Jeffrey S. Ross, M.D. Department of Pathology and Laboratory Medicine Albany Medical College

1. The Evolving Role of the Molecular Pathologist: Opportunities and ChallengesPersonalized Medicine, Companion DiagnosticsCME & P.A.C.E.® Symposium at 2012 ASCO Jeffrey S. Ross, M.D. Department of Pathology and Laboratory Medicine Albany Medical College Albany, NY rossj@mail.amc.edu

2. “A Perfect Storm” in the Healthcare Industry Driving Personalized Oncology New Customer Dynamics • $ Out of pocket • Informed Pharmaceutical Industry Dynamics • Mergers • Patent expirations New Communication Mechanisms • Wireless networking • Broadband Healthcare Industry Dynamics Advances in Medicine • Genomics • Proteomics • Imaging technologies • Devices and biosensors • Molecular Medicine • Nanotechnologies Changing Economics • Generics cost shift to consumer • Pricing Source: Craig Fitzgerald, Various industry sources

3. Personalized Oncology: Key Issues • Which patients will benefit most? • Improved efficacy? • Reduced toxicity? • Cost/benefit ratio? • What time should the test(s) be performed? • What sample to test? • Tumor for Expression Profiling or Sequencing? • Blood for Germline Sequencing? • CTC? • What technique to use? • Gene sequence (DNA)? Germline? Tumor Cells? • Gene dosage (DNA)? • Gene expression (mRNA)? • Protein expression (IHC) and Proteomics? • Which algorithm or mathematic model should be used to make predictions? • Are the tests regulated by the FDA? EMA? CLIA? • Will the test(s) be reimbursed or will the patient have to pay out of pocket? • Will the added test cost reduce overall expenditures by driving better clinical outcomes?

4. Roles for Anatomic Pathologists in Molecular Pathology and Personalized Medicine • Providing slide-based biomarker services • IHC • FISH • CISH • Preparing biopsies and resections for extraction-based biomarker services • RT-PCR • Genotyping by traditional sequencing techniques • Next Generation Sequencing

5. Comparison of Molecular Pathology Test Platforms (1)

6. Comparison of Molecular Pathology Test Platforms (2)

7. Pathologist Roles in Preparing Clinical Samples for mRNA Profiling and DNA Genotyping/NGS • Selecting the best tissue block when multiple blocks are available • Highest percentage of malignant cells • Selecting viable areas and avoiding necrosis and wound healing/severe inflammation • Recommending or performing sample enrichment when necessary

8. Small Samples and FNAs • A FFPE section with a 4 X 4 mm minimum surface area with minimum of 40 micron thickness measurement is recommended • For FNAs • Sample is pun into a cell pellet and then converted to an FFPE tissue section • Enrichment by macrodissection rarely possible • Local IHC Procedures • Local pathologists should be discouraged from performing excessive numbers of immunostains that deplete the small amounts of tissue needed for RNA/DNA analysis • Critical importance for NSCLC biopsies • Limit immunostains for the squamousvs non-squamousdeision

9. Selected Common Examples of Slide Based Molecular Pathology for Solid Tumors • Breast Cancer • ER and PR by IHC • HER2 by IHC and FISH/CISH • NSCLC • SCC vsAdenocarcinoma • ALK IHC Status • EGFR IHC in SCC for cetuximab • ERCC1 for platinin resistance • CRC • Mis-match Repair (MSI) IHC for hMLH1, hMSH2, hMSH6

10. Improving ER Testing • Open Issues • Can IHC be standardized? • Do we want simple positive or negative results only? • Can mRNA testing select the best hormonal therapy options? • How do we predict tamoxifen resistance in ER IHC+ tumors? • Can we “personalize” hormonal therapy

11. The ASCO-CAP 2010 Guidelines for Hormone Receptor IHC Testing • Did not require IHC staining intensity in slide scoring • Focused on pre-analytic issues • Pre-analytic guidelines differ from HER2 guidelines (72 vs 48 hrs max fixation) • CAP Certification + Proficiency testing required • Did not establish a cut-off staining percentage for ER or PR positive status

