Pediatric High-grade Gliomas Research Articles

HGG-41. COMBINATORIAL BH3-MIMETIC STRATEGY FOR OVERCOMING RADIORESISTANCE IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND High-grade gliomas (HGGs) are a leading cause of cancer-associated death in children. Radiation therapy (RT) continues to be the only standard-of-care treatment across HGG subtypes, but disease frequently recurs following only a transient response. We have recently identified that HGGs utilize the pro-survival BCL-2 family of proteins to evade cell death after radiation, and we demonstrated that effective inhibition of BCL-2 with the CNS-penetrant drug venetoclax could be utilized to disrupt this resistance and augment radiation-induced cell killing. While this treatment significantly prolonged survival in orthotopic mouse models of pediatric HGG, tumors invariably developed resistance to single-agent venetoclax and recurred. METHODS We profiled tumors from venetoclax-resistant animals for alternate BCL-2 family binding. We then assessed susceptibility to MCL1 inhibition in venetoclax-treated or treatment naïve culture models. Synergy was tested in vitro, and mitochondrial ROS production and induction of apoptosis was assessed using the MCL1-specific agent S63845. Finally, venetoclax and S63845 were tested in combination following fractionated radiotherapy in orthotopic models of DIPG. RESULTS MCL1 is an acquired dependency following treatment with RT + venetoclax. S63845 synergizes with venetoclax following RT to drive mitochondrial ROS production and apoptosis. The combination of venetoclax and S63845 significantly prolongs survival in xenograft models of DIPG. CONCLUSIONS Combinatorial targeting with BH3 mimetics augments radiation-induced cell killing and may open new therapeutic avenues for pediatric HGG.

HGG-18. ONC201 COMBINED WITH THE ONCOLYTIC ADENOVIRUS DELTA-24-RGD AS A NEW THERAPEUTIC REGIME FOR PEDIATRIC HIGH-GRADE AND DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Pediatric high-grade (pHGGs) and diffuse midline gliomas (DMGs) patients have the worst survival rates in childhood cancer. The oncolytic adenovirus Delta-24-RGD and the imipridone ONC201 are two of the most promising therapeutic agents in the field, having demonstrated safety and effectiveness in clinical trials. Therefore, we evaluated whether the Delta-24-RGD/ONC201 combination could increase the therapeutic benefit in pHGGs and DMGs. METHODS A battery of human pHGG, DMG, and murine DMG cell lines was used for the study. Viral replication was assessed by hexon titration. Characterization of combination response was assessed by MTS, RNAseq, Seahorse, and immunofluorescence. In vivo experiments were performed to assess the efficacy of the proposed combination. The tumor microenvironment infiltration was evaluated by flow cytometry. RESULTS ONC201 cotreatment with Delta-24-RGD did not affect the virus replication capability in vitro. Cytotoxicity studies showed that cotreatment was either synergistic or additive. Importantly, cells treated with the combination maintain the mitochondrial damage caused by ONC201, showed a significant reduction of IGF1/IGF1R and PDGF pathways, and increased nuclear DNA damage. Remarkably, Delta-24-RGD/ONC201 treatment in human pHGG and DMG xenograft-bearing mice showed a safe toxicity profile and a significant increase in the median overall survival compared to each agent alone. Combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment towards a proinflammatory phenotype. We observed an increase in both lymphoid and myeloid lineage in the combined treatment versus either single treatment. Interestingly, Delta-24-RGD/ONC201 produces a higher activation in CD8+ T cells, higher activation and proliferation in CD4+ T cells, and less immunosuppressed regulatory T cells. CONCLUSIONS Our data supports that the Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone by potentiating nuclear DNA damage and improving the antitumor (immune) response. Altogether, our results reveal the potential of this combination for future clinical trials.

NURS-20. A PATIENT AND FAMILY EDUCATION PROJECT: COMPREHENSIVE RESOURCE FOR MEK INHIBITOR-INDUCED PARONYCHIA

Abstract BACKGROUND Targeted therapies for pediatric tumors of the central nervous system have recently brought promising results as well as new challenges in the clinical management of these patients. FDA approval of MEK inhibitors has prompted a significant increase in use of these agents in pediatric neuro-oncology patients. MEK inhibitors are commonly used for treatment of pediatric low and high-grade gliomas, as well being a proven treatment for plexiform neurofibromas. Cutaneous reactions to targeted therapy occur frequently, with paronychia arising in more than half the patients. METHODS Despite providing a robust MEK inhibitor skin care in-service at the start of treatment, the multidisciplinary neuro-oncology team identified a need for specific paronychia education for patients undergoing therapy with MEK inhibitors. The education content was developed following a thorough literature review of MEK inhibitors and associated side effects. Evidence-based information was selected to create a clear, concise, one-page educational resource. RESULTS The educational flyer addressed five key educational objectives including defining and reviewing paronychia as an expected side effect of MEK inhibitor therapy, discussing strategies for prevention and management of paronychia, and identifying when to contact a healthcare provider for paronychia support. Content included written and pictorial definitions to aid in recognition of paronychia, bulleted list and visual guide for preventive nail maintenance, recommendations and instructions for over-the-counter management strategies, review of therapies that may be prescribed, and specific guidelines on when to seek prompt medical attention. This flyer served as a valuable educational tool, equipping individuals undergoing MEK inhibitor therapy with knowledge about paronychia, prevention, and effective management. CONCLUSION MEK inhibitor-induced paronychia is an undesirable side effect that can impact adherence to treatment. By empowering patients and families with this educational resource, healthcare providers may enhance patient engagement, promote adherence to treatment, and optimize quality of life.

