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MAF file for copy number variations identified in the cohort

Treatment history for patients enrolled on the PancSeq protocol.

Supplementary Figure S1: Overview of workflow and data generation. Supplementary Figure S2: Analysis of neoplastic cellularity. Supplementary Figure S3: Recurrent copy number alterations. Supplementary Figure S4: Mutational signature analysis. Supplementary Figure S5: Analysis of PDAC gene expression signatures. Supplementary Figure S6: Clinically relevant alterations in the cohort.

Supplementary Experimental Methods

MAF file for mutations identified in the cohort

This standard operating procedure was established by the Center for Cancer Genomics at Dana-Farber Cancer Institute, the Brigham and Women's Hospital and the Klarman Cell Observatory at the Broad Institute, to standardize the collection of fresh metastatic breast cancer biopsies and their allocation to various bulk and single cell assays, including whole exome and bulk RNA-sequencing, single-cell RNA sequencing, and spatial profiling of RNA and protein. The use of a well defined workflow has allowed us to generate high quality data from these different assays, by implementing efficient modes of communication, minimizing the time elapsed from sample collection to preservation or processing, and ensuring optimal transportation conditions. Visual Abstract

Seventy percent of breast cancers express the estrogen receptor (ER), and agents that target the ER are the mainstay of treatment. However, virtually all people with ER+ breast cancer develop resistance to ER-directed agents in the metastatic setting. Beyond mutations in the ER itself, which occur in 25–30% of people treated with aromatase inhibitors1–4, knowledge about clinical resistance mechanisms remains incomplete. We identified activating HER2 mutations in metastatic biopsies from eight patients with ER+ metastatic breast cancer who had developed resistance to aromatase inhibitors, tamoxifen or fulvestrant. Examination of treatment-naive primary tumors in five patients showed no evidence of pre-existing mutations in four of five patients, suggesting that these mutations were acquired under the selective pressure of ER-directed therapy. The HER2 mutations and ER mutations were mutually exclusive, suggesting a distinct mechanism of acquired resistance to ER-directed therapies. In vitro analysis confirmed that the HER2 mutations conferred estrogen independence as well as—in contrast to ER mutations—resistance to tamoxifen, fulvestrant and the CDK4 and CDK6 inhibitor palbociclib. Resistance was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib. Activating HER2 mutations are shown to confer resistance to ER-directed therapies in patients with ER+ metastatic breast cancer. Drug resistance caused by HER2 mutations was overcome by combining ER-directed therapy with a HER2 kinase inhibitor.

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC. Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096–111. ©2018 AACR. See related commentary by Collisson, p. 1062. This article is highlighted in the In This Issue feature, p. 1047

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing., Identifying the mutational landscape of tumours from cell-free DNA in the blood could help diagnostics in cancer. Here, the authors present ichorCNA, software that quantifies tumour content in cell free DNA, and they demonstrate that cell-free DNA whole-exome sequencing is concordant with metastatic tumour whole-exome sequencing.

BackgroundLittle is known about mechanisms of resistance to PARP inhibitors and platinum chemotherapy in patients with metastatic breast cancer andBRCA1/2 mutations. Further investigation of resistance in clinical cohorts may point to strategies to prevent or overcome treatment failure.Patients and MethodsWe obtained tumor biopsies from metastatic breast cancer patients withBRCA1/2deficiency before and after acquired resistance to PARP inhibitor or platinum chemotherapy. Whole exome sequencing was performed on each tumor, germline DNA, and circulating tumor DNA. Tumors underwent RNA sequencing, and immunohistochemical staining for RAD51 foci on tumor sections was performed for functional assessment of intact homologous recombination.ResultsPre- and post-resistance tumor samples were sequenced from 8 patients (4 withBRCA1and 4 withBRCA2mutation; 4 treated with PARP inhibitor and 4 with platinum). Following disease progression on DNA-damaging therapy, four patients (50%) acquired at least one somatic reversion alteration likely to result in functional BRCA1/2 protein detected by tumor or circulating tumor DNA sequencing. Two patients with germlineBRCA1deficiency acquired genomic alterations anticipated to restore homologous recombination through increased DNA end resection: loss ofTP53BP1in one patient and amplification ofMRE11Ain another. RAD51 foci were acquired post-resistance in all patients with genomic reversion, consistent with reconstitution of homologous recombination. All patients whose tumors demonstrated RAD51 foci post-resistance were intrinsically resistant to subsequent lines of DNA-damaging therapy.ConclusionsGenomic reversion inBRCA1/2was the most commonly observed mechanism of resistance, occurring in 4 of 8 patients. Novel sequence alterations leading to increased DNA end resection were seen in two patients, and may be targetable for therapeutic benefit. The presence of RAD51 foci by immunohistochemistry was consistent with BRCA1/2 protein functional status from genomic data and predicted response to later DNA-damaging therapy, supporting RAD51 focus formation as a clinically useful biomarker.