12. ER mRNA Testing*: Concordance Between IHC Status and mRNA Levels 25 20 15 10 5 0 Relative ER mRNA Expression + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - - - - - + - + - - IHC > 20% ER + IHC ER - ER Status by IHC on Core Needle Biopsies * Measured by either RT-PCR (eg Oncotype Dx) or by Microarray (eg TargetPrint)

13. HER-2 TestingIs CISH the “Kish of Death” for FISH and IHC? • Is IHC still the international method of choice for screening with 2+ cases triaged to FISH? • Will primary FISH testing ever become the standard? • Will mRNA detection gain in popularity? • Will the recently approved CISH (SISH) assay become the preferred method? • Has the ToGA Trial and Gastic/GEJ trastuzumab approvals changed how HER2 testing is done? • Will trastuzumab-DM1 and pertuzimab require HER2+ testing prior to use?

14. The ASCO-CAP Guidelines for HER2 Testing* • Key Issues • 20% of testing in the field incorrect • Required higher thresholds for HER2 positive status • 10% to 30% IHC 3+ required • FISH ratio increased from 2.0 to 2.2 • Serious concerns for patients who are HER2 positive by trastuzumab package insert guidelines but not positive by ASCO-CAP criteria not receiving anti-HER2 therapy • Criteria for HER2 positive differ from the gastric/GEJ approvals • Some laboratories no longer use the ASCO-CAP guidelines and have reverted to the package insert guidelines * Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast ancer. J Clin Oncol. 2006 Dec 11;

15. IHC: Positive Control on Same Slide with Sample from Each New Patient It has been estimated that 3-4% of all US HER2 testing is falsely negative.

16. HER2 Gene Copy Number Alteration Validation • Increased HER2 gene copies detected by NGS • HER2 FISH Positive Breast Invasive Duct Carcinoma Demonstrating High HER2 Copy Number • HER2 Protein 3+ Expression by IHC ERBB2 RARA

17. Chromogenic In Situ Hybridization Overall FISH/CISH Concordance = 98% NOT AMPLIFIED: 2 SIGNALS PER NUCLEUS HIGHLY AMPLIFIED DETAIL • CISH • Chromogenic ISH • Light microscope ready • All the advantages of the DNA target in paraffin sections, but does not require fluorescent detection • FDA approval 7/2/08

18. mRNA Oncotype Dx™ mRNA TargetPrint Genomic HealthRedwood City, CA Agendia Amsterdam, NE 2005 2008 Not Approved Not Approved On the market On the market RT-PCR (FFPE) Agilent Array (Fresh tissue) RNA RNA Centralized at company headquarters Same HER2 Gene Mutation Testing Next-Gen Sequencing Foundation Medicine Cambridge, MA Others 2010 Not Approved Homebrews Hybrid Capture Sequencing (FFPE) DNA Centralized in commercial and academic labs Di-merization HERmark™ Monogram Biosciences/USLabs/LCASouth San Francisco, CA 2008 Not Approved On the market VeraTag™ Capillary electrophoresis (FFPE) Protein dimers Decentralized in clinical and commercial labs ELISA Serum HER2 Advia Centaur™ Siemens Healthcare DiagnosticsDeerfield, IL 2002 Approved (PMA) On the market Sandwich Immunoassay (serum) Protein (serum) Decentralized in clinical and commercial labs CTC Based Assays Veridex OncoVista Others 2004 Not Approved On the market FISH RT-PCR (whole blood) DNA RNA Centralized and decentralized Emerging HER2 Tests

