(P 01) EXTRASKELETAL EWING SARCOMA OF THE EXTREMITIES AND TRUNK IN < 25 YEARS OLD PATIENTS: A RETROSPECTIVE ANALYSIS OF A MONO-INSTITUTIONAL SERIES.

Objective: Extra skeletal Ewing's sarcoma (EES) is a malignant tumour belonging to the undifferentiated round cell sarcoma tumours (USRCS). Although current international recommendations indicate that EES should be treated with the same principles as for bone Ewing Sarcoma (Bone-ES), the optimal treatment strategy of EES remains uncertain. We have retrospectively analysed the clinical features, treatments, and outcomes of patients aged minor of 25 years with EES treated in our centre. Moreover, we compared the genetic profile of EES and Bone-ES to highlight differences between these entities.

Methods: Nineteen EES patients (12-25 years), treated at the Istituto Ortopedico Rizzoli of Bologna (Italy), from 1988 to 2022, were identified. Diagnosis was reviewed according to the WHO classification 2020. All cases were evaluated for specific chromosomal translocations involving EWSR1 or FUS. The extra-skeletal origin was determined on radiological imagining. Patients with visceral or head-neck primary location were excluded. Approval by the Local Ethical Committee was obtained. The clinic-pathological variables analyzed include: sex, age, location, depth, size, synchronous metastasis, baseline serum lactate dehydrogenase, tumor’s initial presentation (primary, unplanned/recurrence), local treatment, surgical margins, systemic treatment, histopathological response after chemotherapy (according to the Bologna System). Overall survival (OS), and Event-free survival (EFS) were estimated by Kaplan-Meier. Univariate (log rank test) and multivariate analysis (cox proportional hazard model) were done to explore prognostic factors. We performed RNA sequencing in 30 Small Round Cell Sarcomas (SRCSs) including, 18 Ewing (12 Bone-ES and 6 EES), 9 CIC-DUX4-rearranged sarcomas (CDS), 3 BCOR::CCNB3 sarcoma (BCS). Paired-end libraries were synthetized using TruSeq RNA Library Prep Kit for Enrichment and loaded on NovaSeq 6000 platform (Illumina), and were aligned to the GRCh38 human genome by STAR algorithm. FeatureCounts tool and DEseq2 were used to assign aligned reads to genes and to perform normalization applying variance stabilization transformation (vst). Uniform Manifold Approximation and Projection (UMAP) for non-linear dimensionality reduction was employed for visualizing sarcoma groups in two dimensions based on 8000 genes with highest variance.

Results: Sixteen patients (84%) presented as a primary tumor, 3 (16%) a recurrent disease and 2 (10%) as “unplanned excision”. Five patients (26%) were metastatic at diagnosis. The cumulative 5-year OS rate was 67% (95%CI 39-83) (Figure 1). Size (HR=9 P=0.019) and tumor’s initial presentation (HR=12 p=0.013), resulted statistically correlated to OS (Figure 2A-B). Local recurrence occurred in 1 patient (5%), at 4 months from diagnosis; distant metastases occurred in 6 patients (32%), at a mean time of 21.5 months (95% CI 5-38): 4 cases in the lung (66%), 1 in lung and abdomen (17%), 1 in the bone (17%). The 5-year EFS rates was 52% (95% CI 28-72) (Figure 3A). Statistical evaluation revealed tumor’s initial presentation and size to be independent risk factors associated to EFS (HR=16 p=0.003 and HR=6 p=0.033, respectively) (Figure 3B-C). Histologic response to neoadjuvant chemotherapy (Ewing-like protocol) was analyzed with the Bologna System score. Overall, GR had a better survival (100% vs 50% of PR, p=0.1397). By dimensionality reduction using UMAP, the four SCRSs subtypes showed a clear separation, we did not observe differences in the transcriptional profile between EWSs arising in bone compared to soft tissue EWSs (Figure 4).

Conclusion: Recurrent/unplanned tumors and big dimension resulted to be independent prognostic factor related to OS and EFS. Based on the genes with highest variance, unsupervised analysis highlighted a similar transcriptional profile in EWS Sarcoma beside tumor’s tissue origin. This result supports the use of similar therapeutic approach among EWSs sarcomas.