Pediatric Brain Tumors Research Articles

High Expression of GABAA Receptor β Subunit Genes Is Associated with Longer Overall Survival in Medulloblastoma

Background/Objectives: Most of the rapid inhibitory neurotransmission in the brain is mediated through activation of the γ-aminobutyric acid (GABA) type A (GABAA) receptor, which is a ligand-gated ion channel. GABAA receptor activation via GABA binding allows for an intracellular influx of Cl− ions, thus inducing cellular hyperpolarization. Each GABAA receptor consists of a combination of five subunits, and several subunits have been proposed as biomarkers and therapeutic targets in cancer. Here, we show the expression of genes encoding β subunits of the GABAA receptor, namely GABRB1, GABRB2, and GABRB3, across the four different molecular subgroups of medulloblastoma (MB), which is the most common malignant pediatric brain tumor. We also show the associations of GABAA receptor β subunits with MB patients’ overall survival (OS). Methods: The expression of genes encoding GABAA receptor β subunits was analyzed using a previously described dataset comprising 763 MB tumor samples. Patients were classified into high- and low-gene-expression groups, and the Kaplan–Meier estimate was used to examine the relationship between gene expression levels and patient OS. Results: High GABRB1 expression was associated with better OS within each of the four molecular subgroups. The GABRB2 gene showed higher transcript levels in Group 3 MB compared to all other subgroups, and high expression was associated with better prognosis in Group 3 tumors. GABRB3 expression was significantly higher in Group 3 and Group 4 MB, and high expression of GABRB3 genes was associated with longer OS in the sonic hedgehog (SHH) subgroup. The high expression of GABRB1, GABRB2, and GABRB3 is associated with longer patient OS in a subgroup-specific manner. Conclusions: These results indicate a role for GABAA receptors containing β subunits in influencing MB progression.

Abstract B002: A therapeutically targetable positive feedback loop between long noncoding RNA-HLX-2-7, HLX, and MYC that promotes childhood cancer group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is categorized into four main molecular subgroups: wingless pathway (WNT)-activated, sonic hedgehog pathway (SHH)-activated, group 3 (G3), and group 4 (G4). Although the driver pathways for G3 and G4 MB are unclear, most of these tumors are characterized by c-Myc and MYCN signatures, respectively. Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma formation and progression. We have identified lncRNA, lnc-HLX-2-7, as an oncogene and a potential therapeutic target in group 3 (G3) medulloblastomas. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of its host coding gene HLX, a sense overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium oxide nanoparticle–coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-ASO-lnc-HLX-2-7) and lipid nanoparticle-coated HLX (LNP-si-HLX) inhibits tumor growth by 40-50% in an intracranial medulloblastoma xenograft mouse model (p<0.01). Combining nanoparticle-coated lnc-HLX-2-7 and HLX with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with monotherapy (p<0.01). Thus, the lnc-HLX-2-7–HLX–MYC axis is important for regulating G3 medulloblastoma progression, providing a strong rationale for using lnc-HLX-2-7 and HLX as therapeutic targets for G3 MBs. Citation Format: Ranjan R Perera. A therapeutically targetable positive feedback loop between long noncoding RNA-HLX-2-7, HLX, and MYC that promotes childhood cancer group 3 medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B002.

Abstract B071: Identifying the clinical utility of CSF as a source of liquid biopsy in pediatric brain tumors

Abstract Pediatric tumors of the central nervous system (CNS) are a leading cause of cancer-related childhood mortality. Along with imaging, it is essential to molecularly characterize tumor tissue for the diagnosis, treatment, and prognosis of these cancers. Biopsies of deeply-seeded lesions may not be possible or may carry significant risks. On the other hand, liquid biopsy, using cerebral spinal fluid (CSF), provides a minimally invasive alternative to identify and monitor tumor DNA at crucial time points, from diagnosis to follow-up. This allows for molecular diagnosis, identification of therapeutic targets, monitoring response to therapy, and early detection of recurrence or progression. The aim of our study was to assess the practicality and usefulness of liquid biopsies in a diverse group of CNS tumor patients. We used a combination of methods, including a targeted hybridization capture panel and low-pass whole genome sequencing (LP-WGS) based on the sample characteristics. We analyzed a total of 243 CSF samples from patients with CNS tumors including: low-grade glioma (LGG; N=57), high-grade glioma (HGG; N=53), medulloblastoma (MB; N=40), germinoma (CNS-GCT; N=29), and no prior biopsy (N=42). We found somatic mutations in 88% of CSF samples from HGG and 67% from LGG with known tumor DNA variants and sufficient sequencing quality. It's important to note that in LGG patients, samples collected from lumbar puncture were positive only in disseminated cases, while a higher positivity rate was found in cases where CSF was collected from ventricular spaces. Moreover, we observed positive copy number alterations (CNA) in CSF samples collected from HGG, embryonal tumors, and CNS-GCT patients at the time of diagnosis and follow-up with a sensitivity of 64%. Within our sample cohort that had an unknown or query tumor due to no prior biopsy, we were able to detect diagnostic, targetable mutations and/or a positive CNA profile in 30% of cases. This supports the utility of liquid biopsy as a diagnostic tool in in CNS lesions. This tool may also assist in monitoring for relapse in high-grade tumors. For example, in a case study, we were able to detect recurrent MB by indication of a positive CNA profile in keeping with the primary tumor CNV, six years post-surgical resection. In CNS- GCT patients, we found that LP-WGS was highly sensitive at diagnosis as we have observed similar CNV in both CSF and tumor DNA. In summary, the utility of our liquid biopsy platform in the context of CNS cancers is an impactful tool that can aid clinicians in diagnosis, treatment and monitoring disease in a minimally invasive manner. Citation Format: Liana Nobre, Mansuba Rana, Yoshiko Nakano, Ian Burns, Robert Siddaway, Richard Yuditskiy, Cyril Li, Andrew Bondoc, Anthony Liu, Uri Taboori, Cynthia Hawkins. Identifying the clinical utility of CSF as a source of liquid biopsy in pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B071.

