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Allen M. Gown, M.D

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Allen M. Gown, M.D “Next Generation Immunohistochemistry” A Window Onto The Molecular Biology of GI Tract Tumors Allen M. Gown, M.D. Medical Director and Chief Pathologist PhenoPath Laboratories Seattle, Washington Clinical Professor of Pathology, University of British Columbia HematoxylinH & & E Eosin Immuno histo chemistry Immunohistochemistry The application of antibodies with predefined specificities to tissue coupled with the use of detection systems permitting visualization of the target Albert Coons American pathologist and immunologist 1912-78 J Clin Pathol 27:14-20, 1974 Taylor CR and Kledzik G Hum Pathol 12:590-6, 1981 The application of ‘immunostains’ provides an independent method of cell identification against which traditional subjective morphologic criteria may be compared: histopathology may thereby be transformed from something of an art to more of a science. Cell Type Analysis Has Driven IHC Development • Immunohistochemistry can identify cell type with greater certainty than H&E-based morphologic patterns • Most of tumor classification based upon cell type (e.g., squamous cell carcinoma, neuroendocrine carcinoma, acinar cell tumor, etc.) • Cell (tumor) type is a surrogate for predicting the behavior of tumor Immunohistochemistry Cell Type Analysis Has Driven IHC Development Marker Normal Tissues Tumor Colorectal CDX-2 Colorectal epithelium adenocarcinoma SALL4 Germ cells Germ cell tumor kit Interstitial cells Cajal GISTs CD20 B cells B cell lymphoma Villin GI tract epithelium GI tract adenoCAs Beta cells of Insulin Insulinoma pancreatic islet Neuroendocrine cells Synaptophysin Carcinoid tumor of intestine And now for something completely different…. Next Generation Immunohistochemistry PROVIDING A WINDOW ONTO THE MOLECULAR ALTERATIONS UNDERLYING CANCERS AND THUS IDENTIFYING APPROPRIATE THERAPIES SCIENCE VOL 339 29 MARCH 2013 Major Genetic Alterations in Cancer Mutation Translocation Deletion Amplification Methylation Major Genetic Alterations in Cancer Mutation Mutant Protein Translocation Loss of Expression Deletion Abnormal Localization Amplification Overexpression Methylation Expression of Fusion Proteins Major Genetic Alterations in Cancer Mutation Mutant Protein Translocation Loss of Expression Deletion Abnormal Localization Amplification Overexpression Methylation Expression of Fusion Proteins Examples of Gene Mutations Identifiable by Immunohistochemistry • Mutant protein (e.g.,BRAF) • Loss of expresssion (e.g, MMR, SDH) • Abnormal localization (e.g., ß-catenin) • Overexpression (e.g., p53) RAS/RAF/MEK/ERK Pathway BRAF • Second RAF paralogue • Proto-oncogene encoding a serine/threonine kinase that transduces regulatory signals through the RAS/ MEK/ERK pathology • This pathway hyperactivated in ~30% of human malignancies • Gain of function mutations result in aberrant activation of ERK signaling (thyroid papillary carcinoma, melanoma, colon carcinoma, others) • Mutant BRAF acts as an oncogene, promoting tumor cell viability and cell growth BRAF Mutations • Activating mutations mostly in CR3 domain, in P-loop and activating segment of the kinase domain • Most common activating mutation is thymine—>adenine in nucleotide position 1799, resulting in substitution of valine by glutamate BRAF and Colorectal Cancer • BRAF mutations (predominantly V600E) occur in 8-10% of colorectal adenocarcinomas • BRAF and KRAS mutations are mutually exclusive • Patients with BRAF mutated tumors have significantly shorter median progression-free and median overall survival than patents with wild type BRAF tumors BRAF and Colorectal Cancer Targeted therapies in the RAS/ MEK/ERK pathology BRAF V600E Mutation • Most common BRAF mutation • Melanoma (40-60%) • Papillary thyroid carcinoma (45%) • Low grade serous ovarian carcinoma (35%) • Colorectal adenocarcinoma (5-15%) Significance of BRAF Mutation in Colorectal Cancer • Associated with proximal location, higher age, female, MSI-H, high tumor grade, mutinous histology • Associated with reduced overall and disease- free survival; role in MSI-H setting uncertain • Poor prognosis in all groups of advanced colorectal cancer • Meta-analysis of 26 studies show mortality HR = 2.25 Significance of BRAF Mutation in Colorectal Cancer • BRAF V600E mutation occurs in two thirds of MSI sporadic tumors, almost never in setting of Lynch Syndrome • BRAF V500E in non-MSI tumors has particularly poor prognosis, mutually exclusive with KRAS mutation, but appears to predict worse or no response to EGFR targeted therapies Significance of BRAF Mutation in Colorectal Cancer • American National Comprehensive Cancer Network now recommending BRAF testing in setting of wild type KRAS metastatic CRC Genes, Chromosomes & Cancer 52:748-52, 2013 • N = 31 colon cancers (14 BRAF