Brain Tumor In Adults Research Articles

CNSC-54. CENTRAL AND BOUNDARY-DRIVEN GROWTH PATTERNS DOMINATE RESPECTIVELY IDH WILD-TYPE AND MUTANT GLIOMAS

Abstract Diffuse gliomas are the most prevalent primary malignant brain tumors in adults and are characterized by invasive growth and poor prognosis. Tumor cells infiltrate the brain parenchyma and spread to distant sites. The aim of this study is to characterize the spatial heterogeneity, mode, and direction of tumor development to identify key areas for localized treatment. Neuronavigation was employed to collect n=134 image-guided samples from n=16 adult patients with diffuse gliomas, including n=7 IDH wild-type (IDHwt) and n=9 IDH mutant (IDHmut) tumors from regions with and without MRI abnormalities. Collected samples were subjected to targeted and shallow whole-genome sequencing. Somatic variants were used to determine the evolutionary processes governing heterogeneity. Examination of the dN/dS ratio, Tajima’s D score, and MOBSTER model suggested that all tumors are consistent with neutral evolution, as previously reported. Considering that genetic heterogeneity was a result of stochastic mutations, we used phylogeographic relationships between samples to dissect the spatiotemporal development of these tumors. Evolutionary (patristic) and cartesian (Euclidean) distances between sample pairs from the same patient were correlated in n=2/9 IDHmut and n=2/7 IDHwt patients, suggesting that migratory or punctuated evolutionary processes play a major role in tumor diffusion. An examination of spatial diffusion revealed that the latest descendant appeared peripherally in n=5/9 IDHmut patients, suggesting boundary-driven growth. In the remaining cases, three patients exhibited centrally-driven growth, whereas a fourth patient displayed a mixed pattern. In contrast, in n=4/7 IDHwt patients, descendants appeared closer to the tumor center, suggesting centrally-driven growth. In the remaining three IDHwt patients, one patient showed a diffusion that was consistent with boundary-driven growth, while the other two patients showed a mixed pattern. Taken together our data suggest that IDHwt tumors tend to develop centrally, whereas IDHmut tumors preferably develop outward. Our data could aid in tailoring localized treatment based on projected growth patterns.

CSIG-27. GLIOBLASTOMA CELL STATE PLASTICITY AND THERAPEUTIC RESISTANCE IS MEDIATED BY BRD2

Abstract GBM is a highly aggressive primary brain tumor in adults, notable for its heterogeneity, high recurrence rates, and increased resistance to therapy. Multi-omics analyses have consistently identified three distinct transcriptionally defined cell states – proneural (PN), classical (CL), and mesenchymal (MES) – in IDH-wildtype GBM, with high plasticity among these states in response to diverse stimuli. Transition of GBM cells to a MES cell state is particularly associated with high invasiveness, resistance to therapy, and recurrence. However, the underlying mechanisms driving the MES transition remain poorly understood. In this study we identified the PTEN/NF-kB/BRD2 pathway as key driver of the MES transition, with BRD2 acting as a crucial epigenetic modulator for cell state transitions in GBM. We demonstrate that PTEN plays a critical role in regulating the chromatin binding of BRD2, BRD4 and p65/RelA. Additionally, acetylation of RelA at lysine 310 is required for BRD2 localization to the promoters of MES genes, which is essential for their expression. Loss of BRD2 leads to transition of GBM cells from MES to PN cell state and displayed enhanced sensitivity to ionizing radiation (IR). Further, bromo domains (BDs) of BRD2 are important for MES transition, as mutations in these domains resulted in transitioning of GBM cells from MES to other cell states. Furthermore, we show that BD2-specific BET inhibitors displace BRD2 from MES gene promoters, abrogate MES transition, and enhance GBM sensitivity to IR. Using a brain-penetrant BD2 inhibitor, we effectively reduced GBM cell invasion and tumor-associated microglia/macrophage (TAM) abundance in orthotopic xenograft models. These findings establish BRD2 as a key regulator of MES transition and therapeutic resistance in GBM models, paving the way for the use of bromodomain-specific BET inhibitors in targeting MES GBM.

