MolecularDiagnosisofCancer-12-12-13final

癌症的分子诊断顾海华,Ph.DCell:15058751720email:[e-mail]癌症的分子诊断用癌症的分子特证来对癌症组织和细胞进行检测，以此来对癌症分类并为制定有效的治疗方案提供依据有效的治疗方案1）彻底治愈（早发现）2）控制病情并且延缓死亡期3）靶向治疗：有效治疗和降低副作用4）降低治疗费用1.癌症的发病机理和特征2.癌症的分子诊断的重要性4.癌症分子诊断和个体化治疗3.用于癌症分子诊断的技术癌症的发生是由于细胞里DNA的突变而引起的体细胞突变生殖细胞突变GenomeInstabilityofCancerWikipediaDiverseSomaticMutationsUncoveredbyExomeSequencinginColorectal（结直肠）Tumors*Singlebasemutations(92%)*Insertion,Deletions,orDuplications(Theremaining8%).Woodetal,2007,SCIENCE,VOL318:1108-*Mutatedgenemountains(5).*Mutatedgenehills(largenumbers~80)DNA和RNA变异的检测是癌症的分子诊断中一个重要部分EightHallmarksofCancer避免免疫击毁持续细胞分裂的信号传导躲避生长抑制体激活侵袭和转移抵抗细胞死亡诱导血管生成细胞能量代谢调节失控促进永久复制致癌基因EGFR,BCR-ABL,Her2抑癌基因BRCA1/2,PT53EnhancedGlucoseUptake癌症分子诊断与癌症特征致癌基因BCR-ABL2.Whyis癌症分子诊断needed?a.预测：遗传性癌症（BRCA1/2,TP53)b.早期诊断：及时治疗c.预后：Breastcancer:ER+;TNBCd.验证对治疗的反应e.传统的肿瘤检测特异性和灵敏性差f.肿瘤的异质性-个体化治疗传统的肿瘤检测和治疗方法1）视，摸，X光射线,CT,MRI,活检，病理切片评估2)手术后的化疗，放疗副作用大，易复发，抗药性(denovo)用于癌症分子诊断的技术PCR/DNAsequencingcDNAMicroarrays*FISH（荧光原位杂交）Highthroughputsequencing•Genomewidesequencing*RNAseq(transcriptomeprofiling)*Exome-sequencing*CGH（比较基因组杂交）a.DetectingDNAandRNAmutationsandcopynumberchangesincancercellsCNV:CGH,FISHRNA-Seq(TranscriptomeProfiling)a.Cloonan,N.Nat.Methods5,613–619(2008)b.Mortazavi,A.,Williams,B.A.,McCue,K.,Schaeffer,L.&Wold,B.Nat.Methods5,621–628(2008)c.Shendure,J.,Mitra,R.D.,Varma,C.&Church,G.M.Nat.Rev.Genet.5,335–344(2004).naturemethods|VOL.5NO.7|JULY2008|585转录组分析1.Discovernoveltranscripts:Newalternativesplicingspecies（新的选择性的剪接RNA产物）FusiontranscriptsduetochromosomaltranslocationNewtranscriptionalunitsNewmutationsandSNP(单核苷酸多态性)2.Highlyrelatedgenescanbeanalyzedeasily3.Detectlowabundanttranscriptswithquantitation(digital;spikedwithknownconcentrationofRNAstandard)4.ResultscanbereproducedbetweenlaboratoriesandacrossplatformsAdvantagesofRNA-SeqoverMicroarraysforGeneExpressionAnalysisExomeSequencing(AKAtargetedexomecapture)180,000exonsinthegenome.Theseconstituteabout1%ofthehumangenomeorabout30megabases.WikipediaAGGUAGGUTCTCCACAComparativeGenomicHybridization（CGH）Wikipedia检测基因组中不同DNA拷贝数变化ChromosomalRegionGainandLossinANeuronalTumorSamplebyCGHWikipediaWikipedia检测染色体上基因拷贝数变化b.Detectingalteredpropertiesofcancercells*Immunohistochemistry(IHC):*Cellspecificsecretedproteins/peptides:ELISA:PSAinprostatecancer*PET-CT(higherglucoseuptake):影像成像技术*Circulatingtumorcells(CTC)(metastasis):流式细胞仪CellsearchCTCTest(ApprovedbyFDA)用于癌症分子诊断的技术Immunohistochemistry(IHC)（免疫组化）H202ELISA(Enzyme-LinkedImmunosorbentAssay)(酶联免疫吸附试验)DetectionofTumorMarkersbyELISACA-125(aglycoprotein)iscomponentofocularsurface,therespiratorytractandthefemalereproductivetractepithelia.It’sleveliselevatedinbloodofovariancancerpatients(butnotveryspecific).PSAisamemberofthekallikrein-relatedpeptidasefamilyandissecretedbytheepithelialcellsoftheprostategland.It’sleveliselevatedinbloodofprostatecancerpatients.1.PET-CT:0.5cm的结节(~90%)Cancercellstakeupalotofglucose2.常规CT:1cm的结节PET-CT(Positronemissiontomography–computedtomography)(正电子发射断层扫描)2-[18F]-fluoro-2-deoxy-D-glucose(FDG)2-氟-2-脱氧-D-葡萄糖PET-CT(正电子发射断层扫描)检测肿瘤的原理脂肪和核酸合成的底物有氧肝糖CELLSEARCH®CTCTest(Veridex)A7.5-mLsampleofbloodisplacedinaspecialtube,centrifugedtoseparatesolidbloodcomponentsfromplasma,thenplacedintheCellTracks®AutoPrep®System.Usingferrofluidnanoparticleswithantibodiesthattargetepithelialcelladhesion(EpCAM),CTCsaremagneticallyseparatedfromthebulkofothercellsintheblood.Useantibodiestostaincytokeratins(CK8,18,19)toconfirmthepresencesofCTC.UseCD45antibodytodepletecontaminatedbloods.ThepresenceofCTCsintheperipheralbloodsisassociatedwithdecreasedprogressionfreesurvivalanddecreasedoverallsurvivalinpatientstreatedformetastaticbreastormetastaticcolorectalcancer.ThePresenceofCTCsinthePeripheralBloodscanPredictOverallSurvivalofBreastCancer癌症分子诊断和个体化治疗1.肿瘤细胞基因组的不稳和异质性2.制定更好的治疗方案和方法c.NSCLC:EGFRmutation,EML4-ALKb.BreastCancer:ER,Her2a.CML:-Imatinibresistance-BCR-ABLmutationsMostofthecancersaredrivenbymutationsofdifferentgenesOnlysomerarecancer(i.e.CML)ismainlydependentonasinglegenemutationFigure2.23aTheBiologyofCancer(©GarlandScience2007)ChromosomalTranslocationinChronicMyelogenousLeukemia(CML)Figure4.15aTheBiologyofCancer(©GarlandScience2007)BCR-ABListheResultofReciprocalChromosomalTranslocationBCR-ABLFigure4.15bTheBiologyofCancer(©GarlandScience2007)DifferentFormofBCR-ABLisAssociatedwithDistinctTypesofLeukemiaDifferentpartsofBCRSameABL(B)癌症分子诊断NeedsPCRtoidentitydifferentFusionconjunctionsanddifferentFormsofBCR-ABLImatinibMakesCMLfromaDeadlyCancerintoaTrueChronicDisease(Thebestexampleoftargetedcancertherapy)JNCIJNatlCancerInst(2011)103(7):553-561.Gleevec/ImatinibforTreatingChronicMyelogenousLeukemia(CML)NCI/NIHwebsiteASmallmoleculeinhibitor/ATPCompetitorBCR-ABLBCR-ABLImatinib抗药性1.ActivationoftheBCR-ABLIndependentpathwaysBreakpointODS/TkinaseDBLSH3SH2NLSDNABDABDYkinaseBCR|ABLRaspathwayPI3KpathwayJAK/STATpathwayGrowthfactorreceptorsReactiveoxygenspeciesTyrkinases/phosphatasesandtheirsubstratesCytoskeletonassociatedproteins…othertargetsImatinib抗药性1.ActivationoftheBCR-ABLIndependentpathways(minor)2.Mut