Brainstem Tumors Research Articles

Surgical management of a large cystic trochlear nerve schwannoma mimicking a brainstem glioma: a case report

IntroductionIntracranial schwannomas represent a rare group of intracranial tumors, with purely motor nerve schwannomas being the rarest of them. The anatomical proximity of these tumors to the brainstem may present a radiological challenge in differentiating them from intra–axial brainstem tumors, which can influence further decision–making and treatment options.MethodsWe report on a 47–year–old male patient who was diagnosed with a large cystic intracranial tumor with radiological features suggestive of an intrinsic brainstem glioma.ResultsAfter discussing treatment options and risks based on a presumed radiological diagnosis, microsurgical treatment via lateral–suboccipital craniotomy in semi–sitting position, under continuous intraoperative neuromonitoring was performed. Intraoperative findings proved that the tumor was an extra–axial schwannoma originating from the left trochlear nerve. Gross total removal of the lesion was achieved.ConclusionDue to their rarity, non–specific symptoms and the possibility to mimicking intra–axial brainstem tumors on imaging, these tumors may present a diagnostic challenge and should be taken into account during treatment decision-making.

DDDR-11. INHIBITING OXIDATIVE PHOSPHORYLATION AND HYPOXIA WITH ATOVAQUONE – A STRATEGY TO RADIOSENSITIZE DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is a uniformly fatal paediatric brainstem tumour with median survival of less than 1 year. Radiotherapy has been the only effective treatment for decades, but most DMGs recur within several months due to radioresistance. The hypoxic tumour microenvironment is a main feature of solid tumours including gliomas and a major contributor to radioresistance. Therefore, alleviating tumour hypoxia to enhance the effectiveness of radiotherapy is a potential therapeutic strategy to improve the survival outcomes of DMG patients. We found evidence suggesting increased mitochondrial oxidative phosphorylation (OXPHOS) in DMG. Hence, we aimed to target OXPHOS by decreasing the oxygen consumption rate (OCR) of DMG cells, which in turn will alleviate hypoxia by sparing more oxygen and subsequently improve the radiosensitivity of DMG cells. We performed a high-throughput screening to identify potent OCR inhibitors from a library of 1963 FDA-approved drugs. The most promising OCR inhibitor identified was atovaquone, a drug used for treatment of pneumocystis pneumonia and malaria. We found that atovaquone inhibited mitochondrial metabolism of DMG cells by specifically targeting the mitochondrial complex III. Increased mitochondrial reactive oxygen species after exposure to atovaquone suggested that it increases oxidative stress. The treatment alleviated hypoxia and decreased the expression of hypoxia-inducible factor-1a in 3-dimensional DMG neurospheres and improved the radiosensitivity in several DMG cultures. To overcome the poor bioavailability of commercially available atovaquone that results in low therapeutically effective brain concentrations, we tested its efficacy against the amorphous solid dispersion (ASD) atovaquone formulation which appears to enhance atovaquone levels in the brain. We found that both the formulations inhibited OCR and hypoxia at similar doses and enhanced DMG radiosensitivity. The combination of ASD atovaquone and radiation significantly improved survival in orthotopic DMG mouse model. Collectively, this data provides evidence of atovaquone as a potential radiosensitizer for DMG.

Additive Effects of Cu-ATSM and Radiation on Survival of Diffuse Intrinsic Pontine Glioma Cells.

Diffuse intrinsic pontine gliomas (DIPG) are highly aggressive and treatment-resistant childhood primary brainstem tumors with a median survival of less than one year after diagnosis. The prevailing standard of care for DIPG, radiation therapy, does not prevent fatal disease progression, with most patients succumbing to this disease 3-8 months after completion of radiation therapy. This underscores the urgent need for novel combined-modality approaches for enhancing therapy responses. This study demonstrates that the cellular redox modulating drug, copper (II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) dose-dependently (1-3 μM) decreased clonogenic cell survival in SU-DIPG50 and SU-DIPG36 cell lines during 6 h of exposure but had no significant effect on survival in normal human astrocytes (NHA). Additional significant (>90%) decreases in DIPG clonogenic survival were observed at 24 h of Cu-ATSM exposure. However, NHAs also began to show dose-dependent 10-70% survival decreases at this point. Notably, 3 μM Cu-ATSM for 6 h resulted in additive clonogenic cell killing of DIPG lines when combined with radiation, which was not seen in NHAs and was partially inhibited by the copper chelator, bathocuproinedisulfonic acid. Cu-ATSM toxicity in DIPG cells was also inhibited by overexpression of mitochondrial-targeted catalase. These results support the hypothesis that Cu-ATSM is selectively cytotoxic to DIPGs by a mechanism involving H2O2 generation and copper and being additively cytotoxic with ionizing radiation.

