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Figure S5 shows the confirmation of differential gene expression between RNA seq samples.

Supplementary Table S1

Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in high-risk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n = 104) and patient-derived xenografts (n = 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT+, and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT+, or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT–expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets.Significance:These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

Purpose:PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG).Patients and Methods:Patients (3–25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN).Results:Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2–18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7–14; TP53wt 13.3 mo; 95% CI, 11.8–NA; P = 3.4e−2), genome instability (P = 3.1e−3), and RT resistance (P = 6.4e−4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome.Conclusions:Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

Relapsed neuroblastomas are enriched with activating mutations of the RAS–MAPK signaling pathway. The MEK1/2 inhibitor trametinib delays tumor growth but does not sustain regression in neuroblastoma preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP1 as an additional driver of relapsed neuroblastomas, as well as a mediator of trametinib resistance in other cancers. Here, we used a highly annotated set of high-risk neuroblastoma cellular models to modulate YAP1 expression and RAS pathway activation to test whether increased YAP1 transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven neuroblastomas. In NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K) cell lines, trametinib caused a near-complete translocation of YAP1 protein into the nucleus. YAP1 depletion sensitized neuroblastoma cells to trametinib, while overexpression of constitutively active YAP1 protein induced trametinib resistance. Mechanistically, significant enhancement of G1–S cell-cycle arrest, mediated by depletion of MYC/MYCN and E2F transcriptional output, sensitized RAS-driven neuroblastomas to trametinib following YAP1 deletion. These findings underscore the importance of YAP activity in response to trametinib in RAS-driven neuroblastomas, as well as the potential for targeting YAP in a trametinib combination.Significance:High-risk neuroblastomas with hyperactivated RAS signaling escape the selective pressure of MEK inhibition via YAP1-mediated transcriptional reprogramming and may be sensitive to combination therapies targeting both YAP1 and MEK.

Table S1 includes MYCN amplification and chromosome 11q status across a panel of 16 neuroblastoma cell lines. Table S2-4 include differentially-expressed genes identified in each of the three differential expression analyses. Table S5 includes unique differentially expressed genes between NLF sgCon + DMSO and NLF YAP1-/- #4 + Tram.

This file includes figure legends corresponding to Supplementary Figures S1-6.

SUPPLEMENTARY TABLES, FIGURES AND FIGURE LEGENDS

Supplementary Data

One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN-mediated process have been elucidated. Here we show that the growth arrest-specific 7 (GAS7) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN-amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN. Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell–cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression.Significance:Heterozygous deletion or MYCN-mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma.See related commentary by Menard, p. 2815

Supplementary Figure from Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003

ImportanceHigh-risk neuroblastoma is a complex genetic disease that is lethal in 50% of patients despite intense multimodal therapy. Our genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) within theBARD1gene showing the most significant enrichment in neuroblastoma patients, and also discovered pathogenic (P) or likely pathogenic (LP) rare germline loss-of-function variants in this gene. The functional implications of these findings remain poorly understood.ObjectiveTo define the functional relevance ofBARD1germline variation in children with neuroblastoma.DesignWe correlatedBARD1genotype withBARD1expression in normal and tumor cells and the cellular burden of DNA damage in tumors. To validate the functional consequences of rare germline P-LPBARD1variants, we generated isogenic cellular models harboring heterozygousBARD1loss-of-function (LOF) variants and conducted multiple complementary assays to measure the efficiency of DNA repair.Setting(N/A)Participants(N/A)Interventions/Exposures(N/A)Main Outcomes and MeasuresBARD1expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associatedBARD1germline variants.ResultsBoth common and rare neuroblastoma associatedBARD1germline variants were significantly associated with lower levels ofBARD1mRNA and an increased burden of DNA damage. Using neuroblastoma cellular models engineered to harbor disease-associated heterozygousBARD1LOF variants, we functionally validated this association with inefficient DNA repair. TheseBARD1LOF variant isogenic models exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA doublestrand break sites, and enhanced sensitivity to cisplatin and poly-ADP ribose polymerase (PARP) inhibition.Conclusions and RelevanceConsidering that at least 1 in 10 children diagnosed with cancer carry a predicted pathogenic mutation in a cancer predisposition gene, it is critically important to understand their functional relevance. Here, we demonstrate that germlineBARD1variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications, and these findings may also extend to other cancers harboring germline variants in genes essential for DNA damage repair.Key PointsQuestionHow do neuroblastoma patient BRCA1-associated RING domain 1 (BARD1) germline variants impact DNA repair?FindingsNeuroblastoma-associated germlineBARD1variants disrupt DNA repair fidelity. Common risk variants correlate with decreasedBARD1expression and increased DNA double-strand breaks in neuroblastoma tumors and rare heterozygous loss-of-function variants induceBARD1haploinsufficiency, resulting in defective DNA repair and genomic instability in neuroblastoma cellular models.MeaningGermline variation inBARD1contributes to neuroblastoma pathogenesis via dysregulation of critical cellular DNA repair functions, with implications for neuroblastoma treatment, risk stratification, and cancer predisposition.

