San Antonio Breast Cancer Symposium 2016 San Antonio Breast Cancer Symposium – December 6-10, 2016 Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer (MBC) San Antonio Breast Cancer Symposium 2016 Ofir Cohen, Dewey Kim, Jorge Buendia, Esha Jain, Seth Wander, Coyin Oh, Ada Waks, Nelly Oliver, Karla Helvie, Lori Marini, Asaf Rotem, Max Lloyd, Dan Stover, Victor Adalsteinsson, Samuel Freeman, Gavin Ha, Carrie Cibulskis, Kristin Anderka, Cory Johannessen, Ian Krop, Levi Garraway, Eric Winer, Nancy Lin, Nikhil Wagle This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

Resistant ER+ Metastatic Breast Cancer (MBC) Background: San Antonio Breast Cancer Symposium – December 6-10, 2016 Resistant ER+ Metastatic Breast Cancer (MBC) In spite of tremendous advances in the treatment of ER- positive breast cancer, patients frequently develop resistance to these therapies These resistant tumors remain the most common cause of breast cancer death, yet mechanisms by which this resistance develops are poorly understood There is an urgent need to develop new therapies for patients who no longer respond to these therapies. Knowledge of the molecular and genomic landscape of ER+ metastatic breast cancer is underexplored ESR1 LBD mutations Metastatic breast cancer is breast cancer that has spread beyond the tissues of the breast. An estimated 150,000 women and men in the U.S. are living with metastatic breast cancer. The median survival is about 3 years – and more than 40,000 people in the U.S. die from metastatic breast cancer each year, accounting for 14% of all cancer deaths among women in the U.S. Though treatments for metastatic breast cancer are improving, there is currently no cure for metastatic breast cancer. Breast cancers that express the estrogen receptor (ER+ breast cancers) account for the vast majority of cancer cases. In spite of tremendous advances in the treatment of ER+ breast cancer using therapies directed against the estrogen receptor (ER), patients frequently develop resistance to these therapies. These resistant tumors remain the most common cause of breast cancer death, yet mechanisms by which this resistance develops are poorly understood. One of the reasons for this is that our knowledge of the molecular and genomic landscape of resistant ER+ MBC remains underexplored. Much more work is required to fully understand all of the clinically relevant resistance mechanisms in breast cancer patients treated with ER-directed therapies. Moreover, there is an urgent need to develop new therapies for patients who no longer respond to these therapies. Until recently, most studies of breast cancer tumor samples have studied primary, treatment-naïve breast cancers (such as The Cancer Genome Atlas project, TCGA). However, mortality and morbidity is largely the result of metastatic and drug resistance tumors. This represents a clinically important population of patients that is largely uncharacterized by comprehensive exome and transcriptome sequencing. Our current research is part of a growing effort by many researchers to start closing the gap by better understanding the genomic underpinning of the metastatic and resistance states. Toy et al., 2013

San Antonio Breast Cancer Symposium – December 6-10, 2016 Objectives GOAL: to improve our understanding of resistant ER+ metastatic breast cancer by using next-generation sequencing technology to directly characterize tumor samples from patients who have developed resistant MBC What is the genomic landscape of ER+ metastatic breast cancer following therapies that target the ER? What are the evolutionary changes that can occur between the initial ER+ primary breast cancer and the resistant metastatic breast cancer? How can we integrate DNA and RNA sequencing into our understanding of metastatic breast cancer and resistance? In order to make advances in our understanding of metastatic breast cancer, we need to be able to study tumor specimens from patients, ideally specimens that are linked to clinical information. The goal of this research is to improve our understanding of resistant ER+ breast cancer by using cutting-edge genomic technology to directly characterize tumor samples from patients who have developed resistant breast cancer. Specifically, we are asking the following questions: What is the genomic landscape of ER+ metastatic breast cancer following therapies that target the ER? What are the evolutionary changes that can occur between the initial ER+ primary breast cancer and the resistant metastatic breast cancer? How can we integrate DNA and RNA sequencing into our understanding of metastatic breast cancer and resistance? This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

Study design and genomic methods San Antonio Breast Cancer Symposium – December 6-10, 2016 Study design and genomic methods 149 metastatic biopsies with ER+ metastatic breast cancer 44 matched primary tumors 64 biopsies collected 2006-2015 85 biopsies collected 2015 Nearly all patients had received at least 1 ER-directed therapy (tamoxifen, AI, fulvestrant) prior to biopsy Whole Exome Sequencing (WES) of tumor and germline 149 metastatic biopsies, 44 matched primary tumors Transcriptome Sequencing (RNA-seq) of tumor 128 metastatic biopsies 44 matched from 40 patients. The overarching goal of this work is to utilize genomic studies to improve our understanding of endocrine-resistant, ER+ metastatic breast cancer. To accomplish this, we have used massively parallel sequencing (also known as “next-generation sequencing”) to sequence the whole exome (the genes encoding all of the proteins in the cancer cell) and transcriptome (all of the genetic messages in the cell that direct the expressions of proteins) of from breast cancer samples from patients with resistant, ER+ metastatic breast cancer. This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

