Musculoskeletal Disorders Page of Fig SNHG14 was upregulated but miR2861 was downregulated in serum and hMSCs from patients with osteoporosis a Expressions ofSNHG14 in the serum of nonosteoporosis people and osteoporosis patients n b The negative relationship between the expression ofSNHG14 and miR2861 in the serum of osteoporosis patients n c Expression of SNHG14 in hMSCs of nonosteoporosis people andosteoporosis patients n d Expression of miR2861 in hMSCs of nonosteoporosis people and osteoporosis patients n p Fig SNHG14 was targeted by miR2861 a Common binding sequences between SNHG14 and miR2861 b Expression of miR2861 mRNA inhMSCs c Dualluciferase reporter assay d Expression of miR2861 mRNA in hMSCs e Expression of SNHG14 mRNA in hMSCs N p 0cDu BMC Musculoskeletal Disorders Page of Fig shSNHG14 suppressed osteoblast differentiation of hMSC a The expression of SNHG14 mRNA in hMSCs b ALP activities in shSNHG14 orshNC transfected hMSCs on day day and day c Osteoblast differentiation assessed through osteoblast marker genes of RUNX2 OSX ALPand OC normalized to actin on day day and day d ALP and Alizarin Red staining on day N p MiR2861 suppressed osteoblast differentiation of hMSCTo further evaluate the effects of miR2861 on hMSCosteoblast differentiation we induced hMSCs to differentiate to osteoblasts after transfection with miR2861mimic or miRNC Overexpression of miR2861 significantly suppressed osteoblastic differentiation which wasindicated by decreased ALP activity Fig 4a lower expression levels of RUNX2 OSX ALP and OC Fig 4b 0cDu BMC Musculoskeletal Disorders Page of Fig MiR2861 suppressed osteoblast differentiation of hMSC a ALP activities measured at day day and day of osteoblastdifferentiation b osteoblast differentiation assessed by the mRNA expression of RUNX2 OSX ALP and OC day day and day c ALP andAlizarin Red staining results on day N p and reduced in vitro matrix mineralization Fig 4c inmiR2861mimic transfected hMSCs in contrast to cellstransfected with miRNCThe effects of SNHG14 on osteoblast differentiation ofhMSC were attenuated by miR2861Whether miR2861 could attenuate the effects ofSNHG14on osteoblast differentiation of hMSCFigure 5a illustrated that shSNHG14 decreased ALP activity but the effects were attenuated by cotransfectionwith miR2861 inhibitor Figure 5b demonstrated thatdownregulation of miR2861 greatly lowered osteoblastic differentiation induced by shSNHG14sinceshSNHG14 decreased osteogenesisAKT2 was targeted by miR2861Finally the mechanisms by which miR2861 functionedto affect the differentiation of hMSCs were exploredOur bioinformatics analysis and luciferase assay resultsshowed that AKT2 could bind with miR2861 Fig 6aand b In addition overexpression of miR2861 decreased the expression levels of AKT2 and downregulation of SNHG14 reduced the expression of AKT2Fig 6c and dDiscussionsOsteoblastic differentiation from hMSCs many originates from many cell events that are affected by variousmolecular and cellular procedures during the development of bone and skeleton It is crucial to reveal important factors that mediate this phenomenon and to studythe underlying mechanisms Owing to the successfulfindings from the previous studies different lncRNAshave been shown to participate in the osteoblast differentiation by targeting corresponding cell signaling pathways One study revealed the expression profiling of 0cDu BMC Musculoskeletal Disorders Page of Fig The effects of SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861 a ALP activities in cells transfected withcontrol shSNHG14 or shSNHG14 miR2861inhibitor at day b Expression of osteoblast marker genes of RUNX2 OSX ALP and OC at day N p lncRNAs in C3H10T12 mesenchymal stem cells undergoing early osteoblast differentiation [] LncRNA H19promoted osteoblast differentiation via the TGF1Smad3HDAC signaling pathway by deriving miR675[]Various lncRNAs and miRNAs are dysregulated during the hMSCs differentiation of osteoblast [ ] Inour study we found a similar phenomenon We firstlyanalyzed the expression of SNHG14 and miR2861 inserum and hMSCs of osteoporosis patients Comparedto nonosteoporosis participants the expression levels ofSNHG14 in serum and hMSCs of osteoporosis patientswere greatly elevated The expression of miR2861 wasdrastically downregulated in hMSCs of osteoporosisgroup A negative relationship was established betweenthe expression of SNHG14 and miR2861 in serum ofosteoporosis group Similar to previous studies we identified that lncRNA SNHG14 was upregulated but miR was downregulated in serum and hMSCs from patients with osteoporosisFig AKT2 was targeted by miR2861 a Shared binding sequences between AKT2 and miR2861 b Dualluciferase reporter assay c and dWestern blot assay of AKT2 protein expression levels N p 0cDu BMC Musculoskeletal Disorders Page of With the common shared binding sequences lncRNAscould target their specific miRNAs and exert the biological roles in the pathogenesis of many cellular procedures [] For examplelncRNA DGCR5 acts as atumor suppressor in papillary thyroid carcinoma via targeting miR2861 [] We first confirmed the commonbinding sequences between SNHG14 and miR2861 Cotransfection of SNHG14 ²UTR with miR2861 led tothe suppression of luciferase activities compared withthat of SNHG14 MUT Moreover shSNHG14 elevatedthe expression levels of miR2861 The relative expression levels of SNHG14 were also lowered in cells transfected with miR2861 As far as we know we are thefirst to reveal that SNHG14 is targeted by miR2861 during the hMSCs differentiation to osteoblastAccording to previous reports ALP is highly expressedin osteoblast which is an important indicator for maturedifferentiation of osteoblast [] Osteoblastspecific genesRUNX2 Osterix ALP and OC are also critical genes to indicate the existing of osteoblast [ ] To investigatethe effects of SNHG14 on hMSC osteoblast differentiation we induced hMSCs differentiation to osteoblastsafter transfection with shSNHG14 or shNC The expression of SNHG14 was suppressed in cells transfected withshSNHG14 Suppression of SNHG14 markedly loweredosteoblastic differentiation which was indicated by lowerexpression levels of the osteoblastspecific genes RUNX2Osterix ALP and OC decreased ALP activity and in vitromatrix mineralization by Alizarin red staining inshSNHG14 transfected hMSCs compared with cells transfected with shNC Similar to previous reports [ ] wealso observed that silencing of SNHG14 could suppresshMSCs differentiation to osteoblastsA novel regulation role of Runx2miR3960miR2861was demonstrated in mouse osteoblast differentiation []MiR2861 was found to promote osteoblast differentiationby increasing the expression of Runx2 [] To investigatethe effects of miR2861 on hMSC osteoblast differentiationwe induced hMSCs to differentiate to osteoblasts aftertransfection with miR2861mimic or miRNC Overexpression of miR2861 greatly suppressed osteoblastic differentiation which was indicated by lower expression levelsof the osteoblastspecific genes RUNX2 OSX ALP andOC and decreased ALP activity and reduced in vitromatrix mineralization in miR2861mimic transfectedhMSCs compared to cells transfected with miRNC Different from the previous study [] we noticed that miR2861suppressed osteoblast differentiation of hMSC Moreoverwe observed that the effects of SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861ConclusionsIn our data confirmed that the induction effects of SNHG14 on osteoblast differentiation of hMSCswere attenuated by miR2861 SNHG14 could induceosteogenic differentiation of hMSC in vitro by targetingmiR2861Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s12891020035069Additional file AbbreviationhMSCs Human bone marrow mesenchymal stem cellAcknowledgmentsNot applicableIndividual persons dataNot applicableAuthors contributionsMD BW SF YL XM XF contributed to data analysis drafting or revising the gave final approval of the version to be published and agree to beaccountable for all aspects of the workFundingThere is no funding sourceAvailability of data and materialsThe analyzed data sets generated during the study are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe ethics review board of the Orthopedic Hospital of Shenyang ShenyangCity Liaoning Province approved our study Written informed consent wasobtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived January Accepted July ReferencesAggarwal S Pittenger MF Human mesenchymal stem cells modulateallogeneic immune cell responses Blood Sonoyama W Liu Y Fang D Yamaza T Seo BM Zhang C Liu H GronthosS Wang CY Shi S Mesenchymal stem cellmediated functional toothregeneration in swine PLoS One 200611e79Nuttelman CR Tripodi MC Anseth KS Dexamethasonefunctionalized gelsinduce osteogenic differentiation of encapsulated hMSCs J Biomed MaterRes Part A Dawson E Mapili G Erickson K Taqvi S Roy K Biomaterials for stem celldifferentiation Adv Drug Deliv Rev Nguyen MK Jeon O Krebs MD Schapira D Alsberg E Sustained localizedpresentation of RNA interfering molecules from in situ forming hydrogels toguide stem cell osteogenic differentiation Biomaterials Eskildsen T TaipaleenmÃki H Stenvang J Abdallah BM Ditzel N Nossent AYBak M Kauppinen S Kassem M MicroRNA138 regulates osteogenicdifferentiation of human stromal 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Mizuno M Kuboki Y Osteoblastrelated gene expression of bone marrowcells during the osteoblastic differentiation induced by type I collagen JBiochem Jang WG Kim EJ Kim DK Ryoo HM Lee KB Kim SH Choi HS Koh JTBMP2 protein regulates osteocalcin expression via Runx2mediated Atf6gene transcription J Biol Chem Salingcarnboriboon R Tsuji K Komori T Nakashima K Ezura Y Noda MRunx2 is a target of mechanical unloading to alter osteoblastic activity andbone formation in vivo Endocrinology Lu YF Liu Y Fu WM Xu J Wang B Sun YX Wu TY Xu LL Chan KMZhang JF Long noncoding RNA H19 accelerates tenogenic differentiationand promotes tendon healing through targeting miR29b3p and activatingTGF1 signaling FASEB J Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
Neurogenesis From Neural CrestCells Molecular Mechanisms in theFormation of Cranial Nerves andGangliaKarla MndezMaldonado12 Guillermo A VegaLpez34 Manuel J Aybar34 andIv¡n Velasco15 Instituto de Fisiologa Celular Neurociencias Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico Departamento de Fisiologa y Farmacologa Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autnomade Mxico Ciudad de Mxico Mexico Instituto Superior de Investigaciones Biolgicas INSIBIO CONICETUNT SanMiguel de Tucum¡n Argentina Instituto de Biologa Dr Francisco D Barbieri Facultad de Bioqumica Qumica yFarmacia Universidad Nacional de Tucum¡n San Miguel de Tucum¡n Argentina Laboratorio de Reprogramacin CelularInstituto Nacional de Neurologa y Neurociruga Manuel Velasco Su¡rez Ciudad de Mxico MexicoThe neural crest NC is a transient multipotent cell population that originates in thedorsal neural tube Cells of the NC are highly migratory as they travel considerabledistances through the body to reach their ï¬nal sites Derivatives ofthe NC areneurons and glia of the peripheral nervous system PNS and the enteric nervoussystem as well as nonneural cells Different signaling pathways triggered by BoneMorphogenetic Proteins BMPs Fibroblast Growth Factors FGFs Wnt proteins Notchligands retinoic acid RA and Receptor Tyrosine Kinases RTKs participate in theprocesses of induction speciï¬cation cell migration and neural differentiation of the NCA speciï¬c set of signaling pathways and transcription factors are initially expressed inthe neural plate border and then in the NC cell precursors to the formation of cranialnerves The molecular mechanisms of control during embryonic development havebeen gradually elucidated pointing to an important role of transcriptional regulatorswhen neural differentiation occurs However some of these proteins have an importantparticipation in malformations of the cranial portion and their mutation results in aberrantneurogenesis This review aims to give an overview of the role of cell signaling and of thefunction of transcription factors involved in the speciï¬cation of ganglia precursors andneurogenesis to form the NCderived cranial nerves during anogenesisKeywords cranial nerve peripheral nervous system hindbrain cell signaling transcriptional regulatory networktrigeminal nerve facial nerve vagus nerveAbbreviations BMP bone morphogenetic proteins CN cranial nerve CNS central nervous system DRG dorsal rootganglia FP ï¬oor plate hESCs human embryonic stem cells Msx Muscle segmentrelated homeobox NC neural crestNCCs neural crest cells NT neural tube PA pharyngeal arches Pax Paired box PNS peripheral nervous system rrhombomere RA retinoic acid RTK receptor tyrosine kinase Shh Sonic hedgehog Sox Srybox VSMC vascular smoothmuscle cells Wnt Wingless and Int1 WRPW TrpArgProTrp Zic Zinc ï¬nger protein of cerebellumEdited byMichael PiperThe University of QueenslandAustraliaReviewed byRoberto MayorUniversity College LondonUnited KingdomRebecca McLennanStowers Institute for MedicalResearch United StatesCorrespondenceManuel J AybarmanuelaybarfbqfunteduarIv¡n Velascoivelascoifcunammx These authors have contributedequally to this workSpecialty sectionThis was submitted toStem Cell Researcha section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted June Published August CitationMndezMaldonado KVegaLpez GA Aybar MJ andVelasco I Neurogenesis FromNeural Crest Cells MolecularMechanisms in the Formationof Cranial Nerves and GangliaFront Cell Dev Biol 103389fcell202000635Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alINTRODUCTIONDuring the embryonic development of vertebrates one of themain events after the gastrulation process is neurulation whichallows the formation of the neural tube NT The neuralectoderm generates not only the central nervous system CNSbut also another set of cells between the NT and the nonneural ectoderm located in the most dorsal part of the NT calledthe neural crest NC Hall SimµesCosta This versatile and plastic cell population was ï¬rst described byWilhelm His years ago Hall The NC is one of themost important features that separate vertebrates from otherchordate anisms It arises at the posterior and lateral bordersof the neural and nonneural ectoderm the neural plate borderFigure Cerrizuela the embryo NCCs are divided into cranialNC cells NCCs are multipotent and give rise to several celltypes depending on the site of origin along the anteroposterioraxis oftrunkincluding cardiac vagal and sacral A Minouxand Rijli SimµesCosta and Bronner VegaLopez Cranial nerves CN transmit sensory and motorinformation between the brain and tissues of the head andcervical region The CN are formed from the contribution oftwo specialized embryonic cell populations cranial NC andectodermal placodesOrigin of the Neural CrestNCCs which are multipotent delaminate from their origin andmigrate throughout the body to diï¬erentiate into several celltypes including cells of the peripheral nervous system PNSmelanocytes cranial cartilage and bone neuroendocrine cellsand several other phenotypes B In humans at least cell types have been deï¬ned as NC derivatives Vickaryousand Hall Proper NC migration relies on environmentalcues such as EphEphrins Smith Semaphorin3F Gammill Versican Szab thechemokine Stromal cellderived factor Theveneau or Robo2 Shiau The migration patternsof NCCs have been clearly described for model anisms likebirds frogs and mice In all vertebrates cranial NCCs emergefrom the forebrain midbrain and hindbrain regions Coulyand Le Douarin Serbedzija Depending ontheir axial origin cranial NCCs will either migrate through thefacial mesenchyme and into the frontonasal process or willpopulate the branchial arches Noden Lumsden Serbedzija The sensory module of the PNSin the cranial region is composed of an array of paired gangliaadjacent to the hindbrain that transduce the perception of touchpain temperature position and special sensory informationfrom the periphery to the CNS Cranial NCCs migrate to formsensory ganglia such as the trigeminal V the facial VII theglossopharyngeal IX the vagus X CN and also to form themotor ganglia for the oculomotor III and accesory XI CNTable and Figures acomplex and multistep processinitially directed by cell signaling molecules including BoneMorphogenetic Proteins BMPs Wnts Wingless and Int1NC formation isNeural CrestDerived Cranial NervesFibroblast Growth Factor FGF and retinoic acid RA Thesesignals reveal the tissue interactions into the ectodermal cellpopulations the neural plate the nonneural ectoderm and theunderlying mesoderm in a highly coordinated manner VegaLopez It has been proposed that NC speciï¬cationoccurs during gastrulation as a consequence of the action oftwo successive gradients of secreted signals A combinationof intermediate levels of activity of BMP and Wnt signalingacting on the ectoderm to induce and specify NC precursorsat the neural plate border and a subsequent requirement ofboth signals is needed for maintenance of speciï¬cation duringneurulation Aybar and Mayor Steventon Inchick embryos it was shown that NCCs are speciï¬ed as earlyas the blastula stage Prasad It was demonstratedthat during gastrulation Pax7 expression is restricted to cellslocated in a region in the medial epiblast which are NCfated andcontribute to the neural folds and later to migrating NCCs Basch The inhibition of Pax7 function in chicks inhibitedthe expression of key NC markers such as Snai2 OMIM Sox9 Srybox OMIM Sox10 OMIM andHNK1 beta13glucuronyltransferase like OMIM Basch and BronnerFraser This evidence suggests thatthe neural plateprospective ectoderm interaction at the neuralplate border might not be a requisite for NC speciï¬cation orinduction and that neural plate border formation and NCinduction might be separable eventsThe variousresearch works carried outto study theorigin of NCCs have identiï¬ed genes anized into a generegulatory network that participate in and control the inductionspeciï¬cation and diï¬erentiation of NC SimµesCosta An example of this are the transcription factors involvedin induction such as FoxD3 Forkhead Box D3 OMIM Snai2 and Sox9 SaukaSpengler and BronnerFraser GarcaCastro and coworkers identiï¬ed a novel preneuralborder state characterized by early Wntβcatenin signalingtargets that displayed diï¬erent responses to BMP and FGFsignaling from the neural border genes in human cells Leung These preborder genes Gbx2 Gastrulation brainhomeobox OMIM SP5 OMIM Zic3 OMIM and Zeb2 OMIM had their induction andpeak of expression before the classical neural plate borderspeciï¬er genes such Msx12 Muscle segmentrelated homeobox OMIM Pax37 OMIM and Zic1 OMIM Such speciï¬er genes together withsignaling molecules direct the expression of NCspeciï¬c geneslike AP2 OMIM FoxD3 Snai2 Sox9 and Sox10Speciï¬ers regulate NC eï¬ector genes involved in migrationSox9 Sox10 Cad7 and diï¬erentiation [Col1a Collagen typeI alpha OMIM Ngn1 Neurogenin OMIM MitfMicrophthalmiaassociated transcription factor OMIM] in human NC development Betters The NC population migrates to diï¬erent regions of the mouseembryo from the NT after the epithelialmesenchymal transitionmaintaining its multipotential character until completingdiï¬erentiation in its ï¬nal destination Baggiolini To study the ontogeny of the NC diï¬erent model anismsboth in vivo and in vitro have been used Several proteinsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Neural crest origin regions in human and mouse embryos and some of its cranial derivatives A The topleft part of the scheme shows the origin of theneural crest cells green that migrate through the embryo On the topright side the level of axial origin see axial color key of different regions of the neural crest isrepresented in developing mouse or equivalent human embryos the migration of neural crest is represented in green inside the embryos and the direction ofmigration is marked with black arrows Depending on their axial level of origin and migratory pathways neural crest cells adopt different fates and contribute tovarious tissues and ans B The main cranial derivatives labeled in green are shown Abbreviations d days E mouse embryonic stage NCCs Neural CrestCells s somite St human stage VSMC vascular smooth muscle cellsincluding transcription factors as well as epigenetic modiï¬ersthat take part in the speciï¬cation and diï¬erentiation of the NChave been described The study of transcription factors and ofthe signaling pathways in which they participate is importantto understand the diï¬erentiation programs and how thesemultipotent cells are committed to a speciï¬c destination On theother hand transcriptome analysis during the development of theNC from speciï¬cation to migration Meulemans and BronnerFraser and a more recent study covering the migrationto the diï¬erentiation of the NC show the importance of theinteraction between the diï¬erent transcription factors and thesignaling pathways at every stage of NC development SimµesCosta However these authors acknowledge thatit is diï¬cult to have a complete global map since only a fewtranscriptional regulators have been characterized and little isknown about the function of the products of the eï¬ector genesacting on NC migration Betancur SimµesCosta andBronner VegaLopez NC and cranial placodes are thought to appear togetherduring the evolution of vertebrates to give rise to speciï¬c sensorystructures of the head Northcutt and Gans Northcutt The components of the sensory nervous system of the headare derived from the NC and from an embryonic cell populationdeveloping in close proximity the cranial sensory placodes theolfactory lens otic trigeminal epibranchial and paratympanicplacodes A series of events induce develop and anizethese cell precursors which through reciprocalinteractionswith NCCs build the functional sensory system in vertebratesSteventon Singh and Groves Migrating NCCsarrive ï¬rst at the site of ganglia development ie the trigeminalFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cFrontiersinCellandDeveopmentallliBoogywwwfrontiersniAugustlVoumeAlrticeTABLE Contributions of neural crest cells and placodes to ganglia and cranial nervesCranial nerveGanglion and typeOrigin of neuronsReferencesCNI Olfactory Ensheating gliaof Olfactory nervesCNIII Oculomotor mCiliary visceral efferentCNV Trigeminal mixTrigeminal general afferentCNVII Facial mixSuperior general and special afferentCNVIII Vestibulocochlear sCNIX Glossopharyngeal mixCNX Vagus mix Superiorlaryngeal branch and recurrentlaryngeal branchCNXI Accessory mInferior geniculate general and special afferentSphenopalatine visceral efferentSubmandibular visceral efferentAcoustic cochlear special afferent and Vestibularspecial afferentSuperior general and special afferentInferior petrosal general and special afferentOtic visceral efferentSuperior general afferentInferior nodose general and special afferentVagal parasympathetic visceral efferentNo ganglionTelencephalonolfactory placode NCCs at forebrainNCCs at forebrainmidbrain junction caudal diencephalonand the anterior mesencephalonNCCs at forebrainmidbrain junction from r2 into 1st PAtrigeminal placodeHindbrain NCCs from r4 into 2nd PA 1st epibranchialplacode1st epibranchial placode geniculateHindbrain NCCs 2nd PAHindbrain NCCs 2nd PAOtic placode and hindbrain from r4 NCCsHindbrain NCCs from r6 into 3rd PA2nd epibranchial placode petrosalHindbrain NCCs from r6 into 3rd PAHindbrain NCCs from r7r8 to 4th and 6th PAHindbrain NCCs 4th and 6th PA 3rd nodose and 4thepibranchial placodesHindbrain NCCs 4th and 6th PAHindbrain from r7r8 to PA NCCs 4th PABoyd Muller ORahilly andM¼ller Barraud Noden Couly Wahl Lee dAmicoMartel and Noden Forbes and Welt DamicoMartel and Noden DamicoMartel and Noden Lumsden Barlow and Northcutt Begbie andGraham Barlow Krimm Sandell Narayanan and Narayanan DamicoMarteland Noden ORahilly and M¼ller Barlowand Northcutt Narayanan and Narayanan DamicoMarteland Noden Muller and ORahilly ORahilly and M¼ller Abbreviations CN Cranial Nerve m purely motor nerve mix mixed nerve sensory and motor NC neural crest PA pharyngeal branchial arch r rhombomere s purely sensory nerve There is no known ganglionof the accessory nerve The cranial part of the accessory nerve sends occasional branches to the superior ganglion of the vagus nerveMndezMadonadoletalNeuralCrestDerivedCranailNerves\x0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Contribution of neural crest cells to the formation of cranial nerves I III V VII VIII IX X and XI These selected cranial nerves are formed by thecontribution of cranial placodes and neural crest cells indicated in green Neural crestderived Schwann cells produce peripheral myelination of cranial nerves IIIXIIThe sensory nerves are the olfactory I the optic II and the vestibulocochlear VIII The motor nerves are the oculomotor III the trochlear IV the abducens VIand the accessory XI The remaining are mixed nervesganglion but the diï¬erentiation of these cells is delayed untilthe migration and diï¬erentiation of the corresponding placodalcells in chicks Covell and Noden Placodal speciï¬cationand development as well as its contribution to the assembly ofplacodal derivatives is a complex and wideranging topic thatis beyond the scope of this review We will focus on discussingthe main signaling pathways and relevant transcription factorsinvolved in the speciï¬cation of cranial NCCs precursors theirdiï¬erentiation to form CNs and ganglia that are exclusively NCderived and the alterations caused by the mutations of certaingenes that are important for the neurogenesis of NC derivativesSIGNALING PATHWAYS INVOLVED INCRANIAL NEURAL CRESTDEVELOPMENTareseveralsignaling pathwaysThereand transcriptionfactors that are known to regulate NC and CN formationduring development We discuss some important pathwaysinvolved in cranial NCCs induction and speciï¬cation in closerelationship with the cranial ganglia and nerves derived from theNC Figure BMPsBone morphogenetic proteins are proteins that control severalimportant steps in the formation and diï¬erentiation of the CNSof vertebrates These proteins act in diï¬erent regions of theCNS to regulate fate proliferation and diï¬erentiation Aftergastrulation the presence of BMPs and the activation of thissignaling pathway are essential for the diï¬erentiation of thenonneural ectoderm whereas the inhibition of this pathway isrequired for the proper formation of the neural plate It has beenproposed that the later activation of BMPs receptors participatesin the induction of the NC through a very ï¬ne regulation wherethe presence of BMPs at a speciï¬c time will give rise to the NCin mouse and human Embryonic Stem Cells ESCs Figure 3BMizuseki Leung Seminal studies in Xenopus have shown that there is an activitygradient of BMPs controlled by their antagonists and that anintermediate level is needed to induce the formation of the NCLaBonne and BronnerFraser Marchant Barth Tribulo Thus the BMP antagonistsChordin OMIM and Noggin OMIM areexpressed in a spatiotemporal manner thatinï¬uences theformation of the NC In mouse at embryonic day E Nogginis expressed in the neural folds and in the dorsal region afterthe closure of the NT The expression of Chordin is low atthe level of the neural plate and in the paraxial mesodermThese antagonists participate in the induction of NC as wellas in delamination but also protect from apoptosis induced byBMP during migration and diï¬erentiation of NCCs Importantlyit was observed that the decrease in the expression of theseBMP antagonists alters the PNS derived from the NC andcraniofacial skeletal elements Noggin knockout mice presentedall cranial nerves but the vagus X and glossopharyngeal IX aredisanized and fused Doubleknockout mice of Noggin andChordin lack CN and only a structure similar to the trigeminalganglion V is present Anderson In the chickembryo the activity of BMP signaling during the formation ofNC precursors is modulated by CKIPSmurf factors throughthe regulation of Smad degradation resulting in intermediateFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Gene regulatory network involved in neural crest contribution to the formation of cranial nerves The cranial ganglia and cranial nerves are formed inprecise positions along the dorsoventral and anteroposterior axes of the midbrainhindbrain region A The drawing represents a human embryo at stage days somites equivalent to mouse day E9510 somites and chick stage h somites The cell signaling pathways that providedevelopmental cues to neural crest precursors are colorcoded when these factors diffuse the target regions are indicated with arrows with the same color In panelB an idealized scheme of the hindbrain shows the cell signaling gradients and the genes that establish the dorsoventral pattern C The illustration of the human days stage and chick stage hindbrain rendered ï¬at to eliminate cerebral ï¬exures The levels of origin of the neural crest cells NCCs and placodeswhich contribute to the formation on cranial nerves are indicated on the left NCCs from the corresponding rhombomeres also populate other embryo structures in asegmental fashion and generate different craniofacial derivatives The positions of the cranial ganglia and the otic vesicles are indicated on the right side thecontribution of NCCs is indicated in green The segmental nested expression of HOX genes is colorcoded On the right signaling pathways and the expression oftranscription factors involved in cranial nerve CN formation are indicated Adapted from Lumsden and Keynes Noden Yamamoto and Schwarting BallyCuif and Wassef Takahashi and Osumi and M¼ller and ORahilly Abbreviations CN cranial nerve FP ï¬oor plate Mmesencephalon NCCs neural crest cells OV otic vesicle r rhombomere PA pharyngeal archeslevels of BMP activity required for proper NC formationPiacentino and Bronner In contrast placode progenitorshave diï¬erential BMP signaling requirements as they can bespeciï¬ed under low or no BMP signaling Thiery A study of human ESCs hESC showed that if BMPs areblocked with Noggin for h on days or of thediï¬erentiation protocol there is a dramatic decrease in theinduction of human NCCs However if the inhibition is madeon day the inhibition is partial so the participation of BMPsat the beginning of the induction of the NC is very importantwhile the inhibition of this pathway promoted the expressionof neural genes such as SOX1 OMIM HES5 OMIMFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial Nerves and SOX2 OMIM Leung Thisprotocol produced sensory peripheral neurons and it will beof interest to investigate if such neurons can contribute to thesensory CN after grafting them in experimental animals aswell as the eï¬ect of modulating BMPs on peripheral neurondiï¬erentiation Interestingly BMP antagonism upregulates theseneural stem cell markers but several reports indicated that Sox1Hes5 and Sox2 are involved in the suppression of neuronaldiï¬erentiation by maintaining neural stem and progenitorcells in an undiï¬erentiated state in mammalian cells Kan BaniYaghoub The generation ofneurons from stem cells depends on the decrease of Sox13expression caused by proneural proteins However if Sox13target genes were repressed independently of proneural activityneural progenitor cells diï¬erentiated prematurely and someneuronal features emerged These results demonstrate a dualrole of proneural proteins in the acquisition of a deï¬nitiveneuronal fate and indicate that the proneural proteindirectedrepression of Sox13 expression is a required and irreversiblestep in the commitment to neuronal diï¬erentiation in severalspecies including mammals Guillemot Farah Bylund BMP4 OMIM and Smad proteins have beeninvolved in an interesting mechanism called retrograde signalingin trigeminal ganglia from rats Ji and Jaï¬rey Thismechanism elicits a speciï¬c transcriptionalresponse thatcontributes to the speciï¬cation of diï¬erent subpopulations ofsensory neurons in the trigeminal ganglia CN V As axonsfrom the neurons oftrigeminal ganglia grow and extendinto their three main peripheral axonal branches ophthalmicmaxillary and mandibular that innervate the correspondingregions of the face they encounter BMP4 which results in aretrograde signal that leads to transport back transcription factorsSMAD1 and from axons to the somata where nuclearaccumulation of the phosphorylated and transcriptionally activeSmad forms contributes to neuronal speciï¬cation and gangliapatterning Nohe Ji and Jaï¬rey BDNF BrainDerived Neurotrophic Factor OMIM signaling wasalso found to regulate axonal levels of SMAD1 and inconcert with BMP4 for patterning of the trigeminal gangliaJi and Jaï¬rey Hippo PathwayGenetic studies have demonstrated that Hippo signaling is crucialin an size regulation controlling cell number by modulatingcell proliferation and apoptosis processes Huang Hippo is a criticalfactor for proliferation and epithelialmesenchymal transition during embryonic development andcancer In the neural tube of the mouse chicken and frogYAP YesAssociated Protein OMIM is expressed inthe ventricular zone progenitor cells and colocalizes with theneural progenitor cell marker Sox2 Milewski Cao It has been observed that the ectopic expressionof one ofthis pathwayTAZ Transcriptional Coactivator With PDZBinding MotifOMIM in mammalian cells stimulates cell proliferationthe transcriptional regulators ofreduces the inhibition by contact and promotes the epithelialmesenchymal transition Lei A relationship between this signaling pathway and the classicalNC genes such as interaction with Pax3 is through TAZ andthe phosphoprotein YAP65 These proteins participate as coactivators of Pax3 It has been suggested using transgenicmice that Tead2 TEA Domain Family Member OMIM is an endogenous activator of Pax3 in NCCs Milewski Through expression assays Pax3 and Yap65were colocalized in the nucleus of NC progenitors in thedorsal region offorSchwann cell proliferation and diï¬erentiation in a stagedependent manner Nuclear TAZYAP complexes activate cellcycle regulators to promote Schwann cell proliferation whiledirecting diï¬erentiation regulators in cooperation with Sox10 formyelination in rodents Deng the NT HippoTAZYAP are criticalNeuroï¬bromatosis Nf2 OMIM is a tumorsuppressor that inhibits YAP during dorsal root ganglia DRGdevelopment Merlin encoded by the NF2 tumorsuppressivegene was identiï¬ed through genetic studies in mouse embryosand proved to be an important upstream regulator of theHippoYap pathway Neuroï¬bromatosis is an inherited diseasecharacterized by the development of bilateral Schwann celltumors originated from CN VIII Mouse with speciï¬c Schwanncellinactivated Nf2 alleles developed schwannomas and SChyperplasia McClatchey Giovannini Merlin has also been shown to act as a suppressor ofmouse neural progenitor proliferation by inhibiting TAZYAPpathway activity Lavado The mechanism bywhich Merlin regulates YAP activity might involve p21 Proteinactivated kinase PAK1 OMIM activation whichinduces phosphorylation of Merlin thus abrogating its scaï¬oldfunction for YAP and LATS12 OMIM andthereby attenuates YAP phosphorylation by LATS12 in mousecells Sabra it has been suggested that nuclearexport signals of Merlin mediate YAP nuclear export in epithelialmammalian cells Furukawa Hindley and coworkers investigated the role of HippoYAPsignaling in NC development and neural diï¬erentiation Theyshowed thatthe activity of YAP promotes an early NCphenotype accompanied by premature migratory behavior andthat HippoYAP interacts with RA signaling in hESCs Hindley A recent study demonstrates that YAP is necessaryfor the migration of a premigratory pool of NCCs sincethey incorporated YAP signaling into a BMPWntdependentmolecular network responsible for the migration of trunklevelNC in avians Kumar Notch SignalingNotch is a family of conserved receptors whose activation isinduced by speciï¬c ligands Delta1 OMIM Delta OMIM Delta4 OMIM Jagged1 OMIM and Jagged2 OMIM through interactionwith four possible receptors Notch14 Perdigoto and Bardin Once the Notch receptors are activated through thecellcell interaction proteolytic cuts are carried out resultingin the release ofthe Notch Intracellular Domain NICDFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesMumm NICD translocate to the nucleus andforms a transcriptional complex together with the DNA bindingprotein CBF1 C promoter binding factor OMIM This complex recognizes the speciï¬c sequence CTGTGGGAAin itstarget genesfor example Hes1 OMIM Kageyama Notch1 receptor is present during development oftherhomboencephalon at E95 in mice showing strong expressionwithin the hindbrainincluding the trigeminal geniculatepetrosum and nodose placodes which give rise to CN VVII IX and X respectively and is also expressed in the oticand olfactory vesicle Reaume A study wherehuman induced pluripotent stem cells were induced toward NCdiï¬erentiation showed that when Notch signaling is blockedusing a Îsecretase inhibitor DAPT or shRNA for JAGGED the genes specifying NC [DLX5 Distalless homeobox OMIM PAX3 SNAI2 SOX10 and TWIST1 OMIM] are downregulated However the ectopic expression ofNICD1 increased its expression demonstrating that Notch alsoparticipates signiï¬cantly in NC induction Noisa Mead and Yutzey evaluated the function of Notch signaling inmurine NCderived cell lineages in vivo They demonstratedthat cellautonomous Notch has an essential role in properNCCs migration proliferation and diï¬erentiation with criticalimplications in craniofacial cardiac and neurogenic developmentMead and Yutzey Sonic HedgehogSonic Hedgehog Shh signaling is involved in the correctdevelopment of NC and therefore in the generation of its cellularderivatives Figure 3B Shh is a member of the family ofthe secreted Hedgehog proteins Sonic Shh OMIM Indian Ihh OMIM and Desert Hedgehog Dhh OMIM Shh regulation during NC diï¬erentiation is crucialduring head and face morphogenesis Mutant mice and humanslacking Shh present holoprosencephaly and cyclopia due to thelack of separation of the forebrain lobes Chiang It is suggested that Shh inhibition maintains Pax3 expressionso the lack of Shhmediated regulation for Pax3 inductionpromotes the constitutive induction of NC generating theaforementioned phenotypes A subset of Fox genes regulated byShh signaling is important during lip morphogenesis in miceEither Shh addition or Foxf2 OMIM overexpressionwas shown to be suï¬cient to induce cranial NCCs proliferationEverson On the other hand enhanced Shh signaling in mousemediated by lossoffunction Ptch1WigWig of the Shh receptorPatched1 Ptch1 OMIM suppressed canonical Wntsignaling in the CN region This critically aï¬ected the survivaland migration of cranial NCCs and the development ofplacodes as well as the integration between NC and placodesKurosaka Ptch1WigWig mutants exhibited severelydisanized trigeminal CNV and facial nerves CNVII thatdid not develop properly and failed to project to their appropriatetargettissues Kurosaka High levels of Shhsignaling have been correlated with Moebius Syndrome whichis characterized by cranial nerve defects including trigeminalabducens CNVI and facial alterations concurrent with othercraniofacial defects Verzijl VegaLopez NCCs migration is particularly sensitive to Shh levels since inmice lacking Shh these cells continue their migration beyondthe normal position and fuse medially condensing into a singlemidline ganglion Fedtsova Mutation in the mouseHedgehog acyltransferase Hhat OMIM gene producedhypoplasia and aberrant fusion of cranial ganglia CN V VII IXand X and aï¬ected NC and placode gene markers expressionsuggesting that a regionalized action of the Hedgehog signalingis required for proper cranial ganglia and nerve developmentand patterning Dennis In vitro analyses showedthat Shh increased the number of cranial NC progenitors fromquail embryos yielding neural and mesenchymal lineages Shhcan decrease the neuralrestricted precursors without aï¬ectingsurvival or proliferation These data also suggestthemesenchymalneural precursor was able to yield both the PNSand superï¬cial skeleton Calloni thatReceptor Tyrosine Kinase RTK FamilyHumans have known RTKs which fall into subfamiliesA few years ago a systematic work summarized the contributionof the mouse model to the understanding of the role of a subsetof RTKs in regulating the activity of NCCs in developmentFantauzzo and Soriano With respect to its downstreamsignaling RTKs induce the activation of various pathwaysincluding PLCÎ PI3K MAPK JNK Shc Erk and the JAKSTATpathways In this section we discuss insights pointing tomechanisms of action of some RTK families in relation tothe development of the cranial NC that have emerged fromrecent evidenceEph ReceptorsEphrin ligands and Eph erythropoietinproducing humanhepatocellular carcinoma receptors comprise an increasinglywell studied family of signaling molecules Ephrins bind totwo families of transmembrane tyrosine kinase receptors EphAand EphB While Atype Ephrins preferentially bind to EphAreceptors Btype Ephrins do so to EphB receptors In Xenopusthe streams of NCCs going to the second branchial arch expressEphrinB2 whereas cells reaching the third arch express EphB1disruption of EphEphrin signaling results in aberrant migrationof NCCs causing mixing of the streams in the branchial pouchesSmith Eph receptor functions are best characterizedin the mouse nervous system where they are involved inneuronal development and axon guidance Wilkinson Xuand Henkemeyer migration and proliferation Conover H | 2 |
distribution and reproduction in any medium provided the original work isproperly citedLacrimal gland neoplasms comprise up to of all orbital masses clinically and histologically Much of our current coreknowledge regarding lacrimal gland tumors stems from prior study of their more common counterparts the salivary glandsThe prognosis for each lacrimal gland tumor is contingent upon proper clinical evaluation and ultimately the histopathologicdiagnosis We describe a case of an invasive carcinoma expleomorphic adenoma CaexPA with a cystadenocarcinomacomponent arising from the lacrimal gland in the absence of any previously diagnosed pleomorphic adenoma benign mixedtumor or prior incisional surgery This case illustrates the importance ofincludingimmunohistochemistry and genetic testing to narrow a diï¬erential diagnosis and potentially aid or guide therapy in the futureOur ï¬nding suggests that carcinoma of the lacrimal gland may be derived from previously undiagnosed and perhaps evensubclinical pleomorphic adenomathe histopathologic assessment IntroductionMalignant mixed tumor of the lacrimal gland also knownas carcinoma expleomorphic adenoma CaexPA is thethird most frequent epithelial lacrimal gland tumor afterpleomorphic adenoma PA and adenoid cystic carcinomaACA [] Clinically aï¬ected patients tend to be to years older than those with PA and often present with aninsidiously progressive mass of a lacrimal fossa that suddenly becomes symptomatic Other reported clinical settings are patients without any previously known lacrimalgland tumor who develop a rapidly growing mass associated with pain and patients with one or more recurrencesof PA who undergo malignant transformation [] Diagnostic imaging is critical for the clinical diagnosis of a lacrimalgland neoplasm Computed tomography CT and magnetic resonance imaging MRI are ideal but MRI remainsthe preferred method to visualize surrounding tissue anddetect radiographic features concerning aggressive malignancy [] The incidence of a malignant transformation ofPA increases with the duration of the tumor Prior studiesreport that approximately less than of PA undergoesmalignant change within years after the ï¬rst treatmentand by the end of years [] CaexPA can arisein patients without a known preexisting lacrimal glandtumor The malignant component is most often adenocarcinoma not otherwise speciï¬ed however other histologicsubtypes have been described such as adenoid cystic carcinoma ductal carcinoma mucoepidermoid carcinoma andsquamous cell carcinoma 0cCase Reports in PathologyHerein we report a case of an invasive CaexPA with anepithelial component consistent with a cystadenocarcinomain the absence of any previously diagnosed PA and supportedby immunohistochemical and molecular studies Case PresentationA 64yearold male patient presented to the emergencydepartment due to left lateral canthal pain tearing and ipsilateral hearing loss over four months The initial examinationrevealed a visual acuity of bilaterally The pupils werereactive without an aï¬erent pupillary defect Intraocularpressures were and mmHg respectively Confrontational visual ï¬elds and color plates were unremarkable Therewas a complete abduction restriction of the left eye Theexternal examination revealed edematous upper and lowereyelids predominantly overlying the lateral orbital rim associated with temporal sloping and a nontender palpable andï¬xed mass of the temporal fossa a There wasptosis of the left upper eyelid with a reduced marginal reï¬exdistance of two and a half millimeters mm Exophthalmometry measured mm and mm with a base measurement of mm The anterior segment was otherwisenormal The fundus exam revealed symmetrically sharp andpink disc margins without pallor or edemaMaxillofacial CT scan with contrast showed a lytic lesion ofthe left orbital wall with associated heterogeneous soft tissuemass measuring cm medially displacing the left lateralrectus muscle b Magnetic resonance imaging of thebrain and orbits revealed an enhancing ltrating mass of theleft lateral orbital wall extending into the left supra zygomaticmasticator space c A core guided needle biopsywas performed and the hematoxylineosin HE stainedslide revealed a moderately diï¬erentiated adenocarcinomainvolving ï¬brous connective tissue and demonstrating a cribriform architectural pattern with moderate cytologic atypia andindividual cell necrosis Figures 2a and 2bPositron emission tomography and CT of the chest abdomen and pelvis did not reveal any underlying malignancy orevidence of metastases Subsequently the patient underwentleft orbital exenteration with eyelid sparing Grossly the specimen included orbital contents frontal bone portions of thefrontal sinus and zygomatic bone Serial sectioning revealeda cm multilocular cystic mass involving the lacrimalgland fossa abutting the globe superotemporallyHistopathologically the HEstained sections disclosedpredominantly neoplastic cystic structures in the proximityof the lacrimal gland acini measuring to mm in diameterltrating ï¬brous connective tissue and bone cA small focus of pleomorphic adenoma was identiï¬ed associated with a lowgrade ductal carcinoma in situ The invasivecystic component revealed intraluminal papillary architecture and cribriform arch formations of the lining epitheliumd The neoplastic epithelium was composed ofmedium to largesized cuboidal cells with intercalated ductcell appearance eosinophilic cytoplasm and apocrine features Small foci of invasive solid components were observeddemonstrating cribriform architecture moderate to severenuclear pleomorphism and up to mitotic ï¬gures per highpower ï¬eld Columnar cells with pseudostratiï¬ed nucleiwere also present with moderate nuclear atypia Foci of ruptured cysts with hemorrhage granulation tissue lymphocyticltrate and dystrophic calciï¬cation were also seen No lymphovascular or perineural invasion was identiï¬edImmunohistochemical studies using monoclonal antibodies were performed with appropriate positive controls encompassing Gata3 mouse monoclonal antibody predilute Cell Marque Rocklin CA gross cystic disease ï¬uidprotein GCDFP15 mouse monoclonal antibody dilute Thermo Fisher Scientiï¬c Fremont CA androgen receptor AR rabbit monoclonal antibody dilute Cell Marque Rocklin CA SOX10 rabbit polyclonalantibody dilute Cell Marque Rocklin CAprostatic speciï¬c antigen PSA mouse monoclonal antibody predilute Cell Marque Rocklin CAthyroid transcription factor TTF1 mouse monoclonalantibody predilute at micrograms Ventana Medical Services Tucson AZ p63 mouse monoclonal antibody predilute Biocare Medical Concord CA p53mouse monoclonal antibody predilute at microgramsVentana Medical Services Tucson AZ high molecularweight cytokeratin HMWK903 mouse monoclonalantibody predilute Cell Marque RocklinCA CAM mouse monoclonal antibody predilute Ventana Medical Services Tucson AZ estrogen receptor ERmouse monoclonal antibody diluted VectorLaboratories Burlingame CA progesterone receptor PRmouse monoclonal antibody prediluted Leica BiosystemsNewcastle UK Her2neu rabbit monoclonal antibody predilute Ventana Medical Services Tucson AZ and Ki67rabbit monoclonal antibody predilute Ventana MedicalServices Tucson AZ Immunohistochemical studies wereperformed on an automated stainer Leica BiosystemsInc BOND III System Buï¬alo Grove IL All antibodiesand testing were performed in a CLIAcertiï¬ed laboratoryThe invasive component of the tumor was positive fata3 AR HMWK903 and CAM52 and focally positivefor GCDFP15 while negative for p63 ER PR SOX10 PSAand TTF1 stains Figures 2e2h p53 was positive in lessthan of the tumor cells The Ki67 proliferative index ofthe tumor cells was low p63 and SOX10 stainshighlighted the PA and the in situ component of the tumorDetection of HER2 status by immunohistochemistry wasequivocal and reï¬ex testing using ï¬uorescence in situhybridization FISH was negative for HER2neu geneampliï¬cation based on the updated guidelines of theAmerican Society of Clinical OncologyCollege of AmericanPathologists criteria for HER2neu testing in breast cancerMolecular proï¬ling using a nextgeneration sequencingNGS based assay Foundation One was performed onthe lacrimal gland tumor paraï¬nembedded tissue to identify genomic alterations within cancerrelated genes Thefollowing genomic alterations were detected HRAS G13RPIK3CA E542K and BCORL1 H215fs Microsatellitestatus MDstable and tumor mutation burden TMB low1MutsMbThe combined histopathologic immunohistochemicaland molecular ï¬ndings supported the diagnosis of invasive 0cCase Reports in PathologyaabcbcFigure a Clinical photo of the patient at time of presentation direct view b Computed tomography maxillofacial axial cut Illustrating a cm mass of the lateral orbit eroding the lateral orbital wall with a soft tissue component extending into the orbit causing proptosis andnonaxial displacement of the globe c MRI brain and orbit T1 fatsuppressed image with gadolinium showing a lobulated mass withmixed cystic and solid components and inhomogeneous enhancement involving the left lateral orbital wall and suprazygomatic leftmasticator space Additionally the mass demonstrates edema and enhancement suggesting ltration by the massCaexPA disclosing a carcinomatous component with apredominant cystic growth pattern and focal solid areasreminiscent of a cystadenocarcinoma The surgical marginswere negative In addition the microscopic examination ofthe left eye globe revealed pseudoexfoliation syndrome andFuchs endothelial dystrophyThe patient underwent postoperative adjuvant chemoradiation followed by excision of the eyelids with no residualtumor Followup examination showed no evidence of recurrence or metastatic disease nine months after completingadjuvant therapy DiscussionMuch remains unknown about both the natural histologicprogression and malignant transformation of CaexPAWe know that lacrimal gland neoplasms comprise oforbital masses clinically [ ] and of orbital masseshistologically [ ] and nearly half of epithelial tumorsare malignant [] Although uncommon malignant transformation of PA can occur with incomplete excision InvasiveCaexPA can represent malignant transformation of PAmany of which develop recurrence or distant metastasis tothe lung bone abdomen and CNS [] This casehighlights the importance of an immunohistochemical andgenetic evaluation in complex lacrimal tumorsThe malignant epithelial component of CaexPA hasmorphological varieties including adenocarcinoma adenoidcystic carcinoma squamous cell carcinoma mucoepidermoid carcinoma and ductal carcinoma It could be likelya mixture of subtypes [] Rarely the only evidence ofpleomorphic adenoma is the presence of large areas of hyalinized stroma composed of myoepithelial cells and a fewductal structuresIn the current case the carcinomatous component of thetumor discloses predominantly an ltrative cystic growthpattern reminiscent of a cystadenocarcinoma previouslydescribed in neoplasms of the salivary and lacrimal glandsThe term cystadenocarcinoma of the salivary gland hasevolved It encompasses a variety of tumors depicting a cysticpattern of growth to a subset of papillary and cystic malignant neoplasms that have indolent behavior This is alsoobserved in other lowgrade salivary gland carcinomashowever it is important to note that they can demonstrateltrative growthlocal recurrence and metastasize toregional lymph nodes at the time of diagnosisPreviously Foss [] in a review of cases ofcystadenocarcinoma of the salivary gland used the followingdiagnostic criteria occurrence within a salivary gland invasive growth a predominantly cystic pattern ofgrowth with or without a papillary component and theabsence of acinar or mucoepidermoid diï¬erentiation or evidence of origin in a PA In the same review the predominantcell type varies among tumors and includes small cuboidalintercalated ductlike cellslarge cuboidal cells and tallcolumnar cells The subgroups of the large cuboidal cells 0cCase Reports in PathologyababcHMWCK903Androgen receptordedp53ep63fcfghghFigure a Moderately diï¬erentiated adenocarcinoma involving cores of ï¬brous connective tissue HE 2x b High power view of thetumor revealed a cribriform pattern moderate nuclear pleomorphism and scattered mitoses HE 40x c Neoplastic cystic structures inclose proximity to lacrimal gland tissue star in a hyalinized stroma and lymphoid proliferation HE 2x d Tumor that arose from afocus of pleomorphic adenoma star depicting cystic structures with small papillae HE 4x Inset papillary projections lined by largecuboidal cells with moderate nuclear atypia and apocrine features HE 20x e Tumor cells were strongly positive for HMWK903 4xf Nuclear expression of androgen receptor was present in the tumor 20x g p53 positive in less than of tumor cells 4x h p63was negative within the invasive component of the tumor 4xhave central nuclei abundant eosinophilic cytoplasm largenucleoli and apocrine features which are similar to thoseobserved in this case The fourth group has a combinationof cell types Furthermore Foss suggest that tumorswith predominantly columnar cells are associated withincreased metastatic potentialThe cystic growth pattern characteristic of this tumor isoften associated with an ammatory response due toruptures of the dilated structures as noted in this case Cystformation in neoplasias of the salivary and lacrimal glandscan behave as a mimicker of an ammatory process Pakdel report a case of a spontaneous rupture of a PAmasquerading as orbital cellulitis [] Histologically aruptured cystic space of a PA surrounded by a monocyticltrate and foreign bodytype granulomas is described Inthis paper the authors consider the spontaneous rupture ofthe cystic space as an underlying mechanism for the acutepresentation of this tumorCystadenocarcinoma can have a broad diï¬erentialdiagnosis The principal considerations include papillary cystic acinic cell adenocarcinoma secretory carcinoma mucoepidermoid carcinoma MEC and ductal carcinoma Aciniccell carcinoma and secretory carcinoma are typically indolent monotypic tumors that can disclose a papillary cysticarchitecture Histologicallysecretory carcinoma sharesnearly identical growth patterns to acinic cell carcinoma 0cCase Reports in Pathologybut instead shows a multivacuolated eosinophilic cytoplasmoften with luminal and intracytoplasmic mucin and no truezymogen granules Immunohistochemically secretory carcinoma is S100 and mammaglobinpositive and typicallynegative for DOG1 while acinic cell carcinoma shows theopposite staining proï¬le Additionally secretory carcinomaharbors t1215p13q25 resulting in an ETV6NTRK3 genefusion [] MEC is usually composed of a mixture ofpredominantly epidermoid squamoid cells abundant intermediate cells ranging from small basal cells with basophiliccytoplasm to larger cells with eosinophilic cytoplasm andmucous cells Welldiï¬erentiated MEC is a circumscribedtumor that can disclose glandular and cystic structures linedby a single layer of mucussecreting cells however the intermediate and highgrade tumors show solid nests or sheets ofcells composed of primarily epidermoid cells with a scant cystic component and obvious invasion severe pleomorphismnecrosis and increased mitosesOn the other hand primary ductal adenocarcinoma ofthe salivary gland SDC originates from the excretoryportion of the salivary duct is a rare aggressive malignantepithelial tumor and accounts for only of epithelial lacrimal gland tumors Histologically the tumor is highly ltrative and usually solid with occasional cystic areas disclosingbreastlike ductal carcinoma features with central necrosisOccasionally cystadenocarcinoma with large cuboidal cellseosinophilic cytoplasm and highgrade nuclear atypia bearssome similaritiesto ductal adenocarcinoma Howeverpapillarycystic invasive growth is not usually seen in ductaladenocarcinoma [] The invasive component features trabeculae ducts and sheets of neoplastic cells in a desmoplasticstroma with perineural and vascular invasion The latter isnot commonly observed in cystadenocarcinoma Immunohistochemically the tumor cells are positive for a low molecular weight cytokeratin CAM52 CK7 CEA EMA andGCDFP15 Other than epithelial markersthis tumorexpresses AR in up to of invasive cases HER2 positivityin of cases and p53 overexpression in all reportedcases The Ki67 proliferative index is over The tumoris ER and PR negativeFurthermore in this case the tumor also demonstratescytomorphologicalfeatures similar to that of a low tointermediategrade ductal carcinoma in situ of the breast Thelatter ï¬ndings might correlate with the previously describedlowgrade cribriform cystadenocarcinoma of the salivary glandalso known as lowgrade salivary ductal carcinoma and salivaryductal carcinoma in situ currently categorized as intraductalcarcinoma low grade and high grade respectively These typesof tumors show a variety of growth patterns both solid and cystic ranging from cribriform to solid to micropapillary and arereminiscent of lowgrade ductal carcinoma in situ of the breastFocal ltration may be noted []The exact pathogenesis of CaexPA remains controversial Prior studies have demonstrated that PA and CaexPA of the salivary and lacrimal glands share similar genomicproï¬les and frequently overexpress the PLAG1 oncoprotein[ ] Harrison [] in their review indicate that thedevelopment of CaexPA follows a multistep model of carcinogenesis with the progressive loss of heterozygosity at 8qthen 12q and ï¬nally 17p Alterations including ampliï¬cationgene fusion and translocations in 12q genes such as HMGICHMGA2 and MDM2 may play a role in the malignant transformation In the same study the authors also mention thatloss of 17p is usually common in CaexPA indicating tumorsuppressor gene p53 loss as this tumor evolves Additionallyit appears that CaexPA and other malignant epithelialtumors other than ACC do not harbor MYB gene rearrangements or fusions It is important to note that there does notappear to be a correlation between rearrangement statusand clinical outcome []Additional mutational analysis of the lacrimal gland carcinomas has been also evaluated [] demonstrating that RASKRAS NRAS PIK3CA and MET mutations are frequent indiverse epithelial neoplasms of the lacrimal gland with thehighest proportion of mutations found in adenoid cystic carcinoma PIK3CA and MET mutations can coexist with RASmutationsPIK3CA and HRAS mutations are detected in this casewhich correlates with alterations already described in lacrimal gland carcinomas however alterations in MDM2HMGA2 NTRK3 p53 PLAG1 and ETV6 among others werenot observedIn summary this case demonstrates an invasive CaexPA with a malignant epithelial component that resemblesthe cystadenocarcinoma mixed cell type described by Foss [] Immunohistochemically the tumor was ARpositive while negative for ER PR and Her2 with expression of p53 in less than of the tumor cells Theprevalence of AR varies between the diï¬erent subtypes ofsalivary gland carcinomas SGC In recent medical literature data AR expression has been detected in as many as of SDC [] Dalin [] in the same study alsoindicate that adenocarcinoma and acinic cell carcinoma ofthe salivary gland express AR in and of the casesrespectivelyOur ï¬ndings emphasize the importance ofThe current classiï¬cation of the salivary gland tumors[] and the combined histopathologic and immunohistochemical features suggest that this tumor could representthe results of the natural course of a lowgrade cystadenocarcinoma with focal transformation into an intermediatehighgrade invasive ductal carcinoma expleomorphic adenomafurtherexploration of CaexPA pathogenesis especially the extentof disease and the histologic subtype In additiontheimmunohistochemical and genetic testing provides important support for the diagnosis as well as potentially guidesfuture therapy and prognostic evaluation Correctly identifying the type of malignant epithelial component is a signiï¬cant factor in the survival of aï¬ected patients To ourknowledge this patient represents the ï¬rst case in whichCaexPA of the lacrimal gland reveals an indolent epithelial malignancy with a low proliferative index arising froman undiagnosed PA after many likely consecutive molecular alterationsLastly due to the rarity of these tumors arising from thelacrimal gland further studies are necessary to evaluate theirbiologic behavior and determine any correlation betweenCaexPA and pseudoexfoliation syndrome 0cCase Reports in PathologyEnd Results International Journal of Ophthalmology vol no pp [] F A Jakobiec J R Bily and R L Font Orbit in OphthalmicPathology An Atlas and Textbook W H Spender Edpp WB Saunders Co Philadelphia 4th edition[] M H Devoto and J O Croxatto Primary cystadenocarcinoma of the lacrimal gland Ophthalmology vol no pp [] R H Simpson Salivary duct carcinoma new developmentsmorphological variants including pure in situ high gradelesions proposed molecular classiï¬cation Head and NeckPathology vol Supplement pp [] R D Foss G L Ellis and P L Auclair Salivary gland cystadenocarcinomas a clinicopathologic study of cases TheAmerican Journal of Surgical Pathology vol no pp [] L Barnes J W Eveson P Reichart and D Sidransky Pathology and Genetics of Head and Neck Tumours IARC [] J Antony V Gopalan R A Smith and A K Y Lam Carcinoma ex pleomorphic adenoma a comprehensive review ofclinical pathological and molecular data Head and NeckPathology vol no pp [] F Pakdel N Pirmarzdashti S Soltani Z Nozarian F AAmoli and A Kassaee Spontaneous rupture of lacrimalgland pleomorphic adenoma Ophthalmic Plastic and Reconstructive Surgery vol no pp e41e43 [] R R Seethala Head and neck pathology Surgical PathologyClinics vol no [] S L von Holstein A Fehr M Persson Lacrimal GlandPleomorphic Adenoma and Carcinoma ex PleomorphicAdenoma Genomic Proï¬les Gene Fusions and Clinical Characteristics Ophthalmology vol no pp [] T Y Chen M G Keeney A V Chintakuntlawar et alAdenoid cystic carcinoma of the lacrimal gland is frequentlycharacterized by MYB rearrangement Eye vol no pp [] M Dalin P Watson A Ho and L Morris Androgen receptor signaling in salivary gland cancer Cancers vol no p Conflicts of InterestNone of the authors declare any competing interests Currently Dr Del Valle Estopinal is the Director of OphthalmicPathology and Assistant Clinical Professor of the Departments of Ophthalmology and Pathology at University ofCalifornia Irvine The City Dr S Orange CA Authors ContributionsDr Maria Del Valle Estopinal was the ocular pathologistthat performed the histopathologic immunohistochemicaland genetic evaluation Dr Bryant Carruth wastheoculoplasticstrained ophthalmologist who performed thecore biopsy The other authors participated in the clinicalcare and preparation of this manuscript All authors readand approved the ï¬nal paperAcknowledgmentsThe authors acknowledge the cooperation of the patient Theyacknowledge the help of the SUNY Upstate Departmentof Pathology and Eye Plastic and Reconstructive Surgeonsof Central New York in the contribution to evaluationand treatment of this patientReferences[] C L Shields J A Shields R C Eagle and J P Rathmelllacrimal glandClinicopathologic review of cases oflesions Ophthalmology vol no pp [] R L Font J O Croxatto and N A Rao Tumors of the Eye andOcular Adnexa American Registry of Pathology WashingtonDC [] W Harrison P Pittman and T Cummings Pleomorphicadenoma of the lacrimal gland a review with updates ontransformation and molecular genetics SaudimalignantJournal of Ophthalmology vol no pp [] SO Baek YJ Lee SH Moon YJ Kim and YJ JunPrimary adenocarcinoma of the lacrimal gland Archives ofPlastic Surgery vol no pp [] D Bell M C Sniegowski K Wani V Prieto and B EsmaeliMutationallacrimal gland carcinomas andimplications for treatment Head Neck vol Supplement pp E724E729 landscape of[] J A Shields C L Shields J A Epstein R Scartozzi and R CEagle Jr Primary epithelial malignancies of the lacrimalgland the Ramon L Font lecture Ophthalmic Plastic Reconstructive Surgery vol no pp [] Lacrimal Gland Tumor Study Group An epidemiologicalsurvey of lacrimal fossa lesions in Japan number of patientsand their sex ratio by pathological diagnosis Japanese Journalof Ophthalmology vol no pp [] S L von Holstein M H TherkildsenJ U PrauseG Stenman V D Siersma and S Heegaard Lacrimal glandlesions in Denmark between and Acta Ophthalmologica vol no pp [] W M Hassan M S Bakry H M Hassan and A S AlfaarIncidence of orbital conjunctival and lacrimal gland malignant tumors in USA from Surveillance Epidemiology and 0c' | 2 |
recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junÃn virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ ] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavirritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm | 0 |