19. Squamousvs Non-Squamous (Adenocarcinoma) in NSCLC • At least 75% of cases can be resolved on H & E • IHC to decide Squamousvs Non-Squamous consumes vital tissue reserves for clinical trial biomarkers • Over-use of IHC markers must be discouraged • Adenocarcinoma • TTF-1 (Best), Napsin, PE-10 • Squamous Carcinoma • P63 (Best), CK5/6, 34βe12 • Desmocolin-3 (Need More Testing) • Cocktails – Nuclear/Cytoplasmic Antibodies • Adenocarcinoma – TTF-1/Napsin • Squamous – P63/CK5/6

20. EML4-ALK Rearrangement in NSCLC • Crizotinib • Oral ALK4 receptor kinase inhibitor • Phase I Trial on NSCLC patients with EML4-ALK rearrangement • “echinoderm microtubule-associated protein-like 4” – “anaplastic lymphoma kinase” • 10/19 (53%) had a partial response • Seen in 5-13% of adenocarcinomas • More common in non-smokers • All ELM4-ALK Positive NSCLC are Negative for EGFR and KRAS mutations • ELM4-ALK translocation can be detected by FISH or NGS • ALK IHC (Cell Signaling Ab) being developed as a CDx for crizotinib Shaw et al. J ClinOncol. 27;2009:4247-4253.

21. EGFR IHC Testing in Squamous Cell NSCLC • May be developed as a CDx for selection of NSCLC SCC for treatment with cetuximab • ECCO/ESMO data indicated that “H Score” IHC scoring must be used and tumors must have a score > 200 • EGFR IHC not currently required for HNSCC case selection for cetuximab use

22. ERCC1 Testing in NSCLC • ERCC1 as a predictive marker in cisplatin-based adjuvant chemotherapy in resected NSCLC has been suggested in large, retrospective analyses • Confirmation needed in large prospective studies • Data mainly for cis-platinin, data for carbo-platinin lacking • Hypothesis that low/negative ERCC1 IHC expression is predictive of platinum-based chemotherapy-sensitivity has not yet been fully validated • ERCC1 by RT-PCR efficacy varies due to the quality of material • IHC may vary on inter-observer variability and the target lesion chosen for examination • Blood-based SNPs methods determining ERCC1 polymorphisms holds promise • The role of ERCC1 in NSCLC patient-tailored chemotherapy although far from being firmly established may possibly prove to play an important role in tailored chemotherapy for NSCLC

23. Detecting MSI in CRC with IHC • Routine use in many CRC treatment institutions • Mis-match Repair Defective CRC defined as Loss of expression of 2 or more mis-match repair enzymes (MLH1, MSH2, MSH6, others) • Associated with improved prognosis and potential for reduced treatment intensity stage for stage • IHC results also may indicate HNPCC

24. Selected Common Examples of mRNA Profiling for Solid Tumors • Cancer of Unknown Primary Origin • Non-Hodgkin’s Lymphoma • Breast Cancer • Molecular Portraits • Tumor Grading • Predicting prognosis and guiding management • Pharmacogenomics

25. Technology Platforms For High Throughput Analysis Of mRNA Expression cDNA arrays Oligonucleotide arrays Multiplex RT-PCR Probe length Hundreds to thousands of base long cDNA 20-75 base oligonucleotides 15-30 base PCR primers Probes per array 100 –5,000 >20,000 Few hundred Probes per transcript 1-2 1- >10 1 Examples of sources Clontech, Agilent, Incyte, core laboratories Affymetrix, Amersham, Mergen, Agilent, core laboratories Qiagen, Applied Biosystems, Roche Molecular Systems, Genomic Health Detection system Chemiluminescence Radioisotope Chemiluminescence Chemiluminescence Gel electrophoresis

26. Challenges that Face Genomic Microarrays • Over-fitting The Data • Discovery Research without a hypothesis • Initial excitement followed by failure to reproduce the results • Test results may vary with the extent of the disease • Lack of Currently Approved Rules of Evidence • No standardized approach to data validation • Multivariate Analysis finds independent significance but association with disease outcome was actually due to chance • “Too many genes, not enough patients”

27. Microarray Classification of Non-Hodgkin’s Lymphoma Staudt. Cancer Cell Vol. 2, No. 5, 11/02: 363 - 366

28. Site of Origin for Metastatic AdenocarcinomaDennis et al Cancer Res 2002;62:5999-6005 Expression Profiles of 61 Genes by SAGE Established Tumor Markers by RT-PCR in Common Adenocarcinomas

29. Molecular Portrait of Breast Cancers Basal–like HER-2 “Normal Luminal B Luminal A Sørlie et al. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10869-10874.

