BACKGROUND:Lung metastases in patients with metastatic rhabdomyosarcoma (RMS) have not been treated uniformly across Europe. This provides comparison of the impact of whole lung irradiation (WLI). METHODS:Lung-metastatic patients included in the Cooperative Weichteilsarkom Studiengruppe-IV 2002, European Pediatric Soft Tissue Sarcoma Study Group MTS 2008 or the Soft Tissue Sarcoma Registry received four- or six-drug chemotherapy, surgery, and/or irradiation (radiotherapy) of the primary tumor. Treatment of lung lesions consisted of metastasectomy, WLI, or no local treatment according to protocols. RESULTS:A total of 238 patients with lung-metastatic RMS were included. Half of these patients (n = 119/238) had lung metastases only (median age, 6.6 years), mainly classified as embryonal RMS (n = 93/119, 78%). Lung-only patients underwent metastasectomy (n = 17) and/or WLI (n = 31) or no local treatment of lung metastases (n = 71). Early complete response of lung metastases was associated with favorable 3-year overall survival (p = .009). A trend toward improved event-free survival after WLI could be identified in patients ≥10 years old (hazard ratio [HR], 2.7; 95% CI, 0.8-9.6), but not in patients under 10 years old (HR, 0.86; 95% CI, 0.44-1.66). Lung-relapse-free survival (lung-RFS) was not influenced by WLI or metastasectomy in the univariable and multivariable analyses. When analyzing all lung-metastatic patients (including those with metastases in other sites, n = 238), WLI in patients older than 10 years was a significant prognostic factor (HR, 2.2; 95% CI, 1.0-4.9). CONCLUSIONS:The analysis failed to show a significant benefit of WLI in patients with lung-metastatic RMS, apart from the subgroup of patients older than 10 years. Lung-RFS was not influenced by WLI.

Abstract Background Accurate subgrouping of medulloblastoma is critical for risk stratification and development of novel therapies. While DNA methylation profiling has advanced subgroup classification, functional kinase activity profiles may provide additional insights beyond (epi-)genetic data and reveal directly actionable therapeutic targets. Methods Serine/threonine kinase (STK) activity profiling was performed on 50 medulloblastoma samples (HIT cohort) using PamChip arrays, quantified via BioNavigator software (PamGene) and integrated with previously published STK profiling data from 50 medulloblastoma samples (Netherlands cohort)(Zomerman et al. 2018). Unsupervised clustering was conducted on datasets separately and combined to identify peptide phosphorylation profiles. Medulloblastoma subgroups were determined by DNA methylation analysis. Results In the HIT cohort, the main cluster predominantly consisted of Group 3 and Group 4 MB samples, unlike the other clusters (p = 0.033). As published, there were two clusters in the Netherlands cohort, one including SHH-activated and a subset of Group 3 MB samples, the other Group 4 and other Group 3 samples. Upon combination of the two datasets, two stable clusters were identified with excellent separation of SHH from Group 3/Group 4 samples (p < 0.001). Clinical variables (age, evidence of metastatic disease, use of radiotherapy, age of the sample) were independent from the clusters. Conclusion Functional kinase activity profiling using PamGene consistently identifies two main clusters in medulloblastoma, aligning with known molecular subgroups. This suggests that kinase activity is correlated with medulloblastoma subgroups, and that kinase pathways may have subgroup-specific roles. This data may facilitate the identification of therapeutically relevant kinase targets to support development of precision therapies in medulloblastoma. To this end, we are currently performing upstream kinase analysis aiming to identify subgroup-dependent kinases. 1. Zomerman WW, Plasschaert SLA, Conroy S, Scherpen FJ, Meeuwsen-de Boer TGJ, Lourens HJ, et al. Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma. Cell Rep. 2018;22(12):3206–16.

PURPOSE:Detection of bone marrow disease has been a major component of rhabdomyosarcoma (RMS) staging with bilateral bone marrow aspirates/biopsies (BMAB) from the iliac crests considered the diagnostic gold standard. This study's goal was to determine if 2-18F-labeled fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG PET/CT) or PET/magnetic resonance imaging (MRI) may replace BMAB in RMS staging. METHODS:Patients were recruited in Europe and North America. Medical records, FDG PET/CT or FDG PET/MRI (PET), and BMAB reports were collected retrospectively. Images were re-reviewed by nuclear medicine physicians at the patients' home institutions if PET reports indicated increased FDG uptake at bone/bone marrow sites or if tumor cells were detected in bone marrow samples. RESULTS:PET imaging and BMAB were performed before chemotherapy and tumor resection in 301 patients with FDG-avid RMS. Bone marrow metastases were detected by PET and BMAB in 54, by PET only in 24, and by BMAB only in one of 301 patients with FDG-avid primary tumors. Absence of bone marrow metastases was confirmed by PET and BMAB in 222 of 301 patients. These observations translated into 98% sensitivity and 90% specificity for PET in detecting bone marrow metastases when BMAB was considered as gold standard. FDG-avid marrow disease was patchy (1-5 foci) in one third of cases. Patients with marrow disease indicated by PET and BMAB had more FDG-avid bone marrow foci, more metastatic sites, and lower survival than those with marrow disease indicated by PET only, suggesting more advanced disease stages. CONCLUSION:Bone marrow sampling may be omitted in patients without evidence of FDG-avid bone marrow disease by PET. If there are FDG-avid bone marrow lesions, further investigation by MRI and/or biopsy should be considered.