DDDR-05. SPATIALLY RESOLVED DRUG SENSITIVITY PROFILING IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is the most common malignant primary brain tumor in adults, characterized by poor overall survival and an urgent need for more effective treatment strategies. Macroscopic heterogeneity, as observed through MR imaging, divides the tumor into distinct regions: a tumor-cell dense contrast-enhancing tumor core and a non-contrast-enhancing infiltration zone that appears hyperintense on T2/FLAIR imaging. The infiltration zone contains both infiltrating tumor cells and non-malignant cells from the local brain microenvironment. Although genomic and transcriptomic differences between the tumor core and the infiltration zone have been reported, functional differences, such as differential drug sensitivities, remain largely unknown. METHODS To investigate this, we collected region-specific glioblastoma patient tissue samples from both the contrast-enhancing tumor core and the non-contrast-enhancing but 5-aminolevulinic acid (5-ALA) fluorescent infiltration zone during surgery. Using pharmacoscopy, a microscopy-based single-cell drug screening platform, we assessed drug responses across regions and patients. RESULTS To determine whether drug responses within a single tumor region were more similar compared to responses across different regions we collected multiple tissue samples from each tumor region in seven patients. We identified region-specific drug responses for temozolomide and lomustine, among other drugs, in some of the patients. However, drug sensitivities were not consistent within the same region across different patients. Thus, across patients, the region-specific drug responses were obscured by patient heterogeneity. To address intra- and inter-tumor drug response heterogeneity, we developed a complementarity score based on region-specific drug response data of 60 drugs across 23 paired samples. The complementarity score identified promising drug combinations that demonstrated strong anti-tumor activity across tumor regions and patients. CONCLUSIONS Monotherapy approaches have largely failed in glioblastoma, despite extensive molecular profiling efforts aimed at identifying tumor-specific vulnerabilities. Identifying drug combinations that synergistically target the locoregional heterogeneity of glioblastoma may open up new avenues of personalized therapy.

TMIC-34. INFILTRATIVE BEHAVIOR OF PATIENT-DERIVED GLIOBLASTOMA CELL LINES IN REGION-SPECIFIC HUMAN BRAIN ORGANOIDS

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults. GBMs are known for their highly invasive and proliferative properties and are associated with a dismal prognosis (~12-18 months post-diagnosis). Neuroanatomical disparities in the occurrence of GBMs suggest that in addition to microenvironmental impacts, tumor cells are impacted by the cellular and molecular diversity across varying regions of this nervous system. This idea has been difficult to study due to the lack of human-specific models that effectively recapitulate the biological behaviors of GBMs in region-specific microenvironments. To address these challenges, we engrafted GFP-tagged patient-derived glioma cell lines into human induced pluripotent stem cell (hiPSC)-derived organoids patterned to mimic the most forebrain (dorsal cortex) and hindbrain (spinal cord) regions of the nervous system. Immunostaining assays at 2, 7, 14, and 21 days post-engraftment compared the extent of infiltration, as measured by the distance traveled by glioma cells, in both regions. We found that glioma cells infiltrated a significantly greater distance in the dorsal cortical organoids compared to the spinal cord organoids. This suggests a more conducive environment for glioma infiltration in the forebrain vs. hindbrain at baseline. We are currently determining if these regional biases in infiltration capacity are modified with either optogenetic stimulation of the host brain organoids or in the presence of interregional interactions in an assembloid system. Thus, using this fully human-specific platform, we aim to gain insights into the infiltrative behaviors of GBM cells, which will advance our understanding of this devastating disease.

