Molecular characterization of osteosarcoma and Ewing sarcoma in pediatric patients reveals novel RNA-based intervention points

Ewing sarcoma (EW) and osteosarcoma (OS) are the two most common bone sarcomas (BS) in the pediatric population. BS are heterogeneous diseases characterized by distinct clinical and pathologic features. The high metastasis recurrence and extremely poor clinical prognosis pose the urgent need for new treatment strategies for these young patients. Within the national clinical trial SAR_GEN-ITA (ClinicalTrial.gov id:NCT04621201), we performed whole exome and total RNA sequencing of 28 and 18 OS and EW samples, respectively, from pediatric patients. The analysis of somatic mutations and copy number alterations confirmed the low mutational burden and high genomic instability of these BSs. Differential expression analysis revealed a significant overexpression of genes that contribute to epithelial to mesenchymal transition and are targets of KRAS in OS and EW samples, respectively. Using available expression data of osteoblast samples as controls, we identified 4,025 and 3,890 differentially alternative splicing (AS) events in OS and EW respectively. By analysis of trans- and cis-acting elements, we identified splicing factors, such as SRSF1 and U2AF1, responsible for the somatic rewiring of AS lanscape of BS. Amongst other oncogenic and prognostic splicing events, the inclusion of the nonsense mediated-determinant micro-exon 30 of the cancer gene FLNA emerged as the strongest candidate in both OS and EW samples. Furthermore, since AS events are a source of neoantigens to a greater extent than single point mutations, we found 454 novel junctions that gave rise to 2,755 neoepitopes, most of them being private to patients. Together, our findings revealed novel candidate intervention points for lowly mutated pediatric BS tumors that could be exploited with RNA-based approaches and immunotherapies.