TPS4270 Background: The DNAJB1-PRKACA fusion transcript was detected as the oncogenic driver of tumor pathogenesis in fibrolamellar hepatocellular carcinoma (FLC) and other cancers, e.g. oncocytic neoplasms of pancreas and bile duct. We and others have shown that the fusion protein can be targeted by T cell-based immunotherapy: Application of Fusion-VAC-XS15 a peptide-based T cell activator including the TLR1/2 agonist XS15 emulsified in Montanide ISA 51 VG in two FLC patients was well tolerated without systemic side effects and induced long-lasting T-cell response accompanied by disease remission with a progression-free survival of up to 80 months and 60 months in both patients (Bauer et al. Nat. Commun., 2022). Based on these promising data, Fusion-VAC-XS15 combined with Atezolizumab is currently under evaluation in the advanced or metastatic situation since October 2023 (Hackenbruch et al. Front Oncol., 2024; NCT05937295). For localized FLC, surgical resection still represents the only curative treatment option but shows high relapse rates which underscores the high medical need for adjuvant treatment options. We here present the FusionVAC22_02 trial, which evaluates Fusion-VAC-XS15 as an adjuvant therapeutic in FLC patients who have reached complete remission. Methods: FusionVAC22_02 is a Phase I open label, multicentric clinical trial evaluating immunogenicity along with safety, toxicity and first signs of clinical efficacy of Fusion-VAC-XS15 as adjuvant treatment, in 20 patients with FLC or other cancers with proven DNAJB1-PRKACA fusion protein, and lacking adjuvant treatment options. One key eligibility criterion is achievement of complete remission (e.g. due to surgery, radiotherapy, local intervention or systemic treatment) according to RECIST1.1. Of note, a history of liver transplantation or prior immune-mediated side effects (e.g. after treatment with checkpoint-inhibitors) are no exclusion criteria. Fusion-VAC-XS15 is applied twice in a 4-week interval, with an optional booster 56 days after the second application, followed by a 6-month follow-up. Primary objectives include assessment of immunogenicity in terms of peptide-specific T cell responses, as well as evaluation of safety and toxicity. Safety assessment is based on the frequency of adverse events according to CTCAE v5.0. Clinical efficacy is determined by RECIST1.1 assessment on imaging. Recruitment started in July 2025, nine FLC patients from various parts of the world have been included and treated so far, four of which have reached the primary endpoint. Clinical trial information: NCT06789198 .

Background CD19-directed chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape for pediatric B-cell acute lymphoblastic leukemia (B-ALL), yet relapses driven by antigen escape remain a major limitation. Dual-targeting CAR approaches recognizing CD19 and CD22 have shown promising clinical activity, but sustained remissions are limited by insufficient CAR T-cell persistence. Methods CAR22.19, a fully human tandem CD19/CD22 CAR, was developed and administered under a named-patient program to nine heavily pretreated pediatric patients with relapsed or refractory B-ALL. Treatment indications were CD19-negative blast population (n=5), relapse after CD19 CAR T (n=3) and/or restricted access to approved CAR T-cell products (n=3). Autologous and donor-derived CAR22.19 T-cells (CART22.19) were manufactured using a good manufacturing practice-compliant, semiautomated fresh-in-fresh-out process. Safety and efficacy were assessed through standardized clinical monitoring, measurable residual disease analysis, and CAR T-cell kinetics. Results Preclinical validation demonstrated antigen-specific cytotoxicity and dual antigen activity. Clinically, CART22.19 were well tolerated, with no treatment-related deaths and no grade ≥3 neurotoxicity, while grade ≥3 cytokine release syndrome occurred in 38.5% (5/13) of infusions and resolved with standard interventions. An initial complete molecular remission was achieved in 78% (7/9) of patients, with a 12-month overall survival rate of 55.6% (95% CI, 20.4-80.5%). Complete remission in CD19⁻CD22⁺ disease underscores the functional contribution of the CD22-targeting domain, whereas all patients refractory to prior CD19 CAR T-cell therapy relapsed early despite retained CD19⁺CD22⁺ expression. Limited in vivo persistence may represent a contributing factor to treament failure. Notably, durable remission and sustained functional persistence of CART22.19 was achieved in one patient refractory to autologous CART22.19 following infusion of donor-derived CART22.19 after reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (alloHSCT) in nonremission. Conclusions CART22.19 therapy demonstrated a favorable safety profile and promising clinical activity in a high-risk pediatric population, with dual targeting enabling disease control in CD19-negative leukemia. Nonetheless, limited CAR T-cell persistence may represent an important obstacle to sustained remission. Our findings support further clinical development of CART22.19 and indicate that donor-derived CAR T-cells following RIC alloHSCT may represent a potential therapeutic strategy to enhance persistence and improve outcomes in heavily pretreated pediatric patients.

Pediatric Hodgkin lymphoma (HL) is highly curable, and reducing the treatment intensity in patients who respond well to induction therapy is a key strategy for minimizing long-term adverse effects. Biomarkers that identify good responders at diagnosis would enable further de-escalation of the treatment. Circulating microRNAs (miRNAs) have shown promise as noninvasive indicators of therapeutic response in hematological cancers, yet their association with early metabolic response on quantitative 18F-Fluorodeoxyglucose-Positron Emission Tomography (18F-FDG-PET) in pediatric HL has not been defined. Here, we investigated the potential of circulating miRNAs to predict the response to induction therapy in pediatric HL. Small RNA sequencing of serum samples from 35 patients revealed 24 Hodgkin lymphoma-associated miRNAs that were differentially expressed between adequate and inadequate responders. Subsequent quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation demonstrated significantly elevated miR‑148a‑3p levels at diagnosis in inadequate responders to induction therapy than in adequate responders (p=0.009). These results indicate that circulating miR‑148a‑3p may enhance current predictive approaches by identifying high‑risk patients less likely to achieve rapid metabolic remission.