HGG-09. CONTRASTING CLINICAL COURSES OF THREE PEDIATRIC PATIENTS WITH PDGFRA AMPLIFIED RADIATION-INDUCED HIGH-GRADE GLIOMA

Abstract BACKGROUND Radiation-induced gliomas (RIG) can occur in 3% of patients receiving cranial radiation. Often, these gliomas are high-grade, treatment refractory, and share common molecular profiles like PDGFRA amplification, regardless of their previous malignant origin. Despite similar genetic alterations, the clinical course of these tumors is varied and not well-studied. Herein, we describe three patients with a PDGRFA-amplified RIG with a particular focus on their contrasting disease course. CASE DESCRIPTION: Our first patient is a now-deceased male diagnosed with medulloblastoma at 5 years of age, treated with surgical resection and craniospinal radiation followed by chemotherapy per COG ACNS0332. Ten years later, he was diagnosed with diffuse pediatric type high-grade-glioma (HGG) in the pons with PDGFA, KIT, MTAP amplification, and CDKN2A/B loss. His condition deteriorated quickly, and he succumbed to his disease in 8 weeks after family decided against further therapies. Our second patient is a now 20-year-old female, initially diagnosed with a craniopharyngioma at 6 years of age and treated with surgical resection and adjuvant radiation therapy. Eleven years later, she developed a diffuse pediatric type HGG in the pons with PDGFRA, MDM2, and MYCN amplification. She was treated with radiation therapy and has had stable disease on serial imaging for 3 years. Our third patient is a now-deceased male diagnosed with a right temporal melanotic neuroectodermal tumor at 5 years of age, treated with surgical resection and craniospinal radiation followed by chemotherapy per POG99701. Fifteen years later, he developed a diffuse pediatric-type HGG in the cerebellum/pons with PDGRFA amplification. He was treated with proton radiation and concurrent oral Temozolomide per COG ACNS0423. His disease progressed on treatment, and he died prior to initiation of a PDGFRA kinase inhibitor (avapritinib). CONCLUSIONS Further investigation into the underlying factors that drive the differing disease course in patients with RIG is needed.

HGG-43. DIFFUSE PEDIATRIC HIGH GRADE GLIOMA SUCCESSFULLY TREATED WITH MULTIMODAL THERAPY

Abstract BACKGROUND Pediatric high grade glioma (HGG) have a dismal prognosis despite aggressive treatment. In this report, we discuss a patient with HGG successfully treated with combination of resection, radiotherapy, chemotherapy, and biologic therapy. RESULTS An 8-year-old male presented with intermittent headaches, vomiting and fatigue. Imaging demonstrated four solid/cystic enhancing intra-axial lesions within the left cerebral hemisphere- two within the left frontotemporal region, one in the superior parietal region, and one in the temporal lobe, all with perilesional edema and resultant subfalcine herniation with midline shift. The patient underwent subtotal resection of the frontotemporal and parietal lesions. Histopathology was consistent with glioblastoma. Whole exome sequencing revealed alterations in TP53, ATRX, PTEN, TSC2 as well as subclonal mutation in NF1. The tumor was unable to be further classified by DNA methylation. However histopathologic and molecular findings support the diagnosis of diffuse pediatric-high grade glioma, H3-wildtype and IDH-wildtype. The patient received adjuvant 59.4 Gy focal proton therapy with concurrent temozolomide and maintenance chemotherapy with temozolomide and lomustine for 6 cycles. End of therapy imaging demonstrated complete resolution of all sites of disease. Due to high likelihood of recurrence, the patient was subsequently started on targeted therapy with everolimus and ribociclib which continue to be well tolerated without dose adjustments. The patient remains stable and without recurrence 2.5 years since diagnosis, and 15 months since initiation of biologic therapy. Additionally, the patient underwent testing for cancer predisposition which revealed presence of TP53 heterozygous mutation consistent with Li-Fraumeni syndrome. The patient remains without other malignancies on surveillance imaging per LFS guidelines. CONCLUSION Our case report illustrates the value of both chemotherapy and biologic therapy in treatment of HGG. In addition to this, it emphasizes the importance of germline testing for cancer predisposition in patients with high grade tumors.