PDOC-04 MULTIDISCPLINARY APPROACH TO PEDIATRIC POSTERIOR FOSSA TUMORS, OUR CLINICAL EXPERIENCE: CASE SERIES

Abstract Paediatric brain tumor is second only to leukemia in this age group and more than 60% are located infratentorial. The first treatment option is surgical resection with aim of complete total resection, the proximity to fourth ventricle and CSF pathways make these children prone to develop obstructive hydrocephalus and multiple other complications related to pre and post operative. Chemotherapy and radiotherapy as adjuvant management is also important after surgery to selected cases. The aim of this case series was to review the demographic and clinical presentation of posterior fossa tumor children prior to definitive management, duration to adjuvant therapy after surgery and complications related to surgical management. This was a two-year retrospective review from March 2022 to march 2024, all paediatric less than 18 years of age with brain tumor admitted to our neurosurgery pediatric ward were selected. Late presentation to hospital was observed in most of the patients, obstructive hydrocephalus that warrant an emergent CSF diversion was noted in majority of the children. All cases require surgery were performed more than 5 days after admission to hospital, histological results report majority of the tumor to be pilocytic astrocytoma, medulloblastoma and ependymoma, all brain stem tumors on MRI image were not operated and were offered chemotherapy. Complications related to surgery include CSF leak, wound dehiscence and re occurrence of tumor were observed in more than 5 cases. Delayed chemotherapy and radiotherapy was also noted in this study. This single institution study concluded that a prospective study is important and a well-defined protocol at assessing novel ways and approaches of management, is required to all children with posterior fossa tumor, this will minimise the complications observed in this study.

466P Re-irradiation therapy for pediatric brainstem tumours: 20 years of clinical experience

466P Re-irradiation therapy for pediatric brainstem tumours: 20 years of clinical experience

Upper brainstem pediatric low-grade gliomas: review and neuroendoscopic approach.

Pediatric brain tumors, particularly those affecting the brainstem, present a significant challenge due to their intricate anatomical location and diverse classification. This review explores the classification, anatomical considerations, and surgical approaches for pediatric brainstem tumors, focusing on recent updates from the World Health Organization (WHO) classification. Brainstem tumors encompass a spectrum from diffuse gliomas to focal intrinsic and exophytic types, each presenting unique clinical and surgical challenges. Surgical strategies have evolved with advancements in neuroimaging and surgical techniques, emphasizing approaches such as neuroendoscopy and tailored incisions to minimize damage to critical structures. Despite the complexities involved, recent developments offer promising outcomes in tumor resection and patient management, highlighting ongoing advancements in neurosurgical care for pediatric brain tumors.

H3K27M diffuse midline glioma is homologous recombination defective and sensitized to radiotherapy and NK cell-mediated antitumor immunity by PARP inhibition.

Radiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune responses. H3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1 inhibition by AZD9574 with concurrent RT. H3K27M mutation caused an HRR defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG tumor cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574. The HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of best-in-class PARP inhibitors with RT in DMG patients. H3K27M DMG are HRR defective and selectively radiosensitized by PARP inhibitor.PARP inhibitor with RT enhances NKG2D ligand expression and NK cell-mediated lysis.NK cells are required for the therapeutic efficacy of PARP inhibitor and RT. Radiotherapy is the cornerstone of H3K27M-mutant diffuse midline glioma treatment, but almost all patients succumb to tumor recurrence with poor overall survival, underscoring the need for RT-based precision combination therapy. Here, we reveal HRR deficiency as an H3K27M-mediated vulnerability and identify a novel mechanism linking impaired RT-induced histone H1 polyubiquitination and the subsequent RNF168/BRCA1/RAD51 recruitment in H3K27M DMG. This model is supported by selective radiosensitization of H3K27M DMG by PARP inhibitor. Notably, the combination treatment results in NKG2D ligand expression that confers susceptibility to NK cell killing in H3K27M DMG. We also show that the novel brain penetrant, PARP1-selective inhibitor AZD9574 compares favorably to olaparib when combined with RT, prolonging survival in a syngeneic orthotopic model of H3K27M DMG. This study highlights the ability of PARP1 inhibition to radiosensitize and induce an NK cell-mediated antitumor immunity in H3K27M DMG and supports future clinical investigation.