Seventy percent of breast cancers express the estrogen receptor (ER), and agents that target the ER are the mainstay of treatment. However, virtually all people with ER

Background: While great strides have been made in the treatment of ER+ MBC, therapeutic resistance is nearly universal. The genomic evolution of ER+ breast cancer in the metastatic setting under the selective pressure of multiple lines of therapies is not well understood. To address this, we analyzed the clonal dynamics of serial metastatic samples (mets) to evaluate how tumors evolve and to identify acquired resistance mechanisms. Methods: We performed WES on 462 clinically annotated samples from 208 patients (pts) with ER+ MBC, including 67 primary tumor biopsies, 229 metastatic biopsies and 160 blood samples (cfDNA). Pts with multiple mets included cases with temporally concordant metastatic tumor and blood samples (48 pts) and cases with serial mets obtained over the course of treatment in the metastatic setting (69 pts). Treatments given between the serial mets included CDK4/6 inhibitors (23 pts), and selective estrogen receptor degraders (19 pts), among others. Results: In the temporally-concordant mets, we found that cfDNA mutations (muts) largely overlap with muts found in tumor biopsies, capturing >85% of clonal tumor muts. However, we observed a higher level of heterogeneity in cfDNA compared to biopsies (p.value< 1.05e-19, Welch test) and a subset of high-confidence muts that were only detected in cfDNA, including in clinically important genes such as ESR1, PIK3CA, KRAS, and ERBB2. Analysis of serial mets was used to elucidate the evolutionary dynamics within the metastatic setting under the selective pressure of treatment. The median duration between mets was 112 days and the median number of inter-biopsy unique treatments was two. Most tumors continued to evolve within the metastatic setting, with 50 out of 69 pts (72%) acquiring a meaningful sub-clone (50% increase in relative cancer cell fraction) and 31 out of 69 (45%) acquiring muts in known cancer genes, including a subset acquiring a plausible resistance alteration such as alterations that dysregulate ER (5 out of 69 pts, 7%; ESR1 mut, FOXA1 amplification (amp), NCOR1 bi-allelic deletion (del)), ERBB (4%; ERBB2 amp, ERBB3 mut), RAS (4%; KRAS mut, NRAS amp, NF1 del), FGF/FGFR (12%; FGFR2 mut, FGFR1/2 amp, FGF3 amp), and cell cycle (13%; RB1 del, CDK4 amp, AURKA amp, CDKN2A del). Finally, in pts who had multiple mets, we observed several cases of evolutionary convergence toward equivalent resistance mechanisms including convergent RB1 loss as a mechanism of resistance to a CDK4/6 inhibitor and convergent BRCA2 reversion following resistance to a PARP inhibitor. Conclusions: This study demonstrates that ER+ MBC continues to evolve under the selective pressure of treatments in the metastatic setting. These findings elucidate the challenge of studying high complexity and heavily treated tumors, while also highlighting some commonalities in the evolutionary trajectories selected by these treatments. The multiplicity of clinically relevant genomic alterations acquired in these advanced stages highlights the need for serial biopsies and the potential to inform post-progression therapeutic choices through targeting the acquired dependencies in post-progression tumors. Citation Format: Cohen O, Buendia-Buendia J, Wander S, Nayar U, Mao P, Waks A, Kim D, Freeman S, Adalsteinsson V, Helvie K, Livitz D, Rosebrock D, Leshchiner I, Dellostritto L, Garrido-Castro A, Jain E, Periyasamy S, Mackichan C, Lloyd M, Marini L, Krop I, Garraway L, Getz G, Winer E, Lin N, Wagle N. Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-02.

Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor. Citation Format: Utthara Nayar, Ofir Cohen, Christian Kapstad, Adrienne Waks, Seth A. Wander, Corrie Painter, Samuel Freeman, Priyanka Ram, Nicole Persky, Lori Marini, Karla Helvie, Nelly Oliver, Cynthia X. Ma, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4952.

1054Background: HR+/HER2- primary breast tumors demonstrate less anti-tumor immune activity than HER2+ or triple-negative BC. However, minimal data exist about the TME of HR+/HER2- met tumors, part...

12016Background: Combining CDK4/6i with endocrine therapy results in prolongation of disease control in HR+ MBC, though resistance invariably occurs. There is a critical need to understand mechanis...