CNSC-03. LINKING CELL FATE DETERMINATION IN THE EMBRYONIC FOREBRAIN TO PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric high-grade gliomas are treatment resistant, usually fatal cancers encompassing diffuse midline glioma, K27-altered (DMG), and diffuse hemispheric glioma, G34R/V (DHG). Most patients harbour canonical and non-canonical mutations in histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and stalled differentiation states as oligodendroglial progenitor cells (OPC) in DMG and GABAergic interneuron precursors in DHG. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes GABAergic interneuron cell fate and migration during early neurogenesis. Methods & Results Our lab has shown DLX2 promoter occupancy of Gad1/Gad2, and early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of DLX2 with these target sequences reduces reporter gene expression in vitro, and the Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3 and loss of H3K27 acetylation, as well as a reduction in migration, invasion, and colony formation in vitro. To validate in human pHGG, we first probed an RNA-seq database containing 62 patient-derived pHGG cell lines which demonstrated an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DHG cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2-expressing DMG cell lines underwent stable transfection of a DLX2-mCherry overexpression construct in conjunction with a SOX2-BFP overexpression construct, confirmed by qPCR and immunoblot. Analysis of histone H3 marks, K27M, and OPC signature genes will be compared to OPC genes from Dlx1/Dlx2 DKO mouse ganglionic eminences. CONCLUSIONS Manipulation of DLX2 in pHGG provides a model system for assessing the contribution of a homeobox factor to cell fate decisions regulating differentiation and tumorigenicity.

METB-15. ROLE OF DNA METHYLATION PROFILING IN THE MANAGEMENT OF PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS: A SINGLE INSTITUTIONAL SERIES

Abstract BACKGROUND DNA methylation profiling has now been integrated into the WHO classification of CNS tumors and serves as confirmatory means to diagnose CNS tumors in conjunction with genetic sequencing/microarray analysis. The practical impact on patient care in the retrospective and prospective settings is less known. METHODS Methylation analysis was performed on a cohort of previously and newly diagnosed children with CNS tumors at Children’s Hospital Orange County from 2022-2023. Data regarding histologic diagnosis, integrated molecular diagnosis, methylation classification, timing of result completion and functional impact of methylation results on patient diagnosis/management was evaluated. RESULTS Forty patients (21 boys, ages 5 weeks-17 years) with previously diagnosed (N=11) or newly diagnosed (N=21) CNS tumors were evaluated by methylation analysis at one or more institutions. The most common tumor types were diffuse type pediatric high-grade glioma (N=6) and ependymoma PFA (n=4). Methylation analysis revealed no match in two cases (ALK-EML4 fusion embryonal tumor and recurrent low grade glioneuronal tumor) and had insufficient DNA extraction in one patient with low grade glioneuronal spinal cord tumor. Among the 5 tumors with duplicate methylation analyses at two institutions, three Ependymoma PFA subgroups were concordant while two showed differing classifications (diffuse type pediatric high-grade glioma RTK1 subtype vs. non- classifiable and medulloblastoma group 4 vs. non-WNT, non-SHH). In patients with previously diagnosed CNS tumors, methylation either led to a change in classification or modification of diagnosis in 7 out of 11 patients. The average time from surgery to the receipt of methylation results for newly diagnosed patients was 24 days (range 4-90 days). CONCLUSION Methylation analysis was beneficial in both the previously diagnosed and newly diagnosed setting in patients with previously difficult to classify tumors following conventional molecular analysis. Changes in tumor classification or modifications in diagnosis were observed in the majority of patients.

HGG-13. INFANT-TYPE HEMISPHERIC GLIOMA (IHG): AN INDIVIDUAL PATIENT DATA META-ANALYSIS

Abstract BACKGROUND Infant-type hemispheric gliomas (IHG) are epigenetically distinct pediatric high-grade gliomas characterized by fusions in receptor tyrosine kinase (RTK) genes. METHODS We performed a methodical literature search, including 30 publications (22 case reports), to identify patients who met the diagnostic criteria of IHG based on the 2021 WHO Classification of CNS Tumors. Individual patient data were obtained from published literature and/or via the authors of the publications. Survival analysis was conducted using the Kaplan-Meier method, and multivariate analysis was performed to investigate the effect of clinical and molecular variables on outcomes. RESULTS Hundred-fifty-five previously reported and one unpublished IHG were identified: 131 (84%) had fusions in RTK genes, of which ALK was most prevalent (62/131), followed by NTRK1/2/3 (30/131), ROS1 (30/131), and MET (9/131). Twenty-five patients, with either no identified RTK fusion (6/156) or not assessable fusion (19/156), had methylation scores ≥ 0.9 for IHG (using the Molecular Neuropathology brain tumor classifier versions ≥ 11b4). Surgery followed by adjuvant chemotherapy in 69% (67/97) was the most common primary treatment used in our cohort. The 3-year EFS and OS were 55% (95%CI: 45-67) and 81 % (95%CI: 73-89). 41 patients with relapsed or progressive tumors received various second-line treatments including surgery, chemotherapy, radiotherapy, and targeted therapy. Based on multivariate analysis, complete resection resulted in better EFS (p = 0.05) and OS (p = 0.009), and the presence and type of RTK gene fusion were not associated with clinical outcomes. CONCLUSION Our results show that despite favorable OS, patients with IHG often show early progression, indicating that the primary optimal treatment for IHG is yet to be established. Our analysis further indicates that achieving a safe, complete resection may play an important role in treating these patients. A comprehensive analysis of the salvage regimen is required to understand their role in OS.