Frameless Stereotactic Biopsy of Brainstem Tumors Using the Stealth Autoguide: A Technical Note.

In the molecular era of neuro-oncology, it is increasingly necessary to obtain tissue for next-generation sequencing and methylome profile for prognosis and targeted oncological management. Brainstem tumors can be technically challenging to biopsy in the pediatric population. Frame-based and frameless techniques have previously been described and proven to be safe and efficacious in children. Recent cranial robotic guidance platforms have augmented the fluency of frameless stereotactic approaches, but the technical nuances of these procedures in children are not often discussed. We present a technical workflow for frameless stereotactic biopsy of brainstem tumors in children using the Medtronic Stealth Autoguide cranial robotic guidance platform and examine safety and efficacy of this surgical approach. A minimally invasive, frameless, transcerebellar approach is described, including operative steps and workflow. We assessed operative times, diagnostic accuracy and yield, and complication rates. Five patients underwent biopsy with the technique described. The youngest patient in our series was of 2 years. The intended target was achieved on postoperative imaging in all cases, and diagnostic tissue was obtained in all 5 patients. One patient had a clinically insignificant hemorrhage. Frameless stereotactic biopsy of the brainstem can be performed safely, efficiently, and accurately using the Medtronic Stealth Autoguide robotic platform in children as young as 2 years.

Re-irradiation for progressive Diffuse Intrinsic Pontine Glioma (DIPG): The Spanish experience

IntroductionDiffuse intrinsic pontine glioma (DIPG) is the most common malignant brainstem tumour in children. Despite advances in understanding its biology, current treatments have shown minimal impact on overall survival in this fatal disease. Focal radiotherapy (RT) is the only treatment proven to improve symptoms and extend progression-free survival. Albeit palliative, re-irradiation (rRT) has emerged as the best alternative for progressive disease. This study presents the Spanish experience with re-irradiation in DIPG. ResultsBetween April 2015 and December 2023, 44 paediatric patients with progressive DIPG underwent rRT in 16 Spanish institutions. Median time from diagnosis to progression was 9.9 months (range, 4.2–24.3 months). Median dose of rRT was 20 Gy (range, 18–40 Gy) in 2 Gy fractions (range, 1.3–4 Gy). Twenty-two patients (50 %) received other treatments besides RT. Clinical improvement was seen in 77.3 %, and radiological improvement in 60 %. Treatment was well tolerated (1 case toxicity >grade 2 related to rRT). Median overall survival was 15.5 months (range, 8.2–63.2 months), with a median time from rRT to death of 4.2 months (range, 0.6–10.3 months). Longer time between diagnosis and rRT (>10 months) and dose of rRT >20 Gy were statistically significantly correlated with better overall survival. There was no survival benefit in patients receiving additional treatments. ConclusionsRe-irradiation is safe and effective in progressive DIPG patients, not only improving symptoms but also prolonging survival. However, the ideal candidates for rRT remain undefined, as well as the best irradiation scheme. Prospective studies are needed.

Addressing barriers in diffuse intrinsic pontine glioma: the transformative role of lipid nanoparticulate drug delivery.

