Department of Pathology and Laboratory Medicine Albany Medical College Albany, NY

1. Massively Parallel Sequencing in NSCLC: Comparison to Traditional Hot Spot Analysis for Selection of Approved and Novel Targeted Therapies JS Ross, A Parker, M Jarosz, S Downing, R Yelensky, D Lipson, P Stephens, G Palmer, M Cronin, CE Sheehan Department of Pathology and Laboratory Medicine Albany Medical College Albany, NY Foundation Medicine, Inc. Cambridge, MA

2. Background (1) • Next Generation DNA Sequencing (NGS) has recently been applied to FFPE cancer biopsies and major resections (Ross JS et al. J ClinOncol 29: 2011) • Current Hot-Spot Genotyping only detects: • Mutations restricted to specific exons and codons • NGS detects: • Whole exome mutations in numerous cancer related genes • Insertions and deletions • Translocations and fusions • Copy number alterations (amplifications)

3. Background (2) • Recently, biomarker testing has emerged as a major driver of the selection of therapy for non-small cell lung cancer (NSCLC) • Currently, “hot-spot” DNA sequencing and FISH are used to select therapies for NSCLC: • EGFR genotyping for tyrosine kinase inhibitor (erlotinib) • EML4:ALK translocation testing for crizotinib • The emergence of comprehensive genomic profiling by NGS has led investigators to question whether more thorough gene sequencing techniques could discover potential targets for the treatment of relapsed and metastatic NSCLC not currently searched for in current routine practice

4. 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

5. Design (1) • DNA was extracted from 4 x 10 m FFPE sections from 49 primary NSCLC (28 female; 21 male; mean age 68 years; 24% Stage I; 13% Stage II; 5% Stage III; 16% Stage IV; 46% Stage unknown) • The exons of 145 cancer-related genes were fully sequenced using the IlluminaHiSeq 2000 (Illumina, San Diego, CA) and evaluated for point mutations, insertions/deletions (indels), specific genomic rearrangements and copy number alterations (CNA) • A total of 606,676-bp content was sequenced and selected using solution phase hybridization, to an average coverage of 229×, with 84% of exons being sequenced at ≥100× coverage • This assay captures and sequences 2,574 coding exons representing 145 cancer-relevant genes (genes that are associated with cancer-related pathways, targeted therapy or prognosis), plus 37 introns from 14 genes that are frequently rearranged in cancer

6. Design (2) • To maximize mutation-detection sensitivity in heterogeneous NSCLC biopsies, the test was validated to detect base substitutions at a ≥10% mutant allele frequency with ≥99% sensitivity and to detect indels at a ≥20% mutant allele frequency with ≥95% sensitivity, with a false discovery rate of <1% • Samples included 5% fluid cell blocks; 5% regional lymph nodes; 3% pericardial biopsy and 87% lung biopsies or resections • There were 46 adenocarcinomas (34 acinar, 19 lepidic, 2 mucinous, 1 papillary), 1 large cell carcinoma, and 2 squamous cell carcinomas • Results were compared with commercial laboratory allele-specific PCR genotyping on the same tissue blocks

7. Cancer Genome Profiling Workflow <14-21 days

8. Increasing Coverage To 500x Allows For >99% Sensitivity To Detect Mutant Alleles >5%, With No False Positive Mutation Calls Sensitivity vs Allele Frequency at 500X Coverage (1Mb panel) 10% 5% • Deep coverage is required for clinical grade samples

9. Lower Coverage Misses Relevant Mutations

10. Genomic Alteration Categories Category A:  Approved / standard alterations that predict sensitivity or resistance to approved / standard therapies Category B:  Alterations that are inclusion or exclusion criteria for specific experimental therapies Category C: Alterations with limited evidence that predict sensitivity or resistance to standard or experimental therapies Category D: Alterations with prognostic or diagnostic utility Category E: Alterations with clear biological significance in cancer (i.e. driver mutations) without clear clinical implications Highly Actionable “Page 1” Actionable in Principle “Page 2” Prognostic “Page 3” Biologically Significant “Page 4”

11. Initial Cohort Results (1) • For EGFR status, the NGS result was concordant with commercial laboratory genotyping in 23/23 (100%) cases • In 22/23 (96%) NSCLC samples, NGS revealed 53 total genomic alterations, including : • 14 (64%) base substitutions • 2 (9%) INDELs • 6 (27%) CNA • 0 (0%) rearrangements • Genomic alterations associated with sensitivity or resistance to targeted therapies for NSCLC were found in 16/22 (73%) of cases including: • KRAS 4 STK11 3 JAK2 2 PIK3CA • BRAF 2 EGFR 1 NF1 1 TSC1 1 TSC2 1 CCNE1 1 PTCH 1 CDK4 1 CCND1 1 BRCA2 1 CDKN2A 1 ATM

12. Initial Cohort Results (2) • In comparison with the COSMIC database, NGS results were similar for most genes except for • a lower rate of EGFR mutations (9% vs. 21%) • a higher rate of KRAS mutations (41% vs. 16%) • an unprecedented rate of JAK2 mutations (14% vs. 0%) • 7/22 (32%) of the NSCLC had 2 or more potentially actionable alterations after NGS

