乳腺癌her2检测指南2009版课件

中国乳腺癌Her-2检测指南（2009版）解读,北京协和医院 卢朝辉,中华医学会病理学分会“西部行”讲学,HER2 介绍,HER2过表达和细胞增殖,PI3K / Akt Ras / MEK / MAPK (STAT),CoA,TF,CoR,Proliferation Migration,HER4,HER2, human epidermal growth factor receptor 2; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; STAT, signal transducer and activator of transcription; CoA, co-enzyme A; TF, tissue factor; CoR, co-repressor,HER2 阳性率 (1)-早期或没有分期,Study,n,Stage,HER2 positive, %,Acosta 2001 Ross 2003 Owens 2004a Press 2005b Francis 2008 Gown 2008a Ferno 2006 Penault-Llorca 2008 UK NEQAS,9307 5227 16,092 2502 6512 6604 5043 2079 30,720,I-IV I-III I-IV I-IV NS NS NS I-III I-IV,20 24 23 26 17; 13 20 14 16 15,aData from high-throughput laboratories; bBCIRG reference laboratory IHC, immunohistochemistry; FISH, fluorescence in situ hybridisation; NS, not specified; NEQAS, National External Quality Assessment Scheme,HER2-positivity rates (either early breast cancer or unspecified),Method,IHC / FISH NS FISH only FISH only IHC 3+; 2+ IHC / FISH IHC / FISH IHC / FISH FISH,HER2阳性率 (2)晚期,Study,n,HER2 positive, %,Ross 2003 Dybdal 2005 Penault-Llorca 2005 TRM 2006 UK NEQAS 2008,279 5998 699 413 812,26 23 30 32 17,HER2-positivity rates in metastatic breast cancer studies,Method,NS IHC IHC IHC / FISH FISH,TRM, Tumor Registry Munich,HER2阳性乳腺癌生存率低,Herceptin 提高了HER2阳性MBC的预后,Dawood et al 2008,通过荟萃分析：和HER2阴性的患者相比HER2阳性患者使用赫赛汀后下降了44%的死亡风险(multivariate analysis adjusted for patient and tumour characteristics: HR 0.56; 95% CI 0.45, 0.69; p0.0001),HR, hazard ratio; CI, confidence interval,BC治疗前都应该了解HER2状态,HER2检测确定病人HER2状态后能让病人得到最优的治疗方案,Goldhirsch et al 2006; Dowsett et al 2007; Wolff et al 2007,HER2检测法则,ISH, in situ hybridisation,Hanna & Kwok 2006,IHC 3+不需要再用ISH确定HER2状态，因为IHC 3+和FISH +吻合率较高(eg chromosome 17 polysomic cases, Hofmann et al 2007) ISH方法包括FISH，CISH，SISH,-,+,Eligible for Herceptin,Retest with ISH,-,+,Patient tumour sample,Eligible for Herceptin,Eligible for Herceptin,HER2检测方法,HER2检测方法,CISH, chromogenic ISH; SISH, silver-enhanced ISH,HER2检测方法的优缺点(1),Performed in majority of pathology laboratories Relatively easy, quick and cheap; can be automated IHC-stained slides can be stored and re-assessed Cell morphology can be seen in same section Less affected by preanalytical factors and handling than IHC Score interpretation more quantitative than for IHC Identifies HER2-positive tumours (gene amplified) within IHC 2+ cases Automation available,Susceptible to variations in testing protocol Score interpretation subjective and semi-quantitative Costly (more expensive than IHC) Signal decays over time Areas of