1、 个体化医疗的现状与未来个体化医疗的现状与未来四四.生物标志物研究生物标志物研究吕林莉 M.D.,Ph.D.东南大学医学院Outline 生物标志物的概念 如何评价生物标志物？生物标志物的研究方法？生物标志物的概念生物标志物的概念什么是生物标志物什么是生物标志物(biomarker)？“measurable and quantifiable biological parameters”-a Medical Subject Heading(MeSH)term,1989“A characteristic that is objectively measured and evaluated as a
2、n indicator of normal biological processes,pathogenic processes or pharmacological responses to a therapeutic intervention.”-Biomarker Definitions Working Group,2001,NIHFeatures of a Useful BiomarkerHigh sensitivity and specificityEasy accessible sampleCorrelation with histological scoringChange in
3、advance of clinical signsTranslational from research to clinical use 不同水平生物标志物不同水平生物标志物DNAPrimary transcriptmRNATranscriptionproteinTranslationRNA processingNucleusBiomarker Examples Cholesterol is one of the most well-known biomarkers of cardiovascular health Physical measurements:body temperature(
4、fever);blood pressure(stroke risk)Other biomarkers:blood sugar level(diabetes)antigens(hepatitis)proteins(heart attack)genetic variations(Huntingtons disease)生物标志物的临床应用生物标志物的临床应用Ludwig JA et al.Nature reviews 2005,5:845-856 目前临床很多疾病的诊断依赖病理诊断，但不能作为常规筛查、监测手段 众多疾病缺乏早期、特异性生物标志物 治疗缺乏个体化方案生物标志物应用现状生物标志物应用
5、现状Clin J Am Soc Nephrol 3:18951901,2008.Biomarkers for chronic kidney diseaseAre we treating sub-populations?疾病疾病药物药物无反应率无反应率抑郁SSRIs,SNRIs,TCAs40-60%哮喘-adrenergics,LTD44-75%糖尿病Sulfonylurea,Biguanides,Glitazones50-75%肿瘤(乳腺癌 肺癌)Various70-100%From Kalow,Tyndale&Meyer,Pharmacogenomics,2001Novel biomarke
6、rs are needednEarly,accurate diagnosis -Individualized therapy and improved treatment outcomesnBetter defined populations will allow more specific drugs-Better efficacy-Fewer side effects“The use of biomarkers will change medical practice from a population-based approach to an individualized approac
7、h”Felix Frueh,Associate Director of Genomics at CDER,FDAEvolution of the biomarkers researchHigh plasma cholesterol and cardiovascular diseasesElevated plasma cholesterol Centre Moderate+severe Severe Italy 57.8%(93/161)19.3%(31/161)France 48.8%(189/387)17.3%(67/387)Nearly 50 percent of all future m
8、yocardial infarction and stroke events occur in those with normal or below normal lipid levels.EUROASPIRE Study Group,1997Additional biomarkers(inflammation)Hs-CRP and cardiovascular risk Hs-CRP is the most widely studied biomarker of inflammation in cardiovascular risk.Since the early 1990s with th
9、e development of highly sensitive assays for its measurement,correlations of hs-CRP with both cardiovascular risk factors and future cardiovascular events has been possible.CRP and LDLC levels and the risk of cardiovascular diseasesC-Reactive Protein(mg/L)3.0 160LDL-cholesterol(mg/dL)3.02.01.00.0Mul
10、tivariable Relative RiskIncreased CRP levels are associated with increased risk of cardiovascular events independently of LDL-C levelsRidker PM et al.,200227,939 womenHigh CRP-high LDLHigh CRP-low LDLLow CRP-low LDLLow CRP-high LDLPorbability of Event-free SurvivalYears of Follow-up0.990.980.970.960
