Obrien et al., Imatinib Compared with Interferon and Low-Dose Cytarabine for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia.

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1 Clinical Cancer Genotyping Long Phi Le, MD/PhD Department of Pathology Diagnostic Molecular Pathology Laboratory Translational Research Laboratory Massachusetts General Hospital Boston, MA

2 The case for broad cancer genotyping Our current approach Next generation sequencing SOLiD/ Ion Torrent Pilot

3 The Old Paradigm in Cancer Treatment

4 The New Paradigm in Cancer Treatment

5 t(9;22) in CML Blast crisis Obrien et al., Imatinib Compared with Interferon and Low-Dose Cytarabine for Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia. N Engl J Med Well Cycle 4 Cycle 8 Cycle 12 Cycle 16 Cycle 20 Cycle 24 Cycle 28 Cycle 32 Cycle 36 Cycle 40 Cycle 44

6 HER2 Amplification in Breast Cancer FISH detects HER2amplification in 15-25% of ductal carcinomas Predicts responsiveness to Trastuzumab Romond EH et al., Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer. N Engl J Med 353:

7 Molecular Markers and Targeted Therapy EGFR: Erlotinib/ Gefitinib 20% Lung adenocarcinomas KRAS: Cetuximab resistance 36-50% Colon adenocarcinoma BRAF V600E: PLX % Melanoma ALK: Crizotinib 3-5% Lung adenocarcinoma BRAF 1799 T>A V600E

8 Targeted Molecular Therapy in Active MGH Trials DRUG MECHANISM OF ACTION ACTIVE MGH STUDIES Axitinib PDGFR, VEGFR Inhibitor 1 AZD-2171/Cediranib VEGFR Inhibitor 4 AZD-6244 MEK Inhbitor 1 Bevacizumab Anti-VEGF 15 BI-2536 Plk-1 Inhibitor 1 Cetuximab Anti-EGFR 6 Dasatinib Bcr-Abl, Src Kinase Inhibitor 2 Erlotinib hydrochloride EGFR Inhibitor 6 Enzastaurin hydrochloride PKC beta inhibitor 1 Gefitinib EGFR Inhibitor 4 Lapatinib ditosylate EGFR, HER2 Inhibitor 1 Neratinib EGFR, HER2 Inhibitor 1 PF MET Inhibitor 1 Rituximab Anti-CD20 3 Sirolimus mtor Inhibitor 1 Temsirolimus mtor Inhibitor 1 Trastuzumab Anti-HER2 1 CP Anti-IGF-1R 1 EXEL-7647 EGFR, HER2, EphB4, VEGFR Inhibitor 2 EXEL-2880 MET, VEGFR-2 Inhibitor 1 AZD-6244 MEK Inhibitor 1 Everolimus mtor, FKBP12 Inhibitor 1 Tandutinib PDGFR, KIT, Flt3 Inhibitor 1 Sorafenib Multi-kinase Inhibitor 2 Sunitinib Flt3, VEGFR, KIT, PDGFR Inhibitor 11 Vatalanib VEGFR-2, KIT, PDGFR Inhibitor 2 Volociximab Integrin Inhibitor 1

9 Metastatic Colorectal Cancer & KRAS Testing Based on systematic reviews of the relevant literature, 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. -Allegra et al. J Clin Oncol 2009

10 US Growth in Genetic Testing

11 Molecular Diagnostic Techniques Molecular Cytogenetics Microarrays PCR and QPCR Sequencing

12 More With Less Genomic TL ng/panel DNA TL ng/panel DNA

13 Considerations in Clinical Genotyping Platform Clinical Test Archived FFPE tissue Analytical sensitivity 2 week turnaround time Report in patient s record Performed in a CLIA lab Actionable Targets Predicts response/resistance Clinches diagnosis Yields prognosis Stratify patients for trials Adaptability for new targets Logistics Clinical patient coordinator Accessioning Automation Eventually test all tumors Other Economics Finances and billing Bioinformatics Potential for research

14 Distribution of KRAS Mutations COSMIC, Wellcome Trust Sanger Institute, 2010

15 SNaPshot Genotyping Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis ddntp loci of interest ddntp ddntp Electrophoretic Output Relative fluorescence A B C D E F Increasing molecular weight