invasive carcinoma may be difficult to identify Few pathologists and technologists are trained in the methodology and its interpretation,IHC,Advantages,Disadvantages,Method,FISH,Relatively new technology so less experience than for FISH Results more difficult to interpret by inexperienced staff Some methods require separate slides for HER2 gene and CEP17 copy numbers Separate slides needed for HER2 gene and CEP17 copy number,Less affected by pre-analytical factors and handling than IHC Quick to perform Requires only standard light microscope Staining remains stable for a long period of time Cell morphology can be seen in same section Fully automated CISH method with automated image analyses Other advantages same as for CISH Fully automated CISH method with automated image analysis Both HER2 and CEP17 brightfield viewable on same slide Other advantages same as for CISH,CISH SISH Dual SISH,Advantages,Disadvantages,Method,HER2检测方法的优缺点(2),Results more difficult to interpret by inexperienced staff,IHC,IHC,Water bath 95C (40 minutes),Peroxidase block (5 minutes),Apply primary Ab (30 minutes),Apply chromogenic substrate (10 minutes),Apply visualisation reagent - secondary Ab polymer (30 minutes),View with light microscope,Tumour tissue fixed on slides,HercepTest protocol,HercepTest PathWay InSite HER2 / neu,Kit,Dako Ventana BioGenex,Manufacturer,A0485 CB11 or 4B5 CB11,Antibody,Commercially available IHC kits,IHC读片,IHC 1+ Barely perceptible membrane staining in 30% of tumour cells; cells only stained in part of membrane,Images courtesy of Dako,IHC 0 No staining or membrane staining in 30% of tumour cells,IHC 2+ Weak / moderate complete membrane staining in 30% of tumour cells,IHC 3+ Strong complete membrane staining in 30% of tumour cells,IHC常见问题,Poor fixationa Heterogeneous staining,aImage courtesy of W Hanna; bimage from Dako,Cytoplasmic staining, IHC 0b Diffuse homogeneous stain specifically confined to the cytoplasm,Dot artefactb Brown dots represent cytoplasmic staining, not membrane staining,Edge artefacta Staining along edge of tissue while the centrally located tumour is devoid of staining,很多别的因素能影响IHC判读,ISH原理,HER2 gene,Probe,Prepare target DNA,Denature target DNA,Probe binds to denatured DNA and emits a signal,ISH: HER2基因扩增判读,FISH positivea,Fluorescence DNA probe,CISH positiveb,Digoxigenin-labelled DNA probe with chromogenic detection (DAB signal),SISH positivec,Dinitrophenol-labelled DNA probe with chromogenic detection (silver signal),aImage courtesy of W Hanna; bimage from Invitrogen; cimage from Ventana,Dual SISH / Red Amplified