“Next Generation Immunohistochemistry” · 2019. 4. 26. · Comparison of NG-IHC (VE1) v....

“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

Hematoxylin & EosinH & E

Immuno histochemistry

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

CDX-2 Colorectal epithelium Colorectal 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

Insulin Beta cells of pancreatic islet Insulinoma

Synaptophysin Neuroendocrine cells of intestine Carcinoid tumor

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


Mutation

Translocation

Deletion

Amplification

Methylation

Loss of Expression

Abnormal Localization

Overexpression

Expression of Fusion Proteins

Mutant Protein


Mutation

Translocation

Deletion

Amplification

Methylation

Loss of Expression


Overexpression


Mutant ProteinMutation Mutant Protein

Loss of Expression


Overexpression

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


• 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


• 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

• 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

Genes, Chromosomes & Cancer 52:748-52, 2013

Cancer 119:2765-70, 2013

VE1

pBR1

BRAF V600EBRAF-WT BRAF V600E

Sinicrope FA et al., Cancer 119:2765-70, 2013

VE1

pBR1

BRAF V600E BRAF-WT BRAF-WT

• 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%)

Am J Surg Pathol 37:1592-1602, 2013

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


BRAF V600E MLH1

• 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

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

Adakapara CA et al., Histopathology 63:187–193, 2013

INCOMPLETE SENSITIVITY

INCOMPLETE SPECIFICITY

Comparison of NG-IHC (VE1) v. Molecular Analysis for Detection of BRAF V600E

Paper N No. Mutant

HIER Platform Scoring Sensitivity Specificity

Adackapara et al., 2013 52 17

Citrate pH 6

Manual S, M, W 71% 74%

Affolter et al, 2013 31 14

EDTA pH 9

Ventana Binary 100% 100%

Capper et al., 2013 91 11 pH 8 Ventana

Binary (>80%)

100% 91%

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%

Toon et al 2013 201 38 ? ?

Binary (>75%)

98% 100%

Comparison of NG-IHC (VE1) v. Molecular Analysis for Detection of BRAF V600E

Paper N No. Mutant

HIER Platform Scoring Sensitivity Specificity

Adackapara et al., 2013 52 17

Citrate pH 6

Manual S, M, W 71% 74%

Affolter et al, 2013 31 14

EDTA pH 9

Ventana Binary 100% 100%

Capper et al., 2013 91 11 pH 8 Ventana

Binary (>80%)

100% 91%

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%

Toon et al 2013 201 38 ? ?

Binary (>75%)

98% 100%

Why This Discordance?

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

• 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

s u m m a r y

Examples of Mutations Leading to Loss of Protein Expression

INI-1/SMARCB1Rhabdoid tumors

(and others)

Mismatch Repair (MLH1, MSH2, MSH6, PMS2)

Coloretal adenocarcinoma

E-cadherinLobular breast

cancer

Succinic dehydrogenase

Subset of gastrointestinal stromal tumors

PTENEndometrial, 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

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

HNPCC (Lynch Syndrome) Hereditary Non-polyposis Colorectal Cancer

Reasons for MMR IHC




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

http://www.helsinki.fi/bioscience/mmrandcancer/mmrgenetics.html

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 Are Microsatellites?

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

What Is Microsatellite Instability (MSI)?

New Nomenclature

dMMR pMMR

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)

Are MSI-H tumors distinct?

Histologic Patterns of MSI Adenocarcinomas

from Bellizzi AM and Frankel WL, Adv Anat Pathol 16:405-17, 2009

Mucinous Signet Ring

Medullary

MucinousMedullary

TIL Pushing Border

IHC vs. MSI Testing

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 MSI

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)

Immunohistochemical localization “integrates” what happens at the genomic level to MMR genes

Identifies genotypically distinct variants of colorectal adenocarcinoma with important clinical implications

MMR IHC and Colorectal Adenocarcinoma

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

NO ADJUVANT CHEMOTHERAPY

N=570

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

ADJUVANT CHEMOTHERAPY

N=570

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

J Clin Oncol 28:3219-26, 2010

dMMR pMMR

n = 457

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




Keratins [OSCAR]

Keratin

Keratin 7

Keratin

Keratin 20

CDX-2

CDX-2

Villin

Villin

Immunophenotype

Keratins [OSCAR] Uniformly positive

Keratin 7 Negative

Keratin 20 Negative

CDX2 Negative

Villin ?Focally positive

Synaptophysin Negative

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

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

MMR IHC Interpretation Caveats

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

• 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)