DIPG-27. MATCHED PRIMARY-RELAPSE PEDHGG PATIENT-DERIVED XENOGRAFT MODELS TO IDENTIFY THERAPY RESISTANCE PROFILES

Abstract BACKGROUND We established patient-derived xenografts (PDXs) from matched primary-relapse pairs of pediatric high-grade gliomas (pedHGGs) in collaboration with the Swedish Childhood Tumor Biobank. Further, we used human iPSC derived cortical organoids to evaluate interactions between glial and tumor cells under treatment pressure potentially lost in xenograft models. Using this setup, we hope to follow tumor relapse evolution and to identify novel mechanisms behind treatment evasion and relapse formation. METHODS Tumoral singel cell suspensions from matched primary-relapse pairs are injected orthotopically in immunodeficient NOG mice. We establish in vitro 2D models in stem cell conditions from the first generation of animals transplanted. Lentivirus-based labelling with GFP/Luciferase was done on PDX-derived cell lines to allow cell tracking and to quantify efficacy of drug screening efforts. Co-culture of these cells with normal cells in 3D assembloids were performed using 150-250 days old cortical organoids. Single-cell RNA sequencing and bulk RNA sequencing (RNA-seq) on patient biopsies, their corresponding PDXs from first generation of mice, and PDX-derived cell lines, was performed to track potential changes in vivo and in vitro and to further validate primary-relapse specific evolution. RESULTS 2D and 3D cell cultures offered efficient labeling and we quantified differences in sensitivity to standard therapy between primary and relapse samples. PDX models and assembloids highlighted pedHGG growth patterns, modes of invasion and cell interactions, especially astrocyte-tumor cell interactions. These 3D models also allow for further validation of targeted drug testing based on multiomics data (WGS, methylation and RNA-seq) from the patient biopsies. CONCLUSION PedHGG primary-relapse in vitro 2D cell line/ in vivo 3D assembloid models together with PDXs is a robust approach to study different aspects of relapse-specific tumor biology. Our results offer insights aiding the development of novel therapeutics inhibiting relapse mechanisms already in the first line of treatment.

HGG-26. MLH1, MSH2, MSH6, AND PMS2 IMMUNOHISTOCHEMISTRY REPRESENTS A HIGHLY SENSITIVE SCREENING METHOD FOR MISMATCH REPAIR DEFICIENCY SYNDROMES IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric central nervous system (CNS) tumors can occur as first manifestations of cancer predisposition syndromes (CPS) resulting from pathogenic variants in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. Early detection of MMR deficiencies (MMRD) may warrant the therapeutic use of checkpoint inhibitors and enable genetic consulting for the patient’s families. METHODS We prospectively screened 128 pediatric high-grade gliomas (pedHGG) for MMR protein expression by immunohistochemistry (IHC) as part of the HIT-HGG-2013 clinical trial. We compared IHC results to independently collected patient information on CPS including MMRD to test the method’s efficiency in screening for Lynch syndrome and Constitutional MMRD syndrome CMMRD. RESULTS From 128 successfully tested tumors, ten cases (10/128, 7.8%), showed loss of expression of at least one of the MMR proteins. In seven of the ten affected patients (7/128, 5.5%), genetic testing uncovered heterozygous (6/10) or homozygous (1/10) pathogenic germline variants in MMR genes (MLH1, MSH2 or MSH6) confirming the diagnosis of Lynch syndrome respectively CMMRD. In three cases (3/128 2.3%), MMR alterations were restricted to tumor tissue. The group of diffuse midline gliomas, H3 K27-altered, (67/128, 52.3%) did not show MMR alterations, while either somatic or germline MMR mutations were detected in two of eight patients with diffuse hemispheric glioma, H3 G34-mutant. In 88 patients (88/128, 68.8%) either clinical or molecular-genetic information on CPS could be provided. All ten patients with signs of MMRD were successfully detected by IHC from tumor tissue. CONCLUSIONS IHC for MMR proteins represents a highly sensitive screening method for MMRD in pedHGG. Considering the occurrence of at least 11.5% (7/61) of MMRD in pediatric patients with diffuse high-grade glioma excluding DMG, MMRD IHC should be part of routine diagnostics as cost-effective, broadly available and robust screening method.

HGG-15. SYNERGISTIC EFFECTS OF COMBINED MEK/CDK4/6 INHIBITOR TREATMENT ON BRAFV600E POSITIVE HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric high-grade glioma (pHGG) is associated with poor overall survival and standard care has remained unchanged for the past decade. BRAFV600E mutations occur in 5-10% and dabrafenib in combination with mekinist is approved in this subtype. Nevertheless, acquired resistance to targeted therapeutics is a common setback in highly aggressive tumors. Homozygous CDKN2A/B loss co-occurs in approximately 60% of this pHGG subtype. Taking advantage of those combined molecular alterations and exploiting acquired resistance mechanisms is of utmost importance to increase overall survival in these patients. METHODS Four HGG cell models with BRAFV600E mutation and homozygous CDKN2A/B loss were tested for the effects of CDK4/6 inhibitor monotherapy (palbociclib, ribociclib, abemaciclib) and/or combination treatment with trametinib on cell survival, cell cycle, apoptosis and senescence. RESULTS Abemaciclib showed the highest efficacy of CDK4/6 inhibitors comparable to trametinib monotherapy in our cell models. Abemaciclib led to a cell cycle arrest and combined treatment with trametinib induced distinctly increased levels of apoptosis in our tumor models when compared to monotherapy and a great proportion of surviving cells were senescent. One cell model was orthotopically implanted in mice and abemaciclib and combined treatment with trametinib showed a slightly increased response when compared to trametinib alone. CONCLUSIONS Summarizing, treatment with abemaciclib showed promising therapeutic effects in HGG cell models with BRAFV600E mutation and homozygous CDKN2A/B loss comparable to trametinib alone. Levels of apoptosis were distinctly higher with combinatorial abemaciclib and trametinib treatment when compared to monotherapy alone and senescence was induced in a great number of surviving cells. In vivo abemaciclib was more efficient when compared to trametinib alone. Currently we are working on in vivo experiments and the effect on sustained induction of senescence and mechanisms of tumor cell growth after discontinuation of therapy and in a combinatorial metronomic approach and in combination with radiotherapy.