Supratentorial ependymomas (ST-EPN) are often life-threatening brain tumors that are characterized by significant molecular heterogeneity. Hypoxia contributes to this heterogeneity, but the extent and nature of this relationship remain unclear. This poses challenges for effective treatment strategies, demanding a more holistic and detailed understanding of the underlying biology. In this study, we employed single-nucleus (n = 63) and spatial (n = 30) transcriptomics to delineate the cellular and spatial landscape of ST-EPN. Characterizing the transcriptional consensus programs of this entity revealed two previously undescribed programs that are associated with the remodeling of the extracellular matrix and the ZFTA fusion identity. These programs were related to a highly unfavorable outcome and were exclusive to ZFTA fusion-positive tumors. Developing a set of spatial scoring algorithms that are sensitive to mixed spot transcriptomes enabled the identification of the role of hypoxia as a potential driver of spatial organization. Together with recurrent spatial associations inferred between programs, this informed a generalized model of the higher-order architecture. The functionally and spatially defined zones of this model displayed distinct immune presence, with the highest seen in the hypoxic zone. These results provide insights into the molecular heterogeneity and spatial organization of ST-EPNs, which may aid in the development of targeted future treatments.Significance: The characterization of transcriptional and spatial heterogeneity in supratentorial ependymoma provides a resource that can inform investigations into potential targets for therapeutic interventions aimed at tumor cells within distinct microenvironments.

ABSTRACT:BackgroundCurrent treatment strategies for pediatric intracranial ependymoma do not consider molecular heterogeneity. Here, we evaluated molecular group-specific determinants of outcome and developed an improved risk stratification model. METHODS:Patients aged 0-21 years with localized intracranial ependymoma were enrolled into the prospective clinical trial E-HIT2000. Treatment included maximum safe surgery, local radiotherapy, and chemotherapy, stratified according to age, histology and, following a major amendment, residual tumor. Clinical data were analyzed in a pooled molecularly annotated cohort with data from patients treated analogously within subsequent registries. RESULTS:For 291 trial patients, the 5-year progression-free survival (PFS) and overall survival (OS) were 62 ± 3% and 81 ± 2%, respectively. For the molecularly annotated pooled cohort (n = 228), 5-year PFS/OS were: posterior-fossa group A ependymoma (EPN-PFA) (n = 146): 45 ± 4%/77 ± 4%; posterior-fossa group B ependymoma (EPN-PFB) (n = 19): 90 ± 7%/100%; supratentorial ependymoma, ZFTA fusion-positive (EPN-ZFTA) (n = 59): 64 ± 7%/86 ± 5%; supratentorial ependymoma, YAP1 fusion-positive (EPN-YAP1) (n = 4): 50 ± 25%/100%. Patients with EPN-PFA without molecular risk factors (1q gain, and/or subtype EPN-PFA1c/d/e, 2a), with complete resection, and postoperative radiotherapy showed favorable outcomes (5-year PFS/OS 75 ± 10%/92 ± 7%). For patients with EPN-PFA with molecular risk factors, prognosis was poor irrespective of residual tumor status (5-year PFS/OS: 33 ± 6%/64 ± 6%). Among EPN-ZFTA, 11/59 tumors were classified as EPN-ZFTA with alternative fusions, associated with inferior PFS (5-year PFS/OS: 36 ± 15%/91 ± 9%). For EPN-ZFTA-RELA, homozygous deletions of CDKN2A were associated with unfavorable outcomes (4-year PFS/OS: 19 ± 16%/57 ± 18% vs. 79 ± 7%/97 ± 3%, P = .0001). Finally, we developed a novel stratification model that discriminates standard and intermediate risk patients from those at high risk (P < .0001 for PFS and OS). CONCLUSIONS:These results strongly suggest the inclusion of molecular parameters into stratification and the use of distinct treatment strategies within future ependymoma trials.

PURPOSE:The International Soft Tissue Sarcoma Database Consortium is a collaboration of the North American and European pediatric oncology cooperative groups that aims to provide treatment recommendations for pediatric patients' sarcoma diagnoses. METHODS AND MATERIALS:The International Soft Tissue Sarcoma Database Consortium radiation oncology committee has developed international consensus guidelines for the use of radiation for local therapy in pediatric patients with metastatic rhabdomyosarcoma (RMS) based on grade and quality of evidence. Specifically, the guidelines address management based on disease burden, disease location, and local therapy options that focus on radiation techniques. RESULTS:Patients who present with metastatic RMS at initial diagnosis should be strongly considered for definitive therapy to the primary site and radiation to involved regional lymph nodes after neoadjuvant chemotherapy. When feasible, local treatment of all sites of disease is recommended. Evaluation of the location, size, and extent of disease and patient prognosis should be used when deciding which radiation dose and modality therapy is most appropriate for metastatic disease. CONCLUSIONS:Although the evidence is limited, these consensus guidelines highlight consensus positions regarding the best practice treatment to assist providers as they navigate treatment decisions for their pediatric patients with metastatic RMS.

We report a retrospective single-center analysis of pediatric patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia focusing on relapses outside of the typical locations, bone marrow, central nervous system, or testes. The purpose of the analysis was to describe the relapse patterns after immunotherapy with the bispecific antibody blinatumomab. The cohort includes 90 patients suffering a total of 167 relapses. The majority of relapses, 103/167 (61.7%), were isolated bone marrow relapses, while 34/167 (20.4%) were combined bone marrow and extramedullary relapses (EMR), and 30/167 (18.0%) were classified as isolated extramedullary relapses (IEMR). EMR included 36/64 (56.3%) central nervous system relapses, 10/64 (15.6%) testicular relapses, and 18/64 (28.1%) relapses occurred in other extramedullary sites (OEMR). Seven in 64 (4.2%) of these OEMR presented as isolated OEMR without bone marrow involvement at the time of diagnosis. Multivariate analyses did not show significant differences in EMR rates for patients undergoing hematopoietic stem cell transplantation, patients receiving total body irradiation or patients suffering acute or chronic graft-versus-host disease. Blinatumomab treatment, however, displayed a significant association with OEMR (HR 3.169; 95% CI [1.032-9.728], p = 0.044) and an increased risk for isolated OEMR (HR 13.322; 95% CI [2.276-77.989], p = 0.004) in univariate and multivariate analyses. These results suggest a higher rate of OEMR after treatment with blinatumomab in pediatric ALL, and require confirmation by prospective trials as well as consented diagnostic and therapeutic algorithms.