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth-leading cause of cancer-related death in the United States and is projected to become the second leading cause by 2030. Most patients present with advanced disease and die within 12 months of diagnosis. Recent genomic studies of primary pancreatic cancer resection specimens have identified several molecular alterations and genomic subtypes of the disease that may guide precision medicine approaches to clinical management. However, the molecular landscape of metastatic PDAC has been less well characterized. Moreover, biopsy-driven studies in metastatic PDAC have been historically very challenging due to the aggressive course of this disease as well as the low-volume and heterogeneous nature of biopsies that makes deep molecular characterization difficult. Insufficient genomic analysis of a patient’s tumor early in their disease course is a major barrier to enrollment on clinical trials of targeted therapies. To address these limitations, we have implemented a multi-disciplinary clinical and research biopsy protocol to enable real time comprehensive molecular characterization of metastatic PDAC biopsy specimens. We have performed core needle biopsies of metastatic lesions in the liver or peritoneal cavity in 42 patients at the time of initial presentation. A low rate of complications was observed, with only a single patient having a self-limited hemorrhagic complication after liver biopsy. On average, 4-6 separate biopsy specimens were collected from each patient for histopathology and genomic analysis. Whole exome sequencing (WES) was performed in a CLIA-certified laboratory and a comprehensive molecular report of somatic alterations and selected pathogenic germline variants was returned to the referring clinician with a typical turn-around time of 3-5 weeks. We observed a striking incidence of recurrent germline and somatic alterations in DNA-damage repair genes, such as BRCA2, ATM and CHEK2. We also observed alterations in genes with known therapeutic implications, such as BRAF, RNF43, STK11 and ROS, and in select cases, these results guided choice of second or third line therapy. In parallel to WES, we performed RNA sequencing on bulk tumor tissue and readily identified expression signatures defining multiple subtypes of tumor and stroma that may have prognostic or therapeutic implications for tumor- or stroma-directed therapies. Collectively, these results demonstrate the feasibility and value of real-time genomic characterization of metastatic PDAC and provide a path forward for improved stratification and enrollment of PDAC patients on molecularly defined clinical trials. Citation Format: Andrew J. Aguirre, Scott Carter, Nicholas Camarda, Arezou Ghazani, Jonathan Nowak, Annacarolina Da Silva, Lauren Brais, Dorisanne Ragon, Devin McCabe, Lori Marini, Kristin Anderka, Karla Helvie, Nelly Oliver, Ana Babic, Paul Shyn, Douglas Rubinson, Anuj Patel, James Cleary, Nadine McCleary, Matthew Kulke, Thomas Clancy, Leona Doyle, Jason Hornick, Christine Ardito-Abraham, Ruth Yu, Michael Downes, Ronald Evans, Richard A. Moffitt, Jen Jen Yeh, William C. Hahn, Charles Fuchs, Robert Mayer, Nikhil Wagle, David Tuveson, Levi A. Garraway, Brian M. Wolpin. Real-time genomic characterization of metastatic pancreatic cancer to enable precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2017-3036

Background: While great strides have been made in the treatment of estrogen receptor-positive (ER+) metastatic breast cancer (MBC), therapeutic resistance invariably occurs. A better understanding of the underlying resistance mechanisms is critical to enable durable control of this disease. Methods: We performed whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) on metastatic tumor biopsies from 88 patients with ER+ MBC who had developed resistance to one or more ER-directed therapies. For 27 of these patients, we sequenced the treatment-naïve primary tumors for comparison to the resistant specimens. Tumors were analyzed for point mutations, insertions/deletions, copy number alterations, translocations, and gene expression. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of all metastatic samples demonstrated several recurrently altered genes whose incidence differed significantly from primary, treatment-naïve ER+ breast cancers sequenced in the TCGA study (TCGA). These include ESR1 mutations (n=17, 19.3%; 32.86 fold enrichment, q.value Comparing to matched primary samples from the same patient, many alterations were found to be acquired in several cases, including for ESR1, ERBB2, PIK3CA, PTEN, RB1, AKT1, and others. Initial analysis of RNA-seq data from metastatic samples (n=59) allowed classification of individual resistance mechanisms into broader resistance modes based on the observed transcriptional state. Conclusions: We present a genomic landscape of resistant ER+ MBC using WES and RNA-seq. Multiple genes were recurrently altered in these tumors at significantly higher rates than in ER+ primary breast cancer. When compared with matched primary tumors from the same patient, alterations in these and other genes were often found to be acquired after treatment, suggesting a role in resistance to ER-directed therapies and/or metastasis. Potential resistance mechanisms appear to fall into several categories; integrating RNA-seq data may enhance the ability to identify these categories even when genomic alterations are not identified. Multiple clinically relevant genomic and molecular alterations are identified in metastatic biopsies– with implications for choice of next therapy, clinical trial eligibility, and novel drug targets. Citation Format: Cohen O, Kim D, Oh C, Waks A, Oliver N, Helvie K, Marini L, Rotem A, Lloyd M, Stover D, Adalsteinsson V, Freeman S, Ha G, Cibulskis C, Anderka K, Tamayo P, Johannessen C, Krop I, Garraway L, Winer E, Lin N, Wagle N. Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-01.

542Background: BRCA-d cancers are deficient in homologous recombination and susceptible to tx that cause DNA damage (platinum chemotx and poly (ADP-ribose) polymerase inhibitors (PARPi)). MoR to DN...