Myc 9aaTAD activation domain binds to mediator of transcription with superior high affinity.

The overexpression of MYC genes is frequently found in many human cancers, including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. Using our prediction algorithm, the nine-amino-acid activation domain (9aaTAD) has been identified in all four Yamanaka factors, including c-Myc. The predicted activation function was experimentally demonstrated for all these short peptides in transactivation assay. We generated a set of c-Myc constructs (1-108, 69-108 and 98-108) in the N-terminal regions and tested their ability to initiate transcription in one hybrid assay. The presence and absence of 9aaTAD (region 100-108) in the constructs strongly correlated with their activation functions (5-, 3- and 67-times respectively). Surprisingly, we observed co-activation function of the myc region 69-103, called here acetyl-TAD, previously described by Faiola et al. (Mol Cell Biol 25:10220-10234, 2005) and characterized in this study as a new domain collaborating with the 9aaTAD. We discovered strong interactions on a nanomolar scale between the Myc-9aaTAD activation domains and the KIX domain of CBP coactivator. We showed conservation of the 9aaTADs in the MYC family. In summary for the c-Myc oncogene, the acetyl-TAD and the 9aaTAD domains jointly mediated activation function. The c-Myc protein is largely intrinsically disordered and therefore difficult to target with small-molecule inhibitors. For the c-Myc driven tumors, the strong c-Myc interaction with the KIX domain represents a promising druggable target.

Abstract B012: M-PACT: Methylation-based predictive algorithm for CNS tumor liquid biopsies

Abstract Background: DNA methylation-based classification has been transformative in the molecular diagnostics of pediatric central nervous system (CNS) tumors. Current diagnostic pipelines integrate histopathology and molecular tools in accordance with the WHO classification of CNS tumors which rely on the availability of tissue specimens. However, some tumors are not amenable to neurosurgical resection due to their high-risk locations and tissue collection is not routinely performed at relapse. In addition, molecular biomarkers are largely lacking for longitudinal disease monitoring in pediatric neuro-oncology. Liquid biopsies (LBs) have emerged as a minimally invasive, longitudinal source of tumor-derived cell-free DNA (cfDNA) ideally suited for detecting minimal residual disease (MRD). In addition, LBs provide a sensitive means of studying tumor evolution at high temporal resolution. Success rates of LB analyses in neuro-oncology have varied drastically between studies, warranting the deployment of more robust and reproducible assays. Methods: We applied a novel cfDNA methylation-based workflow to a large cohort of cerebrospinal fluid (CSF) samples collected from pediatric brain tumor patients (n>200 patients). Enzymatic methylation sequencing (EM-seq) was adapted for CSF-derived cfDNA inputs in the picogram range, enabling the acquisition of genome-wide cfDNA methylation profiles across the cohort. Utilizing non-oncological cfDNA profiles and CNS tumor DNA methylation datasets as a reference, we developed an innovative computational pipeline that incorporates DNA methylation imputation and deep learning of a neural network. In addition, deconvolution of cfDNA profiles facilitated classification of low tumor burden samples, including those with balanced genomes that would have been missed using previous generation assays. Results: Our CSF-based classifier, M-PACT (Methylation-based Predictive Algorithm for CNS Tumors), yielded accurate tumor detection and entity prediction (AUC=0.9) in our benchmarking cohort (n>100 CSF samples). Proof-of-concept studies showed the potential of this methodology to track tumor evolution in serial CSF samples at both the genetic and epigenetic level, illuminating potential mechanisms driving treatment resistance and recurrence. Conclusions: Collectively, this study provides the framework for robust methylation- based tumor detection and classification of CSF LBs. M-PACT can be applied to aid in tumor diagnostics and to detect MRD during follow-up, warranting prospective validation of its broad applicability and clinical utility in future trials. Citation Format: Tom T. Fischer, Kyle S. Smith, Katie Han, Anna Kostecka, Hong Lin, Daniel Senfter, Tatjana Wedig, Nathalie Schwarz, Natalia Stepien, Sibylle Madlener, Christine Haberler, Esther Hulleman, Sandeep K. Dhanda, Stefan M. Pfister, Santhosh Upadhyaya, Amar Gajjar, Giles W. Robinson, Joonas Haapasalo, Hannu Haapasalo, Kristiina Nordfors, Johannes Gojo, Kendra K. Maass, Kristian W. Pajtler, Paul A. Northcott. M-PACT: Methylation-based predictive algorithm for CNS tumor liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B012.

