免疫组化法检测系统测试概念 Pre-analytic 测试选择标本类型, 采集, 运输时间固定 ( 类型和时间 ) 组织处理 ( 类型和温度 ) 组织切片 Tissue should be fixed in 10% neutral-buffered formalin. Goldstein NS, H

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1 Updates of Applied Immunohistochemistry in Diagnostic Pathology and Clinical Management of Neoplastic Disease Z.G. Liu, MD Medical Director of IHC and Molecular Diagnostics 什么是免疫组化? 基于切片的检测特殊蛋白质, 糖分子等的免疫化学免疫组化不是简单的特染, 更近似于载玻片上的 ELISA IHC 检测问题 预后和预测指标 HER2 检测 (20% 的误差率 ) 实验室内部的变数 实验室间病理会诊重复所有 IHC 检测? 为何缺乏 IHC 规范化? 技术经验不足免疫组化技术抗原修复一抗检测系统手动与自动化工作人员没有足够的时间为 QA / QC 紧缩的预算缺乏病理学家的支持行业支持不足 IHC Assay Total Test Concept Preanalytic Test selection Specimen type, acquisition, transport time Fixation: type and time Tissue processing, type, and temperature Analytic AR procedure Protocol, control selection Reagent validation Technical staff training/certification Laboratory certification Postanalytic Control evaluation Interpretation of results Reporting of results Pathologist, experience, and CME Arch Pathol Lab Med 2000;124;

2 免疫组化法检测系统测试概念 Pre-analytic 测试选择标本类型, 采集, 运输时间固定 ( 类型和时间 ) 组织处理 ( 类型和温度 ) 组织切片 Tissue should be fixed in 10% neutral-buffered formalin. Goldstein NS, Hewitt SM, Taylor CR, et al.: Recommendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol 2007;15: breast tissue should be fixed for a minimum of 6 hours and no longer than 48 hours Wolff AC, Hammond ME, et al.: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor 2 testing in breast cancer. Arch Pathol Lab Med 2007;131:18-43 CAP-ASCO Practical Issues Cold ischemic time (time from removal a tumor mass (if identified grossly) or breast tissue cut surface is exposed to formalinpreferably ASAP from removal recommended <1 hr Time and duration of fixation time should be recorded in report (in OR/ office/ clinicdefault in-lab time) and may included formalin steps on processor CAP-ASCO Guidelines 大样本处理 Breast excision/ resection specimens should be fixed in NBF from a minimum of 6 to maximum of 48 hours for Her-2 testing (core biopsies same- revised 2011); (8 hours minimum; 72 maximum for ER-PR) Any alternative fixative should be validated against fixation in NBF to yield equivalent results- 95% concordance 冷缺血时间在一小时内 固定在 10% 福尔马林缓冲液 (1:20 的比例 ) 片切 不超过 2-3 毫米的组织厚度 记录冷缺血时间, 在福尔马林的时间 2

3 免疫组化法检测系统测试概念 Analytic 抗原恢复程序选择对照试剂验证 ( 抗体 ) 技术员培训 / 认证实验室认证 (CAP) Antigen Retrieval 抗原恢复 Enzyme digestion Trypsin Pepsin Protease K Ficin Heat-induced epitope retrieval (HIER) Low ph High ph Low temperature (overnight) 个性化抗原恢复 Cyclin D1 - HIER 类癌 - 嗜铬粒蛋白 the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Fresh slide vs. stored slides Xie R, Chung J-Y, Ylaya K, et al.: Factors influencing the degradation of archival formalin-fixed paraffin-embedded tissue sections. J Histochem Cytochem 2011;59:

4 一抗的选择 Internet (Histonet, IHC Resource Group, UK NEQAS, NordiQC, etc.) Medical journals Meetings (USCAP, CAP, ASCP) Company recommendation A. maximum sensitivity p16 immunohistochemistry as an alternative marker to distinguish atypical lipomatous tumor from deep-seated lipoma. ALT Lipoma B. maximum specificity First Antigen Second Antigen Prostate basal cell p63 HMW Keratin Breast myoepithelial cell p63 Myosin Neuroendocrine lineage Syn Chromo/CD56 Lung adeno TTF1 Napsin A Squamous p63/40 CK5 GI CDX2 Villin Validation 抗体验证 Test known positive and negative cases How many cases do you run (5, 10, 25 or more)? Compare results with previous IHC method, or with morphology, flow cytometry, molecular, microbiology CAP AP Checklist ANP Positive tissue controls are used for each antibody. Positive controls assess the performance of the primary antibody. ANP Appropriate negative controls are used. Negative controls must assess the presence of nonspecific staining in patient tissue as well as the specificity of each antibody. Control Slides Positive controls- internal and external Positive controls on same slide Multi-tissue control blocks Negative controls: tissue/ patient and antibody 4

5 Negative Control Policy Negative reagent controls run with avidinbiotin detection Negative reagent controls run with infectious disease antibodies Internal cells or tissue elements must be evaluated for nonspecific staining when no negative control present Pathologist can order a negative control slide (if needed) after reviewing initial slides Negative controls is no longer needed in polymer based systems, (2012) Tissues with pigment- sentinel LN, skinespecially when using DAB Tissue in which no negative control elements are present 免疫组化法检测系统测试概念 Post-analytic 对照评估结果解释报告结果病理学家 ( 经验和 CME) 石蜡块的存储 成功的 IHC 标准化要求 适当的固定和处理 实现所有 IHC / ISH 测试需要验证和不断的监视 参加水平测试 ( PT) 一个团队的努力, 技术人员和病理学家 IHC 质量控制 提供反馈 ( 好或坏 ) 给做免疫组化染色的个人 IHC 与鉴别诊断和其他技术比较改变染色条件测试诊断明确的病例 如果出现错误, 首先重复而不改变染色条件 胃腺癌 Her2 status 5

6 BRAF 突变的特异性单克隆抗体 重复染色 ER-negative in low-grade breast CA Controls (internal/external) weak Prognostic/predictive results contradicts with previous results Two contradictory markers positive 黑色素瘤 IHC 棕色 革命 未分化肿瘤的分类淋巴瘤和白血病的免疫分型反应性与癌前病变和癌的鉴别确定转移性肿瘤的原发部位肿瘤预后和预测生物标志 CLINICAL USE OF IMMUNOHISTOCHEMISTRY Most common antibodies used in the pathology lab 6

