BACKGROUND:Reirradiation is used for children and adolescents with recurrent ependymoma after prior surgery and focal irradiation. This study assessed the long-term benefits and risks of a second course of fractionated radiotherapy, patient selection, factors associated with progression-free survival (PFS) and overall survival (OS), and the role of craniospinal irradiation (CSI) at first recurrence. METHODS:From July 1994 to January 2024, 150 pediatric ependymoma patients at St. Jude Children's Research Hospital received a second course of fractionated radiotherapy. Sixty-four were enrolled in a prospective trial. Inclusion required conventional fractionation (≥50.4Gy) for both courses, with CSI for metastatic disease. Kaplan-Meier estimates measured PFS and OS, Cox models assessed associations with covariates, and competing risks analysis evaluated necrosis and death from complications. RESULTS:At 10 and 20 years, PFS/OS for the cohort were 19.5%/34.3% and 9.0%/13.1%, respectively. Females had significantly better outcomes, and survival varied by initial failure pattern. No significant difference in PFS (p = 0.1351) or OS (p = 0.2705) was found between focal irradiation and CSI for patients with local failure. Necrosis was higher with proton therapy versus photons; grade 3 necrosis after reirradiation occurred in 19.9%. The 10-year cumulative incidence of death from complications or secondary tumors was 8.5%. CONCLUSIONS:A second course of conventionally fractionated radiotherapy can extend survival in recurrent pediatric ependymoma, but prognosis remains poor, especially for those with combined local and distant failure or adverse features. CSI did not improve outcomes for local failures. Proton therapy increased necrosis risk, emphasizing the need for careful selection and follow-up.

BACKGROUND:The utility of chemotherapy for childhood ependymoma is uncertain without a prior randomized trial. METHODS:ACNS0831, a multicenter phase 3 randomized study conducted through the Children's Oncology Group (COG), included patients 1-21 years with newly diagnosed intracranial ependymoma. Patients with complete/near-total resections (GTR/NTR) or complete response (CR) to induction therapy were randomized to radiation (RT) alone or RT followed by chemotherapy (RT-CHEMO). Primary outcomes were event-free survival (EFS) and overall survival (OS). Due to anticipated noncompliance with chemotherapy, an "as treated" analysis was planned. Patients with subtotal resection (STR) were non-randomly assigned to RT-CHEMO. Grade 2 supratentorial tumors with GTR or CR to induction therapy were observed. RESULTS:Of 449 eligible patients, 325 with GTR/NTR or CR were randomized. Five-year EFS was 63.7% (95% CI: 55.1%-71.1%) for RT only (n = 161) versus 69.2% (60.8%-76.3%) for RT-CHEMO (n = 164) (1-sided log-rank P = .299, HR = 0.866). Five-year OS was 86.9% (79.8%-91.6%) for RT only versus 88.3% (81.8%-92.6%) for RT-CHEMO (1-sided log-rank P-value = .172, HR = 0.757). The "as treated" and "as randomized" analysis results were similar. Sixty-three subjects with STR were assigned to RT-CHEMO; 5-year EFS was 33.6% (22.1%-45.5%) and OS 74.0% (60.5%-83.5%). Supratentorial grade 2 tumors with GTR or CR were observed with a 5-year EFS of 66.9% (49.0%-79.7%) and OS of 100%. Molecular classification was provided for 94% (n = 422) of all subjects. CONCLUSIONS:Primary analysis showed no benefit for maintenance chemotherapy. Further follow-up is important to assess its effect on late relapses. This is the largest cohort of molecularly classified ependymomas treated on a Phase 3 randomized trial.