Tumor-Promoted Changes in Pediatric Brain Histology Can Be Distinguished from Normal Parenchyma by Desorption Electrospray Ionization Mass Spectrometry Imaging

Background: Central nervous system (CNS) tumors are the second most frequent type of neoplasm in childhood and adolescence, after leukemia. Despite the incorporation of molecular classification and improvement of protocols combining chemotherapy, surgery, and radiotherapy, CNS tumors are still the most lethal neoplasm in this age group. Mass spectrometry imaging (MSI) is a powerful tool to map the distribution of molecular species in tissue sections. Among MSI techniques, desorption electrospray ionization (DESI-MSI) has been demonstrated to enable reliable agreement with the pathological evaluation of different adult cancer types, along with an acceptable time scale for intraoperative use. Methods: In the present work, we aimed to investigate the chemical profile obtained by DESI-MSI as an intraoperative surgical management tool by profiling 162 pediatric brain biopsies and reporting the results according to the histopathology and molecular profile of the tumors. Results: The 2D chemical images obtained by DESI-MSI allowed us to distinguish tumor-transformed tissue from non-tumor tissue with an accuracy of 96.8% in the training set and 94.3% in the validation set after statistical modeling of our data using Lasso. In addition, high-grade and low-grade tumors also displayed a distinct chemical profile when analyzed by DESI-MSI. We also provided evidence that the chemical profile of brain tumors obtained by DESI-MSI correlates with methylation-based molecular classes and specific immunophenotypes found in brain biopsies. Conclusions: The results presented herein support the incorporation of DESI-MSI analysis as an intraoperative assistive tool in prospective clinical trials for pediatric brain tumors management in the near future.

Foundation models for fast, label-free detection of glioma infiltration.

A critical challenge in glioma treatment is detecting tumour infiltration during surgery to achieve safe maximal resection1-3. Unfortunately, safely resectable residual tumour is found in the majority of patients with glioma after surgery, causing early recurrence and decreased survival4-6. Here we present FastGlioma, a visual foundation model for fast (<10 s) and accurate detection of glioma infiltration in fresh, unprocessed surgical tissue. FastGlioma was pretrained using large-scale self-supervision (around 4 million images) on rapid, label-free optical microscopy, and fine-tuned to output a normalized score that indicates the degree of tumour infiltration within whole-slide optical images. In a prospective, multicentre, international testing cohort of patients with diffuse glioma (n = 220), FastGlioma was able to detect and quantify the degree of tumour infiltration with an average area under the receiver operating characteristic curve of 92.1 ± 0.9%. FastGlioma outperformed image-guided and fluorescence-guided adjuncts for detecting tumour infiltration during surgery by a wide margin in a head-to-head, prospective study (n = 129). The performance of FastGlioma remained high across diverse patient demographics, medical centres and diffuse glioma molecular subtypes as defined by the World Health Organization. FastGlioma shows zero-shot generalization to other adult and paediatric brain tumour diagnoses, demonstrating the potential for our foundation model to be used as a general-purpose adjunct for guiding brain tumour surgeries. These findings represent the transformative potential of medical foundation models to unlock the role of artificial intelligence in the care of patients with cancer.

Innovative pan-tumor target strategy for CAR-T therapy: cancer-specific exons as novel targets for pediatric solid and brain tumors.