Non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) are a heterogeneous malignancies with different histopathological characteristics. Distinct molecular findings help to classify NRSTS into subtypes. Further new molecular subtypes give insight into the heterogeneity of these rare tumours. Over the past 25 years, five large international prospective clinical trials have been conducted to improve prognosis for pediatric, adolescent, and young adult patients (< 25 years) with NRSTS and rare soft tissue neoplasms. The overall cure rate is around 70% but varies dramatically between the different entities. New treatment approaches are still needed for some histotypes and for metastatic tumors to improve outcome.The European paediatric soft tissue sarcoma study Group (EpSSG) proposes guidelines developed by an European NRSTS group supported by the European Reference Network on Paediatric Cancer (ERN PaedCan). This consensus summarizes the standard of care, diagnostic work up, multimodal treatment and surveillance recommendations for pediatric, adolescent, and young adult patients with NRSTS and rare soft tissue neoplasms, according to the Consensus Conference Standard Operating Procedure methodology. The unique features of selected histotypes are discussed.

Background: Previous work from our group demonstrated an association between immunohistochemical detection of Human cytomegalovirus (HCMV) late antigen and poor event-free survival (EFS) in pediatric medulloblastoma. Whole-genome sequencing (WGS) further identified increased abundance of HCMV-aligned reads at the UL88 locus, particularly in Group 3 tumors, a molecular subgroup associated with aggressive clinical behavior and poor prognosis. Methods: We performed an integrated multi-omics analysis of pediatric medulloblastoma using WGS (n = 39) and RNA sequencing (RNA-seq; n = 28) datasets. RNA-seq data were filtered using stringent alignment criteria (MAPQ ≥ 20) and compared with fetal brain (n = 12), adult brain (n = 12), and HCMV-infected cell culture controls (n = 3). Only high-confidence uniquely aligned reads were retained to reduce nonspecific and multi-mapped viral alignments. Sequencing reads were aligned to the HCMV Merlin reference genome (NC_006273.2) using a standardized analytical pipeline. A subset of 28 cases with matched tumor WGS, tumor RNA-seq, and germline WGS data was used for integrated multi-omics analyses. Orthogonal validation analyses were performed in Group 3 tumors using independent genomic and transcriptomic approaches. Exploratory survival analyses were conducted in a combined cohort (n = 84) integrating genomic and immunohistochemical datasets. Results: Recurrent low-level HCMV-aligned molecular signals were identified across medulloblastoma datasets. Reads aligning to UL76, UL88, and UL99 were the most consistently detected HCMV-associated late-gene signals across RNA-seq and WGS datasets. A composite HCMV late-gene signature (UL76-UL88-UL99) showed higher levels in Group 3 tumors than in other molecular subgroups (p < 0.05 in WGS analyses). Orthogonal analyses demonstrated concordant low-level HCMV-associated genomic and transcriptomic signals enriched in tumors with MYC-associated activation and chromosome 17 imbalance. In the combined cohort (n = 84), elevated HCMV-associated signal assessed by immunohistochemistry and genomic profiling was associated with reduced EFS (median 55 vs. 147 months; log-rank p < 0.001). The subgroup classified as HCMV-high Group 3 demonstrated the strongest association with adverse outcome in exploratory multivariable analyses (HR = 6.43, p = 0.002). Conclusions: This study identifies recurrent low-level HCMV-associated genomic and transcriptomic signals across pediatric medulloblastoma datasets, with preferential enrichment in biologically aggressive Group 3 tumors. Although the extremely low abundance of viral-aligned reads precludes definitive evidence of productive viral infection, the reproducible detection of HCMV-associated molecular signatures across independent sequencing platforms supports further investigation into a potential oncomodulatory association in pediatric medulloblastoma. Additional validation using optimized viral detection methodologies, independent cohorts, and mechanistic studies will be necessary to clarify the biological and clinical significance of these findings.

INTRODUCTION:We evaluated the survival rate/survivor characteristics following first progression/relapse of metastatic rhabdomyosarcoma (M1 RMS), using pooled European and US collaborative group data from the INternational Soft Tissue saRcoma ConsorTium (INSTRuCT). METHODS:Patients with first diagnosis of M1 RMS aged 0-40 years were identified within the INSTRuCT database (Upfront Cohort; UC). The First Event Cohort (FEC) included UC patients with first event of disease progression/relapse. Clinical features and survival of FEC patients were described. RESULTS:UC included 1095 eligible M1 RMS patients. 5-year Overall and Event Free Survival were 32.0% (95% Confidence Interval (CI) 29.2-34.9) and 27.5% (95% CI 24.8-30.2) respectively. Median time to event was 13.9 months (range 1 day-172.6 months). Among UC patients, 727 with first event of progression/relapse were included in FEC. 3-year Overall Survival for FEC from first event was 8.0% (95% CI 6.1-10.2). Thirty-four (4.7%) FEC patients were alive with > 3 years follow up ("disease free") and 16 (2.2%) with < 3 years follow up. FEC patients alive > 3 years were significantly more likely than deceased FEC patients to have: younger age (p = 0.0031); no locoregional lymph node involvement (p = 0.0013); fewer metastatic sites (p = 0.006); no bone and/or bone marrow disease (p < 0.001 for each); lower Oberlin scores (p < 0.0001); time to first event > 18 months (p < 0.0001). Univariate and multivariable analyses conducted in FEC to investigate factors impacting OS showed that Oberlin score ≥ 2 (Hazard Ratio (HR) 1.295, 95% Confidence Limits (CL) 1.07-1.57, p = 0.0074) and involvement of loco-regional lymph nodes at diagnosis (HR 1.28, 95% CL 1.08-1.52, p = 0.0053) were associated with worse outcome. CONCLUSIONS:Outcomes following first progression/relapse of M1 RMS are dismal. Survivors had fewer adverse prognostic features at first presentation and later first events. Further work is required to predict survivors of first relapse more reliably.

