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個(gè)體化醫(yī)療的現(xiàn)狀與未來(lái)四.生物標(biāo)志物研究呂林莉M.D.,Ph.D.東南大學(xué)醫(yī)學(xué)院 Outline生物標(biāo)志物的概念如何評(píng)價(jià)生物標(biāo)志物？生物標(biāo)志物的研究方法？ 生物標(biāo)志物的概念 什么是生物標(biāo)志物(biomarker)？“measurableandquantifiablebiologicalparameters”--aMedicalSubjectHeading(MeSH)term,1989“Acharacteristicthatisobjectivelymeasuredandevaluatedasanindicatorofnormalbiologicalprocesses,pathogenicprocessesorpharmacologicalresponsestoatherapeuticintervention.”--BiomarkerDefinitionsWorkingGroup,2001,NIH FeaturesofaUsefulBiomarkerHighsensitivityandspecificityEasyaccessiblesampleCorrelationwithhistologicalscoringChangeinadvanceofclinicalsignsTranslationalfromresearchtoclinicaluse 不同水平生物標(biāo)志物DNAPrimarytranscriptmRNATranscriptionproteinTranslationRNAprocessingNucleus BiomarkerExamplesCholesterolisoneofthemostwell-knownbiomarkersofcardiovascularhealthPhysicalmeasurements:bodytemperature(fever);bloodpressure(strokerisk)Otherbiomarkers:?bloodsugarlevel(diabetes)?antigens(hepatitis)?proteins(heartattack)?geneticvariations(Huntington’sdisease) 生物標(biāo)志物的臨床應(yīng)用LudwigJAetal.Naturereviews2005,5:845-856 目前臨床很多疾病的診斷依賴病理診斷，但不能作為常規(guī)篩查、監(jiān)測(cè)手段眾多疾病缺乏早期、特異性生物標(biāo)志物治療缺乏個(gè)體化方案生物標(biāo)志物應(yīng)用現(xiàn)狀 ClinJAmSocNephrol3:1895–1901,2008.Biomarkersforchronickidneydisease Arewetreatingsub-populations?疾病藥物無(wú)反應(yīng)率抑郁SSRIs,SNRIs,TCAs40-60%哮喘?-adrenergics,LTD44-75%糖尿病Sulfonylurea,Biguanides,Glitazones50-75%腫瘤(乳腺癌肺癌)Various70-100%FromKalow,Tyndale&Meyer,Pharmacogenomics,2001 NovelbiomarkersareneededEarly,accuratediagnosis-IndividualizedtherapyandimprovedtreatmentoutcomesBetterdefinedpopulationswillallowmorespecificdrugs-Betterefficacy-Fewersideeffects “Theuseofbiomarkerswillchangemedicalpracticefromapopulation-basedapproachtoanindividualizedapproach”FelixFrueh,AssociateDirectorofGenomicsatCDER,FDA Evolutionofthebiomarkersresearch HighplasmacholesterolandcardiovasculardiseasesNearly50percentofallfuturemyocardialinfarctionandstrokeeventsoccurinthosewithnormalorbelownormallipidlevels.EUROASPIREStudyGroup,1997%ofMI Additionalbiomarkers(inflammation)Hs-CRPandcardiovascularriskHs-CRPisthemostwidelystudiedbiomarkerofinflammationincardiovascularrisk.Sincetheearly1990swiththedevelopmentofhighlysensitiveassaysforitsmeasurement,correlationsofhs-CRPwithbothcardiovascularriskfactorsandfuturecardiovasculareventshasbeenpossible. CRPandLDL–ClevelsandtheriskofcardiovasculardiseasesC-ReactiveProtein(mg/L)<1.01.0-3.0>3.0<130130-160>160LDL-cholesterol(mg/dL)3.02.01.00.0MultivariableRelativeRiskIncreasedCRPlevelsareassociatedwithincreasedriskofcardiovasculareventsindependentlyofLDL-ClevelsRidkerPMetal.,200227,939women HighCRP-highLDLHighCRP-lowLDLLowCRP-lowLDLLowCRP-highLDLPorbabilityofEvent-freeSurvivalYearsofFollow-up0.990.980.970.960.001.0002468Evolutionofthebiomarkersresearch:CRPandLDL-Clevelsandevent-freesurvivalamongwomen27,939womenThemedianvalueswereasfollows:C-reactiveprotein:1.52mg/LLDLcholesterol:123.7mg/dLor:3.20mmol/LCRPandLDL-Ccouldgivebetterprognosticinformationthanthetwomarkersseparately.RidkerPMetal.,2002 如何評(píng)價(jià)生物標(biāo)志物？ 