HER2c,Dinitrophenol-labelled DNA and centromeric probes with chromogenic detection (silver and Alk-Phos Red),多体,The CEP17 probe identifies the centromere of chromosome 17 Polysomy means there are 2 CEP17 signals (green) and in consequence 2 HER2 gene signals (orange) detected per nucleus This can result in false-negative interpretation of ISH,Normal 2 CEP17 2 HER2 genes Gene amplification 2 CEP17 2 HER2 genes Polysomy 2 CEP17 2 HER2 genes,Gene amplification vs polysomya,aData are signals per nucleus,Image courtesy of J Rschoff and M Hofmann,FISH methodologies,Formalin-fixed, paraffin-embedded tissue section specimens,FISH probe mix,Wash,Mount on slides,View with fluorescence microscope,HER2 FISH pharmDx protocol,Hofmann et al 2008,FISH 判读,FISH negative (no amplification) Ratio of HER2 gene (orange) to CEP17 (green) signals is 2.0,FISH positive Ratio of orange to green signals is 2.0,Images courtesy of W Hanna using PathVysion,FISH判读常见问题,Under-fixed tissuea Staining is patchy; probe signals may be weak or absent,Over-fixed tissuea Persistent autofluorescence; probe signals may be weak or absent,Pretreatment under-digestionb High autofluorescence; very strong counterstain uptake,aImages from Abbott; bimage from Ventana,Many other factors can affect FISH interpretation,CISH,CISH methodology is similar to FISH but can be visualised with a light microscope,CISH判读,CISH negative (no amplification),CISH positive (6 dots, clusters or a mixture of these in 50% of tumour cells),Images from Invitrogen SPoTLight HER2 CISH kit,CISH判读常见问题,Poor fixationa Formalin pigmentation is obscuring the HER2 staining,aImages from Invitrogen; bimage courtesy of W Hanna,Over-counterstaininga Counterstain too strong,Small air bubbles in mounta,Artefactb Signals outside the nucleus,Many other factors can affect CISH interpretation,SISH,Fully automated, rapid (10 hours) methodology SISH detection system produces silver signals that are stable over long periods of time Automation allows high throughput, consistency and reduced risk of errors A rapid semi-quantitative method is used initially and verified with a quantitative method in equivocal cases SISH assays Her2 Silver- ISH single colour (Ventana Medical Systems) Her2 Dual Colour ISH (Ventana Medical Systems),SISH判读,SISH negative,SISH positive,Images from Ventana INFORM HER2 DNA,SISH判读常见问题,Weak or absent counterstain Under-counterstaining / pretreatment over-digestion due to sub-optimal tissue preparation,Oxidising, fading and / or disappearance of the SISH signal May be due to certain brands of mounting medium,Slide-drying