Caterina Parlato

Sarcomas in pediatric and adolescent–young adult (AYA) populations represent rare and biologically heterogeneous tumors with complex genetic underpinnings. Genomic profiling reveals subtype-specific alterations and therapeutic targets. Such tumors still represent an unmet clinical need due to limited treatment options and poorer outcomes, especially in advanced stages. Here, we present the SAR-GEN2016 and SAR-GEN_ITA clinical trials, conducted across 12 Italian centers, which enrolled 201 patients, including 158 bone and soft-tissue sarcoma samples collected at diagnosis or relapse. Whole-exome sequencing was successfully performed on 120 tumor samples. The most representative histotypes were osteosarcoma (n = 53), Ewing sarcoma (n = 39), rhabdomyosarcoma (n = 13), and synovial sarcoma (n = 5), and the genomic analyses were mainly focused on these subtypes. Overall, our cohort showed genomic differences between subtypes, highlighting how genomic complex sarcomas and fusion-driven sarcomas are distinct entities. The genomic complex histotypes, such as osteosarcoma, were characterized by a lower tumor mutational burden (TMB) and higher copy-number variation burden with enrichment of the CN2 signature. Recurrent and metastatic Ewing sarcomas have a higher TMB compared with treatment-naïve primary tumors, along with increased intratumoral heterogeneity. Oncogenic pathway analyses revealed dysregulation of the RTK–RAS and NOTCH pathways across subtypes, particularly in metastatic and recurrent tumors. In 71 of 120 analyzed samples (59%), at least one potentially actionable genomic alteration was identified, and 16% of those patients with relapsed disease received a matched targeted therapy based on the molecular profiling results. All findings were classified as ESCAT tier II or III. Our findings support the value of integrating genomic and clinical data to accelerate translational research in rare tumors.Significance:Pediatric and AYA sarcomas are rare with poor outcomes in advanced stages and limited treatment options. Through the SAR-GEN2016 and SAR-GEN_ITA multicenter trials, we performed whole-exome sequencing on 120 tumor samples with matched normal tissue from 158 patients with bone and soft-tissue sarcoma. Our integrative genomic analysis supports the genomic stratification and precision oncology in rare pediatric sarcomas.

Assessment of ITH using the Shannon diversity index score. Box plots show the distribution of ITH scores (y-axis), in which each dot corresponds to an individual tumor sample. The ITH scores were assessed across sarcoma subtypes, disease status, and tumor location, with meaningful differences observed in osteosarcoma (OS) and Ewing sarcoma (EW) but not in RMS or SS. ITH distribution stratified by (A) tumor location in OSs and (B) disease status in EWs.

Oncogenic signaling pathways. A–D, Mirrored circular bar plots depict the overall enrichment of oncogenic pathways in osteosarcoma (OS; A), Ewing sarcoma (EW; B), RMS (C), and SS (D). Each bar represents a single pathway, with the height indicating the proportion of samples harboring corresponding somatic alterations. Somatic variants are shown in yellow and CNVs in light blue, with the corresponding percentage of affected samples. E and F, The heatmaps provide a detailed view of (E) variants and (F) CNVs in genes of each pathway stratified by disease status and, grouped by disease subtype. Top color bar indicates disease status. Bar plots on the right represent the proportion of each alteration type across pathways, cells, and corresponding color code.

Five patients received a matched targeted therapy based on the molecular profiling results.

Details of sample according to histotypes.

Clinical and sample overview of Sargen and Sargen-ITA patients

ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)

Significant cytobands identified in osteosarcoma (primary and recurrent) and Ewing’s sarcoma (primary, recurrent, metastasis) with the respective genes

CNV across sarcoma subtypes. A, CNV burden is shown with a box plot highlighting significant differences across all tumor samples from osteosarcoma (OS), Ewing sarcoma (EW), RMS, and SS, without stratification by disease status. A statistically significant difference was observed between EW and OS samples (two-tailed Wilcoxon test). B–D, Recurrent and significant CNVs were identified in OS and EW samples using GISTIC2 for primary (B and C) and metastatic (D) tumors. The cytobands are visualized using the R package maftools. Blue bars indicate deletions, red bars indicate amplifications, and gray bars represent nonsignificant CNVs. The G-score represents the amplitude and frequency of the CNVs across tumors of interest. E, Distribution of CN signatures across the cohorts, stratified by disease status for each tumor subtype. Left, signature names. Each dot represents a mutational signature. The color of the dot indicates the number (N) of samples harboring the signature, whereas the size of the dot reflects the percentage of samples carrying that signature. Signatures are grouped horizontally based on their classification and vertically by disease. The color bar at the bottom indicates the disease status.