13. NSCLC: Actionable Genomic Alterations Cetuximab/Panitum resist. Gefitinib, Erlotinib, others MEK inhibitors (sens. and resist.)/Vemurafenib resist. mTOR inhibitors PI3 kinase, mTOR inhibitors Vemurafenibsens./Cetuximab resist. CDK4/6 inhibitors DNMT inhibitors JAK2 inhibitors PARP inhibitors Dasatinib mTOR inhibitors MEK/ERK inhibitors CDK inhibitors CDK4/6 inhibitors Notch inhibitors Nutlins FGFR inhib PARP inhib Tubulins. Genes with Actionable Alterations Genes with Alterations, Actionability Unknown

14. Multiple ‘Potentially Actionable Alterations in a Single Sample NSCLC sample SM92 BRAFc.1397G>T p.G466V PIK3CA c.1035T>A p.N345K CDK4Gene amplification MDM2 Gene amplification JAK2 c.1849G>T p.V617F NSCLC sample SM87 EGFRGene amplification MDM2 Gene amplification NSCLC sample SM51 BRAFc.1397G>C p.G466A STK11 c. 493G>T p.E165* KRASc.34G>T p.G12C

15. Expanded Cohort Results: Initial Gene Rearrangement Detected

16. Novel RET:KIF 5B Rearrangement in NSCLC (11.3Mb Pericentric Inversion) KIF5B RET Break Break KIF5B-RET ATG ATG • Translation 32,316,377 bps 43,611,118 bps Kinesin Coiled coil Tyrosine kinase • KIF5B(exons 1—15)RET(exons 12—20) ATG ATG ATG RET-KIF5B KIF5B-RET • Novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET in lung adenocarcinoma Lipson et al. Nature Med, Feb, 2012

17. KIF5B-RET Transformed Cells are sensitive to Multi-Targeted Kinase Inhibitors • Sunitinib, but not gefitinib inhibited RET phosphorylation • KIF5B-RET expression in Ba/F3 cells led to oncogenic transformation • Cells were sensitive to sunitinib, sorafenib, and vandetanib • Not sensitive to gefitinib • Hypothesis: RET kinase inhibitors should be tested in prospective trials for therapeutic benefit in NSCLC patients with KIF5B-RET rearrangements

18. Expanded Cohort NSCLC Gene Rearrangements Identified by NGS • EML4:ALK • In FISH + tumors • In FISH – tumors • RET:KIF5B

19. Percentage Of Samples With Actionable Alterations Across Major Tissue Types (224 Total Cases) • 71% cases carried ≥1 plausibly actionable alterations • 32 % cases carried ≥2 plausibly actionable alterations N=94 N=76 N=31 N=29 N=24

20. Comparison of NGS with Traditional Hot-Spot Genotyping in NSCLC, CRC, Breast Cancer and Melanoma Also Detected by Hot-Spot Genotyping N = 111

21. Novel Genomic Alterations* Discovered in NSCLC by NGS in an Expanded Cohort * Novel alterations discovered in tumor cell (somatic) sequence only as determined by comparison with the COSMIC database. Gene variants of undetermined significance which may represent germline variants are not included in this list.

22. NSCLC EGFR Activating Mutation • Sample: SM58 • Mutation: EGFR_c.2573T>G_p.L858R • Freq=32%, depth=53 • 79 year old white female non-smoker • FNA of lung mass: NSCLC • FNA sample cytocentrifuged and converted to an FFPE section • Very small numbers of viable tumor cells • Extensive tumor cell necrosis • Genotyping by allele-specific PCR showed identical activating EGFR mutation

23. Acquired Resistance to EGFR-TKI Sensitivity to gefitinib and erlotinib Resistance to gefitinib and erlotinib Nutlins • By NGS, the resistance clone was seen in 6% of cells and the sensitizing mutation in 25%

24. NSCLC: JAK2 Mutation Detected by NGS Low power of pleural biopsy positive for adenocarcinoma • Sample: SM86 • Mutation: JAK2_c.1849G>T_p.V617F • Freq=4%, depth=205 • 77 year old white female • Lung adenocarcinoma diagnosed by pleural biopsy • Patient diagnosed with polycythemiavera c.1849G>T p.V617F High power view shows adenocarcinoma of the lung. Rare capillaries not blood filled. No nucleated RBC or blasts seen. G T A T G T G T C T G T G G A ValCys Val Cys Gly

25. Multiple CNAs in Adenosquamous Carcinoma • Sample: SM92 • Mutation: CDK4 amp (6.6x), MDM2 amp (3.3x) • 77 year-old white male • Left lower lobe • Adenosquamous Carcinoma (composite tumor) • pT2 pN0 pMx CDK4 MDM2 Low power view of lobectomy specimen High power view of tumor with adenocarcinoma glands to the left and squamous carcinoma to the right

26. Conclusions • Deep sequencing (NGS) of clinical NSCLC samples is completely concordant with traditional hot-spot genotyping • NGS uncovers an unexpected number of genomic alterations that could influence therapy selection for NSCLC • Broad-based, deep sequencing of cancer-related genes results in sensitive detection of all classes of genomic alterations in NSCLC and can reveal actionable genomic abnormalities that inform treatment decisions