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MAF file for copy number variations identified in the cohort

Treatment history for patients enrolled on the PancSeq protocol.

Supplemental Tables and Figures

PancSeq protocol gene list for CLIA-certified somatic and germline analysis from whole exome sequencing data.

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

We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic.Significance:FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355

Characteristics of biopsies obtained on PancSeq protocol.

We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic.Significance:FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355

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

Background: BTCs are aggressive cancers with a poor prognosis. In preclinical models, MEK inhibition modulates the tumor immune microenvironment and enhances responses to programmed death-ligand 1 (PD-L1) inhibition. We report a randomized, open-label, multicenter phase 2 trial of atezolizumab (anti-PD-L1) as monotherapy or in combination with cobimetinib (MEK inhibitor) in BTC (NCT03201458). Methods: Eligible patients had advanced BTC [intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer (GBC)], with 1-2 lines of prior therapy in the metastatic setting, measurable disease by RECIST v1.1, and ECOG performance status ≤1. Patients randomized to Arm A received atezolizumab 840 mg IV Q2w. Patients randomized to Arm B received oral cobimetinib 60 mg daily (21 days on/7 days off) plus atezolizumab 840 mg IV Q2w. The primary endpoint was progression free survival (PFS) using the Kaplan-Meier method and compared between groups under the assumption of Cox proportional hazards, stratified for primary tumor site. Secondary endpoints included objective response rate (ORR), safety and tolerability, and overall survival (OS). Results: 86 patients were enrolled at 23 centers in the United States; 77 patients were randomized and received at least one dose of study therapy (Arm A: n=37, ICC=21, ECC=7, GBC=11; Arm B: n=38, ICC=22, ECC=8, GBC=8). Median age was 63 (range 44-86), and 48 (62%) were female. The trial met its primary endpoint, with a median PFS of 3.65 months (cobimetinib+atezolizumab) vs 1.87 months (atezolizumab monotherapy) (p=0.027). OS data are not mature at the time of analysis. There was 1 PR (3.2%), 13 SD (41.9%), and 17 PD (54.8%) in the combination arm and 1 PR (2.9%), 10 SD (29.4%), and 23 PD (67.6%) in the atezolizumab monotherapy arm. Two patients in the combination arm remain on therapy 15+ months from enrollment. One patient in each treatment arm had known mismatch repair deficiency (MMRd), of whom 1 had PD as a best response and the other withdrew prior to response evaluation. Grade 3-4 treatment-related adverse events were similar in both arms, and there were no treatment-related deaths. 4 (10%) of patients receiving atezolizumab monotherapy and 8 (22.2%) receiving cobimetinib+atezolizumab discontinued therapy due to adverse events. Changes in tumor CD8, CD4, FoxP3, PDL1, and MHC expression from paired tumor biopsies will be presented at the conference. Conclusions: We report the first randomized trial of immunotherapy in BTC. The combination of atezolizumab plus cobimetinib met its primary endpoint and significantly prolonged PFS as compared to atezolizumab monotherapy in BTC. The combination of atezolizumab and cobimetinib had manageable toxicity and warrants further investigation in BTC. Citation Format: Mark Yarchoan, Leslie Cope, Robert A. Anders, Anne Noonan, Laura W. Goff, Lipika Goyal, Jill Lacy, Daneng Li, Anuj Patel, Aiwu R. He, Ghassan Abou-Alfa, Kristen Spencer, Edward Kim, Stephanie Xavier, Amanda Ruggieri, S. Lindsey Davis, Autumn McRee, Paul Kunk, Qingfeng Zhu, Andrea Wang-Gillam, Andrew Poklepovic, Helen Chen, Elad Sharon, Gregory B. Lesinski, Nilo Azad. A multicenter randomized phase 2 trial of atezolizumab as monotherapy or in combination with cobimetinib in biliary tract cancers (BTCs): A NCI Experimental Therapeutics Clinical Trials Network (ETCTN) study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT043.

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