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

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 11.10 The Biology of Cancer (© Garland Science 2007) DNA Mismatch Repair System

MLH1 PMS2 MLH2 MSH6 DNA Mismatch Repair

DNA mismatch repair promotes genomic stability by correcting base- base and small insertion/deletion mispairs that arise during DNA replication and recombination http://www.helsinki.ﬁ/bioscience/mmrandcancer/mmrgenetics.html What Are Microsatellites?

Repetitive segments of DNA two to ﬁve nucleotides in length scattered throughout the genome both in noncoding as well as coding regions Regions are inherently unstable and susceptible to mutations What Is Microsatellite Instability (MSI)?

The presence of a discrepancy between the size of microsatellites in DNA from tumor compared with nontumor tissue Usually results from loss of gene expression of one or more MMR genes that would normally correct these errors

MLH1 MSH2 MSH6 PMS2 New Nomenclature

dMMR pMMR Are MSI-H tumors distinct?

MSI-H tumors more likely arise on the right side MSI-H tumors more likely to occur in people with positive family history of colorectal cancer MSI-H tumors more likely to be cribriform, solid, signet ring, high grade (’medullary’), mucinous Lymphocytic inﬁltration most important feature for predicting MSI-H (nodular “Crohn-like” peritumoral or TIL) Histologic Patterns of MSI Adenocarcinomas

Mucinous Signet Ring Medullary Mucinous

Medullary TIL Pushing Border

from Bellizzi AM and Frankel WL, Adv Anat Pathol 16:405-17, 2009 IHC vs. MSI Testing IHC MSI

Cost $$ $$$

Analyte Protein DNA

How much tumor required Very little Very little

Requirements Tumor only Tumor + normal

Possibility of contamination by normal No Yes

Turnaround Next day 2-7 days

Identiﬁes involved gene Yes No

Assay sensitive to ﬁxation Yes No

adapted from Bellizzi AM and Frankel WL, Adv Anat Pathol 16:405-17, 2009 IHC v. MSI Testing

Concordance high in most studies

High concordance possible even with just two antibodies (e.g., MLH1, MSH2) but even higher with four (MLH1, MSH2, PMS2, MSH6)

Potential shortcoming if IHC is inability to detect missense mutations that nevertheless result in immunoreactive but nonfunctional protein Rigau V et al. Arch Pathol Lab Med 127:694-700, 2003 MSI-Type Colorectal Adenocarcinomas

Hypermethylation of MLH1 promoter CpG islands But such tumors may be better characterized as “CpG Island Methylator Phenotype High” (CIMP-H) MMR IHC and Colorectal Adenocarcinoma

Immunohistochemical localization “integrates” what happens at the genomic level to MMR genes Identiﬁes genotypically distinct variants of colorectal adenocarcinoma with important clinical implications NO ADJUVANT CHEMOTHERAPY

N=570

Ribic CM et al. NEJM 349:247-57, 2003 ADJUVANT CHEMOTHERAPY

N=570

Ribic CM et al. NEJM 349:247-57, 2003 J Clin Oncol 28:3219-26, 2010

n = 457 Outcome of Patients with Stage III Colorectal Adenocarcinoma Treated with Adjuvant 5-FU

dMMR pMMR Conﬂicting Data on Predictive Role of MSI in 5-FU Response in Colorectal Adenocarcinoma

Vilar E and Tabernero J, Cancer Discovery May 2013 502-11 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

Keratins [OSCAR] Keratin 7 Keratin Keratin 20 Keratin CDX-2 CDX-2 CDX-2 CDX-2 Villin Villin Immunophenotype

Keratins [OSCAR] Uniformly positive

Keratin 7 Negative

Keratin 20 Negative

CDX2 Negative

Villin ?Focally positive

Synaptophysin Negative MSH-2 MSH-6 MLH-1 PMS-2 Am J Pathol 159:2239-2248, 2001

“Minimally differentiated” or “medullary” carcinoma 87% show reduced or absent CDX2 60% showed MSI phenotype Am J Clin Pathol 140:561-6, 2013

CDX2-/K20- phenotype associated with older age, higher stage, LN metastases, “medullary” histology, BRAF mutation, CIMP-H status. Patients have worse survival compared with those expressing CDX2 and/or K20 This is a poor prognosis subgroup MMR IHC Interpretation Caveats

There must be complete loss of MMR expression in the tumor cell population There can be variegated and incomplete immunostaining owing to ﬁxation issues as well as intrinsic variation (e.g., MSH6) Don’t overcall dMMR if there is no staining within the non-neoplastic elements 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) Gastrointestinal Stromal Tumors (GISTs)

• Originally thought to be smooth muscle tumors (“leiomyoblastoma”) or autonomic nerve tumor (“GANT”) • Related to interstitial cells of Cajal (ICC) • Both GISTs and ICCs express KIT, CD34, and DOG1 KIT and PDGFRA Mutations in GISTs

from Marrari A et al, Arch Pathol Lab Med 136:483-9, 2012 c-kit IHC is a Cell Type Speciﬁc Marker!