IMMU-61. THE LAST OF US: A FUNGI BASED DENDRITIC CELL VACCINE

Abstract BACKGROUND Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults. The current standard treatment for newly diagnosed GBM involves maximal surgical resection followed by radiotherapy and temozolomide. Despite these interventions, the median overall survival for GBM patients remains approximately 15 months, highlighting the critical need for innovative therapeutic approaches. OBJECTIVE Our laboratory has previously developed a subcutaneous autologous tumor vaccine incorporating irradiated (IR) tumor cells with phagocytosis-stimulating ligands (Mannan-BAM), toll-like receptor (TLR) agonists, and an immunostimulant anti-CD40 antibody (collectively termed MBT). This combination has shown efficacy in mouse models of colon carcinoma, breast cancer, melanoma, and, more recently, gliomas. Our study aimed to create a dendritic cell (DC) vaccine using this strategy. METHODS To evaluate whether MBT could induce the maturation of DCs in vitro, we harvested BMDCs from mice and co-cultured them in the presence of MBT-tagged irradiated tumor cells. We then analyzed the maturation markers using flow cytometry and assessed cytokine production via ELISA. To test the efficacy of this approach in vivo, we utilized two different murine glioma models (SB28 and GL261). RESULTS Using flow cytometry, we observed a significant upregulation of important co-stimulatory molecules. Additionally, ELISA analysis demonstrated that MBT-treated DCs significantly increased the secretion of Type 1 cytokines while limiting the secretion of the regulatory cytokine IL-10. MBT-primed DCs combined with immune checkpoint inhibitor PD-1 increased mouse survival in a glioma model compared to WT mice. However, mice eventually succumbed to cerebral edema. Histology demonstrated significant treatment effects. CONCLUSION Our results demonstrate that MBT effectively matures DCs ex-vivo. Further research is required to immunophenotype the response to vaccination in glioma models and explore ways to overcome resistance.

NCOG-41. COMPARATIVE ANALYSIS OF MORTALITY AND SHORT-TERM SIDE EFFECTS IN PATIENTS WITH GLIOBLASTOMA MULTIFORME TREATED WITH TEMOZOLOMIDE VERSUS TEMOZOLOMIDE PLUS BEVACIZUMAB CHEMOTHERAPY

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor in adults with limited treatment options and poor prognosis. Temozolomide (TMZ) chemotherapy is the standard treatment for GBM, but alternative therapies have shown limited success. Bevacizumab (BEV), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), is used to restrict GBM growth and spread. This study evaluates the efficacy of combining TMZ with BEV compared to TMZ alone in managing GBM. METHODS A retrospective cohort study was conducted using TriNetX database data, focusing on patients diagnosed with GBM and receiving chemotherapy. Cohorts were defined by treatment: TMZ plus BEV versus TMZ alone. Propensity-score matching balanced baseline characteristics, including demographics and health conditions. Primary outcomes included mortality rates and short-term side effects within the first year of treatment, such as cognitive communication deficits, headache, dizziness, insomnia, and nausea/vomiting. RESULTS After matching, 922 patients were included in each cohort. There were no significant demographic differences post-matching. The mortality rate was higher in the TMZ plus BEV group compared to TMZ alone (51.30% vs. 36.98%, RR: 1.387, 95% CI: 1.249-1.541). However, TMZ plus BEV was associated with a lower incidence of cognitive communication deficits (1.95% vs. 4.34%, RR: 0.45, 95% CI: 0.26-0.779) and dizziness (7.27% vs. 10.52%, RR: 0.691, 95% CI: 0.513-0.93). No significant differences were found in headache, insomnia, or nausea/vomiting rates between the groups. CONCLUSION This study indicates that combining bevacizumab with temozolomide may increase mortality in GBM patients but reduce certain side effects such as cognitive deficits and dizziness. These findings emphasize the need to carefully consider the benefits and risks of different chemotherapy regimens for GBM and highlight the necessity for further research to optimize treatment strategies and improve patient outcomes.