Background To achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. Methods We performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n = 40/579) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on pediatric high-grade gliomas (pHGGs) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. Results ALT is common in pHGGs (n = 24/63, 38.1%), but occurs infrequently in other pediatric brain tumors ( Conclusions We demonstrate that ATRX is mutated in only a subset of ALT+ pHGGs, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with the development of ALT in patients with pHGG.

SummaryPediatric brain and spinal cancer are the leading disease-related cause of death in children, thus we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children’s Brain Tumor Network and Pacific Pediatric Neuro-Oncology Consortium created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to create OpenPBTA, an open collaborative project which establishes over 40 scalable analysis modules to genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals that TP53 loss is a significant marker for poor overall survival in ependymomas and H3 K28-altered diffuse midline gliomas and further identifies universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas. OpenPBTA is a foundational analysis platform actively being applied to other pediatric cancers and inform molecular tumor board decision-making, making it an invaluable resource to the pediatric oncology community.In BriefThe OpenPBTA is a global, collaborative open-science initiative which brought together researchers and clinicians to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Shapiro, et. al create over 40 open-source, scalable modules to perform cancer genomics analyses and provide a richly-annotated somatic dataset across 58 brain tumor histologies. The OpenPBTA framework can be used as a model for large-scale data integration to inform basic research, therapeutic target identification, and clinical translation.HighlightsOpenPBTA collaborative analyses establish resource for 1,074 pediatric brain tumors NGS-based WHO-aligned integrated diagnoses generated for 641 of 1,074 tumors RNA-Seq analysis infers medulloblastoma subtypes, TP53 status, and telomerase activity OpenPBTA will accelerate therapeutic translation of genomic insights

BackgroundPediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target.MethodsWe investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing.ResultsWe identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2–4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed.ConclusionTaken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.

BackgroundTo achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available.MethodsWe performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n=40/579) of these tumors and completed additional validation by ultrabright telomeric fociin situon a subset of these tumors. We used CCA to validateTelomereHunterfor computational prediction of ALT status and focus subsequent analyses on pediatric high-grade glioma (pHGG) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations.ResultsALT is common in pHGG (n=24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (ATRXmutations occur in 50% of ALT+ pHGG and in 30% of ALT-pHGG. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. Conclusions: We demonstrate thatATRXis mutated in only a subset of ALT+ pHGG, suggesting other mechanisms ofATRXloss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with development of ALT in patients with pHGG.Key PointsATRX alterations are frequent, but not required, for an ALT phenotype in pHGGspHGG patients with germline mismatch repair variants have higher rate of ALT + tumorsTelomereHunteris validated to predict ALT in pHGGsImportance of the StudyWe performed orthogonal molecular and computational analyses to detect the presence of alternative lengthening of telomeres in a highly characterized cohort of pediatric brain tumors. We demonstrate that many pHGG utilize ALT without a mutation in ATRX, suggesting either loss of function of ATRX via an alternative mechanism or an alternate means of development of ALT. We show that germline variants in MMR genes are significantly associated with ALT in pHGG. Our work adds to the biological understanding of the development of ALT and provides an approach to stratify patients who may benefit from future ALT-directed therapies in this patient population.