HGG-52. MULTI-INTEGRIN TARGETING EFFECTIVELY SUPPRESSES DIFFUSE MIDLINE GLIOMA MIGRATION

Abstract Diffuse midline gliomas (DMGs) are aggressive tumors characterized by infiltration into normal midline brain tissue, hindering surgical resection and contributing to overall morbidity and mortality. We established a novel two-step pooled whole genome CRISPR-Migration screen by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds, a common feature in glioblastoma (GBM). Comparing DMG and GBM primary cell lines (n=5), genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all cell lines. Multi-institutional transcriptomic data revealed an association of expression of these focal adhesion genes with survival primarily in K27M-DMG and pediatric high-grade glioma compared to lower-grade brain tumors, and ITGB1 expression increased with glioma grade. Single-cell RNA-seq and spatial transcriptomic data demonstrated higher expression of ITGB1 in OPC-like/Mesenchymal cells associated with leading edge and microvasculature in both K27M-DMG and GBM tumors. Unexpectedly, knockout of ITGB1 (ITGB1-KO), primarily responsible for cell adhesion to laminin, 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 impaired mitochondrial fusion. Orthotopic implantation of control and ITGB1-KO models of H3K27M-DMG and adult GBM in mouse brains revealed two distinct phenotypes: (1) ITGB1-KO reducing proliferation and significantly increasing survival; and (2) ITGB1-KO leading to upregulation of ITGB3 and ITGB5 in vivo without impacting survival. Cilengitide (αvβ3 and αvβ5 inhibitor) ablated migration of all ITGB1-KO models on vitronectin (secreted by pericytes surrounding blood vessels) in vitro. Ongoing combinatorial therapy with an experimental anti-Integrin Beta 1 antibody (AIIB2, CNS delivered) and Cilengitide is underway with promising early results. In summary, multi-integrin inhibition provides a feasible avenue to curb glioma spread in the brain through impaired focal adhesion as well as blunting mitochondrial glucose metabolism.

DIPG-06. FUNCTIONAL MULTI-OMIC PROFILING OF THE LANDSCAPE OF DIFFUSE MIDLINE GLIOMAS TO IDENTIFY THERAPEUTIC VULNERABILITIES

Abstract BACKGROUND Diffuse midline glioma (DMG) is a devastating pediatric high-grade glioma (pHGG) located along the midline structures of the brain and spine. Palliative radiotherapy is the only approved treatment outside of clinical trials, with patients given 9–11-months survival post-diagnosis. Over the last 10-years, it has become common for patients to be prescribed precision therapies targeting genetic alterations, however largely, DMG harbor genetic alterations that are untargetable. METHODS To improve DMG patient outcomes, we are performing multi-omic analysis on 210 DMG, normal controls and other pHGG samples to functionally characterize the genomic landscape of these tumors and understand the protein-controlled functional consequences of recurring somatic alterations. RESULTS Genomic sequencing has been performed on 40/210 pHGG samples, identifying 290 unique mutations and several recurrent chromosomal alterations. Additionally, proteomic/phosphoproteomic profiling identified both unique and similar features of post-translational landscapes across genotypes. Examining H3.3K27M DMG cell-lines, SU-DIPG-XIII and UON-VIBE5, genomic sequencing identified 11 and 6 mutations respectively, however, only UON-VIBE5 possessed a genomically-predicted target, a PDGFRA-D842V mutation, potentially targetable by Avapritinib. Conversely, investigation of proteomes/phosphoproteomes revealed several potential therapeutic vulnerabilities. SU-DIPG-XIII possessed high enrichment of HDAC/BRD and CDK pathways and UON-VIBE5, ATM-centric pathways. In vitro proliferation assays of Avapritinib treated UON-VIBE5 failed to elicit a response at clinically relevant doses, while all proteomically-predicted therapies (HDAC – Fimepinostat, BRD – JQ1, CDK – Ribociclib and ATM – WSD-0628) successfully influenced tumor cell growth whilst leaving microglia controls cells unaffected. Avapritinib and WSD-0628 (CNS-active ATM inhibitor) drug predictions were further validated in the RA055 DMG model, which possesses similar genomic and proteomic features to UON-VIBE5. Avapritinib alone failed to influence tumor growth, while WSD-0628 notably reduced cell proliferation. The combination of these drugs, however, synergistically inhibited RA055 proliferation. CONCLUSIONS These findings thus provide preliminary evidence supporting the need to take a multi-omic approach in treating DMGs.