TMOD-31. SINGLE-CELL PROFILING AND CHARACTERIZATION OF PATIENT-DERIVED XENOGRAFT GLIOBLASTOMA MODEL IN HUMANIZED MICE

Abstract Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults, and the current standard therapy has limited efficacy. To develop effective therapies, it is desirable to develop an animal model that can replicate the tumor heterogeneity and immune microenvironment of human GBM. Here, we describe a novel GBM mouse model established with a patient-derived xenograft (PDX) in humanized mice harboring a nearly complete human immune environment. Humanized mice were generated by engrafting human CD34+ hematopoietic stem cells from umbilical cord blood into immunocompromised mice (NSG, NSG-SGM3, and NOG-EXL) after myeloablation. NSG-SGM3 is a triple transgenic mouse encoding human IL3, GM-CSF, and SCF on the NSG strain background. NOG-EXL is a NOG strain with human IL3 and GM-CSF expression. These cytokines foster the development of the human myeloid lineage and its differentiation. NSG-SGM3 and NOG-EXL were superior in immune cell reproducibility, especially myeloid cells. Radioresistant PDX GBM cells established after repeated irradiation were injected into the forebrain of humanized mice and the same immunocompromised mice without humanization. Human PDX GBM cells formed aggressive tumors with similar growth speed in the humanized mice compared to non-humanized mice; however, their dissemination to the spinal cord was significantly suppressed in humanized mice, suggesting that human immune cells impact the spinal dissemination of GBM cells. Single-cell RNA sequencing analysis revealed infiltration of human CD4+ T, CD8+ T, NK cells, and macrophages, as well as infiltration of regulatory T and exhausted CD8+ cells into the brain tumor, reflecting the immunosuppressive landscape of recurrent human GBM. Furthermore, we found that tumor heterogeneity was increased in humanized mice compared to non-humanized mice. There was a notable rise in neural progenitor-like cell populations, indicating tumor adaptation to immune pressures. This novel GBM-PDX humanized mouse model might be highly beneficial for analyzing the interaction between human tumor cells and human immune cells, and evaluating the effect of immunotherapies.

CNSC-29. CIRCADIAN SIGNALING IN GLIOBLASTOMA SYNCHRONIZES TUMOR DAILY RHYTHMS AND REGULATES TUMOR PROGRESSION

Abstract Glioblastoma (GBM) is the most common primary brain tumor in adults with a poor prognosis despite aggressive therapy. A recent, retrospective clinical study found that administering Temozolomide in the morning increased patient overall survival by 6 months compared to evening. Previous work has shown that murine and human models of GBM have cell-intrinsic circadian rhythms in expression of the core clock genes Bmal1 and Per2. This suggests that GBM has circadian rhythms that entrain to the hosts’ central clock. Understanding the mechanisms underlying circadian entrainment and tumor growth in GBM is essential to determine whether regulation of tumor biology and susceptibility to therapies varies with time of day. Here, we tested the hypothesis that daily host signaling regulates tumor growth and synchronizes circadian rhythms in GBM. We found that GBM cells transduced with luciferase reporters to record clock gene expression have intrinsic circadian rhythms, with Bmal1 peaking 8-12 h before Per2 in murine and human cell lines in vitro and in vivo. Further, intrinsic circadian rhythms in clock gene expression in GBM entrain to the host through glucocorticoid signaling, regardless of tumor type or host immune status. We found daily glucocorticoids (i.e., Dexamethasone) promoted or suppressed GBM growth depending on time of day of administration and on the clock gene, Bmal1. Blocking circadian signals, like VIP or glucocorticoids, dramatically slowed GBM growth and disease progression. Our results suggest that GBM tumors entrain to the circadian circuit of the brain and depend on clock-controlled cues like VIP and glucocorticoids to grow at specific times of day. This work may inform personalized circadian medicine to improve individual GBM patient outcomes.