Summary Tumor-associated macrophages (TAMs) play an important role in tumor immunity and comprise of subsets that have distinct phenotype, function, and ontology. Transcriptomic analyses of human medulloblastoma, the most common malignant pediatric brain cancer, showed that medulloblastomas (MBs) with activated sonic hedgehog signaling (SHH-MB) have significantly more TAMs than other MB subtypes. Therefore, we examined MB-associated TAMs by single-cell RNA sequencing of autochthonous murine SHH-MB at steady state and under two distinct treatment modalities: molecular-targeted inhibitor and radiation. Our analyses reveal significant TAM heterogeneity, identify markers of ontologically distinct TAM subsets, and show the impact of brain microenvironment on the differentiation of tumor-infiltrating monocytes. TAM composition undergoes dramatic changes with treatment and differs significantly between molecular-targeted and radiation therapy. We identify an immunosuppressive monocyte-derived TAM subset that emerges with radiation therapy and demonstrate its role in regulating T cell and neutrophil infiltration in MB.

Pediatric central nervous system (CNS) cancers are the leading disease-related cause of death in children and there is urgent need for curative therapeutic strategies for these tumors. To address the urgency, Children’s Brain Tumor Network (CBTN) has advanced an open science model to accelerate the research discovery for pediatric brain tumors. In first phase of Open Pediatric Brain Tumor Atlas (OpenPBTA) effort CBTN together with Pacific Pediatric Neuro-Oncology Consortium (PNOC) with support of Gabriella Miller Kids First Data Resource Center (KFDRC) created and comprehensively characterized over 1000 clinically annotated pediatric brain tumors. In the second phase of the OpenPBTA effort, through resource awards and collaboration across KFDRC, the NCI Childhood Cancer Data Initiative (CCDI), NCI’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), NCI Center for Cancer Research and additional partnered institutions and foundations, CBTN has expanded OpenPBTA to support high throughput molecular characterization for an additional 1900 pediatric brain tumor patients and their families. This includes the processing and characterization of over 8000 specimens across >50 brain tumor diagnoses. The cohort expansion builds on >1000 previously characterized samples with a portfolio of multimodal data including whole genome sequencing, RNA sequencing, miRNA sequencing, methylation sequencing, proteomics, lipidomics and/or metabolomics. Molecular data is linked to patient longitudinal clinical data, imaging data (MRIs and radiology reports), histology slide images, and pathology reports. To inform novel discovery and clinical implementation of genomic approaches for diagnostic/therapeutic purposes, the data is deposited the cloud-based research environment of the NCI’s CCDI and the KFDRC to provide near real-time integration, dissemination, processing, and sharing of associated petabyte-scale harmonized data. The approach leverages the DRC platform’s cloud-based computational environment in CAVATICA. Processed annotations are facilitated via CAVATICA-enabled shareable pipelines and can be explored through PedcBioPortal, a data visualization/analysis application further integrating additional public and deposited datasets. This expansion phase of OpenPBTA is released with no embargo period and provides one of the largest deeply characterized cohorts of pediatric brain tumor samples and associated clinical data for >3000 pediatric brain tumor patients. CBTN’s open-science, rapid-release model aims to advance novel biomarkers and therapeutic exploratory research, supporting new clinical trial development and accelerated discovery on behalf of changing the outcome for kids with brain tumors. Citation Format: Mateusz P. Koptyra, Komal Rahti, Yuankun Zhu, Bailey Farrow, Daniel Miller, Adam Kraya, Yiran Guo, Peter Madsen, Nicholas Van Kuren, Xiaoyan Huang, Miguel A. Brown, Jennifer L. Mason, Meen Chul Kim, Allison P. Heath, Brian M. Ennis, Bo Zhang, Jena V. Lilly, Jo Lynne Rokita, Christopher Friedman, Ximena P. Cuellar, Catherine A. Sullivan, Noel Coleman, Trang Duros, Thinh Q. Nguyen, Emmett C. Drake, Zeinab Helili, Beth A. Frenkel, Gerri R. Trooskin, Ariana Familiar, Karthik Viswanathan, Christopher M. Beck, Madison L. Hollawell, Valerie P. Baubet, Cassie Kline, Mariarita Santi, Tatiana S. Patton, Stephanie Stefankiewicz, Arya Kamnaa, Ryan A. Velasco, Dani Cardona, Phillip J. Storm, Adam C. Resnick, o/b/o Children's Brain Tumor Network. Expansion of the Pediatric Brain Tumor Atlas: Children's Brain Tumor Network, Kids First Data Resource and Childhood Cancer Data Initiative Open Science effort. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3566.

