(P 429) SARCOMA FUSION GENE ANALYSIS YIELDS PROMISING HLA ANTIGEN FIT IN EWING SARCOMA AND DESMOPLASTIC SMALL ROUND CELL TUMOR

Objective: EWS gene fusions are cancer-specific events in both Ewing sarcoma and Desmoplastic Small round Cell Tumor (DSRCT). We sought to determine whether or not EWS sarcoma gene fusions code for potentially immunogenic peptides that span the fusion breakpoint by analyzing patterns of immunogenicity (common or uncommon) in the context of a Ewing sarcoma or DSRCT patient’s individual major histocompatibility (MHC) class I HLA-A, -B, and C type. Our overall objective was to identify potential candidate antigens to be coded for in EWS-fusion adenovirus and mRNA vaccines suitable for the majority of Ewing sarcoma and DSRCT patients.

Methods: EWS breakpoint and HLA data was obtained from Cleveland Clinic Molecular Pathology NGS laboratory and Caris Life Sciences (Irving, TX). In-frame analysis was performed to determine fusion DNA, RNA, and peptide sequences spanning the EWS breakpoint for each patient. Then, we looked for similarities and differences of peptides encoded by EWS-FLI1, EWS-ERG, EWS-FUS, and EWS-WT1 gene fusions. We then analyzed nested sets (27/HLA allele) of fusion peptides (9-11 mer) spanning the EWS-FLI1, EWS-ERG, EWS-FUS, and EWS-WT1 breakpoints in the context of individual predicted HLA binding of HLA-A, -B, and -C alleles from >45 patients with Ewing sarcoma and DSRCT. Patterns of excellent, good, and poor EWS-fusion peptide antigen fit were then characterized.

Results: : Almost every patient had at least one predicted EWS-fusion peptide that was predicted to be presented by MHC. Interestingly, after analysis of >45 patients, common patterns emerged for each EWS- fusion HLA-fit. EWS-FLI1 type 1 and EWS type 2 had 5 and 4 good fits, respectively. Less common EWS-FLI1 fusions also had good fits. EWS-ERG had 4 fusion antigens that appeared to be presented; EWS-WT1 had 3 breakpoint-spanning peptides that were predicted to bind MHC. Although 9-mer EWS fusion antigens were the most common, MHC also was predicted to bind some, 10- and 11 -mer peptides too. A total of 22 “best” candidate EWS-fusion peptides could be potential targets that would be relevant to> 80% of Ewing sarcoma and DSRCT patients. Validation using experimental means may be used to examine these candidates in more detail.

Conclusion: : A surprisingly small, but potentially useful subset of EWS-FLI1, EWS-ERG, EWS-FUS, and EWS-WT1 fusion peptides are predicted to be highly immunogenic in the context of individual class I HLA binding. This finding has guided the design of EWS vaccine constructs that should be relevant to the majority of high-risk Ewing sarcoma and all DSRCT patients. If our method is applied to other fusion-associated sarcomas, it will also likely yield potential vaccine information for common and uncommon sarcoma fusions including other round cell sarcomas such as PAX7-FOXO1 and PAX3-FOXO1 alveolar rhabdomyosarcoma, SS18-SSX synovial sarcoma, BCOR-CCNB3 sarcoma, and CIC-DUX sarcoma.