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

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.

Abstract 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.

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.

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.

Details of sample according to histotypes.

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

Clinical and sample overview of Sargen and Sargen-ITA patients

Overview of SAR-GEN2016 and SAR-GEN_ITA prospective multicentric trials. A, Workflow of the clinical trials, illustrating the process from patient enrollment through sample collection, genomic profiling, and analysis, to the multidisciplinary discussion at the MTB for therapeutic decision-making, with excluded samples indicated at each stage. B, Combination of percent stacked barcharts and box plot, from left to right: number of samples for each sarcoma subtype, tissue type of the biopsy (fresh or FFPE), disease status at the moment of the surgical procedure (primary, recurrent, and metastasis), purity of the tumor calculated after sequencing, and sex and age of the samples. EW, Ewing sarcoma; NA, not available; OS, osteosarcoma. [A, Created in BioRender. Grieco, M. (2025) https://BioRender.com/c7w0fbz.]

Genomic landscape of the four sarcoma subtypes. A and B, Box plots show the distribution of TMB on the y-axis, in which each dot represents an individual tumor sample. The horizontal dotted line at 1 mutation per megabase (mut/Mb) denotes the threshold used to separate lowly and highly mutated tumors. A, TMB distribution across all tumor samples of osteosarcoma (OS), Ewing sarcoma (EW), RMS, and SS. Statistically significant difference was observed between OS and EW samples (two-tailed Wilcoxon test). B, TMB distribution stratified by disease status (primary, recurrent, and metastasis) within the EW cohort. Significant differences were observed between primary and recurrent tumors (two-tailed Wilcoxon test) and between primary and metastatic tumors (two-tailed Wilcoxon test). C–F, Bar plots summarizing the top 25 mutated genes for each sarcoma subtypes, based on all tumor samples and without stratification by disease status. Genes are listed on the left side of each plot, with the corresponding percentage of mutated samples shown on the right. The percentages represent the number of unique samples harboring a mutation divided by the total number of samples in each sarcoma subtype (OS = 53, EW = 39, RMS = 13, and SS = 5). The x-axis shows the total number of mutations identified per gene. Color code represents the type of variants. G, Summary of the SBS and small indel signatures, stratified by disease status for each tumor subtype. On the left, the signature names. Each dot represents a mutational signature. The color of the dot (N) indicates the number of samples in which the signature is present, whereas the dot size represents the percentage of samples carrying that signature within that cohort. Signatures are grouped horizontally based on their classification and vertically by disease. The color bar at the bottom indicates the disease status.

ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)

Genes detected to be involved in oncogenic pathways

Background:Wiskott-Aldrich Syndrome (WAS) is characterized by eczema, infections, and severe bleeding, but may also include autoimmunity and malignancy. Subjects with X-linked thrombocytopenia (XLT) can display a mild phenotype, although severe complications may occur at any age. WAS and XLT are caused by mutations of the WAS gene. However, retrospective studies have shown conflicting results about their genotype-phenotype correlation and their relative risk of complications. Methods:To evaluate the outcome of patients with WAS or XLT, since January 2004, patients with identified WAS mutations were enrolled in the WAS/XLT IPINet registry at diagnosis and annually evaluated until December 2018 by participating AIEOP-IPINet centers; data were prospectively collected by each participating center throughout a web-based centralized system and then retrieved from the registry for the analysis. This prospective study enrolled 117 patients (according to Zhu criteria, 92 were affected by WAS and 25 by XLT) with appropriate hematological features and documented WAS mutation. Findings:The median follow-up was 6 years (range 1-30 years), resulting in 1110 patient years. At diagnosis, only the patients with WAS presented invasive infections, such as sepsis, meningitis, cerebral abscesses, herpetic infections, and candida infections, while patients with XLT did not present invasive infections. The most common autoimmune manifestations in patients with WAS were hemolytic anemia (20%) and vasculitis (9.3%), inflammatory bowel disease (5%), arthritis (4%), nephropathy (2%), and coeliac disease (1%). Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) was performed in 71 (61%), autologous hematopoietic stem cell gene therapy (HSC-GT) in 10 (8.5%), splenectomy in 16 (14%) patients, while 26 patients (22%) received none of these therapies. The overall survival at 25-year follow-up was 75% for patients with WAS after HSCT. Considering the cut-off date year 2000, it improved to >80%. Patients with WAS treated by haploidentical HSCT with αβTCRT-/CD19 B-cell depletion or gene therapy showed 100% survival at 5 years. The overall survival rate at the 20-year follow-up of the 25 patients with XLT was 83% but with a cumulative incidence of 100% and 19% of infections and autoimmunity, respectively, at the 15-year follow-up. Interpretation:The evidence of the heterogeneity of WAS and XLT outcomes could be instrumental to draw updated recommendations for the management of the patients affected by these rare conditions. It would be desirable to expand the tools to estimate the risk of infectious and autoimmune events in patients with XLT and the impact of their treatment, including HSCT over time. Funding:This study was funded by the European Union-Next Generation EU-NRRP M6C2-Investment 2.1 Enhancement and strengthening of biomedical research in the NHS, Ministero della Salute (PNRR-MR1-2022-12376594). A.S., A.A., P.C., F.F., C.M.P., C.F., D.L., G.S., M.D., M.C., P.A., B.A., P.F. are part of the European Reference Network on Rare Primary Immunodeficiency, Autoinflammatory and Autoimmune Diseases (ERN-RITA, project 739543).