" ethnopharmacological relevance herba patriniae has been used for thousands of years in china as a traditional chinese medicine with heatclearing and detoxicating effects it is applied widly for the treatment of rheumatoid arthritis diarrhea acute hepatitis pelvic inflammatory disease and ulcerative colitis in clinic two species namely patrinia scabiosaefolia fisch ps and patrinia villosa juss pv from the caprifoliaceae family are considered as herba patriniae in the pharmaceutical industry aim of the review this paper aims to comprehensively outline the traditional uses botanical description phytochemistry pharmacology toxicology quality control pharmacokinetics and patents of herba patriniae and elaborate the samedifferent characteristics between ps and pv materials and methods detailed information of herba patriniae was collected from various online databases pubmed web of science google schola china national preproof 0c knowledge infrastructure database national intellectual property administration prc national medical products administration and those published resources msc thesis and books results a total of compounds have been identified in herba patriniae including triterpenoid saponins flavonoids anic acids irids and volatiles a very distinct difference was observed that ps is rich in triterpenoid saponins and volatiles while pv contains more flavonoids two source species of herba patriniae gave similar pharmacological effects on anticancer antiinflammatory antioxidant antimicrobial sedative and hypnotic effects but there were no reports were on antipruritic proangiogenic and antidiarrheal effects for ps and no studies on antidiabetic effects for pv generally herba patriniae showed nontoxic in the clinical dose but mild side effects such as temporary leukopenia dizziness and nausea could be found when large and excessive dosage is used a variety of compounds have been quantified for the quality control of ps and pv the variety growth environment growth time and harvest time not only affected the contents but also the pharmacological activities of the bioactive compounds in the past year patents for compositions containing pv and ps have been filed mainly involving human health hygiene agriculture and animal husbandry unfortunately the research on pharmacokinetics is insufficient only the prototype components and metabolites were repored after intragastric administration of total flavonoids extract from pv in rats herba patriniae has displayed a significant medicinal value in clinic but the differences in phytochemistry pharmacological effects and the content of compounds have been found between two official recorded species about side effects and pharmacokinetic characteristics the differeces between two species have not been well studied for a better clinical use of herba patriniae it is urgent to establish systematic pharmacology quality control pharmacokinetics and clinical researches on the samedifferent characteristics between ps and pv keywords herba patriniae traditional uses phytochemistry pharmacology quality control preproof 0c cells a549 human lung cells cancer aspartate polysaccharide mixture ast list of abbreviations 3t3l1 preadipocytes 5fuhct8 human ileocecal adenocarcinoma cells a2780 human ovarian cancer cells a375s2 human melanoma cells a498 human renal abts carcinoma 'azinobis3ethylbenzothiazoline6sulphonic acid ags human gastric cancer cells akt protein kinase b alt alanine aminotransferase ap acute pancreatitis ap3 aminotransferase bax bcl2associated x protein bcl2 bcell lymphoma2 bclxl b cell lymphoma factor xl bel7402 human hepatoma cells bv2 mouse microglia cells caco2 human colon cancer cells cox2 cyclooxygenase2 crc colorectal cancer dai disease activity index dpph 22diphenyl1picrylhydrazyl ec50 half maximal effective concentration emt epithelialmesenchymal transition fak focal adhesion kinase gcms gas chromatographymass spectrometer glut4 glucose transporter gsh glutathione h2o2 hydrogen peroxide hela human cervical cancer cells hepg2 human hepatoma cells hl60 human promyelocytic leukemia cells ho1 heme oxygenase1 hplc high performance liquid chromatography hsp heat shock proteins hsp heat shock proteins ht1080 human fibrosarcoma cells ht29 human colon carcinoma cells huvecs human umbilical vein endothelial cells ic50 inhibitory concentration icam1 intercellular adhesion molecule icr institute of cancer research il1 interleukin1 beta il6 interleukin il8 interleukin inos inducible nitric oxide synthase irs insulin receptor substrate k562 human malignant myeloid cells ldh lactate dehydrogenase lps lipopolysaccharides mcf7 human breast cancer cells mda malondialdehyde mdamb231 human breast cancer cells mpo myeloperoxidase mrna messenger ribonucleic acid nfκb nuclear factor κb ngf nerve growth factor no nitric oxide nqo1 quinine oxidoreductase nrf2 nuclear factor erythroid 2related factor o2 superoxide anion oh hydroxyl radical pcna proliferating cell nuclear antigen pid pelvic inflammatory disease ps patrinia scabiosaefolia fisch pv patrinia villosa juss raw2647 mouse leukemic monocyte macrophage ros reactive oxygen species rsv respiratory syncytial virus sars severe acute respiratory syndrome sd sprague dawley sgc7901 human gastric cancer cells smmc7721 hepatocellular carcinoma cells stat3 signal transducer and activator of transcription sw480 human colon carcinoma cells tc50 half toxic concentration tcm traditional chinese medicine tgf transforming growth factor beta ti drug treatment index tnfα tumor necrosis factor alpha taoc total antioxidant capacity tsod total superoxide dismutase u14 mice cervical cancer cells u266 human multiple myeloma cancer cells u937 human lymphoma cells uc ulcerative colitis uv ultraviolet preproof 0c table of contents introduction traditional uses botany phytochemistry triterpenoid aglycones and triterpenoid saponins flavonoids anic acids irids volatiles other compounds pharmacology anticancer effect antiinflammatory effect antioxidant effect antimicrobial antiviral and antifungi effects sedative and hypnotic effects others toxicity quality control pharmacokinetics patented formulations and perspectives acknowledgements conflict of interest author contribution references preproof 0c introduction herba patriniae as known as bai jiang cao in chinese is a traditional chinese medicine tcm originally recorded in shen nongs herbal classic as a middle grade medicinal material which has been used for thousands years besides korean ancient pharmacopaea donguibogam also record its medical value and it has been used for more than years in korea jeon et al it possesses the tcm properties of pungent and bitter in flavor and slightly cold in nature and has been classified to the stomach large intestine and liver meridians xiao two official species of patrinia scabiosaefolia fisch ps and patrinia villosa juss pv figure were considered as herba patriniae in chinese pharmacopoeia edition and chinese provincial pharmacopoeias these two plants have been widely used for more than years with good biological activities of clearing heat and detoxification eliminating carbuncle and expelling pus dispelling blood stasis and relieving pain through an analysis of ancient and modern literatures herba patriniae was mostly used in intestinal carbuncle lung carbuncle gynecological epigastric pain postpartum blood stasis and eczema in ancient times chen and han modern pharmacological studies have found that it has effects of anticancer antiinflammation antipathogenic microanisms antioxidation sedation and hypnosis wang et al 2019a nowadays herba patriniae is widely used in the respiratory system digestive system genitourinary system gynecology dermatology and other multidisciplinary diseases in clinical practice zhu and jiang and the number of applied patents increases every year httppsssystemcnipagovcn in view of its high content of amino acids vitamins minerals and other nutrients herba patriniae is not only regarded as a potherb with healthy value but also processed into tea products su et al zeng et al zhong et al in the past decades an increasing number of scholars have studied the chemical constituents and pharmacological effects of herba patriniae interestingly based on these studies we found that there are many differentsame characteristics between ps and pv both of them are official species for herba patriniae but differentiated clinical uses of them in different diseases may be better for the clinical outcome unfortunately we cannot found a comprehensive and updated review on the samedifferent characteristics of the two sources of herba patriniae and actually these two species also have not been differentiated in clinical uses therefore this review aims to systematically summarize the similarities and differences from the preproof 0c aspects of the traditional uses botanical description phytochemistry pharmacology and quality control of these two species of herba patriniae as well as being evidences for their clinical application and further research figure two species of herba patriniae a patrinia villosa juss b patrinia scabiosaefolia fisch a httpwwwcvhaccnspmcshcsh0005548 b httpwwwcvhaccnspmsyaufsyauf010108 traditional uses herba patriniae has a wide geographical distribution mainly in east asia and north america he et al some plants such as sonchus arvensis l sonchus asper vill sonchus oleraceus l etc may be confused as herba patriniae lu and hence these adulterants of herba patriniae should be exclude when clinical use traditionally according to records of shen nongs herbal classic ç¥åæ¬èç» compendium of materia medica æ¬ è 纲 ç® and synopsis of the golden chamber éå®è¦ç¥ tai ping sheng hui fang 太平å£æ æ¹ pu ji fang æ® æµæ¹ sheng ji zong lu å£æµæ»å½ qian jin yi fang åéç¿¼æ¹ and qian jin fang åéæ¹ ancient doctors have used the whole herbs and roots of herba patriniae for disease treatment such as the stomach intestine liver gallbladder and gynecological diseases tian and tian zhu and jiang herba patriniae was recorded in chinese pharmacopoeia edition for the treatments of appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain swollen wellingabscess and clove sores pharmacopoeia committee of the ministry of health of p r china in addition herba patriniae is also preproof 0c recorded in the standards of traditional chinese medicine in many provinces of china table in miao nationality herba patriniae is also called jia jiang le and used to treat rheumatoid arthritis colds and diarrheal qiu wang in dong medicine lu yi medicine drug control institute of yunnan chuxiong health bureau and dai medicine shi ps is called nyangt ngeec liongc bail jangl she wei long and pa hong respectively its whole herb is used to treat infantile diarrhea schizophrenia and infantile tinea capitis respectively ps is also called ba gai bao in zhuang medicine and its root is used to treat icteric hepatitis furuncles and snakebites shi pv is called bitter vegetable by she nationality biological products identification institute of the ministry of health and pao zi tong by tujia nationality peng and guan its whole herb can be used to treat appendicitis intestinal febrile symptoms constipation mammary abscess blister carbuncle and qi stagnation pv is also called ba gai lan and hong pa in zhuang medicine biological products identification institute of the ministry of health and dai medicine shi respectively and its root is used to treat jaundice hepatitis furuncle local ulceration caused by snake injury and infantile convulsion moreover in korea people usually use the roots or whole plants of ps and pv as a traditional herbal medicine to treat appendicitis inflammation wound healing edema abscesses endometritis and abdominal pain after childbirth kang et al yang et al in recent years herba patriniae has been extensively applied in clinical practice in china especially in gynecology such as postpartum pain mastitis dysmenorrhea and tubal obstructive infertility liu 2019a it is noteworthy that herba patriniae is one of the most important ingredients in many prescriptions of tcm which is effective in diarrhea he acute hepatitis song pelvic inflammatory disease zhang 1997a typhoid fever paratyphoid fever sun ulcerative colitis liu anal cryptitis shi pelvic endometriosis yan and qiu acute pancreatitis he et al 2019b itching wang and wang gastroesophageal reflux disease benign prostatic hyperplasia rhinosinusitis mumps and phlebitis kong and zhao zhu and jiang a powder composed of coicis semen radix aconiti lateralis preparata and herba patriniae is a classic prescription for treating intestinal carbuncle in the synopsis of the golden chamber which is clinically used to treat chronic appendicitis chronic pelvic inflammatory disease and chronic prostatitis ji in addition a powder containing herba preproof 0c patriniae in the prescription is also used to treat sinusitis acute purulent tonsillitis and recurrent upper respiratory tract infection qin and diao zhu and jiang moreover it showed significant efficacy in the treatment of psoriasis vulgaris yan et al keshan disease scientific research cooperation group of herba patriniae in yan'an city for the prevention and control of keshan disease and chronic pelvic inflammation jia in the form of tablets in the chinese pharmacopoeia edition there are chinese herbal medicine prescriptions containing herba patriniae among which kangfu xiaoyan shuan and yifei qinghua gao are used to treat gynecological diseases and respiratory diseases respectively while longqing pian nankang pian niaosaitong pian and qianliexin jiaonang are used to treat genitourinary diseases state pharmacopoeia commission of p r china a summary of the traditional and traditional and clinical preparation of herba patriniae in china is given in table the tender stems and leaves of herba patriniae are rich in nutrients fresh in taste and grow in the mountains without environmental pollution it is a highquality vegetable that urban and rural residents like to eat pv tea is also abundant in hubei province and fujian province jiang 2019a xu et al herba patriniae is not only used in human health but also in agriculture fishery and animal husbandry interplanting herba patriniae in the newly reclaimed tea garden can increase the natural vegetation and reduce soil erosion and surface runoff caused by rainstorm erosion in the rainy season chen the combination of herba patriniae and other medicinal plants can be used to treat poisoned wound of cattle by agkistrodon acutus bitting crawling bee disease liver and skin diseases of turtle and fish and postpartum abdominal pain in cattle chen li shi zhao preproof 0c table the information of herba patriniae in national and local standards in china standards standard of traditional chinese medicine in hunan province standard of traditional chinese medicine in shandong province standard of traditional chinese medicine in heilongjiang province standard of traditional chinese medicine in liaoning province standard of traditional chinese medicine in sichuan province standard of traditional chinese medicine in guizhou province chinese pharmacopoeia edition application acute appendicitis diarrhea enteritis hemorrhagic leucorrhea red eye pterygium postpartum abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles acute appendicitis diarrhea hemorrhagic leucorrhea postpartum blood stasis abdominal pain swelling and pain of eye hepatitis boils and carbuncles acute appendicitis diarrhea dysentery postpartum blood stasis abdominal pain conjunctivitis boils and carbuncles acute appendicitis and its abdominal pain postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles dosage g standardsetting department hunan food and drug administration g g g g g g shandong medical products administration heilongjiang medical products administration liaoning food and drug administration sichuan food and drug administration guizhou medical products administration pharmacopoeia committee of the ministry of health of p r china preparation name yiyi fuzi baijiang san èè¡éåè´¥é
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±æ±¤ table traditional and clinical preparation of herba patriniae in china formulation main compositions powder coicis semen aconiti lateralis radix praeparata herba patriniae decoction portulacae herba isatidis folium arnebiae radix herba patriniae persicae semen carthami flos paeoniae radix rubar powder powder artemisiae argyi folium angelicae sinensis radix paeoniae radix alba dipsaci radix achyranthis bidentatae radix herba patriniae herba patriniae angelicae sinensis radix chuanxiong rhizoma paeoniae radix alba cinnamomi cortex powder herba patriniae moutan cortex cinnamomi cortex siphonostegiae herba aucklandiae radix decoction herba patriniae notopterygii rhizoma et radix dianthi herba aurantii fructus cinnamomi cortex persicae semen decoction herba patriniae reference jin gui yao lüe ãéå®è¦ç¥ã zhang 1997b surgery of chinese medicine ãä¸å»å¤ç§å¦ã beijing traditional chinese medicine hospital tai ping sheng hui fang ã太平å£æ æ¹ã volume wang pu ji fang ãæ®æµæ¹ã volume zhu tai ping sheng hui fang ã太平å£æ æ¹ã volume wang sheng ji zong lu ãå£æµæ»å½ã volume zhao qian jin yi fang ãåé翼æ¹ã volume sun preproof 0cpreparation name baijiang tang è´¥é
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±é¥® changyong tang è ç汤 decoction chenzhou sheyao pian é´å·èè¯ç chure jili wan é¤çèºè丸 tablets pills danggui xi tang å½å½æ´æ±¤ danggui yin å½å½é¥® ganyan chongji èçå²å decoction decoction electuary formulation main compositions decoction herba patriniae rhei radix et rhizoma persicae semen decoction herba patriniae cinnamomi cortex siphonostegiae herba moutan cortex aucklandiae radix decoction herba patriniae angelicae sinensis radix bambusae caulis in taenias rehmanniae radix moutan cortex glycyrrhizae radix et rhizoma herba patriniae zingiberis rhizoma recens poria coicis semen platycodonis radix liriopes radix salviae miltiorrhizae radix et rhizoma paeoniae radix alba rehmanniae radix pv tribuli fructus rhei radix et rhizoma herba patriniae cinnamomi cortex ginseng radix et rhizoma aconiti lateralis radix praeparata coicis semen coptidis rhizoma astragali radix abri herba angelicae sinensis radix aurantii fructus immaturus paeoniae radix alba tetrapanacis medulla angelicae sinensis radix angelicae pubescentis radix angelicae dahuricae radix sanguisorbae radix herba patriniae angelicae sinensis radix herba patriniae dipsaci radix paeoniae radix alba rehmanniae radix bambusae caulis in taenias bupleuri radix angelicae sinensis radix paeoniae radix alba paeoniae radix rubra citri reticulatae pericarpium aurantii fructus curcumae radix cyperi rhizoma salviae miltiorrhizae radix et rhizoma scrophulariae radix artemisiae scopariae herba isatidis radix herba patriniae huangdan tang é»ç¸æ±¤ decoction jiedu dihuang wan 解æ¯å°é»ä¸¸ pills gualou san powder artemisiae scopariae herba gardeniae fructus lonicerae japonicae flos forsythiae fructus herba patriniae isatidis radix paeoniae radix rubra paeoniae radix alba bupleuri radix perillae caulis platycodonis radix sojae semen germinatum rehmanniae radix astragali radix trichosanthis radix scutellariae radix liriopes radix mantidis ootheca rhei radix et rhizoma ginseng radix et rhizoma gardeniae fructus cistanches herba peucedani radix cimicifugae rhizoma paeoniae radix alba anemarrhenae rhizoma vaccariae semen polygalae radix herba patriniae jujubae fructus trichosanthis semen herba patriniae asari radix et rhizoma zingiberis rhizoma magnoliae officinalis cortex platycodonis radix ginseng radix et rhizoma saposhnikoviae radix reference sheng ji zong lu ãå£æµæ»å½ã volume zhao sheng ji zong lu ãå£æµæ»å½ã volume zhao sheng ji zong lu ãå£æµæ»å½ã volume zhao qian jin fang ãåéæ¹ã volume ï¼sun ï¼ gu jin ming fang ãå¤ä»åæ¹ã yan and liu qian jin fang ãåéæ¹ã volume ï¼sun ï¼ qian jin fang ãåéæ¹ã volume ï¼sun ï¼ sheng ji zong lu ãå£æµæ»å½ã volume zhao study on the treatment of common diseases with traditional chinese medicine ã常è§ç
çä¸ å» æ²» ç ç 究 ã teaching and research group of traditional chinese medicine the first affiliated hospital of xi'an medical college lin zheng yi an yi fang ã临è¯å»æ¡å»æ¹ãsun sheng ji zong lu ãå£æµæ»å½ã volume zhao sheng ji zong lu ãå£æµæ»å½ã volume preproof 0c preparation name æ èæ£ lanwei xiaoyan wan éå°¾æ¶ç丸 lanweiyan heji éå°¾çåå formulation main compositions pills lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix decoction lonicerae japonicae flos taraxaci herba herba patriniae forsythiae fructus rhei radix et rhizoma paeoniae radix rubra toosendan fructus aucklandiae radix persicae semen lanweiyan tang éå°¾ç汤 lanwei yihao xiaoyan wan éå°¾ä¸å·æ¶çç lenge xiaoji tang 棱èªæ¶ç§¯æ±¤ lishi zhiyang pu yao ç湿æ¢çæè¯ lidan tuihuang tang å©èéé»æ±¤ neibu wuxiang wan å
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ºæ±¤ qumai wan ç¿éº¦ä¸¸ decoction pills decoction powder decoction pills decoction pills yinqiao hongjiang jiedu tang decoction rhei radix et rhizoma moutan cortex persicae semen paeoniae radix alba salviae miltiorrhizae radix et rhizoma bupleuri radix lonicerae japonicae flos forsythiae fructus herba patriniae coicis semen lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix talci pulvis sargentodoxae caulis sparganii rhizoma curcumae rhizoma salviae miltiorrhizae radix et rhizoma paeoniae radix rubra corydalis rhizoma moutan cortex persicae semen coicis semen sargentodoxae caulis herba patriniae kochiae fructus bombyx batryticatus dictamni cortex angelicae dahuricae radix schizonepetae spica artemisiae scopariae herba herba patriniae alumen glycyrrhizae radix et rhizoma talcum cinnabaris artemisiae scopariae herba herba patriniae isatidis radix curcumae radix gardeniae fructus aquilariae lignum resinatum olibanum aucklandiae radix caryophylli flos dipsaci radix rehmanniae radix praeparata paeoniae radix alba magnoliae officinalis cortex herba patriniae ginseng radix et rhizoma poria cervi cornu salviae miltiorrhizae radix et rhizoma lycopi herba paeoniae radix rubra persicae semen carthami flos olibanum myrrha vaccariae semen citri reticulatae pericarpium toosendan fructus foeniculi fructus angelicae dahuricae radix herba patriniae taraxaci herba dianthi herba realgar vaccariae semen rehmanniae radix ephedrae herba imperatae rhizoma herba patriniae saposhnikoviae radix achyranthis bidentatae radix rhei radix et rhizoma lonicerae japonicae flos forsythiae fructus sargentodoxae caulis herba patriniae moutan cortex gardeniae fructus paeoniae radix rubra persicae semen coicis semen corydalis rhizoma toosendan fructus olibanum myrrha reference zhao beijing chinese traditional patent medicine specification ãå京å¸ä¸æè¯è§èã volume beijing municipal bureau of health selected data of acute abdomen treated by integrated traditional chinese and western medicine ãä¸è¥¿å»ç»åæ²»çæ¥è
¹çèµæéç¼ã affiliated hospital of guangzhou college of traditional chinese medicine lin zheng yi an yi fang ã临è¯å»æ¡å»æ¹ã sun compilation of traditional chinese medicine preparations ãä¸è¯å¶åæ±ç¼ã cao obstetrics and gynecology ã å¦ äº§ ç§ å¦ ãshanghai college of traditional chinese medicine selection of medical prescriptions of cixi and guangxu ãæ
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" the widely recognized anticancer potential of aspirin has created a broad interest to explore theclinical benefits of aspirin in cancer therapy however the current understanding of the molecular mechanismsinvolved in the anticancer potential of aspirin remains limitedmethods cancer stemness assays which contained aldh side population chemoresistance sphere formationand tumorigenesis were performed to validate aspirin function in vitro and in vivo histone modification assay wasperformed to check the effect of aspirin on histone methylation as well as the activity of hdac and kdm6abinhibitor in vivo assay was performed to evaluate therapeutic effects of various inhibitor combination mannersresults in regards to in vitro studies aspirin diminishes cancer cell stemness properties which include reducing thealdh subpopulation side population chemoresistance and sphere formation in three cancer types in regards toin vivo studies aspirin decreases tumor growth and metastasis and prolongs survival in addition our resultsshowed that aspirin inhibits inflammationrelated stemness gene expression especially icam3 identified by a highthroughput sirna platform in regards to the underlying molecular mechanism of action aspirin reduces histonedemethylase kdm6ab expression that mediates histone methylation and suppresses gene expression via a coxindependent manner in regards to therapeutic strategies aspirin combined hdm inhibitors icam3 downstreamsignaling srcpi3k inhibitors or icam3 inhibitor lifitigrast prevents cancer progression in vivos the aforementioned findings suggest a molecular model that explains how aspirin diminishes cancercell stemness properties these findings may provide novel targets for therapeutic strategies involving aspirin in theprevention of cancer progressionkeywords aspirin histone methylation cancer stemness icam3 cox therapeutic strategies correspondence shenwenzhi2011126com1department of pathology and institute of precision medicine jining medicaluniversity hehua road jining chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang stem cell research therapy page of cancer stem cell csc was found as the chief culprit toinitiate tumor occurrence to enhance tumor malignancyand to cause tumor recurrence whereby the maintenanceof cancer cell stemness mainly depends on the tumormicroenvironment or also called the niche [] currently the specific properties of csc were identified likehigh aldh1 activity aldehyde dehydrogenase sidepopulation chemoresistance and other cd moleculescd44 cd133 or markers sox2 oct4 nanoglgr5 positive in cancer clinical therapy which targetedhighly tumorigenic cscs may provide new targets orinsight for cancer therapy however unfortunately cscshad demonstrated a relative resistance to conventionalchemotherapy and radiotherapy moreover the cancer cellstemness and the resultant tumor initiationmalignancycould be maintained by the tumorassociated inflammation factors within the tumor microenvironment niche[ ] our previous work presented a mediumthroughput sirna screen platform to identify inflammation genes that regulate cancer cell stemness and obtainedseveral novel candidates agents that target these genesmay inhibit both inflammation and cancer cell stemnessat the same timeaspirin a nonsteroidal antiinflammatory drugiscommonly used as an antipyretic analgesic antiinflammatory and antithrombotic agent [ ] recentobservational and epidemiological studies have shownthat regular prolonged use of aspirin reduces the riskfor several cancers eg colorectal esophageal breastlung prostate liver and skin cancers [] althoughthe benefits of aspirin for cancer patients have beenwidely appreciated the mechanism remains unclear previous studies attribute the anticancer potential of aspirin to the inhibition of cyclooxygenase2 cox2which is upregulated in various cancer cells [ ] ofnote an increasing body of evidence suggests that aspirin exhibits anticancer effects in a coxindependentmanner histone modification is a reversible process mediated bythe epigenetic enzymes [ ] histone methylation andacetylation are two important chemical modifications thatact in transcriptional activation or inactivation chromosome packaging and dna damagerepair [ ] histone demethylases hdms and histone deacetylaseshdacs are the key enzymes that remove methyl andacetyl groups respectively to regulate gene transcriptionin this regard aspirin was reported to affect hdacs expression and suppress progression of some cancers like aspirin mediates h3k27 acetylation to prevent coloncarcinogenesis and aspirin cooperates with p300 to activate h3k9 acetylation further to promote colorectal cancer cell apoptosis [ ] however the specific rolesand mechanisms of aspirinmediated histone methylationin cancer stemness remains insufficient thus we studiedthe role of aspirin on histone methylation and the attendantand cancerprogressioneffects on cancercellstemnessthe aldh subpopulationour results indicated that aspirin diminishes various cancer cell stemness properties which include reducingside populationchemoresistance and sphere formation in three cancertypes in vitro aspirin inhibits tumor growth and metastasis as well as prolongs survival in vivo aspirininhibits inflammationrelated stemness genes especiallyicam3aspirin reduces histone demethylasekdm6abexpression which mediates histone methylation respectively with a coxindependent manner and aspirin hdm inhibitors aspirin icam3downstream signaling srcpi3k inhibitors and aspirin icam3 inhibitor lifitigrast all reduce cancer progressionin vivo the abovementioned findings demonstrate apromising mechanism of action and potential therapeutic strategy of aspirin in the prevention of cancerprogressionmethodscell culturemdamb231 breast cancer cell and a549 lung cancercell were purchased from atcc hepg2 was obtainedfrom the chinese academy of sciences mdamb231breast cancer cell and hepg2 liver cancer cell were cultured in dmem medium a549 lung cancer cell was cultured in medium allculture media weresupplemented with fbs gibco and were grown at °c in co2 incubators all cells were passaged forless than months before renewal from frozen earlypassage stocks and tested to ensure thatthey weremycoplasma negativecytotoxicity assayaspirin was purchased in sigma cat a2093 and dissolved in dmso mdamb231 a549 and hepg2 cellswere cultured in 96well plate and treated with variousconcentrations and mm for a549 cells and mm for mdamb231 cells and and mm forhepg2 cells of aspirin for h cell activity was testedby applying cck8 kit dojindo china following themanufacturers instructionsaldefluor assaythe aldeflour assay kit stemcell technologies vancouver canada was used to measure aldh enzymaticactivity in three cancer cell lines mdamb231 a549and hepg2 in brief cells were treated with aspirin for h and cells were suspended in aldeflour 0czhang stem cell research therapy page of assay buffer containing aldh1 substrate and incubated for min at °c cells treated with the specific aldh inhibitor deab served as the negative control stained cellswere analyzed on bd facs calibur flow cytometer bdbiosciences san jose ca data analysis was performedusing flowjo software tree star inc ashland orside population assaymdamb231 a549 and hepg2 cells treated with aspirin for h were harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of cellsml hoechst dye wasadded at a final concentration of μgml μgmla549 and μgml hepg2 in the presence or absence of μm fumitremin c ftc the followingsteps were described previously [ ]cell apoptosis assaythe mdamb231 a549 and hepg2 cells were treatedwith aspirin and cisplatin ddp μgml for h simultaneously then harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of cellsml apoptotic cells were stainedwith propidiumiodide and annexinvfitc bd biosciences flow cytometry analysis was performed by facscalibur cytometer bd biosciences in which a minimum of events were recordedsphere formation assaycells were collected and rinsed to remove serum thendissociated to singlecellsuspension in serumfreedmemf12 medium supplemented with iumlpenicillin μgml streptomycin ngml human recombinant epidermal growth factor hregf ngmlhuman recombinant basic fibroblast growth factorbfgf and b27 supplement invitrogen cells weresubsequently cultured in ultralow attachment 24wellplates at a density of cells per well then treatedwith aspirin all the time until the spheres were formedanimal studyfemale balbc mice at weeks were separated randomly into several groups n ¥ 4t1luci cellswere inoculated sc into each mouse at the right axillafor lung metastasis assay at days after injection micewere treated intraperitoneally with aspirin mgkg aspirin mgkg every days and dmso used as thecontrol for chemoresistance assay at days after injection mice were first treated intraperitoneally with cisplatin mgkg then treated with aspirin mgkgaspirin mgkg every days and dmso used as thecontrolnodscid mice at weeks were separated randomly into several groups n ¥ a549luci cellswere inoculated sc into each mouse for inhibitor treatment assay days after tumor cells injection micewere first treated intraperitoneally with aspirin mgkgor kdm6ab inhibitor gsk j1 mgkg or src inhibitor a new specific highefficient src inhibitor [ ] mgkg or pi3k inhibitor ly294002 mgkg orlifitegrast mgkg dmso used as the controltumor volume mm3 was measured with calipers andcalculated by using the standard formulalength width22 the individual measuring the mice was unaware of the identity of the group measured primarytumor tissues were harvested and separated into threeparts one was formalin fixed paraffin embedded andsectioned for ihc staining the other two were brokenby the tissue homogenizer then used for rna trizoland protein ripa lysis buffer extraction animal usecomplied with nankai university and jining medicaluniversity animal welfare guidelines western blottingthe western blot steps were described previously [ ]the special primary antibodies used in this assay arelisted in supplementary table s1 all western data wasthe representative image of three biologically independent repeats the results were quantified using image jsoftware national institutes of health baltimore mdand analyzed by graphpad prism5 software graphpadsoftware san diego ca usanuclear fractionation analysiscells were harvested and the cytoplasmic and nuclearfractions were separated and extracted with an nepernuclear and cytoplasmic extraction kit thermo fisherscientific inc ma usa h3k3me marker proteinswere detected by western blotimmunofluorescencecells grow on glass slides and tumor tissue slices werefixed in paraformaldehyde and labeled with primaryantibodies overnight at °c followed by incubation withspeciesappropriateroomtemperature for h nuclei were stained with dapi andimages were obtained using a leica dm4000 uprightmicroscope or confocal fluorescence microscopy nikontokyo japan [ ]antibodiessecondaryatchromatin immunoprecipitation assaythe assay was performed with an ezzyme chromatinprep kit millipore according to the manufacturersprotocol antihistone modification marker antibodieswere used to precipitate dna crosslinked with histone modification markers respectively and normal rabbitigg was used in parallel as a control enriched dna wasthen used as a template to assess the binding intensity of 0czhang stem cell research therapy page of histone modification markers to putative binding sitesin the icam3 promoter primers used in this assay arelisted in supplementary table s2immunohistochemistryimmunohistochemistry was performed on tumor tissuesections from the mice primary antibodies raise againstthe target proteins at a dilution overnight the expression levels of the proteins were evaluated accordingto the percentage of positive cells in each tumor tissuesections the images were recorded by olympus bx51epifluorescent microscopy under a or objective olympus co tokyo japan changes in apoptosis in the three cancer cell lines usingthe cell apoptosis assay after cisplatin ddp treatmentour results indicated that apoptosis increases in theaspirintreated groups versus controls and thereby reduces cisplatin chemo resistance fig 1e f in orderto determine the effects of aspirin on cancer cell stemness we next investigated the changes in cell sphere formation in the three cancer cell lines using the sphereformation assay our results showed that sphere formation decreases in the aspirintreated groups versus controls fig 1g h the abovementioned findings suggestthat aspirin diminishes cancer cell stemness propertiesin vitrostatistical analysisall data were analyzed using graphpad prism5 softwaregraphpad software san diego ca usa values wereexpressed as means ± sem p values were calculatedusing a twotailed students t test two groups or oneway anova more than groups unless otherwisenoted a value of p was used as the criterion forstatistical significance an asterisk indicates a significantdifference with p two asterisks indicate a significant difference with p and three asterisks indicatea significant difference with p [ ]resultsaspirin diminishes cancer cell stemness properties in vitroin order to establish the proper working concentrationsof aspirin in various cancer cells we determined theic50 of aspirin in a549 lung cancer cells mdamb231breast cancer cells and hepg2 liver cancer cells using acytotoxicity assay our results showed a 107mm ic50in a549 lung cancer cells a 43mm ic50 in mdamb breast cancer cells and a 97mm ic50 in hepg2liver cancer cells fig s1 based on the ic50 we choseworking concentrations of and mm aspirin fora549 lung cancer cells and mm aspirin formdamb231 breast cancer cells and and mmaspirin for hepg2 liver cancer cells in our studiesthe aldh subpopulation decreasesin order to determine the in vitro effects of aspirin oncancer cell stemness we investigated aldh subpopulation changes in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing the aldh staining assay our results indicatedthatin theaspirintreated groups versus controls fig 1a b inorder to determine the effects of aspirin on cancer cellstemness we next investigated the changes in the sidepopulation in the three cancer cell lines using the sidepopulation assay our results indicated that the sidepopulation decreases in the aspirintreated groups versuscontrols fig 1c d in order to determine the effects ofaspirin on cancer cell stemness we next investigated theaspirin diminishes cancer cell metastasis and stemnessproperties in vivoin order to determine the effects of aspirin on cancercell metastasis and stemness in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice seven days after implantation we ip injected themice with mgkg aspirin mgkg aspirin ordmso control group times per week fig 2a ourresults showed thattumor volume decreases in theaspirintreated groups versus the control fig 2b however we found that the body weight did not change inthe aspirintreated groups versus the control fig 2c inaddition we found that the survival time increases in theaspirintreated groups versus control fig 2d with respect to the effect of aspirin on cancer cell metastasiswe found thatlung metastasis decreases in aspirintreated groups versus the control fig 2eg with respect to the effect of aspirin on cancer cell stemnessproperties we found thatthe immunocytochemicalstaining of sox2 and oct4 stemness markers decreasesin the aspirintreated groups versus dmso controlsfig 2h i the abovementioned findings suggest thataspirin diminishes cancer cell metastasis and stemnessproperties in vivoaspirin reduces cancer cell chemoresistance in vivoin order to determine the effects of aspirin on cancercell chemoresistance in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice eight days after implantation we ip injected themice with mgkg cisplatin mgkg aspirin mgkgcisplatin mgkg aspirin or dmso control groupevery days fig 2j our results showed that tumor volume decreases in the cisplatinaspirintreated versus thedmso control fig 2k however we found that thebody weight did not change in the cisplatinaspirintreated versus the dmso control fig 2l in additionwe found that the survival time increases in the cisplatinaspirintreated groups versus the dmso controlfig 2m we also found that the rate of tumor growth 0czhang stem cell research therapy page of absllec evitisop hdladeabctrl2mm5mm 4mm10mm ctrleddp10ugml2mm5mm 4mm10mm agpehagpeha549ctrl 5mm 10mm ctrl 2mm 4mm sllec evitisop hdlasllec evitisop hdlahepg2fctrl 5mm 10mm slliec ssotpopaa549 slliec ssotpopactrl 5mm 10mm ctrl 2mm 4mm hepg2 slliec ssotpopactrl 5mm 10mm cblockctrl2mm5mm 4mm10mm agpeha549d noitlaupopeds ia549 noitlaupopeds ictrl 5mm 10mm ctrl 2mm 4mm fig see legend on next pagehepg2h noitlaupopeds ictrl 5mm 10mm rebmun erehpsgctrl2mm5mm 4mm10mm agpehhepg2ctrl 5mm 10mm ctrl 2mm 4mm rebmun erehpsrebmun erehpsctrl 5mm 10mm 0czhang stem cell research therapy page of see figure on previous pagefig aspirin restrains cancer cell stemness properties in vitro a aldh staining assay was performed to check aldh subpopulationpercentage in the three cancer cell lines with or without aspirin treatment b statistic results of aldh subpopulation percentage were shown cside population assay was performed to detect sp percentage in three cancer cell lines with or without aspirin treatment d statistic results of sppercentage were shown e facs was performed to detect cell resistance to cisplatin and the percentage of apoptotic cells was shown f statisticresults of apoptosis cells percentage were shown g sphere formation assay was performed to check the cell sphere formation ability in the threecancer cell lines with or without aspirin treatment scale bars μm h statistic results of sphere amounts were shownwas slower in the cisplatinaspirintreated versus thedmso control fig 2n with respect to the effect of aspirin on cancer cell stemness properties we found thatthe immunocytochemical staining of sox2 and oct4stemness markers decreases in the cisplatinaspirintreated groups versus dmso controls fig 2o p theabovementioned findings suggest that aspirin reducescancer cell chemoresistance in vivoaspirin inhibits the expression of inflammationrelatedstemness genes in vitro and in vivoour previously published report established a mediumthroughput sirna screening platform that identifies inflammation genes that regulate cancer cell stemness specifically we identified several novel candidate genesthat decrease oct4 expression and the aldh subpopulation both of which characterize stemness fig 3ain order to determine whether aspirin decreases theexpression of these novel candidate genes to further diminish cancer cell stemness we investigated the expression of novel candidate genes and stemness markerssox2 and oct4 in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing western blot our results showed that icam3ccl16 pde3a prtn3 sox2 and oct4 protein expression decreases in the aspirintreated groups versuscontrols fig 3b fig s2a we also found that icam3ccl16 pde3a prtn3traf6 bcar1 il1a il1bnfkb1 ikbkb sox2 and oct4 mrna expression decreasesin the aspirintreated group versus controlfig 3c moreover in icam3 ccl16 pde3a prtn3traf6 bcar1 il1a il1b nfkb1 sox2 and oct4the protein expression decreasesindicated byimmunofluorescencestainingaspirintreatedmbamd231 fig 3d and a549 cells data not shownversus the controlasin thein order to confirm the above in vitro results we theninvestigated mrna and protein expression in tumorsfrom and 46day aspirintreated miceversus control using qpcr and western blot our resultsdemonstrated that icam3 pde3a prtn3 traf6bcar1 il1a il1b nfkb1 ikbkb sox2 and oct4mrna expression decreasesin the aspirintreatedgroups versus control fig 3e in addition we foundthat icam3 pde3a prtn3 traf6 bcar1 il1ail1b nfkb1 sox2 and oct4 protein expressionsimilarly decreases in the aspirintreated groups versuscontrol fig 3f fig s2b the abovementioned findingsexpression ofsuggesttheinflammationrelated stemness genesin vitro andin vivoaspirin decreasesthataspirin mediates histone methylation to regulate targetgenes expressionin order to determine the mechanism underlying the action of aspirin we explored the regulatory effect of aspirin on histone methylation markers in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using western blot our resultsindicated thatthe expression of h3 trimethylationmarkersie h3k43me h3k93me h3k273meh3k363me and h3k793me increases in the aspirintreated higher concentration groups versus controlfig 4a s3a we also found that the expression of histone demethylases ie kdm6a and kdm6b decreasesin the aspirintreated higher concentration groups versuscontrol fig 4a in addition we studied the protein expression of h3k43me h3k93me h3k273meh3k363me h3k793me and h3 in a549 lung cancercells mdamb231 breast cancer cells and hepg2 livercancer cells using immunofluorescence ourresultsshowed that the protein expression of the h3 methylation markers within the nucleus increases in the aspirintreated groups versus control fig 4b to further support this we extracted the nuclear proteins of eachgroup and detected these h33me markers the resultsalso showed that the expression of h33me markers wasincreased within the nucleusin the aspirintreatedgroups versus control fig 4c s3bin order to identify the role of h3 methylation in regulating selected inflammationrelated stemness genes wemeasured the amount of icam3 dna fragments in h3modification marker pulldowned dnas in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using the chipqpcr assaywe selected icam3 since our previous studies demonstrated that icam mediates cancer cellinflammationand stemness ourtheamount of icam3 dna fragments in the various h3methylation marker pulldowned dnas decreases in alllines fig 4c the abovementionedthree cancer cellfindingssuggesthistoneresults demonstrated thatreducesthataspirin 0cbmcemuovl romutdliavvrus noitcarfctrlasp 50mgkgasp 100mgkgdayscg thgew ydobictrlasp 50mgkgasp 100mgkg days of treatmentectrlasp50 asp100ctrlasp 50mgkgasp100mgkggsedoni ssatsat emgnulfhctrlasp asp ctrlasp asp xostcoi sllec xos sllec tcoctrl asp50asp100ctrl asp50 asp100ctrl asp50 asp100zhang stem cell research therapy page of a4t1luci injectionaspirin injectionsurvivalday timej4t1luci injectionaspirin cisplatin 2mgkg injectionsurvivalday kmcemuovl romutctrlcisplatinasp 25cisplatinasp50cisplatin dayslg tihgew ydobctrlcisplatinasp 25cisplatinasp50cisplatin days of treatmentmliavvrus noitcarfncisplatin treatedctrlasp asp50yadyadctrlcisplatinasp 25cisplatinasp50cisplatintimefig see legend on next pageop sllec xoscisplatin treatedctrlasp asp50xostcoctrl asp25 asp50cisplatin treated sllec tcoctrl asp25 asp50cisplatin treated 0czhang stem cell research therapy page of see figure on previous pagefig aspirin suppresses cancer cell metastasis and stemness in vivo a schema of the metastasis model established by subcutaneousimplantation of 4t1luci cells into the 4th pair of mammary fat pad of balbc mice b tumor growth curve of 4t1luci with or without aspirintreatment c the body weight of balbc mice in the course of aspirin treatment d the survival curve of balbc mice inoculated with 4t1luciwith or without aspirin treatment e the representative luciferase images showing the 4t1luci tumors at the primary site and lung metastasissites with or without aspirin treatment f representative he staining images of 4t1luci tumors metastasis to the lung with or without aspirintreatment scale bars lower panel μm g statistic results of metastasis loci of 4t1luci tumors metastasis to the lung with or without aspirintreatment h immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment representativeimages with magnification were shown scale bars μm i statistic results of sox2 or oct4 positive cells in 4t1luci primary tumors withor without aspirin treatment j schema of the chemoresistance model established by subcutaneous implantation of 4t1luci cells into the 4thpair of mammary fat pad of balbc mice k tumor growth curve of 4t1luci with or without aspirin treatment in the presence of cisplatin l thebody weight of balbc mice in the course of aspirin treatment in the presence of cisplatin m the survival curve of balbc mice inoculated with4t1luci with or without aspirin treatment in the presence of cisplatin n the representative luciferase images showing 4t1luci tumors at theprimary sites with or without aspirin treatment on day before cisplatin administration and day after cisplatin administrationo immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment in the presence of cisplatinrepresentative images with magnification were shown scale bars μm p statistic results of sox2 or oct4 positive cells in 4t1luciprimary tumors with or without aspirin treatment in the presence of cisplatindemethylase ie kdm6a and kdm6b expressionwhich mediates histone methylation and thereby inhibits gene expression in vitroaspirin mediates h3 methylation to regulate icam3expression in vivoin order to confirm the above in vitro results we nextexamined h3 methylation marker expression in tumorsfrom aspirintreated mice versus control using immunocytochemistry our results demonstrated that the h3methylation marker immunostaining within the nucleusincreases in the aspirintreated group versus controlfig 4d e we also found that the amount of icam3dna fragments in the various h3 methylation markerpulldowned dnas decreasesin the aspirintreatedgroup versus control indicating that icam3 expressionis blocked fig 4f these findings suggest that aspirinmediates h3 methylation and thereby regulates icam3expression in vivoside populationaspirin mediates h3 methylation to regulate icam3expression via a coxindependent mannerin order to determine the role of cox in aspirinmediated h3 methylation and targeted gene expressionwe knocked down cox1 and cox2 expression in a549cells respectively fig 5a s3c and examined thealdh populationand chemoresistance the results showed that the aldh population fig 5b e and side population fig 5c f were decreased in shcox1 or shcox2 cells treated with aspirincompared to shcox1 or shcox2 cells treated withdmso and also the aldh population and side population were decreased in shcox1 or shcox2 cellstreated with aspirin compared to shctrl treated with aspirin moreover the apoptosis was increased in shcox1or shcox2 cells treated with ddp and aspirin comparedto shcox1 or shcox2 cells treated with ddp anddmso ctrl and also the apoptosis was increased inshcox1 or shcox2 cells treated with ddp and aspirincompared to shctrltreated with ddp and aspirinfig 5d g in addition western blot results displayedthat the h3 trimethylation markers were increased andthe histone demethylases ie kdm6a and kdm6bwere decreased in shcox1 or shcox2 cells treated withaspirin compared to shctrl treated with aspirin fig 5hs3d accordingly as the new target genes icam3 expression was decreased in shcox1 or shcox2 cellstreated with aspirin versus shctrl treated with aspirinfig 5i s3e these findings suggest that aspirin mediates h3 methylation and thereby regulates icam3 expression via a coxindependent manneraspirin combined with hdm kdm6ab or icam3signaling inhibitors diminish cancer progression in vivoour previous work proved that icam3 could mediatesrcpi3k signaling to promote cancer cell stemness inorder to investigate the use of aspirin combined withhdm kdm6ab or icam3 signaling inhibitors as thetherapeutic strategies we implanted a549luciferasecells into the fourth fat pad of male nodscid micetwentythree days after implantation we injected ip themice with mgkg aspirin mgkg aspirin mgkg kdm6ab inhibitor gsk j1 mgkg aspirin mgkg src inhibitor mgkg aspirin mgkg pi3kinhibitor ly294002 mgkg aspirin mgkg lifitigrast icam3 inhibitor or dmso control group every days fig 6a our results showed that tumor size andtumor volume decreases in the aspirintreated groupand the aspirin inhibitortreated groups versus dmsocontrol fig 6b c however we found that the bodyweight did not change significantly in the aspirintreatedgroup and the aspirin inhibitortreated groups versusdmso control fig 6din addition we found that the survival time increasesin the aspirintreated group and the aspirin inhibitortreated groups versus dmso control fig 6e these 0czhang stem cell research therapy page of fig see legend on next page 0czhang stem cell research therapy page of see figure on previous pagefig aspirin inhibits the expression of inflammationrelated stemness genes in vitro and in vivo a schematic representation of the sirna screenleft summary of the results from the rnai screen right b western blot examining the expression of inflammatory candidates and stemnessproteins sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirin treatment c quantitative pcr examining the mrnaexpression of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirintreatment d immunofluorescence staining of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cellswith or without aspirin treatment scale bars μm e quantitative pcr examining the mrna expression of inflammatory candidates andstemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treated with aspirin for different survival days f western blotexamining the expression of inflammatory candidates and stemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treatedwith aspirin for different survival dayssuggestresultsthat aspirin combined with hdmkdm6ab or icam3 signaling inhibitors diminishcancer progression in vivo and may serve as the therapeutic strategiesproposed model of aspirin inhibits cancer cell stemnessand cancer progressionbased on our findings we propose the following modelfig aspirin inhibits histone demethylase hdm expression which then mediates histone methylationh3k43me h3k93me h3k273me h3k363meh3k793me respectively these h3 methylations theninhibit the expression of various inflammationrelatedstemness genes previously identified by highthroughputsirna screening il1a il1b icam3 ccl16 traf6pde3a prtn3 nfkb1 ikbkb bcar1 using theicam3 gene as a representative of the inflammationrelated stemness genes by the aspirinmediated h3modifications restrain icam3 promoter activity andcause icam3 expression is inhibited thus aspirin maydiminish cancer cell stemness properties and cancer progression in vitro and in vivo by inhibiting the expressionof various inflammationrelated stemness genes mostinterestingly the above process was not depending oncox expression as the therapeutic strategies aspirincombined various inhibitors suppressed tumor progression effectivelydiscussionthe widely recognized anticancer potential of aspirin aclassical nonsteroidal and antiinflammatory drug hascreated a broad interest to explore the clinical benefitsof aspirin in cancer therapy [] previous findingsby many investigators have establishe | 0 |
"the kinetics and localization of the reactions of metabolism are coordinated by the enzymes that catalyze themthese enzymes are controlled via a myriad of mechanisms including inhibitionactivation by metabolitescompartmentalization thermodynamics and nutrient sensingbased transcriptional or posttranslational regulationall of which are influenced as a network by the activities of metabolic enzymes and have downstream potential toexert direct or indirect control over protein abundances considering many of these enzymes are active only whenone or more vitamin cofactors are present the availability of vitamin cofactors likely yields a systemsinfluence overtissue proteomes furthermore vitamins may influence protein abundances as nuclear receptor agonistsantioxidants substrates for posttranslational modifications molecular signal transducers and regulators ofelectrolyte homeostasis herein studies of vitamin intake are explored for their contribution to unraveling vitamininfluence over protein expression as a body of work these studies establish vitamin intake as a regulator of proteinabundance with the most powerful demonstrations reporting regulation of proteins directly related to the vitaminof interest however as a whole the field has not kept pace with advances in proteomic platforms and analyticalmethodologies and has not moved to validate mechanisms of regulation or potential for clinical applicationkeywords proteomics big data vitamin metabolism precision nutrition molecular nutritionintroductionregulatory mechanismscellular metabolism is a system of chemical reactions inwhich cells harness the energy stored in the chemicalbonds of substrate molecules to perform their biologicalfunctions maintain homeostasis or to synthesize buildingblocks for structural maintenance or cellular division thekinetics of these reactions are dependent on the activity ofthe proteins which catalyze them thus proteins are keymodulators of metabolismmetabolic activity also exerts network control over itselfby a diverse array of mechanisms which finely tune proteinexpression responses via nutrient sensing machineries products or intermediates of a metabolic pathway caninhibit or activate metabolic enzymes eg malate inhibitsthe succinate dehydrogenase complex and fructose correspondence nv83cornelledudivision of nutritional sciences cornell university ithaca ny usa26bisphosphate activates phosphofructokinase theoxidative status of a cell can drive the directionality ofredox reactions and impact abundances of redox reactioncatalyzing proteins eg the keap1nrf2 network responds to oxidative stress by upregulating expression ofantioxidantfunctioning proteins splicevariant or isozyme expression can impact relative pathway utilization atmetabolic network nodes eg splice variants and isozymesof pyruvate and lactate dehydrogenase respectively impactthe bridge between glycolysis and the tricarboxylic acidtca cycle [ ] additionally local metabolite concentrations and thermodynamics can dictate the directionalityof reactions catalyzed by compartmentspecific isozymeseg reductive activity of isocitrate dehydrogenase can beconfined to the cytosolspecific isozyme the impactsof the abovementioned regulations are closely monitoredby nutrient sensing proteins which initiate molecularevents altering protein activation and expression eg the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cjeong and vacanti nutrition metabolism page of serinethreonine kinase ampactivated protein kinasemammalian target of rapamycin and sterol regulatoryelementbinding protein are part of overlapping proteinnetworks that orchestrate proteinexpression and posttranslational modification responses to nutrient availability[ ] considering that many metabolic enzymes do notfunction in isolation and as detailed in the sections thatfollow require vitamin cofactors to stabilize intermediatesdonateaccept electrons shuttle substrates and hold reactants in close proximity vitamin status is a critical consideration when examining proteinmediated regulation ofmetabolism and the impacts of metabolism on proteinexpressionin addition to their potential regulatory roles ascofactors vitamins orchestrate other direct or indirectmechanisms influencing protein abundance retinoicacid vitamin a interacts with nuclear receptors impacting gene transcription ascorbic acid vitamin cimpacts oxidative status and associated protein networks and is reported to exhibit epigenetic regulation overprotein expression vitamin d regulates calcium signaling machinery activates nuclear receptors and exertshormonal regulation over protein expression [ ]and niacin vitamin b3 and biotin vitamin b7 can beincorporated as posttranslational modifications impacting protein function [ ]herein studies on systemic intake dietary injectionoral gavage of vitamins and their impacts on tissueproteomes are examined and their contributions tounraveling vitaminbased regulation of protein expression and tissue function are explored the currentwork is intended to provide informationto understand each vitamins figs and molecularfunctions and highlight its role as a cofactor or substrate in the reactions of central metabolism fig tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12and s13 finally this work is intended as a resourcefor identifying regulation of proteins related to vitaminmetabolism in published works the public domain ofproteomic data sets is ever expanding but is rarelysearched for effects related to vitamin metabolism tothat end all proteins are specified by their hugogene nomenclature committee hgnc gene symbolor the hgnc gene symbol of the human orthologwhen identified in another species and proteins requiring a vitamin as a cofactor or substrate are tabulated tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10s11 s12 and s13proteomics platformsproteomics platforms ofthe discussed studies areprovided to place them on a technological timelineplatforms are described with the terms orbitrap qtofquadrupole timeofflight tripletof triple time offlight qqq triplequadrupole 2dgems twodimensional gel electrophoresis mass spectrometry and2dge in brief orbitrap platforms are the workhorses ofmodern proteomics because their high achievable massresolutions combined with high sensitivity are bestsuited for maximizing the number of proteins identifiedin a complex sample [ ] though qtof and tripletof instruments capable of maintaining mass resolutionfig fat soluble vitamin structures 0cjeong and vacanti nutrition metabolism page of fig water soluble vitamin structuresat higher scan speeds hold a substantial influence inthis arena within the categories of orbitrap qtof andtripletof there are major technological advances notdiscussed here qqq platforms are best suited for quantifying a predetermined list of proteins lower scan speedsand mass resolution render them less capable thanorbitrap qtof or tripletof systems for nontargetedapplications advances in nanoflow liquid chromatography coupled directly to mass spectrometry haveimproved proteomic depth by orders of magnitude overthat achievable by 2dgems where the upstream selection of protein spots predates the modern definition ofnontargeted proteomics similarlyidentifying differentially intense protein spots using 2dge alone is considered an important milestone in the development ofproteomics but is rarely discussed outside the topic of thefields historyvitamin regulation of tissue proteomesvitamin avitamin a exists in alcohol aldehyde acid and esterforms known as retinol retinal retinoic acid and retinylesters respectively fig several carotenoids areprecursors to vitamin a including α and βcarotene βcarotene is converted to two molecules of retinalby beta carotene oxygenases bco1 or bco2 retinal is an important component of rhodopsin rho aprotein in rod cells responsible for detecting low levelsof light thus night blindness is telltale characteristic of vitamin a deficiency retinoic acid serves as asignaling molecule acting through nuclear retinoic acidrara rarb rarg and retinoid x rxra rxrbrxrg receptors which regulate growth and differentiation [ ] cellular and anismal trafficking ofvitamin a is dependent on retinolretinoic acid bindingproteins rbp family crabp1 crabp2 and retinolesterification via lecithin retinol acyltransferase lrat retinal is oxidized to retinol via aldehyde dehydrogenases aldh family and retinol is oxidized to retinoicacid by retinol dehydrogenases rdh and dhrs families in addition to inducing night blindness vitamin adeficiency adversely impacts cellular growth bone development and antibodybased immune responses in an orbitrapbased study of mouse embryo headstoxic levels of prenatal retinoic acid exposure intendedto model an established risk factor for craniofacial birthdefects are reported to induce abundance alterations inproteins associated with craniofacial development and 0cjeong and vacanti nutrition metabolism page of fig schematic of vitamin involvement in reactions of central carbon metabolism the depicted lipid bilayer represents the inner mitochondiralmembrane abbreviations defined in the abbreviations section vitamins specified by alphanumeric designations 0cjeong and vacanti nutrition metabolism page of neural crest processes in a parallel tripletofbased study of gerbil plasma and 2dgemsbased studyof gerbil liver and white adipose tissue a few dozen protein abundances linked to a handful of biological processesare reported to respond to dietary retinol βcarotene lutein or lycopene though process or pathway enrichmentanalyses are not reported as the authors discuss plasmawas not depleted of common highly abundant proteinsupstream of analysis by mass spectrometry which areknown to adversely impact data quality in anorbitrapbased study of plasma from nepalese childrendozens of proteins are associated with circulating carotenoid abundances potentiating development oflowcostantibodybased tests for carotenoid deficiencies apair of 2dgemsbased studies link tissue function toprotein abundance responses to vitamin a status in micebrains and bovine muscle vitamin b1thiamine vitamin b1 is composed of linked pyrimidineand thiazole rings decorated with methyl amine andalkylhydroxyl functional groups fig thiamineistransported through the plasma membrane viathiamine transporters slc19a2 and slc19a3 andthen twice phosphorylated on the alkylhydroxyl functional group by thiamine pyrophosphokinase tpk1rendering it active as thiamine diphosphate tdp tdp is a cofactor for enzymes catalyzing the oxidativedecarboxylation of ketoacids including the pyruvatedehydrogenase complex pdha pdhb pdhx dlatdld the oxoglutarate dehydrogenase complex ogdhdlst dld and the branched chain keto acid dehydrogenase complex bckdha dbt dld it is also acofactor for transketolase tkt in the nonoxidativebranch of the pentose phosphate pathway independent from its role as a cofactor thiamine is believed toregulate ion transport activity in the nervous system vitamin b1 deficiency is marked by a broad range ofneurological respiratory and cardiovascular pathophysiologies and is termed beriberi symptoms of beriberi aredifficult to directly link to the molecular functions ofvitamin b1 in a 2dgemsbased study of type diabetic andhealthy control subjects authors report treatment withthiamine reduces albumin alb abundance in urine indicating the vitamin serves a protective role of kidneyfunction in a qtofbased study of rat thalamiunder thiamine deficiency glyceraldehyde3phosphatedehydrogenase gapdh is the most upregulated protein fold while regulated proteins are most enrichedin the synaptic vesicle cycle pathway according to thekegg database proteomic changes are accompaniedby diminished performances on cognitive tests vitamin b2riboflavin vitamin b2 is composed of an isoalloxazinering and a bound ribitol fig it is activated byriboflavin kinase rfk forming flavin mononucleotidefmn and by flavin adenine dinucleotide synthase flad1 forming flavin adenine dinucleotide fad bound fmn or fad serves as an electron carrierfor redoxreactioncatalyzing proteins flavoproteinsincludingcomplexsdha sdhb sdhc sdhd the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldacylcoa dehydrogenases acads and methylenetetrahydrofolate reductase mthfr dehydrogenasethesuccinateriboflavin deficiency in humans predominantly occursin combination with that of other nutrients howeveranimal studies link it to impaired fetal and intestinaldevelopment [ ]iron absorption and lipidmetabolism [ ]in a qtofbased study of duck livers riboflavin deficiency is accompanied by a reduced abundance of smallchainspecific acylcoenzyme a dehydrogenases acadsfor which riboflavin serves as a cofactor and concordantelevation of hepatic small chain fattyacid lipid contentdramatic decreases in protein abundance are reported forinpp1 involved in inositol signaling thrsp purportedregulator of lipid metabolism bdh2 a regulator of lipidmetabolism fxn involved in mitochondrial ironsulfurcomplex assembly and ndufs1 a subunit of electrontransport chain complex i in a qtofbased study ofmaternal riboflavin deficiency reductions in fetal duck hepatic tca cycle betaoxidation and electron transport chainproteins are reported with idh3a being the lone memberof these pathways whose abundance increases vitamin b3niacin vitamin b3 is inclusive of nicotinic acid and nicotinamide fig which are converted to their mononucleotide forms by nicotinate phosphoribosyltransferase naprt and nicotinamide phosphoribosyltransferase namptrespectively both forms of the mononucleotide aresubsequently converted to their adenosine dinucleotideforms by nicotinamidenicotinic acid mononucleotidenmnat1 nmnat2 nmnat3adenylyltransferasesnicotinamide adenine dinucleotide nad is a cofactorform of the vitamin whereas nicotinic acid dinucleotide issubsequently converted to nad by nad synthase nadsyn1 nad is reduced to nadh by oxidative reactions of glycolysis the tca cycle and βoxidation andsubsequently serves as a redox equivalent carrier to theelectron transport chain and to regenerate reducedascorbic acid vitamin c glutathione and thioredoxin nad can also be phosphorylated by nadkinases nadk nadk2 to form a distinct redox shuttlingcofactor nadp nadp is reduced by reactions in the 0cjeong and vacanti nutrition metabolism page of oxidative pentose phosphate pathway g6pd pgd andother enzymes eg me1 me3 idh1 idh2 to nadphnadph provides reducing equivalents for biosynthetic reactions in fatty acid cholesterol and deoxyribonucleotidesynthesis outside its role as a reducing equivalentshuttle nad provides adenine dinucleotide phosphateadp ribose for synthesis of the second messenger cyclicadenosine monophosphate camp via the activity of adenylate cyclases adcy family nad also providesadpribose and polyadpribose for post translationalmodifications of proteins via activity of adpribosyl transferases art family and adp ribose polymerases parpfamily [ ] camp and protein polyadpribosylationare important mediators of cell signaling and proteinexpression niacin is synthesized from tryptophan butin small quantities relative to a healthydietary intake deficiency known as pellagraismarked by dermatitis and severe gastrointestinalneurological pathophysiologies which are fatalif untreated no proteomic studies on systemic intakeof vitamin b3 were found at the time of writing thisreviewvitamin b5pantothenic acid vitamin b5 is composed of a moleculeof pantoic acid bound to βalanine fig itsprimary metabolic function is as an acylcarrier pantothenic acid is a substrate in the first reaction of coenzyme a coa biosynthesis catalyzed by pantothenatekinases pank1 pank2 pank3 pank4 coa isa substrate for enzymes catalyzing the oxidative decarboxylation of ketoacids including the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldthe oxoglutarate dehydrogenase complex ogdh dlstdld and the branched chain keto acid dehydrogenasecomplex bckdha dbt dld [] acyl speciesare activated by conjugation with coa and are substratesin or products of glycolysis the tca cycle fattyacidsynthesisβoxidation cholesterol synthesis ketogenesisbranchedchain amino acid catabolism and proteinacetylationoglcnacylation finally phosphopanthetheine product of pank proteins activities is acofactor of the acyl carrier protein domain of fatty acidsynthase fasn vitamin b5 deficiency is rare andusually accompanied by that of other nutrients burning of the feet and numbness in the toes is a characteristic manifestation along with variety of other symptoms no proteomic studies on systemic intake ofvitamin b5 were found at the time of writing this reviewvitamin b6vitamin b6 has aldehyde alcohol and amine forms fig of which the phosphorylated aldehyde form pyridoxalphosphate acts as a cofactor to over enzymes allthree forms of vitamin b6 are phosphorylated by pyridoxalkinase pdxk both the phosphorylated alcohol andamine forms pyridoxine phosphate and pyridoxaminephosphate are converted to pyridoxal phosphate bypyridoxine phosphate oxidase pnpo pyridoxalphosphate is a cofactor for enzymes catalyzing decarboxylase reactions in gammaaminobutyric acid gad1 gad2 and serotonindopamine biosynthesis ddc aswell as for enzymes catalyzing transamination reactionseg got1 got2 gpt gpt2 cysteine synthesiscth heme synthesis alas1 alas2 carnitinesynthesis3hydroxy6ntrimethyllysine aldolase geneunidentified andsphingolipid synthesis sptlc1 sptlc2 pyridoxalphosphate is also an important cofactor for enzymes ofonecarbon metabolism shmt1 and shmt2 andglycogen catabolism pygl and pygm vitamin b6deficiency is rare because of its availability in many foodsand pathophysiologies can be diverse niacin synthesis kynuin a tripletofbased study of streptozotocininduceddiabetic rat hippocampi pyridoxamine treatment prevented longterm recognition memory impairment andregulated protein abundances in a number of diversepathways notably upregulating half of the proteins involved in ubiquinol biosynthesis in a 2dgemsbased study of mice hippocampi the abundances ofphosphoglycerate mutase pgam1 and cannabinoidreceptorinteracting protein cnrip1 are reported tobe elevatedreduced respectively upon administration ofpyridoxine proteomic changes are accompanied by improved novel object recognition the plasma membrane byvitamin b7biotin vitamin b7 is composed of a fusedring structurebound to a valeric acid side chain fig it istransported acrossthesodiumdependent solute carriers slc5a6 and slc19a3[ ] as a cofactorposttranslational modificationbiotin covalently binds lysine residues it is a cofactor for pyruvate carboxylase pc acetylcoa carboxylase acaca propionylcoa carboxylase pcca andthe methylcrotonylcoa carboxylase complex mccc1mccc2 histones are also biotinylated regulatinggene expression the posttranslational modification occurs via the activity of holocarboxylase synthetasehlcs biotin deficiency is rare and has wide ranging pathophysiologies eating raw egg whites can preventitsabsorption leading to deficiency because of its affinityfor avidin a chemical in egg whites that is denaturedupon cooking this observation led to the vitaminseventual discovery no proteomic studies onsystemic intake of vitamin b7 were found at the time ofwriting this review 0cjeong and vacanti nutrition metabolism page of vitamin b9the term folate vitamin b9 is inclusive of a group ofcompounds composed of a pteridine ring linked to paraaminobenzoic acid with a mono or polyglutamate tailfig in its reduced form tetrahydrofolate aonecarbon unit crosslinks as ch or ch2 aminegroups on the ring structure and aminobenzoic acid orbinds the secondary amine as a formyl group on theaminobenzoic acid group [ ] this onecarbonunit is utilized in the synthesis of purines and thymidineconversion of homocysteine to methionine interconversion of serine and glycine and catabolism of histidinereactions collectively termed onecarbon metabolism[ ] at the cellular level onecarbon metabolismis tightly regulated by compartmentalization [ ] while wholebody folate homeostasis is predominantly maintained by the liver through the enterohepaticcycle folate deficiency induces megaloblastic macrocyticanemia and fetal neural tube defects purportedly via itsadverse impact on nucleotide synthesis [ ] lowintake of folate is also linked to cardiovascular disease[ ] neurodegenerative disease [ ] alzheimers disease [ ] and cancer [] in an orbitrapbased study of follicle fluid of womenundergoing in vitro fertilization the folate supplementedgroup is reported to have elevated abundances of apolipoproteins from high density lipoproteins and reducedreactive protein c crp the study is performed onwomen who did not become pregnantin aqtofbased study of a folatedeficiencyinduced intestinal neoplasia mouse model the combinatorial impactsof folate deficiency and methylene tetrahydrofolate reductase heterozygous deletion mthfr are reported toimpact protein abundances spanning diverse cellularfunctions however of samples are discarded as outliers and the simultaneous examination of mthfr anddietary folate deficiency does not allow proteomic adaptations to be attributed to either in isolation in a2dgemsbased study of adult rats aortic calmodulincalm1 calcium signaling protein abundances arepositively correlated with folate dose while abundancesof triose phosphate isomerase tpi1 glycolysis transgelin tagln cytoskeleton and glutathione stransferasealpha gsta3 reductive detoxification respond inversely in an 2dgemsbased study of rat liversprdx6 and gpx1 are reported to be elevated whilecofilin cfl1 is reported to be depleted under folatedeficiency other studies report protein abundancedifferences due to folate intake in rat urinary exosomesqqqbased human plasma 2dgems fetal brain tissue from pregnant mice fed ethanol2dgems pregnant rat livers 2dgems fetal rat livers 2dgems adult rat livers andbrains 2dgems and livers of piglets born tofolate deficient mothers 2dgems vitamin b12cobalamin vitamin b12 encompasses a group of molecules with four linked pyrrole ring derivatives forming a corrin ring and a cobalt atom bound at thecenter of the corrin ring the cobalt atom also binds a56dimethylbenzimidazole nucleotide and a functionalgroup fig the identity of the functionalgroup distinguishes the vitamin b12 compounds ascyanocobalamin hydroxycobalamin hydrocobalaminnitrocobalamin deoxyadenosylcobalamin also called adenosylcobalamin and methyl cobalamin [ ] methylcobalamin serves as a coenzyme in the conversion ofhomocysteine to methionine by methionine synthase mtrin the cytosol and adenosylcobalamin is required forconversion of lmethylmalonylcoa to succinylcoa bymethylmalonylcoa mutase mut in mitochondria vitamin b12 deficiency is closely related to folatedeficiency and can lead to megaloblastic anemia by impairment in the activity of methionine synthase mtr 5methyl tetrahydrofolate cannot be converted toonecarbon donors required for purine and thymidinesynthesis without vitamin b12 as a cofactor thus interfering with dna synthesis and erythrocyte production vitamin b12 deficiency is also linked to neurological disorders independent of anemia ruoppolo and colleagues performed a 2dgemsbased study of lymphocytes isolated from methylmalonicacidemia with homocystinuria cobalamin deficiency typec mmachc patients an inborn error in metabolismmarked by inactivity of the mmachc gene productreceiving a standard treatment of hydroxycobalaminbetaine folate and carnitine protein products of me2glud1 and gpd2 genes involved in anaplerosis andredox equivalent shuttling are upregulated while variant of protein pyruvate kinase muscle isozyme pkmand lactate dehydrogenase b ldhb are downregulatedrelative to lymphocytes isolated from healthy control donors in a 2dgebased study of adult rat cerebralspinal fluid protein abundance shifts are reported topeak after several months on a cobalamin deficient dietmodest shifts or after a total gastrectomy more severeshifts and return to near control values at later timepoints in a 2dgemsbased study glutathione stransferase p gstp1 abundances are diminished andglutathione peroxidase gpx1 abundances are elevated in rat pup kidneys under maternal vitamin b12deficient and maternal folate deficient conditions suggesting maternal dietary intake of these vitamins impacts offspring kidney redox homeostasis mechanisms 0cjeong and vacanti nutrition metabolism page of in a similar 2dgemsbased study of maternal vitaminb12 deficiencythe same group reports that severaldozen rat kidney pup proteins revert to control levelsupon administration of vitamin b12 at birth additionally diminished abundance of betaoxidation proteinsin kidneys of pups born to vitamin b12 deficientmothers is accompanied by elevated ppara apositive regulator offatty acid oxidation suggestingattempted compensation at the cellular levelvitamin cvitamin c ascorbic acid is absorbed at the brushborder and distributed to cells throughout the body bythe sodiumdependent plasma membrane solute carriersslc23a1 and slc23a2 the oxidized form of vitamin c dehydroascorbate is also transported via plasmamembrane glucose transporters slc2a1 slc2a3 andslc2a4 also known as glut1 glut3 and glut4 and reduced intracellularly to ascorbic acid byglutathione and the activity of thioredoxin reductases txnrd1 txnrd2 or txnrd3 vitamin c is a cofactor in the function of prolyl andlysyl hydroxylases which consume oxygen and alphaketoglutarate to form the hydroxylated amino acid residueand succinate the fe2 of these enzymes is restoredfrom fe3 by oxidation of vitamin c in the presenceof oxygen prolyl hydroxylases egln1 egln2 egln3also known as phd2 phd1 phd3 respectively hydroxylate the hif1a protein providing a necessary signal for itsdegradation and preventing a hypoxic response at the cellular level prolyl and lysyl hydroxylase activities arealso necessary for posttranslational modifications to formfunctional collagen lysyl hydroxylases includeplod1 plod2 and plod3 vitamin c serves anearly identical function in reducing fe3 as a cofactor fortrimethyllysine dioxygenase tmlh which catalyzes thefirst reaction in carnitine biosynthesis carnitine isessential for fatty acid catabolism in the mitochondria asonly fatty acyl carnitines formed via the activity of carnitine palmitoyl transferases cpt1a cpt1b and cpt1ccross the inner mitochondrial membrane through thesolute carrier slc25a20 vitamin c similarly servesas a cofactor for tyrosine hydroxylase th which catalyzes the first reaction in catecholamine eg dopamineepinephrine and norepinephrine synthesis additionally vitamin c serves and as a general antioxidant vitamin c deficiency leads to the condition knownas scurvy with symptoms largely attributed to malformedconnective tissue due to improperly folded collagen in a orbitrapbased study on a pig model of hemorrhagicshock vitamin c administration is reported to impactplasma protein abundances in the complement pathwayand those in polytrauma related processes including thestabilization of adamts13 abundance an importantregulator of clot formation an orbitrapbased studyof endoplasmic reticulum enriched fractions of livers inwerner syndrome mouse models identifies around adozen proteins whose abundances are impacted by administration of vitamin c a qtofbased study ofzebrafish reports upregulation of glutamate dehydrogenase glud1 and downregulation of pyruvate kinasemuscle isozyme pkm upon administration of vitamin cin a vitamin e deficient in a qqqbased study of human plasma ascorbic acid concentrationis reported to be inversely related to vitamin d bindingprotein gc abundance 2dgemsbased studiesidentify protein abundance regulations in mouse modelsof sarcoma metastases in the liver and tumor nodules of adenocarcinoma due to administration of vitaminc another 2dgemsbased study reports polypeptide abundance shifts in hemodialysis patient plasma uponvitamin c supplementation vitamin dvitamins d2 and d3 are respectively distinguished by theirergosterol and cholesterol backbones though onlyvitamin d3 is synthesized in animals both can be converted to active forms exposure of 7dehydrocholesterolan intermediate in cholesterol synthesis to ultravioletradiation in the skin and subsequent isomerization produces cholecalciferol vitamin d3 fig whether dehydrocholesterol is derived from cholesterol via activityof 7dehydrocholesterol reductase dhcr7 or synthesizedde novo in the skin is disputed 7dehydrocholesterolis successively hydroxylated by activity of cytochrome p450enzymes eg cyp2r1 and cyp27b1 in the liver and kidney to its active 125oh2 cholecalciferol [125oh2d3]form transport of vitamin d and its metabolitesoccurs bound to vitamin d binding protein gc ergocalciferol is the vitamin d2 equivalent of cholecalciferol and is activated analogously 125oh2d3 influences cellular function via nuclearreceptordependent and nuclear receptorindependentmechanisms the former involves 125oh2d3boundvitamin d receptor vdr forming a heterodimer complex with a retinoid x receptor rxra rxrb rxrgand subsequently binding vitamin d response elementsregulating transcription of genes largely involved modulating calcium and phosphorous transport andmaintaining homeostasis by regulating their absorptionin the kidneysintestines and bones [ ] therapidonsetreceptorindependent of 125oh2d3 are mediated by a membraneassociated rapid response steroid binding protein identifiedextracellularimpactsnuclear 0cjeong and vacanti nutrition metabolism page of as pdia3 and diversely impact cell growth survivaland immune response deficiency in vitamin d impairs bone mineralizationcausing rickets in infantschildren and osteomalacia inadults vitamin d deficiency is also linked tocardiovascular diseases [ ] cancer [ ]neurologicalimpairments [ ] and autoimmunediseases [ ] though underlying mechanisms arenot completely understoodin an orbitrapbased study of mouse fetal and postnatal lung tissue maternal vitamin d deficiency is reflectedin total proteome adaptations which are unexpectedlystrongest at postnatal day opposed to fetal time pointsimpacted proteins include several associated with lungdevelopment an orbitrapbased study of a mousebrain tissue model of remyelination in multiple sclerosisreports calcium binding protein abundances to be upregulated upon treatment with 125oh2d3 consistentwith the vitamin's regulatory role over calcium absorption in an orbitrapbased study of serum fromoverweight adults vitamin d deficiency is reported todifferentially affect abundances of proteins related toblood coagulation in males and females howeverabundances of these proteins are likely impacted by theproduction of serum from whole blood the authors report quantifying proteins table an impressiveanalytical depth for serum in a 2dgebased studyvitamin d deficient children are reported to have diminished serum abundances of adiponectin adipoq in a separate 2dgebased study the same groupreports fetuinb fetub to be elevated in the plasma ofobese vitamin d deficient children compared with theirvitamin d sufficient counterparts however theauthors do not directly identify fetub and rely on comparison of their findings to those of another study two 2dgemsbased studies of rat left ventricular andaortic tissueidentify proteins whose abundances respo | 0 |
thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples edgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients prognosis was veriï¬ed using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identiï¬cation of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore its very important to ï¬nd eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression proï¬les of speciï¬c cancers are easy to obtainwhich helps us better ï¬nd more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to deï¬ne and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenveriï¬ed However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identiï¬cation of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionproï¬les of THCA patients were accessed from TCGA databaseand modules associated with THCA were identiï¬ed by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression proï¬les of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentiï¬cation and Conï¬rmation ofTHCAAssociated GenesedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionproï¬les In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset glmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentiï¬cation of THCAAssociatedModulesAs shown in A a total of DEGs were identiï¬edlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of log k and log Pk were calculated for linearmodels construction respectively is the squared value of thecoeï¬cient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the ï¬rst time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and signiï¬cance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were veriï¬ed to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identiï¬cation of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology ï¬tting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene signiï¬cance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore GHR GPR125 Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was veriï¬ed Figure 2E From the heatmaps of the expression proï¬les of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3AC THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3DFFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patientsprognosis F Heatmap of the genes expression proï¬lesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model AC Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset DF ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identiï¬cationof the important genes during tumorigenesis and the deï¬nitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq proï¬les andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been veriï¬edto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is ï¬rstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction Itsreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereveriï¬ed as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further veriï¬ed by ROC and KaplanMeier curvesAs the expression proï¬les of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and its necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the ï¬nalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci Carling T Udelsman R Thyroid cancer Annu Rev Med 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope Zhao QJ Zhang J Xu L Liu FF Identiï¬cation of a ï¬velong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentiï¬cation ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is speciï¬cally expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identiï¬cation of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConï¬ict of Interest The authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestCopyright Zhao Zhang Shao and Fang This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c' | 2 |
" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically cold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12 these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments doseresponse and timecourse were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23 and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturers protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering à ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as à compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17 thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3adad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once à or twice à to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n4 micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only à pre exposure and à pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5bd similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5eg as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5egdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans ac to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive à and à ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations ag balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot eg g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with no evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw | 0 |
"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et | 0 |
Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learnings groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center DiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patients life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images []Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined []Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the identityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called BONENAVI version The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed CADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing à convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the networks weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physicians scores Taking a closer lookat the results it can be concluded that networks performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physicians further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patients whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center Diagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatients cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution à pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center DiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patientscategory for example malignant_ and healthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value was assigned for malignant_ prefixes whereas the value for healthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Pythons randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po | 2 |
pathogenesis of multiple myeloma MM is not completely known Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets Herein we explored the function and the working mechanism of circular RNA circ_0007841 in MM progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was employed to detect the expression of circ_0007841 microRNA3383p miR3383p and bromodomain containing BRD4 Cell proliferation ability was analyzed through cell counting kit8 CCK8 assay colony formation assay and flow cytometry Transwell assays were conducted to measure the migration and invasion abilities of MM cells Cell apoptosis was also assessed by flow cytometry The interaction between miR3383p and circ_0007841 or BRD4 was confirmed by dualluciferase reporter assay and RNApull down assayResults Circ_0007841 was highly expressed in bone marrow BMderived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs Circ_0007841 promoted the proliferation cell cycle and metastasis and impeded the apoptosis of MM cells miR3383p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR3383p BRD4 could directly bind to miR3383p in MM cells and miR3383p exerted an antitumor role through targeting BRD4 Circ_0007841 promoted the activation of PI3KAKT signaling via miR3383pBRD4 axis Exosomes generated from mesenchymal stromal cells MSCs elevated the malignant behaviors of MM cells via circ_0007841Conclusion Circ_0007841 acted as an oncogene to promote the proliferation cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR3383p to upregulate the expression of BRD4Keywords Multiple myeloma circ_0007841 miR3383p BRD4 ExosomeBackgroundMultiple myeloma MM is a kind of hematologic cancer featured by malignant proliferation of plasma cells [] The therapeutic strategies for MM patients include chemotherapy radiotherapy and targeted therapy [] However MM is still incurable by current treatment Correspondence wy1782126com Department of Hematology The Fifth Affiliated Hospital of Zhengzhou University No3 Kangfuqian Street Zhengzhou Henan ChinaFull list of author information is available at the end of the methods Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patientsNoncoding RNAs ncRNAs are a class of RNAs that are unable to code proteins generally and they are abundant in human genome to regulate cellular processes including proliferation metastasis and apoptosis [] Circular RNAs circRNAs are a kind of ncRNAs that characterized by covalently closed loop structure [] CircRNAs are more stable than linear RNAs and they The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of are resistant to exonuclease due to their loop structure [] CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs miRNAs sponges to modulate the abundance of downstream genes linked to proliferation metastasis and apoptosis [ ] The roles of circRNAs in hematological cancers have been reported before [ ] For instance circCBFB contributed to the proliferation ability while suppressed the apoptosis of chronic lymphocytic leukemia cells through targeting miR607FZD3Wntbetacatenin signaling [] However the functions of circRNAs in MM remain to be uncoveredMiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs mRNAs [] The dysregulation of miRNAs was involved in the pathogenesis of MM [ ] We concentrated on the role of miR3383p miR3383p suppressed the development of many cancers [] As for MM Cao et a0al reported that miR3383p suppressed the proliferation and accelerated the apoptosis of MM cells via CDK4 [] Nevertheless the function of miR3383p in MM is largely unexploredBromodomain containing BRD4 is a crucial epigenetic protein and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [] Zheng et a0al claimed that H19 accelerated the development of MM through upregulating BRD4 via sponging miR1523p [] Here the direct interaction between miR3383p and BRD4 was first found in MM and the function of BRD4 in MM was investigatedIn this study circ_0007841 was found to be abnormally upregulated in MM Lossoffunction experiments revealed that circ_0007841 silencing blocked the proliferation cell cycle progression migration and invasion while induced the apoptosis of MM cells The underlying mechanisms behind the oncogenic role of circ_0007841 in MM were further exploredMaterials and a0methodsPatientsPlasma cells from MM patients n and healthy volunteers n in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841 miR3383p and BRD4 via qRTPCR and Western blot assayCell cultureMM cell lines H929 and OPM2 and normal plasma cell line nPCs were purchased from BeNa Culture Collection Beijing China and maintained in Roswell Park Memorial Institute1640 RPMI1640 medium Gibco Carlsbad CA USA added with fetal bovine serum FBS Gibco unitsmL penicillin and a0μgmL streptomycin Cell culture plates were placed in a CO2 incubator at a0°C and cells were collected in the log phase of growthQuantitative realtime polymerase chain reaction qRTPCRAfter measuring the concentration using NanoDrop Invitrogen Carlsbad CA USA RNA sample a0ng was used to synthesize complementary DNA cDNA with ReverTra Ace qPCR RT Kit for circ_0007841 BRD4 and U6 Takara Dalian China and AllinOne¢ miRNA glyceraldehyde3phosphate dehydrogenase GAPDH First stand cDNA Synthesis Kit for miR3383p GeneCopoeia Rockville MD USA U6 served as the internal control for miR3383p while GAPDH acted as the internal reference for circ_0007841 and BRD4 PCR amplification reaction was conducted with SYBR Green PCR Master Mix Applied Biosystems Foster City CA USA on an ABI thermocycler Applied Biosystems The quantification of circ_0007841 miR3383p and BRD4 was carried out with the ÎÎCt method The specific primers in this study were synthesized from Sangon Biotech Shanghai China and listed as below circ_0007841 ²CTA ACA TCT GTG AAA CCA TCGT3² Forward Reverse ²TCA TCA CAT ACA CGA TAG ACTGG3² miR3383p Forward ²UCC AGC AUC AGU GAU UUU GUUG3² Reverse ²CAA CAA AAU CAC UGA UGC UGGA3² BRD4 Forward ²GTG GTG CAC ATC ATC CAG TC3² Reverse ²CCG ACT CTG AGG ACG AGA AG3² U6 Forward ²CTC GCT TCG GCA GCACA² Reverse ²AAC GCT TCA CGA ATT TGC GT3² GAPDH Forward ²GCG ACA CCC ACT CCT CCA C3² Reverse ²TCC ACC ACC CTG TTG CTG TAG3²and interfering RNAs siRNAs including sicirc_00078411 Cell transfectiontargeting Three small ²UGU circ_0007841 sicirc_00078412 UAG UUG CAA UGA AGA GAG3² si²UAA UGA UCA UGC CAA AUA CUC3² circ_00078413 ²UCA CAU ACA CGA UAG ACU GGC3² its negative control siNC circ_0007841 overexpression plasmid circ_0007841 its control Vector BRD4 overexpression plasmid BRD4 its control pcDNA miR3383p mimics miR3383p its control miRNC miR3383p inhibitor inmiR3383p and its control inmiRNC were obtained from Genepharma Shanghai China MM cells were seeded into 24well plates at a density of à cellswell overnight and transfection was conducted with Lipofectamine Invitrogen 0cWang a0et a0al Cancer Cell Int Page of Cell counting kit CCK8 assayMM cells were plated in 96well plates at the density of à cellswell and cultured overnight After transfection for indicated time points a0h a0h a0h or a0h MM cells were incubated with μL CCK8 Sigma St Louis MO USA for a0h The absorbance at a0 nm was detected by a microplate reader BioTek Winooski VT USAColony formation assayA total of cells were seeded onto the 6well plates to settle down The culture medium was replenished every d After 2week incubation the colonies were immobilized using poly methanol Sangon Biotech for a0 min followed by staining using crystal violet Sangon BiotechFlow cytometry for a0cell cycle and a0apoptosis detectionFor cell cycle analysis MM cells were collected using cold phosphate buffer saline PBS and then immobilized using cold ethanol solution overnight Prior to propidium iodide PI Solarbio Beijing China staining RNase was used to remove RNA in the samples The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur BectonDickinson San Jose CA USA and analyzed using Cell Quest software BectonDickinsonFor apoptosis analysis after transfection for a0 h MM cells were collected with PBS and then these cells were suspended in binding buffer Annexin Vcombined fluorescein isothiocyanate Annexin VFITC Solarbio and PI Solarbio were added to the reaction mixture and MM cells were simultaneously incubated with Annexin VFITC and PI at a0°C for a0min in a dark room The apoptotic MM cells were identified by FACSCalibur BectonDickinson and analyzed using Cell Quest software BectonDickinsonTranswell assaysIn transwell migration assay cell suspension MM cells suspended in μL serumfree medium was added into the upper chambers Costar Corning NY USA A total of μL culture medium with FBS was added into the lower chambers FBS acted as the chemotactic factor in this study After 24h incubation MM cells remained in the upper surface were removed with the cotton swab and the migrated MM cells were fixed with paraformaldehyde Sigma for a0 min and stained with crystal violet Sigma The number of migrated MM cells in five random visual fields was counted by the microscope Olympus Tokyo JapanIn transwell invasion assay the upper chambers were precoated with μL Matrigel Sigma to mimic the extracellular matrix The detection of cell invasion was conducted through using these precoated transwell chambers following the similar procedureBioinformatic prediction and a0dualluciferase reporter assayThe targets of circ_0007841 and miR3383p were predicted by circinteractome and targetscan software respectivelyThe wildtype partial sequence in circ_0007841 that predicted to bind to miR3383p along with the mutanttype sequence with miR3383p in circ_0007841 that was synthesized through using Sitedirected gene mutagenesis kit Takara Dalian China was amplified and cloned into pGL3 luciferase reporter vector Promega Madison WI USA termed as circ_0007841 WT or circ_0007841 MUT MM cells were cotransfected with a0 nM miRNC or miR3383p and a0 ng circ_0007841 WT or circ_0007841 MUT After 48h transfection MM cells were harvested and the luciferase activity was detected with the dualluciferase reporter assay system kit Promega using the luminometer Plate Chameleon V Hidex Finland according to the manufacturers instructions Firefly luciferase activity in each group was normalized to Renilla fluorescence intensityThe wildtype fragment of BRD4 ² untranslated region ²UTR that predicted to bind to miR3383p and the mutant type fragment of BRD4 ²UTR were also amplified and inserted into pGL3 luciferase reporter vector Promega to generate BRD4 ²UTR WT and BRD4 ²UTR MUT Cotransfection of MM cells with BRD4 ²UTR WT or BRD4 ²UTR MUT and miRNC or miR3383p was conducted following the similar procedureRNApull down a0assayRNApull down assay was conducted to test the interaction between circ_0007841 and miR3383p Biotin RNA Labeling Mix Roche Shanghai China was used in this study The wildtype and mutanttype binding sites in circ_0007841 that were predicted to bind to miR3383p were biotinylated to obtain Biocirc_0007841 WT and Biocirc_0007841 MUT MM cells were disrupted and incubated with BioNC Biocirc_0007841 WT or Biocirc_0007841 MUT The abundance of miR3383p was measured by qRTPCRWestern blot assayProteins were obtained using whole cell lysis buffer Roche Basel Switzerland for a0min on the ice Protein samples were quantified using Pierce BCA Protein Assay kit Thermo Fisher Scientific Rockford IL USA Then a0 µg of proteins were run on sodium dodecyl sulfate 0cWang a0et a0al Cancer Cell Int Page of polyacrylamide gel electrophoresis SDSPAGE gel and transferred to the polyvinylidene fluoride PVDF membrane Millipore Billerica MA USA After blocking with wv nonfat dry milk for a0h primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody ab205718 Abcam Cambridge MA USA The protein bands were measured using the enhanced chemiluminescent ECL system Beyotime Shanghai China according to the manufacturers instructions Gray analysis was conducted to quantify the expression of proteins using ImageJ software Primary antibodies including antiBRD4 ab128874 antiphosphorylatedphosphatidylinositol 3kinase antipPI3K ab70912 antiPI3K ab32089 antipAKT serinethreonine kinase pAKT ab38449 antiAKT ab64148 antiCD63 ab59479 antiCD81 ab79559 and antiβactin ab8226 were purchased from AbcamExosome isolationExosome isolation kit Qiagen Frankfurt Germany was used to extract exosomes from the culture medium of MM cells according to previous studies [ ]Statistical analysisAll statistical data in three independent experiments were shown as mean ± standard deviation SD Data were analyzed using GraphPad Prism The differences between two groups or among more than two groups were assessed through using Students t test or oneway analysis of variance ANOVA followed by Tukeys test The comparison between groups was considered significant when P value less than Linear correlation was analyzed using Spearmans correlation coefficientResultsCirc_0007841 elevates the a0malignant behaviors of a0MM cellsCirc_0007841 was abnormally upregulated in bone marrow BMderived plasma cells from MM patients compared with that in healthy individuals Fig a01a Meanwhile the level of circ_0007841 was higher in MM cell lines than that in normal plasma cell line nPCs Fig a01b The dysregulation of circ_0007841 in MM attached our attention Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells As mentioned in Fig a0 1c and d the level of circ_0007841 was downregulated with the transfection of sicirc_00078411 sicirc_00078412 or sicirc_00078413 Among these three siRNAs sicirc_00078411 was chose for the following assays due to its highest knockdown efficiency Fig a01c d Cell proliferation was assessed through CCK8 assay colony formation assay and flow cytometry According to the results of CCK8 assay sicirc_00078411 transfection significantly inhibited the proliferation of MM cells Fig a0 1e f The number of colonies was markedly reduced with the knockdown of circ_0007841 compared with siNC group Fig a0 1g The cell cycle of MM cells was arrested in G1S transition in sicirc_00078411 group than that in siNC group Fig a01h These findings together demonstrated that circ_0007841 silencing hampered the proliferation ability in MM cells Whats more circ_0007841 interference notably suppressed the migration and invasion of MM cells via transwell migration and invasion assays Fig a01i j The apoptosis rate of MM cells was increased in sicirc_00078411 group compared with that in siNC group Fig a01k Overall circ_0007841 accelerated the proliferation cell cycle progression and metastasis and inhibited the apoptosis of MM cellsmiR3p could directly interact with a0circ_0007841 in a0MM cellsTo address the mechanism by which circ_0007841 functioned in MM cells circinteractome website was used to seek the targets of circ_0007841 As shown in Fig a0 2a miR3383p possessed the complementary sites with circ_0007841 The luciferase activity was dramatically reduced in circ_0007841 WT group when cotransfected with miR3383p suggesting the target relationship between circ_0007841 and miR3383p in MM cells Fig a02b c We also constructed mutant type luciferase plasmid circ_0007841 MUT to investigate if UGC UGG in circ_0007841 was the binding sequence with miR3383p The luciferase intensity remained unaffected in circ_0007841 MUT group with the cotransfection of miRNC or miR3383p Fig a02b c suggested that circ_0007841 bound to miR3383p via its UGC UGG sequence RNApull down assay revealed that miR3383p could be pulleddown when using Biocirc_0007841 WT proving the target relationship between miR3383p and circ_0007841 Fig a0 2d e An obvious decrease in the level of miR3383p was observed in BMderived plasma cells from MM patients in contrast to that in normal volunteers Fig a02f Additionally there was a prominent reduction in the expression of miR3383p in MM cell lines than that in nPCs cell line Fig a0 2g The expression of miR3383p was negatively correlated with the level of circ_0007841 in BMderived plasma cells from MM patients Fig a02h The overexpression efficiency of circ_0007841 was high in MM cells and circ_0007841 accumulation caused a notable decrease in the level of miR3383p in MM cells Fig a02i j In summary circ_0007841 could inversely regulate the expression of miR3383p through direct interaction 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 elevates the malignant behaviors of MM cells a The enrichment of circ_0007841 was examined in BMderived plasma cells of MM patients and normal volunteers by qRTPCR b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRTPCR c d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with siNC sicirc_00078411 sicirc_00078412 or sicirc_00078413 by qRTPCR ek MM cells were transfected with siNC or sicirc_00078411 e f CCK8 assay was employed to assess the proliferation ability of MM cells g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells i j The metastasis ability of MM cells was evaluated by transwell assays k The apoptosis of MM cells was analyzed by flow cytometry P P P P if circ_0007841 exerted Circ_0007841 plays an a0oncogenic role through a0targeting miR3p in a0MM cellsTo disclose its oncogenic role through targeting miR3383p we conducted rescue experiments through cotransfecting H929 and OPM2 cells with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p As mentioned in Fig a03a sicirc_00078411 transfection increased the level of miR3383p and the introduction of inmiR3383p reversed the influence of circ_0007841 silencing in the expression of miR3383p Sicirc_00078411mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR3383p via CCK8 assay Fig a03b c Circ_0007841 silencing restrained the colony formation ability while the addition of miR3383p inhibitor partly recovered the colony formation ability in MM cells Fig a0 3d Additionally cell cycle of MM cells was arrested at G1S transition in sicirc_00078411 group and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of inmiR3383p Fig a03e f The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841 and the metastasis ability was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a0 3g h Sicirc_00078411induced apoptosis of MM cells was 0cWang a0et a0al Cancer Cell Int Page of Fig miR3383p could directly interact with circ_0007841 in MM cells a miR3383p was predicted as a candidate target of circ_0007841 by circinteractome software b c Dualluciferase reporter assay was conducted to verify whether miR3383p could bind to circ_0007841 in MM cells d e RNApull down assay was performed to confirm the target relationship between miR3383p and circ_0007841 in MM cells f g The expression of miR3383p was detected in BMderived plasma cells of MM patients and healthy volunteers MM cells and nPCs cells by qRTPCR h The correlation between the expression of miR3383p and circ_0007841 was analyzed using Spearmans coefficient i j The abundance of circ_0007841 and miR3383p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRTPCR P P P P attenuated by the addition of inmiR3383p Fig a0 3i Overall circ_0007841 could promote the malignant potential of MM cells through sponging miR3383pBRD4 is a0validated as a0a a0target of a0miR3p in a0MM cellsBRD4 was predicted as a direct target of miR3383p by targetscan database and the wild type or the mutant type binding sequence between miR3383p and BRD4 was shown in Fig a04a As exhibited in Fig a04b c the luciferase activity was markedly decreased in miR3383p and BRD4 ²untranslated region ²UTR WT cotransfected group while miR3383p transfection had no effect on the luciferase activity in BRD4 ²UTR MUT group compared with that in miRNC and BRD4 ²UTR MUT cotransfected group suggesting the interaction between BRD4 and miR3383p BRD4 was conspicuously upregulated in BMderived plasma cells of MM patients compared with that in healthy individuals Fig a0 4d Meanwhile BRD4 was also found to be upregulated in MM cell lines than that in nPCs cells Fig a0 4e The expression correlation between BRD4 and circ_0007841 or miR3383p was analyzed using Spearmans correlation coefficient As shown in Fig a0 4f g there was an inverse correlation between the levels of BRD4 and miR3383p while the expression of BRD4 was positively correlated with the level of circ_0007841 miR3383p overexpression significantly downregulated the expression of BRD4 in MM cells suggesting the negative regulatory relationship between BRD4 and miR3383p in MM cells Fig a04h Circ_0007841 and miR3383p were cotransfected into MM cells to uncover the relationship among circ_0007841 miR3383p and BRD4 As presented in Fig a0 4i circ_0007841 overexpression upregulated the level of BRD4 and the expression of BRD4 was decreased in circ_0007841 and miR3383p cotransfected group Collectively BRD4 was a target of miR3383p and circ_0007841 could elevate the expression of BRD4 through sponging miR3383pBRD4 overexpression attenuates the a0effects of a0miR3p accumulation on a0MM cellsmiR3383p and BRD4 were cotransfected into MM cells to explore whether miR3383p exerted an antitumor role in MM cells through targeting BRD4 As shown in Fig a0 5a the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was downregulated by the accumulation of miR3383p miR3383p overexpression inhibited the proliferation cell cycle and metastasis of MM cells and these inhibitory effects were attenuated by the 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 plays an oncogenic role through targeting miR3383p in MM cells ai MM cells were transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p a The level of miR3383p was examined in MM cells by qRTPCR assay b c The proliferation of MM cells was measured through conducting CCK8 assay d The proliferation capacity in transfected MM cells was assessed by colony formation assay e f The percentage of MM cells in G0G1 S or G2M phase was analyzed using flow cytometry g h The migration and invasion abilities of MM cells were evaluated by transwell assays i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry P P P addition of BRD4 overexpression plasmid Fig a0 5bh The apoptosis of MM cells was induced by the transfection of miR3383p and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells Fig a0 5i In conclusion miR3383p accumulation restrained the malignant behaviors of MM cells through targeting BRD4Circ_0007841 activates PI3KAKT signal pathway through a0targeting miR3pBRD4 axisThe activation of PI3KAKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis Herein we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841miR3383pBRD4 axis in the See figure on next pageFig BRD4 is validated as a target of miR3383p in MM cells a The complementary sites between miR3383p and the ²UTR of BRD4 were predicted by targetscan software b c The luciferase activity was measured in H929 and OPM2 cells transfected with miRNC or miR3383p and BRD4 ²UTR WT or BRD4 ²UTR MUT d The protein level of BRD4 in BMderived plasma cells of MM patients and healthy volunteers was detected by Western blot assay e The level of BRD4 in H929 OPM2 and nPCs cells was evaluated by Western blot assay f g The linear relationship between BRD4 and miR3383p or circ_0007841 was analyzed using Spearmans coefficient h The expression of BRD4 was detected in MM cells transfected with miRNC or miR3383p by Western blot assay i The protein level of BRD4 was detected in MM cells transfected with Vector circ_0007841 circ_0007841 miRNC or circ_0007841 miR3383p by Western blot assay P P P P 0cWang a0et a0al Cancer Cell Int Page of 0cWang a0et a0al Cancer Cell Int Page of Fig BRD4 overexpression attenuates the effects of miR3383p accumulation on MM cells ai MM cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 a qRTPCR was employed to measure the expression of BRD4 in MM cells b c CCK8 assay was applied to assess the proliferation ability of MM cells d Colony formation assay was performed to analyze the influences of miR3383p and BRD4 on the proliferation of MM cells e f Flow cytometry was conducted to detect the cell cycle of MM cells g h Transwell assays were performed to detect the metastasis of MM cells i The apoptosis rate of MM cells was examined by flow cytometry P P P activation of PI3KAKT signaling Circ_0007841 silencing downregulated the level of BRD4 and the level of BRD4 was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a06a b The activation of PI3KAKT signaling was suppressed with the silencing of circ_0007841 and the addition of inmiR3383p recovered the phosphorylation levels of PI3K and AKT Fig a06a c Meanwhile H929 and OPM2 cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 As mentioned in Fig a06d e miR3383p overexpression downregulated the level of BRD4 and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells The addition of BRD4 alleviated the inhibitory influence of miR3383p overexpression on the activation of PI3KAKT signaling in MM cells Fig a0 6d f Taken together circ_0007841 accelerated the progression of MM through miR3383pBRD4PI3KAKT axisMesenchymal stromal cells MSCsgenerated exosomes accelerate the a0malignant potential of a0MM cells via a0circ_0007841MSCs exert crucial roles in the progression of MM Herein we explored whether exosomes derived from MSCs could regulate the proliferation cell cycle metastasis and apoptosis of MM cells via circ_0007841 MSCs were isolated from the adjacent tissues of MM and normal tissues The expression of circ_0007841 was higher in MSCs and MSCsderived exosomes from adjacent tissues than that in normal tissues Fig a07a b The markers of exosomes CD63 and CD81 were notably upregulated in exosomes of MSCs instead of cell lysate Fig a07c As mentioned in Fig a07d we established a working model as previously described to explore if MSCsderived exosomes could regulate the proliferation cell cycle motility and apoptosis of MM cells [] In this model only exosomes could be transmitted through the filter to the upper chambers As presented in Fig a07ek sicirc_00078411 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 activates PI3KAKT signal pathway through targeting miR3383pBRD4 axis ac Western blot assay was performed to detect the levels of BRD4 and PI3KAKT signalingrelated proteins in MM cells transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p and gray analysis was used to assess the abundance of these proteins df The expression of BRD4 and PI3KAKT signalingassociated proteins in MM cells transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 was examined by Western blot assay P P P transfection inhibited the malignant behaviors of MM cells in Mock sicirc_00078411 group compared with that in Mock siNC group Besides MSCsderived exosomes MSCs siNC group promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells than that in Mock siNC group and these effects were attenuated by the silencing the circ_0007841 suggested that MSCsderived exosomes could promote the progression of MM via circ_0007841 Whats more the exosomes generated from MSCs accelerated the activation of PI3KAKT signaling while this effect was counteracted with the transfection of sicirc_00078411 Fig a0 7l Collectively MSCsderived exosomes could facilitate the progression of MM via circ_0007841DiscussionMM is an incurable cancer currently Because many MM patients were diagnosed at late stage the treatment outcomes of MM patients were unsatisfactory [] Therefore finding crucial markers in MM is urgent to improve the prognosis of MM patientsCircRNAs are featured by closely loop structure and they are widely distributed in human tissues Due to the stability and the universality of the distribution circRNAs are identified as ideal biomarkers for human cancers and other diseases [] For example the high expression of circ_0004277 was associated with the better prognosis of AML patients [] Xia et a0al claimed that circCBFB was highly expressed in chronic lymphocytic leukemia and circCBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [] Circ_0007841 was found to be overexpressed in BMderived plasma cells of MM patients and MM cells Further studies suggested that circ_0007841 promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells These findings | 2 |
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim | 0 |
" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir15a5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a wellknown oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir15a5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at Ëc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at Ëc with co2reverse transcriptionquantitative pcr rtqpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at Ëc for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at Ëc for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions Ëc for min followed by cycles at Ëc for sec and Ëc for sec and a final extension at Ëc for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir15a5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at Ëc for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 siyap1 and the negative control targeting a nonspecific sequence siscramble were provided by thermo fisher scientific inc siha and c33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of siyap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck solution was added to each well and cultured for h at Ëc the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin vfitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdualluciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h posttransfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at Ëc overnight each membrane was probed with primary antibodies against yap1 cat no and βactin cat no at Ëc overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir15a5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir15a5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir15a5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir15a5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir15a5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir15a5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir15a5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir15a5p inhibits the invasion and migration of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c33a cells were significantly inhibited by mir15a5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir15a5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir15a5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcriptionquantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase activity was detected by a commercial caspase activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir15a5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdnayap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir15a5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells cotransfected with firefly luciferase constructs containing the yap1 wt or mut 'utrs and mir15a5p mimics mimics nc mir15a5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir15a5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir15a5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir15a5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s | 0 |
"We also evaluated chest CT findings to determine the involvement of emphysema. The percentage of the COPD group with involvement of emphysema in the chest CT findings was almost twice as high as that of the non-COPD group (38.8% vs 20.3% respectively). Patient characteristics among non-COPD and COPD patients All cases (n?=?270) Non-COPD (n?=?123) COPD (n?=?147) p value Cases 100 (270) 45.6 (123) 54.4 (147) 0.0001 # Age years a 70.1 (3988) 67.9 (3982) 71.9 (5187) 0.0001 # Sex male 73.7 (199) 62.6 (77) 83.0 (122) 0.0001 # History of smoking 78.9 (213) 65.9 (81) 89.8 (132) 0.0001 # COPD managed b 8.5 (23) 1.6 (2) 14.3 (21) 0.0001 # COPD-related systemic comorbidities 55.9 (151) 52.8 (65) 58.5 (86) 0.390 Diabetes 19.3 (52) 16.3 (20) 21.8 (32) 0.280 Ischemic cardiac disease 7.4 (20) 2.4 (3) 11.6 (17) 0.004 # Hypertension 38.1 (103) 37.4 (46) 38.8 (57) 0.900 Hyperlipidemia 11.9 (32) 8.1 (10) 15.0 (22) 0.092 n indicates number. aData are shown as mean (range). bindicates the patients who had been diagnosed as COPD before bronchoscopy. All other data are shown as% (numbers). #p?<?0.05. COPD: chronic obstructive pulmonary disease. Population of non-COPD and COPD in Japanese patients with lung cancer. Schematic presentation of the percentage of non-COPD (n?=?123) and COPD (n?=?147) among Japanese patients with lung cancer. Patients with COPD were classified by GOLD grade that is grade 1 (n?=?95) grade 2 (n?=?41) grade 3 (n?=?11) and grade 4 (n?=?0). Physical assessment variables among non-COPD and COPD patients All cases (n?=?270) Non-COPD (n?=?123) COPD (n?=?147) p value BMI (kg/m 2 ) a 22.1 (2.8) 22.0 (2.8) 22.2 (2.9) 0.749 spirometric variables %VC a 105.9 (20.6) 104.8 (20.8) 106.7 (20.5) 0.520 FEV1 (ml) a 2062 (610) 2302 (555) 1861 (583) 0.0001 # FEV1/FVC a 67.3 (12.6) 77.8 (6.3) 58.9 (10.3) 0.0001 # %FEV1 predicted a 98.2 (25.7) 109.4 (21.1) 88.9 (25.4) 0.0001 # %IC a 85.9 (19.5) 83.6 (17.8) 87.9 (20.6) 0.111 chest CT finding emphysema 30.4 (82) 20.3 (25) 38.8 (57) 0.001 # n indicates number. aData are shown as mean (SD). All other data are shown as% (numbers). #p?<?0.05. BMI: body mass index; VC: vital capacity; FEV1: forced expiratory volume in 1 second; FVC: forced vital capacity; IC: inspiratory capacity; GOLD: the Global Initiative for Chronic Obstructive Lung Disease. Association of COPD prevalence with lung cancer characteristics in Japanese patients undergoing bronchoscopy To evaluate the association of COPD with characteristics of lung cancer the pathological findings EGFR mutation status clinical staging and decision for thoracic surgery were compared between the COPD group and the non-COPD group (). Characteristics of lung cancer status among non-COPD and COPD patients All cases (n?=?270) Non-COPD (n?=?123) COPD (n?=?147) p value Pathology 0.0001 # Adenocarcinoma 53.7 (145) 69.9 (86) 40.1 (59) ## Sq 27.0 (73) 17.9 (22) 34.7 (52) ## NSCLC 10.4 (28) 4.9 (6) 15.0 (21) ## SCLC 6.7 (18) 5.7 (7) 7.5 (11) Large 2.2 (6) 1.6 (2) 2.7 (4) Clinical stage 0.046 # 1A 25.6 (69) 30.8 (38) 21.1 (31) 1B 13.7 (37) 13.0 (16) 13.6 (20) 2A 9.6 (26) 13.0 (16) 7.5 (11) 2B 7.8 (21) 9.8 (12) 6.1 (9) 3A 11.9 (32) 8.9 (11) 14.3 (21) 3B 5.6 (15) 4.1 (5) 6.8 (10) 4 17.0 (46) 16.3 (20) 17.7 (26) ND 8.9 (24) 4.1 (5) 12.9 (19) ## Thoracic surgery 0.0001 # Yes 138 (51.1) 64.2 (79) 40.1 (59) EGFR mutation status 0.001 # Yes 14.8 (40) 21.1 (26) 9.5 (14) ## No 55.2 (149) 59.3 (73) 51.7 (76) ND 30.0 (81) 19.5 (24) 38.8 (57) ## n indicates number. All data are shown as% (numbers). ND indicates not determined. #p?<?0.05. ##indicates a significant difference compared with the non-COPD group. COPD: chronic obstructive pulmonary disease; Sq: squamous cell carcinoma; NSCLC: non-small cell lung carcinoma; SCLC: small cell lung carcinoma; Large: large cell carcinoma; EGFR: epidermal growth factor receptor. Regarding pathologic findings the incidence of adenocarcinoma was significantly lower in the COPD group than in the non-COPD group. In the present study EGFR mutation was observed only in the patients with adenocarcinoma (40/145 cases; 27.6%). In contrast the number of cases in which EGFR mutation status was not determined was significantly higher in the COPD group than in the non-COPD group. Although determination of the clinical stage should be essential in order to propose the therapeutic options for lung cancer some cases in which clinical staging had not been completed were observed in the study population. The number of these cases was significantly higher in the COPD group than in the non-COPD group. In contrast the proportion of patients with each classification in the clinical staging did not differ between the two groups besides the cases in which the clinical staging was not determined. Among the total study population the number of thoracic surgeries performed was significantly lower in the COPD group than in the non-COPD group. Critical impact of the severity of airflow obstruction on the decision to propose thoracic surgery with curative intent To explore whether or not the severity of airflow obstruction might affect the decision to propose thoracic surgery with curative intent patients at stage 3B and 4 were excluded from the analysis because they were not eligible for thoracic surgery. In addition patients for whom the clinical staging had not been completed were also excluded. As a result we evaluated data from 185 patients with lung cancer at stage 1A to 3A who underwent spirometry and bronchoscopy. These patients were subdivided into those who underwent thoracic surgery (135 cases) and those who did not (50 cases). The characteristics and spirometric data for the patients with or without thoracic surgery are summarized in Tables 4 and 5. The characteristics of lung cancer among these groups are also shown in . Univariate analysis identified a significantly lower frequency of thoracic surgery among the patients of greater age and with more severe airway obstruction and advanced clinical staging. Univariate analysis also identified a significantly higher frequency of thoracic surgery among patients with adenocarcinoma. Finally all of the factors with a significant association in the univariate analysis were applied to the multivariate model to identify variables independently associated with the decision for thoracic surgery. Multivariate analysis identified more severe airway obstruction advanced clinical stagings and higher age as independent factors affecting the decision on thoracic surgery ()." | 1 |
" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus piv respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecaloral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled toilet plume in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as the quintessential perpetrator of inflammation to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1 generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a gray zone about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a one size fits all consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should never worry about action only inaction 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o | 0 |
among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patients health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patients quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal disquadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology 20e1 lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202adenocarcinomastech coloproctol 0c' | 0 |
"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Marys Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturers recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patients space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patients blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patients space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to DisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturers recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturers instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c" | 2 |
" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the consensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as phagosomeautoimmune thyroid disease antigen processing andpresentation b cell receptor signaling pathway intestinal immune network for iga production inflammatorybowel disease primary immunodeficiency th1 andth2 cell differentiation th17 cell differentiation natural killer cell mediated cytotoxicity and nfkappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didnt involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science 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"peptide vaccine CTL clone lung cancer Introduction Lung cancer is one of the main causes of cancer-related mortality. Approximately 85% of lung cancers are diagnosed as non-small cell lung cancer (NSCLC) and the overall survival (OS) rate for advanced NSCLC is poor. The 5-year survival rate is 5% for stage IIIb NSCLC and <1% for stage IV NSCLC (1). Treatment for NCSLC is determined by the patients clinical and tumor characteristics performance status (PS) the histological subtype and tumor genotype/phenotype. Recently there have been many studies concerning agents that target molecular changes such as mutations in the epidermal growth factor receptor (EGFR) and the fusion oncogene EML4-ALK in which the echinoderm microtubule-associated protein-like 4 (EML4) is fused with the intracellular domain of anaplastic kinase (ALK) (24). Although significant advances have been made in the treatment of NSCLC using molecular targeted therapies such as erlotinib and crizotinib the median OS for patients with advanced NSCLC remains low (56) and acquired resistance to target agents is a major clinical problem. Therefore the development of novel therapies is needed (7). Immunotherapy manipulates the immune system to control and eradicate cancer. Many recent studies provide evidence suggesting that immunotherapeutic manipulations are viable in many tumor types including lung cancer. Numerous trials of peptide vaccines autologous cellular therapy T cell-directed antibody therapy and monoclonal antibody therapy for lung cancer have been carried out around the world (810) and some of them have shown favorable results (1113). The EML4-ALK fusion gene was identified in NSCLC patients by a team led by Professor H. Mano. This fusion gene was formed as the result of a small inversion within the short arm of chromosome 2 that joins differing portions of the EML4 gene with a portion of the ALK gene (1415). As a result of this fusion constant dimerization of the kinase domain of ALK is induced and its catalytic activity increases consequently. The EML4-ALK fusion gene is mainly identified in young never/former light smokers with NSCLC (16). It is estimated that approximately 5% of all NSCLC cases have this fusion gene. A few reports have also identified EML4-ALK in other cancers namely breast cancer and colorectal cancer (1718). For the most part the EML4-ALK fusion gene and other mutations such as those in EGFR and KRAS are mutually exclusive (19). The chromosomal inversion does not always occur in the same location and multiple EML4-ALK variants have been identified (19). At least 11 variants have been reported. The most common variants are E13;A20 (variant 1) and E6a/b;A20 (variant 3a/b) which have been detected in 33% and 29% of NSCLC patients respectively (14). PF-02341066 (crizotinib) is an ALK inhibitor currently under clinical development. Kwak et al conducted an open-label multi-center two-part phase I trial and found a remarkable 57% overall response rate and a 72% 6-month progression-free survival rate (20). In spite of the marked antitumor activity of crizotinib ALK-positive cancers invariably gain resistance to crizotinib. In the case of ALK-positive cancers as well as EGFR-mutant lung cancer resistance develops on average within the first 2 years of therapy (21). The main resistance mutations are L1196M a gatekeeper mutation and C1156M. In addition to ALK mutations other known mechanisms for acquired resistance include ALK amplification (2122) and EGFR activation (2324). To overcome resistance new ALK inhibitors are currently in early phase studies (25). Novel combinatorial strategies to overcome crizotinib resistance and further improve the clinical outcome are needed. We focused on this new fusion array as a novel target of immunotherapy. There are several methods to detect EML4-ALK NSCLC including polymerase chain reaction (PCR) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) (19). These methods detect high-level EML4-ALK fusion gene expression. Passoni et al identified two HLA-A*02:01-restricted ALK-derived peptides that induce peptide-specific CTL lines (26). We focused on the EML4 array as a novel epitope of immunotherapy. We identified a candidate 9- or 10-amino acid array of novel epitopes using the Bioinformatics and Molecular Analysis Section (BIMAS) software and analyzed its potential as a new immunotherapy epitope with respect to its ability to induce anticancer activity. We then induced and generated a peptide-specific CTL clone from peripheral blood lymphocytes of HLA-A*02:01-positive healthy donors. We report here that an EML4-ALK-derived peptide-specific human CTL clone recognized peptide-pulsed T2 cells and HLA-A*02:01-positive and EML4-ALK-positive tumor cells pretreated with IFN-?. Furthermore we showed that immunotherapy with this novel epitope peptide has potential for treatment of EML4-ALK-positive NSCLC. Materials and methods Peptides Human EML4-ALK-derived peptides carrying binding motifs for HLA-A*02:01-/HLA-A*24:02-encoded molecules were identified by HLA-peptide binding predictions using the BIMAS program (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html). We purchased a total of seven EML4-ALK-derived peptides carrying HLA-A*02:01 binding motifs and two peptides carrying HLA-A*24:02 binding motifs from Geneworld (Tokyo Japan). Cell lines The H2228 human lung adenocarcinoma cell line and EML4-ALK fusion protein variant 3 (E6; A20) were kindly provided by Professor S. Yano (Kanazawa University). T2 is a lymphoblastoid cell line that lacks TAP function and has HLA-A*02:01 molecules that can easily be loaded with exogenous peptides. T2A24 is the same cell line but with HLA-A*24:02 instead. T2 and T2A24 cells were cultured in RPMI medium supplemented with 10% heat-inactivated FBS. HLA-A*02:01/HLA-A*24:02 binding assay In order to determine the binding ability of the predicted peptides to HLA-A*02:01/HLA-A*24:02 molecules an in vitro cellular binding assay was performed as reported previously (27). Briefly after incubation of the T2/T2A24 cells in culture medium at 26°C for 18 h cells were washed with PBS and suspended in 1 ml Opti-MEM (Invitrogen Carlsbad CA USA) with or without 100 ?g peptide and then incubated at 26°C for 3 h and at 37°C for 3 h. After washing with PBS HLA-A*02:01/HLA-A*24:02 expression was measured by flow cytometry using a FITC-conjugated and HLA-A*02:01-/HLA-A*24:02-specific monoclonal antibody (mAb) and the mean fluorescence intensity was recorded. Generation of dendritic cells CD14+ cells were isolated from human peripheral blood mononuclear cells (PBMCs) using human CD14 microbeads (Miltenyi Biotec Bergisch Gladbach Germany). Immature dendritic cells (DCs) were generated from CD14+ cells using interleukin (IL)-4 (10 ng/ml; PeproTech Inc. Rocky Hill NJ USA) and granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng/ml; PeproTech) in RPMI-1640 medium supplemented with 10% FBS. Maturation of DCs was induced by prostaglandin E2 (PGE2; 1 ?g/ml; Sigma St. Louis MO USA) and tumor necrosis factor (TNF-)-? (10 ng/ml; PeproTech). Induction of EML4-ALK-derived peptide-specific CTLs from PBMCs CD8+ cells were isolated from PBMCs using human CD8 microbeads (Miltenyi Biotec Bergisch Gladbach Germany). CD8+ cells (2106) were stimulated by peptide-pulsed irradiated autologous mature DCs (1105). Autologous DCs were prepared from a limited supply; artificial antigen presenting cells (aAPCs) (K562/A2 or A24/CD80/CD83) were alternatively used for further examination. After 1 week these cells were stimulated twice per week by peptide-pulsed irradiated artificial APC-A2 or artificial APC-A24 cells (1105). Supplementation with 10 IU/ml IL-2 (Proleukin; Novartis Pharmaceuticals Basel Switzerland) and 10 ng/ml IL-15 (PeproTech) was performed every 3 to 4 days between stimulations (28). IFN-? ELISPOT assay Specific secretion of IFN-? from human CTLs in response to stimulator cells was assayed using the IFN-? ELISPOT kit (BD Biosciences) according to the manufacturers instructions. Stimulator cells were pulsed with peptide for 2 h at room temperature and then washed. Responder cells were incubated with stimulator cells for 20 h. The resulting spots were counted using an ELIPHOTO counter (Minerva Tech Tokyo Japan). HIV-gag (7785) (SLYNTYATL) was used as an irrelevant peptide in the CTL assay. Generation of CTL clones Cultured cells were incubated with peptide-pulsed T2/T2A24 cells at a ratio of 2:1 for 3.5 h at 37°C. CD107a-specific antibodies (BioLegend San Diego CA USA) were included in the mixture during the incubation period. CD8+CD107a+ cells were sorted using a FACSAria II cell sorter (BD Biosciences). Sorted CTLs were stimulated and the CTL clones were established as described previously (29). Flow cytometry H2228 cells with or without pretreatment with 100 U/ml IFN-? (PeproTech) for 48 h were harvested and stained with anti-HLA-A2 Ab-FITC (MBL Japan) and analyzed using a FACSCanto II flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo software. Cytotoxicity assay The cytotoxic capacity was analyzed using the Terascan VPC system (Minerva Tech Tokyo). The CTL clone was used as the effector cell type. Target cells treated with 100 U/ml IFN-? (PeproTech) 42 h previously were labeled through incubation in calcein-AM solution for 30 min at 37°C. The labeled cells (1104) were then co-cultured with the effector cells for 46 h. Fluorescence intensity was measured before and after the culture period and specific cytotoxic activity was calculated as described previously (29). HLA-A*02:01 blocking of T-cell activity was tested by pre-incubating the target cells with anti-HLA-A -B -C mAb (W6/32) or an isotype control mAb (mIgG2a?; BioLegend San Diego CA USA). Results Identification of HLA-A*02:01-/HLA-A*24:02-restricted EML4-ALK-derived peptides As candidate EML4-ALK- derived and HLA-A*02:01-/HLA-A*24:02-restricted CTL epitopes we selected nine peptides with highly predicted scores for HLA-A*02:01/HLA-A*24:02 binding calculated using BIMAS software (Tables I and II) and evaluated their ability to bind to HLA-A*02:01/HLA-A*24:02 molecules. All nine peptides were able to bind HLA-A*02:01/HLA-A*24:02 molecules (Fig. 1). Generation of an EML4-ALK-derived peptide-specific CTL clone from human PBMCs We next assessed the capacity of EML4-ALK-derived peptides to generate peptide-specific CTLs in vitro from human PBMCs of HLA-A*02:01/HLA-A*24:02 healthy donors. CTLs were induced by three stimulations with DCs or artificial APCs loaded with the EML4-ALK-derived peptides. CTLs were tested for specificity for each peptide using the IFN-? ELISPOT assay. Peptides A B and C could induce peptide-specific CTLs that were able to specifically recognize T2 cells pulsed with each peptide but not T2 cells without peptides (Fig. 2). Peptides B and C were able to induce CTLs from only one donor (healthy donor 3 for peptide B and healthy donor 4 for peptide C) but peptide A was able to induce CTLs in three of four donors (healthy donors 2 3 and 4). Based on this result we used peptide A for further examinations. Next we obtained one CTL clone from peptide A-specific CTLs that was able to specifically recognize T2 cells pulsed with peptide A but not T2 cells pulsed with an irrelevant HIV-gag peptide using single cell sorting with a CD107a antibody. The population of CD8+CD107a+ cells represented 0.984% of all stimulated cells (Fig. 3A). These cells were sorted as single cells in each well of a 96-well plate. Twenty-one days after cell sorting peptide specificity was assessed using the IFN-? ELISPOT assay (Fig. 3B). The established clone reacted to the T2 cells pulsed with peptide A but not to T2 cells pulsed with the irrelevant HIV-gag peptide. These results indicate that a peptide A-specific CTL clone was successfully established from PBMCs from a healthy donor. The EML4-ALK-specific CTL clone recognizes HLA-A*02:01+ lung carcinoma cells with the EML4-ALK variant 3a/b incubated with IFN-? We next evaluated the ability of the EML4-ALK-specific CTL clone to recognize the cancer cell line H2228 which expresses HLA-A*02:01 and EML4-ALK using the IFN-? ELISPOT assay. Even though the EML4-ALK-specific CTL clone failed to recognize H2228 cells it did recognize those pretreated with 100 U/ml IFN-? 48 h prior to examination (Fig. 4A). We examined the effect of IFN-? on H2228 cells. Incubating target cells with IFN-? for 48 h increased the expression of MHC class I molecules on the cell surface (Fig. 4B). This result indicates that the peptide A-specific CTL clone was able to recognize H2228 cells because of increased expression of MHC-class I on the H2228 cell surface. Specific IFN-? production by the peptide A-specific CTL clone was detectable in H2228 cells treated with IFN-?. The specificity was abolished by an anti-HLA-class I mAb but not by an isotype control suggesting that the observed production was HLA-A2 restricted (Fig. 4C). A cytotoxicity assay was also performed. The peptide A-specific CTL clone was able to specifically lyse H2228 cells pretreated with IFN-? 48 h prior to examination. This specific lysis was blocked by the anti-HLA-class I mAb but not by the isotype control. These results indicate that the peptide A-specific CTL clone showed cytotoxicity and the ability to produce IFN-? against HLA-A*02:01+ EML4-ALK+ NSCLC cell lines (Fig. 5). Discussion In the present study we identified a new tumor-associated CTL epitope (peptide A) derived from EML4-ALK which binds to HLA-A*02:01 molecules and we were able to establish a peptide-specific CTL clone from human PBMCs that specifically recognized cognate peptide-pulsed T2 cells and HLA-A*02:01 tumor cells expressing EML4-ALK that had been pretreated with IFN-?. EML4-ALK-positive lung cancers are highly sensitive to ALK inhibition. However as with trastuzumab or gefitinib (3031) patients typically gain resistance within 1 to 2 years of starting therapy (23). We aimed to overcome these difficulties with immunotherapy. We identified a glypican-3 (GPC3)-derived peptide and showed that GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies than in those with low frequencies (32). This indicates that the ability to induce a peptide-specific CTL clone is important for effective immunotherapy. We also revealed that GPC3 is an ideal target for anticancer immunotherapy since it is specifically overexpressed in hepatocellular carcinoma (HCC) (3335). In the present study we chose a peptide array from EML4-ALK from which we were able to induce a peptide-specific CTL clone. EML4-ALK is a strong oncogene overexpressed in cancer cells of NSCLC breast cancer kidney cancer and colon cancer (17). We performed RT-PCR and assayed the EML4 DNA levels of certain lung cancer cell lines. H2228 cells express EML4 moderately but at higher levels than other lung cancer cell lines. EML4 expression has been reported as highly expressed in CD8+ T cells. RT-PCR showed that EML4 DNA levels were high in PBMCs and CD8+ T cells. Because of a lack of suitable antibodies we could not perform western blotting. However our success at inducing a peptide A-specific CTL clone from CD8+ T cells indicated that the CTL clone had no cytotoxicity against CD8+ T cells. This CTL clone could not recognize cancer cell lines without the ability to increase the amount of HLA class I presented on cell surfaces. Further examination is needed to achieve higher tumor reactivity. Combination chemotherapy or radiation therapy plus immunotherapy was recently reported to have a synergistic effect (36). Moreover some mechanisms of synergy between radiation therapy chemotherapy and immunotherapy have been revealed (37). In one of the mechanisms these therapies upregulated tumor antigens and MHC moieties. These results suggest that combination therapy could be used to make tumor cell lines more susceptible to this peptide A-specific CTL clone-mediated cytolysis (3841). In addition this treatment may be able to overcome resistance to ALK inhibition. Some resistance mechanisms for targeting drugs have been examined. The most commonly identified causes of resistance are point mutations such as L1196M (4244) G1269A (22) and S1206Y (21). These point mutations occur in the tyrosine kinase domain which plays an important role in oncogenesis. Our peptide array was selected from EML4 which has no correlation with these point mutations. It is possible that this treatment is effective for tumor cells resistant to ALK inhibitors. In this study we identified a new epitope peptide derived from the EML4-ALK fusion gene. We successfully induced an HLA-A*02:01-restricted peptide-specific CTL clone that demonstrated cytotoxicity for EML4-ALK-positive tumor cells. This is a new epitope-based vaccine therapy design for EML4-ALK-positive cancer cells. In order to obtain a stronger effect further analysis is needed. Acknowledgements We thank Professor S. Yano for providing the H2228 cell line which possesses the EML4-ALK fusion gene Professor H. Mano for providing the EML4-ALK fusion DNA and Professor N. Hirano for providing artificial APCs. This study was supported in part by Health and Labor Science Research Grants for Clinical Research and Third Term Comprehensive Control Research for Cancer from the Ministry of Health Labor and Welfare Japan and the National Cancer Center Research and Development Fund (25-A-7). References 1 Silvestri GA Tanoue LT Margolis ML The noninvasive staging of non-small cell lung cancer: the guidelines Chest 123 147S 156S 2003 12527574 2 Reck M What future opportunities may immuno-oncology provide for improving the treatment of patients with lung cancer? 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cancer: results from a randomized double-blind multicenter phase II study J Clin Oncol 30 2046 2054 2012 22547592 13 Topalian SL Hodi FS Brahmer JR Safety activity and immune correlates of anti-PD-1 antibody in cancer N Engl J Med 366 2443 2454 2012 22658127 14 Soda M Choi YL Mano H Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer Nature 448 561 566 2007 17625570 15 Bonanno L Favaretto A Rugge M Role of genotyping in non-small cell lung cancer treatment: current status Drugs 71 2231 2246 2011 22085382 16 Fukui T Yatabe Y Mitsudomi T Clinicoradiologic characteristics of patients with lung adenocarcinoma harboring EML4-ALK fusion oncogene Lung Cancer 77 319 325 2012 22483782 17 Lin E Li L Guan Y Exon array profiling detects EML4-ALK fusion in breast colorectal and non-small cell lung cancers Mol Cancer Res 7 1466 1476 2009 19737969 18 Robertson FM Petricoin EF III Cristofanilli M Presence of anaplastic lymphoma kinase in inflammatory breast cancer 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epithelial- mesenchymal transition and resistance to HER-2-targeted therapies in HER-2-positive luminal breast cancer PLoS One 8 e71987 2013 23991019 32 Sawada Y Yoshikawa T Nakatsura T Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival Clin Cancer Res 18 3686 3696 2012 22577059 33 Nakatsura T Yoshitake Y Nishimura Y Glypican-3 overexpressed specifically in human hepatocellular carcinoma is a novel tumor marker Biochem Biophys Res Commun 306 16 25 2003 12788060 34 Okabe H Satoh S Nakamura Y Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression Cancer Res 61 2129 2137 2001 11280777 35 Saito-Hisaminato A Katagiri T Nakamura Y Genome-wide profiling of gene expression in 29 normal human tissues with a cDNA microarray DNA Res 9 35 45 2002 12056413 36 Weir GM Liwski RS Mansour M Immune modulation by chemotherapy or immunotherapy to enhance cancer vaccines Cancers 3 3114 3142 2011 24212948 37 Hodge JW Ardiani A Gameiro SR The tipping point for combination therapy: cancer vaccines with radiation chemotherapy or targeted small molecule inhibitors Semin Oncol 39 323 339 2012 22595055 38 Garnett CT Palena C Hodge JW Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes Cancer Res 64 7985 7994 2004 15520206 39 Gelbard A Garnett CT Hodge JW Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis Clin Cancer Res 12 1897 1905 2006 16551875 40 Kaneno R Shurin GV Shurin MR Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells Cell Oncol 34 97 106 2011 41 Ramakrishnan R Assudani D Gabrilovich DI Chemotherapy enhances tumor cell susceptibility to CTL-mediated killing during cancer immunotherapy in mice J Clin Invest 120 1111 1124 2010 20234093 42 Choi YL Soda M Yamashita Y EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors N Engl J Med 363 1734 1739 2010 20979473 43 Katayama R Khan TM Benes C Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK Proc Natl Acad Sci USA 108 7535 7540 2011 21502504 44 Lovly CM Pao W Escaping ALK inhibition: mechanisms of and strategies to overcome resistance Sci Transl Med 4 120ps2 2012 Figure 1 EML4-ALK-derived peptides bound to HLA-A2 or HLA-A24 molecules. In vitro cellular peptide binding assays for HLA-A*02:01 (A) or HLA-A*24:02 (B) were performed using a FACS system. Figure 2 IFN-? release by in vitro-induced anti-EML4-ALK CTLs. CD8+ T cells from four healthy donors were stimulated with EML4-ALK-derived peptide-pulsed autologous DCs and aAPCs. CTLs induced by EML4-ALK-derived peptides (1105) were stimulated with T2 cells pulsed with or without 1 ?M EML4-ALK-derived peptides. IFN-?-producing CTLs were detected by IFN-? ELISPOT assay. DCs dendritic cells; aAPCs artificial antigen presenting cells; CTLs cytotoxic T cells. Figure 3 Peptide A-specific CTL clone established from anti-EML4-ALK CTL. (A) Peptide A-specific CTL clones established using CD107a single cell sorting. Peptide A-specific CTLs (1105) were incubated with peptide-pulsed T2 cells (5104) with CD107a-specific antibodies for 3.5 h at 37°C. CD8+CD107a+ cells were sorted using a FACSAria II cell sorter. Square CD8+CD107a+ cells that are peptide A-specific CTL clones. (B) Recognition of peptide-pulsed T2 cells by peptide A-specific CTL clones. A peptide A-specific CTL clone (1104 cells) was incubated with stimulator cells that had been pulsed with 1 ?M peptide A or HIV-gag peptide. IFN-?-producing CTLs were detected by IFN-? ELISPOT assay. CTLs cytotoxic T cells. Figure 4 Recognition of lung carcinoma cells expressing HLA-A*02:01 and the EML4-ALK fusion gene by the peptide A-specific CTL clone. The peptide A-specific CTL clone recognized H2228 cells pretreated with IFN-? 48 h prior to the assay. (A) The peptide A-specific CTL clone (1104 cells) was incubated with H2228 cells with or without IFN-?. IFN-? production was detected by IFN-? ELISPOT assay. (B) IFN-? increased expression of HLA-A2 presented on H2228 cells. Incubation of H2228 cells with 100 U/ml IFN-? for 48 h increased HLA-A2 presentation on the cells. Dotted line HLA-A2 on H2228 cells without IFN-?. Black line HLA-A2 on H2228 cells incubated with IFN-? (higher than on H2228 cells without IFN-?). Dashed line and shaded region: no staining of H2228 cells with/without IFN-?. (C) Inhibition of IFN-? production by an anti-HLA-class I mAb. Blocking experiments were performed using an HLA-A -B -C-specific mAb (W6/32) or an isotype control mAb (mIgG2a?). The peptide A-specific CTL clone was incubated with H2228 cells (HLA-A*02:01+/EML4-ALK+) pretreated with IFN-? 48 h prior to examination. IFN-?-producing CTL clones were detected by IFN-? ELISPOT assay. The bar graph shows the percentage of inhibition. CTL cytotoxic T cell. Figure 5 Cytotoxic activity of the peptide A-specific CTL clone against H2228 cells. The peptide A-specific CTL clone was incubated with H2228 cells pretreated with IFN-? 48 h prior to the assay at various E/T ratios and specific lysis was assessed. Blocking experiments were performed using the HLA-A -B -C-specific mAb (W6/32) or the isotype control mAb (mIgG2a?). CTL cytotoxic T cell. Table I HLA-A2 peptide binding predictions of the BIMAS program. Peptide name Peptide sequence Binding scorea A RLSALESRV 69.552 B AISEDHVASV 90.183 C TVLKAALADV 51.79 D KLIPKVTKT 59.989 E YLLPTGEIV 237.82 F MLIWSKTTV 118.238 G VMLIWSKTTV 315.95 a Binding scores were estimated using BIMAS software (http://www-bimas.cit.nih.gov/mobio/hla_bind/). Table II HLA-A24 peptide binding predictions of the BIMAS program. Peptide name Peptide sequence Binding scorea H NYDDIRTEL 369.6 I VYFIASVVVL 200 a Binding scores were estimated using BIMAS software (http://www-bimas.cit.nih.gov/mobio/hla_bind/). Diagn Pathol Diagn Pathol Diagnostic Pathology 1746-1596 BioMed Central 24972450 4085714 1746-1596-9-128 10.1186/1746-1596-9-128 Research Overexpression of both platelet-derived growth factor-BB and vascular endothelial growth factor-C and its association with lymphangiogenesis in primary human non-small cell lung cancer Liu Jiannan 1 2 [email protected] Liu Chuanyong 1 [email protected] Qiu Liyun 3 [email protected] Li Juan 1 [email protected] Zhang Pei 1 [email protected] Sun Yuping 1 [email protected] 1Department of Oncology Jinan Central Hospital Affiliated to Shandong University No. 105.Jiefang Road Jinan Shandong 250013 P.R. China 2Department of Oncology Yuhuangding Hospital Yantai Shandong 264000 P.R. China 3Department of Pharmacology Jinan Central Hospital Affiliated to Shandong University Jinan Shandong 250013 P.R. China 2014 27 6 2014 9 128 128 1 4 2014 13 6 2014 Copyright © 2014 Liu et al.; licensee BioMed Central Ltd. 2014 Liu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0) which permits " | 1 |