One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN-mediated process have been elucidated. Here we show that the growth arrest-specific 7 (GAS7) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN-amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN. Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell–cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression. Significance: Heterozygous deletion or MYCN-mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma. See related commentary by Menard, p. 2815

Background Gene fusion events are significant sources of somatic variation across adult and pediatric cancers and are some of the most clinically-effective therapeutic targets, yet low consensus of RNA-Seq fusion prediction algorithms makes therapeutic prioritization difficult. In addition, events such as polymerase read-throughs, mis-mapping due to gene homology, and fusions occurring in healthy normal tissue require informed filtering, making it difficult for researchers and clinicians to rapidly discern gene fusions that might be true underlying oncogenic drivers of a tumor and in some cases, appropriate targets for therapy. Results We developed annoFuse, an R package, and shinyFuse, a companion web application, to annotate, prioritize, and explore biologically-relevant expressed gene fusions, downstream of fusion calling. We validated annoFuse using a random cohort of TCGA RNA-Seq samples (N = 160) and achieved a 96% sensitivity for retention of high-confidence fusions (N = 603). annoFuse uses FusionAnnotator annotations to filter non-oncogenic and/or artifactual fusions. Then, fusions are prioritized if previously reported in TCGA and/or fusions containing gene partners that are known oncogenes, tumor suppressor genes, COSMIC genes, and/or transcription factors. We applied annoFuse to fusion calls from pediatric brain tumor RNA-Seq samples (N = 1028) provided as part of the Open Pediatric Brain Tumor Atlas (OpenPBTA) Project to determine recurrent fusions and recurrently-fused genes within different brain tumor histologies. annoFuse annotates protein domains using the PFAM database, assesses reciprocality, and annotates gene partners for kinase domain retention. As a standard function, reportFuse enables generation of a reproducible R Markdown report to summarize filtered fusions, visualize breakpoints and protein domains by transcript, and plot recurrent fusions within cohorts. Finally, we created shinyFuse for algorithm-agnostic interactive exploration and plotting of gene fusions. Conclusions annoFuse provides standardized filtering and annotation for gene fusion calls from STAR-Fusion and Arriba by merging, filtering, and prioritizing putative oncogenic fusions across large cancer datasets, as demonstrated here with data from the OpenPBTA project. We are expanding the package to be widely-applicable to other fusion algorithms and expect annoFuse to provide researchers a method for rapidly evaluating, prioritizing, and translating fusion findings in patient tumors.

Purpose: PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Patients and Methods: Patients (3–25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). Results: Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2–18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7–14; TP53wt 13.3 mo; 95% CI, 11.8–NA; P = 3.4e−2), genome instability (P = 3.1e−3), and RT resistance (P = 6.4e−4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. Conclusions: Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.

Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in high-risk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n = 104) and patient-derived xenografts (n = 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT+, and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT+, or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT–expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets. Significance: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, decreases self-renewal and migration. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7i and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.

Relapsed neuroblastomas are enriched with activating mutations of the RAS–MAPK signaling pathway. The MEK1/2 inhibitor trametinib delays tumor growth but does not sustain regression in neuroblastoma preclinical models. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP1 as an additional driver of relapsed neuroblastomas, as well as a mediator of trametinib resistance in other cancers. Here, we used a highly annotated set of high-risk neuroblastoma cellular models to modulate YAP1 expression and RAS pathway activation to test whether increased YAP1 transcriptional activity is a mechanism of MEK1/2 inhibition resistance in RAS-driven neuroblastomas. In NLF (biallelic NF1 inactivation) and SK-N-AS (NRAS Q61K) cell lines, trametinib caused a near-complete translocation of YAP1 protein into the nucleus. YAP1 depletion sensitized neuroblastoma cells to trametinib, while overexpression of constitutively active YAP1 protein induced trametinib resistance. Mechanistically, significant enhancement of G1–S cell-cycle arrest, mediated by depletion of MYC/MYCN and E2F transcriptional output, sensitized RAS-driven neuroblastomas to trametinib following YAP1 deletion. These findings underscore the importance of YAP activity in response to trametinib in RAS-driven neuroblastomas, as well as the potential for targeting YAP in a trametinib combination. Significance: High-risk neuroblastomas with hyperactivated RAS signaling escape the selective pressure of MEK inhibition via YAP1-mediated transcriptional reprogramming and may be sensitive to combination therapies targeting both YAP1 and MEK.