Embryonal tumor with multilayered rosettes (ETMR) is a lethal embryonal brain tumor entity. To investigate the intratumoral heterogeneity and cellular communication in the tumor microenvironment (TME), we analyze in this work single-cell RNA sequencing of about 250,000 cells of primary human and murine ETMR, in vitro cultures, and a 3D forebrain organoid model of ETMR, supporting the main findings with immunohistochemistry and spatial transcriptomics of human tumors. We characterize three distinct malignant ETMR subpopulations - RG-like, NProg-like and NB-like - positioned within a putative neurodevelopmental hierarchy. We reveal PDGFRβ+ pericytes as key communication partners in the TME, contributing to stem cell signaling through extracellular matrix-mediated interactions with tumor cells. PDGF signaling is upregulated in chemoresistant RG-like cells in vivo and plays a role in recruiting pericytes to ETMR TME by finalizing a signaling cascade which promotes the differentiation of non-malignant radial glia cells, derived from our 3D model, into pericyte-like cells. Selective PDGFR-inhibition blocked the lineage differentiation into pericytes in vitro and reduced the tumor cell population in vivo. Targeting ETMR-pericyte interactions in the TME presents a promising therapeutic approach.

Background:Recent advances in reconstruction of the shoulder girdle and scapula had a significant impact on functional outcome in adults who underwent oncologic scapulectomy. In children and adolescents, scapula tumors are rare. Moreover, the growing skeleton and high functional demands in this age group may hinder transferability of the promising results achieved in adults. This study aims to explore the functional outcome and different reconstructive options used in children undergoing (partial) scapulectomy. Methods:A single-center retrospective analysis of scapula tumors in children was performed. Furthermore, a systematic review and synthesis of qualitative and quantitative studies were conducted to investigate the functional outcome of children and adolescents undergoing Malawer II or Malawer III resection. Results:In total, 3 patients were deemed eligible for the single-center retrospective analysis. The 3 children (2 boys, 1 girl, aged 4 - 11 years) all had Ewing sarcoma of the scapula. Two patients underwent Malawer II resection and had a better functional outcome than the 1 child that underwent Malawer III resection. Concerning the systematic review, of the 714 initial search results, 17 studies were eligible for inclusion. In total, 47 patients were extracted from the 17 studies. The analysis showed that patients who underwent Malawer III resection had a significantly better functional outcome if a reconstructive surgery was performed. Patients who underwent glenoid-preserving Malawer II resection showed similar results with or without reconstruction. Conclusion:Children and adolescents undergoing Malawer III resection benefit from a reconstructive procedure other than humeral suspension. Reconstruction using either endoprosthesis or extracorporeal irradiation and reimplantation provide similar functional outcome after Malawer III resection. In Malawer II resection, reconstructive procedures do not influence functional outcome.

Background:The presence of both regional and distant lymph node metastases (LNM) in paediatric and adolescent/young adult (AYA) patients with soft tissue sarcomas (STS) significantly impacts clinical outcomes. However, reported rates of LNM vary widely across the literature and are often accompanied by substantial uncertainty. We aimed to quantitatively synthesise global proportions of LNM across different histological subtypes and tumour sites in this population. Methods:In this meta-analysis, we systematically searched MEDLINE, Scopus, and Web of Science from inception until May 1, 2024 (updated on June 1, 2025) for studies published in English that reported LNM rates in patients with STS aged 0-21 years. Eligible study designs included cohort studies, case-control studies, case series, and randomised controlled trials. Patient-level data were not requested from study authors. LNM had to be confirmed clinically, by imaging, or histologically. We excluded reviews, editorials, and case reports with fewer than three patients. We conducted a random-effects Bayesian meta-analysis using a logit transformation to synthesise LNM proportions. The posterior distributions of LNM prevalence were summarised by the posterior mean and 95% credible intervals (CrIs). Study quality was assessed across seven domains: confounding, participant selection, exposure classification, deviations from intended exposures, missing data, outcome measurement, and selective reporting. Findings:Of 3969 records screened, 263 articles were included in the data synthesis. These comprised 147 studies on rhabdomyosarcoma (RMS), 106 on non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), and 10 on mixed RMS/NRSTS cohorts, representing 53,093 patients with STS. The pooled posterior mean proportion of LNM in patients with RMS (n = 41,547) was 0.228 (95% CrI: 0.202-0.255), with the highest rates observed in patients with alveolar RMS (posterior mean proportion: 0.370; 95% CrI: 0.276-0.473). Subgroup analysis by RMS primary site revealed the highest LNM rates in the perianal/perineal region (0.466; 95% CrI: 0.397-0.537), extremity (0.281; 0.210-0.363), and non-parameningeal head/neck region (0.259; 0.167-0.376). Among patients with NRSTS (n = 11,546), the pooled posterior mean proportion of LNM was 0.111 (95% CrI: 0.092-0.133), with desmoplastic small round cell tumour (0.440; 0.335-0.552), clear cell sarcoma (0.212; 0.163-0.275), and malignant rhabdoid tumour (0.199; 0.141-0.273) showing the highest rates. Most analyses had moderate-to-high heterogeneity (95% CrI for tau: 0.7443-1.1139). Interpretation:Our Bayesian meta-analysis synthesises global evidence on the prevalence of LNM in paediatric and AYA patients with STS, highlighting the significant heterogeneity in LNM rates by histological subtype, particularly in NRSTS, and by tumour location, especially in RMS. Future studies should aim to standardise lymph node staging protocols and reporting practices to improve classification accuracy and enhance comparability across studies. Funding:None.