7 IHC Heat Maps Convention 结肠 CA Barrett 的化生膀胱腺癌卵巢粘液性 CA 小肠 CA 神经内分泌肿瘤 CDX2 Courtesy Dr. Gown for all heat maps CDX-2 Expression in Normal Tissues Werling RW et al., Am J Surg Pathol 27:303-10, 2003 * WT-1 卵巢浆液性 CA 间皮瘤血管瘤肾母细胞瘤原发性腹膜 CA 促纤维增生蓝色圆形细胞瘤睾丸支持细胞瘤性索间质肿瘤 AML 后肾腺瘤 Immunophenotype Band WT1 7

8 CK7 在大多数上皮细胞和肿瘤阳性鳞状 CA, 基底细胞 CA, 前列腺 CA,RCC 结肠 CA, HCC 通常阴性 CK20 几乎仅限于胃肠道上皮细胞和肿瘤在结肠 CA 阳性, 而多数的非结肠 CA CA with GI 表现型 ( 卵巢粘液性 CA, 膀胱腺 CA) Merkel 细胞癌尿路上皮 CA CK7 and CK20 CK7+/CK20+: TCC, Pancreatic CA, Ovarian mucinous CA, Merkel cell CA Ck7+/CK20-: Lung CA, Breast CA, Nonmucinous ovarian CA, mesothelioma,endometrial CA CK7-/CK20+:Colonrectal CA CK-/CK20-: SCC, RCC, HCC, Prostate CA SCC CK5 AND p63 尿路上皮 CA 肌上皮细胞层 ( 乳房 ) 基底细胞层 ( 前列腺 ) 皮肤附件肿瘤 CD10 滤泡性淋巴瘤肾细胞 CA 伯基特氏淋巴瘤肝癌的 毛细胆管 胃肠道肿瘤中的 刷状缘 子宫内膜间质肉瘤尿路上皮 CA 胰腺实性假乳头状肿瘤肌上皮细胞 TTF1 肺腺癌肺类癌肠道类癌阴性小细胞 CA 除未分化之外所有类型甲状腺癌阳性脉络丛乳头状瘤 8

9 Immunophenotype Band TTF-1 CLONE SPT24 TTF-1 Shows higher sensitivity for pulmonary adenocarcinoma as compared to 8G7G1/1 But positivity can be seen on colonic adenocarcinoma and mammary carcinoma CEA 肺腺癌所有的胃肠道肿瘤卵巢粘液性癌, 膀胱肿瘤腺癌宫颈腺癌 ( 子宫内膜 CA -) 胆管 CA CD45 所有良性淋巴组织和绝大多数淋巴瘤阴性的肿瘤 / 细胞 :R / S 细胞,90% 的浆细胞,20% 的前体 B 细胞肿瘤 ; CD45/CD43 screen 未分化肿瘤 CD43 所有 T- 细胞淋巴瘤间变性大细胞淋巴瘤浆细胞瘤 Most B 细胞淋巴瘤阴性 CLL MALT PAX5 所有的 B- 细胞淋巴瘤 ( 浆细胞瘤除外 ) 何杰金氏淋巴瘤 9

10 胃肠道间质瘤精原细胞瘤 / 无性细胞瘤 AML/ CML 肥大细胞瘤黑色素瘤子宫内膜癌 CA 甲状腺 CA Merkel 细胞瘤腺样囊性癌脑胶质瘤血管平滑肌脂肪瘤 CD117 CD34 血管肿瘤 DFSP 胃肠道间质瘤 AML,MDS 淋巴母细胞性白血病孤立性纤维瘤脂肪肿瘤 VIMENTIN 灵敏度! 阴性可排除肾细胞 CA, 甲状腺乳头状 CA, 黑色素瘤和子宫内膜 CA 间皮瘤 New Antibodies ERG Erythroblast transformation-specific (ETS) family of transcription factors Protein required for platelet adhesion to subendothelium Translocations: TMPRSS2-ERG,NDRG1- ERG (prostate CA), EWS-ERG(Ewing s), FUS-ERG (AML) ERG stain in 1880 tumors Tumor type ERG positive Total Epithelioid hemangioendothelioma Kaposi sarcoma Extramedullary myeloid tumor 7 19 Ewing sarcoma 2 29 Prostate AC Lung large cell CA 1 42 Mesothelioma 1 27 Miettinen M, et al. Am J Surg Pathol. 2011;35:

11 Adenosis IHC Endothelium vwf 50-75% Not specific HG-PIN FVIIIrA 80% Least specific CD34 90% More specific ASAP CD31 Fli1 90% 94% Very specific Most specific ERG 96% Very sensitive/ specific Tomlins SA, et al. Arch Pathol Lab Med. 2012;136: Kahn et al Miettinen et al, 2011 Folpe et al DOG-1 Labeled 100% of PDGFRA-mutated GIST All schwannomas and fibromatoses are DOG1-negative DOG-1 + on 36% of KIT-negative tumors Claudin 1 Useful in meningioma,perineurioma, low-grade fibromyxoid sarcoma Widely distributed in normal tissues (membranous) Protein product is a membrane protein and component of tight junction strands West RB, et al. Am J Pathol. 2004;165: Liegl B, et al. Am J Surg Pathol (3): TLE1 滑膜肉瘤 TEL1 Transducer-like enhancer of split (TLE1), important in Wnt-APC pathway Sensitivity higher than all other IHC markers of SS, but lower than FISH (t(x;18). Rarely positive in MPNST 11

12 淋巴造血标记抗体 病变 / 细胞系 抗原 True Histiocytes CD163 Langerhans Cells Langerin Hairy Cell Leukemia Annexin A1, CD11c B-cell Lymphoma CD19 Myeloid CD33 Hodgkin s lymphoma TARC IgG-4 related entities IgG4 ALCL 间变性大细胞淋巴瘤 p80 (new clone) TARC Thymus and Activation-Regulated Cytokine Known Antigen Distribution in Normal Tissue Distribution in tumor cells Utility 激活相关的细胞因子 None Reed-Sternberg and Hodgkin s cells Hodgkin s lymphoma differential diagnosis TARC Peh SC, Kim LH, Poppema S. Am J Surg Pathol Jul;25(7): 毛细胞白血病的简单诊断分析 : 免疫细胞化学检测膜联蛋白 A1(ANXA1) Immunohistochemical expression of Langerin in Langerhans cell histiocytosis Falini B, et al. Lancet Jun 5;363(9424): Lau SK, Chu PG, Weiss LM. Am J Surg Pathol Apr;32(4):