HGG-22. LINKS BETWEEN M6A RNA METHYLATION, R-LOOPS AND DNA DAMAGE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND Paediatric high-grade gliomas (pHGG) are aggressive malignant tumours of the central nervous system that unfortunately carry poor prognoses. R-loops, which are DNA:RNA hybrids, are a known source of genomic instability in eukaryotic cells. The RNA strand of R-loops can be m6A methylated at double-strand break sites in order to facilitate DNA repair. Therefore, we investigated whether dysregulation of m6A methylation and R-loop homeostasis at DNA damage sites deregulates DNA repair pathways in pHGG. METHODS Immunocytochemistry was performed to assess global m6A, R-loop, γH2AX and several DNA repair protein levels in pHGG cell lines compared to human astrocytes. Colocalisation between m6A and R-loops, R-loops and γH2AX, m6A and γH2AX, R-loops and 53BP1, R-loops and PARP1, R-loops and total ATM, and R-loops and phosphorylated ATM was assessed in pHGG cell lines and human astrocytes. RESULTS Immunocytochemistry analysis revealed that levels of global m6A, R-loop, γH2AX and several DNA repair proteins were generally higher in pHGG cell lines, thereby indicating that cellular homeostasis is dysregulated. Furthermore, a high degree of colocalisation between m6A and R-loops was observed. R-loops were found to colocalise with γH2AX, and m6A was found to colocalise with γH2AX, thereby corroborating the notion that m6A methylated R-loops are likely present at DNA damage sites. Finally, R-loops were also found to colocalise with a variety of DNA repair proteins, including 53BP1, PARP1, total ATM, and phosphorylated ATM, where different Pearson’s correlation coefficient values were obtained, thereby suggesting that R-loops associate with DNA repair proteins to different extents. CONCLUSIONS The link between m6A methylation, R-loops and DNA damage in relation to the pathogenesis of pHGG is currently being investigated. Experiments are also being performed where we are investigating m6A readers to see whether their aberrant expression contributes to disease pathogenesis via deregulating DNA repair pathways.

DIPG-33. COMBINING CLONAL TRACKING WITH SINGLE CELL RNA SEQUENCING REVEALS TEMPORAL SHIFTS IN CELL STATES ASSOCIATED WITH TRAMETINIB RESISTANCE IN MAPK ALTERED DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) is an aggressive and incurable paediatric high-grade glioma with median overall survival of 9-12 months. A distinct subset of DMG harbour alterations in the MAPK pathway, and we have previously shown that DMG-MAPK may exhibit sensitivity to MEK inhibitors but develop resistance under long-term exposure to drug. Elucidating these resistance mechanisms could spearhead better treatment strategies for these patients. METHODS To study clonal evolution under trametinib, we used CloneSifter lineage tracking technology to uniquely barcode 300,000 DMG-MAPK cells which were then treated with a GI80 dose of trametinib for 11 weeks. We extracted DNA to quantify barcode enrichment at endpoint across six technical replicate experiments and leveraged CloneSifter’s compatibility with single cell RNA sequencing (scRNA-seq) to couple clonal identity with transcriptional states in ~90,000 trametinib treated cells at varying timepoints. RESULTS DNA sequencing revealed 0.5% (1500/300,000) of the initial barcodes were enriched across trametinib replicates and were also detectable by scRNA-seq. Conducting cNMF analysis on the single cell dataset showed populations of trametinib treated cells associated with each other in a temporal manner, whereby early and late-stage resistant populations were defined by neuronal and stem cell like markers, respectively. A time-dependent enrichment of CloneSifter barcodes was also observed, suggesting dynamic expansion of clones over the treatment window. Notably, week 11 trametinib treated cells showed higher expression of resistance driver genes related to EMT, stemness and p38/MAPK upregulation. CONCLUSIONS Utilizing the sequence-specific reporter construct built in to the CloneSifter system allows for retrieval of resistant clones marked by enriched barcodes under treatment prior to drug exposure, enabling further characterization of monoclonal populations of trametinib-resistant cells. In summary, we have employed novel tools to study resistance development in DMG-MAPK that could be exploited to devise rational treatment strategies for these patients in an evolutionary-guided manner.