常用評(píng)價(jià)指標(biāo)（一）敏感性（二）特異性（三）Youden指數(shù)（四）陽(yáng)性似然比（五）陰性似然比（六）陽(yáng)性預(yù)報(bào)值（七）陰性預(yù)報(bào)值（八）ROC曲線 ECG診斷試驗(yàn)的結(jié)果ECG診斷結(jié)果心肌梗塞合計(jì)出現(xiàn)不出現(xiàn)陽(yáng)性陰性合計(jì)416(TP)9（FP）425104(FN)171（TN）275520180700(N)一、敏感性（Sensitivity):TP/(TP+FN)=TPR(truepositiverate)TRP=Sen=416/(416+104)=0.8該指標(biāo)只與病例組有關(guān)，反映了診斷試驗(yàn)檢出病例的能力 ECG診斷試驗(yàn)的結(jié)果ECG診斷結(jié)果心肌梗塞合計(jì)出現(xiàn)不出現(xiàn)陽(yáng)性陰性合計(jì)416(TP)9（FP）425104(FN)171（TN）275520180700(N)二、特異性（Specificity)Spe=Truenegativerate(TNR)=TN(FP+TN)=171/(171+9)=0.95該指標(biāo)只與對(duì)照組有關(guān)，反映了診斷試驗(yàn)排除非病例的能力。 靈敏度與特異度的優(yōu)缺點(diǎn)優(yōu)點(diǎn)：靈敏度與特異度不受患病率的影響，其取值范圍均在（0,1）之間，其值越接近于1，說(shuō)明其診斷準(zhǔn)確性越好。缺點(diǎn)：當(dāng)比較兩個(gè)診斷試驗(yàn)時(shí)，單獨(dú)使用靈敏度或特異度，可能出現(xiàn)矛盾。解決辦法：將兩指標(biāo)結(jié)合：Youden指數(shù)、陽(yáng)性似然比、陰性似然比等 ECG診斷試驗(yàn)的結(jié)果ECG診斷結(jié)果心肌梗塞合計(jì)出現(xiàn)不出現(xiàn)陽(yáng)性陰性合計(jì)416(TP)9（FP）425104(FN)171（TN）275520180700(N)三、Youden指數(shù)，=Sen+Spe-1=TPR-FPR=0.8-0.05=0.75Youden指數(shù)取值范圍在（0，1）之間，其值越接近1，診斷準(zhǔn)確性越好。 ECG診斷試驗(yàn)的結(jié)果ECG診斷結(jié)果心肌梗塞合計(jì)出現(xiàn)不出現(xiàn)陽(yáng)性陰性合計(jì)416(TP)9（FP）425104(FN)171（TN）275520180700(N) ECG診斷試驗(yàn)的結(jié)果ECG診斷結(jié)果心肌梗塞合計(jì)出現(xiàn)不出現(xiàn)陽(yáng)性陰性合計(jì)416(TP)9（FP）425104(FN)171（TN）275520180700(N) 醫(yī)生最關(guān)心的問題：1.試驗(yàn)陽(yáng)性時(shí)患病的概率多大？2.試驗(yàn)陰性時(shí)不患病的概率多大？ 陽(yáng)性預(yù)測(cè)值是在診斷試驗(yàn)陽(yáng)性的受試者中，標(biāo)準(zhǔn)診斷有病的病例（真陽(yáng)性）所占的比例 陰性預(yù)測(cè)值則是在診斷試驗(yàn)為陰性的受試者中，標(biāo)準(zhǔn)診斷證實(shí)無(wú)病的受試者（真陰性）所占的比例。 ECG診斷結(jié)果心肌梗塞合計(jì)出現(xiàn)不出現(xiàn)陽(yáng)性陰性合計(jì)416(TP)9（FP）425104(FN)171（TN）275520180700(N) 陽(yáng)性預(yù)報(bào)值與陰性預(yù)報(bào)值 ROC曲線 ROC（receiveroperatingcharacteristic的縮寫，譯為“接受者工作特征”）ROC曲線研究歷史1950’s雷達(dá)信號(hào)觀測(cè)能力評(píng)價(jià)1960’s中期實(shí)驗(yàn)心理學(xué)、心理物理學(xué)1970’s末與1980’s初診斷醫(yī)學(xué)ROC的涵義與起源 不同診斷界值時(shí)靈敏度與特異度間的平衡(tradeoff)0204060801005060708090100特異度靈敏度百分率（％） ReceiverOperatingCharacteristiccurveAreaUnderCurve(AUC)-GraphedCurve1=.50?Purechance…nobetterthanrandomguessCurve3isbetterthanCurve2Curve4=1.0?TotallySensitive?completelyaccurateclassificationofeffectivelyandless-effectivelyinstructedstudents 完美與無(wú)用的ROC曲線真陽(yáng)性率即靈敏度假陽(yáng)性率即1－特異度機(jī)率線(chanceline)(diagonalreferenceline) 診斷準(zhǔn)確度較低（<0.7）0.00.20.40.60.81.00.00.20.40.60.81.0FPRTPRA＝0.664A＝0.830診斷準(zhǔn)確度較高（>0.9）0.00.20.40.60.81.00.00.20.40.60.81.0FPRTPRA＝0.938ROC曲線下面積（Area）與診斷準(zhǔn)確度高低高0.90-1.00=excellent(A)中0.80-0.90=good(B)0.70-0.80=fair(C)低0.60-0.70=poor(D)0.50-0.60=fail(F) ROC曲線小結(jié)ROC曲線反映了靈敏度與特異度間的平衡(增加靈敏度將降低特異度；增加特異度將降低靈敏度)。在ROC曲線空間，如果曲線沿著左邊線，然后沿著上邊線越緊密，則試驗(yàn)準(zhǔn)確度越高。在ROC曲線空間，如果曲線沿著機(jī)會(huì)線（45度對(duì)角線）越緊密，則試驗(yàn)準(zhǔn)確度越低。ROC曲線下面積是重要的試驗(yàn)準(zhǔn)確度指標(biāo)。 