Identification of FGFR2/3 fusions from clinical cfDNA NGS using a de novo fusion caller.

3545 Background: FGFR2/ 3 rearrangements are promising therapeutic targets, especially in advanced urothelial cancer (aUC) with FDA-approved erdafitinib. Liquid biopsy is an attractive non-invasive method to identify these fusions, but detection in cfDNA is technically challenging due to low tumor shedding levels, short molecules, and wide variation in gene partners. To address this, we developed an assembly-based fusion detection algorithm to call rearrangements in a de novo fashion without reliance on a fixed partner set and applied it to > 15,000 clinical samples. Methods: A cohort of 15,218 patients with mixed cancer types (including 698 aUC patients, as well as breast, cholangiocarcinoma, colorectal, and gastric), plus 276 healthy control samples were previously tested with Guardant360(R), a clinical 74-gene cfDNA NGS-based assay. The median unique molecule coverage was approximately 3,000 molecules sequenced to 15,000x read depth. Samples were reanalyzed in silico using the novel algorithm: in brief, reads aligned to candidate fusion breakpoints were assembled into de Bruijn graphs. Resulting contigs were aligned to the reference and filters were applied to remove technical artifacts. Results: The majority of FGFR2 (86%) and FGFR3 fusion partners (73%) in the mixed cancer cohort were observed only once, consistent with previous reports (Helsten 2016). FGFR3-TACC3 was the most common fusion, occurring in 72% of FGFR3 fusion-positive patients. In 37% of FGFR2 fusion positive patients, the de novo caller detected partners not previously described. In the aUC cohort, FGFR3 fusions were detected in 3.3% of patients, with 8/10 (80%) partner genes/intergenic regions occurring only once, which is in line with previous reports (Nassar 2018). No fusions were identified in 276 healthy control samples. In the mixed cancer cohort, common mutations co-occurring with FGFR2 fusions were FGFR2 N549K, PIK3CA H1047R, and TP53 R175H (5.6% each); KRAS Q61H was observed in 28% of patients with FGFR3 fusions. Conclusions: FGFR2/ 3 fusion partners detected by a highly specific assembly-based de novo fusion caller were heterogeneous and individually rare, highlighting the importance of a de novo approach. We observed an FGFR3 fusion prevalence in cfDNA from aUC patients that is comparable to previous reports for tissue testing, demonstrating an ability to capture targetable genomic rearrangements with plasma-based NGS in this patient population.