artefact Brown or black background throughout the tissue indicates that the slide has dried between steps,Images from Ventana,Many other factors can affect SISH interpretation,双SISH,The dual system uses SISH to detect HER2 + Alk-Phos ISH Red to detect CEP17 Fully automated, rapid (15 hours) methodology SISH detection system produces silver signals that are stable over long periods of time Alk-Phos ISH Red detection system produces chromogenic red signals that are stable over long periods of time Automation allows high throughput, consistency and reduced risk of errors,双SISH判读,Dual SISH non-amplified for HER2 gene or centromere,Dual SISH amplified for HER2 gene and non-amplified for centromere,Images from Ventana,双SISH常见问题,Weak or absent counterstain Under-counterstaining / pretreatment over-digestion due to sub-optimal tissue preparation,Oxidising, fading and / or disappearance of the SISH signal May be due to certain brands of mounting medium,Drying artefact Brown or black background throughout the tissue,Images from Ventana,Many other factors can affect dual SISH interpretation,Plasma blush Pink background in plasma cells,未来的HER2检测,Guidelines on pre-analytical techniques will help ensure optimal quality of testing Implementation of large QA / QC programmes will improve quality of testing (Full) Automation will help improve scoring quality New technologies promise more quantitative read-outs and assessment of dimerisation status caveat: not yet validated in a large clinical trial setting; more data are needed before any conclusions or recommendations for routine use can be given,Her2检测指南的背景,为何需要指南？,Her2 成为乳腺癌预后和化疗方案选择的重要指标 Her2 检测的环节非常多 不同实验室的结果可比性差,各国都制订了her2 检测指南,CAP & ASCO UK Canada 中国 2006版her2检测指南 2009版her2检测指南,免疫组化检测Her-2 阳性率应该有多高？,不同的文献报道不同 10%34% 国内较公认在20%左右 不应低于10%,Her2免疫组化质控是国际性难题,UK-评估: HER2 检测 一月-十二月 2007,2+ IHC Total FISHed - 一些实验室应用 ISH 最为最初的检测 (TC: 60) - 一些实验室 FISHing 3+ cases (TC: 46),jasanicf.ac.uk,结果不明的病例 (2+),Roche 2006 7-41% (18%),jasanicf.ac.uk,UK NEQAS 优化HER-2 检测率: 数据仅仅来自UK (2003- 至今),- Effective Feedback & monitoring of UK labs. - UK labs have to pass the NEQAS assessments to be accredited (CPA),jasanicf.ac.uk,HER-2 评估检测率: 数据来自36个国家 - UK & 海外,jasanicf.ac.uk,Her-2 IHC检测在中国,？,2008年2100例乳腺癌her-2免疫组化检测,14家医院Her-2检测分布柱形图,2008年全国免疫组化Her-2质控(45家医院),中国2009版her2检测指南的主要内容,2009版指南的主要内容,检测的时机 检测的流程 组织标本的制备 染色要求与结果判读 实验室质控要求 报告的内容,检测的时机,初次诊断为乳腺浸润性癌，包括穿刺、活检、术中冰冻及乳腺癌切除标本 复发患者，是否再做her2检测，与临床商议,标本的类型：手术切除标本 粗针穿刺标本 冷冻后的标本,改良根治术乳腺癌标本,新辅助化疗乳腺癌标本,保乳术乳腺癌标本,固定前标本处理,转移性乳腺癌：如果可以取得转移灶活检标本，转移灶活检标本比原始标本更有价值,固定前标本处理,对所有标本在离体1小时以内进行固定(30min ASAP),乳腺标本在手术切除、大体观察和切缘标记后立即切为5mm至10mm厚的薄片并予固定，标本切除和固定之间的时间间隔尽可能短,可使用纱布或滤纸将相邻的组织片分开，保证固定液的充分渗透和固定,如肉眼能确定的肿瘤，尽快取一小块肿瘤和正常乳腺组织放入包埋盒并立刻进入固定液,注：手术切除标本的固定步骤 标本定位，根治术标本，以腋窝的脂肪组织作为外侧的标志，皮肤 朝上，以6点的部位最靠近取材医师（如同取材医师面对患者） 判断乳腺标本的外观特点并测量记录，确定标本的四个象限并标注： 外上、外下、内下和内上。 