Leptomeningeal metastases are the primary source of morbidity and mortality for pediatric medulloblastoma patients. Due to limited surgical sampling of metastases in patients, little is understood of the mechanisms of metastasis. Here, we identify biologically distinct quiescent small metastases (designated as micrometastases) and mitotically active larger metastases (macrometastases). Macrometastases are more metabolically active than micrometastases and contain higher levels of lipids, particularly cholesterol. Macrometastases secrete CXCL12, which attracts lipid-laden macrophages into the tumor. Lipid-laden macrophages upregulate the cholesterol transporter ABCG1, promoting the efflux of free cholesterol, which is then taken up by tumor cells via the HDL receptor SCARB1. CXCL12-driven macrophage recruitment and exogenous cholesterol are sufficient and necessary to drive progression of medulloblastoma leptomeningeal metastases in vivo. High fat diets drive metastatic progression in vivo. Dietary or pharmacological interventions targeting the CXCL12-SCARB1-cholesterol axis represent therapeutic strategies to either prevent or treat medulloblastoma leptomeningeal metastases.

Childhood cancer survivors have a heightened risk of developing subsequent neoplasms (SN) related to therapy. We analyzed whole-genome, exome, and RNA sequencing of 200 breast, meningioma, and thyroid SNs, which developed a median of 26.4 years after childhood cancer, among 160 survivors. Meningioma and thyroid SNs were enriched for driver gene rearrangements compared with de novo tumors, including NF2-disrupting alterations and kinase fusions potentially induced by radiation. Radiation correlated with increased insertion-deletion signature ID5. Nitrogen mustard treatment correlated with elevated "flat" signature SBS5 in breast and meningioma SNs; in vitro, these agents caused an unresolved flat signature associated with multiple flat signatures from the Catalogue of Somatic Mutations in Cancer. In meningioma, platinum therapy correlated with NF2 splice-site variants. Analysis of 19 multisample survivors revealed intrapatient heterogeneity in meningioma, including clonally independent tumors. These results demonstrate the long-term impact of childhood cancer treatment on the genomes of SNs developing in adulthood, which may guide SN treatment and prevention. SIGNIFICANCE:This represents the most comprehensive genomic characterization of SNs from childhood cancer survivors to date, revealing the mutagenic impact of multiple therapies on the SN genome, including the potential impact of nitrogen mustards such as cyclophosphamide. These results may guide the optimization of future cancer treatment regimens to prevent SN development. See related commentary by Bertrums and van Boxtel, p. 1483.

Distinct molecular variants of the brain cancer ependymoma are distributed along the rostral-caudal extent of the central nervous system (CNS). Historically proposed to arise from ventricular ependyma, recent studies have suggested conflicting cellular origins, including the neural radial glia and the roof plate lineages. Using single-cell transcriptomics, immunohistochemistry, and lineage tracing, we demonstrate that ependymomas across all CNS compartments transcriptionally mirror MSX1 +ve pre-neural crest/roof plate (Pre-NC/RP) lineage derivatives. Ependymoma subgroups recapitulate the spatial and molecular diversity of regional Pre-NC/RP populations, while retaining conserved MSX1 expression. Expression of the oncogenic fusion ZFTA-RELA within the murine Pre-NC/RP lineage generated tumors that faithfully resembled human ependymoma. These findings identify a common embryonic cellular origin for ependymomas and reconcile previously conflicting models of tumorigenesis.

BACKGROUND:Ependymoma (EPN) is the third most common pediatric brain tumor with no targeted therapies available to patients. In supratentorial ependymoma, the most frequent driver alteration is a gene fusion between ZFTA and RELA (denoted ZR), leads to constitutive localization of ZR in the nucleus. Because ZR is not currently druggable, we tested whether ZR expression leads to aberrant protein interactions that could represent therapeutic vulnerabilities. METHODS:Using CRISPR-Cas9 pooled screening, we identified many novel druggable ZR interacting proteins including XPO1, CARM1, SMARCA4, and CDK1. We focused on the nuclear export protein (XPO1), given the ability of most XPO1 inhibitors (i.e. Selinexor) to cross the blood brain barrier, FDA approval, and documented safety profiles in children. RESULTS:We found that specific nuclear ZR levels are needed for cell proliferation and are regulated by XPO1. Increased ZR accumulation in the nucleus does not increase oncogenic gene expression but drives tumor cells out of cell cycle, as compared to a defective ZR DNA binding mutant. Treatment of ZR driven patient-derived mouse models with Selinexor impairs cell growth and extends survival of animals in vivo. The combination of Selinexor treatment with Gemcitabine and Ribociclib (used in a clinical trial for relapsed EPN at St Jude Children's Research Hospital (SJDAWN)) further extends mouse survival. CONCLUSION:Our findings demonstrate that ZR interacting proteins constitute therapeutic leads, and that XPO1 is critical for titrating 'goldilocks' levels of ZR nuclear expression. We identify a novel combination therapy of Selinexor, Gemcitabine, and Ribociclib that may be immediately translated into clinical trials for EPN patients that are currently without targeted treatments.