HGG-53. MULTI-DIMENSIONAL INTEGRATIVE PROFILING IDENTIFIES BCL2L1 METHYLATION AS A PREDICTIVE BIOMARKER FOR MCL-1 ACTIVITY IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND Pediatric high-grade gliomas (pHGGs) pose a significant challenge as the most aggressive central nervous system tumors in children. The lack of effective treatment and poor survival rates emphasize the critical need for innovative therapies to improve the prognosis of pediatric patients with pHGG. METHODS We executed CRISPR-cas9 knockout (KO) screenings in 65 pediatric and 10 adult high-grade glioma (HGG) cell lines to explore unique functional dependencies associated with pHGGs. Drug assays were carried out to assess the targetability of a gene dependency of interest in pHGG cell lines. Subsequently, Random Forest machine learning algorithm was employed on ‘omics’ datasets to identify biomarkers indicative of drug response. Finally, biomarkers were confirmed through sequencing-based methods across various pediatric cancers. RESULTS CRISPR-cas9 KO screens revealed 8 crucial genes essential for pHGG growth, namely MCL-1, ATIC, DHFR, EED, HDAC2, PARP1, PDK1, PIK3CA and TYMS. Among these, Myeloid Cell Leukemia (MCL-1) was further characterized as it emerged as the top differential genetic dependency in pHGGs. Consistent with this, MCL1 inhibitors exhibited potent anti-cancer activity on 43% of pHGG cell lines. Multi-feature prediction analysis identified 140 features correlated to response. Strikingly, a previously undescribed methylation site within the BCL2L1 locus, cg00300298, ranked in the top 5% of features. Tissue analysis revealed a wide range of other pediatric brain cancers that harbor the BCL2L1 cg00300298 methylation mark as compared to non-malignant brain tissue. Lastly, we validated the utility of BCL2L1 methylation as a predictive biomarker of MCL1 inhibitor sensitivity across a large panel of pediatric brain cancer cell lines. CONCLUSION Using an integrated genomic approach, we identify MCL1 as a distinct therapeutic target in BCL2L1-methylated pediatric brain cancers. This offers a rational approach for stratifying patients who may benefit from MCL1 inhibitors.

RADT-01. TUMOR VOLUME INCREASE AND NEED FOR RADIOTHERAPY PLAN ADAPTATION IN PEDIATRIC HIGH-GRADE GLIOMA AND DIFFUSE MIDLINE GLIOMA DURING A SIX-WEEK COURSE OF RADIOTHERAPY

Abstract BACKGROUND Pediatric High-Grade Glioma/ diffuse midline glioma (pHGG/DMG) growth during a six-week course of radiotherapy may precipitate geographical miss of target volumes and early disease progression. In a patient cohort, we retrospectively evaluated the evolution of tumor volumes during radiotherapy, the clinical need for plan adaptation, and potential correlation with local disease progression. METHODS Twenty-four pHGG/DMG patients (12M/12F, ages 4-20 years) with gross tumor volume (GTV) after debulking (n=7) or biopsy (n=17) received 30 fractions of Volumetric Modulated Arc Therapy within an adaptive radiotherapy-protocol. The GTV was defined by combined T1+FLAIR MRI sequence abnormalities. A 1-cm margin was applied for Clinical Target Volume (CTV). MRI-based target volume re-evaluation was performed at fraction 10 and 20, defining GTV increase >1mL as growth. The clinical need for plan adaptation was based on tumor volume changes. GTV increase and need for plan adaptation were evaluated against local progression-free survival (LPFS). RESULTS Median time between baseline MRI and radiotherapy start was 10 days (range 3-29). From baseline, GTV growth was observed in 7/24 patients at fraction 10, and in an additional 7/24 patients at fraction 20 (cumulative 14/24), corresponding to a mean volume increase of 21%/3.7mL (range [-7%/-2.7mL] to [+185%/24.4mL]) and 40%/8.7mL (range [-18%/-8.1mL] to [+329%/115.1mL]) after 10 and 20 fractions, respectively. At fraction 10 and 20, respectively 6 and 6 plans needed adaptation in a total of 11 patients. Baseline tumor volumes between patients with/without plan adaptation were equivalent. Median LPFS was 8.3 months (95%CI 8.0-12.7). No significant difference in LPFS was observed between patients with versus without GTV increase or plan adaptation. CONCLUSIONS Between MRI at baseline and at fractions 10 and 20, this adaptive radiotherapy protocol using 1-cm CTV margins demonstrated pHGG/DMG tumor volume increase with a clinical need for plan adaptations in 11/24 patients.

IMG-28. AUTOMATIC BRAIN TUMOR VOLUMETRIC ANALYSIS IN MAGNETIC RESONANCE IMAGING GENERALIZABLE TO PEDIATRIC NEURO-ONCOLOGY

Abstract BACKGROUND The prognosis of brain tumors is variable in clinical practice if it only relies on human interpretation of magnetic resonance imaging (MRI). The automatic segmentation of brain tumors in MRI enables quantitative analysis in support of clinical trials and personalized patient care. We developed benchmarked deep learning-based tools that are generalizable to the volumetric quantification of various tumor types across diverse populations. METHODS We participated in the well-established international brain tumor segmentation challenge (BraTS 2023) and benchmarking competition. The challenge made available 4,500 multi-national brain tumor cases with multi-sequence MRIs, including pediatric high-grade gliomas (PED), i.e., high-grade astrocytoma and diffuse midline glioma, and adult gliomas, brain metastases (MET) and intracranial meningiomas (MEN). Each case comprises four MRI volumes: T1, contrast-enhanced T1, T2, and T2-FLAIR. Manual segmentations were provided to establish ground truth for enhancing tumor (ET), tumor core (TC), and whole tumor (WT). Our framework used a model ensemble strategy based on two state-of-the-art deep learning models: a convolutional neural network (nnU-Net) and a vision transformer (Swin UNETR) and was tested for broader applicability across multiple tumor types. The framework was trained on 99, 1,000, and 165 cases and validated on 45, 141, and 31 unseen cases for PED, MEN, and MET, respectively. Automatic segmentations were evaluated by lesion-wise volume overlap (Dice similarity score, DSC) and Hausdorff distance (HD). RESULTS In the evaluation on independent unseen test datasets, our automatic tool was ranked first for PED, third for MEN, and fourth for MET volumetric analysis. Our method resulted in PED lesion-wise DSC of 0.733, 0.782, 0.817 and HD (mm) of 75.93, 25.54, 24.18 for ET, TC, and WT, respectively. CONCLUSIONS These brain tumor volumetric analysis tools are readily available to be efficiently tested on diverse datasets. Automatic MRI analysis provides consistent quantitative data for multi-institutional protocols and clinical trials.