16 MGH SNaPshot Assay Gene Amino Acid cdna Residue Gene Amino Acid cdna Residue AKT1 49G - E17 E17 KRAS G12-34G KRAS G12-35G APC R C KRAS G13-37G APC Q C KRAS G13-38G APC R C APC T1556fs* _4667insA NOTCH1 L T NOTCH1 L T BRAF V G BRAF V T NRAS G12-34G NRAS G12-35G CTNNB1 D32-94G NRAS G13-37G CTNNB1 D32-95A NRAS G13-38G CTNNB1 S33-98C NRAS Q61-181C CTNNB1 G34-101G NRAS Q61-182A CTNNB1 S37-109T NRAS Q61-183A CTNNB1 S37-110C CTNNB1 T41-121A PIK3CA R88-263G CTNNB1 T41-122C PIK3CA E G CTNNB1 S45-133T PIK3CA E G CTNNB1 S45-134C PIK3CA Q C PIK3CA Q A EGFR G G PIK3CA H C EGFR T C PIK3CA H A EGFR L T PIK3CA G G EGFR E746_A _2249del EGFR E746_A _2250del PTEN R C EGFR Exon 19 deletions PTEN R C ERBB2 Exon 20 insertions PTEN R C FLT3 D G PTEN K267fs*- 800delA IDH1 R C - TP53 R G IDH1 R G - TP53 G G TP53 R C JAK2 V G TP53 R G TP53 R C KIT D A TP53 R G TP53 R C

17 MGH SNaPshot v1.0 Genomic DNA NRAS38 BRAF1799 NRAS182 PI3K263 bcat95 bcat122 EGFR2235_49R TP Melanoma BRAF 1799 T>A V600E

18 Lung Cancers: SNaPshot TP53 5% AKT 1% BRAF 2% HER2 2% CTNNB1 2% ALK 3% PIK3CA 4% NRAS 1% IDH1 <1% No Mutation 42% EGFR 15% KRAS 23% N=650

19 Lung Cancers: Mutation Overlap PIK3CA KRAS 56 isolated (58 total) 1 B-cat TP T790M 5 1 EGFR 36 isolated (50 total) 2 1 BRAF NRAS ALK 13 APC

20 Colorectal Cancers: SNaPshot APC 4% BRAF 7% NRAS 3% No Mutation Identified 34% PIK3CA 6% TP53 21% KRAS 25% N=250

21 Colorectal Cancers: Mutation Overlap 3 PIK3CA 3 BRAF 6 isolated 1 KRAS 20 isolated (36 total) TP53 18 isolated (28 total) 6 APC NRAS 3

22 More Than Just Point Mutations

23 The Future of Clinical Cancer Genotyping Do we have the technology? Is it cost-effective? What to genotype? The challenges? By Angela Canada Hopkins

24 Next Generation Sequencing: The Big Brothers Roche 454 Illumina/Solexa Life Technology SOLiD Helicos

25 Next Generation Sequencing: Uno Tube, Mucho Data Next Generation Sequencing First Generation Sequencing

26 Ten Years of Sequencing Adapted from Nature 1 April 2010

27 Next Generation Sequencing: The Little Brothers Roche 454 GS Jr Illumina GA IIe Life Technology SOLiD PI Life Technology Ion Torrent

28 The Future of Clinical Cancer Genotyping Do we have the technology? Is it cost-effective? What to genotype? The challenges? By Angela Canada Hopkins

29 How Many Genes? Pre-Human Genome Project >100,000 genes Post-Human Genome Project 30,000 genes 20,000-25,000 Human exome ~180,000 exons 1% of the genome ~30Mb

30 Clinical Cancer Genotyping: A Mountain to Climb? Non-Silent Mutations in Pancreatic Cancer Bert Vogelstein: AACR 2010 Meeting Plenary Session Published Cancer Exomes 11 Colorectal Science Breast Science Pancreas Science Gliomas Science Leukemias NEJM, Nature Breast Nature Breast Nature Granulosa Cell NEJM Lung Nature Melanoma Nature Medulloblastomas - Unpublished Mutations per Tumor Mutations per Tumor Non-Silent Mutations in Different Tumors

31 Clinical Cancer Genotyping: Focus! Focus! Bert Vogelstein: AACR 2010 Meeting Plenary Session Review of Literature/Databases 116,432 human cancers 353 histopathologic subtypes 130,072 intragenic somatic mutations 3142 mutated genes Potential Driver Genes 286 tumor suppressor genes (>15% of mutations are truncating) 33 oncogenes (same codon mutated in at least 2 tumors) Mutations per Tumor Mutations per Tumor Genetic Alterations in Pancreatic Cancer Driver Gene Alterations in Pancreatic Cancer

32 A Little Math Desired Analytical Sensitivity 1-5% Typical NGS Error Rate 1-2% Whole Genome Sequencing 30x 1 error >3.3% sensitivity Targeted Sequencing x 0-4 errors in 200 reads 1%-2% error Set threshold at 5% Whole Genome Sequencing at 200x >$60,000!