SURG-31. HYBRID THERAPY FOR NEWLY DIAGNOSED AND RECURRENT GLIOBLASTOMA: STAGED PROCEDURE INTEGRATING OPEN SURGICAL RESECTION WITH LASER INTERSTITIAL THERMAL THERAPY

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults with a median overall survival (OS) of 15.6 months. Despite advancements, 5-year survival rates have not significantly improved. Laser interstitial thermal therapy (LITT) has been employed to treat both newly diagnosed (nGBM) and recurrent GBM (rGBM) with demonstrable safety and efficacy. Traditionally, LITT has been limited to smaller lesions given physical and thermal constraints. OBJECTIVE We present two case examples of a novel, hybrid treatment approach to GBM, utilizing both surgery and laser ablation as part of a single-staged procedure. METHODS Two geriatric (>65 years) patients with nGBM or rGBM were treated with surgical resection followed by LITT the subsequent day. Both patients underwent preoperative magnetic resonance imaging (MRI) showing multifocal disease with a dominant component, causing mass effect in the second case, and an unresectable component. RESULTS The first case underwent surgical resection of a large, multicentric rGBM in the parieto-occipital region, followed by LITT of an unresectable lesion in the inferior temporal gyrus. The second patient underwent surgical resection of the dominant temporal lobe component, and LITT of the insular component. Neither patient had intraoperative or ablation complications. Both patients had stable postoperative neurological exams and were discharged on the day after ablation, with no new deficits at follow-up. CONCLUSION The prognosis for GBM remains dismal despite significant research and attempts at advancements in patient management. Surgical resection is a critical component of the GBM treatment paradigm. However, surgery alone is not sufficient in many patients. LITT has shown promise for improving GBM outcomes, mainly due to its minimal invasiveness, enhanced immune responses, BBB disruption, and cytoreductive potential. We demonstrate a novel, hybrid approach to GBM using hybrid surgical resection and LITT in a single-staged procedure to overcome traditional constraints of LITT.

EXTH-78. A NOVEL BISPECIFIC S100A4/TFR ANTIBODY TO REPROGRAM THE GLIOMA TUMOR MICROENVIRONMENT AND TARGET GLIOMA STEM CELLS

Abstract GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. While immunotherapy is a promising treatment modality for GBM, most clinical trials have thus far only provided a modest benefit to GBM patients. We propose that enhancing the efficacy of immunotherapy for GBM requires approaches that increase T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. S100A4 is a major regulator of stemness, epithelial-mesenchymal transition, and a critical regulator of immune suppressive myeloid and T cell phenotypes in GBM. Targeting S100A4 in either glioma or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising immunotherapy target. We recently developed a S100A4 blocking monoclonal antibody; however, this antibody does not cross the blood brain barrier (BBB) efficiently to confer significant therapeutic benefit, limiting its efficacy in treating GBM. To overcome this challenge, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration, and tested this in a pre-clinical syngeneic, orthotopic mouse model of GBM. Using ELISA, confocal immunofluorescent microscopy, flow cytometry, and bulk RNA sequencing, we report that systemic administration of the S100A4-TfR BsA results in increased immune cell infiltration with a preferential increase in CD4 T cell infiltration, and potent immune remodeling of the tumor microenvironment to a more pro-inflammatory state. Furthermore, the BsA is taken up by glioma cells intracellularly, which is correlated with depletion of intracellular S100A4 and decreased nuclear SOX2 localization, which would reduce stemness of glioma cells. Functionally, in vitro BsA treatment of primary glioma cell cultures shows decreased S100A4 expression, proliferation, and self-renewal. Our results suggest that S100A4-TfR BsA is a novel dual activity inhibitor to simultaneously reprogram the GBM immune landscape and reduce glioma stemness.