13 5A4 (Recently generated) ALK/p80 Prognostic / Predictive Markers Neoplasm Breast Marker ER (SP1) Reactive with ALK + tumor HER2 (SP3) Ki-67 (SP6) Glioblastoma Renal cell Lung/colon Endometrial / Lung / GBM CLL/SLL MGMT CA IX ERCC1 PTEN ZAP70 CNS Tumors Tumor Antigen Germinoma/Seminoma PLAP, ckit, OCT3/4 Sall4 Genogenic IHC --- 使用免疫组织化学方法检测基因的改变 Hemangioblastoma Meningioma Glial Tumors (prognostics) Medulloblastoma Atypical Teratoid/ Rhabdoid Tumor S100, inhibin α, podoplanin EMA, PR, Claudin-1 Ki-67, PH-H3, p53 Synaptophysin, NeuN, Microtubule-assoc Protein, Neurofilaments Loss of INI-1 Takei H et al. Arch Pathol Lab Med 2007;131: Gown AM, Curr Diagn Pathol 8: IHC 可检测的遗传学改变 Gene Genetic Alteration IHC Finding 肿瘤 ERBB2(HER2) Amplification HER2 Overexpression CDH1 MMR genes Mutation, Methylation, & LOH Mutation, Methylation, & LOH Loss of E-cadherin Loss of MLH1,MSH2, MSH6, PMS2 乳腺肿瘤 乳腺小叶癌, 弥漫型胃癌 MSI 大肠癌 CA, HNPCC 22q11.22 LOH Loss of INI1 横纹肌样瘤 APC/B-Catenin Mutation NuclearOverexpre ssion of B-Catenin NPM Mutation Cytoplamic Overexpression 纤维瘤病 AML 13

14 DNA MISMATCH REPAIR SYSTEM MLH1 PMS2 MLH2 MSH6 免疫组化检测 MMR 结肠腺癌 Lynch Syndrome Guide chemotherapy, 5 FU Based Identifying carcinoma of unknown primary MLH1 MSH6 错配修复损失 (MSI) MLH1 突变, 甲基化 IHC 确定的遗传学改变 基因的改变 IHC 的发现肿瘤 t(14;18) BCL2/IGH BCL2 - 过度表达 FL(> 90%), 弥漫性大 B 细胞淋巴瘤的亚型 (20-30%) t(11;14)ccnd1 (BCL1)/IGH 细胞周期蛋白 D1 过度表达 MCL, 浆细胞瘤 t(2;5) ALK/NPM ALK - 异常表达 ALCL, 炎症性肌纤维母 细胞瘤 t(11;22) EWSR1/FLI1 t(11;22) EWSR1/WT1 FLI1 - 异常表达 WT1 - 异常表达 PNET / 尤文氏肉瘤 DSRCT t(x;17)tfe3/aspl TFE3 - 异常表达 ASPS, 小儿 RCC 14

15 变性大细胞淋巴瘤 EGFR 突变特异性单克隆抗体 ALK-1(CD 246) IHC 表现丟失过表达新型融合产品 使用 IHC 测定基因变异 基因的改变 缺失, 突变, 甲基化 基因扩增 易位 Genogenic 免疫组化 替代方法价格便宜, 成本效益较快 ( 相对于分子技术 ) 细胞蛋白异常定位 突变, 易位 适合标准化 Undifferentiated Tumor IHC of undifferentiated tumor 未分化肿瘤 通常是指肿瘤的增殖是 原始细胞 组成的更准确地定义应该是 I don't know what this tumor is based on the H&E appearance 15

16 Types of undifferentiated tumor 上皮性肿瘤 ( 癌 ) 生殖细胞 - 性索间质肿瘤神经内分泌肿瘤黑色素瘤 lymphohematopoetic 肿瘤 ( 淋巴瘤, 白血病, 组织细胞 ) 间充质肿瘤 ( 肉瘤 ) 未分化肿瘤的形态学分类 多形性大细胞肿瘤上皮样大细胞肿瘤梭形细胞肿瘤小圆蓝色细胞肿瘤 LARGE CELL UNDIFFIRENTIATED MALIGNANT NEOPLASM Carcinoma Melanoma Seminoma Epithelioid sarcoma, angiosarcoma, MPNST Lymphoma Epithelial Markers Pan-cytokeratin AE1/AE3? The majority of HCC will be AE1/AE3 negative Some RCC, neuroendocrine and SCC are negative Keratin OSCAR (Our Second Cytokeratin Antibody Rocks!) Much higher sensitivity but Combination of CK-LMW(Cam5.2) and CK- HMW (34betaE12) Tumors co-expression of vimentin and cytokeratin Endometrial CA Renal cell carcinoma Thyroid carcinoma Some poorly differentiated lung carcinoma Any carcinoma undergoing sarcomatoid transformation Germ Cell Tumor Markers SALL4: Yolk Sac Tumor, Embryonal ca, and Seminoma OCT3/4: Embryonal ca and Seminoma CD30 (Ki-1): Embryonal ca D2-40 (podoplanin): Seminoma, in a diffuse strong membranous pattern Glypican-3: Yolk Sac Tumor HLA-G: Intermediate trophoblastic cells of choriocarcinoma 16

