Abstract 4545: Integrative immune-genomic comparison of canonical Ewing sarcoma with Ewing-like mimics identifies potential targets for personalized therapies

Introduction: Ewing sarcoma (ES) is one of the most common types of small round cell sarcoma (SRCS) from the skeletal origin, characterized by small round blue cell morphology and FET-ETS fusions. However, recent NGS-based technology has dramatically increased the characterization of other SRCS entities that share morphological, pathological, and clinical similarity with canonical ES. Currently, these sarcomas are often treated with similar regimens derived from ES. Whether any subsets of these sarcomas might benefit from personalized anti-cancer strategies remains largely unknown. Methods: To explore potential therapeutic vulnerabilities, 51 FFPE samples from 49 patients (median age 31 years, 65.3% male) diagnosed as ES or its mimics by conventional pathology review were sent to DNA (OncoScreen®Plus) and RNA sequencing (OncoRNA, Burning Rock Biotech) for genomic and immune signature profiling. Tumor samples were classified into 5 groups based on their similarity with classic ES: (a) Canonical ES (n=12), defined as ES with EWSR1-FLI1fusion and a primary bone origin; (b) Non-canonical ES, which were found with either non-EWSR1-FLI1 FET-ETS fusion, or ES in extra-skeletal sites (n=11); (c) Non-FET-ETS fusion SRCS (n=10), defined as SRCS with gene fusions other than FET-ETS family; (d) Fusion-negative SRCS (n=4); and (e) non-SRCS, where the diagnosis was primarily suspected as ES but finally re-diagnosed as another histology (n=12). Results: The NGS-based diagnosis rectified the pathological diagnosis in 9/51 samples, including ES misdiagnosed as other (n=3), other misdiagnosed as ES (n=4), Non-FET-ETS fusion SRCS misdiagnosed as ES (n=1) and ALK fusion sarcoma misdiagnosed as SRCS (n=1). In the remaining 42 samples, 23 diagnoses were confirmed by both NGS and FISH, while 11 SRCS were negative in both NGS and FISH assay, yielding a concordance of 81.0% (34/42). In the 8 samples with discordant diagnosis, a definitive diagnosis was achieved by FISH but not NGS in 1 of 8 (12.5%), and vice versa in 7 of 8 (87.5%). Interestingly, genomic profiling revealed that Non-FET-ETS fusion SRCS had high frequencies of TP53 mutations (40%) and copy number loss (including 30% in CD274 and JAK2; 20% in PTCH1, CDKN2A/B, and MTAP) and featured higher levels of homologous recombination deficiency (HRD) (p=0.024) and chromosome instability (p=0.022) than other groups. Remarkably, non-canonical ES demonstrated upregulated PDCD1 expression (p=0.034). The distribution of immune hot