"Background Compared with FISH and qRT-PCR analyses immunohistochemistry (IHC) is the preferred screening test in most pathology practices for ALK-rearrangement detection. With 100% sensitivity and 98% specificity the VENTANA ALK (D5F3) IHC assay has been approved in the EU and some Asian countries for ALK-rearrangement detection. However an automated Ventana IHC platform is not available in most pathology labs. In this study we evaluated the applicability of conventional IHC with D5F3 antibody in routine pathological practice and proposed detection methods and procedures that ensure that patients with ALK+?are not missed. Methods FISH and IHC analyses were performed on 297 lung adenocarcinoma cases. VENTANA IHC and qRT-PCR assay were applied to evaluate ALK-fusion status in the discordant cases of FISH and IHC. The association of ALK+?with clinicopathological characteristics was statistically analyzed. Results IHC had 100% sensitivity and 81.8% specificity for detecting ALK+. Eight ALK-expressed cases were ALK- five of which had ALK fusion detected by qRT-PCR analysis. Three of these five cases showed ALK expression using VENTANA IHC assay. ALK+ was associated with younger age and lymph node metastasis in this Chinese lung adenocarcinoma patient cohort. Conclusions The advantages of low cost and 100% sensitivity allow conventional IHC to serve as a robust diagnostic tool for screening patients with ALK+ especially in pathology labs without a VENTANA IHC platform. For cases in which ALK is weakly expressed qRT-PCR is necessary as a diagnostic test for ALK-fusion detection. Virtual slides The virtual slide(s) for this can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2269448351088278. Immunohistochemistry Fluorescence in situ hybridization qRT-PCR ALK rearrangement D5F3 antibody Lung adenocarcinoma Introduction Lung cancer is the most common cause of cancer death worldwide estimated to be responsible for nearly 1.38 million cancer deaths per year [1]. Despite improvements in the prevention and treatment of lung cancer the overall 5-year survival rate remains at 15% [2]. Efforts have been made to develop new treatment strategies. In recent years rearrangements of the anaplastic large cell kinase (ALK) gene have been discovered in approximately 5% of lung adenocarcinomas resulting in the constitutive expression of a fusion protein - most commonly EML4-ALK - with oncogenic activity [3-7]. Crizotinib a potent and specific small molecule inhibitor of both ALK and c-MET tyrosine kinases [8-10] was approved by the Food and Drug Administration (FDA) for the treatment of non-small-cell lung cancer (NSCLC) patients with ALK gene rearrangement (ALK+). The FDA-approved Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular) was mandated for ALK+?testing in crizotinib trials which in a sense indicates that FISH analysis has been clinically validated. However the FISH detection of ALK gene rearrangement in routine surgical pathology practice remains impractical due to financial and technical problems. Theoretically reverse transcriptase-polymerase chain reaction (RT-PCR) is a standard method for determining the fusion genes but the requirement of fresh frozen tissue samples for extracting RNA has limited its application in clinical practice. IHC is relatively inexpensive and faster and is performed routinely in most surgical pathology practices. Mutation-specific IHC has been demonstrated as a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma [11]. Recently a fully automated VENTANA ALK (D5F3) assay was developed using D5F3 primary antibody (commercialized by Cell Signaling Technology or CST) and VENTANA OptiView DAB detection for use with VENTANA automated platforms. Our group demonstrated that the sensitivity and specificity of the VENTANA ALK assay were 100% and 98% respectively [12]. The VENTANA ALK (D5F3) IHC assay was approved to detect ALK rearrangement in pathology practice in the EU and some Asian countries including China and Japan. However the application of the VENTAMA ALK IHC assay requires a VENTANA automated platform which is not available in most pathology labs. In this study we applied IHC analysis using CSTs D5F3 antibody to detect ALK rearrangement in a Chinese lung adenocarcinoma patient cohort to assess the sensitivity and specificity of IHC analysis. In the third detection method a qRT-PCR assay (Amoy Diagnostics Xiamen China) approved by European Conformity (CE marking) and the China Food and Drug Administration (CFDA) was applied on formalin-fixed paraffin embedded (FFPE) samples to analyze the discordant cases of IHC and FISH. Materials and method Clinical materials and tissue microarray (TMA) construction This study included 297 FFPE samples with lung adenocarcinoma diagnosed at the Cancer Institute and Hospital Chinese Academy of Medical Sciences (CICAMS) in Beijing between January 2009 and March 2012. Among the 297 cases 218 were unselected and 79 cases were not effectively treated using conventional treatment. Among the 218 unselected cases 178 (with enough tissue) were constructed onto seven TMAs to represent biopsies. A 1.5 mm diameter core was taken from the cancer area based on hematoxylin and eosin (H&E)-stained sections of each sample. The remaining 39 unselected cases (without enough tissue) and 79 selected cases were cut into tissue sections. In the cases where tissue sections/cores fell off the slides during FISH or IHC analysis tissue sections were re-cut. The collection of these specimens was approved by the National Cancer Center Ethics Committee. The patients medical records were reviewed to obtain their clinicopathological parameters including age at diagnosis sex smoking history tumor size histological classification and pathological TNM stage. IHC Immunohistochemical staining was performed on 4 ?m-thick FFPE tissue sections or TMAs. Briefly the slides were deparaffinized and antigen retrieval was then performed in a steam cooker for 1.5 minutes in 1 mM EDTA pH 9.0 (Maixin Biological Techology Co. Ltd. Fuzhou China). ALK (D5F3) rabbit monoclonal (Cell Signaling Technology Danvers MA USA) was applied at 1:150 in SigalStain antibody diluent (Cell Signaling Technology Danvers MA USA) for 1 h. Universal secondary antibody (DAKO) was applied for 15 min. Diaminobenzidine or 3-amino-9-ethylcarbazole was used as chromogens and slides were counterstained with haematoxylin before mounting. The criteria for scoring ALK were as follows. First the intensity was graded as 0 negative; 1 weak (light brown); 2 moderate (brown); and 3 strong (dark brown). Second the proportion of positive tumor cells was graded: 0 no positive cells; 1 <10%; 2 11%-30%; 3 31%-50%; 4 51-70%; and 5 >70%. A final score was derived by adding the two primary scores. Final scores of 0 were defined as negative expression (?); scores of 25 as weakly positive expression (+); and scores of 68 as strongly positive expression (++). Fully automated VENTANA ALK (D5F3) IHC analysis was performed as previously described [12]. According to the manufactures scoring algorithm a binary scoring system (positive or negative for ALK status) was adopted to evaluate the staining results. The presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) was considered to be ALK positive while the absence of strong granular cytoplasmic staining in tumor cells was deemed to be ALK negative. FISH FISH was performed on 3 ?m-thick FFPE tumor tissues using a break-apart probe specific to the ALK locus (Vysis LSI ALK Dual Color Break Apart Rearrangement Probe; Abbott Molecular Abbott Park Illinois USA) according to the manufacturers instructions. Tumor cells the nuclei of which had one or more FISH signals of each color were enumerated. A positive cell was defined as one in which the nucleus had split signals (two or more signal diameters apart) or a single orange signal (deleted green signal) in addition to fused and/or split signals. A sample was considered positive if >25 cells out of 50 were positive. If a sample had 5 to 25 positive cells (10 to 50%) another 50 tumor cells were counted. If the average percentage of positive cells in 100 tumor cells was <15% (<15/100) the sample was considered negative. If the average percentage of positive cells was ?15% (?15/100) the sample was considered positive. TMA cores with high backgrounds or very weak signals that affected the signal assessment were excluded from the analysis. Real-time quantitative reverse transcription PCR (qRT-PCR) The EML4-ALK fusion mRNA was detected by qRT-PCR using an AmoyDx EML4-ALK Fusion Gene Detection Kit (Amoy Diagnostics Xiamen China). Briefly total RNA was extracted with an AmoyDx FFPE RNA Kit (Spin Column) from 510 ?m-thick FFPE sections with over 70% tumor cells. For each sample 100500 ng of extracted RNA was used for reverse transcription into cDNA at 42°C for 1 h. Real-time PCR was then carried out in each of the four reactions of the EML4-ALK Fusion Gene Detection Kit according to the manufacturers protocol. Reaction 1 amplifies EML4-ALK variants 12 3a and 3b (variants 1/2/3a/3b); reaction 2 amplifies EML4-ALK variants 4 and 4?; reaction 3 amplifies EML4-ALK variants 5a 5b 5? and 8 (variants 5a/5b/5?/8); and reaction 4 amplifies the reference gene beta-actin. All of the assays were performed on an Agilent Mx3000P QPCR instrument (Agilent Technologies Santa Clara CA). The following PCR procedure was used: an initial denaturation at 95°C for 5 min followed by 95°C for 25 s 64°C for 20 s and 72°C for 20 s to ensure the specificity and 31 cycles of 93°C for 25 s 60°C for 35 s and 72°C for 20 s to perform the data collection. The quantitative judgment was according to the fusion fluorescence signal. Assay reactions achieving Ct values of ?30 cycles were considered positive for one of the variants detected by that reaction mixture. A housekeeping gene (beta-actin) was used to control the integrity of the RNA. Statistical analysis The statistical analysis of the tumors size and age was carried out using Students t tests. The values are shown as mean?±?SD. The relationship between ALK+?and clinicopathological variables was analyzed with the chi-square test. Statistical significance was defined as p?<?0.05. Results Concordance of ALK IHC and FISH Using the newly developed antibody ALK (D5F3) we analyzed ALK expression in 297 lung adenocarcinoma cases. The cases with strongly or weakly positive ALK expression showed readily appreciable cytoplasmic staining (Figures 1A and 1B). In contrast the cases with negative expression did not show any discernable staining (Figure 1C). Strong ALK expression was identified in 32 cases weak expression in 12 cases and no expression in 253 cases (Table 1). Figure 1 Representative cases of IHC staining FISH and qRT-PCR analysis in lung adenocarcinoma. (A-C) ALK IHC staining using CSTs D5F3 antibody. (A) Cytoplasmic reactivity of strong intensity in tumor cells (original magnification x40). (B) Weak to moderate cytoplasmic reactivity in tumor cells (original magnification x100). (C) No staining in tumor cells (original magnification x200). (D-F) FISH analysis using Vysis ALK Break-Apart probes. (D) The ALK+ case in which the majority of cells contained more than one copy of a single green signal without a corresponding orange signal in addition to fused signals using FISH analysis. Green arrow represents more than one copy of a single green signal red arrow represents single red or split red-green signals indicative of ALK-rearrangement and yellow arrow represents touching red-green signals not indicative of ALK-rearrangement. (E)ALK+ case with split red-green signals. (F) NSCLC case without ALK rearrangement. (G-I) VENTANA ALK (D5F3) IHC assay revealed no expression in ALK- patients and strong expression in ALK+ patients. (G) Strong ALK expression (original magnification x20). (H) Unspecific staining (original magnification x40). (I) No ALK expression (original magnification x20). (G-L) Graphs from qRTPCR showing change in the normalized reporter signal (delta Rn) against PCR cycle number. (J)ALK fusion was detected at around 14 cycles of qRT-PCR analysis in a case with strong ALK expression. (K)ALK fusion was detected at around 28 cycles in a case with weak ALK expression. (L) No ALK fusion was detected with endogenous control gene beta-actin expressed normally. Table 1 Correlation of IHC and FISH IHC Total ++ a + b - c FISH+ 31(96.9%) 5(41.7%) 0(0%) 36 FISH- 1(3.1%) 7(58.3%) 242(100%) 250 Total 32 12 242 286 astrongly positive ALK expression. b weakly positive ALK expression. cnegative ALK expression. " | 1 |
"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adultspopulation of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients intakeUsual Macronutrients intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrientsintake and physical activity between different FTO genotypes The post hoc Tukeys test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrientsprotein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableCalorieATP valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele à kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrientsintakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3 and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States JAMA Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab Fredriksson R Hagglund M Olszewski PK 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YF Xu TY Xu XW Su DF Miao CYInvolvement of leptin receptor long isoform LepRbSTAT3 signalingpathway in brain fat massand obesityassociated FTO downregulationduring energy restriction Mol Med May Batterham RL Cohen MA Ellis SM Le Roux CW Withers DJ Frost GS GhateiMA Bloom SR Inhibition of food intake in obese subjects by peptide YY3 N Engl J Med Sep Wardle J Carnell S Haworth CM Farooqi IS ORahilly S Plomin R Obesityassociated genetic variation in FTO is associated with diminished satiety JClin Endocrinol Metab httpsdoi101210jc2008 Velders FP De Wit JE Jansen PW Jaddoe VW Hofman A Verhulst FCTiemeier H FTO at rs9939609 food responsiveness emotional control andsymptoms of ADHD in preschool children PLoS One Nov e49131Karra E ODaly OG Choudhury AI Yousseif A Millership S Neary MT ScottWR Chandarana K Manning S Hess ME Iwakura H A link between FTOghrelin and impaired brain foodcue responsivity J Clin Invest Aug Publishers NoteSpringer Nature remains neutral with regard to jurisdictional 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Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high dietary fat intake are determined byFTO rs9939609 gene polymorphism in selected Indonesian adults Asia PacJ Clin Nutr Mar281183Livingstone MB Robson PJ Black AE Coward WA Wallace JM McKinley MCStrain JJ McKenna PG An evaluation of the sensitivity and specificity ofenergy expenditure measured by heart rate and the Goldberg cutoff forenergy intake basal metabolic rate for identifying misreporting of energyintake by adults and children a retrospective analysis Eur J Clin Nutr Mar573455 Zheng Y Huang T Zhang X Rood J Bray GA Sacks FM Qi L Dietary fatmodifies the effects of FTO genotype on changes in insulin sensitivity JNutr May Hardy DS Racette SB Hoelscher DM Macronutrient intake as a mediatorwith FTO to increase body mass index J Am Coll Nutr 201433256e66 Qi L Kraft P Hunter DJ Hu FB The common obesity variant near MC4Rgene is associated with higher intakes of total energy and dietary fatweight change and diabetes risk in women Hum Mol Genet Nov Zhong T Duan XY Zhang H Li L Zhang HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript levels but does not altertheir DNA methylation profiles in rat white adipose tissues Gene 0c" | 2 |
" oral administration is the most common way to deliver drugs to the systemic circulation or targetans orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver in theearly stages of drug development it is important to predict firstpass metabolism accurately to select candidatedrugs with high bioavailability the caco2 cell line derived from colorectal cancer is widely used as an intestinalmodel to assess drug membrane permeability however because the expression of major drugmetabolizingenzymes such as cytochrome p450 cyp is extremely low in caco2 cells it is difficult to predict intestinalmetabolism which is a significant factor in predicting oral drug bioavailability previously we constructed a mouseartificial chromosome vector carrying the cyp cyp2c9 cyp2c19 cyp2d6 and cyp3a4 and p450 oxidoreductasepor 4cypsmac genes and increased cyp expression and metabolic activity in hepg2 cells via transfer of thisvectorresults in the current study to improve the caco2 cell assay model by taking metabolism into account weattempted to increase cyp expression by transferring the 4cypsmac into caco2 cells the caco2 cells carryingthe 4cypsmac showed higher cyp mrna expression and activity in addition high metabolic activity availabilityfor permeation test and the potential to assess drugdrug interactions were confirmeds the established caco2 cells with the 4cypsmac are expected to enable more accurate prediction ofthe absorption and metabolism in the human intestine than parental caco2 cells the mammalian artificialchromosome vector system would provide useful models for drug developmentkeywords mammalian artificial chromosome chromosome transfer cytochrome p450 intestinal metabolismcaco2 cell correspondence kazukitottoriuacjp1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan2chromosome engineering research center cerc tottori university nishicho yonago tottori japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc biotechnology page of bioavailability is an important area of concern in drugdevelopment poor oral bioavailability has led to drugwithdrawal oral drug bioavailability is often limited bymetabolizing enzymes and efflux transporters in the gut the caco2 cell line derived from human colon carcinoma is a commonly used model for estimating the intestinal absorption of new drug candidates althoughcaco2 cells express a variety of efflux and uptake transporters they have an absence or low levels of cytochrome p450 cyp isoforms such as cyp3a4 andcyp2c that are typically expressed in the human intestinal epithelium therefore caco2 cells are of limited use in evaluating the role of metabolism inintestinal absorption after oral administration to predict the intestinal absorption of drugs more accurately itis necessary to modify caco2 cells to increase their expression of cyp isoformssome studies reported that cyp3amediated metabolism in caco2 cells was enhanced by transfection withboth cyp3a4 and cyp oxidoreductase por [ ] treatment with 1α25dihydroxyvitamin d3 [ ] or the combination of transfection with cyp3a4 and treatment withboth sodium butyrate and 12otetradecanoylphorbol13acetate in contrast few studies aimed at enhancingmultiple cyp isoforms in caco2 cells have been performed honkakoski and his collaborators created caco2cell lines expressing nuclear receptors pregnane x receptor and constitutive androstane receptor [] thesenuclear receptors upregulated the expression of somecyp isoforms in caco2 cells but cyp activities remainedvery low in the absence of 1α25dihydroxyvitamin d3therefore a new approach is needed to introduce multiple cyp isoforms in caco2 cellsmammalian artificial chromosome ac vectors derived from native chromosomes have several advantagesover conventional vectors acs segregate freelyfrom host chromosomes through a set of cell divisionsand are adapted to carry multiple target genes with a desired copy number and mbsized genomic regions withendogenous regulatory elements furthermore acs carrying genes of interest can be transferred into varioustarget celllines via microcellmediated chromosometransfer mmct considering these advantages acshave been used to generate several model cells for pharmacokinetic and toxicokinetic studies previously a lack of cyp3a4 expression in the caco2cell line was addressed through the introduction of exogenous cyp3a4 and por which is a coenzyme ofcyps via a human artificial chromosome hac vectorderived from human chromosome [ ] the haccarrying cyp3a4 and por genes conferred sufficientcyp3a activity to parental caco2 cells to be useful forpredicting the intestinal extraction ratio in humansrecently a mouse artificial chromosome mac vectorconstructed from native mouse chromosome wasused to increase the activity of multiple cyps in hepg2cells which are a liver cancer cell line typically exhibiting low cyp activity in this study four cyp genescyp3a4 cyp2c9 cyp2c19 cyp2d6 and a pene were loaded on the mac 4cypsmac and transferred to hepg2 cells tchepg2 to make the cellsmore suitable as a model to evaluate drugdrug interactions ddis and hepatotoxicity in the initial screeningof candidate drugs the expression and activity ofcyps in tchepg2 were comparable to those in humanhepatocytes and this expression was sustained after along culture period because of the stability of the macin human cells regarding the assessment of ddisthe activity of cyps in tchepg2 was reduced in a concentration and timedependent manner by specific inhibitors which reflects the conditions in primary humanhepatocytes furthermore metabolic toxicity of aflatoxinb1 which is converted to its active metabolite viacyp3a4 and exerts hepatotoxicity through dna damage was clearly recapitulated in tchepg2 cells rather than parental hepg2 cells this study suggestedthat tchepg2 can provide a useful model to assess notonly hepatic metabolism but also cypmediated hepatotoxicity during the early stages of drug development andthe system using the mac can improve the existingcellbased modelin the current study we aimed to utilize previouslyconstructed 4cypsmac to generate a novel caco2 cellline with increased activity of multiple major cyps the4cypsmac was transferred to caco2 cells via mmctto establish caco2 cells carrying the 4cypsmac andthe caco2 4cypsmac cells were examined to determine whether they exhibited sufficient cyp activity foruse in initial drug screeningresultsmmct and analyses of acquired clonescho cells carrying a mac vector with cyp2c9cyp2c19 cyp2d6 cyp3a4 por and gfp genes wereprepared 4cypsmac fig 1a using cho cells asdonor cells and caco2 cells as recipient cells weattempted to generate caco2 cells carrying the 4cypsmac via mmct fig 1a after selection four drugresistant gfppositive clones were obtained caco24cypsmac fig 1b to examine whether the cypand por genes were introduced into the obtainedclones genomic pcr analyses were performed chocells with the 4cypsmac and caco2 cells were usedas positive and negative controls respectively consequently a band of the desired size was observed for eachprimer set in the candidate clones fig 1c next achromosome specimen was prepared from the acquired 0cohta bmc biotechnology page of fig introduction of the 4cypsmac into caco2 cells a transfer of the 4cypsmac into caco2 cells the structure of the mac carrying fourcyps and por is shown at the top a cag promoter was placed upstream of each gene a schematic view of the transfer of the 4cypsmac tocaco2 cells is shown at the bottom the 4cypsmac was transferred from cho cells to caco2 cells through the mmct method b an image ofgfp fluorescence in parental caco2 cells and caco2 4cypsmac cells the gfp fluorescence indicates the presence of the 4cypsmac in thecaco2 cells the white bars indicate a distance of μm c results of genomic pcr analyses to detect four cyp and por transgenes on the macin caco2 cells donor cho cells and caco2 cells are positive and negative controls respectively d images of fish analyses of caco2 cellscarrying the 4cypsmac red and green signals indicate the mac and transgenes respectively the arrow shows the 4cypsmac and the insetshows an enlarged image of the 4cypsmacclones and fish analysis was performed to check thekaryotype fish analysis revealed that a single copy of the4cypsmac existed in the candidate clones fig 1drtqpcr analysis was performed to examine themrna expression level of the introduced cyps and porin the obtained clones compared with that in parentalcaco2 cells the gene expression level was particularlyhigh in the caco2 4cypsmac and caco2 4cypsmac clones fig among the introduced genespor expression was only slightly enhanced in theseclones basal expression of por in parental caco2 cells ishigh as observed in a previous study caco2 4cypsmac showed high expression of the majority of genescompared with caco2 4cypsmac the caco2 cellline consists of a heterogeneous population of cells therefore difference of the gene expression levels between obtained clones may partly depend on the population into which the 4cypsmac has been introducedalthough the other two clones obtained by mmct stillshowed higher expression levels than the parental caco2cells the level of increase was moderate therefore we selected caco2 4cypsmac and caco2 4cypsmac clones for further analyses to evaluate the availability asan improved model system these results suggest that wesuccessfully transferred the 4cypsmac to caco2 cellsand the genes on the mac were highly expressedmonolayer formation of caco2 4cypsmac cellswe seeded caco2 4cypsmac and caco2 4cypsmac cells at a concentration of à cellswell 0cohta bmc biotechnology page of fig gene expression analyses of caco2 cells with the 4cypsmac the expression levels of the four cyps and por in caco2 cells with the4cypsmac the relative expression levels of the four cyps and por genes of the parental caco2 cells and acquired clones were analyzedthrough rtqpcr gapdh was used for normalization mean ± se n in a millicell 24well cell culture insert plate the caco 4cypsmac cells spread across the entire membrane and formed a cell layer while the caco2 4cypsmac cells did not spread and there were gaps in thecell layer fig 3a caco2 4cypsmac appeared toaggregate and form multiple layers rather than spreadand form a single layer it was reported that multilayeredareas appeared in the cell population for late passagecells the teer value was measured using amillicellers fig 3b the teer value is an index oftight junction formation and when the value is almostconstant it is considered that a cell layer has formedwith the exception of the caco2 4cypsmac clonethe caco2 cells and caco2 4cypsmac cellsshowed an increase in teer value untilit plateauedafter d the caco2 cells and caco2 4cypsmac showed almost equivalent teer values because monolayer formation is essential for the permeation test thesubsequenttests were performed using the caco24cypmac cellsculture timedependent change in gene expressiontotal rna was extracted from caco2 cells andcaco2 4cypsmac cells on the 4th 11th and22nd days after seeding we compared the expressionlevel of each gene on each day and confirmed thatthe expression levels of the four cyps and por increased in both the caco2 cells and caco2 4cypsmac cells fig 3c the expression levels of allgenes analyzed were significantly higher in the caco24cypsmac cells on the 22nd day than those inparental caco2 cells the gene expression levelincreased depending on the culture time and the geneexpression levels of the caco2 4cypsmac cellsestablished in the current study were higher thanthose of parental caco2 cells with the exceptions ofcyp3a4 and cyp2d6 the expression levels in parental caco2 cells were higher in all cases until day the parental caco2 cell line appears to have higherpotential to enhance the expression of cyp2c9 andcyp2c19 during differentiation 0cohta bmc biotechnology page of fig monolayer formation assay a images of bright and gfp fluorescence from cells seeded on the membrane of a millicell24 plate in caco24cypsmac cells did not spread throughout the membrane and did not form a cell layer but in caco2 4cypsmac there were no gapsbetween cells and they formed a cell layer the white bars indicate a distance of μm b transepithelial electrical resistance teer values ofcaco2 cells caco2 4cypsmac and caco2 4cypsmac cells c culture timedependent change in gene expression the relative expressionlevel was evaluated in caco2 and caco2 4cypsmac mean ± se n the expression levels in caco2 and caco2 4cypsmac at day were compared with those in humanadult intestine additional file figure s1 in caco24cypsmac the expression levels of cyp2c9 andcyp2c19 were comparable and that of cyp2d6 washigher than in human adult intestine although cyp3a4expression was significantly enhanced in caco2 4cypsmac compared with that in parental caco2 cellsthe expression was stilllower than in human adultintestinecyp metabolic activity measurementa p450glo assay with each specific substrate wasemployed to measure the metabolic activity of cyps inthe caco2 4cypsmac cells which had high geneexpression levels as confirmed through rtqpcr analysis the activities of all four cyps were higher in thecaco2 4cypsmac clone than in caco2 cellsfig 4a the resultsthe introducedindicate that4cypsmac expressed functional cyps and increasedthe total activity of each cyp in the caco2 cells theenhancements in the rates of metabolic activity ofcyp2c9 cyp2c19 and cyp2d6 were generally correlated with those of mrna expression however therewas a gap between the enhancement of the rate ofcyp3a4 mrna expression and that of metabolic activity this may have been because the parental caco2 originally had extremely low expression of cyp3a4mdz permeability testa permeation test was conducted using midazolammdz a cyp3a substrate to examine whether the cellsreflected the behavior of small intestinal epithelial cellsin terms of mdz permeation the penetration test wasperformed on day after cell seeding when the teervalue plateaued we measured the amount of ²ohmdz in each of the donor side apical recipient sidebasal and intracellularly the amounts of ²oh mdz 0cohta bmc biotechnology page of fig activity of each cyp in the caco2 4cypsmac cells a the metabolic activity of each cyp in caco2 4cypsmac cells the relative activityfor each cyp was measured by comparing the parental caco2 cells and the caco2 4cypsmac mean ± se n b permeability test usingmdz the permeability test was performed d after seeding caco2 cells and caco2 4cypsmac whereby μm mdz was added to theapical side and after min the apical intracellular and basal supernatants were collected the ²oh mdz in the supernatant was measuredthrough lcmsms c cyp3a4 inhibition test ketoconazole an inhibitor of cyp3a4 was added to the caco2 4cypsmac and incubated for h a luminescent substrate was measured to detect cyp3a4 activity with different concentrations of ketoconazolein alllayers of the caco2 4cypsmac cells werehigher than in those of caco2 cells fig 4b moreoverer was calculated using eq and the results were and for caco2 and caco2 4cypsmac respectively er indicates the rate of metabolism during cellpermeation cyp3a4 was scarcely expressed in parentalcaco2 cells so the er value was extremely low howevercaco2 4cypsmac cells showed an er of which was higher than in the caco2 cells and mdz wasmetabolized by cyp3a4 when passing through the cellsinhibition testto determine the availability of the established clone forthe assessment of ddis we added ketoconazole an inhibitor of cyp3a4 to the caco2 4cypsmac cells andexamined whether the metabolic activity was reduced ketoconazole at and μm was addedand cells were incubated at °c for h followed by themeasurement of metabolic activity the metabolic activityof cyp3a4 decreased as the inhibitor concentration increased fig 4c the activity of cyp3a4 in caco2 4cypsmac appeared to be sufficient for the inhibition test compared with that in parental caco2 cells in addition to thepermeation test for cyp3a4 inhibition of cyp3a4s function by ketoconazole in caco2 4cypsmac cells was alsoconfirmed this suggests that the established cells could beused for ddi testing of drugs that are substrates ofcyp3a4 therefore the caco2 4cypsmac cells moreaccurately reflect the behavior of cyp3a4 substrates in human epithelial cells than parental caco2 cellsdiscussionin the current study we introduced four cyps and porinto caco2 cells to increase their drug metabolic 0cohta bmc biotechnology page of abilities which are typically low this study was intendedto establish a better human cell model to more preciselyevaluate the behavior of drugs in the small intestine the4cypsmac was successfully introduced into the caco2cells and successfully increased cyp activityin contrastin this studythe gene expression and activity of cyps in tchepg2 carrying the 4cypsmac are either comparableto or higher than those in primary human hepatocytes to the other cypscyp3a4 mrna expression was still low in caco2 carrying the 4cypsmac compared with the level in human adult intestine despite the significant enhancementof mrna expression regarding the genes on the4cypsmac each is present as a single copy becausethe nature of gene regulation is supposed to differ between hepg2 and caco2 changing copy number of thecyp3a4 gene may further optimize the expression profile of caco2 cells to that of human intestinethe established caco2 4cypsmac cells with particularly high gene expression showed high activity in all cypsin the future we will conduct metabolic tests inhibitiontests and permeation tests using drugs that are substratesfor other cyps and investigate whether the caco2 4cypsmac cells reflect the behavior of drugs in small intestinal epithelial cells the cyp expression level in the humansmall intestine is reported to be approximately forcyp3a4 approximately for cyp2c9 approximately for cyp2c19 and approximately for cyp2d6 it will be necessary to evaluate whether the proportion ofcyp expression in the caco2 4cypsmac is close tothat of the human small intestineif cyp metabolic capacity is guaranteed in the established clones the established cell line can be used asnew human small intestine model cells in recent yearssmall intestine model cells prepared from induced pluripotent stem cells have been reported but such cells areconsidered difficult to use for screening large quantitiesof drug candidate compounds however caco2cells are easy to handle therefore it is possible to usecypmodified caco2 cells to test large numbers of candidate compounds as a first screeningwako osaka japan supplemented with fetal bovine serum fbs and μgml g418 parental caco2cells atcc® htb37¢ atcc manassas va usawere maintained in dulbeccos modified eagles mediumdmem wako supplemented with fbs memnonessential amino acids gibco thermo fisher scientific waltham ma usa m hepes gibco mmsodium pyruvate gibco mm glutamax gibcoand penicillinstreptomycin wako caco2 cells withthe 4cypsmac were maintained in the above mediumsupplemented with μgml g418 these cells werecultured at °c in co2microcellmediated chromosome transfertransfer of 4cypsmac from cho cells to caco2 cellswas performed using a standard procedure brieflydonor cho cells were cultured in f12 medium supplemented with fbs and μgml colcemid after h microcells were isolated through centrifugation withdmem containing cytochalasin b and filtration thenmicrocells suspended in phytohemagglutinin p phapdmem were poured onto caco2 cells in a 6cm dishand incubated for min the cells were treated withpolyethylene glycol peg solution g of peg1000 ml of dmem ml of dimethyl sulfoxide for minfollowed by washing with dmem after h of recoveryculture cells were seeded in a 24well collagencoatedplate corning ny usa and maintained with selectionmedium h after seeding thereafter the medium waschanged twice a week to obtain drugresistant clonesbecause the mac carries a gfp gene gfppositiveclones were selected from the drugresistant clonesgenomic pcr analyseswe extracted genomic dna from cell lines using a genomic dna extraction kit with dnasefree rnase gentra systems minneapolis mn usa the primers forthe genomic pcr are listed in additional file tables1 they amplified each gene region on the 4cypsmac we used kod fx takara otsu japan in accordance with the manufacturers instructionsthe mammalian artificial chromosome vector systemwould provide useful models for drug development theestablished caco2 cells with the 4cypsmac are expected to more accurately predict absorption and metabolism in the human intestine than parental caco2 cellsmethodscell culturechinese hamster ovary cho cells jcrb0218 jcrbcell bank nibiohn osaka japan carrying the 4cypsmac were maintained in hams f12 nutrient mixturefish analysestrypsinized cells were incubated for min in m kcland fixed with methanol and acetic acid and thenslides were prepared using standard methods fish analyses were performed using the fixed metaphase of each cellhybrid using digoxigeninlabeled roche germany dna[mouse cot1 dna invitrogen carlsbad ca usa] andbiotinlabeled dna [pac 4cypspor] essentially as described previously chromosomal dna was counterstained using dapi sigmaaldrich st louis mo usaimages were captured using an axioimagerz2 fluorescencemicroscope carl zeiss germany 0cohta bmc biotechnology page of rtqpcrwe extracted mrna using the rneasy mini kit qiagen germany and synthesized firststrand cdna usingthe high capacity cdna reverse transcription kit applied biosystems foster city ca usa the primersare listed in additional file table s1 for rtqpcranalysis tb green premix ex taq takara was usedand relative mrna expression was evaluated throughthe δδct method gapdh was used for normalizationculture timedependent expression level change of fourcypscaco2 cells and caco2 4cypsmac were seededin a 6cm dish at a concentration of à cellswell cells were lysed using trizol invitrogen causa on days and after seeding and rnawas extracted and purified using an rneasy mini kitqiagen thereafter cdna synthesis was performedusing the highcapacity cdna reverse transcriptionkit applied biosystemsactivity test of the four cypswe tested the metabolic activity ofthe four cypsusing the p450glo¢ assay promega madison wiusa the luminogenic substrates used for the testwere luciferinipa cyp3a4 luciferinme egecyp2d6 luciferinh cyp2c9 and luciferinhege cyp2c19 cells wereseeded in 48wellcollagencoated plates corning at a density of à cellswell after h the medium was changedand h later we added transport medium tm containing substrate tm was prepared using hanks balanced salt solution hbss with mm nahco3 mm glucose and mm hepes which was adjusted to ph after incubation we added detectionreagent and measured the luminescence using an infiniteandcyp2c19 required h of incubation while cyp2d6and cyp3a4 required h after the measurementthe cells were washed with pbs dissolved in lysisbuffer and diluted fivefold the same amount of celltiter glo promega was added to μl of the lysateand luminescence measurement was performed tonormalize data to the number of viable cellswako cyp2c9f500 platereadermidazolam mdz permeability testthe obtained clones were assessed in an mdz permeation test each cell was seeded on a 24well cell cultureinsert plate millipore billerica ma usa at a concentration of à cellswell the medium was changedonce a week after seeding and every d after the secondweek transepithelial electrical resistance teer wasmeasured using millicellers millipore before mediumexchange the test was performed d after seedingfor the test tm ph prepared by adding mmnahco3 mm glucose and mm hepes tohbss at ph was used the donor side solutionwas prepared by dissolving μm mdz in tm ph the acceptor side solution was prepared by adding fbs to tm ph on the test day themedium was removed from the culture insert seededwith the cells and the cells were rinsed twice withtm ph tm ph and tm ph wereadded to the apical and basal chambers respectivelyand cells were incubated at °c for min the testwas started by adding the donor side solution to thedonor side chamber and the acceptor side solution tothe acceptor side chamber thirty minutes after thestart of the test the solution in the donor side andacceptor side chambers was collected moreover tomeasure the amount of mdz and ²hydroxy mdz²oh mdz in the cells after the test the cultureinsert was quickly rinsed three times with icecoldtm ph the membrane was cut using a cutterand μl of icecold tm ph was added cellswere detached from the membrane through sonicationand a cell suspension was used as a sample thesesamples were deproteinized and stored at °cuntil measurementlcmsms was used for the measurement of mdzand ²oh mdz in the samples qtrap5500 sciexframingham ma usa and a prominence uflc system shimadzu kyoto japan were combined for measurement the hplc conditions and msms conditionsare shown in additional file table s2 quantitativeanalysis was performed in multiple reaction monitoringmode mass transitions mz were formdz for ²oh mdz and for ²oh mdz d4 data were analyzed usinganalyst software sciexequation formula to calculate extraction ratio erer ¼metabolite donorþreceiverþintracellularpþparent receiverþintracellularððþ þ pmetabolite donorþreceiverþintracellularðþtokyo chemicalinhibition testketoconazoleindustry tokyojapan was used as an inhibitor against cyp3a4 andchanges in metabolic activity were measured using ap450glo assay with luciferinipa cells were seededin a 48well collagencoated plate at à cellswell and the medium was changed after d thenext daythe medium was collected cells werewashed twice with pbs and then μl of tm ph containing ketoconazole tokyo chemical industry at and μm was added toeach set of three wells tm was adjusted to ph byadding mm nahco3 mm glucose and mm 0cohta bmc biotechnology page of received april accepted august referencesbenet l wu c hebert m wacher v intestinal drug metabolism andantitransport processes a potential paradigm shift in oral drug delivery jcontrol release xie f ding x zhang qy an update on the role of intestinal cytochromep450 enzymes in drug disposition acta pharm sin b takenaka t kazuki k harada n kuze j chiba m iwao t matsunaga t abes oshimura m kazuki y development of caco2 cells coexpressingcyp3a4 and nadphcytochrome p450 reductase using a human artificialchromosome for the prediction of intestinal extraction ratio of cyp3a4substrates drug metab pharmacokinet hu m li y davitt cm huang sm thummel k penman bw crespi cltransport and metabolic characterization of caco2 cells expressing cyp3a4and cyp3a4 plus oxidoreductase pharm res schmiedlinren p thummel ke fisher jm paine mf lown ks watkins pbexpression of enzymatically active cyp3a4 by caco2 cells grown onextracellular matrixcoated permeable supports in the presence of1alpha25dihydroxyvitamin d3 mol pharmacol fan j liu s du y morrison j shipman r pang ks upregulation oftransporters and enzymes by the vitamin d receptor ligands 1alpha25dihydroxyvitamin d3 and vitamin d analogs in the caco2 cell monolayer jpharmacol exp ther cummins cl mangravite lm benet lz characterizing the expression ofcyp3a4 and efflux transporters pgp mrp1 and mrp2 in cyp3a4transfected caco2 cells after induction with sodium butyrate and thephorbol ester 12otetradecanoylphorbol13acetate pharm res korjamo t honkakoski p toppinen mr niva s reinisalo m palmgren jjmonkkonen j absorption properties and pglycoprotein activity of modifiedcaco2 cell lines eur j pharm sci korjamo t monkkonen j uusitalo j turpeinen m pelkonen o honkakoskip metabolic and efflux properties of caco2 cells stably transfected withnuclear receptors pharm res kublbeck j hakkarainen jj petsalo a vellonen ks tolonen a reponen pforsberg mm honkakoski p genetically modified caco2 cells withimproved cytochrome p450 metabolic capacity j pharm sci mes to hbss the cells were preincubated for h at °c and 1000fold diluted cyp3a4 substrate wasadded one hour later μl of the supernatant wascollected from the well mixed with μl of detectionreagent and the luminescence value was measuredusing an infinite f500 plate reader wakosupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12896020006378additional file figure s1 comparison of gene expression betweenhuman small intestine and day culture of caco2 and caco2 4cypsmac table s1 primer sequences for genomic pcr and rtqpcrtable s2 lcmsms analysis conditions mdz ²oh mdzabbreviationscyp cytochrome p450 ac artificial chromosome mmct microcellmediated chromosome transfer por p450 oxidoreductase hac humanartificial chromosome mac mouse artificial chromosome ddi drugdruginteraction cho chinese hamster ovary mdz midazolamteer transepithelial electrical resistance er extraction ratioacknowledgmentswe thank satoru iwado at tottori university for technical assistance with theexperiments we also thank dr hiroyuki kugoh dr masaharu hiratsuka drhiroyuki satofuka and dr takahito ohira at tottori university for criticaldiscussions this research was partly performed at the tottori bio frontiermanaged by tottori prefecture we thank edanz group wwwedanzeditingcomac for editing a draft of this manuscriptauthors contributionsall authors conceived and designed the experiments yo and kka performedthe experiments yo sa kko and yk wrote the paper mo and yksupervised the study all authors read and approved the final manuscriptfundingthis work was supported in part by the basis for supporting innovative drugdiscovery and life science research binds from the japan agency formedical research and development amed under grant numberjp18am0301009 ykavailability of data and materialsthe data and materials used andor analyzed during the current study areavailable from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsdr mitsuo oshimura is ceo and a shareholder of trans chromosomics incdr satoshi abe is a member of trans chromosomics inc and the otherauthors declare no conflict of interestauthor details1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan 2chromosomeengineering research center cerc tottori university nishicho yonagotottori japan 3trans chromosomics inc nishicho yonagotottori japan 4laboratory of biopharmaceutics meijipharmaceutical university noshio kiyose tokyo japan oshimura m uno n kazuki y katoh m inoue t a pathway fromchromosome transfer to engineering resulting in human and mouseartificial chromosomes for a variety of applications to biomedicalchallenges chromosom res satoh d abe s kobayashi k nakajima y oshimura m kazuki y human andmouse artificial chromosome technologies for studies of pharmacokineticsand toxicokinetics drug metab pharmacokinet kazuki y hoshiya h takiguchi m abe s iida y osaki m katoh m hiratsukam shirayoshi y hiramatsu k ueno e kajitani n yoshino t kazuki k ishiharac takehara s tsuji s ejima f toyoda a saka | 0 |
previous studies have shown a strong coexistence of colorectal neoplasia crn and cardiovascular diseases cvd this study was aimed to summarize the available evidence on association of cvd risk with early crn detection in asymptomatic populations pubmed web of science and embase were systematically searched for eligible studies published until dec studies exploring the associations of recommended cvd risk assessment methods eg risk scores carotid artery plaque and coronary artery calcium score [cacs] with risk of crn were included metaanalyses were conducted to determine the overall association of cvd risk with the crn a total of studies were finally included the association of carotid artery plaque with the risk of colorectal adenoma ad was weakest pooled odds ratio [or] confidence interval [ci ] participants with cacs100 had about 2fold increased risk of ad than those with cacs0 the pooled ors were ci and ci for the risk of advanced colorectal neoplasia an and ad respectively in participants with framingham risk score frs20 when compared to participants at low risk frs10 frs might help identify subgroups at increased risk for an but further studies are needed keywords cardiovascular disease risk assessment colorectal neoplasiaintroductionboth colorectal cancer crc and cardiovascular diseases cvd are the leading causes of mortality and morbidity worldwide12 previous studies have shown a strong coexistence of colorectal neoplasia crn and cvd probably due to the shared risk factors eg smoking obesity and metabolic syndrome and pathophysiological mechanisms eg chronic inflammation and oxidative stress3current guidelines8 recommend assessing the cvd risk in healthy people using risk estimation scores such as framingham risk score frs1112 procam13 and the pooled cohort equation14 which are based on individuals medical history and easily available laboratory data in addition assessment of subclinical atherosclerosis by imaging modalities could be added as risk modifiers to help make clinical decisions for borderline or intermediaterisk adults8 routine use of imaging modalities is not recommended for cvd risk assessment in clinical practice due to the medical costs or invasiveness but incorporation of imaging data such as the anklebrachial index abi coronary artery calcium score cacs and carotid artery plaques cap could improve the prediction of cvd risk15clinical epidemiology chen this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cchen dovepressvarious risk scores have also been developed for predicting advanced colorectal neoplasia an18 although several studies2526 have reported that elevated blood lipids the well documented cvd risk factor and history of cvd were associated with increased risk of crc the majority of risk scores developed for an did not include them into the models27 recent studies have reported the associations between cvd risk assessment and risk of1 crn higher frs estimating the 10year risk of developing coronary heart disease chd1112 was significantly associated with the higher risk of an frs vs frs10 odds ratio [or] confidence interval [ci] abi was associated with 13fold increased risk of an in a recent study29 cap and cacs were also found to be positively related to the increased risk of adenoma ad and an in several studies30given a number of shared risk factors and mechanisms between cvd and crc and the emerging epidemiological evidence of association between cvd risk and crc there is a possibility that cvd risk assessment could help trigger crc screening therefore the aim of this review was to provide an overview of the cvd risk assessment methods and their associations with the risk of crn fully understanding of the current knowledge and existing gap might promote better prevention and treatment for cvd and crc circulating and urinary biomarkers have either no or only limited value when added to cvd risk estimation score systems834 thus only score models and imaging methods recommended as risk modifiers abi cacs and cap in the guidelines8 were included in this reviewmaterials and methodsthis systematic review was conducted following the procedure recommended by the cochrane collaboration35 and was reported according to the preferred reporting items for systematic reviews prisma checklist36 ethical approval and patient informed consent were not necessary since all the data included in the current study were obtained from previously published studiesand metaanalyses remaining publications and reference lists were scrutinized studies that fulfilled the predefined criteria were includedinclusion and exclusion criteriawe required that included studies meet the following criteria published as an original research in a peer reviewed cardiovascular risk has been assessed using either score models or imaging methods recommended as risk modifiers abi cacs and cap in the guidelines3 only included participants who were considered asymptomatic reported the association of cvd risk assessment results with the risk of crn studies were excluded if they were published as conference proceedings dissertations or s only or were not published in english pico eligibility criteria for this review were presented in the supplementary table s1data extractiontwo authors yc and xc independently performed data extraction of all included studies the following information was ed author publication year study period number of participants age number of males outcome ad an and so on data source medical records questionnaires or both cvd risk assessment and association indexdiscriminatory accuracy or hazard ratio [hr] specificity sensitivity or area under the receiver operator characteristic curve values] in case of any disagreement consensus was obtained by discussionquality assessment in eligible studiesrisk of bias and applicability were assessed according to quality assessment of diagnostic accuracy studies2 quadas237 quadas2 evaluates the risk level of bias composed of four basic components patient selection index test reference standard flow and timing clinical applicability is also assessed for the first three components the risk of bias and concerns regarding applicability for each study was then rated as high low or unclearliterature search strategiespubmed embase and web of science were searched up to december to identify the relevant papers the searched items were presented in the appendix which mainly covers expressions for cvd risk score models recommended imaging modalities crn and discriminatory accuracy or strength of association after removal of duplicates titles and s of records were screened according to the inclusion and exclusion criteria full texts of the statistical analysiswe pooled ors for the same cvd risk assessment index using r statistical software version and the r meta package version for frs and cacs ors were pooled separately for different levels of scores using the lowest level as reference two kinds of outcomes ad and an were reported in the studies using frs for cvd risk assessment and thus ors were pooled separately for different outcomes heterogeneity across studies was evaluated submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen using cochranes q statistic with p value and the i2 statistic if significant heterogeneity was observed i2 or pqstatistics a randomeffects model was used to calculate pooled estimates otherwise a fixedeffects model was used35 twosided p values of or lower were considered to be statistically significantresultsliterature search resultsa total of records were obtained in the initial search including citations from pubmed citations from embase and citations from web of science after removal of duplicates n1609 and exclusion due to our predefined criteria n5727 records were qualified for fulltext assessment fortyfour records were excluded due to the inclusion and exclusion criteria finally a total of studies28 including one study which was identified through crossreferences were included the detailed information of the selection process was presented in figure study characteristicstable summarized the basic characteristics of the included studies published between and of the included studies nine were from korea and the other three studies were from japan austria and turkey respectively the study periods stretched from to with sample sizes ranging from to only one was designed as a prospective study41 and the others were crosssectional studies most studies included participants aged older than years and only one study enrolled subjects aged years32 in addition most studies were predominantly in men with proportions of males among participants ranging from to four cvd risk assessment methods abi cap cacs and frs were used in the included studies all studies explored the role of cvd risk assessment method on the detection of ad and some of risk adenoma3032 and an2829384243focused on colorectal high them also figure flowchart of inclusions of studies about relation of cvd risk to crn note adapted from moher d liberati a tetzlaff j preferred reporting items for systematic reviews and metaanalyses the prisma statement plos med creative commons license and disclaimer available from httpcreativecommonslicensesby40legalcode36 abbreviations cvd cardiovascular disease crn colorectal neoplasiaclinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable basic characteristics of included studies about relation of cvd risk to colorectal neoplasiastudycountrystudy periodnumber of participantsyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj 2019a41lee jy niederseer d basyigit s japankoreakoreakoreakoreakoreakoreakoreakoreakoreaaustriaturkeyage years mean±sd ± ± 530b median± ± ± ± 526b± ± ±male n outcomec data sourcececum intubation ratedcvd risk assessment ad anad hraadad anad hraadadadadad anad anad anmrqmrqmrqmrmrqmrqmrmrmrqmrqmrqmrqnrnrnrnr¥nrabicapcapcapcacscacscacscacscacsfrsfrsfrsnotes ait is a retrospective followup study and all the other studies are crosssectional bsd was not reported cdetected by colonoscopies in all included studies d100 cecum intubation rate participants with failure of cecum intubation were excluded nr not reported studies mentioned that colonoscopies were extended to cecum in the methods section but did not reported the success rate of cecum intubation abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score hra high risk adenoma mr medical records nr not reported q questionnaires sd standard deviationquality assessment of studiesthe results for the quality of included studies using the quadas2 tool are presented in table regarding patient selection one study by kim did not provide detailed information about patient selection31 thus the risk of bias and applicability concerns were rated unclear for this domain in this study otherwise no major risk of bias or applicability concerns were identifiedassociation of cvd risk assessed by different methods with crc risktable described the details of the cvd risk assessment methods in the included studies abi was associated with 13fold ci increased risk of an29 three studies reported the weak association between cap and risk of ad303138 one of them also showed an increased risk of an in the participants with cap but the results were not statistically significant or table risk of bias and applicability judgements in quadas2studyrisk of biasapplicability concernstotalpatient selectionindex testreference standardflow and timingpatient selectionindex testreference standardyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj lee jy niederseer d basyigit s totalnotes _ high risk low risk unclear risksubmit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen table details of the cvd risk assessment methods in the included studies about relation of cvd risk to colorectal neoplasiastudycategoriesboutcome or[ ci]yamaji y kim j abnormal abiabnormal abicap yescap yeskim h cap yescha jm yun ke choi sh cap yescap yescacs cacs cacs cacs cacs cacs cacs cacs cacs yang mh201339cacs kim hb cacs cacs cacs ¥lee yj 2019a41cacs lee jy niederseer d basyigit s frs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highadanadhraadadanadadadhrahrahraadadadadadadadadadadananadadananadadanan[ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]hr [ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]notes ain participants without adenoma cacs0 at baseline compared to cacs0 increased the risk of colorectal adenoma at followup colonoscopy hr ci bthe lowest level was defined as reference abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque frs framingham risk score hra high risk adenoma hr hazard ratio or odds ratio ci confidence interval ci in addition the presence of cap was associated with increased risk of colorectal high risk adenoma or ci four studies reported ors for different levels of cacs with cacs0 as reference32333940 highest cacs levels seemed to be associated with the increased risk of ad with or ranging from to the 10year chd risk estimated by frs was categorized as low risk intermediate risk and high risk ¥ participants with high risk of 10year chd had increased risk of either ad or an in the study by basyigit participants at high chd risk had about 4fold or ci increased risk of an28metaanalyses of available ors for different cvd risk assessment methodsmetaanalyses were performed in the studies that provided ors and their cis for the same cvd risk assessment index the association of cap with the risk of ad was weakest the pooled or ci a medium level of cacs cacs was associated with 134fold increased risk of ad when compared to the lowest category of cacs cacs0 participants with cacs100 had an increased risk of ad and the pooled or was ci the pooled ors were ci and ci for the risk of an and ad respectively in participants with high chd risk frs20 when compared to participants at low chd risk frs10 further details were presented in table and in the supplementary figures s1discussionthis systematic review summarized the associations of recommended cvd risk assessment methods with risk of crn in asymptomatic populations a total of studies including four different methods were identified among these methods frs was most strongly associated with risk of both an and ad participants with frs20 have about 34fold and 23fold increased risk of an and ad respectively when compared to participants at low chd risk frs10 only one study29 reported that abnormal abi greatly increased the risk of an thus it was not included in the metaanalysisboth crc and cvd are thought to develop via a process of insulin resistance inflammation and oxidative clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable metaanalysis of odds ratios for different cvd risk assessment toolsstudycvd risk assessmentcategoriesaoutcomeor cicapcacscacscacsfrsfrsfrsfrsyes vs nocacs vs cacs0cacs vs cacs0cacs vs cacs0intermediate vs low riskhigh vs low riskintermediate vs low riskhigh vs low riskadadadadadadanan note athe lowest level was defined as reference abbreviations ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score or odds ratio ci confidence intervalstress74547 which might partially explain why they share a number of risk factors eg alcohol consumption tobacco use physical activity use of antiinflammatory agents obesity and diabetes mellitus4548 in addition several cellular metabolismrelated pathways eg ampk and pparγ signaling pathways eg wnt signaling pathway and genetic pathways eg lrp6 mutation and tcf7l2 polymorphism are not only associated with accelerated atherosclerosis and an increased risk of cvd but also linked to cancer development and progression7 better understanding of these overlaps might promote shared management of prevention and treatment for both disordersrisk of an in this review the strength of associations between identified cvd risk assessment methods and the risk of crn was generally weak except frs which was modestly associated with frs20 vs frs10 frs was calculated based on age total cholesterol highdensity lipoprotein cholesterol smoking status systolic blood pressure and treatment of blood pressure which are typically available in the medical records44 compared to the more sophisticated risk calculators232449 for predicting an which need variables such as physical activity red meat intake and vegetable consumption frs has relatively higher generalizability and lower recall bias a recent study has recommended the combined preventive screening and research efforts in the prevention of both cvd and cancer50 if participants with highrisk of cvd predicted by frs could be recommended to have a screening for crn which will help increase compliance and uptake of crc screening as persons who are aware of their increased risk are more likely recommendations furthermore it also maximizes the medical values of the comply with to expert information participants obtain from a clinical examination or risk assessment and thus reduces the time and costs for health carehowever there are some issues that merit our attention firstly the included studies are all crosssectional which limits the comparisons between frs and the previously developed risk prediction models for crc secondly frs has its own limitations frs only estimates 10year chd risk for all individuals years or older but not the overall cvd risk in addition it is developed based on the american population while most of study participants are asians in the included studies studies have shown that frs overestimated cvd risk in the asian cohorts51 at last the included studies tended to yield results with wide ci probably due to the limited number of participants the wider the ci the less the precision in summary higher cvd risk might trigger concurrent crc screening which should be further validated on largescale studies and future studies could consider about using the overall cvd risk score models developed from data of local cohorts to predict the risk of crcas for imaging data the association of cap or cacs with risk of ad is not strong enough that imaging index alone might not be useful for informing early detection of crn similarly routine screening with imaging modalities to predict future cardiovascular events is generally not recommended in clinical practice but use of these imaging techniques has been shown to improve cvd risk assessment and serve as a guide for initiating preventive therapies8 a high cacs can help modify the predicted risk obtained from frs alone especially among patients in the intermediaterisk category16 up to now only one risk score developed in the multiethnic study of atherosclerosis mesa study used both cacs and submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen traditional risk factors to predict the 10year chd risk55 inclusion of cacs in the mesa risk score offered significant improvements in risk prediction cstatistic vs p factors in the risk models like smoking behaviors and blood lipids are closely related to the incidence and progression of cvd but they are not direct markers of current status of atherosclerosis this might help explain why the performance of risk models is improved by adding markers with anatomical delineation through imaging technology accounting for the higher performance of the combined use of risk scores and imaging tools on cvd risk assessment further studies could consider about exploring the association of combined form of them with the risk of crcwe also observed that less than half of included studies reported the associations of cvd risk with both risk of an and ad2829384243 colonoscopy is considered to as a valid primary screening tool for crc and is able to detect both ad and an the lower prevalence of an and the limited number of participants in several included studies might limit the power to explore the relation of an with cvd risk which could partly explain why most of studies did not include an as outcome therefore the findings should be carefully interpreted and further validated on largescale studiesour study has some strengths comprehensive search strategies along with welldefined eligibility criteria were used to help identify relevant s in addition two reviewers independently extracted data and assessed the risk of bias in the included studies however several limitations should also be addressed firstly the current meta analysis was based on observational studies there were the possibilities of potential effects of unknown or residual confounding factors on our results secondly as we only considered about established cvd risk models and recommended imaging modalities the potential of other cvd risk assessment index on the detection of crn was not summarized and compared in this study however it is also reasonable to just include these methods since their feasibility and performance for cvd risk prediction have been well approved in the clinical practice thirdly cut off values and group comparisons for the same cvd risk assessment method varied in the included studies which limits the synthesis of results for example the cut off values for cacs are the tertiles of cacs in the study by kim 40 however cacs was categorized into three groups with cut off values at and in the other studies3233 therefore less studies were included in the metaanalysis which might influence the accuracy of the pooled results lastly most of studies were conducted in asian populations which is an inherent limitation of the included studies thus our findings might not be applicable to other populations and needs to be externally validated in racially diverse populationsconclusionsto our knowledge this is the first review that applies metaanalyses to determining the overall association of recommended cv risk assessment methods with the risk of crn in the asymptomatic population frs calculated based on shared risk factors of cvd and crc shows potential to help identify subgroups at increased risk for an whether the combination of frs and imaging index is useful for the optimal evaluation of crn risk remains to be solved in the future studies cvd risk might inform crc screening which needs more research in the future to validate its feasibility and effectivenessabbreviationsabi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque chd coronary heart disease ci confidence interval crc colorectal cancer crn colorectal neoplasia cvd cardiovascular disease frs framingham risk score hr hazard ratio hra high risk adenoma mr medical records nr not reported or odds ratio prisma preferred reporting items for systematic reviews and metaanalyses quadas2 quality assessment of diagnostic accuracy studies2 q questionnaires sd standard deviationfundingthis research was funded by national natural science foundation of china grant number disclosurethe authors report no conflicts of interest in this workreferences bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doidoi103322caac21492 joseph p leong d mckee m et al reducing the global burden of cardiovascular disease part the epidemiology and risk factors circ res doidoi101161circresaha117308903 chan aoo man hj kwok fl et al prevalence of colorectal neoplasm among patients with newly diagnosed coronary artery disease j am med assoc doidoi101001jama29812 clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepress chan aoo lam kf tong t coexistence between colorectal canceradenoma and coronary artery disease results from patients aliment pharmacol ther doi doi101111j13652036200602958x wang sc schulmanmarcus j fantauzzi j et al colon cancer laterality is associated with 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despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has conï¬rmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modiï¬cations nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these ï¬ndingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and eï¬cient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our ï¬ndings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratiï¬ed according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as proï¬les for each hcc patient as previouslydescribed li y zhu each as eventwas quantiï¬ed using the percent spliced in psi value rangingfrom to a commonly used method to reï¬ect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict ï¬lters average psi ¥ percentage of samples withpsi ¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentiï¬cation of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ¤ and log2fc ¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterproï¬ler package version yu the parent genes of deas events were importedinto the string database and used to determine proteinprotein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identiï¬ed by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artiï¬cial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplanmeier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii à lito divide the patients into the high or lowrisk group kaplanmeier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coeï¬cients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at ¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspeciï¬c reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin ampliï¬ed usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by ï¬ow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief à cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was ï¬lled with a culturemedium containing fbs incubation was carried out for h at ¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere ï¬xed with paraformaldehyde stained with crystalviolet and ï¬nally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identiï¬ed deas n the tcga hcc cohortin the current study was stratiï¬ed by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplanmeier curves asdescribed by xiong 2018aresultslandscape of as event proï¬les in hccto systematically characterize the as events and their clinicalsigniï¬cance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identiï¬ed asevents from genes based on their splicing patterns theseas events could be roughly classiï¬ed into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ¥ percentage of sampleswith psi ¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent proï¬les in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentiï¬cation of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as proï¬les of pairednormal and tumor tissues were ï¬nally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents ï¬ltered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes ï¬ltered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas proï¬les were used to identify deas eventually deaswere identiï¬ed from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the ï¬ltered as and deaswas inconsistent the es events accounted for of ï¬lteredas but only of deas however the proportion of apevents rose from of ï¬ltered as to of deasfigure 2b these statistical ï¬ndings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identiï¬ed deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identiï¬ed the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identiï¬ed deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2ac speciï¬c go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identiï¬cation of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identiï¬ed in hcc the blue and the red points represent the deas withstatistical signiï¬cance logfc ¥ adj p b proportions of different as types among ï¬ltered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could inï¬uence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we ï¬rst identiï¬ed sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionproï¬les of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoeï¬cient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapat1393e1593e4948e9941e2008e4387e8636e1393e4574e2519e1528e2202e6860e1522e2379e7699e1780e1843e4211e2609e1994e8164e1036e7765e1089e3569e1065e2135e5986e1038e1611e1110e2586e7736e2117e1072e4524e1151e1829e8136ebetween sfs and speciï¬c as events were illustrated using dotplots figures 5bg for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplanmeier curves for bothos and dfs these results conï¬rmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic signiï¬cance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure proteinprotein interaction ppi analysis of the identiï¬ed differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratiï¬edto form significant kaplanmeier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were ï¬ve and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these ï¬ndings conï¬rm that deas events possessnot only an important biological meaning but also a potentialclinical signiï¬canceconsidering the prognostic value of the aboveidentiï¬ed asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coeï¬cient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coeï¬cients to be less than a ï¬xed value certaincoeï¬cients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coeï¬cients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our ï¬ndings of significant heterogeneity among deas atan individual level which could reï¬ect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown signiï¬cance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height ampliï¬cation is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was veriï¬ed using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classiï¬cation overlap among clustersi iii and iv figures 8bc kaplanmeier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratiï¬cation of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratiï¬cation betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure speciï¬c regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength bg representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and conï¬denceintervals horizontal lines | 0 |