HGG-46. PEDIATRIC RADIATION-INDUCED GLIOMA PATIENT SAMPLES AND NOVEL PATIENT-DERIVED MODELS REVEAL TRANSLATIONALLY RELEVANT TREATMENT SUSCEPTIBILITIES AND POTENTIAL GERMLINE RISK FACTORS

Abstract BACKGROUND Pediatric radiation-induced glioma (RIG) is an incurable secondary high-grade glioma (HGG) afflicting up to 4% of children who previously received cranial radiotherapy (RT) for other malignancies. RIG accounts for up to 10% of pediatric brain tumor deaths. It has no known genetic risk factors nor effective treatment and is uniformly fatal, partly due to extremely limited available models. Our recent molecular characterization of patient samples showed that while RIG and primary pediatric HGG share histological similarities, their genetic alterations and gene expression profiles differ. METHODS We obtained germline DNA sequencing from 28 RIG patients and 112 matched controls who received RT but did not develop RIG. In addition, we developed cell culture and murine orthotopic patient-derived xenograft (PDX) models from four RIGs. We conducted single-cell RNA-seq (scRNA-seq), drug screening/validation, and CRISPR functional screening on our novel models. RESULTS Germline mutations in TP53, PARP10, POLI, DCLRE1A, and MLH3 were more common in RIG patients than controls (p<0.002). Our matched cell culture and PDX models of RIG represent the genetic diversity of the disease. The PDX models form tumors in 30-120 days and closely recapitulate human RIG by histology and scRNA-seq. They show susceptibility across models to DNA-damaging treatments (etoposide, irinotecan, radiation), as well as MEK, PARP, and proteasome inhibition, with orthogonal supporting findings via CRISPR screening. Several two-treatment combinations also show efficacy and synergy across models, including radiation and trametinib in vivo (logrank p=0.029). Discussion: RIG is an understudied but major cause of pediatric CNS tumor mortality that differs genetically from primary pediatric HGG. Our findings suggest specific germline alterations in DNA repair pathways may put some patients at higher risk of developing RIG after cranial RT. We also introduce the first set of patient-derived RIG models in order to advance translational RIG research, with characterization of translationally relevant treatment susceptibilities.

DIPG-53. AN EXCEPTIONALLY RARE CASE OF A DIFFUSE MIDLINE GLIOMA WITH CONCOMITANT H3.1 K27M AND G34R MUTATIONS IN THEHIST1H3C (H3C3) GENE

Abstract BACKGROUND Histone mutations (H3 K27M, H3 G34R/V) define subtypes of paediatric-type diffuse high-grade gliomas (HGG) (diffuse midline glioma (DMG), H3 K27-altered, diffuse hemispheric glioma (DHG), H3 G34-mutant). The WHO classification recognises exceptional cases where these mutations co-occur. We report one such case of a 2-year-old female presenting with neurological symptoms and lethargy. METHODS MRI imaging identified a brainstem lesion; a stereotactic needle biopsy was undertaken, followed by standard diagnostic neuropathology workflows including histology review, panel immunohistochemistry, DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package)) and WES/WGS. Patient-derived in vitro cell cultures were established allowing further characterisation using RNAseq and chromatin immunoprecipitation assays with sequencing (ChIP-seq). RESULTS Histology showed diffusely infiltrating pleomorphic astrocytes, multinucleated cells, and conspicuous mitotic activity; the diagnosis was DMG, H3 K27-altered (immunohistochemistry: H3K27me3 loss, H3K27M positivity). DNA methylation profiling classified the tumour as ‘DMG, H3 K27-altered’ (calibrated score=0.99). WES/WGS revealed concurrent H3.1 K27M and G34R mutations (clonal, in the same reads) of HIST1H3C (H3C3), somatic variants in FGF11 and PIK3CA, and a pathogenic germline NBN variant. The RNAseq profile of the primary tumour and cell cultures clustered with H3 K27M-mutant tumours. Unbiased in vitro drug screening showed no selective sensitivities. ChIP-seq (H3K27ac, H3K27me3, H3K36me3, RNApol2 marks) showed features in keeping with DMG H3 K27M-mutant tumours (H3K27ac loci including OLIG2, IRX1/2, PKDCC). The patient was treated with adjuvant radiotherapy and everolimus, but progressed and passed away 13 months post-diagnosis. CONCLUSION This case is an exceptionally rare, complex variant of histone-mutant paediatric HGG, and the first reported occurrence in HIST1H3C (H3C3). It illustrates that in cis, the H3.1 K27M mutation demonstrates a dominance over molecular and clinical profiles compared to G34R, and highlights the importance of broad molecular profiling to identify such examples for further study.