High-dose methotrexate (HD-MTX) infusions are commonly used to consolidate remission in children with acute lymphoblastic leukemia (ALL). We investigate the potential role of candidate polymorphisms in SLCO1B1 (rs4149056 and rs2306283), ABCB1 (rs1045642), ABCC2 (rs717620), ABCC3 (rs9895420), and ABCC4 (rs7317112) drug transporters genes on HD-MTX pharmacokinetics and patients' outcome (meant both as relapse and drug-related toxicities) in an Italian cohort of 204 ALL pediatric patients treated according to the AIEOP-BFM ALL 2009 protocol. TaqMan SNP genotyping assays determined patient's genotypes. Measurements of HD-MTX plasma concentration were available for 814 HD-MTX courses in 204 patients; MTX clearance was estimated by a two-compartmental linear pharmacokinetic model with first-order elimination and a Bayesian approach, via ADAPT. Independent contributions of age and ABCC4 SNP rs7317112 (A>G, intronic) on MTX clearance were detected in a multivariate analysis (p = 1.57 × 10-8 and p = 2.06 × 10-5, respectively), suggesting a delayed elimination of the drug in older patients and an accelerated one in carriers of the variant GG genotype. After multiple corrections, the association between ABCC2 SNP rs717620 (-24 C>T) and severe hematological toxicity was found (p < 0.005). Moreover, SLCO1B1 SNP rs4149056 (c.521T>C, p.V174A) affected patients' outcomes: carriers of the variant C allele presented a reduced risk of relapse compared to wild-type TT (hazard risk: 0.27, 95% confidence interval [CI]: 0.08-0.90, p = 0.037). Taken together, these data highlighted the importance of variants in drug transporters genes on HD-MTX disposition in the AIEOP-BFM ALL 2009 protocol consolidation phase, and their putative role as predictive markers of outcome.

ABSTRACTBackgroundSeveral studies have shown that the intensity of treatment in Ewing sarcoma has an impact on outcome. The present trial tested the non‐inferiority of intensive, shorter, induction chemotherapy (25 weeks total treatment time) compared to the standard treatment (37 weeks) in non‐metastatic Ewing sarcoma (ES) at onset.ProcedureThis national, multicenter, parallel, randomized, controlled, open‐label, non‐inferiority, phase III trial was conducted in 14 specialized hospitals in Italy. Patients aged 2‐40 years with newly diagnosed localized ES were randomized to receive four courses of induction therapy (one every 21 days) either with a standard arm (Arm A) or with an intensive arm (Arm B). For consolidation therapy, good responders (GRs) in Arm A received nine courses (37 weeks), while Arm B patients received five courses (25 weeks). Poor responders for both arms received four courses followed by high‐dose busulfan/melphalan + autologous stem cell rescue. Follow‐up was 5 years.ResultsIn the study period 2009–2018, 274 patients with ES at onset were screened, 248 were eligible, 15 refused randomization, and 233 were randomized (Arm A: 113; Arm B: 120). Median age was 14 years. Arm B was not inferior to Arm A: 5‐year EFS was 77.5% and 71.6%, respectively (HR vs. Arm A: 0.74, 90% CI: 0.49–1.14). GRs were 54.9% in Arm A and 62.5% in Arm B. Hematological, gastrointestinal, and cardiovascular Grade ≥3 toxicities had higher frequencies in Arm B.ConclusionsIntensive induction therapy showed non‐inferiority in 5‐year EFS when compared with the standard induction therapy. Higher toxicity was reported in Arm B with similar outcome, counterbalanced in GRs with a shorter treatment plan. ClinicalTrials.gov Identifier: NCT02063022.