BIOS-06. MACHINE LEARNING MODEL INDEPENDENTLY IDENTIFIES CLINICAL FEATURES ASSOCIATED WITH WILD-TYPE GLIOBLASTOMA VERSUS MUTANT GRADE 4 ASTROCYTOMA

Abstract BACKGROUND Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, presents significant clinical challenges due to its invasive nature and high recurrence rates. Understanding the clinical demographic factors associated with IDH wild-type (IDH-wt) versus mutant grade 4 astrocytoma (IDH-Mut) is critical for prognosis and treatment strategies. This study aimed to utilize a novel machine learning method to identify key clinical and demographic factors linked to IDH status. METHODS The Cancer Imaging Archive dataset included 617 patients but required preprocessing and cleaning. The cleaning process ensured data completeness and consistency by removing non-relevant variables like date and time, resulting in 20 potential predictors and 317 observations. A random forest model using Gini impurity score was built to classify patient IDH status. The dataset was split into a training set (two-thirds) and a test set (one-third). A variable importance plot identified significant variables based on the mean decrease in the Gini index. RESULTS Our machine learning model achieved 98% accuracy on the test set, outperforming the previously published model of 80% accuracy. A variable importance plot identified age at diagnosis, gender, ethnicity, and race as key predictors of IDH status, with age being the most important. Age was the most important predictor, but race (Caucasian) and ethnicity (not Hispanic or Latino) were also significant. CONCLUSIONS We present a highly accurate machine learning model for differentiating glioma IDH molecular status based only on patient clinical and demographic factors; improving upon prior published models. Future work will use a generalized logistic model to explore variable interactions and predict IDH-wt versus IDH-Mut status. These steps are vital for enhancing predictive accuracy and supporting personalized treatment strategies. Furthermore, we will define associations and interactions between these variables with other biological and therapeutic variables in predicting clinical outcome.

CNSC-47. SENOLYTICS ERADICATE SENESCENT MICROGLIA IN THE BRAIN PARENCHYMA AND IMPROVE SURVIVAL IN OLDER ADULT MICE WITH GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a poor median survival rate. Despite the aggressive standard of care treatment combining surgical resection, chemo-, and radio-therapy, the median overall survival (OS) is only ~15-18 months. Progressively increasing subject age is inversely associated with GBM patient OS such that older adults tend to have significantly worse survival outcomes compared to younger counterparts. Senescent cells accumulate in the body during progressive aging and contribute to worse outcomes in older adult mice with GBM. Senolytics are pharmacologics that cause senescent cells to undergo cell death. OBJECTIVE To comprehensively profile the treatment effect of senolytics combined with or without immunotherapy in mice with GBM across the lifespan. METHODS Young 7-9- and older adult 97-104-week-old wild-type (WT; C57BL/6) and 110-130-week-old INK-ATTAC mice with or without intracranial SB28 cells were treated with or without brain radiation (RT), PD-1 mAb, and an IDO enzyme inhibitor with the senolytics, dasatinib + quercetin, or the AP compound that induces p16INK4A+ senescent cell death in INK-ATTAC mice (n=5/group). The quantification of beta-galactosidase positive cells by flow cytometry was performed on brain-resident neurons, astrocytes, oligodendrocytes, microglia, as well as dendritic cells, macrophages, neutrophils, T cells in the tumor and non-tumor bulk mass. Overall survival was also evaluated. RESULTS Senolytics or AP compound treatment both decrease beta-galactosidase positive microglia in the older adult brain with GBM (p<0.05). Combining radio- and immuno-therapy with senolytics improves overall survival of older adult mice with intracranial GBM (p<0.05). CONCLUSIONS Our ongoing work aims to understand how senescent microglia affect neuronal health and function in the older adult brain with GBM. KEYWORDS Glioma, senescence, senolytic.

