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

1. Comprehensive Genomic Profiling of Breast Cancer By Massively Parallel Sequencing Reveals New Routes To Targeted Therapies JS Ross, CE Sheehan A Parker, M Jarosz, S Downing, R Yelensky, D Lipson, P Stephens, G Palmer, M Cronin Department of Pathology and Laboratory Medicine Albany Medical College Albany, NY Foundation Medicine, Inc. Cambridge, MA

2. 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

3. 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)

4. Background (2) • Biomarker testing has driven the selection of therapy for breast cancer for longer than any other solid tumor • ER/PR testing introduced in 1971 • HER2 testing/trastuzumab approved in 1998 • OncotypeDxTM mRNA profiling in 2004 • Currently, “hot-spot” DNA sequencing is driving the selection of targeted therapies for other solid tumors, but not for breast cancer: • EGFR genotyping for tyrosine kinase inhibitor use in NSCLC in 2005 • KRAS genotyping for anti-EGFR antibody use in CRC in 2007 • BRAF genotyping for BRAF inhibitor use in melanoma in 2011 • 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 metastatic breast cancer not currently searched for in current routine practice

5. Design • DNA was extracted from 4 x 10 m FFPE sections from an initial study-set of 15 primary invasive ductal breast cancers • 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 253×, with 84% of exons being sequenced at ≥100× coverage • The NGS 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 • To maximize mutation-detection sensitivity in heterogeneous breast cancer specimens, 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%

6. Cancer Genome Profiling Workflow <14-21 days

7. 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

8. Lower Coverage Misses Relevant Mutations

9. 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”

10. Results (1) • 15/16 (94%) of the tumors revealed 33 total somatic genomic alterations • Mean of 2.2 alterations per tumor with a range of 0 to 4 alterations per sample • Standard of care alterations consisted of 3(19%) tumors with HER2 copy number increases • The NGS HER2 copy number measurements by NGS in the HER2 amplified cases averaged 80% of the counted HER2 copies on FISH assessment of the same tumor block • Genes co-amplified with HER2 included RARA

11. Results (2) • 10 (63%) of tumors harbored at least one alteration that potentially could have led to clinical trials of novel targeted therapies including copy number increases for: • IGF-1R in 2 (13%) tumors (IGF-1R inhibitors) • MDM2 in 1 (6%) tumor (nutlins) • CCND1 in 3 (19%) tumors (CDK inhibitors) • CCNE1 in 1 (6%) tumor (CDK inhibitors) • CDK4 in 1 (6%) tumor (CDK inhibitors) • FGF1R in 1 (6%) tumor (FGF1R inhibitors) • 5 (31%) of tumors had 1 or more PIK3CA mutations (PIK3CA and mTOR inhibitors) • 6 (38%) of tumors had alterations classically associated with adverse clinical outcome including: • TP53 and PTEN mutations • HER2 copy number increases.

12. Comprehensive Genomic Profiling of Breast Cancers (n=15) Standard-of-care Plausibly Actionable (in trials) Resistance/Negative Predictors

13. 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

14. “Long Tail” Of Genomic Alterations Highlights Potential Benefits Of Comprehensive Profiling in Breast Cancer *

15. Novel Genomic Alterations* Discovered in Breast Cancer 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.

16. HER2 Gene Copy Number Alteration Validation • Increased HER2gene 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. Clinical Dilemmas Potentially Resolved by NGS • ER IHC+ with lack of benefit for hormonal therapy • ESR1 Mutation truncates estradiol binding site of the ER receptor protein • “Functional assay” is negative • HER2 IHC 3+ and FISH- • Activating mutation in the HER2 gene increases HER2 mRNA and HER2 receptor protein levels • No copy number increase

18. Conclusions • Deep massively parallel DNA sequencing of clinical breast cancer samples uncovers an unexpectedly high frequency of genomic alterations that could influence therapy selection for breast cancer • Deep sequencing of genomic DNA can provide a broad cancer-related gene survey at a depth of coverage that provides sensitive detection for all classes of genomic alterations, and when applied to breast cancer patients can reveal actionable genomic abnormalities that inform treatment decisions.