A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1—all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC., Polymorphisms in LMO1 are associated with increased susceptibility to develop neuroblastoma. Here, the authors show that LMO1 directly induces the transcription factor ASCL1, which regulates the differentiation of neurons, demonstrating that ASCL1 is part of the adrenergic neuroblastoma core regulatory circuit.

Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Here we show that the cell surface oncoprotein glypican 2 (GPC2) is highly expressed on multiple lethal pediatric brain tumors, including medulloblastomas, embryonal tumors with multi-layered rosettes, other CNS embryonal tumors, as well as definable subsets of highly malignant gliomas. To target GPC2 on these pediatric brain tumors with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we developed four mRNA chimeric antigen receptor (CAR) T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment. First, we validated and prioritized these CARs using in vitro cytotoxicity and T cell degranulation assays with GPC2-expressing neuroblastoma cells. Next, we expanded the testing of the two most potent GPC2-directed CAR constructs prioritized from these studies to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, locoregional delivery bi-weekly of two to four million GPC2-directed mRNA CAR T cells induced significant and sustained tumor regression in two orthotopic medulloblastoma models, and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells.One Sentence SummaryGlypican 2 is expressed on the surface of multiple pediatric brain tumors and can be successfully targeted with mRNA chimeric antigen receptor T cells.

While much of the somatic coding variation underlying the oncogenic transformation of pediatric high-grade gliomas (HGGs) has been profiled, transcriptional splicing programs of these tumors remain under-explored. Here, we characterize aberrant alternative splicing in pediatric midline HGGs (n = 84). We identified 19,275 recurrent and significant (20% change from control, P < 0.05, FDR < 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. Of those, 27% (n = 5,157) resulted in either a gain or loss of a known protein functional site within 3,294 genes. We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of master splicing regulators, exhibit significantly increased exon 4 inclusion in midline HGGs. This leads to a gain of two known phosphorylation sites in CLK1, increased CLK1 protein expression and hyper-phosphorylation of Serine-rich splicing factors. To assess the impact of this event, we performed differential splicing and expression analyses, comparing tumors with the highest (n= 5) and lowest (n = 5) exon 4 inclusion. We discovered 3,037 genes to be differentially up-regulated in high exon 4 inclusion tumors with an enrichment of cancer-related pathways, including DNA repair, mitotic spindle, myogenesis and EMT. We next integrated these gene signatures with protein-protein interaction networks of kinase and transcription factors and show that increased CLK1 exon 4 inclusion disrupts critical regulatory networks, such as those involving FOXM1, which is implicated in cell cycle and proliferation processes. In summary, we describe aberrant splicing in pediatric HGGs as an additional mechanism that could drive tumorigenesis. Future work will focus on molecular validation and therapeutic targeting of CLK1 in available HGG models. Characterizing tumor-specific splicing variation has the potential to open new therapeutic strategies and understand mechanisms of treatment resistance in children with central nervous system tumors.

Background The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma. Procedure Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models. Results Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1. Conclusions Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.

Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma., Measurement(s)histone H3-K4 methylation • histone H3-K27 methylation • histone acetylation • TF_binding_site • open_chromatin_regionTechnology Type(s)ChIP-seq • ChIP-Seq • ATAC-seqFactor Type(s)type of neuroblastoma cell line • MYCN amplification vs no MYCN amplificationSample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11993883