EXTH-29. EVALUATION OF THE ANTITUMOR EFFECT OF A CHEMOTHERAPY-LOADED DEVICE FOR THE TREATMENT OF PRIMARY BRAIN TUMORS

Abstract Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Tumor recurrence is therefore inevitable, and median survival is just over 14 months. Currently available treatments are inadequate and do not provide optimal tumor control. We believe that treatment localized directly at the tumor site will eliminate a greater number of infiltrating tumor cells while significantly reducing systemic toxicity. For this project, we are collaborating with a team specializing in bioengineering, who have produced a biomaterial enabling the gradual release of various chemotherapeutic agents. Our aim is to use this biomaterial to deliver chemotherapeutic agents to the tumor site through the surgical cavity. Cell-based assays were carried out using glial tumor cells in the presence of the biomaterial containing chemotherapeutics. A survival study was realized with the Fischer F98 rat model, which underwent partial tumor resection followed by injection of biomaterial containing chemotherapy. Histological studies of brains and resected tissues were also carried out to assess the effect of the biomaterial on the brain environment. In vitro assays showed a strong cytotoxic effect against glial tumor cells at 24h, 48h and 72h of biomaterial-drug incubation. The combination of two chemotherapeutic agents showed the best cytotoxic effect on glial tumor cells. We also performed a partial tumor resection and insertion of the chemotherapy-loaded biomaterial into the surgical cavity of the F98 glioma bearing rat. Similar to in vitro studies, the combination of two antineoplastic agents shows a significant improvement in animal survival. In conclusion, the in vitro and in vivo results of this study show that the combination of chemotherapy has the greatest antitumor effect. We hope that these exciting results will lead to the development of a new treatment for patients with glioblastoma and potentially other diseases where complete tumor resection is impossible.

TMIC-18. ROLE OF NEUROTRANSMITTERS IN GLIOBLASTOMA PATHOPHYSIOLOGY: EMERGING INSIGHTS

Abstract INTRODUCTION Glioblastoma (GBM) are the most common primary brain tumor in adults associated with a dismal patient outcome. While it is well-known that glutamatergic neurons can form functional synapses with glioblastoma cells to enhance tumor growth, lesser is known about the effects of activity of other neuronal types on glioblastoma biology, especially among different glioblastoma molecular subtypes. Here, we examined the effects of various neurotransmitters on the growth of glioblastoma cells characterized as classical, proneural and mesenchymal subtypes. METHODS Patient-derived glioblastoma cell lines were exposed to varying concentrations of different neurotransmitters and assessed for cell viability using CCK-8 assay. Transcriptomic data from TCGA (The Cancer Genome Atlas) database was analyzed for correlation of expression of neurotransmitter receptors (NTRs) with patient survival. Using IVY-GAP database, we compared the expression of NTRs at leading-edge versus tumor core of tumors to assess their possible involvement in cell migration and microenvironmental interactions. RESULTS Consistent with previous findings, we observed an increase in cell viability in all GBM cell lines after being incubated with L-glutamate. The classical subtype has a stronger growth response to L-glutamate compared to the other two subtypes. TCGA data revealed the expression of GRIK4 (glutamate ionotropic receptor kainate type subunit 4) as highest in the classical subtype as well as inversely correlated with worse patient survival in this GBM subtype, as opposed to proneural or mesenchymal patient groups. CCK8 assay also suggested that acetylcholine and dopamine increase glioblastoma cell growth. A subset of cholinergic and glutamatergic receptors (CHRM1, GRM2) was found upregulated in infiltrating/leading-edge regions of glioblastoma tumors compared to the tumor core, suggesting possible involvement in glioblastoma-neuron interactions. CONCLUSION Our findings suggest that GBM subtypes may differentially leverage neuro-modulatory mechanisms to support their growth. Future studies will investigate the differential neuronal responses in GBM subtypes using pharmacological/genetic approaches.