用一把长的快刀沿长轴将整个乳腺切成5mm至10mm厚的薄片并予固定,保乳手术标本,全乳房切除标本,Day 1,Day 7,组织应固定于4%中性缓冲福尔马林中648小时，固定液的量为组织体积的10倍 穿刺活检标本固定尽量固定6小时以上,固定剂的选择和固定时间,标本的大体检查,确定标本为哪一侧（左或右）及乳腺切除术的类型 列出标本所含的结构：皮肤、乳头、乳腺、腋窝组织、胸大肌、 胸小肌、胸壁结构 标本重量及大小（皮肤最大长度和宽度） 外观特征：皮肤形状和颜色、皮肤改变的部位及范围（瘢痕，新近的 手术切口、红斑、水肿、变平、皱缩、溃疡）、乳头及乳晕的外观 （糜烂、溃疡、回缩，内陷）、病变的部位及其特征（用病变与乳头 的距离以及时针所指位置或象限来表示）、触摸发现异常加以描述 切面特征：囊肿及扩张导管（大小、部位、数量、内容物）、肿块 （肿块所处象限和距乳头距离，皮肤下的深度，大小，形状，质地， 颜色；有无坏死、出血、钙化；与皮肤肌肉或乳头的关系或附着情况） 如有淋巴结：部位、数量、大小,标本的取材,乳腺：肿瘤取3块；大体检查可见的所有病变都要取材；按顺序至少取一块（外上、外下、内下、内上象限、离肿瘤最近的后切缘）；癌旁组织取一块。 乳头：如果固定后乳头是直立的，如附图所示取材 如果乳头回缩或内陷，通过乳头或乳晕垂直于皮肤表面取材。 胸大肌（如有）：从任何大体可见的异常区域取1块，或如果没有异常发现，则从最靠近肿瘤的部位取1块。 淋巴结：所有淋巴结都要做组织学检查，小淋巴结要全埋，大于0.5cm直径的淋巴结切开。,手术切除标本：,针穿标本：全取,接受新辅助化疗后，肿瘤部位往往产生化疗后改变，大 体上表现为肿块消失、瘤床部位纤维化、边界不清、质地变软。没有明确肿块时： 找到最大径瘤床所在的组织切面 将该切面包含的瘤床全部取材送检 其余切面之瘤床组织选择性取材 瘤床周边组织也应选择性取材,新辅助化疗手术标本的取材,固定后组织处理,酒精梯度脱水、二甲苯透明、石蜡包埋（定期维护自动脱水机；至少每周更换所有溶液（依据标本量而定）；使用高质量的二甲苯或双蒸溶剂；包埋中石蜡温度不应太高（56）以避免损伤热敏感的抗原决定簇） 固定后的脱钙处理（尽量避免使用脱钙后的组织进行IHC 检测） 组织切片,标本来源环节的质控,外院标本、外院蜡块 冰冻过的组织标本，不宜使用 穿刺标本易固定过度 不要使用微波快速固定,切片环节,厚度：35微米 切片后应在6周内检测 烤片温度不宜过高，可70烤4570min，FISH者可65过夜 （烤片设备必须温度可控） 有HE对照,修复染色-IHC,选择抗原修复方法, 哪种加热设备? 哪种缓冲液? 时间多长?,微波炉500W, 15 分钟.,高压锅 121, 15 分钟.,水浴 98, 40 分钟. (标准法),NEQAS推荐：0.01MpH6.0柠檬酸水浴98修复40分钟继之以20分钟冷却,抗原修复所致的染色变化,95-99的EDTA修复液（1mpH8.0）中保持15min（组织切片切记不能暴露在液面之上）室温静置20min,常用IHC 检测的抗体/试剂盒,抗体和试剂盒,类 型,阳性率,CB11 单 抗 25/86（38.1%）,4D5 单 抗 15/86（17.4%）,TAB250 单 抗,A0485 多 抗 25/66（37.8%）,HerceptestTM 多 抗 31/86（38.1%） （抗体为A0485）,HerceptestTM 是经FDA认证用于检测HER2的试剂盒,不同IHC试剂HER2蛋白检测结果有差异,Nils M. Diaz Cancer Control September/October 2001, Vol. 8, No. 5,常规IHC和HercepTest染色一致率仅为%,常规IHC 1+,HercepTest 3+,HercepTest,CB11,TAB250,不同一抗所致的染色变化,HercepTestTM标准检测试剂盒,过氧化物酶阻断剂 即用型兔抗人HER2蛋白抗体 过氧化物酶标记的抗兔多聚复合物 对照片（0，1，3） 阴性对照液 DAB缓冲液 DAB显色剂 抗原修复液 缓冲液,使用HercepTestTM检测HER2,石蜡切片脱蜡至水化：二甲苯 5min2次，100%乙醇3min2次，95% 乙醇3min2次，蒸馏水 1min； 切片放入抗原修复液（Epitope Retrieval Solution）水浴9599修复 40min，室温冷却20min； Wash Buffer 5min3次； 滴加Peroxidase-Bloking Reagent孵育5min，Wash Buffer5min3次； 滴加一抗，37孵育30min，Wash Buffer 5min3次； 滴加Visualization Reagent，37孵育30min，Wash Buffer5min3次； 滴加DAB工作液显色，显微镜下观察，适时终止； 苏木精复染3min，流水冲洗，酸酒精分化45sec，自来水反蓝15min； 常规梯度酒精脱水，二甲苯透明，中性树胶封片。