Chimeric antigen receptor T-cell (CAR-T) immunotherapy has achieved remarkable success in treating chemotherapy-refractory hematological malignancies. However, its efficacy in solid and brain tumors remains limited due to challenges such as insufficient target antigens, poor T-cell adaptability, inefficient tumor site trafficking, and the immunosuppressive tumor microenvironment. To address these challenges, Shaw and colleagues proposed an innovative strategy targeting cancer-specific exons (CSEs) in pediatric solid and brain tumors. Using RNA sequencing data from 16 tumor types, the study identified 157 highly tumor-specific targets, including both known and novel proteins. The researchers validated several targets, including FN1 and COL11A1, demonstrating their therapeutic potential in in vitro and in vivo models. The study's approach of integrating exon-level analysis with a broad search for extracellular matrix proteins offers a new frontier for CAR-T therapy, providing valuable insights for improving immunotherapy in pediatric solid tumors. Although promising, the study also highlights the need for further evaluation of tumor recurrence and CAR-T cell exhaustion. The identification of novel pan-tumor targets may revolutionize CAR-T therapy design and expand its application in pediatric cancer treatment.

EPID-01. PEDIATRIC BRAIN TUMORS IN SUB-SAHARAN AFRICA:A SYSTEMATIC REVIEW AND META-ANALYSIS

Abstract OBJECTIVE brain tumors are a global problem, leading to higher cancer-related morbidity and mortality rates in children. Despite the progressive though slow advances in neuro-oncology care, research, and diagnostics in sub-Saharan Africa (SSA), the epidemiological landscape of pediatric brain tumors (PBTs) remains underestimated. This study aimed to systematically analyze the distribution of PBT types in SSA. METHODS major databases and grey literature were searched for literature on PBTs in SSA published before October 29, 2022. A proportional meta-analysis was performed. RESULTS forty-nine studies, involving 2360 children, met the inclusion criteria for review; only 20 (40.82%) were included in the quantitative analysis. South Africa and Nigeria were the countries with the most abundant data. Glioma not otherwise specified (NOS) was the common PBT in the 4 SSA regions combined. However, medulloblastoma was more commonly reported in Southern-SSA(p=0.01) than in other regions. The prevalence and the overall pooled proportion of the 3 common PBTs was estimated at 46.27% and 0.41 (95%CI 0.32–0.50, 95% prediction interval [PI] 0.11–0.79), 25.34% and 0.18 (95%CI 0.14–0.21, 95%PI 0.06–0.40), and 12.67% and 0.12 (95%CI 0.09–0.15, 95%PI 0.04–0.29) for glioma-NOS, medulloblastoma, and craniopharyngioma, respectively. Sample size moderated the estimated proportion of glioma-NOS (p = 0.02). The highest proportion of craniopharyngiomas was in Western-SSA, and medulloblastoma and glioma-NOS in Central-SSA. CONCLUSION these findings provide insight into the trends of PBT types and the proportion of the top 3 most common tumors across SSA. Although statistical conclusions are difficult due to the inconsistency in the data. We identify critical areas for policy development and collaborations that can facilitate improved outcomes in PBTs in SSA. More accurate epidemiological studies are needed to better understand the burden of the disease and the geographic variation in their distribution, and to raise awareness in their subsequent management.

IMMU-07. THE IMMUNE LANDSCAPE OF PEDIATRIC BRAIN TUMORS: A TRANSCRIPTOMIC ANALYSIS

Abstract Despite standard-of-care therapy, pediatric brain tumors are now the leading cause of cancer-related death in children, highlighting an urgent need for the development of new treatments. Immunotherapy is actively being evaluated as a therapeutic approach to pediatric brain tumors, however, more research is needed to reveal additional antigenic targets that exist in these malignancies. In this study, our team performed a transcriptomic analysis of pediatric brain tumor RNA sequencing data within the Children’s Brain Tumor Network (CBTN) to identify intra and extracellular antigens that are highly expressed across major tumor types. Interestingly, our team observed that most tumors had high gene expression of a group of antigens that were unique to that specific tumor type. Specifically, our analysis revealed that diffuse midline gliomas (DMGs) highly expressed the extracellular antigens CA9, CTCFL, and ST8SIA1, whereas atypical rhabdoid tumors (ATRTs) highly expressed MUC1 and TEK. Other tumors that displayed high gene expression for a variety of antigenic targets included H3-wildtype high-grade gliomas (HGGs), and ependymomas (EPNs). Strikingly, we observed minimal difference in antigen expression in primary tumors when compared to patient-matched recurrences. Beyond antigen expression, we evaluated the expression of antigen processing machinery (APM) which includes major histocompatibility complexes (MHCs). Our findings revealed that DMGs had the lowest level of APM expression, however, these tumors uniquely expressed high levels of the immune checkpoints ADORA2A, CD276, and KLRC1. Contrarily, ATRTs showed a high expression of immune checkpoints CD274 (PD-L1), PVR, and CD80. Other tumors included in our analysis had unique expression patterns of antigen, as well as APM and immune checkpoints. Our findings illustrate that pediatric brain tumors have distinct expression patterns of antigens, APM, and immune checkpoints that are specific to tumor type, emphasizing diverse, rather than general, immunotherapeutic approaches must be considered for childhood brain tumors.