Background Unlike some adult cancers, most pediatric cancers are considered immunologically cold and generally less responsive to immunotherapy. While immunotherapy has already been incorporated into standard of care treatment for pediatric patients with high-risk neuroblastoma, overall survival remains poor. In a mouse melanoma model, we found that radiation and tumor-specific immunocytokine generate an in situ vaccination response in syngeneic mice bearing large tumors. Here, we tested whether a novel immunotherapeutic approach utilizing radiation and immunocytokine together with innate immune stimulation could generate a potent antitumor response with immunologic memory against syngeneic murine neuroblastoma. Methods Mice bearing disialoganglioside (GD2)-expressing neuroblastoma tumors (either NXS2 or 9464D-GD2) were treated with radiation and immunotherapy (including anti-GD2 immunocytokine with or without anti-CTLA-4, CpG and anti-CD40 monoclonal antibody). Tumor growth, animal survival and immune cell infiltrate were analyzed in the tumor microenvironment in response to various treatment regimens. Results NXS2 had a moderate tumor mutation burden (TMB) while N-MYC driven 9464D-GD2 had a low TMB, therefore the latter served as a better model for high-risk neuroblastoma (an immunologically cold tumor). Radiation and immunocytokine induced a potent in situ vaccination response against NXS2 tumors, but not in the 9464D-GD2 tumor model. Addition of checkpoint blockade with anti-CTLA-4 was not effective alone against 9464D-GD2 tumors; inclusion of CpG and anti-CD40 achieved a potent antitumor response with decreased T regulatory cells within the tumors and induction of immunologic memory. Conclusions These data suggest that a combined innate and adaptive immunotherapeutic approach can be effective against immunologically cold syngeneic murine neuroblastoma. Further testing is needed to determine how these concepts might translate into development of more effective immunotherapeutic approaches for the treatment of clinically high-risk neuroblastoma.

BACKGROUND Pediatric high grade glioma (pHGG) remains a fatal disease. Access to richly annotated biospecimens and patient derived tumor models will accelerate pHGG research and support translation of research discoveries. This work describes the pediatric high grade glioma set of the Children’s Brain Tumor Tissue Consortium (CBTTC) from the first release of the Pediatric Brain Tumor Atlas (PBTA). METHODS pHGG tumors with associated clinical data and imaging were prospectively collected through the CBTTC and analyzed as the Pediatric Brain Tumor Atlas (PBTA) with processed genomic data deposited into PedcBioPortal for broad access and visualization. Matched tumor was cultured to create high grade glioma cell lines analyzed by targeted and WGS and RNA-seq. A tissue microarray (TMA) of primary pHGG tumors was also created. RESULTS The pHGG set includes 87 collection events (73 patients, 60% at diagnosis, median age of 9 yrs, 55% female, 46% hemispheric). Operative reports, pathology reports and histology images are available for nearly all cases. Pre- and post-operative MRI images and reports are also available for a subset. Tumor WGS/RNAseq is available for 70 subjects. Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36% H3.3, 47% TP53, 24% ATRX and 7% BRAFV600E variants). In our panel of pHGG, six patients (8 tumors) harbored germline mismatch repair mutations with tumor hyper-mutation. A pHGG TMA (n=77), includes 36 patient tumors with matched sequencing. At least one established glioma cell line was generated from 23 patients (32%). Unique reagents include those derived from a H3.3 G34R glioma and from tumors with mismatch repair deficiency. CONCLUSION The CBTTC and PBTA have created an openly available integrated resource of over 2,000 tumors, including a rich set of pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. While the molecular aberrations underlying WNT- and SHH-driven MBs are relatively well understood, the oncogenic drivers that lead to Group 3/4 MBs are poorly defined, limiting therapeutic progress. In addition to genetic mutations and alterations, cancers display dysregulated transcription and translation. RNA-binding proteins (RBPs) play key roles in both transcription and translation, and a subset of RBPs are differentially expressed in many different cancers. Indeed, we have previously demonstrated an oncogenic role for the RBP LIN28B in neuroblastoma and it is known to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28B and other oncogenes. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. In support of our hypothesis we find that among the four subtypes, LIN28B levels are highest in Group 3 MB, and that overexpression is associated with significantly worse survival. Down-regulation of LIN28B results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. In addition we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally, in order to robustly define the signaling networks downstream from LIN28B that are involved in Group 3 MB metastasis, we have performed who transcriptome RNA-seq profiling of two group 3 cell lines following LIN28B depletion and plan to interrogate a subset of these based on expression change and functional relevance to LIN28B-mediated Group 3 MB metastasis. This work will help define the role for LIN28B in Group 3 MB aggressiveness and pave the way for similar studies in other cancers. Citation Format: Shubin W. Shahab, Jo Lynne Rokita, Kyle Juraschka, Sachin Kumar, Michael Taylor, Robert W. Schnepp, Tobey J. MacDonald, Anna M. Kenney. Targeting the RNA binding protein LIN28B in Group 3 medulloblastoma decreases proliferation and promotes apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3024.

