Novel and established EWSR1 gene fusions and associations identified by next-generation sequencing and fluorescence in-situ hybridization.

EWSR1 is a 'promiscuous' gene that can fuse with many different partner genes in phenotypically identical tumors or partner with the same genes in morphologically and behaviorally different neoplasms. Our study set out to examine the EWSR1 fusions identified at our institution over a 3-year period, using various methods, their association with specific entities and possible detection of novel partners and associations. Sixty-three consecutive cases investigated for EWSR1 gene fusions between 2015 and 2018 at our institution were included in this study. Fusions were identified by either break-apart fluorescence in-situ hybridization (FISH), our clinical RNA-based assay for fusion transcript detection or both. Twenty-eight cases were concurrently tested by FISH and NGS, 24 were tested by FISH alone and 11 by NGS alone. Of the 28 cases with dual testing, 24 were positive by both assays for an EWSR1 gene fusion, 3 cases were discordant with a positive FISH assay and a negative NGS assay, and 1 case was discordant with a negative FISH assay but a positive NGS assay. Three novel fusions were identified: a complex rearrangement involving three genes (EWSR1/RBFOX2/ERG) in Ewing sarcoma, a EWSR1/TCF7L2 fusion in a colon adenocarcinoma, and a EWSR1/TFEB fusion in a translocation-associated renal cell carcinoma. Both colonic adenocarcinoma and renal cell carcinoma had not been previously associated with EWSR1 rearrangements to our knowledge. In a subset of cases, detection of a specific partner had an impact on the histological diagnosis and patient management. In our experience, the use of a targeted NGS-based fusion assay is superior to EWSR1 break-apart FISH for the detection of known and novel EWSR1 rearrangements and fusion partners, particularly given the emerging understanding that distinct fusion partners result in different diseases with distinct prognostic and therapeutic implications.