Routine EWS Fusion Analysis in the Oncology Clinic to Identify Cancer-Specific Peptide Sequence Patterns That Span Breakpoints in Ewing Sarcoma and DSRCT

SIMPLE SUMMARY: EWS-based fusions are aberrantly fused genes that drive Ewing sarcoma and desmoplastic small round cell tumor (DSRCT). Fusion data may be useful for personalized mRNA vaccines but are not yet routinely obtained in clinical practice. We present our workflow for the characterization of EWS driver fusions in a real-world pediatric oncology setting. We use rapid targeted sequencing of the breakpoint and genetic analysis to determine fusion sequences. We report amino acid fusion sequences from the EWS gene and the fusion partner gene (FLI1, ERG, FEV, WT1). Our workflow allows easy discernment of clinically relevant similarities and differences of EWS fusions. This simple analysis allows an understanding of molecular features of driver fusions underlying Ewing sarcoma or DSRCT in a real-world setting. This workflow is being utilized to obtain fusion neoantigen data used in “personalized” cancer vaccine trials under designs that seek to stimulate an immune response against Ewing sarcoma or DSRCT. ABSTRACT: (1) Background: EWS fusion genes are associated with Ewing sarcoma and other Ewing family tumors including desmoplastic small round tumor, DSRCT. We utilize a clinical genomics workflow to reveal real-world frequencies of EWS fusion events, cataloging events that are similar, or divergent at the EWS breakpoint. (2) Methods: EWS fusion events from our next-generation sequencing panel (NGS) samples were first sorted by breakpoint or fusion junctions to map out the frequency of breakpoints. Fusion results were illustrated as in-frame fusion peptides involving EWS and a partner gene. (3) Results: From 2471 patient pool samples for fusion analysis at the Cleveland Clinic Molecular Pathology Laboratory, we identified 182 fusion samples evolved with the EWS gene. They are clustered in several breakpoints: chr22:29683123 (65.9%), and chr22:29688595 (2.7%). About 3/4 of Ewing sarcoma and DSRCT tumors have an identical EWS breakpoint motif at Exon 7 (SQQSSSYGQQ-) fused to a specific part of FLI1 (NPSYDSVRRG or-SSLLAYNTSS), ERG (NLPYEPPRRS), FEV (NPVGDGLFKD) or WT1 (SEKPYQCDFK). Our method also worked with Caris transcriptome data, too. Our primary clinical utility is to use this information to identify neoantigens for therapeutic purposes. (4) Conclusions and future perspectives: our method allows interpretation of what peptides result from the in-frame translation of EWS fusion junctions. These sequences, coupled with HLA-peptide binding data, are used to identify potential sequences of cancer-specific immunogenic peptides for Ewing sarcoma or DSRCT patients. This information may also be useful for immune monitoring (e.g., circulating T-cells with fusion-peptide specificity) to detect vaccine candidates, responses, or residual disease.