11、.001.0002468Evolution of the biomarkers research:CRP and LDL-C levels and event-free survival among women27,939 womenThe median values were as follows:C-reactive protein:1.52 mg/L LDL cholesterol:123.7 mg/dL or:3.20 mmol/L CRP and LDL-C could give better prognostic information than the two markers s
12、eparately.Ridker PM et al.,2002如何评价生物标志物？如何评价生物标志物？常用评价指标常用评价指标（一）敏感性（一）敏感性（二）特异性（二）特异性（三）（三）Youden指数指数（四）阳性似然比（四）阳性似然比（五）阴性似然比（五）阴性似然比（六）阳性预报值（六）阳性预报值（七）阴性预报值（七）阴性预报值（八）（八）ROC曲线曲线ECG诊断试验的结果ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)9（FP）425104(FN)171（TN）275520180700(N)一、敏感性（一、敏感性（Sensitivity):TP/(TP+FN)=TPR
13、(true positive rate)TRP=Sen=416/(416+104)=0.8该指标只与病例组有关，反映了诊断试验检出病例的能力该指标只与病例组有关，反映了诊断试验检出病例的能力ECG诊断试验的结果ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)9（FP）425104(FN)171（TN）275520180700(N)二、特异性（二、特异性（Specificity)Spe=True negative rate(TNR)=TN(FP+TN)=171/(171+9)=0.95该指标只与对照组有关，反映了诊断试验排除非病例的能力。该指标只与对照组有关，反映了诊断试验排
14、除非病例的能力。灵敏度与特异度的优缺点优点：灵敏度与特异度不受患病率的影响，其取值范围均在（0,1）之间，其值越接近于1，说明其诊断准确性越好。缺点：当比较两个诊断试验时，单独使用灵敏度或特异度，可能出现矛盾。解决办法：将两指标结合：Youden指数、阳性似然比、阴性似然比等ECG诊断试验的结果ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)9（FP）425104(FN)171（TN）275520180700(N)三、三、Youden指数，指数，=Sen+Spe-1=TPR-FPR =0.8-0.05=0.75Youden指数取值范围在（指数取值范围在（0，1）之间，其值越
15、接近）之间，其值越接近1，诊断准确性越好。，诊断准确性越好。ECG诊断试验的结果ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)9（FP）425104(FN)171（TN）275520180700(N)ECG诊断试验的结果ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)9（FP）425104(FN)171（TN）275520180700(N)医生最关心的问题：医生最关心的问题：1.试验阳性时患病的概率多大？试验阳性时患病的概率多大？2.试验阴性时不患病的概率多大？试验阴性时不患病的概率多大？阳性预测值是在诊断试验阳性的受试者中，标准诊断有病的病例（真阳阳
16、性预测值是在诊断试验阳性的受试者中，标准诊断有病的病例（真阳性）所占的比例性）所占的比例 阴性预测值则是在诊断试验为阴性的受试者中，标准诊断证实无病的受试者阴性预测值则是在诊断试验为阴性的受试者中，标准诊断证实无病的受试者（真阴性）所占的比例。（真阴性）所占的比例。ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)9（FP）425104(FN)171（TN）275520180700(N)阳性预报值与阴性预报值ROC曲线 ROCROC（receiver operating receiver operating characteristiccharacteristic的缩写，译为
17、的缩写，译为“接受者工接受者工作特征作特征”）ROCROC曲线研究历史曲线研究历史1950s 雷达信号观测能力评价雷达信号观测能力评价1960s中期中期 实验心理学、心理物理实验心理学、心理物理学学1970s末与末与1980s初初 诊断医学诊断医学ROC的涵义与起源的涵义与起源不同诊断界值时不同诊断界值时灵敏度与特异度间的平衡灵敏度与特异度间的平衡(trade off)(trade off)0204060801005060708090100特异度灵敏度百分率（）百分率（）Receiver Operating Characteristic curve Area Under Curve(AUC)-
18、GraphedCurve 1=.50 Pure chanceno better than random guessCurve 3 is better than Curve 2Curve 4=1.0 Totally Sensitive completely accurate classification of effectively and less-effectively instructed students完美诊断试验完美诊断试验0.00.20.40.60.81.00.00.20.40.60.81.0FPRTPR无用诊断试验无用诊断试验0.00.20.40.60.81.00.00.20.4
19、0.60.81.0FPRTPR完美与无用的完美与无用的ROC曲线曲线真真阳阳性性率率即即灵灵敏敏度度假阳性率假阳性率 即即 1特异度特异度机率线机率线(chance line)(diagonal reference line)诊断准确度较低（诊断准确度较低（0.9）0.00.20.40.60.81.00.00.20.40.60.81.0FPRTPRA0.938ROC曲线下面积（曲线下面积（Area）与诊断准确度高低）与诊断准确度高低高 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)0.5
20、0-0.60=fail(F)ROC曲线小结曲线小结lROCROC曲线反映了灵敏度与特异度间的平衡曲线反映了灵敏度与特异度间的平衡 (增增加灵敏度将降低特异度；增加特异度将降低加灵敏度将降低特异度；增加特异度将降低灵敏度灵敏度)。l在在ROCROC曲线空间，如果曲线沿着左边线，然后曲线空间，如果曲线沿着左边线，然后沿着上边线越紧密，则试验准确度越高。沿着上边线越紧密，则试验准确度越高。l在在ROCROC曲线空间，如果曲线沿着机会线（曲线空间，如果曲线沿着机会线（4545度度对角线）越紧密，则试验准确度越低。对角线）越紧密，则试验准确度越低。lROCROC曲线下面积是重要的试验准确度指标。曲线下面