we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magniï¬cation c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magniï¬cation d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magniï¬cation presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the oriï¬ce of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the oriï¬ce of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms []the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patients bhcg level which was found to beelevated miuml and the ï¬nal diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinomaj bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0c' | 0 |
Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations Patients withCostello syndrome present craniofacial abnormalities cardiac defects and cancer predisposition as well as skinabnormalities including papillomas keratosis pilaris and eczematous dermatitis However the mechanisms underlyingthe dermatological abnormalities remain unclear Here we demonstrated that knockin mice expressing an Hras G12Smutation HrasG12S mice are susceptible to develop atopic dermatitis ADlike skin lesions including eczemapruritus elevated serum IgE levels acanthosis and the ltration of mast cells basophils and type2 innate lymphoidcells in the dermis after stimulation with house dust mite allergens Dermatophagoides farinae Dfb Reduced skinbarrier function increased proliferation of phosphorylated ERK pERKpositive epidermal cells and increased Th2typecytokines as well as epithelial cellderived cytokines including IL33 were observed in the skin tissue of HrasG12Smice compared with Hras mice Cultured HrasG12S keratinocytes exhibited increased IL33 expression after Dfbstimulation PD0325901 an MEK inhibitor ameliorated ADlike symptoms in HrasG12S mice showing decreasedproliferation of pERKpositive epidermal cells and decreased expression of IL33 Our ï¬ndings indicate that theepidermis of HrasG12S mice stimulated by Dfb strongly induced IL33 expression and type2 innate lymphoid cellsresulting in ADlike skin lesions These results suggest that the epidermis of HrasG12S mice are prone to developmentof eczematous dermatitis stimulated with house dust mite allergensIntroductionThe skin is a stratiï¬ed epithelium consisting of severallayers of cells in various stages of differentiation In orderto maintain normal skin homeostasis the proliferationdifferentiation and response of epidermal cells to externalstimuli must be tightly regulated1 The RASMAPK signaling pathway plays a crucial role in cell proliferationdifferentiation and apoptosis23 A strong activation of theRASMAPK pathway in skin is known to resultinCorrespondence Yoko Aoki aokiymedtohokuacjp1Department of Medical Genetics Tohoku University Graduate School ofMedicine Sendai Japan2Department of Pediatrics Tohoku University Graduate School of MedicineSendai JapanFull list of author information is available at the end of the Edited by E Candiepithelial cancers and melanoma45 Pigmented lesionshyperkeratosis pruritus curly hair and hyperplasia havealso been observed in vemurafenib a BRAF inhibitortreated patients6 The balance of the RASMAPK signaling pathway could be particularly important for epidermalhomeostasisCFCNoonan syndrome Costello syndrome and cardiofaciocutaneoussyndrome are phenotypicallyoverlapping genetic disorders characterized by craniofacial dysmorphia congenital heart defects and psychomotor retardation7 These syndromes are commonlycaused by germline mutations in components of the RASMAPK pathwaytermed RASopathies which constitutively activate the RASMAPK pathway89 Of thesesyndromes Costello syndrome is characterized by short The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of stature craniofacial abnormalities congenital heart diseases hypertrophic cardiomyopathy and intellectual disability10 Approximately patients with Costellosyndrome develop malignant tumors including rhabdomyosarcoma and bladder carcinoma In we identiï¬ed germline HRAS mutations in patients with Costellosyndrome11 A nucleotide change that cause the substitution of glycine at codon to serine pG12S in oneallele of HRAS has been observed in of Costellosyndrome patients The G12S mutation which has beenidentiï¬ed in somatic cancer is an oncogenic mutationthat activates the downstream pathway Patients withCostello syndrome develop a variety of skin abnormalitiesincluding palmoplantar keratoderma acanthosis nigricans eczema loose skin cutis laxa darker skin colorand papillomata around nose and anus However thepathogenesis of dermatological abnormalities remainsunclearWe have recently generated a strain of knockin miceexpressing an Hras G12S mutation the most frequentmutation in Costello syndrome12 which exhibited facialdysmorphia cardiomyocyte hypertrophy and kidneyanomalies Impaired mitochondrial fatty acid oxidationwas observed in HrasG12S mice fed a highfat diet13Skin abnormalities including papillomas have not beenobserved in young HrasG12S mice weeks old underspeciï¬c pathogenfree conditions In contrast HrasG12Smice over weeks of age or highfat diet fedHrasG12Smice had cutaneous lesions due to scratching Supplementary Fig 1a under the same pathogenicfree condition Although we have not analyzed the histology of skinof HrasG12S mice over weeks of age gross appearances of the skin lesions and scratching behavior suggestthat they are atopic dermatitislike In the current studywe tested to generate experimentally induced dermatitisin HrasG12S mice and found that HrasG12S micedeveloped more severe atopic dermatitis ADlike lesionsthan Hras mice after treatment with house dust miteallergens Dermatophagoides farinae Dfb Furthermorethese ADlike skin lesions in HrasG12S mice werereversedbyan MEK inhibitorPD0325901treatment withResultsDfb ointment induces ADlike skin lesions in HrasG12SmiceWe ï¬rst tested the effect of picryl chloride whichinduce contact dermatitis and imiquimod which inducepsoriasis on the skin of Hras and HrasG12S miceSupplementary Fig 1b c but no difference in skinlesions was observed between them Supplementary Fig1d In contrast the treatment with Dfb ointment developed severe dermatitisincluding severe erythemaOfï¬cial journal of the Cell Death Differentiation Associationhemorrhage scarring and eczema in the dorsal skin ofHrasG12S mice but not in Hras mice Fig 1a andSupplementary Fig 2a The ears of HrasG12S micebecame thick with edema erosion and excoriationFig 1b The dermatitis score was significantly higher inDfbtreated HrasG12S mice than in any other group ofmice SDStreated control Hras mice DfbtreatedHras mice and SDStreated control HrasG12Smice on day Fig 1c and Supplementary Table Other dermatitis parameters including the ear swellingFig 1d and the scratching behavior Fig 1e increasedsignificantly in Dfbtreated HrasG12S mice comparedwith Dfbtreated Hras mice Serum IgE levels weresignificantly higher in HrasG12S mice compared toHras in nontreated baseline ± ngmL vs ± ngmL P Supplementary Fig Although the difference was not statistically significant in SDS treatment groups IgE levels were higher inHrasG12S mice compared to Hras ± ngmLvs ± ngmL P Fig 1f as well as in theDfb treatment groups ± ngmL vs ± ngmL P Fig 1f These symptoms werealso seen in Hras mice but skin lesions are moresevere in HrasG12 mice In both groups of mice the IgEelevations were triggered by Dfb ointmentWe next examined if Dfbinduced dermatitisinHrasG12S mice is caused by the same pathology as inHras miceHistological analysis revealed hyperkeratosis and epidermal hyperplasia in the dorsal skin of DfbtreatedHrasG12S mice Fig 2a The epidermis of HrasG12Smice became thicker than that of Hras mice althoughDfb treatment increased the epidermal thickness in bothHras and HrasG12S mice Supplementary Fig 2b Inthe ADlike skin lesions Dfbtreated HrasG12S micedisplayed increased number of mast cells toluidine blueand tryptase 1 a marked increase in the numbersof T cells CD4 and dendritic cells MHC class IIFig 2a b and Supplementary Fig 2c e Western blottinganalysis revealed that the levels of CD4 protein weresignificantly increased in Dfbtreated mice compared withcontrol mice Supplementary Fig 2d In line with theacanthosis of Dfbtreated HrasG12S mice an increasednumber of phosphohistone H3positivecells wereobserved in the suprabasal epidermis layers of HrasG12Smice Fig 2b Although phosphorylated ERK pERKpositive cells were also increased in the epidermis of Dfbtreated Hras and HrasG12S mice the immunostainedarea in HrasG12S mice was significantly larger than thatin Hras mice Fig 2b and Supplementary Fig 2f g Inaddition we examined the expression of ï¬laggrin andclaudin1 as epidermal barrier markers in AD14 Adecreased expression of claudin1 was observed in Dfb 0cKatata Cell Death and Disease Page of Fig Dfb treatment induces atopic dermatitislike skin lesions in HrasG12S mice a b Skin and ear features of mice on day after treatmentof Dfb ointment HrasG12S mice showed dermatitis by repeated application of Dfb a The severity of ear swelling responses to Dfb was strongerin HrasG12S than Hras mice b c Dermatitis scores of only SDStreated control Hras and HrasG12S and Dfbtreated Hras andHrasG12S mice n per group d Time course of ear thickness from Hras and HrasG12S mice after treatment with SDS or Dfb n pergroup e The number of scratching bouts per min assessed by the video n per group f Serum IgE levels in Hras and HrasG12S mice aftertreatment with SDS or Dfb n per group Data are presented as mean ± SD Signiï¬cance in c d and f was analyzed by oneway ANOVAand the TukeyKramer method P P P and P HrasG12S Dfb vs Hras Dfb P P P and P HrasG12S Dfb vs HrasG12S SDS P P P and P Hras Dfb vs Hras SDSSigniï¬cance in e was analyzed by twotailed Students t test P treated HrasG12S mice compared with control HrasG12Smice Fig 2c d Together these results indicate that Dfbapplied HrasG12S mice exhibited more severe ADlikeskin lesions than Hras mice including acanthosis withhyperproliferation of pERKpositive cells in the epidermisas well as increased ammatory cells and reducedclaudin1 expressionThe skin of Dfbapplied HrasG12S mice shows an increaseof itchassociated factors and ammatory cytokinesTo further characterize the ADlike skin lesions weevaluated the levels of the itchassociated factors andammatory cytokinesin the skin of DfbtreatedHrasG12S mice Itchrelated neuronal markers including skin Tac1 Klk7 and Klk14 mRNA levels or PAR2 andOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see ï¬gure on previous pageFig Histological analysis reveals acanthosis with hyperproliferation of pERKpositive epidermal cells increased ammatory cells andreduced claudin1 expression in the dorsal skin of Dfbtreated HrasG12S mice a Skin tissue stained with HE and TB b c Immunohistochemistryof CD4 tryptase 1 pERK pHH3 and claudin1 in the skin ac Scale bars μm d Lysates from the skin were immunoblotted with antiClaudin1antibody Band intensities were quantiï¬ed and compared among the four groups The expression levels were normalized to GAPDH n in eachgroup Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and the TukeyKramer method P P twotailedStudents t testIl1 proammatory cytokineEndothelin proteins17 were significantly higher thanthose in control HrasG12S and Dfbtreated Hras miceFig 3ac Regarding ammatory cytokines the skinIl4mRNA levels ofTh2related cytokine and epidermalderived cytokinesIl33 and thymic stromal lymphopoietin Tslp were significantly elevated in Dfbtreated HrasG12S mice compared with control HrasG12S and Dfbtreated Hrasmice Fig 3d IL33 leads to the activation of type2innate lymphoid cells ILC2s through ST2 IL33 receptor21 In Dfbtreated HrasG12S mice the skin mRNAlevels of St2 were also significantly increased Fig 3dImmunohistochemistry analysis revealed that DfbtreatedHrasG12S mice showed enhanced expression levels ofIL33 and TSLP in the basal epidermal layers and thesurface of epidermis respectively Fig 3e Likewise theIL33 protein levels were significantly higher in the skin ofDfbtreated HrasG12S mice than in Dfbtreated Hrasmice Fig 3fIncreased numbers of ILC2 and increased IL33 expressionwere observed in the epidermis of HrasG12S miceTo investigate when and how ammatory cytokinesare induced in HrasG12S mice we performed ï¬owcytometry on skin and ear samples days after Dfbapplication that is once the ADlike skin lesions hadbegun to appear Fig 1c The accumulation of basophilsand ILC2s was observed on skin samples of DfbtreatedHrasG12S mice days after Dfb application Fig 4a Asignificant increase in mast cells eosinophils basophilsand ILC2s was also observed in the ears of HrasG12Smice Fig 4b On the other hand these immune cellswere hardly detected in Hras and HrasG12S mice days after SDS application Supplementary Fig The mRNA levels of ammatory cytokines in the skinfrom HrasG12S mice days after Dfb applicationshowed that Il1 and Il33 were significantly elevated inthe skin of Dfbtreated HrasG12S mice compared withDfbtreated Hras mice Fig 5aAtopic dermatitis is characterized by increased serumIgE acanthosis loss of skin barrier function and ltration of immune cells including Th2 cells dendriticcells eosinophils basophils and mast cells2223 On thataccount we next examined the response of immune cellsin these mice HRAS is known to be highly expressed inOfï¬cial journal of the Cell Death Differentiation Associationthe epidermal cells but not in immune cells2425 Indeedno significant difference was found in the population ofimmune cells from the spleen and lymph node LN Theproliferation of naive CD4 T cells and Th2 immuneresponse was comparable between Hras and HrasG12Smice at weeks of age suggesting that ADlike dermatitismay not be caused by different response of immune cellsFig 6a b Then we examined whether an increasedproduction of IL33 and TSLP was observed in primaryepidermal keratinocytes in response to Dfb Six hoursafter Dfb stimulation the mRNA level of Il1 not Tslpwas significantly elevated in cultured epidermal keratinocytes of Hras and HrasG12S mice Fig 5b NotaIl33 in the Dfbstimulatedblykeratinocytes of HrasG12S mice were significantlyincreased compared with those of nonstimulatedHrasG12S and Dfbstimulated Hras keratinocytessuggesting that epidermal keratinocytes with Hras G12Smutation have increased IL33 expression after stimulation with Dfb Fig 5bthe mRNA levels ofPD0325901 reduces skin lesions in DfbstimulatedHrasG12S miceamelioratesTreatment with MEK inhibitorstheabnormalities observed in RASopathyrelated modelmice26 We investigated the effects of an MEK inhibitor PD0325901 on skin lesionsin DfbtreatedHrasG12S mice Supplementary Fig 5a Ten days oftreatment with PD0325901 or saline resulted in a significant improvement of the dermatitis score and earswelling in HrasG12S mice Fig 7a b PD0325901treatment resulted in a marked reduction of epidermalthickness mast cell numbers and pERKpositive epidermal cells as well as recovered expression levels ofclaudin1 Fig 7c d and Supplementary Fig 5b c ThemRNA levels of Il1 Il4 Il33 St2 and Klk14 were significantly lowerin the skin of PD0325901treatedHrasG12S mice than that of vehicle salinetreatedHrasG12S mice Fig 7e and Supplementary Fig 5dThese results suggest that ERK inhibition partially ameliorates Dfbinduced skin lesions in HrasG12S miceDiscussionHere we demonstrated that mice expressing a germlineHras G12S mutation but not Hras mice developed 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see ï¬gure on previous pageFig Expression of itchassociated factors and ammatory cytokines is enhanced in Dfbinduced skin lesions in HrasG12S mice a c dRelative mRNA expression related to neuronal factors a Tac1 and Ngf skin proteases c Klk5 Klk7 and Klk14 and ammation d Il1 Il4 Tslp Il33and St2 in the dorsal skin mRNA levels were normalized to those of 18s rRNA n per group b f Protein extracts from dorsal skin wereimmunoblotted with antiPGP95 antiPAR2 antiEndothelin and antiIL33 antibody n in each group GAPDH same data as in Fig 2d f Bandintensities were quantiï¬ed and compared among the four groups Expression levels were normalized to GAPDH e Immunohistochemistry of IL33and TSLP Scale bars μm Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and the TukeyKramer method P P P and P P twotailed Students t testADlike skin lesions under conditions of Dfb exposureThe levels of IL1 and epithelial cellderived cytokinesIL33 and TSLP were also increased in DfbtreatedHrasG12S mice In addition an increased production ofIL1 IL4 and IL33 as well as ammatory cellsbasophils and ILC2s was observed in the dorsal skin ofHrasG12S mice before the development of ADlike skinlesions Analysis of the underlying mechanism revealedthat Dfbstimulated keratinocytes in HrasG12S miceinduced IL33 production while in naive CD4 T cellsfrom the spleen the Th2 immune response was comparable between Hras and HrasG12S mice Finallythe inhibition of ERK activation by PD0325901 treatmentameliorated the ADlike skin lesions and IL33 production Together these data indicate that germline HrasG12S activating mutation causes ADlike skin lesions viathe ERKIL33 axis Fig cellsepidermalIn the present study after repeated stimulation withDfb HrasG12S mice showed hyperproliferation of pERKpositiveand increased IL33expression in the dorsal skin Increased IL33 expressionwas also observed in primary epidermal keratinocytesfrom HrasG12S mice after Dfb stimulation which issimilar to the reports that the activation of RASMAPKsignaling was associated with increased IL33 expressionin cancer cells2930 Recently IL33 was found to inducethe Th2 ammatory response in allergic diseasesespecially AD31 Excess IL33 is also associated with skinbarrier dysfunction and ILC2 functions which is partiallyregulated by the RASMAPK signaling pathway3233Epithelialspeciï¬c IL33 transgenic mice have been foundto develop ADlike dermatitisincluding acanthosispruritus increased IgE serum levels reduced claudin1expression and increased production of eosinophils mastcells and ILCs3334 Of note IL33 and its receptor ST2are highly expressed in the skinderived ILC2s of ADpatients and lung ILC2s of patients with allergic airwaydiseases respectively3536 Importantly consistent with thephenotype of IL33 transgenic mice and AD patients Dfbtreated HrasG12S mice showed ADlike skin lesionsreduced claudin1 expression increased IL33 expressionhyperproliferation of pERKpositive epidermal cells andincreased ILC2 production Collectively excess IL33could lead to ERK activation resulting in an increase ofILC2s and impaired skin barrier Fig Ofï¬cial journal of the Cell Death Differentiation AssociationThe pathogenesis of skin lesions observed in Costellosyndrome includes dermal connective tissue abnormalities cutis laxa and deep palmer and plantar creaseshyperproliferative skin disease palmoplantar keratoderma cutaneous papilloma and acanthosis nigricansand ammatory skin abnormalities sensitive skineczema and pruritus A previous study showed that inthe skin ï¬broblasts of Costello syndrome elastic ï¬berswere not assembled due to a functional deï¬ciency of theelastinbinding protein as a result of an unusual accumulation ofchondroitin sulfatebearing proteoglycans3738 Regarding the hyperproliferative skin lesions ithas been reported that the root cause of papillomashyperkeratosis and epidermal hyperplasia such as psoriasis is the activation of the RASMAPK pathway39However the pathophysiological mechanism of ammatory skin abnormalities in Costello syndrome remainsunclear In the present study Dfbtreated HrasG12S micedisplayed pruritus and eczema Recently mice withepidermisspeciï¬c BRAFRAF1 deï¬cient also showed ADlike dermatitis which is characterized by increased serumIgE levels and a Th2 response43 The elevated IgE levelshave not been systematically examined or reported inCostello syndrome patients However it is possible thatthe allergic reaction stimulated by house dust mites couldbe involved in the development of ammatory skinabnormalities in Costello syndrome patientsincludingsensitive skin pruritus and eczemaimprove HRASdrivenAt present acitretin has been reported to treat palmoplantar keratosis in patients with Costello and CFC syndrome4445 Several reports have demonstrated that MEKtumorigenesis46inhibitorsimpaired enamel formation in the teeth27 and longtermdepression in the hippocampus in Hras G12V knockinmice28 as well as hyperkeratosis and hyperplasia in theforestomach of BrafQ241R mice26 In the present studyPD0325901 treatment of the ADlike skin lesions inHrasG12S mice was found to reverse these lesions byreducing hyperproliferation of pERKpositive epidermalcells and the production of ammatory cells and cytokines including IL1 IL4 and IL33 Treatment withU0126 an MEK inhibitor in human keratinocytes hasalso been found to restore the reduced expression levels ofclaudin1 and ï¬laggrin and increase ERK activationthrough excess IL333347Indeed reduced claudin1 0cKatata Cell Death and Disease Page of Fig An increase in ILC2s and basophils observed in the skin and ear of Dfbtreated HrasG12S mice a b Flow cytometric analysis of skin aand ear b cells from Hras and HrasG12S mice collected days after Dfb application Eosinophils CD45 SSChi siglecF Basophils CD45siglecF FcεRIα DX5 Mast cells CD45 siglecF FcεRIα DX5 ILC2 CD45 Lin CD3ε CD4 CD8a CD11c FcεRIα NK11 CD19 Ter119 CD5 F4 Gr1 Sca1 GATA3 n in each group Data are presented as mean ± SD Signiï¬cance was analyzed by twotailed Students t test P P expression was improved in Dfbtreated HrasG12S miceafter PD0325901 treatment Recently hypertrophic cardiomyopathy in Noonan syndrome patients were treatedby MEK inhibitor48 So MEK inhibitors could be effectivein patients with RASopathies The most common sideeffect of trametinib MEK inhibitor in human patients isOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig Expression of ammatory cytokines is enhanced in the skin of Dfbtreated HrasG12S mice in the early stage of dermatitisa Relative mRNA expression related to ammation including Il1 Il4 Il13 Tslp Il33 and St2 in the dorsal skin of Hras and HrasG12S mice on day after treatment of Dfb mRNA levels were normalized to those of 18s rRNA n per group b Relative mRNA expression of Il1 Tslp and Il33 inDfbstimulated ngμl h or vehicletreated PBS h keratinocyte from Hras and HrasG12S mice mRNA levels were normalized to those ofGapdh Results represent ï¬ve independent experiments Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and theTukeyKramer method P P and P skin rush and common toxicity associated with vemurafenib BRAF inhibitor is cutaneous abnormalities suchas keratoacanthoma and squamous cell carcinoma by themechanism of paradoxical MAPK pathway activation49Therefore the balance of RASMAPK signaling plays animportant role in the emersion of skin abnormalitiesAdjusting dosage of MEK inhibitors may be effective onskin lesions of patients with Costello syndromeHere Dfbtreated HrasG12S mice exhibited increasedIL33 expression through hyperproliferation of pERKpositive epidermal cells Additionally we show thatPD0325901 treatment ameliorated the ADlike skinlesions in HrasG12S mice under conditions of exposureto Dfb Thus it will be interesting to investigate whethertreatment with IL33 antibody reduces the ADlike skinlesions in HrasG12S mice Our ï¬ndings provide additional perspective that HrasG12S mice will serve as avaluable model to study pathophysiology and potentialtherapeutic approaches in ADMaterials and methodsMiceHrasG12S mice on a C57BL6J background have beendescribed previously13 Male mice were analyzed inthis studyGenotypingThe genomic DNA was extracted from the tail tissueusing a Maxwell Mouse Tail DNA Puriï¬cation KitPromega Madison WI USA or the alkaline lysismethod For the alkaline lysis method a small piece ofeach tail mm was incubated in mM NaOH for min at °C After the addition of M TrisHClpH the extracts were used for PCR Genotyping ofOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig No significant difference was found in Th2 cytokine responses and the proliferation of naive CD4 T cells between Hras andHrasG12S mice a Naive CD4 T cells were isolated from the spleen of Hras and HrasG12S mice at weeks of age After that the cells werestained with CSFE and cultured with mouse antiCD3 monoclonal antiCD28 monoclonal recombinant mouse IL2 recombinant mouse IIL4 andantiIFNγ antibodies for days After incubation the cells were ï¬xed permeabilized and stained with IL5 and IL13 antibodies Flow cytometricanalysis was performed n in each group b After days culture of puriï¬ed naive T cells from spleen of Hras and HrasG12S mice theproliferation index was measured by ï¬ow cytometry n in each group Data are presented as mean ± SD Signiï¬cance was analyzed by twotailedStudents t testHras and HrasG12S mice was performed by PCRusing KOD FX Neo TOYOBO Osaka Japan The primers used for PCR have been described previously1350Induction of dermatitisAtopic dermatitislike skin lesions were induced in malemice at weeks of age according to the manufacturersinstructions The mice were anesthetized with isoï¬uraneand their dorsal hair was removed using an electric clipper2000AD Natsume Seisakusho Tokyo Japan For sensitization mg of Biostir AD Biostir Inc Kobe Japanan ointment of Dfb extract was applied to the shaveddorsal skin and ears Three or four days later hair growthwas removed with an electric clipper and μl of SDS SigmaAldrich St Louis MO USA was applied tothe shaved dorsal skin and ears to disrupt the skin barrierAfter h mg of Biostir AD was applied to theirshaved dorsal skin and ears to induce ADlike skin lesionsThese procedures were repeated twice a week for daysThe mice were sacriï¬ced on day to collect skin and earsamples Supplementary Fig 2a To assess the effect ofDfb ointment to Hras and HrasG12S mice they wererandomly divided into four groups SDStreatedHras SDStreated HrasG12S Dfbtreated Hrasand Dfbtreated HrasG12S using single blinding testEvaluation of dermatitis and ear thicknessThe dermatitis scores were evaluated twice a weekaccording to the development of four symptoms erythemahemorrhage of dorsal skin scarringdryness ofdorsal skin edema of ear and excoriationerosion ofear51 The total dermatitis score maximum score wasdeï¬ned as the sum of individual scores none mild moderate severe for each symptom SupplementaryTable Ear thickness was measured with a digimaticmicrometer CLM115QM Mitutoyo Kanagawa JapanMeasurement of scratching behaviorOn day scratching behavior was monitored by videoGZHM890 JVC Kanagawa Japan for min TheOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see ï¬gure on previous pageFig MEK inhibitor reduces the clinical severity of dermatitis in Dfbstimulated HrasG12S mice a b Gross appearances dermatitis score andear thickness in vehicle saline or PD0325901treated Hras and HrasG12S mice after days of daily injections n per group All of the micein these ï¬gures were treated with Dfb The circle and square show vehicle group without MEKi with same amount of saline c Skin tissuestained with HE and TB in Hras and HrasG12S mice with PD0325901 or vehicle treatment n per group d Immunohistochemistry of pERKand claudin1 in skin c d Scale bars μm e Relative mRNA expression of Il1 Il4 and Il33 in dorsal skin mRNA levels were normalized to those of18s rRNA vehicle group n PD0325901 group n Data are presented as mean ± SD Signiï¬cance was analyzed by oneway ANOVA and theTukeyKramer method P P and P HrasG12S PD0325901 vs HrasG12S vehicle P HrasG12S vehicle vs Hrasvehicle P onetailed Students t testFig Germline HrasG12S mutation causes ADlike skin lesions via ERKIL33 axis Exposure to Dfb allergen induces ADlike skin lesionsincluding eczema acanthosis and pruritus in HrasG12S mice Dfbtreated HrasG12S keratinocytes show increased IL33 expression throughhyperproliferation of pERKpositive epidermal cells Excess IL33 activates basophil and ILC2containing ST2 receptors These activated immune cellsinduce the production of type2 ammatory cytokines such as IL4 Furthermore excess IL33 can activate ERK signaling resulting in reducedclaudin1 expression and skin barrier dysfunction DC dendritic cellsnumber of scratching bouts was assessed by replaying thevideo Each incidence of scratching behavior was deï¬nedas rubbing of ears and dorsal skin with forepaws hindpaws and mouthHistology and immunohistochemistryThe dorsal skins were ï¬xed in neutral bufferedformalin for parafï¬nembedded specimen and paraformaldehyde for frozen specimen Embedded tissueswere sectioned at μm parafï¬nembedded specimensor μm frozen specimens Parafï¬nembedded specimens were stained with hematoxylin and eosin HEand toluidine blue TB Epidermalthickness wasmeasured in ï¬ve randomly selected areas à μmof each HEstained sample Mast cells were counted inten randomly selected areas à μm of each TBstained sample For immunohistochemistry the parafï¬nembedded sections were deparafï¬nized using xylene andrehydrated with ethanol Frozen specimens were driedsufï¬ciently with a dryer Antigens were activated using aHistoï¬ne simple stain kit Nichirei Bio Sciences TokyoJapan The antibodies used are described in Supplementary Table Signals were visualized with a DABSubstrate KitNichirei Bio Sciences Sections werecounterstained with hematoxylin pERK immunostainedareaepidermis was measured in ï¬ve randomlyOfï¬cial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of selected areas à μm of each pERKstainedsampleSpleen and LN immune cell preparation and ï¬owcytometryQuantitative reverse transcriptionPCRTotal RNA extraction and puriï¬cation of keratinocyteswas performed according to the standard proceduresusing an RNeasy Mini Kit Qiagen Hilden Germany andQIAshredder Qiagen The extraction and puriï¬cation ofthe total RNA from the dorsal skin and cDNA synthesiswere performed as previously described26 Quantitativerealtime PCR was performed using THUNDERBIRDSYBR qPCR MIX TOYOBO in a StepOnePlus ThermoFisher Scientiï¬c Waltham MA USA The ampliï¬cationprimers are described in Supplementary Table Eachsample was run in duplicateWestern blottingSkin tissue was homogenized in lysis buffer mM TrisHCl pH and SDS containing phosphatase and protease inhibitor P5726 and P8340 SigmaAldrich Supernatants were collected after centrifugation and the proteinconcentration was determined using a BioRad ProteinAssay BioRad Laboratories Hercules CA Western blotanalyses were performed as previously described26 Brieï¬ythe proteins were transferred onto nitrocellulose membranesand blocked with nonfat milk in Trisbuffered salinewith Tween20 mmolL TrisHCl pH mmolLNaCl and Tween20 for h at room temperature Themembranes were incubated overnight at °C with theantibodies described in Supplementary Table All membranes were visualized using the Western Lightning ECLPlus Kit PerkinElmer Waltham MA USA The bandintensities were quantiï¬ed using NIH ImageJ softwareELISASerum IgE was determined using LBIS Mouse IgEELISA Kit FUJIFILM Wako Shiba | 2 |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | 2 |
Journal of International Medical Research The Authors reuse guidelinessagepubcomjournalspermissions journalssagepubcomhomeimrCase ReportNivolumab plus gemcitabinedexamethasone and cisplatinchemotherapy induce durablecomplete remission inrelapsedrefractory primarymediastinal Bcell lymphomaa case report andliterature reviewGang Huang1 Ju Huang2 Zhili Zhang2Chongchong Xue1 and Yuan Liu2AbstractPrimary mediastinal large Bcell lymphoma PMBCL is an uncommon but aggressive type ofBcell lymphoma Patients with relapsed refractory PMBCL rrPMBCL have limited therapeuticoptions and usually have a relatively poor outcome Immune checkpoint blockade has become apotential treatment for this disease We report here a case of a female patient with rrPMBCLwho was treated with nivolumab plus gemcitabine dexamethasone and cisplatin GDP chemotherapy Complete remission was achieved after four cycles of combined therapy With continuednivolumab maintenance monotherapy she has remained in complete remission for longer than months This is the first report of nivolumab plus GDP chemotherapy inducing completeremission in patient with rrPMBCL This case supplements the limited literature and providesimplications for clinical trial designs regarding the potential use of nivolumab in the treatment ofrrPMBCL1Department of Hematology Yuebei Peoples HospitalShantou University Medical College ShaoguanGuangdong Province China2Guangdong Women and Children Hospital GuangzhouGuangdong ChinaCorresponding authorYuan Liu Medical Genetic Centre Guangdong Womenand Children Hospital No Xingnan Rd PanyuDistrict Guangzhou Guangdong ChinaEmail yuanliu005163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical ResearchKeywordsRelapsed refractory primary mediastinal Bcelllymphoma nivolumab checkpoint blockadegemcitabine dexamethasone cisplatin chemotherapy programmed cell death completeremissionDate received February accepted July IntroductionPrimary mediastinal large Bcell lymphomaPMBCL is an uncommon but aggressivetumor that accounts for to of nonHodgkin lymphoma1 PMBCL is distinguished from diffuse large Bcell lymphomaby virtue of distinct clinical pathologicaland genetic features2 Recently PMBCLwas listed as a separate entity in the latestWorld Health anization classiï¬cation of hematopoieticand lymphoidtumors5 PMBCL has a similar clinical presentation as classical Hodgkin lymphomacHL and PMBCL also shares certain features at the molecular level particularly9p241 alterations and programmed celldeath protein ligand 1ligand PDL1PDL2 expression6 At present management and outcome of PMBCL are still critical and a more serious situation is faced bypeople who are diagnosed with relapsedand refractory PMBCL rrPMBCL19The optimal salvage chemotherapy andautologousforrrPMBCL are of limited efï¬cacy19stem celltransplantRecently agents targeting programmedcell death PD1 and PDL1 have beenimmunotherapy10developed in tumorAntiPD1 therapy with monoclonal antibodies has been approved for the treatmentof several types of solid tumor and cHLThe therapeutic potential of antiPD1 therapy in other malignancies is likely to beapproved soonIn a humanizedimmunoglobulin G1 recombinant monoclonal antibody for the PD1 receptor pidilizumab was approved by the US Food andDrug Administration FDA for treatingandpediatricpatientsthatwithadultrrPMBCL11 Another agenttargetsthe PD1 receptor called nivolumab isa fully humanized immunoglobulin G4monoclonal antibody that has been grantedapproval by the US FDA for treating several solid malignancies and cHL The therapeutic efï¬cacy of nivolumab in patientswith rrPMBCL remains unclearWe report here a patient with rrPMBCLwho received combined treatment with offlabel nivolumab and GDP chemotherapyComplete remission CR was achievedafter four cycles of such combined treatment At the time of this submission thepatient has remained in CR for longerthan months with continued nivolumabmaintenance monotherapyCase reportA 32yearold woman presented to YuebeiPeoples Hospital with intermittent dyspneaand chest pain A positron emission tomography PET scan showed a 10cm mass inthe anterior superior mediastinum with astandardized uptake value of Themass showed unclear margins and compressed the ascending aorta and pulmonarytrunk Small pericardial and left pleuraleffusions were also observedThe mass was diagnosed as PMBCL by asubsequent biopsy Immunohistochemicalstaining showed thatlarge lymphocyteswere positive for CD20 CD79a Pax5BCL6 CD23 CD30and multiplemyeloma1 and negative for CD10 CD3CD5chromogranin Asynaptophysin 0cHuang et alterminalencodingandincludingtwocycles positiveregion inendomysialdeoxynucleotidyltransferase cytokeratin CK CK19 andandS100 Ki67 wassituEpsteinBarrhybridization was negative She was initially treated with six cycles of frontline chemotherapyofrituximab cyclophosphamide doxorubicinvincristine and prednisolone RCHOPand four cycles of doseadjusted etoposidecyclophosphaprednisolonemiderituximabDAEPOCHR were administered Thetimeline of treatment is shown in aShe received tumor resection by thoracoscopic surgery after she continued twodoxorubicinvincristinecycles ofgemcitabine dexamethasonecisplatinumetoposide and rituximabtherapy Her ï¬rst CR was achieved inDecember However monthslater a PETcomputed tomography CT scan showedhypermetabolic lesions located at the leftlung and right adrenal gland but not inthe primary mediastinal site bThe patient reported no physical symptomsand received a repeat tissue biopsy whichconï¬rmed a relapse with PMBCL She wastreated with each cycle of a dexamethasoneifosfamide cisplatin and etoposide regimenand ibrutinib bendamustine and cytarabine therapy A chest CT scan showedFigure Summary of treatment and monitoring the tumor response a Patients timeline chart with thedates of treatment and monitoring the tumor response b Positron emission tomography images Upperpanel a scan of the relapsed hypermetabolic lesions located at the left lung and right adrenal gland beforecombined treatment Lower panel complete remission was achieved after four cycles of nivolumab plusGDP chemotherapyRCHOP rituximab cyclophosphamide doxorubicin vincristine and prednisolone DAEPOCHR doseadjusted etoposide prednisolone vincristine cyclophosphamide doxorubicin and rituximab GDPERgemcitabine dexamethasone cisplatinum etoposide and rituximab CR complete remission PMBCLprimary mediastinal large Bcell lymphoma DICE dexamethasone ifosfamide cisplatin and etoposideIBC ibrutinib bendamustine and cytarabine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical Researchthat the right adrenal gland lesion had partially responded while the lesions in the leftlung had progressed After those cycles ofchemotherapy the patient showed GradeIV myelosuppression and had to receiveblood transfusion treatment Moreover acerebrospinal ï¬uid examination showedthe presence of atypical lymphocytes andno symptoms of infection of the central nervous system were observed Intrathecal chemotherapy cytarabine mg methotrexate mg and dexamethasone mg was thenadministered and no atypical lymphocyteswere detected by repeated cerebrospinalï¬uid analysis These ï¬ndings highly suggested a potential risk of metastasis of thecentral nervous systemtreatmentBecause the disease had progressed withsevere myelosuppression and there were nostandard chemotherapy guidelines or alternative treatment options for the patientother salvage treatments of her refractorydisease needed to be considered Aftermuch discussion with the patient and herfamily she declined autologous hematopoietic stem cell transplantation and receivedcombinedgemcitabine mg dexamethasone mg and cisplatinum mg GDPchemotherapyand the offlabel antiPD1 antibody nivolumab mg After four cycles of combined treatment a repeated PETCT scanshowed thatshe had secondary CRb She received two more cyclesof combined treatment with nivolumab andGDP chemotherapy and then continuedsingle nivolumab maintenance treatment mg Since her ï¬rst dose in May she received doses of nivolumab Shereported moderate fatigue and pyrexia in to days after each administration ofnivolumab Blood tests indicated normalfunction of the liver kidney and thyroidFigure She also had normal bloodlevels of creatinine albumin globulin lactate dehydrogenase aspartate transaminasetotalaminotransferaseofalaninethey2b Neutrophilbilirubin and urea nitrogen during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the ï¬rst four combined therapies because of toxicity of GDPchemotherapy butrecovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2cFurthermore no adverse signs and symptoms were observed in the lungs brainand skin At the time of this submissionshe has remained in CR for longer than months with continued nivolumab maintenance therapyEthics approval was obtained from theethicalcommittee of Yuebei PeoplesHospital Written informed consent wasobtained from the patient for analysis ofthe samples and publicationDiscussionTreatment and outcome are critical in managing PMBCL Because there is no established standard approachthe ï¬rstlinetreatment of PMBCL is generally thesame as that for diffuse large Bcell lymphoma including RCHOP and DAEPOCHR Relapse of PMBCL usually occurs in theï¬rst to months after completion offrontline therapy with a lower incidenceapproximately than diffuselarge Bcell lymphoma19 There are varioussecondlineregimens for patients with rrPMBCL includingthe rituximab ifosfamide carboplatin andetoposide regimenthe rituximab dexamethasone cytarabine and cisplatin regimen and rituximabGDP12 Because of alack of standard guidelines or treatmentoptions for PMBCL the outcome greatlydepends on the patients response to theregimen Thisremains poordespite these secondline salvage chemotherapies and subsequent autologous hematopoietic stem cell transplantation912immunochemotherapyresponse 0cHuang et alFigure Blood test values during the whole treatment process since the first dose of nivolumab The firstfour cycles were nivolumab plus GDP chemotherapy and nivolumab maintenance monotherapy wasadministered since the fifth cycle a Thyroxine thyrotropin FT3 and FT4 levels b Levels of creatininealbumin globulin lactate dehydrogenase aspartate transaminase alanine aminotransferase total bilirubinand urea nitrogen c Neutrophil and platelet countsFT4 free thyroxine FT3 free triiodothyronine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical ResearchIn recent years strategies focusing on thecheckpoint blockade have been developedin tumor immunotherapy10 Therapeuticantibodies targeting the PD1PDL1 axispossess clinical activity and an acceptablesafety proï¬le in treating a growing list oflymphomas13solid tumors and BcellBased on a clinical study of patientswith rrPMBCL pidilizumab was approvedby the US FDA for treatment of adult andpediatric patients with rrPMBCL in Another antibody nivolumab has beengranted approval for treating several solidmalignancies and cHL However studiesregarding application of nivolumab forPMBCL are limited Only ï¬ve reportshave described using nivolumab for treatment of PMBCLrrPMBCL Table asfollows In a phase I study published intwo patients with PMBCL wererecruited and treated with nivolumab atdoses of or mgkg every weeks afterprevious systemic treatments14 No objective responses were observed in this previous study In another phase I study onewithrefractorypatient with PMBCL received combinedtherapy of nivolumab and ipilimumaband died during the therapeutic process15Recently two reports showed the potentialtherapeutic efï¬ciency of nivolumab forpatientsPMBCLrrPMBCL who showed failure with conventional immunochemotherapy1617 Both ofthese two cases had immunerelated adverseeffects during the antibody treatment process One patient with highgrade neutr ia had nivolumab stopped temporarilyand was treated with intravenous immunoglobulin16 The other patient with zosterreactivation was controlled by administration of valacyclovir17 Recently Zinzaniand colleagues showed that combined treatment of nivolumab and brentuximab vedotin had promising antitumor activity and amanageable safety proï¬le in patients withrrPMBCL18 In this phase II study patients were recruited and treated withnivolumab mgkg and brentuximab vedotin mgkg every weeks The objectiveresponse rate was and achievedTable Reports regarding application of nivolumab in primary mediastinal large Bcell lymphomarelapsedand refractory primary mediastinal large Bcell lymphomaNumberof cases DoseCombinedtreatmentAdverse events or mgkg mgkgIpilimumabResponseyesnoNoNo ofpatientsReportsLesokhin AMet al Ansell S et alWright Zet al Yassin R et alZinzani mgkgNoNoHighgrade neutr iaYesZoster reactivationYes mgBrentuximabNeutr iaPL et al vedotinthrombocyt iaand peripheralneuropathy of patientsPresent case mgkgGDPMild fatigue and pyrexiaYeschemotherapyNote means not indicated in the report 0cHuang et alofcyclescombinedCR and achieved partial remission Of patients of them had drugrelatedadverse events and the most common wereneutr ia thrombocyt ia and peripheral neuropathy18 In the present case weattempted several available approaches intreating the patients relapsed disease butfailed to control the progress of the massAfter much discussion with the patient andher family we considered an offlabel nivolumab and GDP chemotherapy as salvagetreatmentfor the patient In September her second CR was achieved afterfourtreatmentCurrently with continued nivolumab maintenance monotherapythe patient hasremained in CR for longer than monthsImmunerelated adverse events that areassociated with checkpoint blockade oftenstart within the ï¬rst few weeks to monthsafter treatment but can occur any time andin any an The most common immunerelated adverse events are hypothyroidismnausea diarrhea pyrexia and fatigue1920In the present case we were concernedabout immunerelated an damage sincethe ï¬rst dose of nivolumab The patientreported moderate fatigue and pyrexiaafter each administration of nivolumaband soon recovered within to daysBlood testing was performed during thewhole therapeutic process and the datawere reviewed and analyzed Blood levelsof thyroxine thyrotropin free triiodothyronine and free thyroxine indicated no thyroiditisFigure 2a Our patient alsoshowed normal metabolic data during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the ï¬rst four combined therapies because of toxicity of GDPchemotherapy but they then recovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2c2b NeutrophilUnlike otherarelymphomas prognosticbiomarkersinPMBCL12 Some serum molecules such aslackinglargelyCCL17 and CD163 are considered aspotential biomarkers for predicting andmonitoring responses and detection ofrelapses in patients with Hodgkin lymphoma1221 The role of serum biomarkers inPMBCL remainsinvestigatedRadiological imaging should only be usedin patients who have new clinical symptomsor signs suggestive of relapse but not inasymptomatic patients922betoTo the best of our knowledge this is theï¬rst reported case of nivolumab plus GDPchemotherapy that induced CR with nosevere immunerelated an damage in apatient with rrPMBCL We also reportthe longest followup observation of successful application of nivolumab in apatient with rrPMBCLThis report supplements the limited literature of nivolumab fortreatment ofPMBCLrrPMBCL and provides implications for clinical trial design regarding thepotential use of nivolumab in treatment ofrrPMBCL Further investigation needs to beperformed for potential application of singleor combined use of nivolumab for patientswith rrPMBCL who experience failure withconventional therapeutic approachesDeclaration of conflicting interestThe authors declare that there is no conï¬ict ofinterestFundingThis research received no speciï¬c grant from anyfunding agency in the public commercial ornotforproï¬t sectorsorcid000000034880ORCID iDYuan LiuReferences Martelli M Ferreri A Di Rocco A et alPrimary mediastinal large Bcell lymphomaCrit Rev Oncol Hematol 0cJournal of International Medical Research Savage KJ Monti S Kutok JL et al Themolecular signature of mediastinallargeBcell lymphoma differs from that of otherdiffuse large Bcell lymphomas and sharesfeatures with classical Hodgkin lymphomaBlood Rosenwald A Wright G Leroy K et alMolecular diagnosis of primary mediastinalB cell lymphoma identiï¬es a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma J ExpMed Mottok A Wright G Rosenwald A et alMolecular classiï¬cation of primary mediastinal large Bcell lymphoma using routinelyavailable tissue specimens Blood Swerdlow SH Campo E Pileri SA et alThe revision ofthe World Healthanization classiï¬cation of lymphoid neoplasms Blood XuMonette ZY Zhou J and Young KHPD1 expression and clinical PD1 blockadelymphomas Blood in Bcell Van Roosbroeck K Ferreiro JF TousseynT et al Genomic alterations of the JAK2and PDL loci occur in a broad spectrum oflymphoid malignancies Genes ChromosomesCancer rearrangements Twa DD Chan FC BenNeriah S et alGenomicinvolving programmed death ligands are recurrent in primary mediastinallymphomaBlood large Bcell Cwynarski K Marzolini MAV BarringtonSF et al The management of primary mediastinal Bcell lymphoma a British Societyfor Haematology Good Practice Paper BrJ Haematol Ribas A and Wolchok JD Cancer immunotherapy using checkpoint blockade Science HematologyOncology Cancer Approvals Safety Notiï¬cations Available online URL wwwfdagovdrugsinformationondrugsapproveddrugsucm610670htmdrugsapproveddrugsucm610670htmwwwfdagovdrugsinformationon Lees C Keane C Gandhi MK et al Biologyand therapy of primary mediastinal Bcelllymphoma current status and future directions Br J Haematol Goodman A Patel SP and Kurzrock RPD1PDL1 immunecheckpoint blockadein Bcell lymphomas Nat Rev Clin Oncol Lesokhin AM Ansell SM Armand P et alNivolumab in patients with relapsed orrefractory hematologic malignancy preliminary results of a phase Ib study J Clin Oncol Ansell S Gutierrez ME Shipp MA et alA phase study of nivolumab in combination with ipilimumab for relapsed or refractory hematologic malignancies CheckMate Blood Wright Z and Brown A Highgrade neutr ia in a patient successfully treated withnivolumab for refractory primary mediastilymphoma Blood Adv nal Bcell Yassin R Hajeer A Alshieban S et al HLAgenotype and response to nivolumab therapy in relapsed refractory primary mediastinal Bcell lymphoma Curr Res Transl Med Zinzani PL Santoro A Gritti G et alNivolumab combined with brentuximabvedotin forrelapsedrefractory primarymediastinal large Bcell lymphoma efï¬cacyand safety from the Phase II CheckMate Study J Clin Oncol Postow MA Sidlow R and Hellmann MDImmunerelated adverse events associatedwith immune checkpoint blockade N EnglJ Med Zinzani PL Ribrag V Moskowitz CH et alSafety and tolerability of pembrolizumab inpatients with relapsedrefractory primarylymphoma Bloodmediastinal large Bcell Jones K Vari F Keane C et al SerumCD163 and TARC as disease response biomarkers in classical Hodgkin lymphomaClin Cancer Res for Cheson BD Fisher RI Barrington SF et alinitial evaluationRecommendationsstagingofHodgkin and nonHodgkin lymphoma theLugano classiï¬cation J Clin Oncol assessmentandresponse 0c' | 2 |
"These studies demonstrate that RRM1 could be a predictive marker of the response to gemcitabine-based chemotherapy in patients with NSCLC [22]. The present study also showed that the DCR was higher in RRM1-positive patients that received docetaxel or vinorelbine rather than gemcitabine-based therapy. In addition docetaxel and vinorelbine each showed a longer PFS than gemcitabine-based therapy. Simon et al. used RRM1 and ERCC1 as molecular determinants and found that RRM1- and ERCC1-tailored selection of first-line therapy could improve response overall survival (OS) and PFS over standard treatments in patients with NSCLC [23]. These studies suggest that responses to cytotoxic chemotherapy vary greatly in patients with NSCLC and individualized therapy based on RRM1 expression may help improve the efficacy of chemotherapeutic agents [24]. Our research was performed retrospectively and this is the major limitation of the study. However the current results provide new information and further insight that can assist clinicians in selecting appropriate and individualized chemotherapy for patients with NSCLC based on RRM1 expression. Several molecular markers have been used as predictive markers of the response to chemotherapy in NSCLC patients. ERCC1 has been used for the prediction of platinum sensitivity in the treatment of NSCLC [6][8]. Park et al. analyzed 217 patients with NSCLC who had received gemcitabine- or taxane-based chemotherapy and found that taxane was associated with a higher response than gemcitabine treatment in patients with EGFR mutations [9]. Another study found that low thymidylate synthase (TS) expression is significantly associated with better clinical outcomes in non-squamous NSCLC patients who were treated with pemetrexed-based chemotherapy [25]. Therefore more prospectively designed studies with combined detection of these markers (RRM1 ERCC1 EGFR and TS) will provide valuable information that will ultimately be used to determine preferable therapeutic approaches for individual patients with NSCLC. In the results of this study suggest that negative RRM1 expression in advanced NSCLC is associated with a higher response rate to gemcitabine-based chemotherapy. Moreover RRM1 may be used as a predictive marker for conventional chemotherapy regimens involving gemcitabine docetaxel and vinorelbine. Additional prospective studies are needed to evaluate the effect of RRM1 expression on the response to various chemotherapeutic regimens in patients with NSCLC. References 1 SchillerJH HarringtonD BelaniCP LangerC SandlerA et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med346: 929811784875 2 PfisterDG JohnsonDH AzzoliCG SauseW SmithTJ et al (2003) American society of clinical oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol22: 33035314691125 3 KellyK CrowleyJ BunnPAJr PresantCA GrevstadPK et al (2001) Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non-small-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol19: 3210321811432888 4 ScagliottiGV De MarinisF RinaldiM Crin²L GridelliC et al (2002) Phase III randomized trial comparing three platinum-based doublets in advanced non small cell lung cancer. J Clin Oncol20: 4285429112409326 5 SchillerJH HarringtonD BelaniCP LangerC SandlerA et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med346: 929811784875 6 RothJA CarlsonJJ (2011) Prognostic role of ERCC1 in advanced non-small-cell lung cancer: a systematic review and meta-analysis. Clin Lung Cancer6: 39340121723790 7 PapadakiC" | 1 |
emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic beneï¬t until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic proï¬les ofHCC contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufï¬ciently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPaciï¬c trial promoted the approvalof sorafenib as ï¬rstline targeted therapy for advanced HCC5ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel ï¬rstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predeï¬ned primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asï¬rstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPaciï¬c trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21 Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as ï¬rstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24 andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical beneï¬t of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer beneï¬ts when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbeneï¬t in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these ï¬ndings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42 which was supported by theï¬ndings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redeï¬ned PFS not conventional PFS buttime until unTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival beneï¬t compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival beneï¬t compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modiï¬cation ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classiï¬cation BCLCguidelines and although the results are difï¬cult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also beneï¬ted fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratiï¬cation would enable the identiï¬cation of thosepatients who will beneï¬t most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second ï¬rstlinetargeted agent for HCC emerged Lenvatinib was approved for theï¬rstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from realworld conditions conï¬rmed the efï¬cacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufï¬cient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88 With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneï¬cial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop ï¬rstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy ï¬nallyconï¬rmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival beneï¬tthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was conï¬rmed by Yoo et al99 in aretrospective study of safety and efï¬cacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefï¬cacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identiï¬ed regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconï¬rms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs and the incrementalcosteffectiveness ratio ICER mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112 compared with regorafenib QALY and ICER RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival beneï¬t in patientswith AFP of ngml or higher118 This was later conï¬rmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the ï¬rstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent ï¬ndingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and conï¬rms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefï¬cacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efï¬cacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efï¬cacy and safety in advanced HCCs as systemicï¬rstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespeciï¬ed statistical signiï¬cance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as ï¬rstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbeneï¬t regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on ï¬rstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efï¬cacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the ï¬rst combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136 Such results were conï¬rmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ammation and rheumatic and musculoskeletal phenotypesincluding ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a ï¬rstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145 where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153 Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as ï¬rstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as ï¬rst155 or secondline inadvanced HCCs156 but unfortunately the heterogeneousnature of the results precludes ï¬rm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufï¬ciently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentiï¬erPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentiï¬erPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164 especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyproï¬le169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneï¬cially employed to produce a therapeutic dualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efï¬cacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR ï¬broblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classiï¬cation of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassiï¬cationG1G6S1S3Msig iC1iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and lowMS1 SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell proï¬lingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespeciï¬ed criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identiï¬ed to be able to predict the effect of or responseto treatment with sorafenib172 Although the molecularclassiï¬cation of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul | 2 |
Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earths species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleeps quotidian normalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brains clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patients desired sleep time or augmenting factors that entrainthe bodys clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon siesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period Catchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances morning larks latephase delays night owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airways soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisordersspecifically sleep walkingalso occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women in males and in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economos encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while asleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in the loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleepor its encephalopathic analogmay affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i | 2 |
"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e | 2 |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | 0 |