17 Sex-Cord Stromal Tumors Inhibin, Calretinin, Melan A Melanomas A great mimicker, usually Ker-, Vim+ Should always be considered when dealing with a PD malignant neoplasm Melanin S-100 protein: high sensitivity, low specificity Sox10 is better HMB45: less sensitive, more specific A103 (Melan-A): highly sensitive and specific Tyrosinase, WT-1, p75 and Mitf,. Small round blue cell tumors 小圆蓝细胞瘤 CD45/43/TDT VIMENTIN AE1/AE3 SYN CHR CD56 Myogenin CD99 DESMIN LYMPHOHEMATOPOIETIC MARKERS CD45 and CD43 as screen makers CD20 and PAX5 as B-cell screen markers T-cell markers: CD2, CD3, CD5, CD7, TDT CD138 and CD56 Plasma cell CD163 better than CD68 as histocytic marker Tumors only CD43 positive Pitfall in lymphhemtopoitic markers Anaplastic large cell lymphoma Acute leukemia (AML, AMML etc) Plasma cell neoplasms Langerhans cell histiocytosis Pax5 in neuroendocrine tumors, Hodgkin lymphoma, and alveolar rhabdomyosarcoma CD79a in acute promyelocytic leukemia (APL) and myelomas Focal CK expression in rare larger cell lymphomas 17

18 Spindle cell, undifferentiated CK (Oscar)+ Carcinoma S100+/Sox10, MiTF Melanoma If everything negative, Unclassifiable sarcoma SPINDLE CELL TUMOR MARKERS DOG1, ERG TLE1 CLAUDIN 1 SMOOTHELIN SOX10 MDM2 CDK4 MYC MUC4 SDH 琥珀酸脱氢酶 Undifferentiated tumor workup Forming DDX based H&E & Clinical history Review the previous pathology if available Don t be rushed by clinician and Do thorough (cost of more stains is usually < cost of rebiopsy; delaying diagnosis is better than wrong diagnosis) If minimal tissue have 10 or more extra unstained slides or ask same tech to cut In pathology, chance also favors the prepared mind Suggested Panel if no DDX Use limited panel to screen Cam5.2 Sox10 CD45 CD43 SYN CHR CK34 MelanA CD30 SALL4 Vimentin AE1/AE3 Vimentin SOX10 CD45/43 Carcinoma Sarcoma Melanoma Lymphoma Vimentin is positive in melanoma and negative in ASPS Keratin can be + in melanomas and some sarcomas Some lymphoid tumors are - for CD45, but + for CD43 18

19 导管腺癌特点 Feature Benign Malignant IHC in pancreatic neoplasms 小叶内导管配置 + Haphazard ducts 分布 + 不完整的导管管腔 + 非典型细胞 + 核的大小, 变化范围 4:1 + 核仁大, 不规则 + 管腔碎屑坏死 + 腺 mitosis + 嗜神经侵袭 + 在脂肪组织中腺体 + 导管腺癌免疫组化 SMAD4 (DPC4) CEA, B72.3, MUC1, DUPAN-2 + >50% p53 and mesothelin + 55% loss of SMAD4 (DPC4) β-catenin: membranous (normal) Ductal Adenocarcinoma: Immunohistochemistry CEA, B72.3, MUC1, DUPAN-2 + >50% p53 and mesothelin + 55% loss of SMAD4 (DPC4) β-catenin: membranous (normal) Pancreatic Neuroendocrine Tumors: Clinical Wide age range Functioning or non-functioning (~40%) 2/3 head of pancreas Unpredictable clinical behavior Most insulinomas benign Most others malignant Metastasis only reliable criterion for malignancy 19

20 Pancreatic Neuroendocrine Tumors: Pathology Gross: Solid, soft Well-circumscribed <0.5 cm microadenoma Microscopic Solid, trabecular, insular, gyriform Intratumoral heterogeneity Often hyalinized stroma Amyloid insulinomas 胰腺神经内分泌肿瘤免疫组化 嗜铬粒蛋白, 突触素 + CD56+( 特异性差 ) 通常多克隆 PAX8+ 通常 ISL1+ CDX-2+ 10% 至 20% World Health Organization Classification: Pancreatic Neuroendocrine Tumor NET, grade 1 < 2 mitoses/10 HPF 2% Ki67 index NET, grade mitoses/10 HPF 3-20% Ki67 index Neuroendocrine carcinoma (grade 3) > 20 mitoses/10 HPF or > 20% Ki67 index Summary 免疫组化在诊断胰腺导管癌 ( 除 SMAD4) 中的作用是有限的 嗜铬粒蛋白, 胰蛋白酶,β-catenin 可用于神经内分泌肿瘤, 腺泡细胞癌 新型转录因子 (PAX8,ISL1) 有助于转移性胰腺癌及神经内分泌肿瘤 MIB-1 为胰腺神经内分泌肿瘤分级的标准染色 卵巢肿瘤的免疫组化 卵巢肿瘤的免疫组化 原发癌之间的区别浆液性癌和移行细胞癌子宫内膜癌透明细胞癌粘液癌原发性与转移性癌癌与性索间质肿瘤癌与生殖细胞肿瘤小细胞癌, 区分两种类型间皮瘤和浆液性癌 20

21 浆液性癌与透明细胞癌 Fact:Stage III / IV CCC 较浆液性癌预后差对以铂类为基础的化疗无疗效 Am J Surg Pathol 2008;32: 透明细胞癌和浆液性癌 IHC HNF1beta 透明细胞癌和浆液性癌 IHC ER WT HNF1beta p53 HNF1beta of CCC Köbel M et al; Am J Surg Pathol 2009;33:14-21 HEPATOCYTE NUCLEAR FACTOR( 肝细胞核因子 ) 1 BETA 卵巢及子宫透明细胞癌阳性 ( 从基因发现表达的研究 ) 子宫内膜异位症阳性 ( 伴有或不伴有透明细胞癌 ) 偶尔其他肿瘤阳性 子宫内膜样癌与浆液性癌 IHC EC SC vimentin + - p16 +/- + Beta catenin + - WT1 - + p53 +/- +/- 21

22 结肠直肠胃乳腺阑尾胆道胰腺子宫内膜和宫颈 卵巢转移癌 卵巢粘液性癌 CK7 + /CK /15 (93%) CK7 + /CK /15 (7%) CK7 - / CK /15 (0%) CK7 - / CK /15 (0%) 结肠粘液癌 CK7 + /CK /11 (9%) - CK7 + /CK /11 (0%) - CK7 - /CK /11 (91%) - CK7 - /CK /11 (0%) Wang NP et al, Am J Surg Pathol 1995 结肠粘液癌与卵巢粘液性癌 原发黏液癌表现出不同程度的 CK20 阳性率 (40-70%) 成熟畸胎瘤发生的粘液性肿瘤 CK7 - CK20+ 右侧大肠和低分化大肠癌通常 CK7+,CK20 - 小肠癌通常 CK7+ 一些阑尾粘液性癌可能会出现 CK7 +( 印戒和混合类型的细胞 ) β-catenin P504S CDX2 乳腺癌与浆液性癌 胰腺癌和卵巢癌形态学和免疫组化 overlap GCDFP 15 Mammaglobulin WT-1 Pax 8 + 乳腺来源 + 乳腺来源 + 浆液性癌 + 浆液性癌 Sister Mary Joseph nodule ** 乳腺癌病人的卵巢肿瘤很可能是新原发卵巢上皮性癌而不是转移癌 ** 22

