Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in many cancers, including intrahepatic cholangiocarcinoma (ICC). The FGFR inhibitor BGJ398 displayed encouraging efficacy in patients with FGFR2 fusion-positive ICC in a phase II trial, but the durability of response was limited in some patients. Here, we report the molecular basis for acquired resistance to BGJ398 in three patients via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), primary tumors, and metastases. Serial analysis of cfDNA demonstrated multiple recurrent point mutations in the FGFR2 kinase domain at progression. Accordingly, biopsy of post-progression lesions and rapid autopsy revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance and was surmountable by structurally distinct FGFR inhibitors. Thus, polyclonal secondary FGFR2 mutations represent an important clinical resistance mechanism that may guide development of future therapeutic strategies.

ATP-competitive fi broblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated effi cacy in 4 patients with FGFR 2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profi les observed clinically for each inhibitor. Our fi ndings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefi t from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show effi cacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefi t in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.

Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in a broad range of cancers and occur in ~20% of ICCs. As seen with other targeted therapies, however, acquired resistance has limited the efficacy of selective FGFR kinase inhibitors such as BGJ398. In a phase II trial of patients with advanced refractory cholangiocarcinoma harboring an FGFR gene alteration, BGJ398 displayed an overall response rate of 22%, but the durability of response was short in some patients. We report the molecular basis of acquired resistance in 4 patients with advanced FGFR2-fusion positive ICC via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), the primary tumor, and metastases. Each patient enjoyed an initial response, but all subsequently progressed within 10 months. Serial analysis of cfDNA revealed multiple point mutations in the FGFR2 kinase domain at progression (Table 1). The gatekeeper mutation, p. V564F, sterically hinders drug binding and was identified in 3 of 4 patients. In patient #1, five different FGFR2 mutations were detected in the post-progression cfDNA but only one, p. K641R, was identified in the post-progression biopsy. A rapid autopsy was performed, and genomic characterization of 12 metastatic lesions revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance that was surmountable by structurally distinct FGFR inhibitors. Thus, our report provides the first genetic evidence of clinical acquired resistance to FGFR inhibitor therapy in patients and informs future strategies for detecting mechanisms of resistance and promoting more durable remissions. Clinical and Molecular Data on Patients with Advanced Refractory CCA treated with BGJ398Patient IDSexAge at Diagnosis (years)FGFR2 Fusion partnerMaximum ResponseProgression Free Survival on BGJ398 (months)Overall Survival since Diagnosis (months)FGFR2 Mutations on cfDNA at progression1F47OPTN28%3.632V564F, N549H, K641R, E565A, L617V2F59ZMYMY450%5.631V564F, N549H/K, E565A, K659M3F69SORBS168%12.650K659M, K714R4M43BICC137%7.418V564F Note: This abstract was not presented at the meeting. Citation Format: Lipika Goyal, Supriya K. Saha, Leah Y. Liu, Giulia Siravegna, Ignaty Leshchiner, Leanne G. Ahronian, Jochen K. Lennerz, Phuong Vu, Benedetta Mussolin, Stephanie Reyes, Pascal Furet, A. John Iafrate, Gad Getz, Diana G. Porta, Ralph Tiedt, Alberto Bardelli, Dejan Juric, Ryan B. Corcoran, Nabeel Bardeesy, Andrew X. Zhu. Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in FGFR2 fusion-positive cholangiocarcinoma patients [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 4114. doi:10.1158/1538-7445.AM2017-4114