21、积是重要的试验准确度指标。生物标志物研究方法生物标志物研究方法 phasePhase1Preclinical ExploratoryPhase2Clinical Assay and ValidationPhase3RetrospectiveLongitudinalPhase4Prospective ScreeningPhase5Disease controlObjectiveTarget biomarker identification,feasibilityStudy assay in people with and without diseaseCase-control studies u
22、sing specimensLongitudinal studies to predict diseaseClinical useSite Biomarker development labBiomarker validation labClinical epidemiologic centersCohort studiesCommunity DesignCross-sectionalCross-sectionalCase-controlprospectiveRCTSample sizesmallsmallmodestmediumlargeVasan RS.Circulation.2006;1
23、13:2335-2362.The Agendia MammaPrint Test首个FDA批准的基因组检测试验 -Feb.2007How they got there？2002 Discovery of 70 gene signature(117 patients)2002 Duplication of results(in another sample set:295 patients)2006 Assay performance 2006 Optimized array format:reproducibility;back to original sample set 2006 Exte
24、rnal confirmation(307 patients,5 hospitals)2007 Approval by FDA生物标志物研究技术 传统研究方法：传统研究方法：PCR,Western blotting,ELISA,et al 新型研究方法：新型研究方法：基因组学技术 蛋白质组学技术：2-DIGE/MS,蛋白质芯片生物标志物研究方法生物标志物研究方法 Question1.What human samples should be collected,and how should they be used?Does this vary between discovery,validat
25、ion and implementation?Answer1.All biological samples are eligible for collection Collected biological material depends on analyte and tissue sourceExamples Biological fluids Serum,plasma,urine,csf Secretions Saliva,seminal fluid Body cavity fluids Pleural fluid,peritoneal fluid,etc Specific tissue
26、material Specialized cells reproductive cells Non-cellular Ideal Biomarker discovery samples should be identical to the projected testing situation (e.g.Do not study plasma for discovery,and then validate or implement assay using serum)Practical set up study with samples that are as close to the tes
27、ting situation as possibleQuestion 2.What is the role of routinely accessible biofluids such as plasma,serum,and urine?What is the role of“proximal”fluids like CSF,synovial fluid,ascites,pancreatic ductal fluid,etc?What is the role of solid tissues?Role of routinely accessible biofluids Very importa
28、nt in the discovery of biomarkers of diseases(systemic vs.organ specific/local)Important for:early detection disease severity tumor burden prognosis monitoring of response to therapy“Proximal fluids”Can reflect disease perturbations in the organs or tissues from which they are secreted Solid tissues
29、 Very important for the development of novel insitu biomarkers Immunofluorescence,immunocytochemistry Imaging mass spectrometryQuestion 3.Must human sample collection be prospective,or can existing repositories be used?What considerations are important in determining the adequacy of repository sampl
30、es?Answer3:Ultimate confirmation of the validity of a biomarker has to be proven in a prospective study Nevertheless,well designed retrospective studies using well characterized samples in repositories can be performed and frequently yield viable candidates生物标志物研究面临的挑战生物标志物研究面临的挑战 The multidisciplinary nature of biomarker discovery&development Complex Multiple disciplines Heterogeneous populations Standards not established Expensive Human resources Multiple technologies
