Anatomic Molecular Pathology: An Emerging Field

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1 Anatomic Molecular Pathology: An Emerging Field Antonia R. Sepulveda M.D., Ph.D. University of Pennsylvania 2008 ASIP Annual Meeting

2 Anatomic pathology (U.S.) is a medical specialty that is concerned with the diagnosis of disease based on the gross, microscopic, and molecular examination of organs, tissues, and cells. Molecular pathology Molecular pathology is an emerging discipline within anatomical pathology which is focused on the use of nucleic acid based techniques such as in situ hybridization, reverse transcription, PCR, and nucleic acid microarrays for specialized studies of disease in tissues and cells. Molecular pathology shares some aspects of practice with both anatomic and clinical pathology, and is sometimes considered a "crossover" discipline.

3 Anatomic Pathology Surgical Pathology Surgical resection specimens Diagnosis, staging Prognostic/predictive markers Biopsies diagnostic, staging Prognostic/predictive markers Cytopathology Cells in biological fluids Fine needle aspiration Autopsy Pathology

4 Surgical Pathology Surgical specimen Sampling Paraffin embedded tissue

5 Surgical Pathology SR08 6XX Diagnosis: Moderately differentiated adenocarcinoma

6 Molecular Tests in Anatomic Tumor tissue Pathology Mutational analysis: EGFR, BRAF, Kras,, LOH Microsatellite Instability & DNA MMR IHC CpG methylation: hmlh1 Chromosomal Translocations Gene expression profiles predictive predictive markers Microarray gene expression profiles MicroRNA profiles Proteomic testing Non neoplastic neoplastic Infectious agents Other Identity tests (STR panels) Other

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8 Tissue Based Molecular Tests for Hereditary Nonpolyposis colorectal Cancer (HNPCC) Workup

9 HNPCC and DNA Mismatch Repair Proteins MutS: MSH2, MSH6,, MSH3 MutL: MLH1, PMS2,, MLH3 MMR proteins function as heterodimers The heterodimers are required for protein stability Hereditary Nonpolyposis Colorectal Cancer (HNPCC): 1 Loss of function/expression of DNA mismatch repair protein in tumors (germline mutation) 2 Mutator Phenotype: Microsatellite Instability (MSI)

10 Algorithm for Laboratory Testing for HNPCC MSI Lindor, NM et al. JAMA, 296 (12), 2006 MLH1 methyl and/or BRAF mut

11 TESTING MSI IN TUMORS PROTOCOL Unstained sections from formalin fixed paraffin embedded tissue Scrape selected areas for DNA extraction PCR amplification with MSI panel DNA fragment characterization by electrophoresis

12 COLORECTAL ADENOCARCINOMA: High Level Microsatellite Instability (MSI H) Loss of hmlh1 Protein Expression N N N N Gologan & Sepulveda et al. Arch Pathol Lab Med 2005; 129: T T T T BAT25 BAT26 D5S346 D17S250

13 Incidence and functional consequences of hmlh1 promoter hypermethylation in colorectal carcinoma 11 of 13 (84%) MSI positive cancers exhibited prominent methylation, compared with 2 of 21 MSI negative cancers (P =0.0001). Methylation specific PCR (MSP) James G. Herman et al. and Stephen B. Baylin PNAS, Vol. 95, Issue 12, , 6875, 1998

14 Distinction of HNPCC and Sporadic Microsatellite Unstable CRC through Quantification of MLH1 Methylation by Real time PCR M Bettstetter et al and W Dietmaier. Clinical Cancer Research 13, , 3228, 2007

15 BRAF Mutation is Frequently Present in Sporadic CRC with Methylated hmlh1, but not in HNPCC BRAF: cytoplasmic serine/threonine kinase Function: mitogen activated protein kinase signaling pathway Most of the mutations in BRAF : thymine to adenine transversion at nucleotide 1796 Substitution of valine for glutamate (V600E) Results in BRAF activating mutation BRAF V600E mutations occur in: Sporadic MSI H H CRC (40 74%) Sporadic MSS CRC 4% to 12% BRAF V600E mutations not detected in HNPCC patients G Deng et al. and YS Kim Clin Cancer Res, 10, , 2004

16 EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) TESTING IN LUNG CANCER

17 Molecular Diagnosis in Lung Cancer NCCN Practice Guidelines (v ) EGFR mutations define a subgroup of NSCLC patients who will benefit from EGFR tyrosine kinase inhibitor (TKI) treatment

18 Distribution of Mutations and Response Rate to TKI EX19 DEL ~90% Not all EGRF mutations associated with response L858R Cancer Science (OnlineEarly Articles). doi: /j x

19 Detection of EGFR Exon 19 Deletions Exon 19 In frame deletions usually 9 24 bp Positive for deletion 208 bp 190 bp = 18 bp Normal

20 Oncotype DX is validated for use in breast cancer patients Multigene Expression Assay (RT PCR) to Predict Recurrence of Tamoxifen Treated, Node Negative Negative Breast Cancer Selection of Patients for Chemotherapy S.Paik et al. NEJM. 351: , 2826, 2004

21 Pathwork Tissue of Origin Test Gene expression microarray based test Helps identify tissue of origin of poorly & undifferentiated cancers 15 tissue types CancerTYPE Molecular Classification Test 92 gene real time quantitative PCR assay Molecular classification of human cancers 39 tumor classes

22 Molecular Diagnosis of Infectious Agents Whipple s disease: Tropheryma whipellii Test: PCR for rrna DNA sequence Confirmation of diagnosis and follow up

23 The Molecular Anatomic Pathology Laboratory Anatomic Pathology Molecular Diagnostics yeinjee.com/.../2007/08/paris louvre pyramid.jpg

24 Career Opportunities in Molecular Anatomic Pathology Environment: Academia, Hospitals, Reference Laboratories and Industry Positions Scientific Directors Research and Clinical Technicians and Technologists Roles: Translational research Test development Validation and clinical testing

25 Acknowledgments: Vivianna Van Deerlin MD, Univ. Pennsylvania