Polyclonal Secondary FGFR2 Mutations Drive Acquired Resistance to FGFR Inhibition in Patients with FGFR2 Fusion-Positive Cholangiocarcinoma

Abstract

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 intralesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation led 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 the development of future therapeutic strategies.Significance: We report the first genetic mechanisms of clinical acquired resistance to FGFR inhibition in patients with FGFR2 fusion-positive ICC. Our findings can inform future strategies for detecting resistance mechanisms and inducing more durable remissions in ICC and in the wide variety of cancers where the FGFR pathway is being explored as a therapeutic target. Cancer Discov; 7(3); 252-63. ©2016 AACR.See related commentary by Smyth et al., p. 248This article is highlighted in the In This Issue feature, p. 235.

Figure 1. Clinical acquired resistance to BGJ398

A-C. Left panels. Axial contrast enhanced CT images in the portal venous phase demonstrating initial response lesions that subsequently progressed despite BGJ398 therapy. Inset shows higher magnification of responsive/resistant lesion. FGFR2-related genetic events detected by Solid Fusion Assay (SFA), RNA sequencing, whole-exome sequencing (WES), or the Guardant360 assay are documented in the text below CT images. Right upper panels, Graphs illustrating serial CA 19-9 serum levels and tumor volume measurements by RECIST v1.1 criteria for Patients 1-3, respectively. The pink box indicates time while the patient was receiving BGJ398. Right-lower panels, Percent allele burden in cfDNA of the indicated mutations from Patient #1 (A), Patient #2 (B), and Patient #3 (C) were monitored over time. The pink box indicates time while the patient was receiving BGJ398. N.D. = not detected.

Figure 2. FGFR point mutations confer resistance to BGJ398 and other FGFR inhibitors

A. In silico model of BGJ398 in binding pocket of FGFR2, demonstrating steric clash in the context of a V564F mutation. B and C. Mutagenesis screen in BaF3 cells which were engineered to express a TEL-FGFR3 fusion protein and subjected to increasing doses of BGJ398. The bar graph in B indicates the number of BGJ398-resistant clones isolated with indicated FGFR3 point mutations, with higher doses of BGJ398 resulting exclusively in colonies harboring the V555M gatekeeper mutation. Proliferation was quantified in C after 3 days with Alamar blue. Corresponding FGFR2 amino acids are indicated after the ‘/’. D. IC50 values for BaF3 cells expressing the indicated constructs and treated with a variety of inhibitors. E and F. Proliferation assays with BaF3 cells expressing the FGFR2 V564F constructs and treated with increasing doses of either BGJ398 (E) or LY287445 (F). G. Phospho-FGFR2 ELISA demonstrating that FGFR2 V564F is resistant to inhibition by BGJ398 compared to wild-type.

Figure 3. Rapid autopsy reveals inter-lesional and intra-lesional heterogeneity of resistance

A. Axial contrast-enhanced CT images and autopsy photographs of seven liver metastases from Patient #'2. B. Heatmap illustrating mutations detected in the indicated autopsy lesions. Three distinct FGFR2 point mutations and four distinct PTEN mutations were identified. C and D. Corresponding images of Liver Met #1 (C) and Liver Met #2 (D) taken from Patient #2's autopsy including heatmaps indicating mutations identified in eight spatially distinct pieces isolated from Responsive Liver Met#1 and eight spatially distinct pieces isolated from Resistant Liver Met #2.