Desmoplastic small round cell tumor (DSRCT) is an aggressive cancer that predominantly affects adolescents and young adults, typically developing at sites lined by mesothelium [1, 2]. DSRCT is genetically defined by a chromosomal translocation that fuses the N-terminus of EWS RNA binding protein 1 (EWSR1) to the C-terminus of Wilms tumor protein (WT1), forming EWSR1::WT1 [3]. This fusion encodes a potent transcription factor and is the only known driver of oncogenic transformation in DSRCT [4]. The lack of a comprehensive understanding of DSRCT biology parallels its dismal survival rate (5%-20%) [1]. These challenges are exacerbated by the absence of clinical trials, the limited systematic collection and analysis of DSRCT biomaterial [1], and the notable lack of specific diagnostic markers, necessitating resource-intensive molecular testing for an accurate diagnosis. Here we first focused on identifying promising candidates for validation as single, fast, and reliable diagnostic DSRCT markers. For this, we performed differential gene expression (DEG) analysis on datasets comprising patient samples from 32 DSRCT and 20 morphological mimics, identifying 23 genes overexpressed in DSRCT (log2 fold change (log2FC) > 2.5; adjusted P-value (Padj) < 0.01; Figure 1A, Supplementary Figure S1A). Secondly, we analyzed EWSR1::WT1 binding sites derived from chromatin immunoprecipitation followed by sequencing (ChIP-seq) data [5] obtained from the JN-DSRCT-1 cell line, identifying 2,065 genomic loci likely regulated by EWSR1::WT1 (Figure 1A). Third, we established JN-DSRCT-1 and SK-DSRCT2 cell lines expressing doxycycline (DOX)-inducible short hairpin RNA (shRNA)-mediated EWSR1::WT1 knockdown (KD) (Supplementary Figure S1B). Differential protein expression (DEP) analysis of these cells identified 104 proteins consistently regulated across both cell lines (log2FC > 1.0 and Padj < 0.01; Figure 1A, Supplementary Table S1). The intersection of these analyses revealed calcium voltage-gated channel auxiliary subunit alpha2delta 2 (CACNA2D2) and IQ motif containing G (IQCG) as potential DSRCT biomarkers (Figure 1A). CACNA2D2 was selected for validation due to its significantly higher expression in DSRCTs compared to IQCG (P < 0.001; Figure 1A). Indeed, DSRCT exhibited the highest expression of CACNA2D2 among all studied morphological mimics and normal tissues (P < 0.001; Supplementary Figures S1C-D). Further ChIP-seq data and motif analyses of EWSR1::WT1 binding coordinates and histone marks in JN-DSRCT-1 and four DSRCT patient samples [5, 6] suggested a direct regulatory role of EWSR1::WT1 through an enhancer interaction at the CACNA2D2 locus (Figure 1B). Notably, KD of EWSR1::WT1 in JN-DSRCT-1 resulted in a loss of the EWSR1::WT1 signal and Histone H3 lysine 27 acetylation (H3K27ac) enhancer marks at the CACNA2D2 locus (Figure 1B). Additionally, chromatin interaction data [6] revealed 19 loops connecting the EWSR1::WT1 binding site to the transcription start site of CACNA2D2, which were depleted upon KD of EWSR1::WT1 (Figure 1C). Super enhancer (SE) analysis further demonstrated that the EWSR1::WT1-bound enhancer exhibited a characteristic SE H3K27ac profile in JN-DSRCT-1, which was lost upon EWSR1::WT1 KD (Figure 1D, Supplementary Table S2). Post-transcriptional and post-translational KD of EWSR1::WT1 in three DSRCT cell line models expressing different EWSR1::WT1 isoforms (Supplementary Figure S2A) resulted in a significant reduction in CACNA2D2 expression (Figures 1E–F, Supplementary Figure S1B, Supplementary Figures S2B–F). Additionally, ChIP-seq data derived from MeT-5A mesothelial cells [6] – the potential cell of origin of DSRCT [7, 8] – ectopically expressing different EWSR1::WT1 isoforms (-KTS, +KTS, or -KTS/+KTS) suggested direct regulation, as evidenced by the co-occurrence of H3K27ac signals and signals for V5- or HA-tagged EWSR1::WT1 isoforms at the CACNA2D2 enhancer region (Supplementary Figure S2G). Notably, MeT-5A cells transfected with a control vector showed no substantial signal at this locus (Supplementary Figure S2G). Publicly available RNA-sequencing (RNA-seq) data from MeT-5A cells [6] expressing different EWSR1::WT1 isoforms showed that CACNA2D2 was differentially expressed in the presence of EWSR1::WT1 (4.1 ≤ log2FC ≤ 5.9, Padj < 0.001) (Supplementary Figure S2H). Finally, quantitative polymerase chain reaction (qPCR) analysis of MeT-5A cells stably expressing a DOX-inducible ectopic EWSR1::WT1 expression cassette confirmed that upon EWSR1::WT1 induction, CACNA2D2 was significantly and highly overexpressed (Supplementary Figure S2I). Taken together, these results emphasize that EWSR1::WT1 is sufficient to drive CACNA2D2 expression. SE analysis of MeT-5A-derived data strikingly showed that the CACNA2D2 enhancer bound by EWSR1::WT1 became a SE upon ectopic expression of EWSR1::WT1− KTS + KTS (Supplementary Figure S2J). To explore whether CACNA2D2 could serve as a surrogate indicator of oncogenic EWSR1::WT1 transformation, we defined a CACNA2D2 gene set and gene signature by performing a correlation analysis of gene expression data from 32 DSRCT patient samples (Supplementary Figure S3A, Supplementary Tables S3-S4). Next, an EWSR1::WT1 signature was computed by performing a combined DEG analysis of newly generated in vivo and in vitro [4] material derived from three DSRCT cell lines upon EWSR1::WT1 KD (Supplementary Figure S3A, Supplementary Table S4). Notably, fast gene set enrichment analysis (fGSEA) of the CACNA2D2 gene set demonstrated a highly significant (Padj < 0.001) and strong positive enrichment for the EWSR1::WT1 signature (normalized enrichment score, NESEWSR1::WT1 = 3.6). Moreover, single sample gene set enrichment analysis (ssGSEA) of expression data from 32 DSRCT patient samples confirmed that the EWSR1::WT1 signature significantly correlated with that of CACNA2D2 (r = 0.75), highlighting a transcriptional interconnection between CACNA2D2 and EWSR1::WT1 in situ (Figure 