V600E +, 17 BRAF V600 E - by pyrosequencing) • Clone VE1 employed on Ventana platform Genes, Chromosomes & Cancer 52:748-52, 2013 • 100% sensitivity (14/14 BRAF V600E positive tumors IHC positive) • 100% specificity (17/17 BRAF V600E-negative tumors IHC negative) • In minority of cases, staining intensity for mutated tumor samples weak or heterogeneous; 8/14 cases diffuse and strong Cancer 119:2765-70, 2013 BRAF-WT BRAF V600E BRAF V600E pBR1 VE1 Sinicrope FA et al., Cancer 119:2765-70, 2013 BRAF V600E BRAF-WT BRAF-WT pBR1 VE1 Am J Surg Pathol 37:1592-1602, 2013 • BRAF IHC may outperform PCR MassArray in routine clinical setting • IHC provided “correct” results in 200/201 (99.5%) of cases, whereas MassArray was “correct” in 195/201 (97%) BRAF V600E MLH1 MSS and V600E BRAF MSS and WT BRAF Toon CW et al., Am J Surg Pathol 37:1592-1602, 2013 BRAF V600E MLH1 MSI and V600E BRAF MSI and WT BRAF Toon CW et al., Am J Surg Pathol 37:1592-1602, 2013 Toon CW et al., Am J Surg Pathol 37:1592-1602, 2013 • Presence of BRAF V600E mutation in MSI colorectal carcinomas virtually excludes Lynch syndrome • Presence of BRAF V600E mutation in MSS colorectal carcinomas predicts poor prognosis Proposed Algorithm MMR IHC BRAF IHC Toon CW et al., Am J Surg Pathol 37:1592-1602, 2013 Histopathology 63:187–193, 2013 • N = 52 colon cancers (17 BRAF V600E+) • Only 12/17 (71%) of BRAF V600E+ tumors were IHC positive • Weak cytoplasmic signal (1+) seein in 6/17 (17%) of BRAF wild type tumors INCOMPLETE SENSITIVITY INCOMPLETE SPECIFICITY Adakapara CA et al., Histopathology 63:187–193, 2013 Comparison of NG-IHC (VE1) v. Molecular Analysis for Detection of BRAF V600E Paper N No. HIER Platform Scoring Sensitivity Specificity Mutant Citrate Adackapara et 52 17 Manual S, M, W 71% 74% al., 2013 pH 6 EDTA Affolter et al, 31 14 Ventana Binary 100% 100% 2013 pH 9 Binary Capper et al., 91 11 pH 8 Ventana 100% 91% 2013 (>80%) Kuan et al., 2014 128 57 pH 8 Ventana S, M, W 100% 94% Sinicrope et al., 2013 75 25 ? Ventana S, M, W 100% 100% Binary Toon et al 201 38 ? ? 98% 100% 2013 (>75%) Comparison of NG-IHC (VE1) v. Molecular Analysis for Detection of BRAF V600E Paper N No. HIER Platform Scoring Sensitivity Specificity Mutant Citrate Adackapara et 52 17 Manual S, M, W 71% 74% al., 2013 pH 6 EDTA Affolter et al, 31 14 Ventana Binary 100% 100% 2013 pH 9 Binary Capper et al., 91 11 pH 8 Ventana 100% 91% Why2013 This Discordance?(>80%) Kuan et al., 2014 128 57 pH 8 Ventana S, M, W 100% 94% Sinicrope et al., 2013 75 25 ? Ventana S, M, W 100% 100% Binary Toon et al 201 38 ? ? 98% 100% 2013 (>75%) Hum Pathol 45: 464-72, 2014 • Prospective study of 103 cases (57 with BRAF V500E mutation) • 100% sensitivity, 94% specificity • Suggest that reason for discordance in other studies may be different epitope retrieval methods Potential Reasons • Different tissues and fixation • Different epitope retrieval methods • Different detection systems and platforms • Different observers • First corollary to Gown’s 2nd Law of IHC Always Employ Antibodies Within The First Six Months of Their Publications That’s When They Are Most Specific! BRAF V600E s u m m a r y • Immunohistochemistry can be employed to see the mutant BRAF protein in the cytoplasm of tumor cells • Immunohistochemistry may or may not be adequately sensitive and specific to replace, or be integrated with, molecular assays Examples of Gene Mutations Identifiable by Immunohistochemistry • Mutant protein (e.g.,BRAF) • Loss of expresssion (e.g, MMR, SDH) • Abnormal localization (e.g., ß-catenin) • Overexpression (e.g., p53) Examples of Mutations Leading to Loss of Protein Expression Rhabdoid tumors INI-1/SMARCB1 (and others) Mismatch Repair Coloretal (MLH1, MSH2, adenocarcinoma MSH6, PMS2) Lobular breast E-cadherin cancer Subset of Succinic gastrointestinal dehydrogenase stromal tumors Endometrial, PTEN breast cancer Reasons for MMR IHC Identifying Lynch Syndrome patients Identifying patients with sporadic ‘MSI tumors’ (who may not require FU-based chemotherapy) Identifying ‘carcinomas of unknown primary’ that are ‘minimally differentiated’ colorectal adenocarcinoma HNPCC (Lynch Syndrome) Hereditary Non-polyposis Colorectal Cancer Autosomal dominant Mutation in MLH1 (~60%), MSH2 (~30%), or MSH6 (~10%) Accounts for 2-5% of colorectal adenocarcinoma Tumors develop at early age, usually found on right side Also develop endometrial adenocarcinoma Synchronous and metachronous colorectal cancers: 40% develop within 10 years without total colonic resection Reasons for MMR IHC Identifying Lynch Syndrome patients Identifying patients with sporadic ‘MSI tumors’ (who may not require FU-based chemotherapy) Identifying ‘carcinomas of unknown primary’ that are ‘minimally differentiated’ colorectal adenocarcinoma Classical ‘Vogelstein’ Pathway of Colonic Adenocarcinoma Progression Figure
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