个体化医疗的现状与未来生物标志物实用课件

1、 个体化医疗的现状与未来四. 生物标志物研究吕林莉 M.D., Ph.D.东南大学医学院 个体化医疗的现状与未来四. 生物标志物研究吕林莉 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？Outline生物标志物的概念生物标志物的概念生物标志物的概念什么是生物标志物(biomarker)？“measurable and quantifiable biological parameters” - a Medical Subject Heading (MeSH) term, 1989“ A characteristic that is objectively measure

2、d and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention.” -Biomarker Definitions Working Group, 2001,NIH什么是生物标志物(biomarker)？“measurablFeatures of a Useful BiomarkerHigh sensitivity and specificityEasy accessible

3、sampleCorrelation with histological scoringChange in advance of clinical signsTranslational from research to clinical use Features of a Useful Biomarke不同水平生物标志物DNAPrimary transcriptmRNATranscriptionproteinTranslationRNA processingNucleus不同水平生物标志物DNAPrimary transcriptBiomarker ExamplesCholesterol is

4、one of the most well-known biomarkers of cardiovascular healthPhysical measurements: body temperature (fever); blood pressure (stroke risk)Other biomarkers: blood sugar level (diabetes) antigens (hepatitis) proteins (heart attack) genetic variations (Huntingtons disease)Biomarker ExamplesCholesterol

5、 生物标志物的临床应用Ludwig JA et al. Nature reviews 2005,5: 845-856生物标志物的临床应用Ludwig JA et al. NatBiomarkers for chronic kidney diseaseCase-control studies using specimensLow CRP- high LDLTRP=Sen=416/(416+104)=0.What considerations are important in determining the adequacy of repository samples?Hs-CRP is the

6、most widely studied biomarker of inflammation in cardiovascular risk.Multiple disciplinesWhat is the role of solid tissues?Ludwig JA et al.Translational from research to clinical use Biological fluids2002 Discovery of 70 gene signature (117 patients)Primary transcript灵敏度与特异度的优缺点Hs-CRP is the most wi

7、dely studied biomarker of inflammation in cardiovascular risk.Spe=True negative rate(TNR)C-Reactive Protein (mg/L) tumor burden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Biomarkers for chronic kidney Clin J Am Soc Nephrol 3: 18951901, 2008.Biomarkers for chronic kidney diseas

8、eClin J Am Soc Nephrol 3: 1895Are we treating sub-populations?疾病药物无反应率抑郁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, 2001Are we treating sub-populationNovel biomarkers are neededE

9、arly, accurate diagnosis -Individualized therapy and improved treatment outcomesBetter defined populations will allow more specific drugs-Better efficacy-Fewer side effectsNovel biomarkers are neededEar“The use of biomarkers will change medical practice from a population-based approach to anindividu

10、alized approach”Felix Frueh, Associate Director of Genomics at CDER, FDA“The use of biomarkers will chEvolution of the biomarkers researchEvolution of the biomarkers reHigh plasma cholesterol and cardiovascular diseasesNearly 50 percent of all future myocardial infarction and stroke events occur in

11、those with normal or below normal lipid levels.EUROASPIRE Study Group, 1997% of MIHigh plasma cholesterol and ca机率线(chance line)(diagonal reference line)Biomarker validation labWhat is the role of routinely accessible biofluids such as plasma, serum, and urine?Ridker PM et al.Very important in the d

12、iscovery of biomarkers of diseases (systemic vs.Disease controlLDL-cholesterol (mg/dL)试验阳性时患病的概率多大？ 2. Biological fluids生物标志物研究面临的挑战C-Reactive Protein (mg/L)Biomarkers for chronic kidney diseaseCirculation.阳性预报值与阴性预报值肿瘤 (乳腺癌 肺癌)High plasma cholesterol and cardiovascular diseases tumor burdenLudwig J

13、A et al.Human resources“measurable and quantifiable biological parameters”Additional biomarkers (inflammation)Hs-CRP and cardiovascular riskHs-CRP is the most widely studied biomarker of inflammation in cardiovascular risk.Since the early 1990s with the development of highly sensitive assays for its

14、 measurement, correlations of hs-CRP with both cardiovascular risk factors and future cardiovascular events has been possible.机率线(chance line)(diagonal refCRP and LDLC levels and the risk of cardiovascular diseasesC-Reactive Protein (mg/L) 3.0 160LDL-cholesterol (mg/dL)3.02.01.00.0Multivariable Rela

15、tive RiskIncreased CRP levels are associated with increased risk of cardiovascular events independently of LDL-C levelsRidker PM et al., 200227,939 womenCRP and LDLC levels and the rHigh CRP-high LDLHigh CRP-low LDLLow CRP-low LDLLow CRP- high LDLPorbability of Event-free SurvivalYears of Follow-up0

16、.990.980.970.960.001.0002468Evolution 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

17、than the two markers separately.Ridker PM et al., 2002High CRP-high LDLHigh CRP-low 个体化医疗的现状与未来生物标志物实用课件如何评价生物标志物？如何评价生物标志物？常用评价指标（一）敏感性（二）特异性（三） Youden指数（四）阳性似然比（五）阴性似然比（六）阳性预报值（七）阴性预报值（八）ROC曲线常用评价指标（一）敏感性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（Sensit

18、ivity): TP/(TP+FN)=TPR (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ECG诊断试验的结果心肌梗塞合 计出现不出现阳 性416ECG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（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

19、的能力。ECG诊断试验的结果心肌梗塞合 计出现不出现阳 性416个体化医疗的现状与未来生物标志物实用课件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，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、You