生物標(biāo)志物研究方法 phasePhase1PreclinicalExploratoryPhase2ClinicalAssayandValidationPhase3RetrospectiveLongitudinalPhase4ProspectiveScreeningPhase5DiseasecontrolObjectiveTargetbiomarkeridentification,feasibilityStudyassayinpeoplewithandwithoutdiseaseCase-controlstudiesusingspecimensLongitudinalstudiestopredictdiseaseClinicaluseSiteBiomarkerdevelopmentlabBiomarkervalidationlabClinicalepidemiologiccentersCohortstudiesCommunityDesignCross-sectionalCross-sectionalCase-controlprospectiveRCTSamplesizesmallsmallmodestmediumlargeVasanRS.Circulation.2006;113:2335-2362. TheAgendiaMammaPrintTest首個(gè)FDA批準(zhǔn)的基因組檢測(cè)試驗(yàn)--Feb.2007 Howtheygotthere？2002–Discoveryof70genesignature(117patients)2002–Duplicationofresults(inanothersampleset:295patients)2006–Assayperformance2006–Optimizedarrayformat:reproducibility;backtooriginalsampleset2006–Externalconfirmation(307patients,5hospitals)2007–ApprovalbyFDA 生物標(biāo)志物研究技術(shù)傳統(tǒng)研究方法：PCR,Westernblotting,ELISA,etal新型研究方法：基因組學(xué)技術(shù)蛋白質(zhì)組學(xué)技術(shù)：2-DIGE/MS,蛋白質(zhì)芯片 生物標(biāo)志物研究方法Question1.Whathumansamplesshouldbecollected,andhowshouldtheybeused?Doesthisvarybetweendiscovery,validationandimplementation? Answer1.AllbiologicalsamplesareeligibleforcollectionCollectedbiologicalmaterialdependsonanalyteandtissuesourceExamples–Biologicalfluids?Serum,plasma,urine,csf?Secretions?Saliva,seminalfluid–Bodycavityfluids?Pleuralfluid,peritonealfluid,etc–Specifictissuematerial?Specializedcells–reproductivecells–Non-cellular Ideal–Biomarkerdiscoverysamplesshouldbeidenticaltotheprojectedtestingsituation–(e.g.Donotstudyplasmafordiscovery,andthenvalidateorimplementassayusingserum)Practical–setupstudywithsamplesthatareasclosetothetestingsituationaspossible Question2.Whatistheroleofroutinelyaccessiblebiofluidssuchasplasma,serum,andurine?Whatistheroleof“proximal”fluidslikeCSF,synovialfluid,ascites,pancreaticductalfluid,etc?Whatistheroleofsolidtissues? RoleofroutinelyaccessiblebiofluidsVeryimportantinthediscoveryofbiomarkersofdiseases(systemicvs.organspecific/local)Importantfor:–earlydetection–diseaseseverity–tumorburden–prognosis–monitoringofresponsetotherapy “Proximalfluids”–CanreflectdiseaseperturbationsintheorgansortissuesfromwhichtheyaresecretedSolidtissues–Veryimportantforthedevelopmentofnovelinsitubiomarkers?Immunofluorescence,immunocytochemistry?Imagingmassspectrometry Question3.Musthumansamplecollectionbeprospective,orcanexistingrepositoriesbeused?Whatconsiderationsareimportantindeterminingtheadequacyofrepositorysamples? Answer3:UltimateconfirmationofthevalidityofabiomarkerhastobeproveninaprospectivestudyNevertheless,welldesignedretrospectivestudiesusingwellcharacterizedsamplesinrepositoriescanbeperformedandfrequentlyyieldviablecandidates 生物標(biāo)志物研究面臨的挑戰(zhàn)Themultidisciplinarynatureofbiomarkerdiscovery&developmentComplexMultipledisciplinesHeterogeneouspopulationsStandardsnotestablishedExpensiveHumanresourcesMultipletechnologies