Molecular characterization of patients with localized Ewing sarcoma targeting discrete prognostic groups: A report from the Children’s Oncology Group.
Authors: Gillani Riaz , Shulman David Stephen , Klega Kelly S. , DelRocco Natalie J. , Krailo Mark D. , Han Ruxu , Slack Jonathan , Bainer Victoria , Armstrong Jessica , Church Alanna J. , Crompton Brian D. , Janeway Katherine A. 
Published: June 2024

11509

Background: Ewing sarcoma (EWS) is an aggressive bone and soft tissue cancer that primarily affects adolescents and young adults. All patients receive intensive therapy, yet 20% of patients with localized disease die and survivors are left with significant morbidity. Molecular biomarkers are needed to inform risk-stratified approaches to therapy. We thus evaluated gene fusions, copy number alterations (CNAs), mutations, immunohistochemistry (IHC), and relapse risk in a large cohort of patients with localized EWS. Methods: We carried out targeted sequencing on a combination of Formalin-Fixed Paraffin-Embedded (FFPE) and frozen tumor specimens from 351 patients with EWS previously enrolled on cooperative group trials (AEWS0031, AEWS1031). Gene fusions were identified using a combination of a targeted DNA assay and an RNA fusion panel. CNAs of chromosome 1q gain, 8 gain, 12 gain, and 16q loss were ascertained using ultra-low pass whole-genome sequencing (~0.1X coverage). Deleterious STAG2 and TP53mutations were identified as part of the targeted DNA assay. Tissue was stained for STAG2 and scored as percent 0, 1+, 2+ and 3+ staining. Tumors with ≥50% 2+ or 3+ were considered as “expressed”, those with ≥50% 0 or a deleterious gene alteration were considered “loss of expression”, and those that fit neither criterion were “indeterminate.” Cumulative incidence of relapse was used as the outcome measure to account for the competing risks of death and secondary malignant neoplasm. Results: 282 of 351 cases (80.3%) were found to have a canonical EWS fusion of EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV, or EWSR1-FEV. 247 cases (87.6%) were evaluable for CNAs, with 20.6% of cases with 1q gain, 50.6% with 8 gain, 21.1% with 12 gain, 17.0% with 16q loss. In univariate analyses, chromosome 1q gain trended toward a higher cumulative incidence of relapse (5-year cumulative incidence of relapse 35%, 95% C.I. [23%,49%] vs. 21%, [16%,28%]; Gray’s test p = 0.063). 277 cases (98.2%) were evaluable for mutations, with 5.1% of cases with TP53 mutations and 7.6% of cases with STAG2 mutations. In univariate analyses, STAG2 mutation was associated with higher cumulative incidence of relapse (53%, [29%,73%] vs. 21%, [16%,26%]; p < 0.001). TP53 mutation was also associated with worse outcome (43%, [17%,67%] vs. 22%, [17%,27%]; p = 0.039). 169 cases (59.9%) were evaluable for STAG2 IHC, and STAG2 loss was associated with higher cumulative incidence of relapse (32%, [20%,45%] vs. 16%, [9.6%,25%]; p = 0.004). Conclusions: TP53 mutation, STAG2 mutation, and STAG2 loss by IHC/ mutation are prognostic among patients with primary localized EWS. We will use these data to develop a framework for molecular risk stratification in localized EWS. Integration of these findings in future clinical trials may allow testing of biologically informed treatment modification in patients with localized EWS.