NIMG-65. SYNTHETIC MRI GENERATION TO FACILITATE AUTOSEGMENTATION OF PEDIATRIC BRAIN TUMORS IN LIMITED DATA SCENARIOS

Abstract BACKGROUND The availability of standard multiparametric MRI sequences, including pre-contrast T1w (T1w-pre), post-contrast T1w (T1w-post), T2w, and FLAIR images, is essential for accurate segmentation of tumor subregions and subsequent response assessment in pediatric brain tumors (PBTs). However, clinical MRI sets are often incomplete due to imaging artifacts or inconsistent acquisition protocols across different centers. This study leverages Generative Adversarial Networks (GANs) to synthesize missing FLAIR and T1w-pre images from T2w and T1w-post images, respectively, to facilitate tumor segmentation in PBTs. METHODS We developed two GAN models based on the pix2pix framework, trained, validated, and tested on FLAIR-T2w and T1w-post-T1w-pre pairs from a multi-histology and multi-institutional cohort of 476 subjects with PBTs from the Children’s Brain Tumor Network (CBTN). The perceptual quality of the synthesized T1w-pre and FLAIR images was evaluated using the Structural Similarity Index (SSI; ideal value = 1). The robustness of the synthesized sequences to replace missing MRI sequences was evaluated by comparing the segmentation performance (Dice score) of our pre-trained autosegmentation model using all real modalities versus replacing real sequences with their synthesized counterparts. RESULTS The T1w-pre and FLAIR synthesis GANs achieved mean SSI values of 0.95 and 0.93, respectively. Median Dice scores for segmentation of whole tumor (WT), enhancing tumor (ET), and tumor core (TC) were calculated for real sequences: 0.90, 0.83, 0.89, replacing real FLAIR with synthetic: 0.86, 0.81, 0.86, and replacing real T1w-pre with synthetic: 0.90, 0.75, 0.89. No statistical significance (p&amp;gt;0.05) was observed in segmentation performance for WT, TC, ET when replacing real T1w-pre with synthetic, and for ET when replacing real with synthetic FLAIR sequences. CONCLUSION This method achieves robust segmentation performance in limited data scenarios by synthesizing missing MRI sequences. Future work includes improving FLAIR synthesis and segmentation performance for WT and ET by implementing and comparing other GAN architectures.

NCOG-17. THE COMBAT PROJECT - CORE POST OPERATIVE MORBIDITY SET FOR PAEDIATRIC BRAIN TUMOURS

Abstract BACKGROUND Brain tumours are associated with high morbidity both from the tumour and the medical and surgical interventions used to treat them. Post operative morbidity reporting following surgery for paediatric brain tumours is poor due to the lack of a common language when reporting harms. One solution is to develop a core outcome set to stipulate the minimum post operative adverse outcomes that should be reported. The COMBAT (Core Post Operative Morbidity Set for Paediatric Brain Tumours) Project will develop a core outcome set that can be applied to post operative paediatric brain tumour patients to harmonise how adverse outcomes are measured and reported, enabling comparison between different trials, studies and clinical services. We aim to generate interest in The COMBAT Project and invite participation in the project from healthcare professionals and researchers experienced in paediatric neuro-oncology for the consensus process. METHODS AND ANALYSIS We will engage with national and international stakeholders including patients and their carers, healthcare professionals and researchers. A systematic review will identify harms reported in post operative paediatric brain tumour patients and how they are measured. Qualitative interviews with patients and carers will identify any harms of importance to them. A Delphi survey will be created from the harms identified. A consensus meeting will be held to finalise the core outcome set. The final core outcome set will be published and disseminated to encourage uptake. DISCUSSION There is a clear need for a common language to harmonise measurement and reporting of harms following surgery for paediatric brain tumour patients. This project will define post operative adverse outcomes that are of critical importance to key stakeholders. It will be used to standardise surgical morbidity outcome measurement and reporting in trials, studies and clinical practice and will inform future research in this field.