The brainstem tumour known as diffuse intrinsic pontine glioma (DIPG), also known as pontine glioma, infiltrative brainstem glioma is uncommon and virtually always affects children. A pontine glioma develops in the brainstem's most vulnerable region (the "pons"), which regulates a number of vital processes like respiration and blood pressure. It is particularly challenging to treat due to its location and how it invades healthy brain tissue. The hunt for a solution is continually advancing thanks to advances in modern medicine, but the correct approach is still elusive. With a particular focus on brain tumours that are incurable or recur, research is ongoing to discover fresh, practical approaches to target particular areas of the brain. To successfully complete this task, a thorough literature search was carried out in reputable databases like Google Scholar, PubMed, and ScienceDirect. The present article provides a comprehensive analysis of the notable advantages of lipid nanoparticles compared to alternative nanoparticle formulations. The article delves into the intricate realm of diverse lipid-based nanoparticulate delivery systems, which are used in Diffuse Intrinsic Pontine Glioma (DIPG) which thoroughly examines preclinical and clinical studies, providing a comprehensive analysis of the effectiveness and potential of lipid nanoparticles in driving therapeutic advancements for DIPG. There is strong clinical data to support the promising method of using lipid-based nanoparticulate drug delivery for brain cancer treatment, which shows improved outcomes.

DIPG-56. BRAINSTEM TUMOR BIOPSY: A SINGLE INSTITUTE EXPERIENCE OF SAFETY AND FEASIBILITY IN A LOW-MIDDLE INCOME COUNTRY (LMIC)

Abstract BACKGROUND Brainstem tumors account for 10-15% of all pediatric central nervous system tumors and carry a dismal prognosis. These include a wide spectrum of histological diagnosis therefore, in most specialized centers stereotactic biopsies for brainstem tumors are considered safe and standard practice. However, this approach continues to pose a challenge in LMIC’s. METHODS We retrospectively reviewed data of patients presenting with brainstem tumors at Aga Khan University Hospital, Pakistan, between 2014 and 2023. In addition to histopathological analysis, whole-genome sequencing and RNA sequencing were also performed on 6 tumors. RESULTS We identified 39 patients with brainstem tumors; amongst these 56.4% were females. Median age at diagnosis was 9 years (IQR: 5.1-11). Most cases presented with walking difficulty (74%) and cranial nerve palsies (46%). Median duration of symptoms was 30 days (IQR: 20-60). On imaging large proportion were intrinsic pontine tumors (53.8%) followed by brainstem (30.8%) and cerebellopontine angle (15.4%). 21 patients underwent stereotactic biopsy with 86% performed after 2016. 43% were intrinsic pontine tumors. Histopathology analysis showed a wide spectrum with diffuse midline glioma in 28% of cases, pilocytic astrocytoma, anaplastic astrocytoma and ganglioglioma in 33%, 14% and 9% of cases respectively. Rare entities identified included embryonal tumor with multilayered rosettes, atypical teratoid rhabdoid tumor and schwannoma (5%). Biopsies were uneventful in 81% of cases. Complication of biopsy in 2 patients included cranial nerve palsy and subdural hemorrhage. No patient died due to complications of biopsy. Surgical and medical management was altered in 9 patients due to availability of histological diagnosis. CONCLUSIONS We present the first study demonstrating safety and feasibility of stereotactic biopsy for brainstem tumors in LMIC. These biopsies should be considered for atypical brainstem tumors and should be performed in well-equipped tertiary neurosurgical facilities by trained neurosurgeons in LMIC.

OTHR-20. MINIMALLY-INVASIVE MOLECULAR DIAGNOSIS OF INFILTRATING BRAINSTEM GLIOMAS THROUGH CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING

Abstract BACKGROUND Accurate molecular diagnosis of infiltrating brainstem tumors, including diffuse midline gliomas and IDH mutant astrocytomas, is essential for prognostication and optimal therapy. Surgical biopsy of the brainstem carries risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally invasive “liquid biopsy” analyzing circulating tumor cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) could represent an alternative molecular diagnostic modality. METHODS We performed MSK-IMPACT, a New York State Department of Health authorized hybridization capture-based next-generation panel DNA sequencing clinical assay on 44 CSF samples from 37 unique patients with brainstem tumors. Ages at time of CSF collection ranged from 1–47 years (median: 29 years). For samples in which diagnostic alterations could not be detected, we performed a series of secondary analyses for known hotspot mutations such as H3F3A, IDH1/2, and BRAF, including manual review for mutant reads below the official clinical reporting threshold, as well as droplet digital PCR (ddPCR). RESULTS Among 44 samples included in this study, 10/44 (23%) samples had mutations detected by MSK-IMPACT using standard clinical calling criteria and 14 of the remaining 34 samples (41%) had supporting evidence of a diagnostically relevant mutation based on manual review. Testing by ddPCR was performed on 11 samples (based on DNA availability), uncovering 4 additional mutations. Overall positive diagnostic yield was 64% (28/44). In biopsied patients, CSF confirmed disease-defining mutations in 80% (16/20) of CSF samples, including H3F3A K28M (9/10; 90%) and IDH1/2 (5/6; 83%). Among 24 unbiopsied patients, a disease-defining mutation was identified in 60% (15/25) of CSF samples. CONCLUSIONS Analysis of CSF cfDNA in patients with brainstem tumors has a high diagnostic yield and obviates the need for tissue biopsy in a majority of patients. A tiered testing approach using next-generation sequencing and ddPCR assays helps maximize diagnostic information and sensitivity.

ETMR-01. BRAIN STEM TUMORS IN CHILDREN LESS THAN THREE MONTHS: CLINICAL AND RADIOLOGIC FINDINGS OF A RARE DISEASE

Abstract BACKGROUND Brainstem gliomas account for 10-20% of all central nervous system pediatric neoplasms. Brain stem tumors in children less than three months old at diagnosis are extremely rare. Our aim is to study a retrospective cohort to improve the understanding of the disease course and guide patient management. METHODS This is a multicenter retrospective analysis across the European Society for Pediatric Oncology SIOP-E HGG/DIPG Working Group linked centers and included patients with a brainstem tumor diagnosed between 2009 and 2020 and aged less than 3 months at diagnosis. Clinical data was collected, and imaging characteristics were analyzed blindly and independently by two experienced neuroradiologists. RESULTS Five cases (female; n=4) were identified of which no patient received any therapy. The epicenter of 2 tumors was in the medulla oblongata alone and in the medulla oblongata and the pons in 3 cases. For patients with tumor in equal parts in the medulla oblongata and the pons (n=3) the extension at diagnosis involved the spinal cord; for the 2 patients with the tumor epicenter in the medulla oblongata alone (n=2) the extension at diagnosis included the pons (n=2) and the spinal cord (n=1). Biopsy was performed in one patient identifying a pilocytic astrocytoma. Two patients died rapidly. In one patient autopsy revealed a high-grade glioma (case 3). Three survivors showed either spontaneous tumor regression (n=2) or stable disease (n=1). The surviving patients were followed-up for 10, 7, and 0.6 years, respectively. One case had the typical imaging characteristics of a dorsal exophytic low-grade glioma. CONCLUSIONS No patient fulfilled the radiologic criteria defining a high-grade glioma. Central neuroradiological review and biopsy (if feasible) may provide useful information with regards to the patient’s management.

IMG-18. DIFFERENTIAL DIAGNOSIS OF POSTERIOR FOSSA TUMORS USING RADIOMICS; A FIRST-LINE DIAGNOSTIC STEP REQUIRED FOR DOWNSTREAM ANALYSES

Abstract BACKGROUND Accurate radiographic diagnosis of pediatric brain tumors (PBTs) that originate in the brainstem and posterior fossa, including medulloblastoma (MB), pilocytic astrocytoma (PA), ependymoma (EPN), atypical teratoid/rhabdoid tumor (ATRT), and diffuse intrinsic pontine glioma (DIPG), is crucial for optimizing surgical approaches and enhancing neoadjuvant therapies. Existing research on the radiographic differential diagnosis of posterior fossa tumors has limitations, including small sample sizes, lack of inclusion of certain histologies especially rarer tumors such as ATRTs, and incomplete analysis of the whole tumor, including peritumoral edema. In this study, we aimed to perform a comprehensive analysis using radiomics and machine learning to differentiate among the common posterior fossa and brainstem tumors. METHODS We employed 927 radiomic features extracted from whole tumor regions within treatment-naïve, standard multiparametric MRI sequences (pre-/post-contrast T1-weighted, T2-weighted, FLAIR) of 264 patients (106 MBs, 78 PAs, 28 EPNs, 27 ATRTs, and 25 DIPGs), collected from the Children’s Brain Tumor Network (CBTN). We adopted a one-versus-rest classification strategy, employing Support Vector Machines combined with the Least Absolute Shrinkage and Selection Operator (LASSO) for feature selection, and implementing nested cross-validation for robustness. RESULTS The performances of the classifiers were evaluated using the Area Under the Receiver Operating Characteristic Curve, yielding values of 0.84 for MBs, 0.84 for PAs, 0.70 for EPNs, 0.75 for ATRTs, and 0.71 for DIPGs. CONCLUSIONS Our method effectively differentiates between various tumor types in the posterior fossa and brainstem, paving the path towards the development of comprehensive diagnostic and prognostic AI tools for pre-treatment histological diagnosis of these tumors. These AI tools can lead to more tailored, risk-adjusted treatments for PBTs, reducing morbidities and improving patient outcomes. Based on our promising initial results, we will expand our dataset to include more samples and incorporate rarer tumor types, as well as piloting in different molecular subclassifications.

