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 CollegeAlbany, NY

Foundation Medicine, Inc.Cambridge, MA

Targeted Therapies for Cancer

Molecular profiling is driving many new targeted cancer therapeutics

Subset of analyzed targets listed; data from BioCentury Online Intelligence Database

• ~500 compounds hitting ~140 targets in development

• Growing number of newly identified potential targets

Background (1)

• Next Generation DNA Sequencing (NGS) has recently been applied to FFPE cancer biopsies and major resections (Ross JS et al. J Clin Oncol 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)

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– Oncotype DxTM 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

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 Illumina HiSeq 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%

Cancer Genome Profiling Workflow

<14-21 days

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)

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Mutant Allele frequency spectrum of known mutations found in a series of clinical samples

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Genomic Alteration Categories

Highly Actionable“Page 1”

Actionable in Principle“Page 2”

Prognostic“Page 3”

Biologically Significant“Page 4”

Category A: Approved / standard alterations that predict sensitivity or resistance to approved / standard therapiesCategory 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

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

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.

Comprehensive Genomic Profiling of Breast Cancers (n=15)Tumor Sample

Number of Alerations

Known and Likely Somatic Non-Synonymous Mutations (mutant allele frequency, sequence coverage depth) and Copy Number Gains (fold change over normal) Potential Actionability

AB_1 0 NoneAB_3 1 CDH1:NM_004360:c.2436_2439delTGAAG:frameshift(0.39,250) None

AB_17 2 MEN1_c.207_207delC_p.D70fs*49(0.04,196), MEN1:NM_130801:c.1322G>A_p.W441*(0.24,202) None

AB_19 4 SMARCA4_c.805delC_p.M272fs*31(0.03,95),

TP53:NM_001126112:c.1028_1028delT:frameshift(0.24,365),IRS2_gain(9x), IGF1R_gain(6x)

IGF-1R Inhibitors

AB_33 1 PTEN_c.370T>A_p.C124S(0.76,452)

AB_35 2 PIK3CA_c.3140A>G_p.H1047R(0.42,455), TP53_c.332T>C_p.L111P(0.49,299) PIK3CA Inhibitors, mTOR Inhibitors

AB_49 1 TP53_c.809T>C_p.F270S(0.11,372) NoneAB_51 3 IGF1R_gain(18x), MDM2_gain(8x), CCND1_gain(4x) IGF-1R Inhibitors, Nutlins, CDK inhibitors

AB_65 2 PIK3CA_c.3140A>G_p.H1047R(0.26,300), TP53_c.488A>G_p.Y163C(0.28,232) PIK3CA Inhibitors, mTOR Inhibitors

AB_67 2 PIK3CA_c.3140A>G_p.H1047R(0.16,535), PIK3CA_c.316G>C_p.G106R(0.03,537) PIK3CA Inhibitors, mTOR Inhibitors

AB_81 2 ERBB2_gain(6x), CCNE1_gain(3x) Lapatinib, Trastuzumab, CDK Inhibitors

AB_83 1 CCND1_gain(4x) None

5014A 4 TP53_c.752T>G_p.I251S(0.34,489), ERBB2_gain(8x), MCL1_gain(5x), CDK4_gain(3x) Lapatinib, Trastuzumab, CDK inhibitors

5016A 4PIK3CA_c.3140A>T_p.H1047L(0.23,1549), TP53_c.396G>C_p.K132N(0.07,282),

PAK3:NM_001128166:c.414G>A_p.M138I(0.06,889), LRP1B:NM_018557:c.11762C>G_p.S3921*(0.23,1528)

PIK3CA Inhibitors, mTOR Inhibitors

5018A 3 PIK3CA_c.1616C>G_p.P539R(0.47,603), PIK3CA_c.3140A>G_p.H1047R(0.53,782),CCND1_gain(6x), FGFR1_gain(5x)

PIK3CA Inhibitors, mTOR Inhibitors, CDK inhibitors, FGFR1 inhibitors

Average: 2.2

Standard-of-care Plausibly Actionable (in trials) Resistance/Negative Predictors

Percentage Of Samples With Actionable Alterations Across Major Tissue Types (224 Total Cases)

N=94 N=76 N=31 N=29 N=24

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71% cases carried ≥1 plausibly actionable alterations32 % cases carried ≥2 plausibly actionable alterations

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

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Novel Genomic Alterations* Discovered in Breast Cancer by NGS in an Expanded Cohort

Total Number of Sequenced Breast Cancers

Total Number of Novel Alterations

Types of Novel Alterations

327 7 (2%) - Chromosomal Inversions (2)- In Frame Deletion (1)- Gene Truncation (1)- Chromosomal Rearrangement (1)- Tandem Duplications (2)

* 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.

ERBB2 RARA

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

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

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.