20、den指数， =Sen+Spe-1=TPR-FPR =0.8-0.05=0.75Youden指数取值范围在（0，1）之间，其值越接近1，诊断准确性越好。ECG诊断试验的结果心肌梗塞合 计出现不出现阳 性416ECG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诊断试验的结果心肌梗塞合 计出现不出现阳 性416ECG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

21、断试验的结果心肌梗塞合 计出现不出现阳 性416Low CRP- high LDL1950s 雷达信号观测能力评价生物标志物研究面临的挑战SSRIs, SNRIs, TCAsEvolution of the biomarkers research : CRP and LDL-C levels and event-free survival among women-Fewer side effectsSpe=True negative rate(TNR)Correlation with histological scoring2006 External confirmation (307 pat

22、ients, 5 hospitals)机率线(chance line)(diagonal reference line)LDL-cholesterol (mg/dL)TP/(TP+FN)=TPR (true positive rate)Role of routinely accessible biofluidsFelix Frueh, Associate Director of Genomics at CDER, FDAPorbability of Event-free SurvivalQuestion 3.Question 2.Circulation.Important for:Youden

23、指数取值范围在（0，1）之间，其值越接近1，诊断准确性越好。ROC曲线下面积是重要的试验准确度指标。医生最关心的问题： 1. 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？Low CRP- high LDL医生最关心的问题： 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（真阳性）所占的比例 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。 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ECG诊断结果心肌梗塞合 计出现不出现阳 性阴 性合计416(TP)

24、9（FP）425104(FN)171（TN）275520180700(N)心肌梗塞合 计出现不出现阳 性416(TP) 9（FP）阳性预报值与阴性预报值阳性预报值与阴性预报值ROC曲线ROC曲线 ROC（receiver operating characteristic的缩写，译为“接受者工作特征”）ROC曲线研究历史1950s 雷达信号观测能力评价1960s中期 实验心理学、心理物理学1970s末与1980s初 诊断医学ROC的涵义与起源 ROC（receiver operating chara个体化医疗的现状与未来生物标志物实用课件不同诊断界值时灵敏度与特异度间的平衡(trade off)

25、0204060801005060708090100特异度灵敏度百分率（）不同诊断界值时灵敏度与特异度间的平衡(trade off)Receiver Operating Characteristic curve Area Under Curve (AUC) - 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

26、 and less-effectively instructed studentsReceiver Operating Characteris完美与无用的ROC曲线真阳性率即灵敏度假阳性率 即 1特异度机率线(chance line)(diagonal reference line)完美与无用的ROC曲线真阳性率假阳性率 即 1特异度What considerations are important in determining the adequacy of repository samples?Change in advance of clinical signsOther biomark

27、ers:ROC曲线下面积（Area）与诊断准确度高低2007 Approval by FDATP/(TP+FN)=TPR (true positive rate)Standards not establishedCRP and LDL-C could give better prognostic information than the two markers separately. early detection27,939 womenEvolution of the biomarkers research : CRP and LDL-C levels and event-free surv

28、ival among women试验阳性时患病的概率多大？ 2.Multiple disciplines生物标志物的研究方法？ Can reflect disease perturbations in the organs or tissues from which they are secretedBiomarker validation lab Body cavity fluidsVery important in the discovery of biomarkers of diseases (systemic vs.诊断准确度较低（0.9）0.00.20.40.60.81.00.00.

29、20.40.60.81.0FPRTPRA0.938ROC曲线下面积（Area）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) 0.50-0.60 = fail (F)What considerations are importROC曲线小结ROC曲线反映了灵敏度与特异度间的平衡 (增加灵敏度将降低特异度；增加特异度将降低灵敏度)。 在ROC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。 在ROC曲线空间，如果曲线沿着机

30、会线（45度对角线）越紧密，则试验准确度越低。 ROC曲线下面积是重要的试验准确度指标。 ROC曲线小结ROC曲线反映了灵敏度与特异度间的平衡 (增加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, feasibilityStud

31、y assay in people with and without diseaseCase-control studies using specimensLongitudinal studies to predict diseaseClinical useSite Biomarker development labBiomarker validation labClinical epidemiologic centersCohort studiesCommunity DesignCross-sectionalCross-sectionalCase-controlprospectiveRCTS

32、ample sizesmallsmallmodestmediumlargeVasan RS. Circulation. 2006;113:2335-2362.phasePhase1Phase2Phase3Phase4PThe Agendia MammaPrint Test首个FDA批准的基因组检测试验 - Feb. 2007The Agendia MammaPrint TestHow they got there？2002 Discovery of 70 gene signature (117 patients)2002 Duplication of results (in another s

33、ample set:295 patients)2006 Assay performance2006 Optimized array format: reproducibility; back to original sample set2006 External confirmation (307 patients, 5 hospitals)2007 Approval by FDAHow they got there？2002 Disc生物标志物研究技术传统研究方法： PCR, Western blotting, ELISA, et al新型研究方法： 基因组学技术 蛋白质组学技术：2-DIG

34、E/MS, 蛋白质芯片生物标志物研究技术传统研究方法：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, validation and implementation?生物标志物研究方法 Question1.Answer1.All biological samples are eligible for collectionCollected biological material depends on an

35、alyte and tissue sourceExamples Biological fluids Serum, plasma, urine, csf Secretions Saliva, seminal fluid Body cavity fluids Pleural fluid, peritoneal fluid, etc Specific tissue material Specialized cells reproductive cells Non-cellularAnswer1.All biological samplesIdeal Biomarker discovery sampl

36、es 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 testing situation as possibleIdealQuestion 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?Question 2.What is the role ofRole of routinely access