LMIC-08. NEUROSURGERY UTILIZATION RATE FOR PEDIATRIC LOW-GRADE GLIOMA: A SIMULATION-BASED ANALYSIS

Abstract BACKGROUND The outcomes of pediatric low-grade glioma (pLGG) are markedly influenced by accessibility to neurosurgical services. Even when clinically recommended, neurosurgical interventions might not be feasible when there is scarcity in qualified medical professionals and preparedness of medical facilities, which is the case in many low- and middle-income countries (LMICs). To aid cancer control planning efforts and guide policy makers and clinicians to prioritize allocation of resources, accurate neurosurgical utilization rate (NUR) estimations are crucial. This study developed a model to estimate the NUR, to facilitate the approximation of neurosurgery needs for pLGG. METHODS A decision tree model was developed to assess the NUR in HICs and LMICs. Patients were classified into five groups based on the anatomic site of the tumor. For each type of cancer, patients had three surgical options, gross total resection, subtotal resection, and biopsy. Anatomic distribution and likelihood to perform surgical resection were retrieved from the literature for both HICs and LMICs. RESULTS Findings showed discrepancy in the anatomic distribution of pLGG in HICs and LMICs. Proportion of patients with cerebral hemisphere, cerebellum, OPHG/diencephalon, brainstem, and spine tumors and their likelihood to undergo surgical intervention were 34% | 28%, 32% | 27%, 17% | 4%, 12% | 3% and 5% | 4% in HICs, and 26% | 22%, 39% | 32%, 26% | 5%, 6% | 2% and 3% | 2% in LMICs, respectively. Overall, NUR in pLGG patients was 66% in HICs and 63% in LMICs. CONCLUSION Despite the limited resources in LMICs, the overall proportion of patients recommended to undergo a surgical intervention is comparable to HICs. For this need to be met, long-term planning need to take place at the policymaking level. It is possible to estimate the NUR for all pediatric CNS tumors following the same approach to more comprehensively estimate the neurosurgical needs of this patient population globally.

DIPG-85. MALT1 IN H3K27-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) is a devastating pediatric brainstem tumor that affects 200-300 individuals in the US per year. Median survival is 9-11 months, and there are virtually no long-term survivors. Despite decades of clinical trials, radiation therapy remains standard of care, extending survival by 2-3 months. Elucidating the mechanisms that drive H3K27-altered DMG pathogenesis in order to uncover therapeutic vulnerabilities is of critical importance. Recent studies implicate MALT1 as a potential therapeutic target in gliomas. MALT1 is the effector molecule of the CARMA-BCL10-MALT1 (CBM) signalosome, a cytoplasmic protein complex that drives downstream pro-survival transcriptional activity. MALT1, which possesses scaffolding and protease functions, promotes cell viability, proliferation, and migration/invasion in multiple cancer types. We aim to evaluate the hypotheses that MALT1 promotes DMG cancer cell proliferation and migration and that MALT1 inhibition will abrogate tumor progression. METHODS/RESULTS We screened a panel of cells including astrocytes, neural stem cells, and DMG cell lines by Western blot and found that CBM complex members BCL10 and MALT1 are present in all cells tested. We next demonstrated that MALT1 is proteolytically active in DMG cell lines SF8628 and HSJD-DIPG-007. Specifically, we showed cleavage of the MALT1 protease substrate HOIL, which was abrogated by treatment with the MALT1 protease inhibitor MLT-748. We next generated multiple dox-inducible MALT1 shRNA DMG cell lines to assess the impact of MALT1 deficiency on DMG biology. Thus far, we find that shRNA knockdown of MALT1 does not impact SF8628 cell proliferation. CONCLUSIONS MALT1 is expressed and proteolytically active in DMG cells. Initial studies show that MALT1 knockdown does not affect proliferation of SF8628 cells. We will next evaluate the effect of MALT1 knockdown on proliferation of additional DMG cell lines, as well as the impact on other malignant features of DMG cells including migration/invasion.