"carcinogenesis is a process of somatic evolution previous models of stem and transient amplifying cells in epithelial proliferating units like colonic crypts showed that intermediate numbers of stem cells in a crypt should optimally prevent progression to cancer if a stem cell population is too small it is easy for a mutator mutation to drift to fixation if it is too large it is easy for selection to drive cell fitness enhancing carcinogenic mutations to fixation here we show that a multiscale microsimulation that captures both withincrypt and betweencrypt evolutionary dynamics leads to a different epithelial tissues are metapopulations of crypts we measured time to initiation of a neoplasm implemented as inactivation of both alleles of a tumor suppressor gene in our model time to initiation is dependent on the spread of mutator clones in the crypts the proportion of selectively beneficial and deleterious mutations in somatic cells is unknown and so was explored with a parameter when the majority of nonneutral mutations are deleterious the fitness of mutator clones tends to decline when crypts are maintained by few stem cells intercrypt competition tends to remove crypts with fixed mutators when there are many stem cells within a crypt there is virtually no crypt turnover but mutator clones are suppressed by withincrypt competition if the majority of nonneutral mutations are beneficial to the clone then these results are reversed and intermediatesized crypts provide the most protection against initiation these results highlight the need to understand the dynamics of turnover and the mechanisms that control homeostasis both at the level of stem cells within proliferative units and at the tissue level of competing proliferative units determining the distribution of fitness effects of somatic mutations will also be crucial to understanding the dynamics of tumor initiation and progressionk e y w o r d scancer evolution initiation metapopulation dynamics neoplastic progression simulationmajor findings competition between epithelial units such as colonic crypts tends to suppress initiation of neoplasms by suppressing mutator clones this suppression of initiation is enhanced when crypts have few stem cells and so are likely to go extinct due to stochastic fluctuations in stem cell numbers this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors evolutionary applications published by john wiley sons ltdevolutionary applications wileyonlinelibrarycom eva 2003 2003 0c 2003 2003 2003 2002 2003 2003introductionthe anization of a population into spatially distinct subpopulations can have a dramatic effect on the evolution of that metapopulation hanski gaggiotti this has implications for both the evolution of anisms and for the effect of tissue architecture on somatic evolution and tissue health in multicellular anisms epithelia are typically divided into subpopulations of tissue stem cells along with the transient amplifying cells and differentiated cells that they produce these subpopulations go by different names in different tissues such as crypts in the intestine or more generally epithelial proliferative units cairns first recognized that the division of stem cells into subpopulations such as crypts acts as a tumor suppressor cairns a mutant stem cell with a reproductive or survival advantage may take over a crypt but is generally constrained from expanding beyond that subpopulation unless it breaches the crypt via a process known as crypt fission which tends to duplicate the mutant crypt cell population however by establishing a population size barrier the mutant clone has to overcome the subpopulation structure of the tissue limits the probability that that clone will acquire further carcinogenic mutations yet clones of mutant stem cells can be observed at scales spanning many crypt diameters especially in compromised tissues such as ulcerative colitis and barrett's esophagus maley salk a fundamental question is therefore how the cryptlevel metapopulation dynamics affect the accumulation of somatic mutations during carcinogenesishere we explore the evolutionary dynamics of mutant stem populations that lead to tumor initiation that is the breach of the crypt barrier allowing clonal expansions of crypts across a tissue as well as of mutant stem cells within and out of a crypt while there may be multiple pathways to tumor initiation it has been shown that the inactivation of a single tumor suppressor gene tsg such as the adenomatous polyposis coli apc gene in colon is sufficient to abrogate crypt homeostasis leading to the formation of aberrant crypts and nascent adenomas humphries wright using an agentbased microsimulation model for both stem cell turnover within a crypt and for the cryptpopulation tissuelevel dynamics we study the role of pretumor evolution in tumor initiation this exploration allows for the selection of mutant crypts across the tissue prior to the inactivation of the tumor suppressor genea form of premalignant field cancerizationwhile the stem cell in which the tumor suppressor is inactivated can proliferate beyond the limit of a single crypt due to crypt bifurcationthe evolution of somatic cells is a complex multiscale process depending on the nature of somatic mutations which may either increase or decrease cell fitness stem cell divisions and differentiation or apoptosis as well as subpopulation eg crypt division and extinction rates there is considerable evidence that carcinogenesis involves both an increase in the rate of epigenetic lesions bielas loeb rubin true loeb breivik ji king weisenberger et al and expansions of clones with a relative fitness advantage over their competitor cells cannataro gaffney townsend maley pepper findlay kassen spencer maley vermeulen williams however it continues to be unclear whether the mutator phenotype is a preinitiation phenomenon in carcinogenesis or is more likely to occur during tumor progression in barrett's esophagus another cryptstructured precancer we found evidence that genomic instability precedes genome doubling and transformation martinez the frequency of deleterious versus beneficial mutations in somatic cells is also unknown though the large number of genes in metazoans devoted to differentiation apoptosis and cell cycle control suggests that the frequency of deleterious mutations may be lower in somatic evolution than anismal evolution rajagopalan nowak vogelstein lengauer recent analysis of somatic mutations in cancer found no evidence of purifying selection except in a few essential genes and strong evidence of positive selection with large selective effects williams suggesting that beneficial mutations are more common than deleterious mutations in somatic evolution martincorena although a definitive answer to these questions can only come from further experimental data a theoretical exploration that recognizes the roles of metapopulation dynamics the mutator phenotype and the proportion of deleterious to advantageous mutations in the process of tumor initiation is called for such an exploration will help the identification of factors that drive the tumor initiation processour model integrates previous efforts to characterize the stem cell dynamics within a crypt cannataro mckinley st mary cannataro mckinley st mary frank iwasa nowak komarova komarova cheng loeffler birke winton potten meineke potten loeffler michor frank may iwasa nowak nowak et al pepper sprouffske maley with models of the dynamics of crypt populations cannataro et al chao eck brash maley luebeck kostadinov maley kuhner loeffler bratke paulus li potten totafurno bjerknes cheng mathematical studies of the stem cell population in the crypt niche suggest that epigenetic alterations that increase the rate of genetic lesions mutator mutations and reduce the fitness of stem cells will tend to drift to fixation if the stem cell population is small whereas carcinogenic mutations that increase the proliferation or survival of a stem cell will tend to spread if the stem cell population is large cannataro komarova michor assuming that most nonneutral somatic mutations are deleterious the accumulation of deleterious mutations may lead to senescence of the intestine over time cannataro however competition between crypts of different fitnesses may significantly change the dynamics of the establishment of a mutator clone through a metapopulation dynamic our in silico experiments suggest that there may have been selection at the level of the anism to minimize the number of stem cells within each subpopulation of birtwell 0c 2003 2002 2003 2003its structured epithelium so as to reduce the probability of tumor suppressor gene inactivation and the initiation of carcinogenesisproliferation rate decreased stem cell loss and caused the mutator phenotypethe following equations and assumptions govern the model 2003 2003methodswe implemented a multiscale model of epithelial tissue architecture with stem cells subdivided into crypts under homeostatic control we examined the time required until the two alleles of a tumor suppressor gene tsg were inactivated in at least one stem cell to represent tumor initiation the model was run at least times for every parameter setting crypts were arranged in a flat hexagonal tissue similar to that observed in colon and contained a population of stem cells as well as an implicitly modeled transient amplifying compartment stem cells divided both symmetrically and asymmetrically symmetric division resulted in two daughter stem cells each having the opportunity during the division event synthesis to acquire a mutation asymmetric division did not result in any new stem cells but did provide an opportunity for stem cell mutation stem cell loss due to cell death or differentiation and stem cell gain due to division events were modeled as a stochastic birthdeath process with parameters that were functions of the stem cell fitness and of homeostatic feedback effects in response to deviations of the crypt cell population from its normal target level equations and a flow chart of the model algorithm is shown in figure s1homeostasis operated at two spatial scales within a crypt if the stem cell population dropped below the target level stem cell division rates increased by a parameterized amount equation if the population rose above the target level stem cell loss rates increased equation the level of homeostatic feedback was proportional to the degree of deviation away from the target equilibrium level equations and we also introduced a mechanism for homeostasis on the hexagonal lattice of crypts if all the stem cells in a crypt died the inhibition on stem cell population growth was released from the neighboring crypts when the stem cell population of a neighbor reached twice the equilibrium level we modeled crypt bifurcation by allocating half of its stem cells to a new crypt in the location of the dead neighborwe included both beneficial mutations that increased the division probability or the survival probability of stem cells as well as deleterious mutations that decreased them these can accumulate indefinitely and affect fitness multiplicatively equation we also implemented a genetic instability mutation that increased the clone's mutation rate 100fold bielas herr kennedy knowels schultz preston ji king the frequency of each mutation type those that changed the cell's fitness the proportion of nonneutral mutations that were deleterious as well as the rate of tsg inactivation was set by parameters each mutation affected proliferation survival or mutation rate parameters by a constant factor half of the deleterious mutations decreased stem cell proliferation and half decreased their survival increased cell loss for the beneficial mutations increased the cell's 2003 2003equationsequation time to stem cell losslet t be a random exponential deviate with distribution function fr t and rate parameter r the time to cell loss due to apoptosis or differentiation is the minimum of the time to cell loss due to background cell death or differentiation and the time to cell loss due to crypt feedbacktcell_loss min 01t1 ¼ fbackground_loss t2 ¼ ffeedback_loss 01 equation homeostatic crypt feedback by differentiationwhen the stem cell population within a crypt expands beyond the homeostatic target level kcrypt\\_size the crypt provides homeostatic feedback via a change in the rate of stem cell loss with a rate parameter equal to the base stem cell loss rate multiplied by the crypt feedback multiplier the crypt feedback rate multiplier is used to calculate the time to stem cell loss due to crypt homeostatic feedback the crypt feedback multiplier is equal to raised to the nth power where n is the excess number of stem cells above the crypt size divided by the kcrypt\\_deviation parameter here kcrypt\\_deviation and kcrypt\\_size is a parameter that we varied across experimentsrfeedback\\_cell_loss rbase_cell_loss2max0ncellskcrypt_sizekcrypt_deviationequation homeostatic crypt feedback by proliferationwhen the stem cell population of a crypt drops below kcrypt\\_size the division rate of the remaining stem cells is increased by a factor that depends on the difference between the current number of stem cells ncellsand kcrypt\\_sizerfeedback_division rbase_division2max0kcrypt_sizencellskcrypt_deviationequation fitness mutation effectskï¬tness is a constant factor representing the effect of a single beneficial mutation on fitness as a first approximation we assume that there are many possible mutations that increase and decrease the fitness of a somatic clone by approximately the same amount and so the effect of n beneficial mutations nbeneï¬cial on stem cell fitness is the constant fitness effect raised to the nth power the effect of n deleterious mutations ndeleterious of small effect is just the inverse of kï¬tness raised to the nth power there is a separate mï¬tness calculated for the division probability and the survival probability of a cell because beneficial and deleterious mutations may affect either of those probabilitiesmï¬tness 01kï¬tness 01nbeneï¬cial 03 kï¬tness 03ndeleteriousbirtwell 0c 2003 2003 2003 2002f i g u r e 2003plots of cumulative hazard functions using the kaplanmeier estimator the tissue was x crypts with stem cells per crypt in panels a through d each colored line represents the function for a specific proportion of deleterious mutations a baseline experiment with default parameter values table b mutation rate reduced to 01x of baseline c mutator phenotype reduced to 01x of baseline d each colored line represents a different number of cells per crypt all proportions of deleterious mutations were included 2003 2003assumptionscrypts consist of stem cells and of transient amplifying cellscrypt density is fixed that is the tissue contains a fixed number of crypts arranged on a hexagonal griddrops below the target level the division rate of each stem cell in the crypt is increased when the number of stem cells grows above the target level the cell loss rate of each stem cell in the crypt is increasedcrypts divide to fill vacant slots left by adjacent crypts that have the number of cells in a crypt transient amplifying compartment gone extinct due to loss of the constituent stem cellsis fixedcrypts attempt to maintain a stable population of stem cells through homeostatic feedback when the number of stem cells the extinction of an adjacent crypt suppresses the homeostatic apoptotic signals allowing the stem cell populations in neighboring crypts to expand once that extinct crypt is replaced the normal birtwell 0cta b l e 2003baseline simulation parameterssimulationmaximum simulation duration in daysstem celldivision rate rbase\\_divisionratio of asymmetric divisions to symmetric divisionsstem cell loss rate rbase\\_cell\\_lossmutation rate per stem cell divisionmutation rate maximumtumor suppressor gene mutation ratetumor suppressor gene mutation rate maximumnumber of transient amplifying cells associated with each stem cellcell division minimum time in dayscell loss minimum time in dayscrypttarget equilibrium level of number of stem cells in a crypt kcrypt\\_sizestandard deviation from equilibrium level the crypt uses this value to determine its level of effect on the cell loss and division rates of the stem cells kcrypt\\_deviationbifurcation threshold factor below which a crypt will not bifurcatecrypt cell loss effect multipliercrypt division effect multipliermultiplier to the division effect for each dead neighbor cryptcanceruncontrolled cell proliferation threshold if a crypt has this threshold times the equilibrium number of stem cells it is considered to be experiencing uncontrolled stem cellnumber of tumor suppressor gene hits that mean cancermutation 2003 2002 2003 2003 years x x x also tested x x default varied from percent of nonneutral mutations that are deleteriousthe factor affecting the loss rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the division rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the cell loss rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the division rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the mutation rate of the stem cell from a mutator mutation also tested homeostatic controls on stem cell numbers of neighboring crypts are restoredcrypt division is triggered by an expansion of the stem cell population of a crypt to twice its homeostatic level as hypothesized by garcia park novelli and wright as long as there is an empty slot adjacent to the enlarged crypta stochastic birthdeath process governs the scheduling of division and cell loss eventsfitness mutations affect in a multiplicative fashion the rate parameters of the birthdeath processthere is a single mutator phenotype that requires only a single mutator mutation additional mutator mutations have no effect on the mutation ratethe loss of the first allele of the tsg has no effect on stem cell fitness 2003 2003results 2003 2003tsg inactivation depends on the emergence of a mutatorat baseline for comparison our tissue was a 5x5 hexagonal lattice of crypts each crypt having stem cells stem cell loss and symmetric division rates were balanced mutations were acquired stochastically with probabilities defined by proportions starting with deleterious mutations beneficial mutations and mutator mutations and ranging in increments to deleterious beneficial and mutator beneficial versus mutator the incidence of tsg inactivation decreased as the proportion of deleterious mutations increased figure 1a table birtwell 0c 2003 2003 2003 2002reducing the base mutation rate to of baseline we observed a marked decrease in tsg inactivation figure 1b as expected similarly reducing the effect of the mutator mutation to 10x the baseline mutation rate instead of 100x produced a significant decrease in tsg inactivation figure 1c we found that the vast majority of tsg inactivation occurred in stem cells that had previously acquired the mutator phenotype figure s2 this assumes that the mutator phenotype can be caused by a single mutation that is otherwise neutral eg overexpression of dna polymerase beta canitrot or a dominantnegative mutation in p53 de vries though this assumption is easily relaxed 2003 2003proportion of deleterious mutations negatively correlates with tsg inactivationnot surprisingly we found that the proportion of deleterious mutations and the incidence of tsg inactivation were negatively correlated figure cell divisions per time remained roughly constant across all proportions of mutations however as the proportion of deleterious mutations decreased the cost of being a mutator also decreased because it accumulated less mutational burden of deterious mutations this resulted in an increased emergence of crypts with fixed mutator stem cell populations conversely across all experiments we observed progressively less tsg inactivation as the proportion of deleterious mutations approached our maximum of turnover levels we observed a reduction in the average number of stem cells per crypt figure s3 the implemented homeostatic control was unable to maintain the target stem cell population size in the face of high turnover rates essentially there is a lag between depletion of the stem cell pool due to cell death and differentiation and replenishment provided by an increase in stem cell division rates with higher levels of cell loss the simulated crypts spend more time further below the target homeostatic number of stem cells as a result there were fewer total stem cells in the simulation and therefore fewer mutations per time allowing less chance for mutator acquisition and tsg inactivation this may or may not be realistic second as the turnover level increased above our baseline the number of mutator crypts present in the tissue at any given time decreased figure since increased turnover should lead to increased opportunities for mutator mutations to arise the decline in mutator crypts was a surprise however the loss of mutator crypts is due to intercrypt competition as described below increased turnover led to increased stochastic fluctuations in stem cell numbers and thereby increased crypt extinction events the reduction in stem cell numbers and mutator crypts combined to produce a reduction in the overall incidence of tsg inactivation as turnover rates increased above baseline if in reality homeostatic control of stem cell numbers prevents increased crypt extinctions with increased stem cell turnover this result would likely not hold however all things being equal increased cell turnover would be expected to increase stem cell number fluctuations 2003 2003increased stem cell turnover initially increased and then decreased tsg inactivationwe modulated stem cell turnover by varying cell loss and symmetric division rates in unison at lower turnover levels we found that increased cell turnover increased tsg inactivation however at turnover levels 2x and 5x our baseline level the incidence of tsg inactivation declined figure due to two factors first at higher 2003 2003the number of stem cells per crypt had a varying effect on tsg inactivationin general fewer stem cells per crypt reduced the rate of tsg inactivation even though the total number of stem cells in the tissue was held constant figure 1d however there is a tradeoff between tsg inactivation and tissue death at very low stem cells per crypt with only one stem cell per crypt there is no opportunity for homeostatic signals within a crypt to compensate for stem cell loss in f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed each bar corresponds to a specific proportion of deleterious mutations the proportion of mutator crypts correlates with the risk of tumor initiation as seen in figure birtwell 0cbase cell loss divison rate base cell loss divison rate 2003 2002 2003 2003base cell loss divison rate base cell loss divison rate f i g u r e 2003the effect of changes in stem cell turnover plots of cumulative hazard functions using the kaplanmeier estimator where each colored line represents the function for a specific proportion of deleterious mutations the baseline division and stem cell loss rates were as seen in figure initially turnover correlated positively with the risk of tumor initiation however at higher turnover rates the risk of tumor initiation decreased due to a reduction in the overall number of living stem cells and decreased incidence of mutator cryptsour model tissues with one stem cell per crypt were mostly unviable and died out before tsg inactivation or the predetermined simulation end timeat and cells per crypt we observed a reduction in the incidence of tsg inactivation figure 1d at all but the lowest proportions of deleterious mutations as was predicted by models of the crypt stem cell niche of single crypts komarova michor figure s4 as in other experiments along with a reduction in the incidence of tsg inactivation the frequency of fixed mutator crypts was reduced as well this shows that selection against mutator cells increases as the number of stem cells increases above some threshold in our model as long as the majority of nonneutral mutations are deleterious supporting the s by michor and komarova komarova michor et al when the stem cell populations are large it is very unlikely that a crypt will go extinct and so there is no intercrypt competition in this case the metapopulation dynamics are reduced to the single crypt dynamicsthe increased risk of tsg inactivation associated with increased stem cells per crypt appeared to plateau after approximately stem cells per crypt the total number of cells in the tissue remained constant as did the total stem cell divisions per time figure s5 the average number of mutations per time increased through stem cells per crypt but then reached a temporary plateau figure there was no statistically significant difference between the average number of mutations per time in the and stem cells per crypt cases as the number of stem cells per crypt increased beyond the average mutations per time decreased except when the proportion of deleterious mutations was the incidence of mutator crypts followed a similar trend figure birtwell 0c 2003 2003 2003 2002f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed as a function of turnover rate each bar corresponds to a specific proportion of deleterious mutations as turnover increased mutator crypts became more rare at higher proportions of deleterious mutationsf i g u r e 2003number of mutations per unit time as a function of cells per crypt and proportion of deleterious mutations where each bar represents a specific proportion of deleterious mutations at most proportions of deleterious mutations mutations per time peaked around stem cells per crypt at deleterious mutations mutations per time reached a minimum at cells per crypt and increased as the cells per crypt grew past we have chosen to explore the case where the total number of stem cells is kept constant assuming that a certain number of selfrenewing tissue stem cells might be required to maintain an epithelial tissue an alternative view is that a fixed number of epithelial units like the crypts might be required to maintain a tissue and that the number of stem cells per crypt could vary by changing the number of differentiated cells produced by each stem cell in this case the risk of tsg inactivation continues to increase with increasing number of stem cells per crypt figure s4 because we held the number of crypts constant but changed the number of cells per crypt in this case the total number of cells in this simulation also changed leading to a large evolving population size of stem cells and thus an increased chance for at least one cell to inactivate both alleles of the tsg 2003 2003partitioning the tissue into crypts imposed a metapopulation dynamicfitness levels measured by the difference between division and loss rates were greater in tissues with smaller crypts even as the total number of stem cells remained constant figure 6a counts of crypt births and crypt life span measurements showed that there was more crypt turnover in smaller crypts figures s6 and s7 this crypt turnover provided an opportunity for fitter phenotypes to spread more easily across the tissue crypt fitness peaked at cells per crypt where there was the most crypt turnover and bottomed out at and cells per crypt after cells per crypt we observed no crypt death and therefore no turnover crypt fitness began to increase again at and cells per crypt through natural selection of stem cells within larger crypts however fitness levels did not increase to the levels seen at cells per cryptthe metapopulation dynamic was observed in the clonal expansion of mutator crypts across the tissue we considered two crypts to have mutator phenotype agreement if they both have a fixed mutator mutation or neither has a fixed mutator we calculated mutator crypt agreement across spatial distance and found that overall agreement decreased as the cells per crypt increased figure 6b further at and cells per crypt closer crypts had increased mutator agreement suggesting clonal expansion of mutator crypts conversely at cells per crypt and above we found that spatial distance correlated less well with mutator agreement indicating that birtwell 0c 2003 2002 2003 2003f i g u r e 2003a crypt fitness measured by the difference between division and cell loss rates across cells per crypt and proportion of deleterious mutations the total number of stem cells remained constant across these experiments the crypt turnover at and cells per crypt allowed fit clones to spread across the tissue resulting in increased overall fitness b mutator agreement by distance class two crypts were in mutator agreement if they both had fixed mutator mutations or neither did overall agreement was higher in the smaller crypts suggesting that mutator clones were able to spread across the tissuewhen there was less crypt turnover mutator crypts arose de novo rather than through cryptlevel clonal expansion 2003 2003discussionin the colon the development of adenomatous polyps frequently involves the inactivation of the apc gatekeeper gene a member of the wntsignaling pathway which represses proliferation and facilitates orderly cell differentiation in the luminal part of the crypts barker goss groden as long as this gatekeeper gene is active mutant stem cell progeny with neoplastic potential is likely eliminated from the crypt and clonal expansion thus averted however when the gatekeeper gene is inactivated the brakes on mutant stem proliferation are removed and mutant cell progeny may undergo focal clonal expansion therefore we asked the question what are the factors that determine the risk of a tsggatekeeper inactivation leading to tumor initiation in a compartmentalized tissue a metapopulationour microsimulations indicate that the base mutation rate figure 1b and the increase in that rate for a mutator clone figure 1c are the main driving forces behind tsg inactivation and thus the initiation of carcinogenesis the proportion of deleterious mutations is important for its effect on selection of the mutator clone we find that if most mutations are assumed to be deleterious then a mutator clone quickly accumulates a large genetic load of deleterious mutations and tends to be driven to extinction in competition with nonmutator cells however if mutations are more likely to be beneficial then a mutator clone can spread more easily which leads to the early inactivation of both alleles of the tumor suppressor gene figure 1a similarly increasing turnover of the stem cells effectively increases the mutation rate and consequently the rate of initiation however when turnover is very high the homeostatic feedback signals cannot maintain the target number of stem cells and so the total stem cell population of the epithelium decreases which in turn decreases the chances for initiation figure since the rate of tsg inactivation is trivially relat | 0 |
Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso and Lucia Altucci Department of Precision Medicine University of Campania Luigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespeciï¬cdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneï¬cial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientiï¬c interest focusing on theidentiï¬cation of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol ï¬setin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modiï¬cations are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modiï¬cations Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT are enzymes classiï¬ed as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identiï¬ed inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespeciï¬c degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair OCallaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context speciï¬city BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deï¬ciency DAngelo Bioactive compounds in the diet can act as antioxidantand antiammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and ï¬setin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health beneï¬ts Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneï¬cial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneï¬cial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and antiammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin ï¬setin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classiï¬cation of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid proï¬le antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidproï¬le antioxidant antiammatoryAntioxidant antiammatoryAnticancer antioxidantantiammatoryAnticancercardiovascularpreventive antiammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesDAngelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonï¬avonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has antiammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger antiammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits proammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modiï¬es SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneï¬cial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneï¬cial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneï¬cial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress ï¬brosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together theseï¬ndings highlight the beneï¬cial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneï¬cialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been conï¬rmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective beneï¬t through improvement ofendothelial functionammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well deï¬ned Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientiï¬c research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efï¬cacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimers disease Parkinsonsdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood ï¬ow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneï¬cial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe ï¬avonoid polyphenol quercetin Que ²²pentahydroxyï¬avone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and antiammatory properties Nabavi Wu In recent years the scientiï¬c community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inï¬ammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inï¬ammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inï¬ammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the bodysantioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseasesammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable conï¬guration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cells antioxidant defense mechanisms moderateoxidative stress can in fact increase the cells antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These ï¬ndings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientiï¬c community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere signiï¬cantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpeciï¬cally in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats conï¬rming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these ï¬ndings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneï¬cial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ammatoryconditions is reported in Table Speciï¬cally a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and antiammatory capacities this ï¬avonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary ï¬brosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclariï¬cation Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneï¬cial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer antiammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneï¬cial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInï¬ammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efï¬cacy of Provex CV Supplement toReduce Inï¬ammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an antiammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a signiï¬cant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These ï¬ndings were also conï¬rmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Speciï¬cally BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and antiammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ammation signaling Speciï¬cally BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is | 2 |
craigpattersontelecaretoj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per loncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatientssaï¬f al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset ogorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseandimplicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 commonduringadversetheoutbreakconï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | 0 |
"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2 chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10 li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p Ë p Ë d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorers instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturers protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at °c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells à were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired students ttest and a p value Ë was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2ce the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3be catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone¢ homogenous caspase37 assay p Ë p Ë d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h be the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p Ë p Ëerianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5ac interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6bd promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7ac the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 ac after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p Ë d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p Ëprotein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18 although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting bd sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p Ë p Ësubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p Ë d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10 consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28 to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a selfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31 anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced | 0 |
"(P>10?7 10?7>P>10?10 10?10>P>10?15 10?15>P>10?20 P<10?20). A SNP is determined to be related with a regulatory region if the SNP or any LD-related SNP (r2 ? 0.8) resides in the ChIP-Seq peaks of the regulatory regions. Regulatory elements include CTCF binding sites DNaseI hypersensitive sites and histone marks from small airway epithelial cells (SAEC) from ENCODE and human alveolar epithelial cells (hAEC) from our laboratory. For each p-value category we calculated the proportions of cis-meQTL SNPs related with regulatory regions. The figures show that the proportions of cis-meQTL SNPs related with regulatory regions increase with the significance of meQTL associations except for the repressive mark H3K27me3. DNA methylation regional associations for lung cancer GWAS SNPs in subjects of European ancestry (a b f and g) Symbols represent the association between established lung cancer GWAS genetic loci in four regions and methylation levels in nearby CpG probes. Y-coordinate P-value for association; x-coordinate genomic location. For each SNP the red solid circle or square represents the methylation probe with the strongest association whereas other methylation probes are colored on the basis of their correlation (measured as r2) to the most-associated probe. For the most-associated probes the P-values in EAGLE discovery set (n=210) and TCGA lung replication data (n=65) are shown. SNP locations are marked by a blue triangle. (ce and hj) show the associations between genotypes and methylation levels of the most associated CpG probes. The box plots show the distribution of the methylation levels in each genotype category with error bars representing the 25% and 75% quantiles. Enrichment of cis-meQTL SNPs for lung cancer risk Analysis based on NCI lung cancer GWAS data (5739 cases and 5848 controls). P-values were produced based on 10000 permutations. AD SQ and SC represent adenocarcinoma squamous cell carcinoma and small cell carcinoma. (a) Enrichment was tested using all cis-meQTL SNPs after LD pruning. (b and c) Strong enrichments were observed for cis-meQTL SNP associated with CpG probes annotated to north shores (b) and gene body (c) regions for SQ. (d) The enrichment in (c) was driven by the cis-meQTLs SNPs impacting CpG probes in non-CpG islands. (e) The enrichment in (d) is driven by the SNPs (or their LD SNPs with r2 > 0.95) overlapping with CTCF binding sites or H3K27me3 mark regions. Replication of EAGLE lung meQTLs in TCGA histologically normal tissue samples. Tissue N All cis associations in EAGLE(34304 associations P<4.010?5) Strong cis associations in EAGLE(12083 associations P<1.010?10) All trans associations in EAGLE(585 associations P<2.510?10) Consistentdirection FDR<0.05 Consistentdirection FDR<0.05 Consistentdirection FDR<0.05 Lung 65 32128 (93.7%) 22441 (65.4%) 11250 (99.3%) 11229 (92.9%) 556 (95.2%) 467 (79.8%) Breast 87 30391 (88.6%) 18762 (54.7%) 11640 (96.3%) 9987 (82.7%) 561 (96.1%) 488 (83.4%) Kidney 142 30975 (90.3%) 23984 (70.0%) 11634 (96.3%) 10783 (89.2%) 558 (95.5%) 506 (86.4%) N is the sample size in replication studies. FDR was calculated based on single-sided p-values. Chromatin marks are enriched on meQTL SNPs. control cis only trans only cis + trans cell line mark proportion proportion fold change proportion fold change proportion fold change SAEC CTCF 11.8% 35.3% 3.0 29.6% 2.5 45.4% 3.8 DnaseI 25.4% 54.0% 2.1 45.8% 1.8 59.6% 2.3 H3K27me3 20.4% 34.1% 1.7 25.4% 1.2 42.9% 2.1 H3K4me3 4.8% 29.7% 6.2 18.0% 3.8 39.9% 8.3 H3K36m3 13.4% 36.8% 2.7 22.8% 1.7 45.4% 3.4 HAEC H3K27me3 17.5% 25.3% 1.4 15.6% 0.9 33.2% 1.9 H3K4me3 7.6% 37.0% 4.9 25.0% 3.3 54.9% 7.2 H3K9-14Ac 17.3% 47.6% 2.8 32.3% 1.9 65.3% 3.8 meQTL SNPs were enriched in chromatin marks including CTCF binding sites DNaseI hypersensitive sites and histone marks from small airway epithelial cells (SAEC) from ENCODE and human alveolar epithelial cells (hAEC) from our laboratory. " | 1 |
collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identiï¬ed in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the antiammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe ï¬nal protein what is more its only amino acid posttranslational modiï¬cation is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains ï¬ve exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by ï¬broblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myoï¬broblasts and locates in the sites ofcollagen matrix deposition in micethe ï¬rst exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ ] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the ï¬rst enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ ]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossiï¬cation and other 0cmediators of ammationcthrc1tgfð½tð½r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1ð½ð¼ð¼ð½integrincxrc4tð½r1p ppsmad23smad4cthrc1dvlpdvlaxinapcð½cateninck1ð¼gsk3dpagt1stabilizationð½cateninpkcð¿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhifð¼vegfecminvasionpsmad23smad4ð½catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespeciï¬c tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury uencing ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver ï¬brosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespeciï¬c the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied []current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the destructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasiondestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 []the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic ï¬broblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ ]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to ï¬bronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation []erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and ï¬bronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac ï¬broblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it brieï¬y cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was ï¬rst discovered more than a decad | 0 |
Understanding of the RelevantRole of LINE1 Retrotransposition inHuman Disease and ImmuneModulationXiao Zhang12 Rui Zhang12 and Jinpu Yu12 Cancer Molecular Diagnostics Core Tianjin Medical University Cancer Institute Hospital National Clinical ResearchCenter of Caner Key Laboratory of Cancer Prevention and Therapy Key Laboratory of Cancer Immunology and BiotherapyTianjin China Tianjins Clinical Research Center for Cancer Tianjin ChinaLong interspersed nuclear element1 LINE1 retrotransposition is a major hallmark ofcancer accompanied by global chromosomal instability genomic instability and geneticheterogeneity and has become one indicator for the occurrence development andpoor prognosis of many diseases LINE1 also modulates the immune system andaffects the immune microenvironment in a variety of ways Aberrant expression of LINE retrotransposon can provide strong stimuli for an innate immune response activatethe immune system and induce autoimmunity and ammation Therefore inhibitionthe activity of LINE1 has become a potential treatment strategy for various diseasesIn this review we discussed the components and regulatory mechanisms involved withLINE1 its correlations with disease and immunity and multiple inhibitors of LINE1providing a new understanding of LINE1Keywords retrotransposons LINE1 regulatory mechanisms cancer immune inhibitorINTRODUCTIONLong interspersed nuclear elements LINEs are the only autonomous and active retrotransposonswhich include LINE1 LINE2 and LINE3 Cordaux and Batzer de Koning Also of LINE2 and LINE3 sequences in the human genome are as a truncated molecularfossil Doxiadis Ardeljan LINE1 retrotransposons are one of the mostabundant and eï¬ective classes of mobile DNAs that account for of the human genomeLander Hancks and Kazazian Fulllength LINE1 is kb and containsa 5cid48untranslated region 5cid48UTR Swergold two reading frames ORF1 and ORF2and a 3cid48UTR punctuated with a polyA tract Babushok and Kazazian Beck Denli revealed a new reading frame ORF0 It is located in the 5cid48UTR of theLINE1 transcript and on the strand opposite of the ORF1 and ORF2 structural genes Antisensepromotor ASP can initiate fusion transcripts and regulate ORF0 to enhance LINE1 mobilityRomanGomez Weber Criscione Both ORFs are required for LINE1 retrotransposition process ORF1 encodes an RNAbindingprotein named ORF1P that has nucleic acid chaperone activity and ORF2 encodes a proteinnamed ORF2P that has endonuclease and reversetranscriptase activities Mathias Feng The ï¬rst step occurs when RNA polymerase II binds to the 5cid48UTR promoterregion of LINE1 and mediates the transcription of fulllength mRNA of LINE1 Lavie The LINE1 mRNA is exported to the cytoplasm where ORF1 and ORF2 are translatedand combined to form a ribonucleoprotein RNP p The RNP is then incorporated intoEdited byTrygve TollefsbolThe University of Alabamaat Birmingham United StatesReviewed bySandra Rose RichardsonThe University of QueenslandAustraliaApiwat MutiranguraChulalongkorn University ThailandJohn LaCavaThe Rockefeller UniversityUnited StatesCorrespondenceJinpu YuyujinputjmuchcomSpecialty sectionThis was submitted toEpigenomics and Epigeneticsa section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted July Published August CitationZhang X Zhang R and Yu J New Understanding of theRelevant Role of LINE1Retrotransposition in Human Diseaseand Immune ModulationFront Cell Dev Biol 103389fcell202000657Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasethe nucleus and the ORF2P endonuclease in the RNP identiï¬esand cuts speciï¬c sequences on the bottom DNA strand atthe consensus site 3cid48AATTTT5cid48 Subsequently the free 3cid48hydroxyl generated at the fracture is utilized by the ORF2P andLINE1 mRNA in the RNP is used as the template for reversetranscription to produce the complementary DNA of the LINE gene Wei Hancks and Kazazian Wang andJordan The distribution of LINE1 in the human genomeis selective LINE1 endonuclease activity and DNA replicationdetermine LINE1 insertion preference Flasch Forexample LINE1 preferentially inserts into nucleosomedepletedDNA primarily as a result of its ATrich sequences Sultana The direction of the DNA replication fork aï¬ects LINE insertion preference because the cleaved strand is usually thelagging strand templateLINE1 elements play a crucial role in the course of speciesformation and evolution On one hand derepressed LINE1functions as a driver of many diseases and even a diagnosticmarker for some diseases Pedersen and Zisoulis On theother it can aï¬ect the developmental processes and uencethe behavior by generating multiple gene products and causingvariable deleterious eï¬ects on the structure of the host genomethrough new insertions deletions and recombinations GarciaPerez LINE1 RNA and protein overexpression isrelated to apoptosis DNA damage and repair cellular plasticityand stress responses and can even promote tumor progressionMorrish Belgnaoui SinibaldiVallebona DNA damage caused by genomewide or intersperse repetitive sequences hypomethylation caninduce ammatory microenvironment Lindqvist Teerawattanapong Here we reviewed the correlationbetween LINE1 and disease as well as immune systemmeanwhile conducted a new exploration in LINE1 inhibitors bycombining its regulation mechanismsFigure shows the relative positions of the 5cid48 untranslatedregion 5cid48UTR the reading frames ORF0 ORF1 andORF2 the 3cid48 untranslated region 3cid48UTR and the Poly A tailORF2 encodes endonuclease EN reverse transcriptase RTand cysteinerich domain C Fulllength LINE1 mRNA wasgenerated using the sense promoter at 5cid48UTR The LINE1mRNA is exported to the cytoplasm where ORF1 and ORF2are translated and combined to form a ribonucleoprotein RNPp The RNP is then incorporated into the nucleus andthe ORF2P endonuclease in the RNP identiï¬es and cuts speciï¬csequences on the bottom DNA strand at the consensus site3cid48AATTTT5cid48 Subsequently the free 3cid48 hydroxyl generatedat the fracture is utilized by the ORF2P and LINE1 mRNA in theRNP is used as the template for reverse transcription to producethe complementary DNA of the LINE1 gene Richardson Kazazian and Moran LINE1 AND DISEASELINE1 and CancerWhen LINE1 retrotransposition is out of control it can lead todiseases More than s focusing on LINE1 and cancerare available in the PubMed archive Rodic theglobal hypomethylation ofLINE1 Hypomethylation and Cancergenome promotesThechromosomalinstability genomic instability and geneticheterogeneity because speciï¬c changes in DNA methylation canaï¬ect a variety of genome sequences especially the intergenicand intronic regions of the DNA resulting in chromosomeinstability and mutations Wilson LINE1 promoterhypomethylation is a biomarkerfor genomewide DNAhypomethylation which is itself a major hallmark of cancerThayer ï¬rst demonstrated the methylation statusof LINE1 in cancer cells Since then LINE1 hypomethylationof tumors has attracted widespread attention Thayer LINE1 hypomethylation was reported to be associatedwith poor survival in more than cases of gastric cancersuggesting its potential as a prognostic biomarker Shigaki This phenomenon was also subsequently foundin lung cancer liver cancer esophageal cancer prostate cancerand endometrial cancer Iwagami Kawano Lavasanifar Ogino analyzed colon cancer samples from two independent prospectivecohorts demonstrating a linear correlation between LINE hypomethylation and aggressive tumor behaviorIt hasbeen reported that global DNA hypomethylation promotesaggressive tumor behavior by amplifying oncogenes or throughabnormal expression of microRNAs Baba In esophageal cancer with high mortality and poor endoscopicscreening sensitivity LINE1 hypomethylation can serve as agood diagnostic biomarker thereby improving 5year survivalShah LINE1 hypomethylation can also be seenin some precancerous lesions For examplein colorectalcancer LINE1 hypomethylation had no significant diï¬erencebetween adenomas and cancerous tissues but it was significantlylower in adenomas than in normal tissues Dawwas Therefore LINE1 hypomethylation also can be used as an earlybiomarker for cancerHoweverthere was no significant diï¬erencein thehypomethylation of LINE1 between the blood samplesof patients with leukemia and those of normalsubjectsBarchitta LINE1 Integrations and CancerMany tumor tissues have been found to present a high levelof LINE1 activity that can rapidly increase their copy numberthrough the copyandpaste mechanism Dunaeva LINE1 can be used as cisregulatory elements to regulatethe expression of host genes Wanichnopparat Pancancer Analysis of Whole Genomes analysis of cancer genomes from histological subtypes revealed thataberrant LINE1 integrations could lead to gene rearrangementRodriguezMartin LINE1mediated rearrangementcan trigger oncogene ampliï¬cation In breast cancer Morse andcolleagues ï¬rst proposed that hypomethylation activates LINE which can utilize the target primed reverse transcriptionpathway to insert into the oncogene MYC causing tumorspeciï¬crearrangement and ampliï¬cation Morse LINE was found to induce the ampliï¬cation of CCND1 oncogenein esophageal tumor by inducing the breakagefusionbridgeFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human DiseaseFIGURE Structure of LINE1 and LINE1 retrotransposition cyclecycles RodriguezMartin LINE1 can mediatethe deletion of tumor suppressor genes It may be through Xinactivation mechanism that LINE1 mRNA forms facultativeheterochromatin in the inactivated region or LINE1 mRNAand premRNA form RISC complex to degrade complementarymRNA Allen Aporntewan In coloncancer Miki reported that LINE1 insertion disrupts thetumor suppressor gene APC which can lead to gene inactivationMiki In lung squamous cell carcinoma wefound that LINE1 insertion into tumor suppressor gene FGGYpromotes cell proliferation and invasion in vitro and facilitatestumorigenesis in vivo Zhang High Expression of ORF1 and ORF2 ofLINE1 and CancerThe activation of LINE1 increases the translation of ORF1 andORF2 which are not expressed in normal somatic tissues ORF1encodes an RNAbinding protein and high expression level ofORF1 was proved to be more common in most of the cancersand therefore as a diagnostic marker In breast cancer highexpression of nuclear ORF1 is associated with distant metastasisand poor prognosis Harris In highgrade ovariancarcinoma the ORF1 level was high and correlated to the lossof TP53 Rodic The expression of both the LINE1ORF1 and cMet protein was significantly increased and peakedin early stage in ovarian cancer suggesting that LINE1 ORF1significantly activates cMet Ko In tumor cellexperiments increased mRNA and protein expression of LINE1ORF1 can result in significant enhancement in cell proliferationand colony formation Tang It is worth noting thatthe expression of ORF1 was heterogeneous and had histologicalspeciï¬city Cancers originating in the endometrium such asbiliary tract esophagus bladder head and neck lung and colonexhibit ORF1 overexpression whereas other cancers such asrenal liver and cervical cancer show little expression of ORF1Ardeljan Recent studies have shown that an ELISAmethod to measure ORF1 in serum can be better in prostatecancer detection Hosseinnejad ORF2 encodes a protein with reverse transcriptase andendonuclease activities High expression of endonuclease inducesdoublestrand DNA breakage that can aggravate DNA damagerepair and increase genomic instability Kines Reverse transcriptase activation can promote cell proliferationthe noncoding RNAand diï¬erentiation and also altertranscription spectrum and otherepigenetic phenotypesresulting in alterations in cell regulatory networkstumordevelopment and other important pathological processes Rodicand Burns Burns Christian ORF2 canexpress early in the tumorigenesis process as it can be detectedby a highly speciï¬c monoclonal antibody mAb chA1L1 in bothtransitional colon mucosa and prostate intraepithelial neoplasiasDe Luca However studies have shown that chA1L1 recognizes both ORF2p and the transcriptional regulatorBCLAF1 so it is not speciï¬c Briggs But recentlytumor proteome proï¬ling studies based on mass spectrometryhave shown that ORF2p was diï¬cult to be detected and afteraï¬nity capture of ORF1p ORF2p has not been detected in stemcell LINE1 proteome analysis Vuong Ardeljan Therefore the detection and application of ORF2 intumors are still worth exploringLINE1 and Metabolic DisordersNew research has shown that LINE1 is also associated with bloodsugar and lipid levels Turcot LINE1 methylation isassociated with type diabetes mellitus T2DM Studies showedthat compared with hypermethylation LINE1 hypomethylationwas associated with a higher risk of worsening metabolic statusindependent of other classic risk factors MartinNunez This discovery highlights the potential role for LINE1DNA methylation as a predictor of the risk of T2DM orFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseaseother related metabolic disorders LINE1 DNA methylation isassociated with increased LDL cholesterol and decreased HDLcholesterol levels and these metabolic changes increase the riskof cardiovascular disease Pearce LINE1 DNAmethylation is also associated with many bloodbased metabolicbiomarkers In fetal neural tissue with neural tube defects it wasfound that the low methylation level of LINE1 was associatedwith the significant reduction of vitamin B12 in maternal plasmaas well as lower folate levels and increased concentrations ofhomocysteine Wang Folic acid and other B vitaminsplay an important role in the biosynthesis of new purines andpyrimidines Therefore the methylation status of LINE1 can be apredictor of some metabolic diseases Current studies have shownthat LINE1 can also regulate metabolism by inserting metabolicgenes It was reported that LINE1 insertions in the FGGY genecan upregulate cytochrome P450 arachidonic acid metabolismand glycerolipid metabolism These metabolic disorders can leadto the occurrence of a variety of diseases and poor prognosisZhang LINE1 and Neurological DisordersLINE1 can aï¬ect the developing brain at diï¬erent stages ofhealth and disease Suarez Ataxia telangiectasiaAT is a progressive neurodegenerative disease caused byataxia telangiectasia mutated ATM gene mutation In researchers found that in nasopharyngeal carcinomas with ATMdeï¬ciency LINE1 retrotransposition increased and ORF2 copynumber increased in AT neurons thus verifying the correlationbetween LINE1 retrotransposition and ATM deï¬ciency Coufal High expression of LINE1 was found in Rettsyndrome caused by mutation of methyl CpG binding protein MeCP2 in the Xlinked gene which was caused by theinclusion of LINE1 5cid48UTR sequence in the MeCP2 targetleading to methylationdependent repression Muotri LINE1 is involved in the aging process In patientswith frontotemporal lobe degeneration LINE1 transcripts werefound to be elevated Li LINE1 hypomethylationhas been observed in most psychiatric studies Increased copynumbers of LINE1 as a result of LINE1 hypomethylation werealso found in patients with schizophrenia bipolar disorder andmajor depressive disorder Liu Li Thelink between LINE1 methylation levels and Alzheimers diseaseis still being studiedLINE1 and Genetic DisordersLINE1 is reported to be related to chromosome disordersThe ï¬rst observation of LINE1 insertion was in whenKazazian observed a new exon of F8 LINE1 insertion inthe Xlinked gene which is a gene encoding coagulation factorVIII in a patient with hemophilia A Kazazian Then a LINE1 insertion was found in the CHM gene of apatient diagnosed with choroideremia The reverse integrationof a LINE1 element into exon resulted in aberrant splicingof the CHM mRNA van den Hurk FurthermoreLINE1 can also promote mobilization of other RNAs intrans Alu and SVA which can be transmobilized leading togene insertions Kemp and Longworth Retrotransposoninsertions were found to account for up to of all NF1mutations Wimmer Neuroï¬bromatosis type I isan autosomal dominant disorder caused by NF1 gene mutationsMessiaen Alu insertion is located bp upstreamof NF1 exon causing the exon to skip and change the reading frame Payer and Burns Only two cases werethought to be a result of independent SVA insertion in SUZ12Paccompanied by 867kb and 1Mb deletions that encompassedthe NF1 gene Vogt In autosomal recessive geneticdisease such as Fanconi anemia caused by SLX4FANCP deï¬ciencyand AicardiGoutieres syndrome AGS of threeprime repairexonuclease mutations LINE1 expression was upregulated andproammatory cytokines were produced through the cGASSTING pathway Brégnard Suarez LINE1 AND IMMUNE REGULATION OFDISEASELINE1 and Autoimmune DiseaseHypomethylated and highly expressed LINE1 has been foundin autoimmune diseases such as systemic lupus erythematosusSLE Sjögrens syndrome SS and psoriasis Schulz Yooyongsatit Mavragani LINE1 RNAis characterized by viral RNA and exists as RNP ps whichcan be recognized by RNA sensors and activate innate immuneresponses Mavragani Cell studies demonstrated thatLINE1 activates the production of IFNβ by RNA pathway Zhao When LINE1 retrotransposition was inhibited byRT inhibitors significant reductions were observed in IFNαIFNβ and IFNγ mRNA levels Brégnard LINE1transcripts and p40 protein a kDa RNA binding proteinthat LINE1 encodes have been detected in SLE and SS patientsIt has been demonstrated that LINE1 can induce the productionof IFNI in vitro by TLRdependent and TLRindependentpathways Mavragani In MRL autoimmunelymphoproliferative syndrome LINE1 ORF2 encoding an RTand its products are associated with an MHC class I molecule onthe cell membrane Benihoud In Fanconi anemia andAGS LINE1 was found to be associated with the activation of theautoimmune system LINE1 also regulates immunity by actingas a cisregulatory element through the mechanism of LINE1mRNA and premRNA forming RISC complex to degrade thecomplementary mRNA Wanichnopparat LINE1 and Tumor ImmunityIn TCGA cancer samples the scientists measured thetranscriptional activity of pathways and found that of immune pathways were significantly negatively correlatedwith LINE1 Jung LINE1 is inversely correlatedwith the expression of immunologic response genes Less LINE activity was found in tumors with high immune activityIn esophageal cancer tissues scientists found that the LINE1methylation level in tumors was significantly positively associatedwith the peritumoral lymphocytic reaction Kosumi The activities of regulatory T cells and PD1 signaling as reportedin cancer immune evasion and chronic ammatory conditionsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasealso have negative correlations with LINE1 It is reported that thenegative correlation between LINE1 and immune activity maybe caused by the destruction of LINE1 inhibition but the speciï¬cmechanism is still unclear LINE1 may also mediate immunetolerance which may change from immune stimulation mode toimmunosuppression mode through continuous IFN signaling ordirectly aï¬ect lymphocyte signalingtumorLINE1 and MetabolismInducedImmunityLINE1 is also associated with blood sugar and lipid levelsAbnormal glucose and lipid metabolism can lead to metabolicreprogramming in tumor cells The most classic metabolismofis Warburg eï¬ect where a large amount ofglucose is absorbed to fulï¬llthe need for proliferationand produce lactic acid Lunt and Vander Heiden The acidic microenvironment caused by lactic acid leads toimpaired Tcell activation and proliferation prevents NK cellactivation stabilizes HIF1α to stimulate the polarization of antiammatory M2 macrophages and inhibits the production ofIFNγ in tumorltrating T cells Husain Colegio Brand Abnormal lipid metabolism intumor cells also can lead to local immunosuppression in themicroenvironment Hao LINE1 can aï¬ect localimmune homeostasis by inserting elements into metabolismrelated genes FGGY is known to encode a protein thatphosphorylates carbohydrates and is associated with obesity andsporadic amyotrophic lateral sclerosis Zhang LINE retrotransposons suppress FGGY leading to lipid metabolismdisturbance and dietinduced obesity in mice Taylor Lung squamous cell carcinoma patients with L1FGGYtissue have a poor prognosis have low levels of CD3 Tcells and have high levels of CD68 macrophages and CD33myeloidderived cells Zhang L1FGGY alsoregulates the abnormal transcription of cytokines related to theimmunosuppressive micromilieuLINE1 INHIBITIONThe correlation between LINE1 and disease as well as immunitywas analyzed Figure The life cycle of LINE1 providesa plethora of ways to target and inhibit LINE1 expressionBanuelosSanchez The inhibition of LINE1 hasbecome a treatment strategy for some diseasesTargeting LINE1 Methylationa CpGFulllength LINE1 transcription is driven bydinucleotiderich internal promoter Hypomethylation of LINE causes the activation of LINE1 which causes retroelementtransposition and chromosomal alteration Saito The hypomethylation of LINE1 has become an important factorin the occurrence and development of diseases so maintainingthe state of LINE1 methylation has become a key methodfor the treatment of diseases Soy isoï¬avone supplementationcan regulate the level of LINE1 methylation in head and necksquamous cell carcinoma HNSCC In a clinical trial of patients with HNSCC who took a soy isoï¬avone supplement mgday orally for weeks before surgery a positivecorrelation was found between LINE1 methylation level anddaily isoï¬avone intake Rozek Some cellbasedstudies and clinical data have shown that LINE1 dysregulationis associated with tumor drug resistance Zhu Lavasanifar It was found in breast cancer cellstreated with paclitaxel that DNMT3a a member of the DNAmethyltransferase family could enhance the methylation level inthe gene by binding to the inner region of the LINE1 gene andthen upregulate the expression level of LINE1 Downregulatingthe expression of DNMT3a can eï¬ectively inhibit the expressionof LINE1 Wang LINE1 retrotransposon silencedalso through histone modiï¬cations Histone demethylaseKDM4B may enhance the LINE1 retrotransposition eï¬cacywhereas depletion of KDM4B reduced itin breast cancerXiang Elevated LINE1 expression was found inPC9 drugtolerant persister DTP cancer cells treated withthe EGFR inhibitor erlotinib HDAC inhibitors can suppressLINE1 in DTP cancer cells Guler Currently DNAmethyltransferase inhibitors and histone deacetylase inhibitorshave entered clinical trials Gaillard Targeting RT ActivityLINE1 elements harbor ORF1 and ORF2 which has reversetranscriptase RT activity and RT inhibition may be anovel noncytotoxic anticancer therapy strategy Sciamanna RT is a key player in retrotransposition andfunctions by transcribing LINE1 mRNA or other RNAs toFIGURE The relationship between LINE1 and diseases and their regulatory mechanismsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human DiseasecDNA at the integration sites Khalid Speciï¬creverse transcription inhibitorsincluding nevirapine NVRand efavirenz EFV which target the HIV1encoded RT andare currently used in AIDS therapy reduce cell proliferationand promotes diï¬erentiation of a variety of cancer cell linesof unrelated histological origin Mangiacasale Landriscina Sciamanna In vivoassays using murine models inoculated with various humancancer cell lines revealed that daily treatment of animals withEFV significantly delayed the progression of tumors Oricchio NVR and EFV dramatically countered L1FGGYabundanceinhibited tumor growth attenuated metabolismdysfunction and improved the local immune evasion in lungsquamous cell carcinomas Zhang EFV hasrecently undergone a phase II clinical trial in patients withmetastatic prostate cancer Houédé Another RTinhibitor F2DABOs has shown antiproliferative activity innude mice helping to promote cell diï¬erentiation and inhibittumor growth Sbardella Later the nucleosidereverse transcription inhibitor abacavir was also shown to inhibitcell growth migration and invasion Carlini Capsaicin is the main chemical component of Asiasari radixand Capsicum annuum as well as the major component of aChinese traditional herbal medicine Shoseiryuto Friedman Capsaicin suppresses LINE1 by inhibiting theRT activity of LINE1 ORF2P which is the LINE1encodedRT responsible for LINE1 activity Nishikawa A recent study revealed that azidothymidine AZT inhibitsthe RT activity of ORF2P in a fetal oocyte attrition modelExperiments showed that AZTtreated oocytes have a reductionof LINE1 ORF1 ssDNA compared with untreated oocytesTharp It is important to note that RT inhibitorsdo not eliminate the tumor but only control its progressionTherefore in addition to the antiAIDS drugs approved by theFDA the combination of Chinese and western medicine can beregarded as an emerging treatmentCombined ImmunotherapyRecent studies suggest that LINE1 hypomethylation may be apositive indicator of immunotherapy DNA methyltransferaseDNMT is an important epigenetic molecule that catalyzedDNA methylation and can induce the development of varioustumors Downregulating the expression of DNMT can eï¬ectivelyinhibitthe expression of LINE1 Wang SoDNA methyltransferase inhibitors DNMTis play an importantrole in the antitumor process DNMTI can improve tumorimmunogenicity promote NK cells and CD8 T cells toplay a cellmediated cytotoxic role and promote immuneresponse to participate in antigen commission by regulatingimmunosuppressive cells Chiappinelli DNMTican enhance the expression of cancertestis CT antigenmaking the tumor more susceptible to CT antigen vaccine Thecombination of decitabine a DNA methyltransferase inhibitorand cancertestiscancergermline antigen NYESO1 vaccinehas a good therapeutic eï¬ect in the primary treatment ofhuman recurrent epithelial ovarian cancer Odunsi A clinical trial has shown that combination therapywith carboplatin and antiprogrammed death1 has a goodtherapeutic eï¬ectin lung cancer because carboplatin caninduce LINE1 expression Langer ThereforeLINE1 can be used as a target of combined immunotherapyin tumor therapyOther InhibitorsRecently a number of other regulatory approaches have beenreported In somatic cells microRNAs miRNAs or miRs alsoregulate the activity of LINE1 Idica MiR128regulates LINE1 activity in somatic cells by targeting the nuclearimport factor transportin1 TNPO1 3cid48UTR which mediatesnuclear import and requires RanGTP for cargo delivery into thenucleus Twyï¬els MiR128 inhibits the expression ofTNPO1 mRNA and protein and TNPO1 deï¬ciency suppressesLINE1 mobilization by inhibiting nuclear import of LINERNP Idica MiR128 also guides the miRNAinduced silencing complex to bind directly to a target siteresiding in the ORF2 RNA of LINE1 Hamdorf At present a novel target of miR128 has been identiï¬ed asheterogeneous nuclear ribonucleoprotein A1 hnRNPA1 whichis required for LINE1 retrotransposition Goodier Fung MiR128 represses hnRNPA1 mRNA andprotein by targeting the CDS of hnRNPA1 which interactswith LINE1 ORF1p via RNA bridge to promote LINE1mobilization Goodier This interaction results intranslational repression of the LINE1 retrotransposition therebyreducing the risk of LINE1mediated mutagenesis Pedersenand Zisoulis Therefore microRNAs can be a target forLINE1 inhibitionAryl hydrocarbon receptor AHR is a ligandactivatedtranscription factor that activates LINE1 expression Teneng AHR is overexpressed in breast and thyroid cancerssuggesting that these tumors also overexpress LINE1 Powell Lai found that biseugenol a novel AHRinhibitor impeded cancer growth and inhibited EMT in gastriccancer cells Lai These ï¬ndings suggest that targetingAHR with small molecule inhibitors may be a novel therapeuticapproach ORF1P phosphorylation by protein kinase A is alsorequired for LINE1 Kinase inhibitors speciï¬cally designed totarget LINE1 ORF1P phosphorylation may be associated withinhibition of LINE1 Bojang Therefore there isroom for drug development research focusing on targeting andinhibiting LINE1 ORF1P phosphorylationCONCLUSIONThe activation of LINE1 retrotransposon is associated with avariety of human diseases and is involved in the occurrenceand progression of disease through retrotranspositiondependentand retrotranspositionindependent mechanisms Currently ithas even become a marker of tumorigenesis and prognosis andis related to immune regulation The eï¬ective inhibition ofLINE1 activation has become a treatment for some diseasesThe inhibition of LINE1 in animal experiments can inhibitFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasethe occurrence and development of tumors so the clinicalapplication of LINE1 inhibitors is imminent In addition toexploring some known inhibitors other mechanisms of LINE inhibition should also be explored We summarized therelationship between LINE1 and diseaserelated immunity andproposed that LINE1 may aï¬ect the immune status of thebody by regulating metabolismleading to poor prognosisMetabolic substances can aï¬ect the immune microenvironmentfor examplelactic acid can lead to immunosuppressivemicroenvironment leading to poor prognosis of tumors Thedysregulation of LINE1 can lead to the disorder of glucoseand lipid metabolism and the inhibition of glucose and lipidmetabolism may reverse the disease progression caused byLINE1 Now the antitumor eï¬ect of regulating the bodysmetabolism has entered clinical trials such as the significanteï¬ect of metformin in the treatment of tumors Therefore themetabolic status of diseases caused by LINE1 can be checkedMetabolic therapy combined with LINE1 inhibitors may inhibitthe progression of LINE1 and may improve the immunemicroenvironment to achieve the optimal therapeutic eï¬ectAUTHOR CONTRIBUTIONSXZ wrote the RZ and JY reviewed and revised the All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by the National Natural ScienceFoundation of China Grant Nos and National Science and Technology Support Program of ChinaGrant No 2018ZX09201015 and Project of Science andTechnology of Tianjin Grant No 18JCQNJC82700REFERENCESAllen E Horvath S Tong F Kraft P Spiteri E Riggs A D High concentrations of long interspersed nuclear element sequence distinguishmonoallelically expressed genes Proc Natl Acad Sci USA 101073pnas1737401100Aporntewan C Phokaew C Piriyapongsa J Ngamphiw C Ittiwut CTongsima S Hypomethylation of intragenic LINE1 repressestranscription in cancer cells through AGO2 PLoS One 6e17934 journalpone0017934Ardeljan D Taylor M S Ting D T and Burns K H The human longinterspersed element1 retrotransposon an emerging biomarker of neoplasiaClin Chem 101373clinchem2016257444Ardeljan D Wang X Oghbaie M Taylor M S Husband D DeshpandeV LINE1 ORF2p expression is nearly imperceptible in humancancers Mob DNA 101186s1310001901912Baba Y Watanabe M Murata A Shigaki H Miyake K Ishimoto T LINE1 hypomethylation DNA copy number alterations and CDK6ampliï¬cation in esophageal squamous cell carcinoma Clin Cancer Res 10115810780432CCR131645Baba Y Yagi T Sawayama H Hiyoshi Y Ishimoto T Iwatsuki M Long interspersed element1 methylation level as a prognosticbiomarker in gastrointestinal cancers Digestion Babushok D V and Kazazian H H Jr Progress | 2 |
emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic beneï¬t until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic proï¬les ofHCC contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufï¬ciently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPaciï¬c trial promoted the approvalof sorafenib as ï¬rstline targeted therapy for advanced HCC5ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel ï¬rstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predeï¬ned primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asï¬rstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPaciï¬c trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21 Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as ï¬rstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24 andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical beneï¬t of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer beneï¬ts when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbeneï¬t in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these ï¬ndings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42 which was supported by theï¬ndings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redeï¬ned PFS not conventional PFS buttime until unTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival beneï¬t compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival beneï¬t compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modiï¬cation ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classiï¬cation BCLCguidelines and although the results are difï¬cult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also beneï¬ted fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratiï¬cation would enable the identiï¬cation of thosepatients who will beneï¬t most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second ï¬rstlinetargeted agent for HCC emerged Lenvatinib was approved for theï¬rstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from realworld conditions conï¬rmed the efï¬cacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufï¬cient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88 With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneï¬cial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop ï¬rstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy ï¬nallyconï¬rmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival beneï¬tthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was conï¬rmed by Yoo et al99 in aretrospective study of safety and efï¬cacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefï¬cacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identiï¬ed regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconï¬rms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs and the incrementalcosteffectiveness ratio ICER mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112 compared with regorafenib QALY and ICER RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival beneï¬t in patientswith AFP of ngml or higher118 This was later conï¬rmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the ï¬rstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent ï¬ndingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and conï¬rms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefï¬cacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efï¬cacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efï¬cacy and safety in advanced HCCs as systemicï¬rstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespeciï¬ed statistical signiï¬cance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as ï¬rstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbeneï¬t regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on ï¬rstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efï¬cacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the ï¬rst combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136 Such results were conï¬rmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ammation and rheumatic and musculoskeletal phenotypesincluding ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a ï¬rstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145 where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153 Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as ï¬rstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as ï¬rst155 or secondline inadvanced HCCs156 but unfortunately the heterogeneousnature of the results precludes ï¬rm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufï¬ciently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentiï¬erPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentiï¬erPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164 especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyproï¬le169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneï¬cially employed to produce a therapeutic dualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efï¬cacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR ï¬broblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classiï¬cation of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassiï¬cationG1G6S1S3Msig iC1iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and lowMS1 SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell proï¬lingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespeciï¬ed criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identiï¬ed to be able to predict the effect of or responseto treatment with sorafenib172 Although the molecularclassiï¬cation of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul | 2 |