23 SMAD4/DPC4 Sex-cord stromal tumors Alpha inhibin: Good marker 55% 的胰腺癌和 35% 的胆汁管和壶腹腺癌具有基因失活而阴性 卵巢, 宫颈, 结肠和阑尾腺癌通常阳性 Calretinin: Highly sensitive but not as specific markerfor sex cord - stromal tumors as compared to inhibin SF-1(steroidogenic factor 1) :100% positive in sertoli cell tumor Germ Cell Tumors CK Vim Glypican3 CD30 PLAP HPL Oct4 D240 Dysg-noma Embryonal York Sac / ChorioCa Int J Gynecol Pathol, Vol. 27, No. 4, October 2008 YST vs Dysgerminoma Dysgerminoma YST OCT4 + - PLAP + + Glypican - + 间皮瘤和浆液性 CA Moc 31 浆液性 CA ER 浆液性 CA Pax8 浆液性 CA Calretinin 间皮瘤 D2-40 间皮瘤 Sall

24 腹水不典型上皮细胞 (Female)??? 卵巢浆液性 CA, GI CA( 胰腺, 结肠 ), 间皮细胞 A. CA (MOC31, CEA)vs. mesothelial (CK5, calretinin) 乳腺癌免疫组织化学 B. MOC31 and/or CEA+: run CA D.Dx (ER, WT1) (CK20, CDX2, CEA, villin) 病理学家的角色转变 决定乳腺癌患者诊断和参与治疗方案的选择 乳腺癌治疗原则 Treatment Thresholds for Endocrine & HER2-Targeted Therapy 选择内分泌治疗的标准? 选择抗 HER2 治疗的标准? 选择使用化疗的标准? 根据肿瘤的形态及生物学特点和对复发风险评估 Goldhirsch Annals of Oncology, 20: (2009) 24

25 Chemo/Endocrine Therapy Decision Making in ER+/HER2- Breast Cancer New Approaches to an Old Problem: Prognosis, Risk, Genomic Profiling & Multigene Assays State-of-the-art molecular technology can be used to analyze global genomic changes in clinical breast cancer samples Genomic activity in early breast cancer Refine breast cancer classification Assess prognosis Assess response to therapy Goldhirsch Annals of Oncology, 20: (2009) Garber. Science. 2004;303: ER+ Breast Cancer (Luminal A) ER KI67 Luminal A 型乳腺肿瘤 Higher level of expression of ER and PR low histologic grade Lower levels of proliferation related genes Ki67 HER2 negative More indolent clinical course May experience late recurrences Better prognosis compared with Luminal-B and other subtypes 25

26 Luminal B 型乳腺肿瘤 Lower levels of ER expression and ER-related genes May be PR negative May over-express HER2 & EGFR Higher histologic grade More aggressive clinical course, worse prognosis More likely lymph node positive Higher expression of proliferation related genes KI-67 > 14% proliferation index, may be useful in separating Lum B from Lum A tumors Luminal-B/Luminal HER2 乳腺癌 Aggressive clinical course Less likely to respond to tamoxifen More likely to benefit from chemotherapy added to endocrine treatment Similar benefit from Herceptin+chemo compared with HER2+/ER- tumors in adjuvant clinical trials Typically - high recurrence score by Oncotype Dx HER2 乳腺癌 HER2+ breast tumors by GEP are ER negative Over-expression of other genes in HER2- amplicon (GRB7, TOP2A) High proliferative index More likely to harbor p53 mutations Higher histologic grade Younger age at presentation Aggressive clinical course, poor prognosis HER2 乳腺癌 Good response to trastuzumab in combination with chemotherapy More likely to show pcr to neo-adjuvant chemotherapy + trastuzumab compared with other subtypes More likely to respond to anthracyclines May be explain by co-amplification of TOP2A 基底亚型乳腺肿瘤 Gene expression profile similar to normal myoepithelial cells No expression of ER and related genes No HER2 expression, High expression of basal cytokeratins (CK5,6,14,17) High expression of proliferation-related genes, EGFR Aggressive clinical course, poor prognosis Increase likelihood of early systemic recurrence Visceral recurrence & brain metastasis more common Hereditary breast cancer BRCA1 mutation generally develop basal-like breast tumors BRCA1 gene normal regulate repair of DNA damage 26

27 Basal type 基底型乳腺肿瘤 BRCA - EGFR 基因突变的特异性单克隆抗体 CK 5/6 EGFR No role for endocrine therapy or trastuzumab 基底亚型乳腺癌治疗策略 Targeting aberrant DNA repair Platinum agents PARP inhibitors (AZD2281; BSI-201) Trabectedin (DNA transcription inhibitor) Antiangiogenesis Bevacizumab Sunitinib EGFR targeting Cetuximab Erlotinib Epigenetic modifications Trichostatin A Butyrate Vorinostat Src inhibitor -Dasatinib Translation of Gene Expression Data into 5 Reagent IHC Test for ER+/N0 Breast Cancer Patients to Determine Prognosis: Mammostrat (Applied Genomics/Clairent) Eur J Cancer Dec;44(18): J Clin Oncol 2006 ; 24 : , Clin Cancer Res. 2008;20: Study # patients Rouzier (2005) Carey (2007)* De Ronde (2010) Esserman (2009) 新辅助化疗的 pcr 率 Pathologic complete response rates (pcr%*) total Basal like HER2 like Lum A Lum B Significance (P value) P < P < P < P < Bhargava (2010)* NR *IHC defined molecular subtypes, NR, not reported, pcr, pathologic complete response *pcr correlates with outcome, surrogate for good prognosis IHC analysis of P53 - NDRG1 - SLC7A5 - CEACAM5 - HTF9C Prognostic index = (1.542 x SLC7A5) + (1.124 x p53) + (1.058 xndrg1) + (0.712 x HTF9C) + (0.504 xceacam) = low-risk group; >0 and < 0.7 = moderaterisk group; >0.7 = high risk group J Clin Oncol 2006 ; 24 : , Clin Cancer Res 2008;14(20) October 15,