,HercepTestTM试剂盒操作步骤,孵育时间的差异：孵育时间必需维持在操 作步骤指示的时间的 1分钟之间,孵育时间会影响敏感度,缓冲液,必需使用试剂盒内所附的缓冲液 所有试剂, 包括缓冲液和cell lines， 必需存放于2-8C冷藏,DAB显色剂所致的染色变化,HercepTest,其他 DAB,Control Cell Lines,0 Cell Line,1+ Cell Line,3+ Cell Line,注意： 在显微镜下监控显色过程，避免过显色造成很 强的背景 在染色过程中，每一步都要充分洗涤，并始终 保持切片潮湿 IHC自动染色系统更易达到标准化，但应进行 严格的对比试验和程序优化 染色必须设阳性对照、阴性对照和空白对照， 被检切片中癌旁组织中正常的乳腺上皮细胞是 最好的阴性内对照,染色的流程,Vantana全自动免疫组化机（PATHWAY） Hercep-test Kit In House Protocol,NordiQC,指南建议使用SFDA认证的试剂,SFDA批准的试剂 IHC 还没有 FISH 2 家 CISH 还没有 如为自行实验室流程，则需要与标准程序验证，并有严格的SOP,存在的主要问题,组织固定：相当大部分医院不合格 染色强度不足，假阴性 胞浆强阳性 抗体的来源不同 结果判读的标准不同（未经过培训） 室内和室间质控缺乏,假阴性假阳性,应当采用新标准（更严格）,Old 0：10% membrane staining, discontinuously +：10% cancer cells, continuously, moderate to strong membrane staining +：10%,New negative：0 or + Borderline：+ positive：+ 30% cancer cells, continuous strong membrane staining,使用ASCO/CAP指南推荐的评分系统,结果判读应注意以下几点： 未用中性缓冲甲醛固定 针吸活检标本固定时间少于1h 切除活检标本固定少于6h或多于48h 先在10物镜下进行判读非常重要 注意细胞膜完全着色的癌细胞比例及着色强度 胞质着色应忽略不计 只评定浸润癌的着色情况 正常乳腺上皮不应着色 对照片出现预想不到的结果等,HER2 IHC检测报告应报告的内容,患者信息：姓名、性别、年龄、门诊/住院号 使用方法（检测法/生产商） 临床医师姓名和联系方式 图像分析方法（如果用了） 检测日期 对照设置情况 标本信息：病理号和蜡块号 样本量是否足够用于评估 标本部位和类型 判读结果（指0，1+，2+，3+） 标本固定液的种类（如果不是4%中性缓冲甲醛应注明） 具有完整包膜着色的浸润性肿瘤细胞的百分比 标本固定前时间（如有记录） 着色的均一性：存在/不存在 标本固定的时间（如有记录） 均一的环周深染模式：存在/不存在 抗体信息（克隆号/生产商） 结论：阳性；不确定；阴性；无法判读,注：如果使用了我国相关机构和（或）美国FDA批准的方法，应该在报告中说明；如果使用了改良的被批准方法，在报告中用说明做了哪些改良并证实其有效性；如果使用未经批准的方法，则应明确地说明实验室对检测的操作和结果负责。各单位病理科可根据本科病理报告的形式，或根据临床医师的需求决定HER2之IHC、IHC和（或）FISH检测的结果是作为乳腺癌病理报告的一部分，还是独立的检测结果发给临床。,注意： 对于2+的病例，应该用FISH、CISH或重复IHC做进一步检 测，也可以选取不同的组织块重新检测或送其他质量控 制良好的实验室进行检测 如仍不能确定，则需要与临床医师沟通已有利于治疗方 案的确定。,不同病理大夫分级的主观性,差别在相邻一个级别之间，甚至2个级别之间 如何减少主观性？ 回顾性专题阅片 （多头镜）集中读片培训 流传阅片,不同实验室之间的染色强度,实验室的着色强度，差异很大 如果均按照最强染色者，阳性率可提高 强度、膜连续性，哪个更重要？,质量控制,影响结果的方面,Time to slicing and fixation,Method of tissue processing,Type of fixation,Equipment calibration,Laboratory procedures,Time of fixation,Assay validation,Staff competence,Type of antigen retrieval,Test reagents,Control materials,Assay conditions,Use of image analysis,Interpretation criteria,Reporting elements,Scoring system,Wolff et al 2007,HER2-testing variation,Post-analytic,Pre-analytic,Analytic,QA / QC 流程减少结果误差,Accredited laboratories follow guidelines developed by these schemes to ensure standardisation of HER2-testing procedures,Internal QC External QA,NordiQC programme,Canadian QA programme,ASCO / CAP,UK NEQAS ICC and ISH scheme,ASCO / CAP, American Society of Clinical Oncology / College of American Pathologists; NordiQC, Nordic Immunohistochemical Quality Control; ICC, immunocytochemistry,Quality control,Tissue sample positive control Ideal positive control, IHC positive (3+)a,aImages courtesy of W Hanna; bImage courtesy of L Bornstein,MDAMB175, IHC negative (1+)b,Control cell line preparations SK-BR-3, IHC positive (3+)a,Patient sample internal controls,Non-tumour