• Is marker both of normal interstitial cells of Cajal as well counterpart tumor, gastrointestinal stromal tumor • Presence of c-kit expression in GIST is not evidence of presence of activating mutation and hence eligibility for imatinib SDH Mutations and GISTs

PNAS 108:314-8, 2011

• Carney-Stratakis syndrome caused by germ line mutations in SDH subunits B, C, or D • Predisposes to GISTs and paragangliomas • Investigated sporadic GISTs in patients lacking kit or PDGFRA mutations (N =34) SDH-Deﬁcient GISTs

Feature SDH Deﬁcient SDH Proﬁcient Age Children and young Older adults Sex distribution Fadults > M F = M Location Stomach Entire GI tract Multinodular Almost always Rare Multifocality Common Rare Histology Epithelioid or mixed Spindle common Lymph node mets Common Rare Course of mets Indolent Aggressive Imatinib sensitivity No Most cases c-kit positive IHC Yes Yes c-kit mutations None ~95% SDH mutations ~50% None

adapted from Doyle LA and Hornick JL, Histopathol 64:53-67, 2014 SDH Mutations and GISTs

Janeway KA et al., PNAS 108:314-8, 2011 SDH Mutation GISTs

Multinodular architecture Loss of Expression of SDH-B Mixed spindle and epithelioid morphology

Doyle LA, Histopathol 61:801-9, 2012 GIST: Phenotype-Genotype Correlations Genotype Sites and Histology Imatinib response

kit exon 11 all locations; usually spindle or mixed Excellent kit exon 9 small and large bowel; spindle or mixed Better at higher dose kit exon 13 usually small bowel; spindle Some kit exon 17 usually small bowel; spindle Some kit exon 8 small bowel; mixed ? PDGFRA exon 19 stomach and omentum; epithelioid Poor PDGFRA exon 12 stomach; epithelioid Variable PDFRA exon 14 stomach; epithelioid Variable SDH deﬁcient* stomach; epithelioid or mixed Poor SDHA-mutant stomach; epithelioid or mixed Poor SDHB/D/D mutant stomach; epithelioid or mixed Poor *including Carney Stratakis and Carney triad Doyle LA and Hornick JL, Histopathol 64:53-67, 2014 Succinate dehydrogenase is an enzyme complex, bound to the inner mitochondrial membrane of mammalian mitochondria and many bacterial cells. It is the only enzyme that participates in both the citric acid cycle and the electron transport chain. Courtesy of Jason L. Hornick, MD PhD Courtesy of Jason L. Hornick, MD PhD

KIT Courtesy of Jason L. Hornick, MD PhD

SDHB Courtesy of Jason L. Hornick, MD PhD

KIT SDHB

Courtesy of Jason L. Hornick, MD PhD Courtesy of Jason L. Hornick, MD PhD Courtesy of Jason L. Hornick, MD PhD KIT exon 11-mutant GIST

SDHB Courtesy of Jason L. Hornick, MD PhD 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) 49 year old male presents with 5 cm mesenteric mass 2 years after partial gastrectomy for GIST

DIFFERENTIAL DIAGNOSES

• Recurrent gastrointestinal stromal tumor

• Other (myoﬁbroblastic process? desmoid?) CD34 CD117 SMActins [1A4] ß-catenin Mesenteric Fibromatosis

• Aggressive ﬁbromatosis, desmoid tumor • All ages • Associated with Gardner syndrome • Abdominal and extra-abdominal (shoulder, chest wall, back) • Deep-seated, poorly circumscribed • Most present with asymptomatic abdominal mass ß-Catenin: Role in Cell Adhesion and Signaling

Axin and APC are negative regulators of Wnt signalling cascade. Axin and APC phosphorylate ß-catenin on APC binding sites, thereby degrading and inactivating the protein.