28 乳腺癌免疫组织化学分类 Luminal A ER+ (+/- PR +) HER2 - AND Ki67 low < 15% Luminal B ER+ (+/- PR +) HER2 +/- AND Ki67 high > 15% (+/- p53 positive) Luminal B/HER2 ER+ (+/- PR +) HER2 + (regardless of Ki67 status (usually high) Her2 ER -, PR -, HER2 + Triple negative (Basaloid) ER -, PR -, HER2 - CK5 +, EGFR + 免疫组化方法用于乳腺癌分子分型 优点 - 广泛的可用性 - 成本低 / TAT 快 - 可结合形态学缺点 - 预分析变量因素 - 定性分析 IHC in breast benign mimics Breast ADH VS. DCIS, FEA vs B9 CK5/6, CK14 stains Breast invasive vs. B9 Myoepithelial cells marker D2-40 lymphatic endothelial cell but react with myoepithelial cells 乳腺癌的诊断中最好的肌上皮细胞抗体 p63 and SMMHC 平滑肌肌球蛋白重链 Calponin S-100 Actins Maspin CD10 p75 D2-40 WT-1 HMW-CK P-cadherin sigma 使用肌上皮细胞标记物的潜在陷井 肌上皮细胞的分布在良性病变和原位癌中可能不是均匀的 针对肌上皮细胞的不同的抗体, 其敏感性和特异性也不同 有些病变的肌上皮细胞的抗原性与正常乳腺结构的肌上皮细胞的抗原性不同 p63 High sensitivity and specificity Not reactive myofibroblast in general Reactive with certain tumor cells Tissue artifact common due to antigen retrieval Combine with SMMHC whenever possible Combine with ck7/ck18, best for micro invasion detection 28

29 原位癌模仿 LVI Immunohistochemisty in diagnosis of UDH, ADH and LG-DCIS CK5/6 CK 34 CK19 ER Cocktail (CK5/CK14, CK7/CK18 and p63)--- ADH5 D2-40 Stain Human Pathology (2008) 39, Differentiation of Breast Epithelial Cells Ck5/6+ Ck8/18/19- Ck14- SMA- Ck5/6+ Ck8/18/19+ Ck14- SMA- Intermediate cell Ck5/6- Ck8/18/19+ Ck14- SMA- Luminal cell UDH UDH Progenitor (stem) cell Ck5/6+ Ck8/18/19- Ck14+ SMA+ Intermediate cell Ck5/6- Ck8/18/19- Ck14+ SMA+ Myoepithelial cell FEA/ADH What is ADH5 cocktail ADH staining with ADH5 Breast marker cocktail CK5,CK14 Brown, basal cells p63 nuclear myoepithelial cells CK7, CK18--RED Distinguish ADH vs. UDH Microinvasion Basal phenotype 29

30 FEA Immunohistochemistry The immunohistochemical profile of flat epithelial atypia mirrors that of low-grade ductal carcinoma. The majority of epithelial cells in flat epithelial atypia stain for CK 19, estrogen receptor, and progesterone receptor. The cells show variable but increased expression of cyclin D1, BCL2 and they are uniformly negative for Her-2/neu. Arch Pathol Lab Med. 2008;132: 软组织肿瘤的免疫组化 IHC 主要用于证明细胞系列分化 软组织肿瘤的免疫组化 此类抗体的特异性相对有限 具有 lineage-specific 的抗体很少 软组织肿瘤 Lineage-Restricted 转录因子 Myogenin( 肌细胞生成素 ) 横纹肌肉瘤 MYO-D1, 横纹肌肉瘤 SOX10, 神经外胚层转录因子, 促纤维增生性恶性黑色素瘤, 神经鞘瘤,MPNST FLI1, 原始神经外胚层肿瘤 (PNET) ERG, 内皮细胞转录因子, 正常的血管内皮细胞血管瘤, 淋巴管瘤, 血管肉瘤和上皮样血管内皮细胞瘤, 前列腺腺癌 软组织肿瘤中与分子异常相关的蛋白 β-catenin 纤维瘤病 MDM2/CDK4, 非典型脂肪瘤 / 去分化脂肪肉瘤 INI1 横纹肌样瘤, 上皮样肉瘤 SDHB/SDHA( 琥珀酸脱氢酶突变 ) 副神经节瘤, 有些 GIST 30

31 β-catenin β-catenin 硬纤维瘤病 70%-90% 低度肌纤维母细胞肉瘤 (30%) 孤立性纤维瘤 (20%) 胃肠道间质瘤, 平滑肌瘤 (0%) 硬纤维瘤病 vs. 低度肌纤维母细胞肉瘤 MDM2 and CDK4 MDM2/CDK4 免疫组化 高分化 / 去分化脂肪肉瘤高敏感, 但并不完全特异 非典型脂肪瘤 / 高分化脂肪肉瘤与良性脂肪瘤或梭形细胞 / 多形性脂肪瘤之鉴别 去分化脂肪肉瘤与其他多形或梭形细胞肉瘤 ( 除了 MPNST,myxofibrosarcoma, 横纹肌肉瘤 ) INI1 正常细胞表达双等位基因失活 ( 纯合子缺失或突变 / 缺失 ) 婴儿期的恶性横纹肌样瘤 ( 肾, 中枢神经系统, 软组织 ) 成人肿瘤如转移性黑色素瘤, 恶性间皮瘤转移性癌, 上皮样血管瘤及脑膜瘤显示横纹肌样瘤细胞学特征但保留 IHI1 表达 90-95% 的上皮样肉瘤有 INI1 丢失 50% 的上皮样 MPNST, 肾髓质癌 31