CTIM-20. IDENTIFYING CANDIDATE ANTIGEN TARGETS FOR PEDIATRIC BRAIN TUMOR IMMUNOTHERAPIES THROUGH COMPREHENSIVE IMMUNOGENOMIC ANALYSIS

Abstract BACKGROUND Identifying tumor rejection antigens continues to be a significant challenge in the development of effective immunotherapies and their application to pediatric brain tumors. METHODS To identify candidate antigen targets for Medulloblastoma (primary MB, n=170; recurrent MB, n=21), Diffuse Intrinsic Pontine Glioma (DIPG, n=10), and Pediatric High-Grade Astrocytoma (HGA, n=12) for adoptive cellular therapy, we conducted a comprehensive immunogenomic analysis of the transcription profiles of the most aggressive pediatric brain tumors and performed in silico antigen prediction across a wide range of antigen classes. IMPORTANCE OF THE STUDY Pediatric brain tumors are heterogeneous diseases, and current standard treatments do not adequately address this variability. Newer and safer patient-specific treatment approaches are needed for high-risk patients who do not respond to standard therapies. Cellular immunotherapy could be crucial for improving survival and reducing morbidity. For an effective immune response against these brain tumors, appropriate tumor antigens must be targeted. While subgroup-specific genetic mutations in medulloblastoma patients have been reported, the immunogenicity of these genetic alterations remains unknown. Using a custom antigen prediction pipeline, we identified potential tumor rejection antigens with significant implications for the development of cellular therapies for MB, DIPG, and HGA. RESULTS Antigen prediction analysis revealed that most patients express few candidate neoantigen targets that pass all filtering criteria. Overall, the proportion of predicted tumor-associated antigens (TAAs) was higher than that of neoantigens and gene fusions for all molecular subtypes, except for SHH, which exhibited a higher neoantigen burden. Notably, cancer-testis antigens and previously unrecognized neurodevelopmental antigens were found to be expressed in most patients across all medulloblastoma subgroups. Although the absolute immune cell content is predicted to be low, immune gene-signature analysis revealed subgroup-specific differences.

EPCO-30. TRANSCRIPTOMIC ANALYSIS IDENTIFIES REGIONAL BIOLOGY AND OUTCOME HETEROGENEITY IN MEDULLOBLASTOMA AND EPENDYMOMA SUBTYPES

Abstract Medulloblastoma, the most common malignant pediatric brain tumor, includes four distinct subtypes: Wnt, Sonic Hedgehog (Shh), Group 3, and Group 4. In contrast, ependymomas, which account for 10% of pediatric brain tumors and 4% of adult brain tumors, are categorized into three types: supratentorial (ST), posterior fossa (PF), and spinal (SP). While molecular subgrouping has significantly advanced the classification of these diseases, the extent of heterogeneity within these subgroups remains unknown. To address this, we collected bulk RNA sequencing (RNASeq) data from 888 medulloblastoma and 370 ependymoma from various institutions in North America and Europe. Our goal was to establish a comprehensive reference landscape for both medulloblastoma and ependymoma. We also included 42 forebrain and 52 hindbrain fetal samples to contrast against the tumor samples. Our analysis revealed that each disease subtype forms distinct clusters. Previously, subtyping was primarily achieved using methylation data; however, we demonstrate that bulk RNASeq data can also accurately identify disease subtypes. By examining gene expression patterns, gene fusions, and copy number aberrations, we validated existing molecular features and discovered new ones for each subtype. Additionally, we developed an innovative pipeline to identify specific biological patterns for a given region within the landscape. This resource coupled with mapping of new patients’ bulk RNASeq data onto the reference landscape, facilitating accurate disease subtype identification for new patients. This extensive dataset, available through the online tool Oncoscape, will assist the scientific community in uncovering novel therapeutic targets and advancing research in pediatric brain tumors.

BIOM-19. METHYLATION PROFILING OF PLASMA CELL-FREE DNA IN PEDIATRIC BRAIN TUMOR PATIENTS

Abstract BACKGROUND Circulating tumor DNA (ctDNA) assays are being evaluated to inform clinical decisions in cancer care in adult patients, but the study in pediatric brain tumor patients is rare partly due to very low levels of ctDNA in blood. Currently, treatment decisions require invasive diagnostic surgical biopsies that carry risks and morbidity. The aim of this study is to utilize methylomes from plasma ctDNA for non-invasive diagnosis in pediatric brain tumor patients. METHODS Using cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), which with low input DNA requirement, we measured methylation profiles of plasma samples from 77 pediatric brain tumor patients and 16 patients with non-neoplastic diseases like epilepsy. Binomial GLMnet classifiers of tumor or tumor subtype were built, in 2000 iterations of 60% training sets. Performance was evaluated in 40% test sets. RESULTS. These 77 pediatric brain tumor patients were a mixture of the most common pediatric brain tumors, include circumscribed astrocytic gliomas (N=23), glioneuronal tumors (N=16), embryonal tumor (N=9), ependymal tumor (N=7), pediatric type diffuse high grade glioma (N= 5), glioma NOS (N=4), mesenchymal tumor (N=4) and other pediatric brain tumors (N=9). The methylation profile differentiated brain tumors from non-neoplastic diseases with 82.9% accuracy (precision = 92.6%, sensitivity = 86.2%, specificity = 66.7 %). For subtype analysis, the methylation profile detected circumscribed astrocytic glioma from non-neoplastic diseases with 85.7% accuracy (precision = 87.5%, sensitivity = 87.5 %, specificity = 83.3 %), and glioneuronal tumors from non- neoplastic diseases with 83.3% accuracy (all precision, sensitivity, and specificity = 83.3 %). CONCLUSIONS The results suggest that methylation profiling of plasma cell-free DNA has the potential to discriminate between pediatric brain tumor patients and patients with non- neoplastic diseases. Larger population cohorts to train even more accurate classifiers will be needed.