PNAS 108:314-8, 2011

SDH-Deficient GISTs

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

Feature SDH Deficient SDH ProficientAge Children and young

adultsOlder adults

Sex distribution F > M F = MLocation Stomach Entire GI tractMultinodular Almost always RareMultifocality Common RareHistology Epithelioid or mixed Spindle commonLymph node mets Common RareCourse of mets Indolent AggressiveImatinib sensitivity No Most casesc-kit positive IHC Yes Yesc-kit mutations None ~95%SDH mutations ~50% None

SDH Mutations and GISTs

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

SDH Mutation GISTs

Multinodular architecture

Mixed spindle and epithelioid morphology

Loss of Expression of SDH-B

Doyle LA, Histopathol 61:801-9, 2012

GIST: Phenotype-Genotype Correlations

kit exon 11 all locations; usually spindle or mixed Excellentkit exon 9 small and large bowel; spindle or mixed Better at higher dose

kit exon 13 usually small bowel; spindle Somekit exon 17 usually small bowel; spindle Somekit exon 8 small bowel; mixed ?

PDGFRA exon 19 stomach and omentum; epithelioid PoorPDGFRA exon 12 stomach; epithelioid VariablePDFRA exon 14 stomach; epithelioid VariableSDH deficient* stomach; epithelioid or mixed PoorSDHA-mutant stomach; epithelioid or mixed Poor

SDHB/D/D mutant stomach; epithelioid or mixed Poor

Genotype Sites and Histology Imatinib response

Doyle LA and Hornick JL, Histopathol 64:53-67, 2014 *including Carney Stratakis and Carney triad

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.

http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Inner_mitochondrial_membranehttp://en.wikipedia.org/wiki/Inner_mitochondrial_membranehttp://en.wikipedia.org/wiki/Mammalianhttp://en.wikipedia.org/wiki/Mitochondriahttp://en.wikipedia.org/wiki/Bacterialhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Citric_acid_cyclehttp://en.wikipedia.org/wiki/Citric_acid_cyclehttp://en.wikipedia.org/wiki/Electron_transport_chain

Courtesy of Jason L. Hornick, MD PhD

KIT


SDHB


KIT


SDHBCourtesy of Jason L. Hornick, MD PhD

KIT exon 11-mutant GIST

SDHBCourtesy of Jason L. Hornick, MD PhD

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?)

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

ß-Catenin

Mesenteric Fibromatosis

Replace with new photos

ß-Catenin

ß-Catenin

GIST

Modern Pathology 18:68-74, 2005

Is Nuclear ß-catenin Expression Found in

Other Tumors?

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

• 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

s u m m a r y

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, 1991 p53

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• 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

• 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

Kobel M et al., J Pathol 222:191-8, 2010

p53 ImmunohistochemistryThree unique immunostaining patterns

Mutated

Mutated

Wild type

p53 and Barrett’s

• N = 154 biopsy specimens with Barrett’s, 32 specimens without dysplasia

• p53 immunohistochemistry assists in diagnosis in difficult cases and predicts progression

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

Suggested Algorithm

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

• Low grade dysplasia currently only accepted predictor for neoplastic progression in Barrett’s esophagus

• Can alterations in p53 improve risk stratification?

Gut 62:1676-83, 2013

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

Kastelein F et al., 2013

Incidence of p53 Overexpression and Absence of Expression

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)


Mutation

Translocation

Deletion

Amplification

Methylation

Loss of Expression


Overexpression


Mutant Protein

Translocation

Overexpression


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

ASPS der(17)t(X;17)(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

Lung cancer Chromosme 2 inversion EML4-ALK ALK


Mutation

Translocation

Deletion

Amplification

Methylation

Loss of Expression


Overexpression


Mutant Protein

Amplification Overexpression

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 ~20-50,000 receptors

HER2 Overexpressed Up to ~2,000,000 receptors

Genentech 2010

Data Presented At SABCS, December 2012

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

Non-amplified

3903 (98.6%)

13

Amplified 57450

(97.2%)

TOTALS 3960 463

N = 9,022 breast cancer cases, 2008-2012

The Role of the Pathologist (Then)

• 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


1

•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

2


•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

What does the Future of Pathology Look Like?

Patients

Oncologists

The New Paradigm of Pathology

BioPharma Targeted Therapies

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

Thank you for your attention

[email protected]

Photograph by Dave Morrow

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