32 pank INI1 Am J Clin Pathol 2009;131: SDHAmutant GIST Wagner et al. Mod Pathol IHC 针对肿瘤融合基因产物 TFE3 腺泡状软组织肉瘤 ; 约 5% 的 PEComas,Xp11 易位的 RCC SDHB SDHA FLI1 Ewing 肉瘤, 血管内皮细胞和肿瘤和淋巴母细胞性淋巴瘤 SDHBmutant GIST ALK 炎症性肌纤维母细胞瘤, 间变性大细胞淋巴瘤小部分肺腺癌 SDHB SDHA 多灶性复发的炎症性肌纤维母细胞瘤 ALK 抑制剂 Crizotinib 治疗 ALK in IMT 3 months after therapy N Engl J Med 2010;363: 免疫组化针对与基因表达相关的蛋白质 DOG1 - 胃肠道间质肿瘤 TLE1 - 滑膜肉瘤 MUC4 - 低度恶性纤维粘液样肉瘤 t(7;16) with FUS-CREB3L2 - 硬化性上皮样纤维肉瘤 32

33 MUC % 的低度恶性纤维黏液样肉瘤 - 软组织神经束膜瘤, myxofibrosarcoma, 孤立性纤维性肿瘤, 韧带样纤维瘤病肌内粘液瘤 TLE1 + 滑膜肉瘤的 80%-90% % 的 MPNST( 通常是脆弱的 ) + SFT( 通常是脆弱的 ) 的 5% 至 10% - 尤文氏肉瘤 Moral of the TLE1 story 新近描述的抗体在六个月内显示最高水平的特异性 没有标记显示 100% 的敏感性和特异性 Differentiation markers in soft tissue Myofibroblast: SMA, Smooth muscle: Desmin, h-caldesmon Striated muscle: Myogenin Nerve Sheath: SOX10, s100 Fat:S100, CD34 Histocytic: CD163 Vascular: CD34, ERG, CD31,D2-40(lymph) Genogenic antibodies Antibody can be used to identify molecular alterations that characterize selected malignancies Antibody as a surrogate for molecular studies, less expensive, short turn around time (TAT) and regular light microscope evaluation 33

34 Genogenic antibodies in sarcoma Fli1 Ewing s sarcoma (? Specificity) TFE3 Alveolar soft part sarcoma ALK Inflammatory myofibroblastic tumor SYT Synovial sarcoma MDM-2/CDK4 Liposarcoma (? Specificity) Immunocytochemical panel for small round cell sarcomas Rhabdomyosarcoma: desmin, musclespecific actin, myogenin, CD99; variable S- 100; ES/PNET: Fli1,CD99; variable S-100, cytokeratin; Mesenchymal chondrosarcoma: CD99; Desmoplastic small round cell tumor: cytokeratin, EMA, WT-1; negative myogenin. Trends in Immunohistochemistry Genogenic immunohistochemistry for diagnosis, and for targeted therapy Methods to develop better monoclonal antibodies with recombinant technology, "technician-free" automation of the IHC procedures Pathologist-free" microscopic image analysis technology for interpretation of high-throughput results Small Round Cell Tumor Ewing sarcoma/pnet Lymphoma Poorly differentiated carcinoma Small cell carcinoma Melanoma Synovial sarcoma, RMS etc. Small Round Cell Tumors CD99 TdT Myog Cam 5.2 Des WT1 ES/PNET % - - A-RMS 15% % + - ALL >90% DSRCT 20%

35 CD 99 can be seen in: Ewing s sarcoma Synovial sarcoma Mesenchymal condrosarcoma Desmoplastic small round cell tumor Small cell osteosarcoma ALL CD99 PAS Desmin + CD99 +/- MSA + SMA - Myogenin + NSE +/- CD34-0 Myogenin in sarcomas Rhabdomyosarcoma 100% Alveolar Embryonal Spindle cell Other sarcomas 0% ES/PNET DSRCT Neuroblastoma etc. Myogenin Part of the Myo-D superfamily of genes Encode for DNA binding proteins activating transcription of skeletal muscle-specific genes Kumar S et al, Mod Pathol

36 Myogenin stain is positive in diffuse pattern Fibromatosis? DFSP? Low grade fibromyxoid sarcoma? Additional tests FISH t(7;16) translocation involving FUS and CREB3L2(L1) - LGFMS Beta-catenin - Desmoid fibromatosis Muc 4 - LGFMS CD34 - DFSP Doyle et al Am J Surg Pathol Volume 35, Number 5, May

37 LGFMS Local recurrence rate less than 10%, Metastatic rate of 6% Overall mortality of 2% Closely related to sclerosing epithelioid fibrosarcoma with same translocation t(7;16)(fus-creb3l2/l1) PEComa Family of related mesenchymal lesions: Angiomyolipoma (AML) Lymphangiomyomatosis (LAM) Clear cell sugar tumor of the lung (CCST) Morphologically and immunophenotypically similar tumors at other sites All share distinctive cell type: perivascular epithelioid cell (PEC) Evidence of myogenic (smooth muscle) and melanocytic differentiation No known normal tissue counterpart PEComa: Histologic Features Nested with delicate vasculature; sheets Predominantly epithelioid, clear to granular eosinophilic cytoplasm Minor spindle cell component Focal association with blood vessel walls 血管周上皮样细胞分化的肿瘤 PEComa 免疫组化 黑素细胞 / 肌源性混合表型 HMB-45 阳性 MITF 阳性 SMA 阳性 ( 上皮 / 透明细胞可阴性 ) Focal S-100 蛋白阳性 <10% MITF PEComa: Criteria for Malignancy Criteria proposed by Folpe (2005) Features associated with malignant behavior (2 or more): Large size (>5 cm) (not consistent in GI tract) Necrosis Mitotic activity (>1 per 50 HPF) High nuclear grade and cellularity Vascular invasion Infiltrative growth 37

38 硬化性肠系膜炎 IgG4 相关的系统性自身免疫性 / 硬化性疾病 自身免疫性 ( 淋巴浆细胞性硬化性 ) 胰腺炎硬化性胆管炎特发性后腹膜纤维化慢性硬化唾液腺炎硬化性淋巴浆细胞性间质肾炎浆细胞肉芽肿肺反应性炎性 psuedotumor >50/HPF, IgG4/IgG >40 Possible link to H. pylori! - Frulloni L, Lunardi C, Simone R, Dolcino M, Scattolini C, Falconi M, Benini L, Vantini I, Corrocher R, Puccetti A. Identification of a novel antibody associated with autoimmune pancreatitis. N Engl J Med Nov 26;361(22): M Distal Jejunum Mass Biopsy Carcinoma Differential Diagnosis Melanoma / clear cell sarcoma PEComa (perivascular epithelioid cell tumor) 38