RBIO-02. DEVELOPMENT OF FLASH-CAR RADIOIMMUNOTHERAPY FOR PEDIATRIC BRAIN TUMORS

Abstract Pediatric brain tumors including medulloblastoma and high-grade glioma are generally refractory to T cell-based immunotherapy, largely due to an immune-hostile microenvironment infiltrated extensively with immunosuppressive macrophages. Ultra-high dose rate (FLASH) radiotherapy holds promise for treating solid tumors, given the potential lower toxicity in normal tissues and possible favorable impact on tumor immunity. Using a genetically engineered mouse model of medulloblastoma and glioma, we show that FLASH radiation stimulates pro-inflammatory polarization in tumor macrophages. Single-cell transcriptome analysis shows that FLASH proton beam radiation skews macrophages towards proinflammatory phenotypes and increases T cell infiltration in the medulloblastoma model. Further bulk transcriptome analyses reveal that FLASH radiation reduces peroxisome proliferator-activated receptor (PPAR)-γ and arginase-1 expression and inhibits immunosuppressive macrophage polarization under stimulus-inducible conditions. Mechanistically, FLASH radiation abrogates lipid oxidase expression and oxidized low-density lipid (oxLDL) generation to reduce PPARγ activity, while standard radiation induces reactive oxygen species (ROS)-dependent PPARγ activation in macrophages. Notably, FLASH radiotherapy improves infiltration and activation of chimeric antigen receptor (CAR) T cells and sensitizes tumors to GD2 CAR T immunotherapy in the autochthonous medulloblastoma and syngeneic glioma models. These findings suggest that FLASH radiotherapy may reprogram macrophage lipid metabolism to reverse tumor immunosuppression and that combination FLASH-CAR radioimmunotherapy may offer exciting opportunities for treatment of pediatric brain tumor.

TMOD-29. BETA INTEGRINS REGULATE MITOCHONDRIALLY-MEDIATED CELL MIGRATION IN H3K27M-DIFFUSE MIDLINE GLIOMA

Abstract Infiltration into the brain tissue is one of the main features of diffuse midline gliomas (DMG), also characterized by the histone mutation H3K27M and overall survival of 12-15 months from diagnosis. To study which genes are critical for H3K27M-DMG tumor cell migration in the brain, we established a novel two-step pooled whole genome CRISPR-Migration screen of metastatic H3K27M-DMG (n=3) and glioblastoma (GBM, n=2) stem cells by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds. Genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all H3K27M-DMG lines. From those, only knockout of ITGB1 (ITGB1-KO) fully ablated in vitro migration in all models. In human tumor RNAseq, expression of ITGB1 correlated with higher glioma grade and worse survival specifically in H3K27M-DMG across pediatric brain tumors. In H3K27M-DMG scRNAseq, increased ITGB1 expression was found in cells with OPC-like/Mesenchymal cell signature associated with leading edge and microvasculature. Unexpectedly, ITGB1-KO led to the decreased expression of multiple mitochondrial NADH dehydrogenase genes, and reduced levels of glycolysis and oxidative phosphorylation (OXPHOS) activity. Preliminary mechanistic data linked this phenotype to increased mitochondrial fusion. Genetic (ITGB1-KO) and pharmacologic (anti-ITGB1 antibody AIIB2, CNS delivered) targeting of ITGB1 in a novel orthotopic model of metastatic H3K27M-DMG (UMPED83) led to reduced proliferation and significant extension of survival (p=0.0003 ITGB1-KO, and p=0.0006 AIIB2). Interestingly, ITGB1-KO in another orthotopic H3K27M-DMG model (SVZ) led to upregulation of other integrins (ITGB3 and ITGB5) without change in survival, resulting in susceptibility to treatment with the ITGB3/5 inhibitor Cilengitide (p=0.006). Treatment with AIIB2 and Cilengitide alone or in combination was ineffective in an H3WT adult GBM orthotopic model suggesting unique H3K27M-DMG targetability. In summary, targeting of integrin pathways offers untapped therapeutic opportunities in H3K27M-DMG by impairing tumor cell survival through altered mitochondrial dynamics.