Regulation of ß-catenin critical to APC’s tumor suppressor effect Nuclear beta catenin

• In nucleus, beta catenin interacts with transcription factors of TCF/LEF family and thus takes part in alteration of gene expression • ß-catenin in nucleus continuously drives transcription of target genes • Lead to increased cell proliferation and/or inhibition of apoptosis ß-Catenin ß-Catenin and Fibromatoses

• Montgomery et al, AJSP, 2002

• Fibromatoses have mutation in APC/ ß-catenin pathway

• Abnormal nuclear accumulation of ß-catenin protein

• Studied expression by IHC in mesenteric ﬁbromatosis, GIST, and sclerosing mesenteritis Meenteric Fibromatosis

Mesenteric Fibromatosis

ß-Catenin Replace with new photos

ß-Catenin GIST

ß-Catenin Is Nuclear ß-catenin Expression Found in Other Tumors?

Modern Pathology 18:68-74, 2005 Ng TL et al., Modern Pathology 18:68-74, 2005 Ng TL et al., Modern Pathology 18:68-74, 2005

Tumors POSITIVE for high level nuclear ß-catenin expression

• Desmoid type ﬁbromatosis (71%)

• Solitary ﬁbrous tumor (40%)

• Endometrial stromal sarcoma (40%)

• Synovial sarcoma (28%) Abnormal localization of ß-catenin to nucleus

• May be mutation of ß-catenin or adenosis polyposis coli (APC) genes

• APC mutations more common in setting of familial adenomatous polyposis

• ß-catenin mutations more common in sporadic aggressive ﬁbromatosis

• Demonstrates that mutation of one gene may result in abnormal localization of another gene product Nuclear beta catenin in colorectal carcinoma • May be a consequence of inactivating mutation of APC tumor suppressor gene which is now unable to down-regulate intracellular beta catenin • OR activation of beta catenin by somatic mutations • Accumulation of nuclear beta catenin may be ﬁrst visible consequence of loss of APC function ‘Chromosomal Instability’ Pathway Early sign of carcinoma?

• Detection of nuclear ß-catenin in adenomas represents expansion of cells - estimate malignant potential? • PJ polyps, which have no APC gene mutations, do not accumulate nuclear ß-catenin Even earlier than adenoma?

[CANCER RESEARCH 61, 8085– 8088, November 15, 2001] ß-Catenin s u m m a r y

• Immunohistochemistry can be employed to see the nuclear ß-catenin protein abnormally located in the nucleus • Nuclear ß-catenin is a surrogate marker for the presence of APC or ß-catenin mutations Examples of Gene Mutations by 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) Identifying Mutated p53 in Human Tumors

Bártek J, Bártková J, Vojtĕsek B, Stasková Z, Lukás J, Rejthar A, Kovarík J,Midgley CA, Gannon JV, Lane DP. Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies. Oncogene 6:1699-1703, 1991p53

Applications to Esophageal AdenoCA p53 • Encoded by TP53 gene • “Guardian of the genome” function as tumor suppressor • Can activate DNA repair proteins following DNA damage (e.g., radiation) • Can arrest cell growth by holding cells at G1/S regulation point • Can initiate process of apoptosis (programmed cell death) if DNA damage proves to be irreparable p53 p53 • Li-Fraumeni syndrome patients have only one functional copy of p53, develop tumors in early adulthood • Mutations can develop following exposure to chemicals, radiation, viruses, etc. • More than 50% of human tumors contain mutations or deletions in TP53 • Most common is missense mutation involving exons 5-8 coupled with loss of wild type allele (LOH) p53 Immunohistochemistry

• Rapid • Inexpensive • Widely available • Surrogate marker for mutational p53 status? p53 • Most (but not all) inactivating mutations result in conformational change of p53 molecule that results in prolonged half-life • Half-life 20 minutes for wild type, hours for mutant proteins • Wild type protein detectable by immunohistochemistry but at low levels that seem to correlate with cell proliferation • Large deletions or truncating mutations may result in apparent loss of p53 expression p53 and Cancer • Missense mutations would be predicted to correlate with nuclear overexpression • Approximately one-third of TP53 mutations are null (nonsense, frameshift, splice site mutations) probably resulting in complete absence of protein expression • Deletions would also predict to result in complete absence of protein expression • Might expect three immunostaining patterns J Pathol 222:191-8, 2010

• DO-7 anti-p53 monoclonal antibody (cross reacts with wild type and mutant) • Scored in three bins: complete absence of expression, focal expression, overexpression (>50%) • Outcome in two different cohorts p53 and Ovarian Cancer

Kobel M et al., J Pathol 222:191-8, 2010 p53 and Ovarian Cancer Kobel M et al., J Pathol 222:191-8, 2010 • Pelvic high grade serous ovarian cancers show either complete loss or overexpression in 88% of cases • p53 overexpression associated with reduced risk of recurrence • Complete absence of expression associated with unfavorable outcome • Suggests functional differences underlying overexpression v. absence of expression p53 Immunohistochemistry Three unique immunostaining patterns