30. Molecular Grading of Breast Cancer • Gene expression profiling data indicates that there are 2 molecular grades of breast cancer • Histologic Grade 2 cases redistribute into Molecular Grades 1 and 2 • Molecular grading has outperformed histologic grading in multivariate analysis of traditional prognostic factors including ER/PR and HER2 status • The Genomic Grade IndexTM has been commercialized by Ipsogen, Inc. Patterns of expression of grade-related genes and their association with histologic grade (HG) and relapse-free survival. GGI score of each tumor is plotted below the corresponding column. Relapse-free survival times in years are indicated below the GGI scores. Sotiriou C, et al.. Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst. 2006 Feb 15;98(4):262-72.

31. Multigene Classifiers and Predictors of Breast Cancer Clinical Outcome* *Ross, JS, Hatzis C, Symmans WF, Pusztai L, Hortobagyi GN. Commericalized multi-gene predictors of clinical outcome in breast cancer. Oncologist. 2008;13:477-493.

32. OncotypeDx MammaPrint Provider/Cost Genomic Health, Inc. $3,647 Agendia, BRV $4,166 Starting Material FFPE Fresh mRNA and FFPE Number of Genes 21 70 Rank of Importance of Pathways Assessed 1 Proliferation 2 ER 3 HER2 1 Proliferation 2 ER 3 HER2 Current Indication Node-Negative ER-Positive Node Negative ER-Positive/Negative Eligible Patient Age Older patients Young and Old Patients Prognostic vs Predictive Prognostic and Predictive Prognostic and Predictive Outcome Prediction Continuous Dichotomous ASCO Biomarkers Guidelines Status Recommended for use Under Investigation Cost Effectiveness Validation Cost-effective in one published study Not reported FDA Status Not submitted Cleared at 510k level Comparison of OncotypeDxTMand MammaprintTM

33. Tissue Requirements for DNA Sequencing • DNA yield from FNAs may not always be satisfactory • Needle core biopsies preferred whenever possible • DNA is typically extracted from FFPE cellblocks from fluids and FNAs • Genotyping DNA in routine practice • EGFR for gefitinib/erlotinib in NSCLC • KRAS for cetuximab in CRC • BRAF for metastatic melanoma • NGS can be performed on FFPE samples • Currently requires 50 ng of DNA (approximately 10,000 cells) • Likely will require less (? 20 ng) as technology improves • Enrichment rarely needed for samples with <20% tumoral DNA present on clinical sample

34. Traditional DNA Sequencing Methods Ross J, Cronin M. Am J ClinPathol, 2010.

35. Next Generation Sequencing • The Human Genome sequence using original Sanger sequencing techniques was completed in 12 years at a cost of $ 3 Billion • The rate-limiting step in Sanger sequencing is the need to separate randomly terminated DNA polymers by gel electrophoresis • NGS bypasses this by physically arraying DNA molecules on solid surfaces and determining the DNA sequence in situ enabled by the development of reversible (chemically or enzymatically) DNA chain terminators • The DNA sequence is determined by measuring which bases are added into an elongating DNA chain, physically anchored to a glass slide or array of beads • In the next few years, the cost of NGS will continue to decrease rapidly and a clinically relevant gene sequence for a tumor may approach $1,000 cost range with turn-around time of only several days