DIPG-35. OVERCOMING THE BLOOD-BRAIN BARRIER CHALLENGE IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are aggressive paediatric brainstem tumours with no known cure. The development of effective treatments is greatly impeded by the failure of most anti-tumour agents to penetrate the blood-brain barrier (BBB). Our research, along with that of others, has revealed that the BBB remains intact in DMG. We employed DMG orthotopic models to explore potential region-specific variations in response to the therapeutic agent temsirolimus as well as confocal microscopy and single-cell RNA sequencing (scRNAseq) to elucidate the impact of DMGs on brain endothelial morphology and pathways governing BBB integrity. We first assessed the effectiveness of the mTOR inhibitor temsirolimus when administered in cortex-injected DMG models compared to brainstem-injected DMG. We found significantly increased survival rates, elevated temsirolimus levels, and reduced tumour cell proliferation in the cortex versus the brainstem. Confocal imaging analysis indicated no structural differences between DMG and Matrigel-injected animals. However, scRNAseq unveiled distinct transcriptomic changes: brainstem-injected DMG exhibited downregulation of genes related to inflammatory and apoptotic pathways, whereas cortex-injected DMG showed downregulation of interferon pathways. To overcome the tightened barrier in the brainstem, we explored three MCL1 inhibitors and SNGR-TNFa (targeting CD13) as potential blood-brain barrier modulators. These inhibitors significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro DMG BBB model, and decreased the protein expression of claudin-5. Moreover, in vivo, we found that administration of a single dose of either SNGR-TNFa or the MCL1 inhibitor S63845 improved penetration of Texas-Red (3KDa) in DMG-engrafted animals, indicating the effective opening of the BBB. In summary, our study revealed that DMG directly affects BBB pathways in endothelial cells, tightening the barrier and reducing treatment efficacy. MCL1 inhibitors and SNGR-TNFa show promise in modulating the BBB and have the potential to enhance therapeutic activity.

DIPG-87. TARGETING GENE-REPRESSIVE EPIGENETIC REGULATION TO INDUCE INTERFERON SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brainstem tumor with no known effective treatment. DIPG is driven by the H3K27M mutant histone, which promotes epigenetic dysregulation, leading to loss of gene-repressive H3K27me3 marks, global DNA hypomethylation, and a distinct gene expression profile. DIPG cells retain histone methylation at histone H3 lysine 9 (H3K9me2/3), suggesting that the tumor may rely on these marks to repress unwanted expression. Our prior work has demonstrated that treatment with the DNA hypomethylating agent decitabine (DAC) elicits expression of cancer-specific antigens and immune activation, including activation of interferon signaling, but modulating gene-repressive H3K9 methylation in DIPG has not been extensively investigated. METHODS We treated four primary patient-derived DIPG neurosphere cell lines with BIX01294 and UNC0642, inhibitors of H3K9 methyltransferases G9a/GLP, and analyzed gene expression changes by RNA-sequencing and Western blot. We assessed the effect of combination treatments on these four cell lines with both DAC and either BIX01294 or UNC0642 and evaluated impact on gene expression, innate immune signaling, and interferon pathway activation. RESULTS We found that treatment with either of the G9a/GLP inhibitors resulted in the upregulation of genes involved in immune signaling. Given the similarities in the immune signaling response elicited from treatment with DAC and the G9a/GLP inhibitors, we evaluated the effect of combination treatments with both DAC and either BIX01294 or UNC0642. These combinations resulted in an increased induction of genes involved in immune signaling, including STING. CONCLUSIONS Pharmacologic inhibition of DNA methylation and histone H3K9 methylation elicits immune activation in DIPG cells. Upregulation of interferon and STING signaling with this combination treatment may be significant in enhancing the immunogenicity of DIPG cells to allow effective immunotherapy.