39 Oscar Melan A Desmin CD34 Cd117 HMB45 IHC results negative negative negative negative negative negative New GIST Marker: DOG1 May be an additional target in the diagnosis and treatment of GIST Highly sensitive and specific for GIST Especially useful for KIT-negative GIST Espinosa et al AJSP 2008 DOG1 65 y/m 20 cm retroperitonial mass 39

40 DX: Dedifferentiated liposarcoma Amplification of MDM2/CDK4 in Soft Tissue Sarcomas Characteristics of atypical lipomatous tumor/well differentiated liposarcoma and dedifferentiated liposarcoma The gene products can be detected by IHC and FISH (preferred for better sensitivity and specificity). If in the correct DDX, positive results can confirm the DX. Could be a promising therapeutic target MDM2 in Liposarcoma Weaver J et al. Mod Pathol 2008 CLINICAL USE OF IMMUNOHISTOCHEMISTRY 65 Year old man with a tumor involving lung and pleura 40

41 间皮与上皮细胞 Basic epithelial panel: CEA, MOC31, BG8 Basic mesothelial panel: Calretinin, WT1, D2-40, CK5 Mesothelioma vs AdenoCa chest Mesothelioma AdenoCA TTF CK5/6 + - Moc Calretinin + - WT1 + - 非典型上皮细胞在胸腔积液 (Female)??? 乳腺癌, 肺癌 CA, 卵巢癌, 胰腺癌, 间皮细胞 Step I CA [MOC31, CEA] vs. mesothelial [WT1, calretinin Vimentin] Step II MOC31 and/or CEA+: go to CA D.Dx [ER, GCDFP-15 and mammaglobin, GATA3,TTF1,NapsinA Adenocarcinoma Moc31 TTF-1 Mesothelioma CK5/6 Calretinin 55 year old man with a lung tumor 肺癌分类 by IHC SCC:CK5, p63 (or p40), Adeno CA: CK7, TTF1, Napsin A, Neuroendocrin: CK7, Synaptophysin,Chromgranin and CD56 Caution: p63 can be positive in neuroendocrine CA (weak, variegated) CK7 can be positive in squamous CA 41

42 Lung Primary vs Met (colon) Lung Colon CK7 + - CK TTF1 + - CDX2 - + *Mucinous ACA of lung is similar as to colon Lung vs. Breast Carcinoma Lung Breast Cytokeratin 7 Cytokeratin 20 TTF-1 Napsin A ER GCDFP-15 Mammaglobin 乳腺球蛋白子宫内膜癌 56 M retroperitoneal mass RCC ACC Pheo AML HCC AE1/AE Cam CD * Inhibin HMB Melan-A Synaptophysin Chromogranin HSA/Arginase PAX * Canalicular pattern 42

43 HCC vs Met Canalicular staining of HCC by polyclonal CEA HCC Met AE1/AE3 - + polyclonal CEA canalicular no CD10 canalicular no Moc HepPar 1/Arginase + - p16, Ki-67, and BD ProExC immunostaining: a practical approach for diagnosis of cervical intraepithelial neoplasia. Immunohistochemistry for SIL P16: most sensitive and less specific BD proexc: Ki67: reserved for conflicts results of p16 and proexc Walts AE, Bose S. Hum Pathol Jul;40(7): Epub 2009 Mar 9. ProEx C (ProEx C) is a recently developed immunocytochemical assay that targets the expression of topoisomerase( 拓扑异构酶 ) II-alpha and minichromosome maintenance protein-2 a b c d 43

44 p16 TEST IS RECOMMENDED IF HGSIL VS. Benign mimics Confirm diagnosis of CIN2 Disagreement about diagnosis of HGSIL Negative biopsy with prior abnormal cytology: HSIL, ASC-H, AGC-NOS, ASC- US/HPV16+ P16 positive stain Strong and diffuse block positive nuclear or nuclear and cytoplasmic of basal layer and extending up at least 1/3 of epithelial thickness CUP 原发病灶不明的肿瘤 Cell Specific Tumor Marker Lung: TTF1, Napasin A Thyroid: TTF1, PAX2, Thyroglobin?, Calcitonin(medullary) Breast: ER,, BRST-2, Mammglobin, GATA-3 RCC: RCC1, Pax2, Pax8 Prostate: P501s, PSA,ERG Colon rectal and GI tumor: CDX-2 Ovarian: WT-1. PAX2, PAX8 Endometrioid CA: vimentin, β-catenin PEComas: SMA, melanocytic markers Specific IHC profiles HCC: CD34, GLYPICAN Meningioma: Claudin PR Barrett s esophagus:cdx2 Hydatidiform mole: p57, FISH (ploidy) Hairy cell leukemia: Annexin Fibromatosis: β-catenin PML: SV40, p53 Kaposi: HHV8 Follicular dendritic cell tumor: CD21 CD35 Inflammatory MFB tumor: SMA (filamentous), +/-EBV, +/-ALK Merkel cell tumor: Ck20+ Monophasic SS: TLE1 CD99, 44

45 BREAST PATHOLOGY ER PR HER2 Ki67 的 E-CADHERIN p63/myosin EGFR VIMENTIN CK5 OSCAR GU PATHOLOGY p504s/p63/ck34 cocktail p63/ck5 cocktail CD44/CK20/p53 cocktail CKIT PLAP CD30 EMA Vimentin CD10 p16 Ki67 p63/ck5 cocktail CEA Beta-catenin EMA Vimentin WT1 CD10 p57 MOC-31 Ber-EP4 (or BG8) Calretinin D2-40 p53 GYN PATHOLOGY CDX2/villin cocktail Synaptophysin Chromogranin CEA CK7 CK20 H. Pylori CMV HSV MLH1 MSH2 GI PATHOLOGY LUNG PATHOLOGY TTF1/Napsin A cocktail p63/ck5 cocktail Synaptophysin Chromogranin CK7 CK20 CDX2/villin cocktail WT1 Calretinin MOC-31 (or BerEP4 or BG8) 45