EXTH-49. DEVELOPMENTAL ANTIGENS CAN TARGET EMBRYONAL PEDIATRIC BRAIN CANCERS IN ADOPTIVE CELLULAR THERAPY

Abstract Mutations in tumor neoantigens have shown promise in immunotherapies for many cancers, yet pediatric brain tumors, which typically have a lower mutational burden, offer fewer opportunities to capitalize on these targeted therapies. Studies indicate that medulloblastoma (MB) and brain stem gliomas (BSG) originate from abnormal reactivation of post-natal developmental processes. This led us to investigate whether proteins expressed during the early post-natal development of the mouse cerebellum and brainstem could be powerful antigens to fight MB and BSG, respectively. We performed both in vitro and in vivo studies in mice with the RNA of developmental antigens from the brain stem and cerebellum in an adoptive cellular therapy (ACT) platform. We evaluated the reactivity and therapeutic efficacy of these treatments as sustainable antigenic targets against BSG and MBs. We demonstrate that T cells activated towards these non-mutated, tissue-specific developmental antigens can recognize distinct subtypes of MB and BSG, and activate specifically without cross-reacting to the normal brain. This conferred a survival benefit in established orthotopic models of these pediatric brain tumor types. Our research exhibits the value developmental antigens can offer when serving as tumor rejection antigens in MB and BSG.

TMET-36. INCREASED METHIONINE CYCLE DRIVES PYRIMIDINE METABOLISM IN LETHAL PEDIATRIC EPENDYMOMAS

Abstract Supratentorial ependymomas (ST-EPN) are pediatric brain tumors found in the cerebral hemisphere, driven by the oncogenic fusion of ZFTA with RELA. Other than surgery and radiotherapy, these tumors do not have effective therapeutic options. Recurrent ST-EPN tumors are highly aggressive and hard to treat, which makes these tumors lethal. To understand the disease mechanism and potentially identify or develop therapeutic targets, we established patient-derived models and interrogated the genetic and metabolic dependencies using cutting-edge, near-genome-wide genetic screening and metabolomic tools. Our integrative transcriptomics and metabolic analysis identify that the ZFTA-RELA fusion drives methionine metabolic reprogramming in the cancer stem cells to regulate permissive epigenetic status and pyrimidine nucleotide synthesis to fuel tumor growth. Limiting methionine or inhibiting MAT2A, a rate-limiting enzyme of methionine metabolism in ST-EPN cells, results in profound cell cycle arrest and make the cells vulnerable to pyrimidine inhibitors compared to other pediatric brain tumors. Thus, for treatment, along with radiotherapy, blocking the methionine cycle by using the methionine limited diet or in combination with pyrimidine metabolism inhibitors can be a novel therapeutic strategy against the aggressive paediatric ST-EPN tumors.

NCOG-16. A PROSPECTIVE OBSERVATIONAL STUDY OF CLINICAL PROFILE, COURSE AND OUTCOME OF PRIMARY BRAIN TUMOR AT A TERTIARY CARE CENTER

Abstract Primary brain tumors include tumor arising from neuro-epithelial cells, cranial nerves, meninges, germ cells, pituitary gland, and blood-forming organs. These tumors can present with either generalized or focal signs and symptoms.General symptoms are due to raised intracranial pressure (headache, vomiting). Focal symptoms are due to the compression of a structure or destruction (focal neurological deficits). The aim of this study is to analyze the clinical profiles of pediatric and adult brain tumor patients. Data of primary brain tumor patients who were diagnosed from 1st December 2019 to December 2021 were prospectively collected and analysed. In our study of 61 patients, the majority of patients had GBM (32.8%) and astrocytoma (31.1%) and medulloblastoma (11.5%). Less common histologies ependymomas (9.8%), meningioma (6.6%), brain stem glioma (3.3%), oligodendrogliomas (3.3%) and germinoma (1.6%). Out of 19 astrocytomas, 10 were of low grade (1 or 2), while 9 were of high grade (3 or 4).The majority (61.7%) of gliomas were of high grade, while the remaining 38.3% were of low grade. The majority (57.5%) of patients received multimodal treatment,26.2%received dual modality (surgery and RT), while the remaining 16.3% received single modality, either surgery or RT. Most (86.9%) of the patients treated with radiotherapy as a part of a single or multimodal treatment. A significant percentage of patients (77.1%) received concurrent and adjuvant temozolamide-based chemotherapy. Primary brain tumors have a different profile in pediatric patients. Among pediatric patients, the median age was 11.8 years, with male predominance. This study concludes that primary brain tumors have a different profile among adults and pediatric patients, with the majority of patients achieving a response to the first line therapy with an overall good tolerance for therapy.