33 Target Exon Capture Microdroplet PCR Solid-phase Capture Solution-phase Capture Reaction Array Metzker ML, Nature Reviews Genetics 2010

34 Cost of Next Generation Sequencing Library Preparation Cost Adapted from Nature 1 April 2010

35 The Future of Clinical Cancer Genotyping Do we have the technology? Is it cost-effective? What to genotype? The challenges? By Angela Canada Hopkins

36 SOLiD Sequencing Pilot Study on SNaPshot Samples PCR amplicons concatenated and sheared for barcoded fragment library construction. Samples pooled prior to emulsion PCR Emulsion PCR used to template each sample on to magnetic beads. Beads modified at 3 end to allow for deposition on to sequencing slide. 50 bp 5 bp BC Deposited slide loaded into one of two available flow cells on SOLiD instrument. 5 mer barcode sequenced first to parse each library Sequence 50 bases sequenced in the forward direction. Mapping and SNP calling performed in CLC Bio Genomics Workbench (ungapped local alignment in colorspace, SNP cutoff at 5%) Courtesy of Life Technologies

37 SOLiD Sequencing Pilot Results Case Total Reads % Mapped Minimum Coverage Maximum Coverage Average Coverage 8,551, ,380, ,700, ,487, ,447, ,424, ,788, ,748, ,260, SOLiD Next Generation Sequencing Variant Calls KRAS c.34g>t (30.1%) EGFR c.2235_2249del15 TP53 c.743g>t (26.0%) KRAS c.34g>a (16.4%) TP53 c.536a>t (10.4%) NRAS c.182a>g (47.7%) TP53 c.880g>t (63.3%) EGFR c.2240_2257del18 KRAS c.34g>c (63.3%) KRAS c.38g>a (22.6%) PIK3CA c.1633g>a (18.8%) TP53 c.818g>a (39.9%) BRAF c.1799t>a (22.1%) PIK3CA c.1636c>a (14.4%) EGFR c.2264c>a (7.4%) TP53 c.743g>a (2.3%) TP53 c.752t>g (1.9%) KRAS c.35g>t (12.0%) TP53 c.713g>t (20.9%) SNaPshot Single Base Extension Genotyping Results KRAS c.34g>t EGFR15 bpdel TP53 c.743g>t KRAS c.34g>a NRAS c.182a>g EGFR18 bpdel KRAS c.34g>c KRAS c.38g>a PIK3CA c.1633g>a TP53 c.818g>a BRAF c.1799t>a PIK3CA c.1636c>a TP53 c.743g>a KRAS c.35g>t

38 KRASExon2: Per Base Error & Coverage Per Base Coverage Average Per Base Error +/- Standard Deviation Per Base Error 95% Confidence Interval Circles = Outliers Outside Per Base Error 95% CI

39 KRASExon2: Per Base Error & Coverage

40 KRASExon2: Per Base Error & Coverage #5 #1 #4 #3

41 TP53 Exon 7: Detection of Compound Heterozygosity #9 #2 #8

42 Detection of Compound Heterozygosity

43 Detection of Compound Heterozygosity 468 mutations 10 mutations Sanger COSMIC Website

44 EGFR Deletion: Mapping Sample #1 Sample #4

45 Digital Genotyping and Structured Data Laboratory (genotype): CDS Variant aavariant Confidence Score BRAF c.1799g>t Allele Frequency BRAF V600E 95 45% (1) Oncogenic (2) Response to PLX4032 Relevance Prevalence Literature 2% Lung ACA 7%Colon ACA 60% Melanoma Flaherty et al. NEJM 2010 Variant Confidence Score = f(quality Value, Coverage, Error, Bi-directionality) Clinical (phenotype): Patient Diagnosis Stage Genotype Treatment Response Literature Jane Doe Metastatic melanoma III BRAF c.1799g>t Plexxikon PLX % Melanoma Flaherty et al. NEJM 2010

46 Clinical Cancer Genotyping: At The Tipping Point Clinicaltargeted sequencing of FFPE DNA initially 100 exons > X coverage Mb data 3-4 week turnaround time $500 raw reagent cost Desired Whole exon coverage Tumor vs. normal? Copy number? RNA expression? Rearrangements? Methylation?

47 Summary Cancer genetics is rapidly expanding with high complexity Molecular profiling will drive cancer management Continued need for higher-throughput cancer genotyping Next gen sequencing poses a huge informatics challenge Clinical next generation sequencing is coming is already here ^

48 MGH Molecular Diagnostics Dora Dias-Santagata Kathy Vernovsky Arjola Cosper Breton Roussel Kristin Bergethon Hannah Stubbs Vanessa Scialabba Sara Akhavanfard Quynh Lam Kenny Fan Jae Han MGH Cancer Center Daniel Haber David Louis Leif Ellisen Darrell Borger Ion Torrent/LifeTech Andrew Felton Jason Meyers Maneesh Jain Simon Cawley Stuart Davidson Mike Lelivelt ABI/LifeTech Kevin McKernan Rosemary Obrien Steve McLaughlin Clarence Lee Nancy Gangemi Timothy Harkins Jeremy Stuart Eric Tsung

49 Questions?