Pediatric brain tumors comprise a heterogeneous molecular and histological landscape that challenges most current precision-medicine approaches. While recent large-scale efforts to molecularly characterize distinct histological entities have dramatically advanced the field’s capacity to classify and further define molecular subtypes, developing therapeutic and less toxic molecularly-defined clinical approaches remains a challenge. To define new approaches to meet these challenges and advance scalable, shared biospecimen- and data-resources for pediatric brain tumors, the Children’s Brain Tumor Network and Pacific Pediatric Neuro-Oncology Consortium, in partnership with the Alex’s Lemonade Stand Foundation Childhood Cancer Data Lab, launched OpenPBTA, a global open science Pediatric Brain Tumor Atlas initiative to comprehensively define the molecular landscape of pediatric brain tumors. The initiative contains multi-modal analyses of research- and clinical-trial based DNA and RNA sequences from nearly 1,000 subjects (with 1,256 tumors) along with their longitudinal clinical data. The OpenPBTA’s open science framework for analysis tests the capacity of crowd-sourced collaborative architectures to advance more rapid, iterative and integrated discovery of the underlying mechanisms of disease across pediatric brain and spinal cord tumors. Since the launch of the project, OpenPBTA has collaboratively created reproducible workflows for integrated consensus SNV, CNV, and fusion calling, enabled RNA-Seq-based classification of medulloblastoma subtypes, and more than 25 additional DNA- and RNA-based analyses. The open-science platform and associated datasets and processed results provide a continuously updated, global view of the integrated cross-disease molecular landscape of pediatric brain tumors. Such biospecimen- and clinically-linked scalable data resources provide unprecedented collaborative opportunities for precision-based, personalized therapeutic discovery and drug development with the upcoming further integration of proteomic sample data (N >300) and drug response datasets, additionally diversifying the multimodal discovery potential of crowd-sourced approaches for accelerated impact for children with brain tumors.

This protocol explains the process of how we collected MYCN, MYC, and Histone ChIP-Seq data, as well as ATAC-Seq data for neuroblastoma cell lines. This protocol is comprised of three sections: Cell Growth and Expansion for care of neuroblastoma cell lines, ChIP-Seq protocol, and ATAC-Seq Protocol. Table 1, within the document, outlines which data was collected for each cell line.

Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, opposes the effects of LIN28B. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7 and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.

BackgroundPediatric high grade glioma (pHGG) remains a fatal disease. Increased access to richly annotated biospecimens and patient derived tumor models will accelerate pHGG research and support translation of research discoveries. This work describes the pediatric high grade glioma set of the Children’s Brain Tumor Tissue Consortium (CBTTC) from the first release (October 2018) of the Pediatric Brain Tumor Atlas (PBTA).MethodspHGG tumors with associated clinical data and imaging were prospectively collected through the CBTTC and analyzed as the Pediatric Brain Tumor Atlas (PBTA) with processed genomic data deposited into PedcBioPortal for broad access and visualization. Matched tumor was cultured to create high grade glioma cell lines analyzed by targeted and WGS and RNA-seq. A tissue microarray (TMA) of primary pHGG tumors was also created.ResultsThe pHGG set included 87 collection events (73 patients, 60% at diagnosis, median age of 9 yrs, 55% female, 46% hemispheric). Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36%H3.3, 47%TP53, 24%ATRXand 7%BRAFV600E variants). A pHGG TMA (n=77), includes 36 (53%) patient tumors with matched sequencing. At least one established glioma cell line was generated from 23 patients (32%). Unique reagents include those derived from aH3.3G34R glioma and from tumors with mismatch repair deficiency.ConclusionThe CBTTC and PBTA have created an openly available integrated resource of over 2,000 tumors, including a rich set of pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.IMPORTANCE OF STUDYHigh-grade gliomas (HGG) remain the leading cause of cancer death in children. Since molecularly heterogeneous, preclinical studies of pediatric HGG will be most informative if able to compare across groups. Given their relatively rarity, there are few readily available biospecimens and cellular models to inform preclinical laboratory and genomic translational research. Therefore, the aim of this CBTTC study was to highlight the panel of pediatric HGG cases whose primary tumors have undergone extensive genomic analysis, have clinical data, available imaging and additional biospecimens, including tumor, nucleic acids, cell lines and FFPE tissue on a tissue microarray (TMA).