1G). These observations were further supported by single-cell (sc)-derived signatures from orthotopically-generated tumors using two DSRCT cell lines with DOX-inducible KD of EWSR1::WT1 at primary (n = 221) and metastatic (n = 221) locations (Figure 1G, Supplementary Table S4). Indeed, ssGSEA of our single-cell data showed highly significant correlation between the NES of our generated EWSR1::WT1 and CACNA2D2 signatures (Figure 1G), regardless of tumor location, implying that CACNA2D2-associated genes are also characteristic features of metastasized DSRCT cells (Supplementary Figure S3B). To delineate the specificity of the interaction between CACNA2D2 and EWSR1::WT1 in DSRCT, we performed ssGSEA using our EWSR1::WT1 and CACNA2D2 signatures on expression data from 20 DSRCT morphological mimics (Figure 1H). Here, non-DSRCT cancer entities showed significantly lower NES and correlation strength for all signatures compared to DSRCT (Supplementary Figures S3C-D). These results further emphasized the high specificity of the CACNA2D2 and EWSR1::WT1 interplay in DSRCT. Moreover, both bulk- and sc-derived CACNA2D2 signatures precisely distinguished DSRCT cell clusters from non-tumor cells in single-cell RNA-sequencing (scRNA-seq) data from four DSRCT patients (n = 11 samples) [9] (Figure 1I, Supplementary Figure S3E). Concordantly, all predicted normal cell types within these tumors exhibited low enrichment of both CACNA2D2 signatures (Supplementary Figures S3F-G). Further, dimensional reduction of CACNA2D2-associated CpG sites in 24 DSRCT patient samples, compared with 192 samples from 13 morphological mimics [10] revealed distinct clustering of all DSRCT samples, which was unique to CACNA2D2 compared to other described EWSR1::WT1-regulated genes or IQCG (Figure 1A, Supplementary Figures S4A-B). Additionally, these CACNA2D2-associated CpG sites exhibited significant (P < 0.001) and specific hypomethylation in DSRCT patient samples, collectively suggesting that the CACNA2D2-associated methylation signature is a distinct and specific feature of DSRCT (Supplementary Figure S4C). To assess the diagnostic utility of CACNA2D2, we optimized a staining protocol for DSRCT cell line xenografts, achieving consistent and robust membranous or cytoplasmatic staining, even uncovering micrometastases (Figure 1J, Supplementary Figure S4D). Finally, we assembled the largest collection of fresh-frozen and paraffin-embedded DSRCT patient samples analyzed to date (n = 61), comprising primary, metastatic, and post-treatment samples, and supplemented it with 249 patient samples from 18 different DSRCT morphological mimics (Supplementary Table S5). CACNA2D2 immunoreactivity was evaluated using a modified Immune Reactive Score (IRS) (Supplementary Material and Methods). Excitingly, DSRCT tumor sections exhibited the highest IRS for CACNA2D2 (IRSmean = 10.5, 6 ≤ IRSDSRCT ≤ 12, P < 0.001) (Supplementary Figure S4E-F), with specificity reaching 98% when applying a cutoff of IRS > 1 (Figure 1K-M, Supplementary Figure S4E). Indeed, even samples derived from CIC- and BCOR-rearranged sarcomas, as well as fusion-positive alveolar rhabdomyosarcomas, showed negligible mean protein expression compared to DSRCT (IRSCIC = 0.21, IRSBCOR = 0, IRSfp-ARMS = 0.56). Furthermore, 100% sensitivity was achieved when applying an IRS cutoff of ≤ 6, implying that DSRCT samples consistently displayed strong staining for CACNA2D2 (Figure 1M). Thus, we recommend a single CACNA2D2 staining for clinically and histologically compatible DSRCT differential diagnosis. If IRSCACNA2D2 ≤ 1, the diagnosis should be reconsidered or re-evaluated using molecular diagnostic procedures (such as fluorescence in situ hybridization, qRT-PCR, or next-generation sequencing), if available (Figure 1N). Conversely, if IRSCACNA2D2 > 1, a diagnosis of DSRCT may be established. Also, CACNA2D2 staining may be used to rule out DSRCT within the broad spectrum of small-round-blue-cell tumors, potentially offering extensive diagnostic utility. Finally, the high, specific, and homogenous membranous expression of CACNA2D2 in DSRCT, combined with the highly specific antibody described here, makes CACNA2D2 an ideal candidate for targeted therapeutic approaches, including drug delivery using antibody-drug conjugates or CAR-T cell therapy. Future studies should investigate the precise role of CACNA2D2 in DSRCT biology, with a focus on its potential contributions in tumor cell fitness, differentiation, and tumorigenic potential. In conclusion, here we developed an extensive toolset for DSRCT research (Supplementary Figure S4G), a validated blueprint for how such resources could be harnessed in other cancer entities, and identified CACNA2D2 as a singular, powerful DSRCT biomarker. Florian Henning Geyer, Florencia Cidre-Aranaz, and Thomas Georg Phillip Grünewald conceived the study. Florian Henning Geyer and Florencia Cidre-Aranaz wrote the paper and drafted all figures and tables. Florian Henning Geyer carried out all in vitro and in vivo experiments and performed all bioinformatic and statistical analyses. Florian Henning Geyer, Alina Ritter, and Thomas Georg Phillip Grünewald performed immunohistochemical evaluation and scoring of tumor samples and TMAs. Florencia Cidre-Aranaz, Roland Imle, and Ana Banito performed and/or coordinated in vivo experiments. Olivier Delattre provided microarray expression data. Seneca Kinn-Gurzo performed in vitro experiments on BER cell lines. Tobias Faehling and Clémence Henon performed single-cell bioinformatic analyses. Karim Aljakouch and Azhar Orynbek performed MassSpec and analyzed MassSpec data. Alina Ritter, Jing Li, Endrit Vinca, Laura Romero-Perez, Martin Sill, and Shunya Ohmura contributed to experimental procedures. Wolfgang Hartmann and Benjamin Friedrich Berthold Mayer provided clinical and/or histological guidance. Enrique De Álava, Juan Díaz-Martín, Stefanie Bertram, Sophie Postel-Vilnay, Martin Ebinger, Monika Sparber-Sauer, Daniel Baumhoer, Carine