ZFTA-RELA is the most recurrent genetic alteration seen in paediatric supratentorial ependymoma (EPN) and is sufficient to initiate tumours in mice1. Despite its oncogenic potential, ZFTA-RELA (ZR) is observed nearly exclusively in childhood EPN, with tumours located distinctly in the supratentorial brain of the central nervous system1. We proposed that specific chromatin modules accessible during brain development would render distinct cell lineage programs at direct risk of transformation by ZR. To test this hypothesis, we performed combined single-nucleus assay for transposase-accessible chromatin and RNA (snMultiome) sequencing of the developing mouse forebrain compared with ZR-driven mouse and human EPN. We demonstrated that specific developmental lineage programs present in transient progenitor cells and regulated by PLAG/L family transcription factors were at risk of neoplastic transformation. Binding of this chromatin network by ZR or other PLAG/L family motifs targeting fusion oncoproteins led to persistent chromatin accessibility at oncogenic loci and oncogene expression. Cross-species analysis of mouse and human ZR EPN revealed significant cell type heterogeneity indicating incomplete neurogenic and gliogenic differentiation, with a small percentage of cycling progenitor-like or radial glial-like cells that established a putative tumour cell hierarchy. In vivo lineage tracing studies identified neoplastic clones that aggressively dominated tumour growth and established the entire EPN cellular hierarchy. These findings identify developmental epigenomic states that are critical for fusion-oncoprotein-driven transformation and show how these states continue to shape tumour progression.

Supplementary Table S1 shows SN sample clinical variables and metadata. Supplementary Table S2 shows a comparison of variables between good-quality vs. excluded samples. Supplementary Table S3 shows a summary of prior treatments across SN patients. Supplementary Table S4 shows a summary of original childhood cancer diagnoses for SN patients. Supplementary Table S5 shows metadata for 33 pediatric cancers with matched FFPE and fresh-frozen exome data. Supplementary Table S6 shows a list of coding-region somatic SNVs used for mutation burden analysis. Supplementary Table S7 shows multivariable analysis comparing SNV burdens between cohorts. Supplementary Table S8 shows multivariable analysis comparing age between cohorts. Supplementary Table S9 shows multivariable analysis comparing SNV burdens between thyroid cancer cohorts among samples with coverage below 100x. Supplementary Table S10 shows cancer-predisposing germline alterations among SN patients. Supplementary Table S11 shows a list of coding-region somatic indels used for mutation burden analysis. Supplementary Table S12 shows multivariable analysis comparing indel burdens between cohorts. Supplementary Table S13 shows SNV signature levels among SNs. Supplementary Table S14 shows multivariable analysis comparing SBS5 burdens in breast SNs stratified by prior treatment. Supplementary Table S15 shows multivariable analysis comparing SBS5 burdens in meningioma SNs stratified by prior treatment. Supplementary Table S16 shows multivariable analysis testing SBS5-cyclophosphamide dose-response relationship in meningioma SNs. Supplementary Table S17 shows variation in cyclophosphamide-containing regimens among meningioma SNs. Supplementary Table S18 shows indel signature levels among SNs. Supplementary Table S19 shows multivariable analysis testing ID5-radiation dose-response relationship in each SN type. Supplementary Table S20 shows multivariable analysis testing ID5-radiation dose-response relationship with all 3 SN types combined. Supplementary Table S21 shows NF2-disrupting structural variants detected in meningioma SNs. Supplementary Table S22 shows multivariable analysis comparing driver alteration frequency between meningioma cohorts. Supplementary Table S23 shows driver fusions in thyroid SNs. Supplementary Table S24 shows multivariable analysis comparing mutation group frequency between thyroid cancer cohorts. Supplementary Table S25 shows multivariable analysis comparing driver alteration frequency between thyroid cancer cohorts. Supplementary Table S26 shows multivariable analysis comparing driver alteration frequency between breast cancer cohorts

Figure S1 shows representative B7-H3 staining intensity scale.