neutrophils account for of circulating leukocytes and are the ï¬rst immune cells recruitedto an ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389ï¬mmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentiï¬cation of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identiï¬ed and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identiï¬ed invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis functionsand antitumorigenicin addition to the identiï¬cation of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classiï¬ed into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimpliï¬cation of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the ï¬eld regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will brieï¬y discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentiï¬ed as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various proammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 ï¬broblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated ï¬broblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophils ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identiï¬ed aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these ï¬ndingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cellneutrophil clusters was found to greatlyincrease metastatic ï¬tness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersitesnetosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within speciï¬cmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedï¬broblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deï¬cient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells speciï¬cally net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of ï¬broblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a signiï¬cantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased ï¬ux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic ï¬exibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicï¬exibility increases the cells ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic ï¬exibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforeï¬cient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic ï¬exibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic ï¬exibility in distinct neutrophil populations may beimportant for cellular functions that can uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic signiï¬cance the neutrophiltolymphocyte rationlr is an important risk stratiï¬cation and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identiï¬ed in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ammation nat rev immunol 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraï¬ckinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identiï¬cation of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep 41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspeciï¬cally target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic ï¬exibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the ï¬guresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389ï¬mmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic ï¬exibility thatfacilitates breast cancer liver metastasis cell rep 15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta n1 versus n2tan cancer cell 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389ï¬mmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps obrien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p | 0 |
CASE REPORTpublished August 103389fbioe202000929SecondGeneration RTQuIC Assayfor the Diagnosis ofCreutzfeldtJakob Disease Patientsin BrazilBreno Jos Alencar Pires Barbosa1 Bruno Batitucci Castrillo1 Ricardo Pires Alvim1Marcelo Houat de Brito1 Helio R Gomes1 S´nia M D Brucki1 Jerusa Smid1Ricardo Nitrini1 Michele C Landemberger2 Vilma R Martins2 Jerson L Silva3 andTuane C R G Vieira3 Department of Neurology Hospital das Clnicas University of S£o Paulo Medical School S£o Paulo Brazil Tumor Biologyand Biomarkers Group International Research Center AC Camargo Cancer Center S£o Paulo Brazil National Centerof Nuclear Magnetic Resonance Jiri Jonas Institute of Medical Biochemistry Leopoldo de Meis National Institute of Scienceand Technology for Structural Biology and Bioimaging Federal University of Rio de JaneiroUFRJ Rio de Janeiro BrazilThe recent development of IQCSF the second generation of realtime quakinginducedconversion RTQuIC using cerebrospinal ï¬uid CSF for the diagnosis of CreutzfeldtJakob Disease CJD represents a major diagnostic advance in the ï¬eld Highly accurateresults have been reported with encouraging reproducibility among different centersHowever availability is still insufï¬cient and only a few research centers have access tothe method in developing countries In Brazil we have had suspected cases of CJDsince when surveillance started Of these were undiagnosed This lack ofdiagnosis is due among other factors to the lack of a reference center for the diagnosisof these diseases in Brazil resulting in some of these samples being sent abroad foranalysis The aim of this research study is to report the pilot use of IQCSF in a smallcohort of Brazilian patients with possible or probable CJD implementing a referencecenter in the country We stored CSF samples from patients with possible probable enetic CJD one case during the time frame of December through June AllCSF samples were processed according to standardized protocols without access tothe clinical data Eight patients presented to our team with rapidly progressive dementiaand typical neurological signs of CJD We used CSF samples from seven patientswith other neurological conditions as negative controls Five out of seven suspectedcases had positive tests two cases showed inconclusive results Among controls therewas one falsepositive a CSF sample from a 5yearold child with leukemia undertreatment The occurrence of a false positive in one of the negative control samplesraises the possibility of the presence of interfering components in the CSF sample frompatients with nonneurodegenerative pathologies Our pilot results illustrate the feasibilityof having CJD CSF samples tested in Brazilian centers and highlight the importance ofinterinstitutional collaboration to pursue a higher diagnostic accuracy in CJD in Braziland Latin AmericaKeywords CreutzfeldtJakob disease prionconversion biomarkersrapidly progressive dementiarealtime quakinginducedEdited byMaria Dos Anjos PiresUniversity of Tr¡sosMontes and AltoDouro PortugalReviewed byAssia AngelovaGerman Cancer Research CenterDKFZ GermanySumit GhoshThe Research Institute at NationwideChildrens Hospital United StatesCorrespondenceBreno Jos Alencar Pires BarbosabrenojbgmailcomTuane C R G VieiratuanebioqmedufrjbrSpecialty sectionThis was submitted toBiosafety and Biosecuritya section of the journalFrontiers in Bioengineering andBiotechnologyReceived May Accepted July Published August CitationBarbosa BJAP Castrillo BBAlvim RP de Brito MH Gomes HRBrucki SMD Smid J Nitrini RLandemberger MC Martins VRSilva JL and Vieira TCRG SecondGeneration RTQuIC Assayfor the Diagnosis of CreutzfeldtJakobDisease Patients in BrazilFront Bioeng Biotechnol 103389fbioe202000929Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alINTRODUCTIONtogroupTSEsconditionsencompasstransmissibleasecondaryspongiformPrion diseases also known asofrareencephalopathiesneurodegenerativeabnormalconversion of a constitutively expressed cellular glycoproteinthe prion protein PrPC into an abnormally folded isoformPrPsc Geschwind Zanusso SporadicCreutzfeldtJakob disease sCJD is the most common priondisease in humans and usually presents as rapidly progressivedementia in combination with variable degrees of multisystemneurologicalimpairment Zerr and Hermann Sinceits clinical and molecular manifestations are heterogeneousand nonspeciï¬c early diagnosis of prion diseases remainschallenging in clinical practice Geschwind and Murray Brain histopathological evaluation andor detection of PrPScare still the standard criteria for establishing a deï¬nitive diagnosisfor CJD CDC However invasiveness when performingthis type of analysis antemortem brings very little beneï¬t to thepatient since these diseases are still incurable Clinical signs andparaclinical tests are the most commonly used approaches duringthe course of the disease and can be used to classify it as a possibleor probable prion disease Brown CDC Among the paraclinical tests brain diï¬usion weightedMRIDWMRI and cerebrospinal ï¬uid CSF analysis have increaseddiagnostic accuracy Eisenmenger Staï¬aroni Bizzi The presence of protein Tauprotein neuronspeciï¬c enolase NSE the astroglial proteinS100B and PrPSc itself are used as biomarkers in CFS for TSEdiagnosis Schmitz Connor Overallthe only protein that is a speciï¬c biomarker for TSE is PrPScPrPSc can be detected in CSF based on its selfpropagating abilityconverting and seeding the aggregation of the nonpathogenicPrPC into PrPScThe realtime quakinginduced conversion RTQuIC assayits experimentalwas developed in and since thenconditions have been improved and tested on a large number ofsamples in several laboratories worldwide Wilham Green It ultrasensitively detects limited amounts of PrPScin CSF and other tissue samples Wilham The recentdevelopment of the secondgeneration IQCSF RTQuIC assayusing CSF for the diagnosis of CJD represents a major diagnosticadvance in the ï¬eld Wilham Orrº In humans RTQuIC analysis showed a sensitivity of and speciï¬city of with encouraging reproducibility amongdiï¬erent centers Franceschini RTQuIC started to be clinically used in and becamea criterion of the Centers for Disease Control and PreventionCDC to diagnose CJD as probable CDC Its highsensitivity and speciï¬city make it an important clinical laboratorytestits global availability is stillinsuï¬cient only a few research centers have access to the methodespecially in developing countriesfor widespread use butSurveillance of TSE cases has been compulsory in Brazil since Martins and suspected CJD cases werereported up to June Of these were undiagnosed deOliveira Cardoso Ministrio da Saºde RTQuIC for the Diagnosis of Brazillian PatientsinimagingperformingexaminationsandDiï¬cultiesneuropathological analyses are found in several medicalunits in the country making diagnosis problematic Somediagnosed patients had samples sent to centers outside thecountry for biomarker analysis allowing for greater coverageof the diagnostic criteria The implementation of a speciï¬ctest in Brazil such as RTQuIC which can provide diagnosisfor diï¬erent TSEs with ease and conï¬dence is urgent to havenotiï¬cation and diï¬erential diagnosis for these diseases Thisis also important to guide medical decisions Here we reportthe pilot use ofthe secondgeneration IQCSF RTQuICassay in a small cohort of Brazilian patients with possibleor probable CJD to implement a reference center for thisanalysis in the countryMATERIALS AND METHODSClinical InvestigationPatients with suspected CJD were admitted for investigationunder a protocol for rapidly progressive dementia RPD aspreviously reported Studart Neto Following a fullneurological examination all patients underwent complete bloodcell count serum electrolytes blood glucose acute Creactiveprotein liver renal and thyroid function tests antithyroid andantinuclear antibody testing as well as treponemal and HIVserology Patients also underwent brain magnetic resonanceimaging [T1 T2 ï¬uidattenuated inversion recovery FLAIRgradient echo and diï¬usion weighted imaging sequences] EEGand CSF cell count biochemistry and g globulin levels CSF protein was assessed in cases of suspected prion diseasesas required by Brazilian norms Selected patients underwentchest and abdomen computed tomography mammography forwomen testicular ultrasound for men and thyroid ultrasoundto rule out paraneoplastic RPD In addition we obtainedonconeural andor neuronal surface antigen antibody testingwhen paraneoplastic or autoimmune encephalitis was suspectedA ï¬owchart for patient inclusion is provided in Figure Three cases had access to molecular analysis of the PRNP genefor polymorphisms in the codon Samples were analyzedusing denaturing highperformance liquid chromatographyTechnical details about this procedure as well as ampliï¬cationreactions and DNA extraction have been previously describedCastro Smid CSF SamplesWe analyzed eight CSF samples from seven patients withpossible probable or genetic CJD referred to the Departmentof Neurology at University of S£o Paulo from December to June One patient named ABT had her CSF testedtwice at diï¬erent times We used CSF samples from sevenpatients with other neurological conditions as negative controlsCSF samples mL were collected by lumbar puncture LPfollowing a standard procedure Two milliliters of the CSFsample were then centrifuged at g for min andstored in polypropylene tubes at ¦C until blind analysis byresearcher TCRGV at UFRJFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsof CJD The patient named ABT had her CSF tested twice atdiï¬erent times ABT1 and ABT2 samples therefore renderinga total of eight CSF samples ABT patient results will be betterreported in Case descriptionWe randomly used CSF samples from seven patients withother neurological conditions as negative controls Five out ofeight suspected samples had positive RTQuIC results in ourhands Figure 2A Clinical diagnosis CSF analysis and RTQuICresults are summarized in Table Samples from patients LRC and DAS were used as positivecontrols given that they were previously analyzed using RTQuICin a reference center outside the country The NationalPrion Disease Pathology Surveillance Center Cleveland OHUnited States Our RTQuIC analyses were also positivecorroborating this result Figure 2A Patient ASM was a geneticCJD positive for the E200K mutation and in our analysis thispatient was also positive presenting a very short lag phase and ahigh ï¬uorescence signal Figure 2AAmong the negative controls there was one false positivesample CT2 Figure 2A The falsepositive case was a5yearold child with leukemia who was receiving intrathecalchemotherapy GBTLI protocol with methotrexate aracitincitrabin dexamethasone during the time frame of the studyIn the following two cases are presented in which the resultsobtained with RTQuIC show interesting aspects related toRTQuIC sensitivity and disease progressionCase ABT was a 72yearold woman with years of formal educationShe had an atypical presentation characterized by a 4monthhistory of rapidly progressive cognitive impairment associatedwith visual hallucinations and gait disturbances with repeatedfalls She had no relevant past medical history or family history ofany neurological conditions On neurological examination shescored on the MiniMental State Examination MMSEand physical tests revealed only a prominent axial syndrome asshe could not sit or stand up without bilateral support There wereno pyramidal signs parkinsonism or visuospatial impairmentA comprehensive investigation with metabolic inï¬ammatoryparaneoplastic and infectious tests was unremarkable Brainmagnetic resonance imaging MRI revealed symmetric diï¬usionweighted image DWI hyperintensities in the basal gangliaFigure a ï¬nding that raised the suspicion of CDJ TheCSF analysis was unremarkable with elevated tau protein levelsand negative The RTQuIC results were inconclusiveonce from wells only two crossed the threshold ABT1sample in Figure 2B Electroencephalogram EEG revealeddisanized electrical brain activity with no periodic wavesThree months later she developed a signiï¬cant worsening withthe need for aid for most activities of daily living severe cognitiveimpairment she could not be tested with the MMSE myoclonusand the presence of prominent frontal reï¬exes on neurologicalexamination At this point a prolonged EEG eventually showedbilateral periodic sharp waves and another lumbar puncturewas performed with a second sample sent for RTQuIC analysiswith a positive result with a high ï¬uorescence ABT2 samplein Figure 2B She eventually died of clinical complicationsFIGURE Flowchart of samples included for RTQuIC analysisRTQuICThe RTQuIC assay was performed using the improvedQuICCSF IQCSF conditions as published Orrº Brieï¬y µL of CSF was added to µL of reaction mixturein each well of a black well plate with a clear bottomNunc The ï¬nal solution contained mM phosphate buï¬er atpH mM ethylenediaminetetraacetic acid tetrasodium saltdihydrate EDTA at pH mM NaCl µM thioï¬avinTThT sodium dodecyl sulfate SDS and mgmLrecombinant Syrian hamster truncated form of prion proteinHa rPrP Samples were tested in quadruplicate threeor four times independently generating a total of or wellsevaluated for each sample The plates were sealed and incubatedin a FLUOstar OMEGA plate reader BMG Labtech Germanyat ¦C with intermittent cycles of shaking s doubleorbital rpm and rest s ThT ï¬uorescence was collected every min using ± nm excitation and ± nmemission wavelengths The threshold was deï¬ned as the averageï¬uorescence for all samples within the ï¬rst h of incubationplus standard deviations SD A sample was consideredpositive when at least two of four replicate wells crossed thisthreshold All IQCSF RTQuIC analyses were performed at theFederal University of Rio de JaneiroUnseeded reaction not shown and random CSF samplesfrom patients with other neurological conditions Table wereselected as negative controls Given the descriptive nature of thisstudy no statistical analyses were performedRESULTSIn the referred time frame seven patients presented to our teamwith rapidly progressive dementia and typical neurological signsFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsTABLE Results of diagnostic investigations in the tested patient cohortSample ageDiagnosisaCSF analysisRTQuIC statusWBCµ LProt mgdLGlu mgdLTaub pgmLCT1 CT2 CT3 HCS HBV RMC ABS LRC DAS ABT1 ABT2 ASM NAJJN GMT TN Control chronic meningitisControl leukemiaControl cranial nerves syndromeControl multiple sclerosisControl HIVControl SAHventriculitisControl chronic meningitisRPD probable CJDRDP probable CJDRPD possible CJDRPD possible CJDRPD genetic CJDRPD probable CJDRPD possible CJDRPD probable CJD1cNANANANegativePositiveNegativeNANANANegativeNegativePositiveNegativeNegativeNegativeNegativeNegativePositivedPositivedNegativePositivePositivePositiveNegativePositiveWBC white blood cells Prot protein levels Glu glucose levels RPD rapidly progressive dementia SAH subarachnoid hemorrhage NA not available aControls andtheir suspected diagnosisinvestigation bReference value for total tau levels was pgmL cThis sample had red blood cells dThese patients also had their CSFsamples sent abroad with positive results The National Prion Disease Pathology Surveillance Center Cleveland OH United States months after the ï¬rst symptoms appeared PRNP gene analysisrevealed codon heterozygous MVin hisCase JJN was a 65yearold male who presented to our clinicwith an 8month history of behavioral changes He had years of education and a medical history of hypertensionand gouty arthritis There was no family history of anyneurological conditions His wife described the ï¬rst symptomsas prominent changesfood preferences with anunusual demand for rice and chicken Two months laterhe developed visual hallucinations mostly described as thepresence of spiders in the ceiling In the ï¬rst evaluationhe was independent of instrumental activities of daily livingNeurological examination revealed an MMSE of withan unremarkable physical examination A laboratory workupincluding metabolicinï¬ammatory and serology studies wasnegative At this point we were surprised by the ï¬nding ofbrain MRI diï¬usion weighted imaging revealing marked bilateralhypersignal in the frontotemporoparietooccipital cortex basalganglia thalamus and less markedly in the hippocampusraising the suspicion of sporadic CreutzfeldtJakob DiseasesCJD Figures 4AB The patient was hospitalized for furtherinvestigation The EEG study revealed bilateral and synchronousslow waves A brain 18FDG PET showed hypometabolism in thetemporal and frontal lobes caudate nuclei and temporoparietalcortex Figure 4C The CSF study was initially unremarkableexcept for the high total tau protein levels whereas ptau and betaamyloid values were within the normal rangeA CSF sample was sentfor autoantibodies and RTQuICtesting The patient was empirically treated with intravenousmethylprednisolone for days and was discharged for outpatientfollowup Atthis point we received CSF results negativefor autoantibodies with an inconclusive RTQuIC responsefrom wells only two crossed the threshold Figure 2CIn the following months he eventually developed cognitivedeterioration and parkinsonism with the presence of morecomplex visual hallucinations He eventually died of clinicalcomplications approximately months after initial symptomsPRNP gene analysis revealed codon heterozygous MVDISCUSSIONsyndromes ofencephalopathiesThe present study reports the results of pilot secondgenerationreferred forRTQuIC testing in a small patient cohortrapidly progressive neurologicalsuspectedprion nature Our center previously reported cases ofrapidly progressive dementia among patients in a 3yearintervalrepresentedthe majority of the samples followed by CJD Studart Neto All cases from that study werediagnosed with CJD according to the University of CaliforniaSan Francisco Modiï¬ed Criteria Vitali which doesnot include CSF testingImmunemediatedAs we described here IQCSF RTQuIC proved to be anextremely important tool in the diagnosis of ABT and JJN casesCases and Both patients presented with rapidly progressiveneurological syndromes and could not initially be classiï¬ed withpossible or probable CJD according to the most recent criteriaCDC Both ABT and JJN showed slightly elevated levelsof total Tau protein but although this biomarker has sensitivity it is only speciï¬c for CJD Connor protein was not elevated in both cases despitetheir genetic subtype MV which is often associated with positive protein levels Manix Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsFIGURE Blind RTQuIC analysis of CSF samples A RTQuIC responses from reactions seeded with µl of CSF from suspected CJD cases LRC DAS ASMJJN GMT TN The RTQuIC responses from reactions seeded with CSF samples from patients with other neurological disorders CT1 CT2 CT3 HCS HBV RMCABS were used as a negative control Each curve represents the mean of four replicate readings of three or four repetitions B Singlewell analysis of CSF samplesfrom patient ABT ABT1 and ABT2 refer to the ï¬rst and second collections respectively C Singlewell analysis of CSF samples from patient JJNIn the case of ABT patient the clinical presentation ofrapid cognitive impairment with visual hallucinations and gaitimpairment was considered atypical In this case the brain MRIwas an important diagnostic clue with DWI hyperintensitiesin the basal ganglia leading to a diï¬erential diagnosis withmetabolic encephalopathies hypoxicischemic lesions or toxicFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsdisease progression To our knowledge this approach performedwith samples of a patient collected at diï¬erent times has not yetbeen tested and this is the ï¬rst report suggesting that RTQuICresults change according to disease evolution A more feasiblehypothesis would be that the ï¬rst sample had some interferencesuch as blood that could mask the positive result Cramm despite a only modest presence of red blood cells in thisparticular sample cellsµL see Table Atypical features for sCJD in JJN Case included a presentation with mild behavioral changes the initialsparing of motor systems with evidence of motor ï¬ndingsin the neurological examination only almost months afterï¬rst symptoms an unremarkable EEG with months ofevolution and the presence of a bilateral T2 hypersignalin the hippocampus The RTQuIC was inconclusive buttogether with MRIit could suggest early diagnosis ofprobable CJD Perhaps with disease progression JJNs CSFsample would obtain a positive RTQuIC result as observedfor ABT patientThe occurrence of one falsepositive case in our RTQuICtest weakens our diagnostic accuracy and underscores the needfor improvements in the protocol Despite having an extremelyhigh speciï¬city IQCSF RTQuIC falsepositive results have beenreported in the literature Hayashi reported thecase of a 61yearold man who presented with rapid cognitiveimpairment myoclonus and recurrent seizures A brain MRIrevealed cortical hyperintensities and the CSF analysis showedelevated and tau levels with a positive RTQuIC Despiteaggressive treatment with corticotherapy the patient died andpostmortem assessment revealed only pathological changes afterconvulsion with no signs of prion disease The authors concludethat convulsion may cause falsepositive RTQUIC results andthat a postmortem evaluation remains the gold standard fordiagnosing similar cases The shaking eï¬ect was analyzed in vitroby Orrº and they showed that long shaking periodsreduced scrapieseeded reaction times but continuous shakingpromoted falsepositive reactionsFIGURE Diffusionweighted images DWI from ABT case revealingenlarged ventricles and symmetric hyperintensities in the basal ganglia Ayellow arrows B shows corresponding apparent diffusion coefï¬cient ADChypointensities in the same territorylesions ie carbon monoxide intoxication Finelli and DiMario A careful clinical assessment made all possibilities lesslikely and CJD became our main hypothesis Despite aninconclusive RTQuIC result for the ï¬rst collected sample thepatient eventually evolved with a more typicalimpairmentand the second CSF sample with a 3month interval yieldedpositive RTQuICThe diï¬erent results obtained with the ABT patient sampleswere very intriguing We hypothesize that this diï¬erence could beattributed to higher loads of PrPsc following disease progressionAlthough disease duration does not seem to be related toRTQuIC results McGuire it is not yet clear whetherthere is a change in the presence of seeds in CSF according toFIGURE A MRI diffusionweighted images from JJN case revealing hyperintensities on frontal temporoparietal and posterior cingulate cortical areas Ayellow arrow B MRI apparent diffusion coefï¬cient maps with corresponding hypointensities in the same regions above B yellow arrow C 18FDG PETCTshows hypometabolism on the bilateral temporoparietal cortex posterior cingulate precuneus and caudate nucleusFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsRegarding our falsepositive case we did not ï¬nd any similarcases reporting the use of intrathecal medications as a possiblereason for a positive RTQuIC result A positive case in a5yearold child without any neurological symptoms wouldnever be expected an 18yearold woman was the youngestperson diagnosed with probable sCJD using RTQuIC Yao Therefore this sample was selected as a negative controlalthough infant CSF was never RTQuIC analyzed Despiteoptimized protocols sample processing issues can always be apossibility for such unexpected resultsThe present study is limited by its pilot nature with a modestsample size Improvements in the establishment of the protocolare necessary requiring a greater number of analyses The useof nasal brushes to collect patient samples for RTQuIC analysisis also valid to improve protocol sensitivity and speciï¬cityHowever this is the ï¬rst study to our knowledge to report speciï¬cbiomarkerbased feasible results performed in a developingcountry for prion diagnosis in addition to pointing out newpossible interferences in the protocol and the need to understandhow the diï¬erent current diagnostic approaches can revealdisease progressionCONCLUSIONThe cases reported here illustrate the importance of usingRTQuIC for patients with neurological syndromes enabling thediagnosis of probable CJD while no other method was suï¬cientto support this diagnosis even with atypical clinical presentationIn addition the identiï¬cation of a false positive in a sample froma leukemic pediatric patient undergoing intrathecal treatmentwith chemotherapy suggests new possible interferences in themethod This will require future investigation of the eï¬ect of thesechemotherapeutic agents for inclusion or not as a limitation forcarrying out the assayCSF samples are often evaluated only once due to theinvasiveness of CSF collection and the absence of curativetreatment for TSEs in addition to rapid disease progressionOne of the cases we report points out the importance ofcarrying out studies that evaluate the progression of the diseaseand that RTQuIC is a useful approach for such studies Inthis case the use of nasal brushes might be prioritized overCSF analysis since this provides greater sensitivity and enablesmore frequent sample collection given the less invasive natureof the procedureFinally our study illustrates the feasibility of having CJDCSF samples tested in Brazilian centers and highlights theimportance of interinstitutional collaboration in order to pursuea greater diagnostic accuracy for CJD in developing countriesREFERENCESBizzi A Pascuzzo R Blevinsetal Evaluation ofM E Mdetecting prion disease with diï¬usion magneticJAMA Neurol101001jamaneurol20201319print]J Grisoli M Lodi R Moscatellia new criterion forimagingofresonance[EpubaheadIt also demonstrates that RTQuIC can be clinically availablefor testing patients from Brazil and other Latin Americancountries and points to the need and feasibility of establishinga national reference center for the diagnosis of these rare butdevastating diseasesDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialETHICS STATEMENTEthical review and approval was not required for the studyon human participants in accordance with the local legislationand institutional requirements Written informed consent toparticipate in this study was provided by the participants legalguardiannext of kin Written informed consent was obtainedfrom the individuals and minors legal guardiannext of kinfor the publication of any potentially identiï¬able images or dataincluded in this AUTHOR CONTRIBUTIONSBB study design patient data collection and manuscript writingBB BC RA MB HG and SB patient data collection andinterpretation JS and RN study design data interpretation andmanuscript critical revision ML and VM genetic analysis JLSand TV RTQuIC facility implementation TV study designRTQuIC evaluations RTQuIC data analysis and manuscriptwriting All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by research grants from the CarlosChagas Filho Foundation for Research Support in the State of Riode Janeiro FAPERJ and the National Council of Technologicaland Scientiï¬c Development CNPq to JLS and TVACKNOWLEDGMENTSWe would like to acknowledge all the patients and their familiesfor their contributions to this studyBrown P Brunk C Budka H Cervenakova L Collie D Green A WHO Manual for Surveillance of Human Transmissible SpongiformEncephalopathies Including Variant CreutzfeldtJakob Disease Geneva WorldHealth anizationCastro R M R P S Landemberger M C Walz R Carlotti C GHuang N Cunha D R High capacity and low costdetection of prion protein gene variant alleles by denaturing HPLCFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsJ Neurosci Methods101016jjneumeth2004CDC CDCs Diagnostic Criteria for CreutzfeldtJakob Disease CJD[Internet] Atlanta Centers for Disease Control and PreventionConnor A Wang H Appleby B S and Rhoads D D Clinical laboratorytests used to aid in diagnosis of human prion disease J Clin Microbiol57e0076919Cramm M Schmitz M Karch A Mitrova E Kuhn F Schroeder B Stability and reproducibility underscore utility of RTQuICfor diagnosis of CreutzfeldtJakob disease Mol Neurobiol 101007s1203501591332de Oliveira Cardoso C A de Albuquerque Navarro M B M Soares B E Cand Oliveira Cardoso T A Avalia§£o epidemiol³gica dos ³bitos pordoen§as pri´nicas no Brasil sob o enfoque da biosseguran§a Cadernos SaºdeColetiva 1015901414462x201500010002Eisenmenger L Porter MC Carswell C J Thompson A Mead S RudgeP Evolution of diï¬usionweighted magnetic resonance imagingsignal abnormality in sporadic creutzfeldtjakob disease with histopathologicalcorrelation JAMA Neurol Finelli P F and DiMario F J Diagnostic approach in patients withsymmetric imaging lesions of the deep gray nuclei Neurologist 10109701nrl0000087718555976aFranceschini A Baiardi S Hughson A G McKenzie N Moda F Rossi M High diagnostic value of second generation CSF RTQuIC acrossthe wide spectrum of CJD prions Sci Rep Geschwind M D Rapidly progressive dementia Continuum 101212con0000000000000319Geschwind M D and Murray K Diï¬erential diagnosis with other rapidprogressive dementias in human prion diseases Handb Clin Neurol 101016b9780444639455000209Green A J E RTQuIC a new test for sporadic CJD Pract Neurol 101136practneurol2018001935Hayashi Y Iwasaki Y Yoshikura N Asano T Mimuro M Kimura A An autopsyveriï¬ed case of steroidresponsive encephalopathywith convulsion and a falsepositive result from the realtime quakinginduced conversion assay Prion Manix M Kalakoti P Henry M Thakur J Menger R Guthikonda B CreutzfeldtJakob disease updated diagnostic criteria treatmentalgorithm and the utility of brain biopsy Neurosurg Focus 39E2Martins V R Gomes H R Chimelli L Rosemberg S and Landemberger M C Prion diseases are under compulsory notiï¬cation in Brazil surveillanceof cases evaluated by biochemical andor genetic markers from to Dement Neuropsychol 101590s198057642008dn10400004McGuire L I Peden A H Orrº C D Wilham J M Appleford N EMallinson G Real time quakinginduced conversion analysisof cerebrospinal ï¬uid in sporadic CreutzfeldtJakob disease Ann Neurol 101002ana23589Ministrio da Saºde Protocolfor Notiï¬cation and Investigation ofCreutzfeldtJakob Disease with a Focus on the Identiï¬cation of the New Variant[Internet] Brazil Ministrio da SaºdeOrrº C D Bongianni M Tonoli G Ferrari S Hughson A GGroveman B R A test for CreutzfeldtJakob disease usingnasal brushings N Engl J Med 101056nejmoa131Orrº C D Groveman B R Hughson A G Zanusso G Coulthart M Band Caughey B Rapid and sensitive RTQuIC detection of humancreutzfeldtjakob disease using cerebrospinal ï¬uid mBio 6e0245114Orrº C D Hughson A G Groveman B R Campbell K J Anson K J MancaM Factors that improve RTQuIC detection of prion seedingactivity Viruses 103390v8050140Schmitz M Ebert E Stoeck K Karch A Collins S Calero M Validation of protein as a marker in sporadic creutzfeldtjakob diseasediagnostic Mol Neurobiol Smid J Landemberger M C Bahia V S Martins V R Nitrini R SmidJ Codon polymorphism of prion protein gene in is nota risk factor for Alzheimers disease Arquivos Neuro Psiquiatr 1015900004282x20130055Staï¬aroni A M Kramer A O Casey M Kang H Rojas J C Orrº C D Association of blood and cerebrospinal ï¬uid tau level and otherbiomarkers with survival time in sporadic creutzfeldtjakob disease JAMANeurol Studart Neto A Soares Neto H R Simabukuro M M Solla D J F Gon§ | 2 |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | 0 |
"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjonThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partners occupation and economic statusPatients occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatients marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patients hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patients family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0c" | 2 |
"Immune checkpoint inhibitors ICIs can induce immunerelated adverse events irAEs includingthyroid dysfunction There are only a few reports on Graves disease induced by ICIs We report a case of newonsetGraves disease after the initiation of nivolumab therapy in a patient receiving gastric cancer treatmentCase presentation The patient was a 66yearold Japanese man who was administered nivolumab mg every weeks as a thirdline therapy for stage IVb gastric cancer His thyroid function was normal before the initiation ofnivolumab therapy However he developed thyrotoxicosis before the third administration of nivolumab Elevatedbilateral and diffuse uptake of radioactive tracer was observed in the 99mTcpertechnetate scintigraphyFurthermore the thyroidstimulating hormone receptor antibody TRAb and thyroidstimulating antibody TSAbtest results which were negative before the first administration of nivolumab were positive after starting thetherapy The patient was diagnosed with Graves disease and the treatment with methimazole and potassiumiodide restored thyroid functionConclusions This is the first complete report of a case of newonset Graves disease after starting nivolumabtherapy confirmed by diffusely increased thyroid uptake in scintigraphy and the positive conversion of antibodiesagainst thyroidstimulating hormone receptor It is important to perform thyroid scintigraphy and ultrasonographyto accurately diagnose and treat ICIinduced thyrotoxicosis because there are various cases in which Graves diseaseis developed with negative and positive TRAb titresKeywords Graves disease Nivolumab Thyrotoxicosis Immune checkpoint inhibitor 99mTcpertechnetatescintigraphy Thyroidstimulating hormone receptor antibodyBackgroundImmune checkpoint inhibitors ICIs such as cytotoxicTlymphocyteassociated protein CTLA4 programmed cell death protein1 PD1 and programmeddeath ligand PDL1 inhibitors have been widely usedas a standard cancer treatment during recent yearsimmunerelatedHowever occasionallycauseICIs Correspondence yhiroshinmsacjp1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo JapanFull list of author information is available at the end of the adverse events irAEs which affect different anssuch as the lung gastrointestinal tract liver nervous system skin and endocrine glands The endocrine irAEsinclude hypophysitis thyroid dysfunction adrenal insufficiency and type diabetes While endocrine irAEs dueto CTLA4 inhibitors such as ipilimumab and tremelimumab mainly include pituitary dysfunction those dueto PD1 inhibitors such as nivolumab and pembrolizumab are mainly related to thyroid dysfunction []The PD1 inhibitorinduced thyroid dysfunction oftenincludes hypothyroidism rather than hyperthyroidism [ ] Thyrotoxicosis following ICI therapy is caused The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYamada BMC Endocrine Disorders Page of spontaneouslyrecover withmostly by thyroiditis syndrome which has been reportedsubsequenttohypothyroidism in many cases[ ] HoweverGraves disease induced by ICI treatment has not beenextensively explored Here we present a case of Gravesdisease shortly after the initiation of nivolumab therapyfor gastric cancerthecancerCase presentationA 66yearold man was diagnosed with stage IVbT4bN0M1 human epidermal growth factor receptor HER2positive gastricat Nippon MedicalSchool Chiba Hokusoh Hospital one and a half yearsbefore the onset of thyrotoxicosis After diagnosis hewas not referred for surgery because of liver metastasiswith a portal tumour thrombus rather the patient received cycles of first line chemotherapy with a combination of tegafurgimeraciloteracil S1 cisplatin andtrastuzumab However the patient presented with progressive disease assessed based on the computed tomography CT and oesophagogastroduodenoscopy OGDevaluations following the first line therapy Hence he received a second line chemotherapy with paclitaxel andramucirumab After cycles of the second line chemotherapy although there was a reduction in tumour sizeafter cycles the patient presented with progressivedisease as assessed by CT At this stage nivolumab mg every weeks was started The patient had anormal thyroid function before the first administrationHowever TSH suppression was observed before the second administration and thyrotoxicosis occurred beforethe third administration of the drug hence nivolumabtherapy was discontinued and the patient was referred toour departmentThe patient had complained of fatigue and shortnessof breath during exertion His height was cm bodyweight was kg heart rate was beats per minute and blood pressure was mmHg There wasno evidence of Graves orbitopathy or pretibial myxedema He and his family members had no history ofandTgAbantibody ngmL Thyroidthyroid diseases Thethyroidstimulating hormoneTSH free triiodothyronine FT3 and free thyroxineFT4 levels were μIUmL pgmL and ngdL respectively Table The titres of thyroidstimulating hormone receptor antibody TRAb andthyroidstimulating antibody TSAb were positive IUL and respectively whereas those of antithyroglobulinantithyroidperoxidase antibody TPOAb were negative IULrespectively The thyroglobulin Tgand IULlevel wasultrasonographyshowed slight goitre Fig 1a and rich blood flow in theparenchyma Fig 1b 99mTcpertechnetate scintigraphywhich was performed on the first consultation day of thepatient at our department showed elevated bilateraland diffuse uptake of the radioactive tracer Fig Wemeasured antithyroid autoantibodiesin preservedserum samples The titres of TRAb and TSAb werenegative before the first administration of nivolumabwhereas they were positive IUL and respectively before the second administration Thus we diagnosed his thyrotoxicosis as newonset Graves diseaseafter the initiation of nivolumab therapy The humanleukocyte antigen HLA typing of the patient showedthe following allelic variants A24022601 B510154 C01021502 DRB104051501 DQA1010203 DQB104010602 DPA10202 and DPB10501We treated the patient with methimazole MMI at adose of mgday and potassium iodide KI at a doseof mgday One month after the initiation of the therapy when the FT3 and FT4 levels of the patient werenormal we discontinued KI Gradually we reduced thedosage of MMI and the continued administration tillthe death of the patient of MMI at a dose of mg everyalternate day stabilised his thyroid function Fig Furthermore as nivolumab was found to be ineffectivebased on the CT and OGD evaluations the patient received irinotecan therapy However after cycles ofchemotherapy the patient was diagnosed with brain metastasis by magnetic resonance imaging MRIforTable TSH FT3 FT4 and Tg levels and TRAb and TSAb titres in our patientTSH μIUmLFT3 pgmLFT4 ngdLTRAb IULTSAb Tg ngmLDay of nivolumab administration NA NANA NANANANANADay first administration of nivolumab Day second administration of nivolumabThe normal range of the thyroid parameters is as follows TSH μIUmL FT3 pgmL FT4 ngdL TRAb IUL TSAb and Tg ngmL 0cYamada BMC Endocrine Disorders Page of Fig Thyroid ultrasonography of the patient a Slight swelling in isthmus b Rich blood flow in parenchymawhich he received gamma knife and steroid therapyThe patient died months after his first visit to ourdepartmentDiscussion and conclusionsWe present a case of newonset Graves disease after theinitiation of nivolumab therapy in a patient receivinggastric cancer treatment Thyrotoxicosisinduced byICIs is mainly a form of destructive thyroiditis Threecases of newonset Graves disease during nivolumabtherapy other than the present case have been reported[] Table Iadarola [] reported a case ofGraves diseaselike hyperthyroidism after the second administration of nivolumab in a patient with left lungIn this case 99mTcpertechnetate scintigcarcinomaraphy in the patient with T3toxicosis showed diffusethyroid uptake of the radionuclide suggesting Gravesdiseaselike hyperthyroidism whereas the TRAb testswere consistently negative Thyroid ultrasonographyshowed a multinodular goitre with a normoechoic pattern and normal vascularity ofthe parenchyma []Brancatella [] reported a case similar to that ofIadarola [] with diffuse thyroid uptake and negative TRAb titre In this case ultrasonography showed anenlargement of the thyroid with a hypoechoic patternand mild hypervascularity Kurihara [] reported acase of simultaneous development of Graves disease andtype diabetes mellitus during nivolumab therapy InFig 99mTcpertechnetate scintigraphy showing elevated bilateral and diffuse uptake of the radioactive tracer 0cYamada BMC Endocrine Disorders Page of Fig Clinical course of the patient MMI methimazole KI potassium iodide Day first administration of nivolumab Day secondadministration of nivolumabTable Comparison of case reports on newonset Graves disease during nivolumab therapyStudyTSHμIUmL FT3pgmLFT4ngdLTRAb IULBeforeNAIadarola []AfterNegativeTSAb BeforeNANAAfterNANANAUSNormalHypervascularNormalRAIU99mTcuptakeHigh99mTcHighRAIUNAHLANANADRB104Brancatella []NANegativeKurihara []NAPositive NAYamada presentcaseUS ultrasonography RAIU radioactive iodine uptake Before before the initiation of nivolumab therapy After after the initiation of nivolumab therapy at theonset of the thyrotoxicosisNegative NegativeHypervascular PositivePositiveHigh99mTcDPB105 0cYamada BMC Endocrine Disorders Page of this case thyrotoxicosis was detected after the sixth administration of nivolumab with positive TRAb titreHowever ultrasonography showed no enlargement ofthe thyroid and a normal vascularisation pattern Thispatient was clinically diagnosed as mild Graves diseaseand treated with MMI [] Unlike these cases our caseis important in terms of confirmation of both positiveTRAb titre and diffuse thyroid uptake in scintigraphyMoreover titres of TRAb and TSAb were convertedfrom negative to positive after starting nivolumab therapy It seems reasonable to presume that Graves diseasewas induced by nivolumab although there is a possibilityof coincidence Furthermore our patient had HLADPB10501 which has been reported to be associatedwith Japanese Graves disease [ ] Although the involvement of HLA cannot be argued based only on asingle case accumulating similar cases might help clarifythe mechanism of development of rare ICIinducedGraves diseaseGraves disease induced by ICIs other than nivolumabhas been rarely reported Azmat [] reported aipilimumabinduced thyrotoxicosis caused bycase ofGraves disease Gan et al[] reported a case oftremelimumabinduced Graves hyperthyroidism Yajima [] reported a case of Graves disease induced bypembrolizumab a PD1 inhibitor In this case TRAbwas positive after the fifth administration of pembrolizumab and thyroid ultrasonography showed a mild increase in the intrathyroidal blood flow A thyroidscintigraphy was not performed because of the iodinetreatment [] The cases of nivolumabinduced Gravesdisease with negative TRAb titre suggest that performingthyroid scintigraphy and ultrasonography can help to accurately diagnose and treat ICIinduced thyrotoxicosisA relationship between thyroid antibodies and PD1inhibitorinduced thyroid dysfunction has not been explained Kimbara [] suggested that patients withpreexisting TgAb and an elevated TSH level at baselineare at a higher risk of thyroid dysfunction induced bynivolumab Osorio [] reported an association between positive thyroid antibodies antithyroglobulin orantimicrosomal antibodies and thyroid dysfunction induced by ICIs In the studies on newonset Graves disease during nivolumab therapy it is interesting to notein two caucasian patients with Graves diseasethatTRAb was negative [ ] Furthermore TRAb waspositive in two Japanese patients including our patient[] Table However additional evidence is requiredto reveal the role of TRAb in the pathogenesis of ICIinduced hyperthyroidismA limitation of our case was radioactive iodine uptakeRAIU was not performed Because imaging with99mTcpertechnetate reflects both blood flow and uptakevia the symporter and does not assess anificationmalignant nodules may appear hyperfunctioning in pertechnetate imaging but hypofunctioning in 123IimagingIn our study tumours were not detected although a partof 99mTc uptake was strongerIn conclusion we reported a case of Graves diseaseshortly after the initiation of nivolumab therapy for gastric cancer Our case presented a typical Graves diseasewith both positive TRAb titre and diffuse thyroid uptakein scintigraphy Moreover our case is valuable in termsof confirming the conversion of TRAb and TSAb fromnegative to positive titres after starting the therapy It isimportant to perform thyroid scintigraphy and ultrasonography because there are cases of nivolumabinducedGraves disease with negative TRAb titre as previouslyreported To revealthe pathogenesis of ICIinducedGraves disease it is necessary to study additional casesof similar natureAbbreviationsCT Computed tomography CTLA4 Cytotoxic Tlymphocyteassociated protein FT3 Free triiodothyronine FT4 Free thyroxine HER2 Humanepidermal growth factor receptor HLA Human leukocyte antigenICI Immune checkpoint inhibitor IrAE Immunerelated adverse eventKI Potassium iodide MMI Methimazole MRI Magnetic resonance imagingOGD Oesophagogastroduodenoscopy PD1 Programmed cell deathprotein1 PDL1 Programmed death ligand RAIU Radioactive iodineuptake Tg Thyroglobulin TgAb Antithyroglobulin antibody TPOAb Antithyroidperoxidase antibody TRAb TSH receptor antibody TSAb Thyroidstimulating antibody TSH Thyroidstimulating hormoneAcknowledgmentsNot applicableAuthors contributionsHY FO and NE interpreted the data drafted the manuscript andparticipated in the endocrinological treatment of the patient HS revised themanuscript TO and SF participated in the gastroenterological treatment ofthe patient All authors have read and approved the final version of themanuscript for publicationFundingNot applicableAvailability of data and materialsThe data that support the findings of this study are stored in NipponMedical School Chiba Hokusoh Hospital Inzai Chiba and available from thecorresponding author on reasonable requestEthics approval and consent to participateThis case report was approved by the ethics committee of Nippon MedicalSchool Chiba Hokusoh HospitalConsent for publicationWritten informed consent was obtained from the patients next of kin forpublication of this case report and any accompanying images A copy of thewritten consent is available for review by the editor of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo Japan 2Department of Gastroenterology Nippon Medical SchoolChiba Hokusoh Hospital Kamagari Inzai Chiba Japan 0cYamada BMC Endocrine Disorders Page of Received April Accepted August ReferencesGonzalezRodriguez E RodriguezAbreu D Spanish Group for CancerImmunoBiotherapy GETICA Immune checkpoint inhibitors review andmanagement of endocrine adverse events Oncologist Michot JM Bigenwald C Champiat S Collins M Carbonnel F PostelVinay S Immunerelated adverse events with immune checkpoint blockade acomprehensive review Eur J Cancer Bertrand A Kostine M Barnetche T Truchetet ME Schaeverbeke T Immunerelated adverse events associated with antiCTLA4 antibodies systematicreview and metaanalysis BMC Med Faje A Immunotherapy and hypophysitis clinical presentation treatmentand biologic insights Pituitary Topalian SL Hodi FS Brahmer JR Gettinger SN Smith DC McDermott DF Safety activity and immune correlates of antiPD1 antibody in cancerN Engl J Med Robert C Schachter J Long GV Arance A Grob JJ Mortier L et alPembrolizumab versus ipilimumab in advanced melanoma N Engl J MedOrlov S Salari F Kashat L Walfish PG Induction of painless thyroiditis inpatients receiving programmed death receptor immunotherapy formetastatic malignancies J Clin Endocrinol Metab Kimbara S Fujiwara Y Iwama S Ohashi K Kuchiba A Arima H et alAssociation of antithyroglobulin antibodies with the development ofthyroid dysfunction induced by nivolumab Cancer Sci Iadarola C Croce L Quaquarini E Teragni C Pinto S Bernardo A et alNivolumab induced thyroid dysfunction Unusual clinical presentation andchallenging diagnosis Front Endocrinol Lausanne Brancatella A Viola N Brogioni S Montanelli L Sardella C Vitti P et alGraves' disease induced by immune checkpoint inhibitors a case reportand review of the literature Eur Thyroid J Kurihara S Oikawa Y Nakajima R Satomura A Tanaka R Kagamu H et alSimultaneous development of Graves' disease and type diabetes duringantiprogrammed cell death1 therapy a case report J Diabetes Investig Dong RP Kimura A Okubo R Shinagawa H Tamai H Nishimura Y et alHLAA and DPB1 loci confer susceptibility to graves disease Hum Immunol Ueda S Oryoji D Yamamoto K Noh JY Okamura K Noda M et alIdentification of independent susceptible and protective HLA alleles inJapanese autoimmune thyroid disease and their epistasis J Clin EndocrinolMetab Azmat U Liebner D JoehlinPrice A Agrawal A Nabhan F Treatment ofipilimumab induced graves disease in a patient with metastatic melanomaCase Rep Endocrinol Gan EH Mitchell AL Plummer R Pearce S Perros P Tremelimumab inducedgraves hyperthyroidism Eur Thyroid J Yajima K Akise Y A case report of Graves' disease induced by the antihuman programmed cell death1 monoclonal antibody pembrolizumab ina bladder cancer patient Case Rep Endocrinol Osorio JC Ni A Chaft JE Pollina R Kasler MK Stephens D Antibodymediated thyroid dysfunction during Tcell checkpoint blockade in patientswith nonsmallcell lung cancer Ann Oncol Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
"high mobility group box hmgb1 is a nonhistone chromatinassociated protein widely distributed in eukaryoticcells and is involved in dna damage repair and genomic stability maintenance in response to stimulus like bacteriaor chemoradiotherapy hmgb1 can translocate to extracellular context as a danger alarmin activate the immuneresponse and participate in the regulation of inflammation and cancer progressionkeywords hmgb1 rage tlr damp inflammation cancerchromatinassociated proteinit washigh mobility group box hmgb1 is a highly conservative nucleoprotein and belongs to the group of nonhistonefirstextracted from calfthymus chromatin in andnamed for its high mobility in gel electrophoresis subsequentinvestigations found that hmgb1 couldtranslocate from the nucleus to the cytoplasm after posttranslational modifications including acetylation phosphorylation and methylation hmgb1 can be expressedat the neuron membrane as well in response to chemoradiotherapy or hypoxia hmgb1 could be transferredto the extracellular context mainly through two waysactive secretion from immunocompetent cells or passiverelease from apoptotic or necrotic cells extracellularhmgb1 transmits danger signals to surrounding cellsby interacting with its classical receptors such as thereceptor for advanced glycation end products rageand tolllike receptors tlr249indepth studies implicated that hmgb1 was a multifunctional protein involved in a variety of cellularsubcellularbiological properties depending on itslocalizationandbinding receptors fig posttranscriptional modification correspondence yizhangzzueducn1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china2state key laboratory of esophageal cancer prevention treatmentzhengzhou university zhengzhou chinafull list of author information is available at the end of the in the nucleus hmgb1 plays a key role in the processof dna replication transcription chromatin remodeling and vdj recombinationthus regulating dnadamage repair and the maintenance of genome stabilityas a dna chaperone cytoplasmic hmgb1 is involvedin immune responses by increasing autophagy inhibitingapoptosis and regulating mitochondrial function atthe membrane hmgb1 promotes axonal sprouting andneurite growth activates platelets and induces cellmigration as a typical damage associated molecular pattern damp extracellular hmgb1 is involved in manyimmune responses by promoting immune cell maturation activation and cytokine production extracellular hmgb1 can also interact with chemokines such ascxcl11 to enhance immune responses as a multifunctional protein hmgb1 exerts different biologicaleffects under different stimuli the deregulation ofhmgb1 is associated with many diseases especiallyinflammatory disorders and cancerhmgb1 in inflammationhmgb1 plays a critical role in the regulation of bothinnate and adaptive immune responses to promoteimmune response to sterile or infectious stimulus insterile inflammation during ischemiareperfusion injuryiri hmgb1 becomes disulfidebonded to increasemacrophage production of proinflammation cytokinesin a tlr4 dependent manner indicating that disulfidebonded hmgb1 can be identified as a diagnosis and the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang and zhang of hematology oncology page of fig the multifunctions of hmgb1 hmgb1 in the nucleus regulates dna damage repair and genome stability as a nonhistone chromatinassociated protein and dna chaperon in the cytoplasm or mitochondria hmgb1 increases autophagy inhibits apoptosis and regulatesmitochondria functions at the membrane hmgb1 promotes axonal sprouting and neurite growth hmgb1 can be transferred to extracellularcontext by two ways active secretion from immunocompetent cells or passive release from apoptotic or necrotic cells participating inimmune responsesin contrasttreatment biomarker for iri when initially secreted byinnate immune cells like macrophages hmgb1 is proinflammatory during the early stages of sepsis howeverthe extracellular hmgb1 could also induce immune tolerance and immunosuppression when released by othersomatic cellsintracellular hmgb1 caninduce protective autophagy and contribute to cellsurvival by delivering lipopolysaccharide lps andpromoting endocytosis hmgb1 activates the noncanonical inflammasome pathway and induces pyroptosis pyroptotic macrophage death may accelerateundesirable immune hyperactivity and immunosuppression which is a potential mechanism associatedwith late mortality from sepsis in hepatic infectious disease the release and activity of hmgb1 as acytokine could be suppressed by glycyrrhizinic acidga by binding with tlr4 hmgb1 regulatesthe hepatitis virusesinduced immunological axis providing a new therapy strategy for the treatment ofacute viral hepatitis in the clinical practice canbesecretedin severe pulmonary inflammatory diseases including covid19 hmgb1inabundance by necrotic pulmonary epithelial cells andinnate immune cells disulfidehmgb1 triggers proinflammatory cytokine release and further exacerbatessevere inflammation therefore hmgb1 mightbeofinflammationtreatmentpotentialtargetaforthethe dual effects of hmgb1 in canceraccording to the alterations of the subcellular locationsreceptors and expression levels hmgb1 is associatedwith the hallmarks of cancer proposed by hanahan andweinberg hmgb1 appears to play paradoxicalroles during the development and therapy of cancer onthe one hand hmgb1 can contribute to tumorigenesisexcessive hmgb1 production caused by chronic inflammatory response seems to be associated with tumorigenesis for example by combining with rage hmgb1plays an important role in regulating oval cells activationand inflammationassociated liver carcinogenesis in mice in established cancers hmgb1 produced by tumorcells may exacerbate inflammationrelated immunosuppression for instance previous research indicated thatlps induced the release of proinflammatory cytokinessuch as il1 il6 and tnfα in a hmgb1dependentmanner to improve colon cancer progression however the underlying mechanism of hmgb1 in the transformation ofinflammation and cancer needs to befurther studied it has been reported that hmgb1 canbe released to extracellular context by necrotic cellsunder hypoxia in growing solid tumor extracellularhmgb1 promotes the release of cytokines such as il6and il8 by activating mapk and myd88dependentnfκb pathways which in turn stimulates tumor cellsproliferation angiogenesis emt invasion and metastasis nucleusand cytoplasmic hmgb1 promotes 0cwang and zhang of hematology oncology page of autophagy and inhibits apoptosis of tumor cells to induce chemotherapy resistance on the other handhmgb1 plays a protective role in the suppression oftumor and tumor chemoradiotherapy and immunotherapy nucleus hmgb1 assists in the regulation of telomere and maintenance of genome stability loss ofhmgb1 results in the instability of genome and leads totumorigenesis thus the roles of hmgb1 in regulationof dna damage repair and cancer etiology indicate thattargeting chromosomal architectural hmgb1 may provide a new perspective for cancer therapy hmgb1located in the cytosol or mitochondria may bind toautophagy associated genes like beclin to regulate cellautophagy and mitophagy absence of hmgb1 resultsin autophagy deficiency and increased apoptosis leadingto tumorigenesis intracellular hmgb1 functions as atumor suppressor by binding tumor suppressor proteinslike rb but it remains to be studied whether hmgb1interacts with other tumor suppressors or oncoproteinsextracellular hmgb1 enhances chemotherapy efficacyby transforming tumor cells from apoptosis to senescence in addition hmgb1 can mediate immunogenic cell death during chemoradiotherapy and enhanceantitumor immunity in response to chemotherapy likeanthracycline or radiotherapy hmgb1 can be rapidlyreleased from dead cells as an alarming molecule uponrelease from necrotic cells or secreted by activated macrophages hmgb1 can recruit inflammatory cells andmediate interactions between nk cells dendritic cellsdcs and macrophages activated nk cells provide anadditional source of hmgb1 which is released into theimmunological synapse between nk cells and immaturedcs promoting the maturation of dcs and the induction of th1 response in addition hmgb1 produced from nsclc cells induced by docetaxel canstimulate t cells for antitumor immune response andimprove immunotherapy effects like cart cells therefore modulating hmgb1 may provide a potentialcombination strategy for cancer chemoradiotherapy andimmunotherapyconclusionhmgb1 is a multifunctional molecule that plays a majorrole in homeostasis as damp molecule hmgb1 playsthe complex roles in various biological processes and isinvolved in the development of many diseases such asautoimmune diseases and cancers hmgb1targetedagents including antibodies and inhibitors have shownbeneficial results in preclinicalinflammatory modelssuch as sepsis these agents await clinical developmentabbreviationshmgb1 high mobility group box rage the receptor for advancedglycation end products tlr tolllike receptors damp damage associatedmolecular pattern iri ischemiareperfusion injury lps lipopolysaccharidega glycyrrhizinic acid covid19 coronavirus disease19 mapk mitogenactivated protein kinase myd88 myeloid differential protein88 nfκb nuclear factor kappalightchainenhancer of activated b cellsemt epithelialmesenchymal transition dcs dendritic cellsacknowledgementsnot applicableauthorsâ contributionsyz designed directed and revised the manuscript sw drafted themanuscript both of the authors read and approved the final manuscriptfundingthis work was supported by the national key research and developmentprogram of china 2016yfc1303500availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china 2state key laboratory ofesophageal cancer prevention treatment zhengzhou universityzhengzhou china 3henan key laboratory for tumor immunologyand biotherapy zhengzhou chinareceived july accepted august referencesgoodwin gh sanders c johns ew a new group of chromatinassociatedproteins with a high content of acidic and basic amino acids[j] febs jâpaudel yn angelopoulou e piperi c balasubramaniam vrmt othman ishaikh mf enlightening the role of high mobility group box hmgb1 ininflammation updates on receptor signalling[j] eur j pharmacol huebener p gwak gy pradere jp et al highmobility group box isdispensable for autophagy mitochondrial quality control and anfunction in vivo[j] cell metab ârivera vargas t apetoh l danger signals chemotherapy enhancers[j]immunol rev âgao q wang sm chen xf et al cancercellsecreted cxcl11 promotedcd8 t cells infiltration through docetaxelinducedrelease of hmgb1 innsclc[j] for immunotherapy of cancer andersson u tracey kj hmgb1 is a therapeutic target for sterileinflammation and infection[j] annu rev immunol âkim hm kim ym hmgb1 lps delivery vehicle for caspase11mediatedpyroptosis[j] immunity âdeng m tang y li w et al the endotoxin delivery protein hmgb1mediates caspase11dependent lethality in sepsis[j] immunity â753e747shi xd yu lj zhang yl et al glycyrrhetinic acid alleviates hepaticinflammation injury in viral hepatitis disease via a hmgb1tlr4 signalingpathway[j] int immunopharmacol saha b tornai d kodys k et al biomarkers of macrophage activation andimmune danger signals predict clinical outcomes in alcoholic hepatitis[j]hepatology baltimore md â andersson u ottestad w tracey kj extracellular hmgb1 a therapeutictarget in severe pulmonary inflammation including covid19[j] molecularmedicine cambridge mass 0cwang and zhang of hematology oncology page of hanahan d weinberg ra hallmarks of cancer the next generation[j] cellâ pusterla t nèmeth j stein i et al receptor for advanced glycationendproducts rage is a key regulator of oval cell activation andinflammationassociated liver carcinogenesis in mice[j] hepatologybaltimore md â yang y yang l jiang s et al hmgb1 mediates lipopolysaccharideinducedinflammation via interacting with gpx4 in colon cancer cells[j] cancer cellint yuan s liu z xu z et al high mobility group box hmgb1 a pivotalregulator of hematopoietic malignancies[j] j hematol oncol mukherjee a vasquez km targeting chromosomal architectural hmgbproteins could be the next frontier in cancer therapy[j] cancer res liu y dong y kong l et al abscopal effect of radiotherapy combined withimmune checkpoint inhibitors[j] j hematol oncol publisherâs notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
immunerelated genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12 Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ¥ ¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficientScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and Chinas low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25 Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ¥ ¥ ¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29 Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34 However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37 there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGFβ can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42 Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47 while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes BH In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine plenty to be done Carcinogenesis 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira ThaÃs C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can | 2 |
"These alleles always augmented germline genotype in UNCeqR thus preventing somatic mutation detections with these alleles even if unobserved in a given germline sequencing. Mutation annotation and analysis Sequence mutations were annotated with a gene a predicted transcript and protein alteration using Annovar (version 8/23/13) (42) and RefSeq gene models. Non-silent mutations referred to non-silent substitution insertion and deletion mutations within translated regions and splice-site mutations. MAFs were compared by one-sided Fisher's exact tests on mutant versus germline read counts with significant results having false discovery rate < 5%. Sequence alignments were visualized using the Integrative Genomics Viewer (43). Germline variant analysis Patient germline variants relative to the reference genome were detected in germline DNA-WES and patient-matched germline RNA-seq using UNCeqRMETA without population polymorphism or mapping artifact allele augmentation P ? 1.1e?9. Germline variant allele fractions were defined and compared between DNA and RNA using the procedure described for somatic mutations. Simulation analysis A novel simulation strategy was followed (diagrammed in Supplementary Figure S2). Using chromosome 2 simulated tumor genomes were generated by randomly sampling 500 sites from exons to define positive mutation sites while the remainder of exon sites served as negative mutations. For the positive sites mutant alleles (substitution insertion or deletion) were randomly sampled at rates 90 5 and 5%. For insertion and deletion alleles allele lengths of 16 were randomly sampled at rates 602095 5 and 1%. Positive mutations were spiked into germline DNA-WES and RNA-seq sequencing by editing a specified MAF of read alignments overlapping the site producing simulated tumor alignments. V characters were used for substitutions and insertions to avoid overlap with germline genotype. Simulated tumor alignments contained a subset of the total positive mutations because the alignment may have minimal or zero depth at some positive sites reflecting reality that a sequencing technology does not cover every site in the genome at high depth and enabling simulated mutations to occur at RNA-seq and DNA-WES uniquely covered sites. Original tumor sequencing served as simulated germline sequencing. Simulated germline sequencing contained the original somatic mutations which had the effects of expanding germline genotype with additional alleles and not triggering variant detection. UNCeqR models were applied to these simulated data. Limiting to sites with at least a germline depth of 10 model detections were compared to the truth to define receiver operating characteristic (ROC) curves (44). A pair of models was compared by their difference in area under the curve over the false positive rate range of 0 to 1 10?5. A P-value was defined using a distribution of differences in area under the curve calculated from 100 permuted models in which the rank of the discrimination threshold (i.e. P-value) between the models at each genomic site was randomly shuffled. Mutation detection by other programs Strelka v2.0.8 (17) was executed on tumor and germline DNA-WES using recommended settings for BWA alignments (strelka_config_bwa_default.ini) DNA-WES (isSkipDepthFilter = 1) and filtering (passed). SNVMix2 (13) was executed upon RNA-seq using default settings. Validation analysis Within exonic regions true positive and false positive mutation detections were defined using patient-matched DNA-WGS alignments based on a published procedure for exome mutation validation (4). Tumor and germline DNA-WGS BAM files were downloaded from ://cghub.ucsc.edu. Specifically tumor and germline DNA-WGS were interrogated at each predicted mutation using samtools (31) with no filtering. True positive mutation predictions met one of two conditions: (1) germline depth ? 10 and read count of predicted mutant allele ?1 in tumor and zero in germline; or (2) germline depth ?10 proportion of mutant allele in germline sequencing not significantly > 2% (proportions test P > 0.25) and proportion of mutant allele in tumor significantly greater than in germline (proportions test P < 0.05). Otherwise false positive mutation predictions had germline DNA-WGS depth ?10 and had depth in tumor DNA-WGS providing ?80% power to detect the mutant allele based the predicted MAF. Power was estimated by a binomial distribution a null probability of 3 10?3 an alpha of 0.05 the observed depth in DNA-WGS and an alternate probability of the predicted DNA MAF. The number of true positives and false positives were tabulated at each model discrimination threshold i.e. P-value or score. The step function of these points (number of false positives versus number of true positives) generated a performance curve in absolute counts that is equivalent to a ROC curve without the denominators of total positives and negatives which were constant and unknown for the validation cohort. Between models performance curves were compared by area under the curve from 0 to 3000 false positives and by the number of true positives (proportional to sensitivity) at fixed numbers of false positives (proportional to 1 ? specificities) of 250 500 and 1000). P-values were calculated to provide evidence for the change in area under the curve and sensitivity estimates using permutation (see Simulation analysis methods). RESULTS Mutation detection models Existing methods to detect somatic mutations are based on either DNA sequencing alone or on RNA sequencing alone and do not integrate more than one type of sequencing (91317). In order to test whether integrating DNA-WES and RNA-seq enables superior somatic mutation detection versus the current standard of DNA-WES alone a new method was developed called UNCeqR. UNCeqR contains different models for detecting somatic mutations based on different sequencing input and statistical modeling. Briefly UNCeqRMETA integrates tumor DNA-WES and RNA-seq UNCeqRDNA uses tumor DNA-WES and UNCeqRRNA uses tumor RNA-seq. UNCeqR software is available at http://lbg.med.unc.edu/tools/unceqr. Evaluation in simulated tumor sequencing To test our hypothesis that somatic mutation detection based on integrated RNA-seq and DNA-WES is superior to that based on DNA-WES alone simulated tumor genomes were generated so that the entire genome space is a completely defined truth of positive and negative somatic mutations. In brief for each patient's sequencing 500 mutant sites were sampled for each site a mutant allele was randomly sampled and then aligned reads in the real RNA-seq and DNA-WES were edited to have the mutant allele at a rate of a fixed MAF (Supplementary Figure S2). By using real sequencing as the basis of the simulation authentic sequencing depths random errors (sequencing and alignment) and patients germline variants were preserved. Sequencing from the lung cancer quadruplet cohort was used for simulation. Patients DNA-WES and RNA-seq had large and similar numbers of sequenced nucleotides (DNA-WES median: 10.6 billion RNA-seq median: 10.2 billion; Kruskal-Wallis P = 0.54) indicating no significant imbalance in total sequencing. UNCeqR models were applied to the simulated tumor sequencing and detected mutations were compared against the truth by receiver operating characteristic curves. In simulations with a 10% MAF (A) the UNCeqRMETA model had significantly superior performance over UNCeqRDNA (difference in area under the curve P < 0.01); in other words UNCeqRMETA achieved a greater true positive rate (greater sensitivity) at the same false positive rate (same specificity) than UNCeqRDNA. In simulations with a 20% MAF (B) UNCeqRMETA continued to be superior to UNCeqRDNA (difference in area under the curve P < 0.01) although the gain in 20% MAF simulations was less (roughly 50% less) than the gain in 10% MAF simulations. This demonstrates that adding RNA-seq improved sensitivity particularly when the mutation signal that is MAF was low. UNCeqRMETA and UNCeqRDNA had large and clear superior performance to UNCeqRRNA which incurred false positives at a higher rate. Alternative ways to integrate RNA and DNA (taking the union or intersection of UNCeqRDNA and UNCeqRRNA) were both inferior to UNCeqRMETA (Supplementary Figure S3). Therefore in simulation UNCeqRMETA achieved superior performance over UNCeqRDNA with the largest gains occurring in mutations with low MAF. . Mutation detection performance in simulated tumor genomes. Model performance is displayed as receiver operating characteristic curves. Sensitivity plateaus below 1 because simulated mutations include sites with zero tumor sequencing depth in DNA and/or RNA (see Simulation analysis methods). Validation by whole genome sequencing To validate the superior performance of integrated DNA-WES and RNA-seq mutation detection (UNCeqRMETA) over DNA-WES only detection (UNCeqRDNA) tumor and germline whole genome DNA sequencing (DNA-WGS) was used as an independent measure of truth for evaluating DNA-WES and RNA-seq mutation detections. Following a published validation procedure (4) mutation detections were interrogated in patient-matched DNA-WGS to determine if a mutation detection was a true positive that is present in the tumor specimen and absent from the germline specimen or false positive that is absent from the tumor specimen or present in the germline specimen. For each mutation model true positives and false positives were summed at each discrimination threshold (e.g. P-value) to generate a performance curve by which true positive rates could be compared at the same false positive rates (see methods for further description). These curves demonstrated that UNCeqRMETA achieved overall superior performance than UNCeqRDNA (difference in area under the curve P < 0.01) and at fixed false positive thresholds (250 500 and 1000) thus validating the result from simulated tumor genomes (Figure 2). Therefore in real tumor sequencing integrated DNA and RNA mutation detection by UNCeqRMETA outperformed DNA-only mutation detection. Figure 2. Validation of mutation detection by whole genome sequencing. The number of true positives and false positives of mutation detection models are plotted as step functions. At fixed false positive totals (250 500 or 1000) each pair of models was compared for differences in number of true positives (*). The published mutation set (46) did not include mutation rankings and was not amenable to rank-based statistical analysis. Other models displayed overall reduced performance relative to UNCeqRMETA and UNCeqRDNA. As another DNA-only control a leading (45) DNA-WES mutation caller from Illumina Strelka (17) was run on the same DNA-WES. Strelka exhibited inferior performance overall smaller true positive rates at fixed false positive rates and never achieved the sensitivity of UNCeqRMETA or UNCeqRDNA (Figure 2). Strelka had greater sensitivity than UNCeqRMETA or UNCeqRDNA at the highest extreme of specificity; however at UNCeqR's minimum false positive rate Strelka's sensitivity was only ?70% of either UNCeqR model. Providing another DNA-only control previously published mutations of this cohort made by heterogeneous pipelines (4691516) had reduced sensitivity than UNCeqRMETA and UNCeqRDNA at the same false positive rate (256 false positives). At this false positive rate indel mutation detections were rare in all models (maximum 1.7%) with UNCeqRMETA and UNCeqRDNA having no significant difference in indel precision (number of true positives divided by the sum of false positives and true positives 92 and 96% respectively) but both having greater indel precision than Strelka (83%) and previously published mutations (82%) (proportions test P < 0.001). Taking the union or intersection of UNCeqRDNA and UNCeqRRNA had higher false positive rates and inferior performance than UNCeqRMETA or UNCeqRDNA (Supplementary Figure S4A). Integrating Strelka with an RNA-seq mutation detector SNVmix did not result in superior performance versus Strelka UNCeqRDNA or UNCeqRMETA (Supplementary Figure S4A). Providing a separate source of validation UNCeqRMETA detected nearly all mutations that were published as validated by targeted resequencing within this cohort (up to 97% depending on the model threshold; Supplementary Figure S5). Repeating this analysis with a slightly increased true positivity definition minimum two confirming tumor WGS DNA reads maintained all findings listed above (Supplementary Figure S4B). Increased mutation signal in RNA-seq To analyze integrated mutation detection across larger cohorts UNCeqR was applied to the lung and breast triplet cohorts (n = 871) and using model thresholds with the same empirically estimated specificity (500 false positives in DNA-WGS validation sequencing marked as triangle point in Figure 2 UNCeqRMETA P-value ? 1.1 10?9 UNCeqRDNA P-value ? 9.3 10?9)." | 1 |