San Antonio Breast Cancer Symposium – December 6-10, 2016 Results: San Antonio Breast Cancer Symposium – December 6-10, 2016 Mutational landscape of ER+ MBC - Significant genes with SNV and indel alterations These are the significant recurrently mutated genes in the MBC cohort Explain plot Many of the genes we see here as significantly mutated are also found to be significantly mutated in primary ER+ breast cancer – for example TP53, GATA3, PIK3CA [Animations with arrows outside these specific genes] We also see genes on this -- additional novel genes are being prioritized by integration with functional genomic screens from our group and (and collaborators) and others N=141 This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

San Antonio Breast Cancer Symposium – December 6-10, 2016 Copy Number landscape of ER+ MBC – Significant Amplification and Deletion peaks San Antonio Breast Cancer Symposium – December 6-10, 2016 Amplifications Deletions ARID1A (1q) FOXP1 (3q) FGFR1 (8p) CDKN2A (9q) MYC (8q) PTEN (10q) FGFR2 (10q) CCND1 (11q) RB1 (13q) FOXA1 (14q) IGF1R (15q) MAP2K4 (17p) ERBB2 (17q) SMAD4 (18q) q.value (significance) q.value (significance) N=149 This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

Acquired Mutations in ER+ MBC San Antonio Breast Cancer Symposium – December 6-10, 2016 ESR1 ESR1 mutations in 24% 92% (12/13) in metastatic samples with matched primaries were acquired. ERBB2 ERBB2 mutations in 8% 83% (5/6) in metastatic samples with matched primaries were acquired. ESR1 – resistance to aromatase inhibitors ERBB2 – ongoing functional work from our group suggests this may also be aresistance mechanism to drugs that target the estrogen receptor. There are several agents in clinical development that can target these mutations RB1 – experimental data suggests that these mutations cause resistance to existing endocrine therapies, but also new CDK4/6 inhibitors RB1 RB1 mutations in 6% 60% (3/5) in metastatic samples with matched primaries were acquired. This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

DFCI Center for Cancer Precision Medicine: Research Biopsies from Patients with ER+ MBC San Antonio Breast Cancer Symposium – December 6-10, 2016 Returned to Physician for Clinical Decision Making Pathology ER, PR, HER2 CLIA Targeted Sequencing Panel Patients with ER+ MBC Whole Exome and Transcriptome Sequencing Studies of Resistance /Discovery of New Targets Metastatic tumor biopsy & blood samples Whole Exome Sequencing cfDNA from serial plasma More than 250 biopsies to date 10-20 new biopsies / month Patients followed longitudinally, with serial blood biopsies every 2 months This study is part of large and integrative effort to better understand the molecular underpinning of ER+ resistance in breast cancer. We will continue to characterize tumor samples from patients with resistant ER+ metastatic breast cancer, including, when possible, multiple samples obtained serially from patients as their therapies change over time. This project also includes both systematic functional studies of resistance and additional ongoing efforts to characterize the patients’ tumors with advanced technologies – namely using blood biopsies for cell free DNA analysis. Ultimately, the goal of this collaborative effort is to integrate the functional and clinical findings into a unified “Resistance Atlas” for ER+ metastatic breast cancer. Cell Line Models and Mouse Models Experimental Studies Additional Molecular Studies and Banking Future Uses This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.

San Antonio Breast Cancer Symposium – December 6-10, 2016 Conclusions & Summary: San Antonio Breast Cancer Symposium – December 6-10, 2016 The mutational landscape of resistant ER+ MBC differs from ER+ primary breast cancer For many genes, including ESR1, ERBB2, PIK3CA, AKT1, and RB1, these alterations can be acquired in the metastatic setting. Experimental data suggests these may represent resistance mechanisms to endocrine therapy Implications for patients: Multiple clinically relevant alterations are identified in metastatic biopsies, with implications for choice of next therapy, clinical trial eligibility, and novel drug targets — highlighting the value of genomic profiling of metastatic biopsies or blood biopsies Implications for drug development: The enrichment of clinically relevant and targetable mutations in the metastatic setting suggests new therapeutic strategies to help overcome or prevent resistance This presentation is the intellectual property of Ofir Cohen. Contact them at (ofirc@broadinstitute.org) for permission to reprint and/or distribute.