36. Comparison of Traditional (Sanger) Sequencing and NGS Ross J, Cronin M. Am J ClinPathol, 2010.

37. Next Generation DNA Sequencing Platforms Ross J, Cronin M. Am J ClinPathol, 2010.

38. EGFR Mutation Testing in NSCLC • Primary used to predict response to anti-EGFR tyrosine kinase small molecule drugs (erlotinib/gefitinib) • No standardized test with multiple methods of gene sequencing in clinical use • Currently, standard of care for EGFR-mutated lung cancer patients should be first-line TKI, using either gefitinib or erlotinib • EGFR mutation correlates with gene amplification • Does not predict the response to anti-EGFR antibodies (cetuximab, panitumumab)

39. EGFR Activating Mutation – NSCLC • Mutation: EGFR_c.2573T>G_p.L858R • Freq=32%, depth=53 • 79 year old white female • FNA of lung mass: NSCLC FNA sample cytocentrifuged and converted to an FFPE tissue block. Very small numbers of viable tumor cells. Extensively necrotic.

40. KRAS Mutation Testing in CRC • Issues • KRAS mutated in 27-53% of CRC • Mutated KRAS identifies 50% of cetuximab/panitumumab resistant CRC • RT-PCR method more sensitive than direct sequencing • ASCO Opinion • - All patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumor tested for KRAS mutations in a CLIA-accredited laboratory • - If KRAS mutation in codon 12 or 13 is detected, then patients with metastatic colorectal carcinoma should not receive anti-EGFR antibody therapy as part of their treatment

41. Pivotal KRAS Data from Randomized Clinical Trials Ross J. Arch Pathol Lab Med, 2011

42. Established Techniques for KRAS Genotyping Ross J. Arch Pathol Lab Med, 2011

43. Biomarkers Linked to Cetuximab/Panitumumab Resistance in CRC Ross J. Biomarkers in Med., 2011

44. KRAS Mutation – CRC • Mutation: KRAS_c.35G>T_p.G12V • Freq=30%, depth=283 • 52 year old white male • KRASG12V mutation by “hot-spot” genotyping at Commercial Laboratory • pT3 pN2 pMx CRC Classic CRC with origin from mucosal surface at lower right

45. Novel ALK Fusion in CRC Detected by NGS pT4pN1pM1 MucinousAdenocarcinoma associated with a serrated sessile polyp A 5,194,955-bp tandem duplication generates an in-frame C2orf44-ALK gene fusion RNA sequencing shows an 89.8-fold increase in expression of ALK beginning at exon 20 relative to exons 1–19. The RNA sequence of the C2orf44-ALK gene fusion shows aberrant splicing Lipson et al. Nature Med, Feb, 2012

46. Vemurafenib in BRAF Mutated Melanoma • Vemurafenibinhibits V600E mutated BRAF tyrosine kinase • BRAF mutation occurs in 50-60% of melanomas • Vemurafenibspecifically blocks the activity of the mutated but not wild-type BRAF mutation and causes apoptosis •  Phase I escalating dose study of 16 BRAF V600E mutated metastatic melanoma patients presented at ASCO 2009 • Of the 16 treated melanoma patients, 9 had partial responses , 6 had stable disease and one had progressive disease as their best responses. • None of 5 non-mutated melanoma patients also treated with vemurafenibhad any responses. • ASCO ‘10: 1/29 BRAF-mutated CRC cases showed a partial response to vemurafenib

47. BRAF V600K Mutation – Metastatic MM • Mutation: BRAF_c.1798_1799GT>AA_p.V600K • Freq=10%, depth=416 • 77 year old white male • Thick melanoma of back • Multiple posterior cervical lymph nodes positive for metastatic melanoma Metastatic Melanoma to a cervical lymph node

48. Molecular Pathology for Clinical Management for Major Solid Tumors

49. Targeted Therapies for Cancer Molecular profiling is driving many new targeted cancer therapeutics • ~500 compounds hitting ~140 targets in development • Growing number of newly identified potential targets Subset of analyzed targets listed; data from BioCentury Online Intelligence Database