objectives paired box protein8 pax8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian cancers between december and may by studying their formalinfixed paraffin embedded blocksresults sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range cervix was the most common cancer site in patients regarding cancer stage there was and of the study population had stage 3b and 2b respectively the histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma scc as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively pax8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated scc all patients diagnosed with well differentiated scc and metastatic adenocarcinoma showed no expression of pax8 a statistically significant was seen for pax8 expression and the different histopathological diagnosis p value keywords female reproductive cancer paired box protein8 immunohistochemical expressionintroductionpaired box protein8 pax8 is a member of the family paired box proteins paxs [ ] pax8 consists of amino acids with a molecular weight of approximately kilo dalton and its molecular properties are located on chromosome 2q13 [] pax8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] during the embryonic period pax8 also plays a significant role correspondence nouh_saadoutlookcom alfarrabi college for science and technology khartoum sudanfull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the m¼llerian ducts [] in a previous experiment the deletion of the pax8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal opening also resulted in poor development of the myometrial tissue several studies have described the immunohistochemical utility of pax8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]in a healthy female reproductive tract pax8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] pax8 was found to be expressed among endometrioid carcinomas transitional the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cali a0et a0al bmc res notes page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] whereas for the ovarian carcinomas pax8 was under expressed considering that few studies have investigated the immunohistochemical expression of pax8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from sudan yet [ ] this study aimed at investigating the immunohistochemical expression of pax8 among sudanese females diagnosed with cervical endometrial and ovarian carcinomasmain textmaterials and a0methodsstudy design and a0population characteristicsthis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from december till may in khartoum state sudan we retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas the retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis the participants demographic data was collected including age place of residence the clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedsections preparation for a0immunohistochemistry stainingtwo sections were cut using rotary microtome leica germany from each histopathological block then one slide was stained by hematoxylin and eosin staining technique the other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry to retrieve pax8 tissues antigen we treated the sections with citrate buffer at ° a0c for a0min in a waterbath then the tissue sections were rinsed first in distilled water and later with tris buffer saline tbs this was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity the slides were then placed in a humid chamber then the slides were drained and rinsed in two successive changes of tris buffer wash buffer for a0 min each nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min then the remaining solution was drained from the slides the sections were then incubated in the primary antibody pax8 antipax8 rabbit antihuman monoclonal antibody ab189249 abcam united kingdom at room temperature in the humid chamber according to the manufacture instructionsobserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the pax8 for the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline pbsresults interpretationsfor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories negative no staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker the slides were interpreted and validated by two expert pathologists blindly of each other results photomicrographs were taken using olympus sp350 camera olympus imaging america inc usastatistical analysisthe statistical analysis of the results was done using ibm spss statistics vs the chisquared test was performed to compare the frequencies of categorical variables statistical significance level was defined as p value at confidence intervalresultscharacteristics of a0the a0study participantsthe study included patients diagnosed with female genital tract cancer patients aged ± a0years range a0years patients were grouped into age groups those aged a0 years constituted half of the study participants the remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively according to patients place of residence patients were originating from the four regions of sudan most of the patients were from western part of sudan followed by from the central part of sudanregarding the site of cancer the cervix was the most commonly involved patients there were and endometrial and ovarian cancer respectively based on the international federation of gynecology and obstetrics figo cancer grading the majority of the study population was diagnosed with stage 3b and 2b cancer and of the patients respectively the were and stage 4b 3a 2a 1b and 4a respectively 0cali a0et a0al bmc res notes page of no statistically significant association between figo staging and age group was found p value histologically there were squamous cell carcinoma scc all of which were cervical cancers and adenocarcinoma scc and adenocarcinoma were further classified into poorly differentiated scc moderately differentiated scc and well differentiated scc endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomabased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type figo staging and cancer histopathological type table a0immunohistochemical expression of a0paxthe immunohistochemical expression of pax8 was shown as a yellowishbrown or brown staining of the nucleus fig a0 based on site of cancer all endometrium carcinoma showed positive expression of pax8 with p value there were only patients who had positive expression of pax8 including adenocarcinoma and scc a statistically significant difference was noted for the pax8 staining and cancer type with p value the analysis of pax8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated scc and metastatic adenocarcinoma had negative results for the pax8 expression while of the endometrium adenocarcinoma were found positive for the pax8 expression a statistically significan was t seen for pax8 expression and the different histopathological diagnosis p value table a0table classification of a0participants demographic and a0clinical diagnosis based on a0age groupage group no total no p value a0years a0years a0years a0yearsresidence of patient central sudan east sudan west sudan north sudansite of cancer cervix endometrium ovarycancer histological type scc adenocarcinomafigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bhistopathological cancer grades well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma 0cali a0et a0al bmc res notes page of fig immunohistochemical expression of pax8 among the different histopathological cancer types and grades the immunohistochemical expression of pax8 is shown as a yellowishbrown or brown staining of the nucleus a well differentiated scc negative b metastatic adenocarcinoma negative c poorly differentiated scc positive d moderately differentiated scc positive e endometrium adenocarcinoma positive f ovarian mucinous cystadenocarcinoma positive g endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cali a0et a0al bmc res notes page of table association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0pax8pax results no total no p valuepositivenegativecancer histological type scc adenocarcinomacancer site cervix endometrium ovaryfigo staging stage stage 2a stage 2b stage 3a stage 3b stage 4a stage 4bcancer histopathological grading well differentiated scc poorly differentiated scc moderately differentiated scc endometrium adenocarcinoma endocervical adenocarcinoma metastatic adenocarcinoma ovarian mucinous cyst adenocarcinomascc squamous cell carcinoma discussionprevious studies on the immunohistochemical expression of pax8 in the normal female reproductive tract showed that pax8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] this study investigated the immunohistochemical expression of pax8 in sudanese patients who were diagnosed with female reproductive tract cancers patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer however previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]regarding the place of residence the majority of patients coming from western sudan this result is in contrary with a previous study in sudan conducted by saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern sudan were higher compared to the other regions in sudan nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresthe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer similar results were seen previously among sudanese females also the high frequency of stages 3b and 2b compared to the other stages were comparable to previous study conducted in sudan this similarity underscores a delayed response among sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentregarding the classification based on the histopathological diagnosis most of the female diagnosed with scc this result was also similar to previous study investigated the prevalence of the different gynecologic cancer in sudan however the expression of pax8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cali a0et a0al bmc res notes page of to the site of cancer development while agrees with another study where pax8 was expressed only in patient interestingly a high frequency of pax8 expression was noted among females diagnosed with endometrium cancer compared to scc this finding is in contrary with a previous report where pax8 was expressed among only of the studied samples also the result was strongly in accordance with other studies [ ] besides that the lack of pax8 expression among those who were diagnosed with well differentiated scc and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]conclusionalthough pax8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated scc and metastatic adenocarcinoma pax8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractlimitations¢ the small sample size investigated in this study reduced the ability of using the expression of pax8 as a diagnostic marker therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemacknowledgementsthe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyauthors contributionseta nsm and ees provided conceptual framework for the study guidance for interpretation of the data and performed data analysis eta ees irs lah and amm performed laboratory work nsm ees msm aay and aa performed the statistical analysis nsm msm ees and aa participated in the manuscript preparation revision and coordination all authors read and approved the final manuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participateethical approval was obtained from the research ethics committee of the faculty of medical laboratory sciences university of khartoum sudan ethical approval no fmlsrec002042 all participant approved to participate by signing an informed consentconsent for publicationnot applicablecompeting interestsno competing interests to discloseauthor details department of histopathology and cytology faculty of medical laboratory sciences university of khartoum khartoum sudan department of histopathology and cytology faculty of medical laboratory sciences national university khartoum sudan alfarrabi college for science and technology khartoum sudan faculty of medicine sinnar university sennar sudan molecular biology department faculty of medical laboratory sciences nile university khartoum sudan faculty of dentistry ibn sina university khartoum sudan department of neurology mayo clinic jacksonville fl usa department of radiology mayo clinic jacksonville fl usa institute of endemic diseases university of khartoum khartoum sudan mycetoma research center university of khartoum khartoum sudan faculty of medicine nile university khartoum sudan received july accepted august references gruss p walther c pax in development cell mansouri a hallonet m gruss p pax genes and their roles in cell differentiation and development curr opin cell biol macchia pe lapi p krude h pirro mt missero c chiovato l souabni a baserga m tassi v pinchera a pax8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis nat genet vilain c rydlewski c duprez l heinrichs c abramowicz m malvaux p renneboog bt parma j costagliola s vassart g autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of pax8 j clin endocrinol metab park s vk c genetics of congenital hypothyroidism j med genet dahl e koseki h balling r pax genes and anogenesis bioessays lang d powell sk plummer rs young kp ruggeri ba pax genes roles in development pathophysiology and cancer biochem pharmacol stoykova a gruss p roles of paxgenes in developing and adult brain as suggested by expression patterns j neurosci mittag j winterhager e bauer k grummer r congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinology bouchard m de caprona d busslinger m xu p fritzsch b pax2 and pax8 cooperate in mouse inner ear morphogenesis and innervation bmc dev biol mittag j winterhager e bauer k grummer rje congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy endocrinolog laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jl a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol wong s hong w hui p buza n comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ld pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jf emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol 0cali a0et a0al bmc res notes page of ozcan a liles n coffey d shen ss truong ld pax2 and pax8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm bmc res notes nesrin r kilic d risk factors for cervical cancer results from a hospital ozcan a liles n coffey d shen ss truong ldjtajosp pax2 and pax8 based casecontrol study int j hematol oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison am j surg pathol nonaka d tang y chiriboga l rivera m ghossein r diagnostic utility of thyroid transcription factors pax8 and ttf2 foxe1 in thyroid epithelial neoplasms mod pathol tacha d zhou d cheng l expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol bowen nj logani s dickerson eb kapa lb akhtar m benigno bb mcdonald jfjgo emerging roles for pax8 in ovarian cancer and endosalpingeal development gynecol oncol k¶bel m kalloger se boyd n mckinney s mehl e palmer c leung s bowen nj ionescu dn rajput a ovarian carcinoma subtypes are different diseases implications for biomarker studies plos medicine 2008512e232 nonaka d chiriboga l soslow ra expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum e pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol laury ar perets r piao h krane jf barletta ja french c chirieac lr lis r loda m hornick jljtajosp a comprehensive analysis of pax8 expression in human epithelial tumors am j surg pathol tong gx devaraj k hamelebena d yu wm turk a chen x wright jd greenebaum ejdc pax8 a marker for carcinoma of m¼llerian origin in serous effusions diagn cytopathol chu pg chung l weiss lm lau sk determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases am j surg pathol brunner ah riss p heinze g meltzow e brustmann h immunoexpression of pax in endometrial cancer relation to highgrade carcinoma and p53 int j gynecol pathol ozcan a shen ss hamilton c anjana k coffey d krishnan b truong ldjmp pax expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study mod pathol aldaoud n erashdi m alkhatib s abdo n almohtaseb a graboskibauer a the utility of pax8 and satb2 immunohistochemical stains in saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth t a fiveyear survey of cancer prevalence in sudan anticancer res saeed me cao j fadul b kadioglu o khalid he yassin z mustafa sm saeed e efferth tjar a fiveyear survey of cancer prevalence in sudan anticancer res mohamed keh ashmeig aaa cervical cancer our experience in sudan philadelphia aacr elhasan lme bansal d osman of enan k abd farag eab prevalence of human papillomavirus type in sudanese women diagnosed with cervical carcinoma j cancer res ther tacha d zhou d cheng ljai morphology m expression of pax8 in normal and neoplastic tissues a comprehensive immunohistochemical study appl immunohistochem mol morphol ord³±ez ng value of pax immunostaining in tumor diagnosis a review and update adv anat pathol gailey mp bellizzi am immunohistochemistry for the novel markers glypican pax8 and p40 δnp63 in squamous cell and urothelial carcinoma am j clin pathol yemelyanova a gown am holmes bj ronnett bm vang r pax8 expression in uterine adenocarcinomas and mesonephric proliferations int j gynecol pathol liang l zheng w liu j liang sx assessment of the utility of pax8 immunohistochemical stain in diagnosing endocervical glandular lesions arch pathol lab med wong s hong w hui p buza njijogp comprehensive analysis of pax8 expression in epithelial malignancies of the uterine cervix int j gynecol pathol de andrade dap da silva vd de macedo mg de lima ma de andrade vm andrade cemc schmidt rl reis rm dos reis r squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer plos one 20191410e0220086publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support 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objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anticancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often carried out in absence of serum or in low serum concentration ie serum conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids however the tumor microenvironment can be teaming with growth factors in this study we assessed the viability and proliferation of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth serumresults the results show that cannabidiol exerts a markedly different effect on the viability of the human ht cancer cell line when cultured in presence of serum in comparison to serum displaying a cytotoxic effect only in the former situation in presence of serum no inhibitory effect of cannabidiol on dna replication of ht cells was detected and a weak inhibition was observed for other cancer cell lines these results indicate that the effect of cannabidiol is cell contextdependent being modulated by the presence of growth factorskeywords paclitaxel colon cancer cannabidiol serumintroductionthe cannabis plant has a therapeutic potential to treat a wide range of diseases including cancer phytocannabinoids are being tested in a0vitro and in a0vivo for the potential to fight different types of cancer cannabis extracts have recently been described to exert a cytotoxic effect on human cancer cell lines however in a0 vitro cancer models present limitations which reduce their predictive validity one of these limitations is to reproduce the nutritional environment of the cells using cell culture media and growth factors many in a0 vitro cancer studies use historical culture media with fetal calf serum fcs however it is usual correspondence albertosainzcgmailcom gh medical barcelona spainfull list of author information is available at the end of the to eliminate or reduce fcs concentrations ie fcs from the media at the moment of drug exposure to avoid confounding effects of growth factors present in serum as in many studies testing the cytotoxic properties of cannabinoids in cancer cells [ ]the deprivation of survival factors from the media can sensitize cells to a subsequent challenge pirkmajer and chibalin showed that the effects of serum starvation in cell cultures are unpredictable according to eastman serum should be kept in cell cultures to avoid both false positive and negative results due to its effects on cell proliferation stipulating the importance of replicating anic conditions to obtain clinically valid resultsin the present study we analyzed the viability response of different cancer cell lines to cannabidiol cbd in presence of a standard concentration of serum in comparison to a low serum concentration the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0csainzcort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoys 5a medium alamarblue® ab invitrogen were bought leibovitzs l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillinstreptomycin and fcs were bought from sigmaaldrich madrid spain cell proliferation reagent wst1 and 5bromo2²deoxyuridine brdu cell proliferation elisa kit were bought from roche sigmaaldrich madrid spain paclitaxel was dissolved in dimethyl sulfoxide and cbd was dissolved in methanol at a0mm and kept at a0°c for a maximum of a0 months when needed paclitaxel and cbd were diluted conveniently in the cell media at the indicated final concentrations cellular controls without cbd or paclitaxel contained cell media without additivescell cultureht29 cells ref htb38 and sw480 cells ref ccl were obtained from american type culture collection ags cells were kindly provided by miguel a pujana catalan institute of oncology idibell barcelona spain and were originally obtained from nuria sala catalan institute of oncology idibell barcelona spain human colon cancer ht29 cells and sw480 cells were maintained in mccoys 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research lhospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillinstreptomycin and a0nm lglutamine a0h before treatment cells were plated in 96well plates at cellswell a0 h later wells in triplicates received cbd and paclitaxel all assays with sw480 and ags cells included fcs while the assays using ht29 cells included either or fcscell viability and a0proliferation assaysfor the viability and proliferation assay based on resazurin and its redoxmediated reduction we used ab and measured the fluorescence of the wells using a plate readerfor the viability and proliferation assay based on cleavage of tetrazolium salts by mitochondrial dehydrogenase we used wst1for the proliferation based on the measurement of dna synthesis we added brdu to cells and detected its incorporation into dna following manufacturer instructionsto assess cell viability dapi was added to the cell suspension a0 min before the analysis by flow cytometry dapi emits higher fluorescence when bound to dna dapi enters rapidly through altered cell membranes allowing the detection of damaged cells the cell population was selected by gating in a forward scatter vs side scatter dot plot excluding aggregates and cell debris samples were analyzed using a gallios flow cytometerstatistical analysisdata was analysed using ibm spss statistics and real statistics using excelwe used shapirowilk test to assess data normality and nonparametrical independent samples kruskalwallis test to identify significant differences between each experimental condition we used dunn test as a posthoc analysis to identify which groups show statistically significant differencesresultsviability and a0proliferation of a0ht cells with a0serum deprivation fcswhen human colon cancer ht29 cells were incubated in media with serum adding cbd at a0µm reduced cell viability as assessed via the resazurin method which is based on evaluating mitochondrial reductive capacity fig a0 1a interestingly when cbd concentrations were a0 µm cell viability increased during the first a0 h differences between or and a0 µm were statistically significant p and p at a0h the increasing viability with cbd a0 µm disappeared while the blocking effect of a0µm cbd was more pronounced fig a0 1a this suggests that cbd can induce mitochondrial stress as reported by others looking at the morphology of cells the treatment with a0µm cbd led to changes in cell form such as massive cellular detachment cell rounding and presence of wrinkled cells characteristic of dead cells fig a0 1b in fact analyzing the presence of dead cells using dapi dye we found an increased percentage in samples incubated with a0 µm cbd when compared to control cells fig a01c thus the loss of mitochondrial activity observed at cbd a0 µm correlated with cell death of note at longer incubation times ie a0days massive cellular death was also observable at a0µm cbd data not shown in summary a0µm cbd shows cytotoxic activity on ht29 cells cultured in fcs 0csainzcort a0et a0al bmc res notes page of fig a ht cells were incubated with fcs and different concentrations of cbd for and h cell viability was assessed by incubation with ab the mean sd of three assays are shown b morphology of ht cells incubated with or without μm cbd for h representative images are shown bar µm c ht cell viability according to dapi staining see the materials and methods section ht cells were incubated without top or with μm cbd bottom for h stained with dapi and immediately analyzed by flow cytometry the cursor identifies dapipositive cells dead cells showing a higher percentage in cbdtreated cells a representative experiment c is shown p viability and a0proliferation of a0ht cells in a0 fcscontrary to the drop in viability of cells in fcs cbd did not inhibit the viability of ht29 cells even after a0days in media containing fcs fig a02a b an apparent increase in ht29 cell viability was observed at a0µm cbd as assessed by ab or wst1 fig a0 suggesting mitochondrial stress we sought to find whether in these conditions cbd could show additive or synergistic antiproliferative effects with the therapeutic drug paclitaxel paclitaxel partially decreased the viability of ht29 cells according to ab measurement but not wst1 thus cbd at a0µm does not grossly affect the viability of ht29 cells after a0days culture in presence of serumto ascertain whether cbd had any effect on proliferation of ht29 cells we measured the incorporation of brdu into dna no changes in dna synthesis were observed after a0days of incubation of ht29 cells with any concentration of cbd fig a02c although paclitaxel in itself did inhibit dna synthesis cbd did not increase the effect of paclitaxel fig a02c in summary cbd up to a0µm do not decrease the viability nor the proliferation of ht29 cells cultured in fcs none of these results showed statistically significant differencesviability and a0proliferation of a0sw480 and a0ags cellsto know whether other cancer cell lines behaved similarly to ht29 showing little or no response to cbd when cultured in fcs we used sw480 another colon cancer cell line and ags a gastric cancer cell lineags cells did not show changes of viability by incubation with cbd up to a0µm though a0nm paclitaxel did decrease their viability fig a0 3a higher paclitaxel concentrations resulted in a severe decrease of ags cells viability data not shown so we used a0nm paclitaxel to observe potential effects of cbd the viability of sw480 cells with cbd and fcs showed a trend to decline fig a03c surprisingly and contrary to ht29 cells a0µm cbd did actually impair dna replication in ags and sw480 cells fig a03b d in fact the inhibition of dna replication was additive to that produced by paclitaxel the assessment of dna replication in sw480 cells 0csainzcort a0et a0al bmc res notes page of showed significant differences between the control sample and a0µm cbd without paclitaxel p any other statistic analysis did not show significant resultsin summary in presence of fcs and during a0days of culture cbd does not affect the viability of ht29 sw480 and ags cells though cbd at a0µm does impair the proliferation of ags and sw480 cellsdiscussionin this study we investigated the effects of cbd and its combination with paclitaxel on the viability of three different cancer cells ht29 sw480 and ags under two different concentrations of serum a standard appropriate for cell growth for ht29 sw480 and ags and a restrictive one of for ht29 only for ht29 cells cbd only reduces cell viability under low fcs with no effects on viability or dna replication when cells were in fcs however for sw480 and ags dna replication was impaired under a0µm cbd with serum moreover the inhibition of dna replication in sw480 and ags cells by cbd and paclitaxel had an additive effectat low cbd concentrations ht29 cells showed a trend towards increased cell viability though the differences were not significant different concentrations of cbd have previously been shown to have opposing effects on cells thus a0µm cbd induces proliferation of t leukemia cells but at higher concentration kills the cells a low concentration cbd increases mitochondrial ca2 augmenting mitochondrial metabolism and cell growth but at high concentration it leads to fig ht cells were incubated for days with fcs and different concentrations of cbd in absence or presence of nm paclitaxel a the viability was assessed by incubation with ab the mean sd are shown n b the viability was assessed by incubation with wst the mean sd are shown n c before harvesting cells were incubated with brdu for h which incorporated into dna and dna synthesis was quantified the mean sd are indicated n fig ags cells and sw480 cells were incubated for days with different concentrations of cbd in absence or presence of nm paclitaxel ags or nm paclitaxel sw480 a c cell viability was assessed by incubation with ab the mean sd of three ags and six sw480 assays are shown b d before harvesting cells were incubated for h with brdu which incorporated into dna and dna synthesis was quantitated the mean sd of three assays ags and assays sw480 are shown p 0csainzcort a0et a0al bmc res notes page of excessive mitochondrial ca2 mitochondrial dysfunction and cell death appropriate culturing conditions are essential for the survival and growth of cells in many studies cell culture conditions are not sufficiently detailed which is essential for study replication one possible solution to address the potential effect of serum could be using culture media without fcs so the media does not need to be altered during drug exposition in any case neither higher serum concentrations nor lower serum concentrations represent the proper microenvironment of a cancer cell in the human body and both approaches could be valid to test the effects of a drug on cell lines the tumor microenvironment is enriched with metabolites including lactate and adenosine [ ] which increases tumor growth and may modulate the therapeutic effect of a drug in tumors that are highly glycolytic increasing mitochondrial activity as exerted by cbd may add metabolic stress to cells forcing them to decreased growth the effect of a drug on cells can be assessed effectively if the experimental conditions of the treatment are the same as the growing conditions before the treatment once growing conditions and treatment conditions differ from more than one variable drug treatment then the resulting effects cannot be associated only to the treatment but to the combination of variableslimitationsour results did not show statistically significant differences with the exception of the assessment of viability of ht29 cells under cbd treatment and the assessment of dna replication of sw480 under a0µm cbd the lack of statistically significant results could be due to the small sample size n for most of the assays our study was also not able to replicate the strongly inhibitory effect of cbd shown in other studies where cannabinoids were tested against cancer cells cultured with fcs fcs contains many growth factors and nutrients and differences in the fcs source could substantially modify the viability proliferation and differentiation of cultured cells there are also other studies where cancer cells were cultured with fcs and treated with cbd or other synthetic cbdlike molecules the results of these studies showed that cbd a0μgml reduced the viability of cancer cells and also had effects on other survival variables [ ] the cell lines used in these studies being different to the ones used in our study could account for the different results observedabbreviationsab alamarblue brdu bromo²deoxyuridine cbd cannabidiol dapi ²diamidinophenylindole fcs fetal calf serumacknowledgementswe would like to thank manuel reina for his expert adviceauthors contributionsschibano pharma ag participated in the idea of the study as and ee designed the study as and ee acquired analyzed and interpreted the data cm provided technical assistance and carried out some experiments as and ee drafted the work all authors read and approved the final manuscriptfundingthis study was partially funded by schibano pharma ag waldsch¶nengrund switzerland and gh medical barcelona spain the design of the study was prepared by as ee and cm and approved by schibano pharma ag and gh medicalavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsas was employee at gh medical while performing this projectauthor details gh medical barcelona spain celltecub department of cell biology physiology and immunology faculty of biology university of barcelona av diagonal barcelona spain received may accepted august references ackermann t tardito s cell culture medium formulation and its implications in cancer metabolism trends cancer https doi101016jtreca n201905004 brand a singer k koehl ge kolitzus m schoenhammer g thiel a matos c bruss c klobuch s peter k kastenberger m bogdan c schleicher u mackensen a ullrich e fichtnerfeigl s kesselring r mack m ritter u schmid m blank c dettmer k oefner pj hoffmann p walenta s geissler ek pouyssegur j villunger a steven a seliger b schreml s haferkamp s kohl e karrer s berneburg m herr w muellerklieser w renner k kreutz m ldhaassociated lactic acid production blunts tumor immunosurveillance by t and nk cells cell metab https 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k buta c cochrane b dirks wg fu j hickman jj hohensee c kolar r liebsch m pistollato f schulz m thieme d weber t wiest j winkler s gstraunthaler g fetal bovine serum fbs pastpresentfuture altex https doi1014573 altex wu hy huang ch lin yh wang cc jan tr cannabidiol induced apoptosis in human monocytes through mitochondrial permeability transition poremediated ros production free radical biol med https doi101016jfreer adbio med201806023publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready 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range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological eï¬cacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was ï¬rstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as antiammatory and immunomodulatory agent [] as MTXcould not only optimize the eï¬cacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location ofammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause antiammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment deï¬ned as control of ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ ]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively ï¬rstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justiï¬cation because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine AdenosineCell membraneCell membrane 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight beneï¬t from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcets disease BD patientssuï¬ering from BDassociated ocular ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcets disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may uence theeï¬cacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs thatuence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no uence on MTX eï¬cacy [] However someresearchers argue that these SNPs have no deï¬nite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters uence the toxicity but not the eï¬cacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the eï¬cacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may uence both eï¬cacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theeï¬cacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theeï¬cacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the eï¬cacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the beneï¬trisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 []ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR []ATIC []TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ammatory infectious and lymphoproliferative [] In the authors opinion all MTX relatedside eï¬ects can be classiï¬ed into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[] e reported frequency of EBV positive in MTXassociated LPDs patients is [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting eï¬cacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the eï¬cacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting eï¬cacy and toxicity of MTXAï¬ecting eï¬cacy and toxicity of MTXAï¬ecting eï¬cacy of MTXAï¬ecting eï¬cacy of MTXAï¬ecting eï¬cacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeï¬ciency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classiï¬ed MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeï¬ciency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patientsgeneral condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its eï¬cacy and an acceptable safety proï¬le asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivitys cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the ï¬rst months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the fetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmithMagenis syndrome [] among patients with speciï¬cmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insuï¬ciency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [] Patients with a history of alcohol intakemight have a greater risk of liver ï¬brosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deï¬ciency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver ï¬brosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular ï¬uid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortiï¬cation enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have uence on the eï¬cacy of MTXAlDabagh found that the reduction in eï¬cacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing uence betweenthe antiammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in speciï¬c patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has antiammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to mgweek whennecessary [ ] In patients with insuï¬cient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of ï¬sh oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX eï¬cacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reï¬ect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectiveï¬rstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a cancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conï¬icts of interestReferences[] R Q H Kloos R Pieters C van den Bos e eï¬ect ofasparaginase therapy on methotrexate toxicity and eï¬cacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp [] R K Bath N K Brar F A Forouhar and G Y Wu Areview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp [] W Wang H Zhou and L Liu Side eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp [] J Smolen and R Landew´e EULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp [] J A Singh K G Saag S L Bridges Americancollege of rheumatology guideline for the treatment ofrheumatoid arthritis Arthritis Rheumatology vol no pp [] M Holdhoï¬ P Ambady A Abdelaziz Highdosemethotrexate with or without rituximab in newly diagnosedprimary CNS lymphoma Neurology vol no pp [] J Peer J M Rowe S Frenkel and E J Dann Testicularlymphoma intraocularvitreoretinal lymphoma and brainlymphoma involvement of three immunoprivileged sites in 0cJournal of Ophthalmologyone patient American Journal of Hematology vol no pp [] S Gangaputra C W Newcomb T L Liesegang et alSystemic immunosuppressive therapy for eye diseases cohort study methotrexate for ocular ammatory diseasesOphthalmology vol no pp [] P W Hardwig J S Pulido J C Erie K H Baratz andH Buettner Intraocular methotrexate in ocular diseasesother than primary central nervous system lymphomaAmerican Journal of Ophthalmology vol no pp [] E Esterberg and N R Acharya Corticosteroidsparingtherapy practice patterns among uveitis specialists Journalof Ophthalmic Inï¬ammation and Infection vol no pp [] K Durrani F R Zakka M Ahmed M MemonS S Siddique and C S Foster Systemic therapy withconventional and novel immunomodulatory agents for ocular ammatory disease Survey of Ophthalmology vol no pp [] S S Gangaputra C W Newcomb M M Joï¬e et alComparison between methotrexate and mycophenolatemofetil monotherapy for the control of noninfectious ocularammatory diseases American Journal of Ophthalmologyvol pp [] V K Ayuso E L van de Winkel A Rothova and J Helenade Boer Relapse rate of uveitis postmethotrexate treatmentin juvenile idiopathic arthritis American Journal of Ophthalmology vol no pp [] S R Rathinam M Babu R undikandy A randomized clinical trial comparing methotrexate and mycophenolate mofetil for noninfectious uveitis Ophthalmologyvol no pp [] A Galor D A Jabs H A Leder Comparison ofantimetabolite drugs as corticosteroidsparing therapy forammation Ophthalmologynoninfectiousvol no pp ocular[] M D de Smet V S Vancs D Kohler D Solomon andC C Chan Intravitreal chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp [] E Kim C Kim J Lee and Y Cho A case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp [] J Smith J T Rosenbaum D J Wilson Role ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp [] CC Chan and D J Wallace Intraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp [] K L Larkin U S Saboo G M Comer Use ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp [] C P Fox E H Phillips J Smith Guidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp [] N G Lambert DJ Wilson D M Albert andW D Chamberlain Intravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofTopical application of methotrexate for inhibition of cornealangiogenesis Graefes Archive for Clinical and ExperimentalOphthalmology vol no pp [] S K Kurup C Gee and C M Greven Intravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi e role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçets disease patients e Egyptian Rheumatologistvol no pp [] S R J Taylor A Banker A Schlaen Intraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re | 2 |
"Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1 mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2 in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9To this end the central premise put forth in this study is that focused ultrasound FUSa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbeccos Modified Eagles Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1 mice were obtained from The Jackson Laboratory 4T1 or E0771 cells à were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume lengthÃwidth22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a sham treatment consisting of exposure to the °C degassed water bath exposure for min Following sham or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to sham or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of vehicle treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day early αPD1 or day delayed αPD1T cell depletionsT cell depletion antibodiesanti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1BC The exceptionally small focus of this system rendered a low ablation fraction of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCsand CD86 DCs in particularsuggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C Sham mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test FH or two way analysis of variance followed by Tukey multiple comparison correction IK nsnot significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2BCBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly à and à reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2DE In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytesin particular CD8 and CD4 T cellsplay an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3BC and EF Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3BC and EF From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6AB Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cellsa reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells BC Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to EF Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to IK Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6 FUS monotherapy n4 GEM monotherapy n9 and combinatorial FUSGEM therapy n6 groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fishers least significant difference LSD without multiple comparisons correction for IK Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fishers LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6CD These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantationthat is days subsequent to final GEM administrationrevealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4BC Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4DE However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1 model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1 mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1 mice relative to their WT counterparts with average 4T1 tumor volume in Rag1 mice being over fivefold higher than that of WT mice on terminati | 2 |
"Nonetheless the tumor TS levels or its polymorphisms in each patient could explain these cases of severe toxicity as it has been suggested in other neoplasms treated with other antifolate drugs [9]. The potential association between different TS genotypes and the toxicity experienced by a European population of patients with NSCLC receiving pemetrexed is also to be studied. Three different types of polymorphisms have been described in the TS gene. In the gene promoter there is a variable number of tandem repeats (VNTR) of 28 pb in the 5?- region. Thus cases of two or three repetitions of this tandem TS gene promoter enhancer region (TSER) have been described [16]. A second type of polymorphism consists in a change in a single nucleotide (G >?C) in one of the sequences of the repetition comprising a single nucleotide polymorphism (SNP) [16]. A third modality of polymorphisms consists in the deletion or insertion of 6 pair of bases (bp) in the 3?-UTR region (untranslated region) [16]. In summary the potential usefulness of TS genotype as an independent prognostic factor or predictor of response to pemetrexed-based regimens in a European NSCLC population has not been studied. Similarly no clear evidence is available about the potential correlation between the different TS genotypes and the toxicity experienced by those patients. Therefore we decided to investigate the three known polymorphisms of TS gene and their correlation with objective response rate (ORR) progression-free survival (PFS) and overall survival (OS) as well as toxicity in European patients with advanced NSCLC treated with pemetrexed-based regimens. Methods Patients and samples Overall 25 consecutive stage III-IV NSCLC patients treated at a single institution from which peripheral blood samples were available were analyzed. All of them received pemetrexed-based regimens in the first second or third line settings according to our institutional therapeutic protocols. After the informed consent was obtained from all patients 10 ml of peripheral blood samples were collected before the administration of the first cycle of a pemetrexed-containing regimen. Blood samples were stored at the Biobank of the University of Navarra and were processed following standard operating procedures approved by the Ethical and Scientific Committees. Tumor ORR to the treatment was assessed using computerized tomography (CT) scans every two pemetrexed-based chemotherapy cycles and categorized according to the Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 as per institutional protocol. The toxicities recorded during pemetrexed-based treatment were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. TS enhancer region genotyping analysis The genomic DNA was extracted from the peripheral leucocytes. The genotypes of the TSER (VNTR) and SNP were determined by polymerase chain reaction (PCR). The variable number tandem repeat (VNTR) of 28 bp polymorphism and the G???C SNP in the first and second repeat were analyzed. A DNA fragment was amplified using previously described PCR conditions and primers [17] and directly sequenced using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems Foster City CA USA). The forward primer 5?-CGTGGCTCCTGCGTTTCC-3? and the reverse primer 3?-GAGCCGGCCACAGGCAT-5? were used. A modification of conventional conditions was necessary. PCR was performed in a reaction mixture with dNTP: 0.35 ?l Buffer: 0.25 ?l MgCl2: 17.5 ?l Tap polymerase: 0.5 ?l H2O: 18 ?l primers 0.1?+?0.1 ?l DMSO: 1.25 ?l and DNA: 2 ?l. The cycling conditions were denaturation 95°C for 10 minutes and 30 cycles at 95°C for one minute then at 64°C for one minute and 72°C one minute and finally seven minutes at 72?ºC. Aliquots of amplified fragments were separated on a 3% agarose gel and the TS VNTR genotype was determined staining 2R (210 base pairs; bp) and 3R (238 bp) alleles. After that we performed a PCR-restriction fragment length polymorphisms (RFLP) by Hae III digestion. The mixture was PCR product: 10 ?l H2O: 7 ?l Buffer 2 ?l and Hae III: 1 ?l. After that we incubated the mixture at 37°C overnight. Aliquots of digested fragments were separated on 12% acrylamide gel and the SNP genotype was determined. The digestion of fragments showed the different genotypes 2RGC: 664746 44 and 7 bp 2RCC: 11346 44 and 7 bp 3RGGCC (3RG): 66474644 28 and 7 bp 3RGCC (3RC): 944746 44 and 7 bp. TS 3?UTR region genotyping analysis The 3?UTR polymorphisms were analyzed by Restriction Fragment Length Polymorphism (RFLP). A fragment containing the 6 bp deletion/insertion was amplified using the reverse primer 5?-CAGATAAGTGGCAGTACAGA-3?and the forward primer 3?-CAAATCTGAGGGAGCTGAGT-5? in 10 ul of reaction mixture with dNTP: 4 ?l Buffer: 5 ?l MgCl2: 4 ?l Tap polymerase: 0.5 ?l H2O: 26.5 ?l primers 4?+?4 ?l and DNA: 2 ?l. The cycling conditions were denaturation 95°C for 10 minutes and 35 cycles at 95°C for 30 minutes then at 57°C for 30 minutes and 72°C one minute and finally seven minutes at 72°C. The fragments were amplified on 2% agarose gel. Afterwards the products were digested with Dra I and the mixture of PCR product: 20 ?l BSA 10%: 0.5 ?l Buffer: 5 ?l H2O: 23.5 ?l and Dra I: 1 ?l. Posteriorly the product was incubated one hour at 37°C. The final digested product was separated in a 3% agarose gel. The different genotypes were deletion 6 bp/insertion 6 bp insertion 6 bp/insertion 6 bp and deletion 6 bp/deletion 6 bp. The expected fragment sizes by genotyping were deletion 6 bp/insertion 6 bp: 148142 88 and 60 bp insertion 6 bp/insertion 6 bp: 88 and 60 bp and deletion 6 bp/deletion 6 bp: 142 bp. We repeated the PCR three times to ensure final results. EGFR mutations analysis As per institutional protocol all patients with advanced NSCLC were tested for EGFR activating mutations before treatment initiation. In brief after having the samples fixed in alcohol and stained by Papanicolau stain DNA was extracted and amplified by PCR technique using EGFR gene exons 1819 20 and 21 specific primers. ABI PRISM® 310 Genetic Analyzer equipment was used for the analysis of the sequencing reactions with both forward and reverse primers. Statistical analysis Fishers exact test was used to investigate the correlation between each genotype and the response to the treatment and the toxicity presented. Kaplan-Meier curves and log-rank test or Tarone-Ware test when indicated were calculated to correlate each genotype with the survival outcomes (PFS and OS). For the subgroup analysis EGFR mutation status and smoking history were considered in order to analyze potential differences in clinical outcome measures (ORR PFS and OS). The SPSS 15.0 software (SPSS Inc. Chicago IL) was employed to perform the statistical analysis. Results Patients characteristics and treatment The clinical and pathological characteristics of the patients included are summarized in . In brief our cohort was mainly composed by males with a median age of 59 years and a past smoking history showing good performance status. Most of the patients showed adenocarcinoma histology (88%) and showed distant metastasis (M1) at onset (72%). Most of the patients received a pemetrexed-based regimen in first line (84%). After a median follow up of 21 months 80% of patients have already progressed and 52% of them have died due to disease progression (). Patients characteristics N pts % Gender ??Female 11 44 ??Male 14 56 Age ??<60 13 52 ??> r =60 12 48 ECOG ??0 9 36 ??1 15 60 ??2 1 4 Tobacco ??Current smoker 4 16 ??Never smoker 7 28 ??Former smoker 14 56 Histology ??Adenocarcinoma 22 88 ??Adenocarcinoma poorly differentiated 2 8 ??Adeno-squamous 1 4 T ??T1-2 12 48 ??T3-4 13 52 N ??N0 6 24 ??N+ 19 76 M ??M0 7 28 ??M1 18 72 Lung metastases ??Presence 7 28 ??Absence 18 72 Liver metastases ??Presence 2 8 ??Absence 23 92 Bone metastases ??Presence 10 40 ??Absence 15 60 Brain metastases ??Presence 8 32 ??Absence 17 68 EGFR ??Wild type 23 92 ??Mutant 1 4 ??Unknown 1 4 Line of treatment ??First/Induction (stage III) 2 8 ??First 21 84 ??Second 1 4 ??Third 1 4 Response ??Response 18 72 ??Progression?+?Stabilization 7 28 Maintenance ??No maintenance 18 72 ??Maintenance 7 28 Progression ??Not progressed 6 24 ??Progressed 19 76 Clinical status ??Alive 12 48 ??Dead 13 52 Eastern Cooperative Oncology Group (ECOG). Epidermal Growth Factor Receptor (EGFR). In addition in 8 out of the 18 subjects showing multiple brain metastases at onset conventional whole-brain radiotherapy (300 cGy) was administered between first and second chemotherapy cycles following our institutional treatment guidelines. Finally 4 out of the 7 patients showing no distant metastases at onset responded to the pemetrexed-based induction chemotherapy. As per institutional protocol all four subjects underwent a 3-D conformal radiotherapy program with concurrent chemotherapy as previously published [18]. Correlation between ORR to the treatment and polymorphisms We studied the potential correlation between the different polymorphisms observed and the response to the treatment obtained (Table 2). For this purpose any kind of radiological response (complete or partial response) was compared to no response to the treatment (disease stabilization or progression). The presence of 3R/3R polymorphism seemed to predict a higher ORR (100%) compared to the rest of the genotypes with a trend toward statistical significance (p =?0.055). In the subgroup analysis a significantly higher ORR to pemetrexed for wild-type EGFR patients showing a 3R/3R genotype (100%) compared to the 2R/2R (77.8%) 2R/3R (33.3%) and 3R/4R (0%) was observed (p =?0.017). Table 2 Overall response rate to the treatment and polymorphisms observed Global distribution of polymorphisms (Pol) Response N (%) Stabilization or progression N (%) p value VNTR 2R/2R 7 (77.8) 2 (22.2) 0.055 3R/3R 7 (100) 0 (0) 2R/3R 4 (50) 4 (50) 3R/4R 0 (0) 1 (100) Pol VNTR (Subanalysis by EGFR status; group of native EGFR-patients) 2R/2R 7 (77.8) 2 (22.2) 0.017 3R/3R 7 (100) 0 (0) 2R/3R 2 (33.3) 4 (66.7) 3R/4R 0 (0) 1 (100) Global distribution of SNP Absence 6 (85.7) 1 (14.3) 0.626 Presence 12 (66.7) 6 (33.3) Global distribution of polymorphisms in 3?-UTR +6/+6 10 (83.3) 2 (16.7) 0.234 +6/-6 6 (54.5) 5 (45.5) -6/-6 2 (100) 0 (0) Pol 3?-UTR (Subanalysis by smoking habit stratification; group of active and former smokers) +6/+6 8 (100) 0 (0) 0.085 +6/-6 4 (50) 4 (50) -6/-6 2 (100) 0 (0) No statistically significant differences were observed comparing the presence and the absence of a SNP G >?C as shown in Table 2. Overall a non-significant correlation between the different 3?-UTR polymorphisms and the ORR was observed. However the genotype +6/+6 seemed to predict a higher ORR among active/former smokers (A/FS) compared to +6/-6 (100% vs. 50%; p =?0.085). Correlation between PFS and polymorphisms Regarding TSER polymorphisms we found a trend toward statistical significance (p =?0.089) in the differences in PFS observed among the different genotypes in favor of the 3R/3R genotype (Figure 1A). Figure 1 Kaplan-Meier curves for progression-free survival (PFS) in months (mo) associated with the different TS polymorphisms. A: TSER genotypes. B: Presence or absence of SNP. C: 3´UTR genotypes. In the case of the absence or presence of a SNP at the third repetition (3R allele) we observed a non-significant increased PFS in the subgroup of patients showing an absence of SNP (Figure 1B). Finally no significant correlations regarding the 3?UTR genotypes and PFS were observed (Figure 1C). Correlation between OS and polymorphisms In this cohort we found a significant correlation between TSER polymorphisms and OS (Figure 2A). The median OS was not reached for 3R/3R genotype patients whereas 2R/3R genotype subjects showed a 70 m OS followed by 3R/4R and 2R/2R genotypes with a median OS of 15 m and 13 m respectively (p =?0.019) (Figure 2A). Figure 2 Kaplan-Meier curves for overall survival (OS) in months (mo) associated with the different TS polymorphisms. A: TSER genotypes. B: Presence or absence of SNP. C: 3´UTR genotypes. No significant differences in OS were observed with regards to the presence/absence of SNP (Figure 2B) or regarding the 3?-UTR polymorphisms (Figure 2C). Correlation between toxicity and polymorphisms The most frequent toxicity was grade (G)1 anemia (28%) and nausea (20%) and G2 leucopenia (40%). The most commom G3-4 toxicities were leucopenia (16%) asthenia (8%) anemia (4%) neutropenia (4%) and dyspnea (4%). Overall we found no significant correlations between the toxicity profiles experienced by the patients and the different TS genotypes (Table 3). Table 3 Correlation between grades of toxicity and different genotypes Global distribution polymorphisms (Pol) No toxicity Grade 1-2 Grade 3-4 p value VNTR polymorphisms 2R/2R 2 (22.2) 4 (44.2) 3 (33.4) 0.545 3R/3R 2 (25) 5 (75) 0 (0) 2R/3R 2 (25) 4 (50) 2 (25) 3R/4R 1 (100) 0 (0) 0 (0) SNP polymorphisms Absence 3 (42.9) 4 (57.1) 0 (0) 0.3 Presence 4 (22.2) 9 (50) 5 (27.8) 3?-UTR polymorphisms +6/+6 3 (25) 6 (50) 3 (25) > 0.05 +6/-6 3 (27.3) 6 (54.5) 2 (18.2) -6/-6 1 (50) 1 (50) 0 (0) Discussion Pemetrexed a multitargeted antifolate drug is essential for the first and second-line as well as maintenance treatment of NSCLC patients with non-squamous histology [6]. TS is the main biological target of pemetrexed. Some studies have suggested that TS expression could be a predictive factor of response in NSCLC [19]. Moreover some VNTR genotypes have been associated with TS expression and activity in other tumor types such as colorectal cancer [17]. In NSCLC patients a correlation between different genotypes and the TS protein expression has been shown [20]. Shintani et al. [20] also confirmed that the TS mRNA levels were significantly higher in lung cancer tissues with the 3R/3R genotype as compared to those with the 2R/2R genotype. Nonetheless definitive studies addressing the correlation of the different genotypes of TS in circulating genomic DNA with response to the treatment PFS or OS in pemetrexed-treated NSCLC European patients are lacking. The potential influence of the EGFR status on those polymorphisms and their correlation with clinical outcome after pemetrexed-based treatment is also unexplored. A recent study by Hu et al. [21] investigated the different TS polymorphisms in genomic DNA of 90 Asian NSCLC patients. In contrast with our findings no specific genotype regarding the TSER or 3?-UTR polymorphisms studied seemed to correlate with a significant difference in ORR PFS or OS. This could be explained by substantial clinical differences between both populations. Our cohort was constituted by Caucasian patients compared to the Asian population studied by Hu et al. In addition our patients were mostly current or former smokers (72%) compared to the Asian population that showed 62% of never smokers. Also in our cohort the subjects mainly received pemetrexed-based chemotherapy as a first line regimen (92%) whereas the cohort studied by Hu et al. [21] was treated with pemetrexed as a second or further line in 62.2% of the cases. These remarkable differences in basic clinical characteristics and in particular the ethnicity between both cohorts are probably also explaining the differences observed in the 3?-UTR genotype frequency between our population and the one studied by Hu et al. In our cohort +6 bp/+6 bp +6 bp/-6 bp and -6 bp/-6 bp genotypes were found in 48% 44% and 8% of the cases respectively. In contrast0.078 47.8% and 44.4% were respectively found in the population studied by Hu et al. [21]. In a previous analysis performed on another Caucasian NSCLC population evaluated at the M.D. Anderson Cancer Center [22] a similar proportion of 3?-UTR genotypes according to our findings was observed (49.2% of +6 bp/+6 bp 42.4% of +6 bp/-6 bp and 8.4% of -6 bp/-6 bp). Additionally the low prevalence of the +6 bp/+6 bp genotype in an Asian population compared to our cohort may be confirmed by a recent study in which from 106 Asian NSCLC patients investigated none of them showed a +6 bp/+6 bp genotype in genomic circulating DNA [23]. Nontheless in this latter study [23] a significantly higher ORR was observed among patients showing a -6 bp/-6 bp 3?-UTR genotype compared to the ORR reported for patients presenting a -6 bp/+6 bp polymorphism (32.2% vs 12.7%; p =?0.008). Accordingly in our cohort a higher ORR in patients showing a -6 bp/-6 bp genotype compared to those presenting a -6 bp/+6 bp polymorphism was also observed (100% vs. 54.5%). However the statistical significance was not reached probably due to the relatively low number of patients included in our analysis. Interestingly enough in the subgroup analysis of our data the +6 bp/+6 bp genotype seemed to predict a higher ORR only among active/former smokers compared to +6 bp/-6 bp (100% vs. 50%; p =?0.085). This novel observation if validated in future studies could be relevant for selecting specific drugs for each patient in a second or third line setting. With regards to the TSER polymorphisms the presence of a 3R/3R polymorphism seemed to predict a higher ORR with a clear trend toward statistical significance (p =?0.055). Moreover that difference was even greater and statistically significant benefiting the subpopulation of wild-type EGFR patients. To our knowledge this is the first time that such observation has been made. An interesting preclinical study by Giovannetti et al. [24] investigated the activity profile of a combination therapy against NSCLC cell lines with different genotypes with erlotinib and pemetrexed. Remarkably pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation. Additionally erlotinib significantly reduced TS expression and activity. Thus when erlotinib and pemetrexed were combined a strong synergism in all NSCLC cells regardless of their genetic signature was observed. This potential crosstalk between the EGFR signaling pathway and the TS expression and activity could in part explain our novel findings showing a significantly higher ORR to pemetrexed in those wild-type EGFR patients harboring a 3R/3R polymorphism. However none of the previous studies have described the EGFR status of the patients analyzed and how that status impacted on the ORR to pemetrexed for certain TS polymorphisms. In terms of survival in the present series after a median follow-up of 21 months PFS was superior for those patients showing a 3R/3R genotype with a trend toward statistical significance as expected considering the higher ORR observed for patients with the same TSER genotype. The relatively low statistical power of our clinical cohort may be accounting for the lack of a full statistical significance observed. Regarding OS the advantage in PFS observed in patients showing the 3R/3R genotype translated into a significantly higher median OS in patients with the same polymorphism compared to the rest. Conversely in the study by Hu et al. [21] no specific genotype was significantly correlated with a superior PFS or OS. As aforementioned the dramatic differences in the populations characteristics between both series might possibly explain this discordance. In our study conversely to the observations made by Wang et al. [23] a significantly superior PFS and OS in patients with the -6 bp/-6 bp 3?-UTR genotype has not been confirmed. Most probably this is also due to the differences in the genotype distribution among populations with markedly different ethnicity and epidemiological characteristics. Finally in accordance with Wang et al. [23] the toxicity profile was not significantly correlated with any TS genotype in our series. As aforementioned this study has some limitations due to its retrospective nature and the low number of patients investigated. Both could be responsible for a low statistical power that may impair our ability to find significant differences between subgroups of patients. Conclusions For the first time in a European population of NSCLC patients receiving pemetrexed the presence of the TSER 3R/3R polymorphism significantly correlated with a superior OS. Moreover this same polymorphism when associated to wild-type EGFR was correlated with a higher ORR to pemetrexed. The presence of the +6/-6 bp 3?-UTR genotype among active or former smokers was correlated to a higher ORR showing a trend toward statistical significance. Finally pemetrexed-induced toxicity was not significantly correlated with a specific TS genotype. These novel data warrant further investigation in larger prospective series and may help to patients selection if finally validated. Abbreviations NSCLC: Non-small cell lung cancer; TS: Thymidylate synthase; EGFR: Epidermal Growth Factor Receptor; VNTR: Variable number of tandem repeats; TSER: Thymidylate Synthase gene promoter enhancer region; SNP: Single nucleotide polymorphism; ORR: Overall response rate; PFS: Progression-free survival; OS: Overall survival; CT: Computerized tomography; RECIST: Response Evaluation Criteria in Solid Tumors; CTCAE: Common Terminology Criteria for Adverse Events; PCR: Polymerase chain reaction; RFLP: Restriction fragment length polymorphism; A/FS: Active/former smokers. Competing interests The authors declare that they have no competing interests." | 1 |
"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Marys Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturers recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patients space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patients blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patients space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to DisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturers recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturers instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c" | 2 |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid 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