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

HeterogeneityofEpidermalGrowthFactorReceptorStatusandMutationsofKRAS/PIK3CAinCirculatingTumorCellsofPatientswithColorectalCancerClinicalChemistryNovember7，2012Studyintroduction•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.Lièvreetal.JClinOncol2008;26:374-9.•5.美国FDA相关网站：•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。•[1]GoldsteinNS,ArminM.EpidermalgrowthfactorreceptorimmunohistochemicalreactivityinpatientswithAmericanJointCommitteeonCancerStageIVcolonadenocarcinoma:Implicationsforastandardizedscoringsystem[J].Cancer,2001,92(5):1331-1346.•[2]BergstromJD,WestermarkB,HeldinNE.Epidermalgrowthfactorre-ceptorsignalingactivatesmetinhumananaplasticthyroidcarcinomacells[J].ExperimentalCellResearch,2000,259(1):292-299.[3]DenningMF,DlugoszAA,ChengC,etal.Cross-talkbetweenepidermalgrowthfactorreceptorandproteinkinaseCduringcalciumnduceddifferentiationofkeratinoytes[J].ExperimentalDermatology,2000,9(3):192-199.Patients4Celllines,cultureconditions,andfluorescenceinSituhybridizationanalysis(MCF,MDA-MB-468,BT-20,MDA-MB-468A)EnumerationofCTCsbyCS(FITC-thefourthchanneloftheCS)SamplepreparationformolecularanalysisafterCellSearchdetectionIsolationofCTCsbymicromanipulationWhole-GenomeAmplificationofDNAfromsingletumorcellswiththeGenomePlexkitWGAofDNAfromsingletumorcellswiththeGenomiPhikitIdentificationofEGFRgeneamplificationsbyPCRonWGAproducts(LINE1reference,EGFRtarget)Sequencingofsingle-cellWGAproductsMaterialsandMethodsResults•ApplicabilityofWGAfromsingle-cellDNA•DetectionofEGFRexpressionandgeneamplificationsonsingleCTCs•MutationalanalysisofsingleCTCs•MutationsindownstreamgenesoftheEGFRsignalingpathwayDiscussion•GenomiPhi-amplifiedDNAwasmoresuitable•Themeanandmediangeneamplificationrates(43.89-and40.29-fold)determinedonGenomiPhiWGAproductsaresimilartothosedeterminedbyqPCRonDNAextracts(approximately107cells)andbyFISHanalysis(30-to40-fold)andareconsistentwithpublisheddataforMDA-MB-468cellpopulations.•Wedetected≥2CTCsin49%ofpatientswithmCRC(24of49)and22%ofpatientswithnmCRC(7of32).Theseresultsaresimilartopreviousfindingsrevealingdetectionratesof≥2CTCsin33%–61%ofpatientswithmCRC(17,30)and26%ofpatientswithnmCRC.•[1]CohenSJ,PuntCJ,IannottiN,SaidmanBH,SabbathKD,GabrailNY,etal.Relationshipofcirculatingtumorcellstotumorresponse,progression-freesurvival,andoverallsurvivalinpatientswithmetastaticcolorectalcancer.JClinOncol2008;26:3213–21.•[2]SastreJ,MaestroML,PuenteJ,VeganzonesS,AlfonsoR,RafaelS,etal.Circulatingtumorcellsincolorectalcancer:correlationwithclinicalandpathologicalvariables.AnnOncol2008;19:935–8.58:1–5.•Although30%–90%ofadvancedprimaryCRCCasesweredescribedtobepositiveforEGFRexpression,CTCswithincreasedEGFRexpressionlevelscouldbedetectedinonly7of33(21%)CTC-positivepatients.•[1]ShankaranV,ObelJ,BensonAB3rd.PredictingresponsetoEGFRinhibitorsinmetastaticcolorectalcancer:currentpracticeandfuturedirections.Oncologist2010;15:157–67.•InadditiontothelowfrequencyofEGFRpositivityinourpatientcohort,notallCTCsofindividualcasescouldbeclassifiedasEGFRoverexpressing,revealingasubstantialheterogeneityinEGFRlevelsamongCTCsfromthesamepatient.ThesevaryingexpressionlevelspresumablyreflectintratumoralheterogeneityofEGFRexpression.•Unliketheoverexpressionoftheimmunotherapytargethumanepidermalgrowthfactorreceptor2(HER2)inbreastcancerpatients,whichismostoftenconnectedwithanamplificationoftheHER2gene,thecorrelationofEGFRproteinlevelsandgeneamplificationandtheirmeaningforEGFRimmunotherapyresponseisstillcontroversial.AsalreadyshownforEGFRproteinexpression,wealsoobtainedaheterogeneousdistributionofEGFRgeneamplificationratesbetweenCTCsofthesamepatientaswellasofdifferentpatients.•[1]NicholsonRI,GeeJM,HarperME.EGFRandcancerprognosis.EurJCancer2001;37(Suppl4):S9–15.[2]CiardielloF,TortoraG.Epidermalgrowthfactorreceptor(EGFR)asatargetincancertherapy:understandingtheroleofreceptorexpressionandothermoleculardeterminantsthatcouldinflu-encetheresponsetoanti-EGFRdrugs.EurJCancer2003;39:1348–54.[3]MoroniM,VeroneseS,BenvenutiS,MarrapeseG,Sartore-BianchiA,DiNicolantonioF,etal.Genecopynumberforepidermalgrowthfactorreceptor(EGFR)andclinicalresponsetoantiEGFRtreatmentincolorectalcancer:acohortstudy.LancetOncol2005;6:279–86.•Foraresponsetoanti-EGFRtherapies,anormalfunctionofdownstreamelementsofthesignalingpathway(e.g.,KRAS,BRAF,andPIK3CA)isessential.•Toovercometheselimitations,wesuccessfullyestablishedaprotocolforthemutationalanalysisofDNAfromsingleCTCs