Mutated Wild type

Mutated p53 and Barrett’s

Kaye PV, et al. Histopathol 54:699-712, 2009

• N = 154 biopsy specimens with Barrett’s, 32 specimens without dysplasia • p53 immunohistochemistry assists in diagnosis in difﬁcult cases and predicts progression Suggested Algorithm

Kaye PV, et al. Histopathol 54:699-712, 2009 Gut 62:1676-83, 2013

• Low grade dysplasia currently only accepted predictor for neoplastic progression in Barrett’s esophagus • Can alterations in p53 improve risk stratiﬁcation? Barrett’s esophagus with low grade dysplasia

Barrett’s esophagus with low grade dysplasia

Esophageal adenoCA with complete loss of p53 Kastelein F et al., 2013 •N = 635 patients •Retrospective study of p53 protein expression as determined by IHC •8% of patients developed high grade dysplasia or adenocarcinoma •More powerful predictor than histologic diagnosis of LGD •Strongly associated with overexpressioln and especially loss of p53 expression Incidence of p53 Overexpression and Absence of Expression

Kastelein F et al., 2013 Gut 63:7-42, 2014

• Marker with greatest body of evidence which can also be applied routinely is p53 • 50-89% positive in Barrett’s dysplasia • Can improve inter observer variability for reporting dysplasia (especially LGD v. atypical reactive [ID]) • Powerful predictor of progression (OR 3-8) Major Genetic Alterations in Cancer

Mutation Mutant Protein

Translocation Loss of Expression

Deletion Abnormal Localization

Ampliﬁcation Overexpression

Methylation Expression of Fusion Proteins Examples of Chromosomal Translocations Identiﬁable by NG-IHC

Tumor Translocation Fusion Generated NG-IHC Target

PNET/ES t(11;22)(q24;q12) EWSR1-FLI1 FLI1

ALCL t(2;5)(p23;q35) NPM-ALK ALK

der(17)t(X;17) ASPS (p11;q25) ASPL-TFE3 TFE3

Synovial sarcoma t(X;18)(p11.2;q11.2) SYT-SSX1 TLE-1

DSRCT t(11;22)(q11;q12) EWSR1-WT1 WT-1

AML t(8;21)(q22;q22) AML1-ETO AML1-ETO

Chromosme 2 Lung cancer inversion EML4-ALK ALK Major Genetic Alterations in Cancer

Mutation Mutant Protein

Translocation Loss of Expression

Deletion Abnormal Localization

Ampliﬁcation Overexpression

Methylation Expression of Fusion Proteins Examples of Ampliﬁed Proteins Identiﬁable by NG-IHC

Tumor Protein ampliﬁed

Breast, gastric cancer HER2

Liposarcoma MDM-2, CDK4

Lymphoma bcl-2

Lung cancer EGFR HER2 Overexpression in Gastric Cancer

Normal HER2 Overexpressed ~20-50,000 receptors Up to ~2,000,000 receptors Genentech 2010

Data Presented At SABCS, December 2012 N = 9,022 breast cancer cases, 2008-2012

Negative (0, 1+) Positive (3+)

Non- 3903 13 ampliﬁed (98.6%) 450 Ampliﬁed 57 (97.2%)

TOTALS 3960 463 The Role of the Pathologist (Then) Next Generation Immunohistochemistry 1

• NG-IHC can be used to identify molecular alterations that characterize selected malignancies • NG-IHC acts as a surrogate for molecular studies, and is less expensive and time consuming and, in some cases, can provide more information Next Generation Immunohistochemistry 2

• NG-IHC can integrate different genotypic changes which result in the same phenotypic changes • NG-IHC can thus expand and better deﬁne categories of disease What does the Future of Pathology Look Like?

•The paradigm shift to molecular based classiﬁcation of tumors will continue and will accelerate

•Molecular oncodiagnostics will play an increasingly large role in tumor analysis The New Paradigm of Pathology

BioPharma Patients Targeted Therapies Oncologists

Published Literature Pathology, Oncology The Changing Role of the Pathologist

“As more drugs that target speciﬁc components of signal- transduction pathways become available and as we increase our knowledge of the complexity of these signalling networks, the burden of selecting the correct drug combinations for each individual cancer patient will ultimately shift to the pathologist, who must identify the underlying defect in each tumor.”

Shaw RJ and Cantley LC. Nature 441:424-30, 2006 [email protected]

Thank you for your attention

Photograph by Dave Morrow