Figure S22 shows IHC evaluation of B7-H3 expression and CD3+ cell infiltration inside syngeneic ZFTA-RELA EPN tumors.

Figure S13 shows sustained or increased cytokine secretion of HER2.CAR T-cells following repetitive stimulation with EPN cells.

Figure S7 shows inverse correlation between B7-H3.CAR T-cell expansion and B7-H3 target antigen density.

Figure S9 shows Type 2 polarization of B7-H3.CAR T-cells over repetitive stimulation with EPN cells.

Figure S4 shows antigen profiling of HER2 in ZFTA-fusion driven EPN cell lines.

DNA methylation-based classification is now central to contemporary neuro-oncology, as highlighted by the World Health Organization (WHO) classification of central nervous system (CNS) tumors. We present the Heidelberg CNS Tumor Methylation Classifier version 12.8 (v12.8), trained on 7,495 methylation profiles, which expands recognized entities from 91 classes in version 11 (v11) to 184 subclasses. This expansion is a result of newly identified tumor types discovered through our large online repository and global collaborations, underscoring CNS tumor heterogeneity. The random forest-based classifier achieves 95% subclass-level accuracy, with its well-calibrated probabilistic scores providing a reliable measure of confidence for each classification. Its hierarchical output structure enables interpretation across subclass, class, family, and superfamily levels, thereby supporting clinical decisions at multiple granularities. Comparative analyses demonstrate that v12.8 surpasses previous versions and conventional WHO-based approaches. These advances highlight the improved precision and practical utility of the updated classifier in personalized neuro-oncology.

PURPOSE:Targeted treatments are desperately needed for ependymomas. Chimeric antigen receptor (CAR) T cells have immense potential to transform patient outcomes. However, CAR T-cell therapy for ependymomas has been largely understudied. In this study, we explore the potential of targeting B7 homolog 3 (B7-H3/CD276) with CAR T cells to treat pediatric ependymomas. EXPERIMENTAL DESIGN:We profiled B7-H3 protein expression in 44 pediatric ependymoma samples by IHC. We generated second-generation human B7-H3.CAR T cells and examined their anti-ependymoma activity in six in vitro and two in vivo xenograft models. We validated findings using HER2-targeted CAR T cells. In addition, we used murine B7-H3.CAR T cells to evaluate in vivo antitumor activity in a fully syngeneic supratentorial ependymoma model. RESULTS:The majority of clinical ependymoma samples (29/44) stained positive for B7-H3, indicating high but heterogeneous expression across patients. In vitro, human B7-H3.CAR T cells had potent anti-ependymoma cytolytic activity, expansion, and persistence, which was inversely correlated with the upregulation of B7-H3 on CAR T cells. We found that CAR T cells favor type 2 cytokines phenotypes after repeated exposure to ependymoma cell lines, which may be driven by C-C motif chemokine ligand 2 secretion by ependymomas. In vivo, there was potent and significant antitumor activity in human xenograft ependymoma models. However, response durability was limited and significantly correlated with the degree of tumor burden. In the syngeneic setting, murine B7-H3.CAR T-cell efficacy against ependymomas was limited and did not extend survival. CONCLUSIONS:Our results support ongoing clinical evaluation of B7-H3.CAR T cells for ependymomas and provide model systems for further studying determinants of anti-ependymoma CAR T-cell treatment efficacy and resistance.

Figure S5 shows phenotypic characteristics of human B7-H3.CAR T-cells.

Figure S21 shows IHC evaluation and quantification of immune cell infiltration inside syngeneic ZFTA-RELA EPN tumors.

Figure S3 shows regulation of B7-H3 transcriptional activity by ZFTA-RELA oncogene.