Ngo, David Horst, Yvonne Versleijen-Jonkers, Armin Jarosch, Sabine Stegmaier, and Thomas Kirchner provided clinical samples. Patrick Joseph Grohar, Thomas Georg Phillip Grünewald, and Jeroen Krijgsveld provided laboratory infrastructure. Florencia Cidre-Aranaz and Thomas Georg Phillip Grünewald supervised the study and data analysis. All authors read and approved the final manuscript. We would like to thank Nadine Gmelin, Stefanie Kutschmann, and Felina Zahnow for their expert technical assistance, and Claudia Schmidt from the Light Microscopy Facility (German Cancer Research Center (DKFZ), Heidelberg, Germany) for her meticulous work in conducting immunohistochemical stainings. We thank the Microarray Core Facility (German Cancer Research Center (DKFZ)) for providing the Gene Expression Arrays and related services. We thank Katharina Bauer, Denise Keitel and Jan-Philipp Mallm from the Single-cell Open Lab (German Cancer Research Center (DKFZ)) for expert support in the preparation of single-cell libraries. We thank the Flow Cytometry Facility team (German Cancer Research Centre (DKFZ)) for their support with cell sorting. We thank Dr. Marc Ladanyi for sharing the SK-DSRCT2 cell line. The authors declare no competing interests. The laboratory of Thomas Georg Phillip Grünewald is supported by grants from the Matthias-Lackas Foundation, the Dr. Leopold und Carmen Ellinger Foundation, the European Research Council (ERC CoG 2023 #101122595), the Deutsche Forschungsgemeinschaft (DFG 458891500), the German Cancer Aid (DKH-70112257, DKH-7011411, DKH-70114278, DKH-70115315), the Dr. Rolf M. Schwiete foundation, the SMARCB1 association, the Ministry of Education and Research (BMBF; SMART-CARE and HEROES-AYA), and the Barbara and Wilfried Mohr foundation. The research team of Florencia Cidre-Aranaz was supported by the German Cancer Aid (DHK-70114111), and the Dr. Rolf M. Schwiete Stiftung (2020-028 and 2022-31). In addition, this work was delivered as part of the PROTECT team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK, the National Cancer Institute, the Scientific Foundation of the Spanish Association Against Cancer And KiKa (Children Cancer Free Foundation). Florian Henning Geyer, Tobias Faehling, Endrit Vinca, and Alina Ritter were supported by the German Academic Scholarship Foundation. In addition, Endrit Vinca was supported by scholarships from the Heinrich F.C. Behr foundation and the Rudolf and Brigitte Zenner foundation, Tobias Faehling by the Heinrich F.C. Behr foundation, and Florian Henning Geyer and Alina Ritter are supported by the German Cancer Aid through the 'Mildred-Scheel-Doctoral Program' (DKH-70114866). This project is co-funded by the European Union (ERC, CANCER-HARAKIRI, 101122595). All views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. In vivo experiments were approved by the government of North Baden and conducted in accordance with ARRIVE guidelines and recommendations of the European Community (86/609/EEC) and UKCCCR (guidelines for the welfare and use of animals in cancer research). Open slides or tissue-microarrays from human formalin-fixed, paraffin-embedded or cryopreserved tissue samples were retrieved from the archives of the Institute of Pathology of the LMU Munich, the Charité Berlin, The Biobank of the Hospital Universitario Virgen del Rocío of Seville, the Hospital Gustave Roussy (Villejuif), the Bone Tumor Reference Center at the University of Basel, the University of Essen, the Cooperative Weichteilsarkom Studiengruppe (CWS) study center, the Klinikum Stuttgart (ethics committee from the Medical Faculty of the Eberhard-Karls University and University Hospital of Tübingen, approval no. 207/2022BO2), the Radboud University Medical Center, the Pathology Institute of the LMU Munich (approval no. 550-16 UE), and the University of Heidelberg (approval no. S-211/2021). The microarray data are deposited at the National Center for Biotechnology Information (NCBI) GEO database with accession codes GSE273438 and GSE273441. All proteomic data is deposited at the PRoteomics IDEntifications database with accession code PXD053786. All other data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background:Despite advancements in treatment, 5-year survival for medulloblastoma (MB) remains 60%-70%. Human cytomegalovirus (HCMV) has been implicated as an oncomodulator in MB, but its impact on survival has not been explored. This study evaluates HCMV expression in pediatric MB tissue and its association with molecular risk groups and survival. Methods:A retrospective study of pediatric MB cases (≤19 years) from 2007 to 2023 was conducted using the WHO 2021 classification, the Northcott brain-tumor classifier, and the SIOP-Europe/ERN PaedCan risk stratification. HCMV immediate-early (HCMV-IE) and late-antigen (HCMV-LA) expression were assessed by optimized immunohistochemistry, and whole-genome sequencing (WGS) data from 20 tumors were analyzed for viral gene expression. Results:Forty-five patients (mean age 8.2 years; 22% ≤3 years) were included: WNT (18%), SHH TP53-wildtype (18%), and non-WNT/non-SHH (64%). Twenty percent of MB belonged to low-risk, 33% to standard-risk, and 47% to high-risk (HR-MB) groups. Four tumors exhibited MYC/N amplification. HCMV-LA positivity was found in 84% of cases, with 53% showing high expression (≥25% of cells). Cox-regression identified HR-MB (HR = 4.197, p = .021) and high HCMV-LA (HR = 4.334, p = .027) as independent predictors of poor outcomes. WGS revealed UL88 as the most abundantly expressed HCMV gene (log2[TPM+1] ≈ 14-15), with maximal expression in Group 3 MB. Conclusions:This study provides the first evidence linking high HCMV-LA expression to adverse outcomes and high-risk molecular features in pediatric MB. UL88 emerged as the most strongly expressed HCMV gene across MB samples. These findings suggest a potential prognostic and therapeutic role for HCMV in MB, warranting validation in larger, prospective cohorts.