Detection of FGFR fusions in intrahepatic cholangiocarcinoma using targeted RNA sequencing.

Abstract

e16185 Background: Comprehensive sequencing efforts have identified fibroblast growth factor receptor (FGFR) translocations in numerous cancers. FGFR2 fusions were recently identified as an actionable therapeutic target in ̃17% of cholangiocarcinoma patients, predominantly found in patients with intrahepatic cholangiocarcinoma. Genomic partners for FGFR fusions are variable. In addition, obtaining high yield tissue biopsies for CCA remains challenging and tumor tissue available for molecular analyses is scarce. When combined, these two factors make detection of FGFR fusions in CCA challenging. An accurate molecular assay to detect FGFR fusions in CCA could improve utilization of FGFR-targeted therapies. Methods: The objective of our study was to develop an approach for targeted RNA analysis to improve sensitivity for FGFR fusion detection from limited tumor material. We adapted the sensitive targeted digital sequencing (TARDIS) approach recently developed in our lab for analysis of RNA. This approach utilizes a combination of open-ended targeted amplification followed by ligation, enabling detection of tyrosine kinase fusions without prior knowledge of the precise sequence of the fusion breakpoint or identity of the fusion partner. For evaluation of analytical performance, we analyzed RNA from a CCA organoid model with a known FGFR2 fusion, and Seraseq Fusion RNA Mix v3 Reference Material with two known FGFR3 fusions. Results: Without prior knowledge the partner or coordinates of the fusion breakpoint, we detected an FGFR2-KIF5C fusion in RNA from a CCA organoid model. The fusion breakpoint coordinates predicted by TARDIS-RNA were validated by comparison with RNA-Seq. To assess sensitivity and quantitative performance of the assay, we analyzed a serial dilution of the reference RNA sample from Seraseq with concentration of RNA molecules representing candidate fusions verified by digital PCR. Using TARDIS-RNA to analyze replicates with 5 ng RNA input, we were able to detect candidate fusions represented by as few as 2.7 fusion molecules on average. Concentrations of fusion molecules measured using the two methods were highly correlated (R = 0.96). Conclusions: These results demonstrate sensitivity and quantitative performance of a targeted RNA sequencing assay to detect and quantify FGFR fusions in tumor specimen from intrahepatic cholangiocarcinoma. On-going work is focused on further evaluating assay performance and characterizing FGFR fusions using additional tissue specimen.