在结直肠癌循环肿瘤细胞中KRAS和PIK3CA的突变以及EGFR异质性的研究精品课件.ppt

1,HeterogeneityofEpidermalGrowthFactorReceptorStatusandMutationsofKRAS/PIK3CAinCirculatingTumorCellsofPatientswithColorectalCancer,ClinicalChemistryNovember7，2012,1,.,2,2,3,Studyintroduction,美国国家癌症综合治疗联盟（NCCN）结直肠癌临床实践指南(2011)明确指出：(1)所有转移性结直肠癌患者都应检测KRAS基因状态；(2)只有KRAS野生型患者才建议接受EGFR抑制剂（如爱必妥和帕尼单抗）治疗。NCCN非小细胞肺癌临床实践指南(2011)明确指出：当KRAS基因发生突变时，不建议使用EGFR-TKIs靶向治疗药物。卫生部颁布的结直肠癌诊疗规范（2010年版）也明确指出：确定为复发或转移性结直肠癌时，检测肿瘤组织KRAS基因状态，以确定合适的治疗方案。1.AmadoRG，WolfM，PeetersM，VanCutsemEetal.JournalofClinicalOncology.April2008.2.VanCutsemetal.ASCOAnnualMeeting2008:abstract2.3.Bokemeyeretal.ASCOAnnualMeeting2008:abstract4000.4.Livreetal.JClinOncol2008;26:374-9.5.美国FDA相关网站：/AboutFDA/CentersOffices/CDER/ucm172905.htm,3,4,EGFR是一种跨膜酪氨酸激酶受体，与配体结合后可引起下游信号传导通路的活化，如：KRASBRAF(4%12%)RAS-RAF-MEK-Erk/MAPKPIK3CA(14%20%)P13K-AKT-PKC-IKK在许多实体瘤中EGFR均有不同程度的表达，表达率最高为头颈部肿瘤，达95%100%。CRC次之，为72%89%，胃癌表达率为41%64%，并且EGFR表达率高的肿瘤恶性度高，侵袭力强，预后差。而且在同一患者中不同的CTCs中，EGFR的表达也存在着异质性。1GoldsteinNS,ArminM.EpidermalgrowthfactorreceptorimmunohistochemicalreactivityinpatientswithAmericanJointCommitteeonCancerStageIVcolonadenocarcinoma:ImplicationsforastandardizedscoringsystemJ.Cancer,2001,92(5):1331-1346.2BergstromJD,WestermarkB,HeldinNE.Epidermalgrowthfactorre-ceptorsignalingactivatesmetinhumananaplasticthyroidcarcinomacellsJ.ExperimentalCellResearch,2000,259(1):292-299.3DenningMF,DlugoszAA,ChengC,etal.Cross-talkbetweenepidermalgrowthfactorreceptorandproteinkinaseCduringcalciumnduceddifferentiationofkeratinoytesJ.ExperimentalDermatology,2000,9(3):192-199.,4,5,Patients4Celllines,cultureconditions,andfluorescenceinSituhybridizationanalysis(MCF,MDA-MB-468,BT-20,MDA-MB-468A)EnumerationofCTCsbyCS(FITC-thefourthchanneloftheCS)SamplepreparationformolecularanalysisafterCellSearchdetectionIsolationofCTCsbymicromanipulationWhole-GenomeAmplificationofDNAfromsingletumorcellswiththeGenomePlexkitWGAofDNAfromsingletumorcellswiththeGenomiPhikitIdentificationofEGFRgeneamplificationsbyPCRonWGAproducts(LINE1reference,EGFRtarget)Sequencingofsingle-cellWGAproducts,MaterialsandMethods,5,.,6,Results,ApplicabilityofWGAfromsingle-cellDNADetectionofEGFRexpressionandgeneamplificationsonsingleCTCsMutationalanalysisofsingleCTCsMutationsindownstreamgenesoftheEGFRsignalingpathway,6,7,Discussion,GenomiPhi-amplifiedDNAwasmoresuitableThemeanandmediangeneamplificationrates(43.89-and40.29-fold)determinedonGenomiPhiWGAproductsaresimilartothosedeterminedbyqPCRonDNAextracts(approximately107cells)andbyFISHanalysis(30-to40-fold)andareconsistentwithpublisheddataforMDA-MB-468cellpopulations.,7,8,Wedetected2CTCsin49%ofpatientswithmCRC(24of49)and22%ofpatientswithnmCRC(7of32).Theseresultsaresimilartopreviousfindingsrevealingdetectionratesof2CTCsin33%61%ofpatientswithmCRC(17,30)and26%ofpatientswithnmCRC.1CohenSJ,PuntCJ,IannottiN,SaidmanBH,SabbathKD,GabrailNY,etal.Relationshipofcirculatingtumorcellstotumorresponse,progression-freesurvival,andoverallsurvivalinpatientswithmetastaticcolorectalcancer.JClinOncol2008;26:321321.2SastreJ,MaestroML,PuenteJ,VeganzonesS,AlfonsoR,RafaelS,etal.Circulatingtumorcellsincolorectalcancer:correlationwithclinicalandpathologicalvariables.AnnOncol2008;19:9358.58:15.,8,9,Although30%90%ofadvancedprimaryCRCCasesweredescribedtobepositiveforEGFRexpression,CTCswithincreasedEGFRexpressionlevelscouldbedetectedinonly7of33(21%)CTC-positivepatients.1ShankaranV,ObelJ,BensonAB3rd.PredictingresponsetoEGFRinhibitorsinmetastaticcolorectalcancer:currentpracticeandfuturedirections.Oncologist2010;15:15767.,9,10,InadditiontothelowfrequencyofEGFRpositivityinourpatientcohort,notallCTCsofindividualcasescouldbeclassifiedasEGFRoverexpressing,revealingasubstantialheterogeneityinEGFRlevelsamongCTCsfromthesamepatient.ThesevaryingexpressionlevelspresumablyreflectintratumoralheterogeneityofEGFRexpression.,10,11,Unliketheoverexpressionoftheimmunotherapytargethumanepidermalgrowthfactorreceptor2(HER2)inbreastcancerpatients,whichismostoftenconnectedwithanamplificationoftheHER2gene,thecorrelationofEGFRproteinlevelsandgeneamplificationandtheirmeaningforEGFRimmunotherapyresponseisstillcontroversial.AsalreadyshownforEGFRproteinexpression,wealsoobtainedaheterogeneousdistributionofEGFRgeneamplificationratesbetweenCTCsofthesamepatientaswellasofdifferentpatients.1NicholsonRI,GeeJM,HarperME.EGFRandcancerprognosis.EurJCancer2001;37(Suppl4):S915.2CiardielloF,TortoraG.Epidermalgrowthfactorreceptor(EGFR)asatargetincancertherapy:understandingtheroleofreceptorexpressionandothermoleculardeterminantsthatcouldinflu-encetheresponsetoanti-EGFRdrugs.EurJCancer2003;39:134854.3MoroniM,VeroneseS,BenvenutiS,MarrapeseG,Sartore-BianchiA,DiNicolantonioF,etal.Genecopynumberforepidermalgrowthfactorreceptor(EGFR)andclinicalresponsetoantiEGFRtreatmentincolorectalcancer:acohortstudy.LancetOncol2005;6:27986.,11,12,Foraresponsetoanti-EGFRtherapies,anormalfunctionofdownstreamelementsofthesignalingpathway(e.g.,KRAS,BRAF,andPIK3CA)isessential.Toovercometheselimitations,wesuccessfullyestablishedaprotocolforthemutationalanalysisofDNAfromsingleCTCsdetectedbyCS.,12,13,ThemainnoveltyofthetechnologydescribedhereisthatitallowsmolecularanalysisofindividualCTCsaftertheyarecapturedandimmunostainedbyCS.Nevertheless,wecouldshowfeasibilityofseveraldownstreamapplicationstofurthercharacterizemolecularfeaturesofsingleCTCsdetectedwithCS,includingimmunocytochemistry,mutationalanalysis,andqPCR.,13,14,return,14,15,EGFRgeneexpressiondetectedbyCS(leftimages)correlateswithEGFRgeneamplificationratesdeterminedbyqPCRandFISH(righttables)withMCF-7(lowEGFRexpression=score01,EGFRamplificationrate0.55/0.7),MDA-MB-468A(moderateEGFRexpression=score2,EGFRamplificationrate1.83/notanalyzed),BT-20(strongEGFRexpression=score3,EGFRamplificationrate6.43/8.2),andMDA-MB-468(strongEGFRexpression=score3,EGFRamplificationrate38.65/30)cells.TheEGFRqPCRwasperformedonDNAextractsfromapproximately107CellsaswellasonWGAproductsfrom10singlecellsafterCS.,continue,10ngPurifiedproduct,15,16,Atleast2CTCsweredetectedin24of49(49%)patientswithmCRCand7of32(22%)patientswithnmCRC.Wefurtherassessed741CTCsfrom33patientswithCRC(27mCRC,6nmCRC)forEGFRproteinexpression.Altogether,1EGFR-positiveCTCcouldbeobservedin7of33(21%)patients,withonly2of33patients(6%)possessingstronglyEGFR-positiveCTCs.WhereasallCTCsdetectedinnmCRCpatientswereEGFR-negative,increasedEGFRlevelswereobservedin7of27(26%)patientswithmCRC.Furthermore,EGFRwasdifferentlyexpressedbetweenCTCsfromthesamepatients,rangingforexamplefromEGFR-negativetostronglyEGFR-positive.,HeterogeneityinEGFRexpressionintheCTCpopulationofpatient9.,continue,16,17,continue,17,18,ToanalyzeCTCheterogeneitymolecularly,wefocusedonbloodsamples(n=5)withmorethan20morphologicallyintactCTCsper7.5mL,whichexplainsinpartthelownumberofsamplesanalyzedbysingle-cellPCR.ThefailuretoanalyzeahighernumberofdetectedCTCsismainlyduetotheinabilitytotransferallCTCsundisturbedfromtheCellSearchcartridgeontoslidesandreidentifythemformicromanipulation.Thus,fromall33patientsanalyzedforEGFRexpressionofCTCs,onlyCTCsfrom3mCRCpatientscouldalsobeanalyzedforEGFRgeneamplificationbyqPCR.,EGFRgeneamplificationratedeterminedbyqPCRin26analyzedCTCsfrompatients6,9,and26.Comp.;CK-PE,cytokeratinphycoerythrin;DAPI,4,6-diamidino-2-phenylindole;APC,allophycocyanin;FITC,fluoresceinisothiocyanate.,return,18,19,FortheestablishmentofatechniquetodetectmutationsonWGAproductsfromsinglecells,weusedMDA-MB-231cellscarryingap53mutation.ToinvestigatetheimpactofcontaminationofasingleCTCwithsurroundingleukocytesduringmicromanipulation,weperformedamutationalanalysisonGenomiPhiWGAproductsfromaMDA-MB-231singlecellsupplementedwith12leukocytes.,Detectionofthep53R280KmutationinasingleMDA-MB-231cell(red).Additionofupto2leukocytes(green)orcell-freeliquidfromtheCScartridge(bluewaves)toasingleMDA-MB-231cellbymicromanipulationdidnotdisturbthedetectionofthep53mutation.,return,19,20,continue,第一步,第二步,20,21,(B),CTCscarryingPIK