DIPG-23. SAFETY AND TECHNICAL YIELD OF BRAINSTEM BIOPSIES IN CHILDREN – AN UPDATED META-ANALYSIS OF 1000 CASES

Abstract BACKGROUND Brainstem tumors represent ~10% of pediatric brain tumors, ~80% of these are diffuse intrapontine gliomas (DIPG, primarily WHO Grade 4 midline glioma, H3 K27M-mutant). Given invariably poor prognosis in these cases, there continues to be immense variation (across the US and internationally) in performing biopsy of these lesions as opposed to making a presumptive diagnosis based on MRI. Several multi-center studies in recent years have aimed to show the safety profile of biopsy but an updated meta-analysis is lacking. METHODS We found 29 studies of pediatric brainstem biopsy in the last 20 years (2003-2023), representing data from 1002 children. We applied meta-analysis of proportions using a random-effects model to generate point estimates, confidence intervals, and measures of heterogeneity. RESULTS 87% of procedures were stereotactic needle biopsies (of these, 62% with a frame, 14% without frame, and 24% robotic.) Biopsy yield (i.e. the rate at which biopsy resulted in a tissue diagnosis) was 94% (95% CI 92-97). Temporary complications were seen in 6% (95 CI 4-8), most commonly including 1) radiographic evidence of bleeding or air without an associated fixed neurological deficit, 2) cranial nerve dysfunction, and 3) delayed awakening. Permanent complications (excluding death) were seen in 1% (95% CI 0.5-2), most commonly including cranial nerve dysfunction and limb weakness. 5 deaths were reported in the entire pooled cohort of 1002 children (0.5%). Gliomas represented 92% of tumors (95% CI 89-95). Of the gliomas for which WHO grading was provided, 68% were high grade (III/IV). CONCLUSION When counseling families on the merits of brainstem biopsy in children, it is reasonable to state that permanent morbidity is rare (< 2%). If biopsy is performed specifically to facilitate enrollment in clinical trials requiring a molecular diagnosis, the risks of biopsy outlined here should be weighed against potential benefits of trial enrollment.

Human Adapted Prosomeric Model: A Future for Brainstem Tumor Classification

This study reevaluates the conventional understanding of midbrain anatomy and neuroanatomical nomenclature in the context of recent genetic and anatomical discoveries. The authors assert that the midbrain should be viewed as an integral part of the forebrain due to shared genetic determinants and evolutionary lineage. The isthmo-mesencephalic boundary is recognized as a significant organizer for both the caudal midbrain and the isthmo-cerebellar area. The article adopts the prosomeric model, redefining the whole brain as neuromeres, offering a more precise depiction of brain development, including processes like proliferation, neurogenesis, cell migration, and differentiation. This shift in understanding challenges traditional definitions of the midbrain based on external brain morphology. The study also delves into the historical context of neuroanatomical models, including the columnar model proposed by Herrick in 1910, which has influenced our understanding of brain structure. Furthermore, the study has clinical implications, affecting neuroanatomy, neurodevelopmental studies, and the diagnosis and treatment of brain disorders. It emphasizes the need to integrate molecular research into human neuroanatomical studies and advocates for updating neuroanatomical terminology to reflect modern genetic and molecular insights. The authors propose two key revisions. First, we suggest reclassifying the isthmo-cerebellar prepontine region as part of the hindbrain, due to its role in cerebellar development and distinct location caudal to the genetically-defined midbrain. Second, we recommend redefining the anterior boundary of the genetically-defined midbrain to align with genetic markers. In conclusion, the authors highlight the importance of harmonizing neuroanatomical nomenclature with current scientific knowledge, promoting a more precise and informed understanding of brain structure, which is crucial for both research and clinical applications related to the human brain.