DNAR-06. EPITRANSCRIPTOMIC TRNA MODIFICATIONS TARGETING ADENOSINE (A) AT POSITION 37 IN GBM

Abstract GBM stands as the most aggressive brain tumor in adults. The median overall survival is less than 2 years despite conventional treatments. Recent advancements in DNA analysis have delineated the molecular characteristics of gliomas, leading to revisions in the WHO classification based on DNA profiles. However, the role of RNA modifications, epitranscriptomics, remains poorly understood in glioma. In this study, we underscore the significance of tRNA modifications targeting adenosine at position 37 in GBM. Transfer RNA isopentenyl transferase 1 (TRIT1) is identified as an isopentenyl transferase catalyzing the conversion of A into i6A at position 37 of tRNA, a nucleotide adjacent to the anticodon region. Additionally, i6A is further modified to 2-methylthio i6A (ms2i6A) by cyclin-dependent kinase 5 regulatory subunit associated-protein 1 (CDK5RAP1) within mitochondrial tRNA. Analysis of TCGA data reveals elevated TRIT1 mRNA expression levels in GBM compared to non-tumor samples. Furthermore, a low expression group of TRIT1 in gliomas demonstrates improved survival outcomes according to data from the CGGA, implicating TRIT1 in glioma progression. Functional assays involving TRIT1 knockdown in human GBM cell lines result in decreased cell viability, indicating its functional significance. Moreover, our data suggest that this phenotype may be mediated through translational control of selenoprotein W (SEPW1) via tRNA^(Ser)Sec modification. GO analysis of TRIT1 and SEPW1 in TCGA data further supports this proposed mechanism. At position 37 of tRNA, we also found that CDK5RAP1 in mitochondrial tRNAs is indispensable for sustaining glioblastoma initiating cell (GIC)-related traits. CDK5RAP1 maintains the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs, independent of translational control of mitochondrial proteins. Notably, CDK5RAP1 abrogates the antitumor effect of i6A by converting i6A to ms2i6A and shields GICs from excessive autophagy triggered by i6A. This work underscores the critical role of tRNA modifications in glioma pathogenesis. We will report these findings alongside RNA modification profile data and clinical data from glioma patients.

IMMU-22. TWIST1-MEDIATED ENDOTHELIAL PLASTICITY DRIVES GLIOBLASTOMA RESISTANCE TO CAR T IMMUNOTHERAPY

Abstract Glioblastoma (GBM) is the most common and most aggressive malignant primary brain tumor in adults. Among the most lethal human malignancies, GBM is highly resistant to cytotoxic treatments and molecularly targeted therapies, and also generally refractory to T cell–based immunotherapies, largely due to their immunologically cold nature, i.e., characterized by a paucity of tumor T cell infiltrates that result from the immunosuppressive microenvironment. Here, we identify a distinct mechanism driven by spatiotemporal interaction of leukocytes with tumor stromal cells, namely, mesenchymal-like population of endothelial cells (ECs) form an immunosuppressive vascular niche that regulates macrophage polarization and induces GBM resistance to CAR T immunotherapy. Our single-cell transcriptome analyses of human and mouse GBM tumors identify a subpopulation of ECs acquire robust immunosuppressive phonotypes to drive alternative macrophage polarization. These ECs are spatially localized proximately to immunosuppressive macrophages and selectively express Twist1. Furthermore, we reveal a Twist1/SATB1-mediated sequential transcriptional activation mechanism, through which Twist1+ tumor ECs produce osteopontin to locally promote immunosuppressive macrophage phenotypes. Genetic or pharmacological ablation of Twist1 reverses macrophage-mediated immunosuppression and enhances T cell infiltration and activation, leading to reduced GBM growth and extended mouse survival. Importantly, pharmacological inhibition and lipid nanoparticle-mediated targeted inactivation of Twist1 overcome GBM resistance to Egfrviii CAR T cell immunotherapy. Thus, these findings uncover a spatially restricted mechanism controlling vasculature-mediated tumor immunity and suggest that targeting endothelial Twist1 may offer exciting opportunities for GBM immunotherapy.

CSIG-06. BRAF GERMLINE MUTATIONS AND PREDISPOSITION TO BRAIN TUMORS

Abstract We report on two BRAF-mutated children and discuss genetic counseling regarding predisposition to brain tumors. Molecular analysis of the PTPN11 gene and twelve other genes in the RAS-MAPK pathway was carried out in a series of 54 dysmorphic patients with suspected Rasopathies. Two heterozygous BRAF germline mutations were detected in two 7-year-old girls, at exon 6 and 12 respectively. Both patients had the distinctive facial appearance of cardio-facio-cutaneous syndrome (CFC) type 1. Patients with CFC are rare and characterized by phenotypic and genetic heterogeneity, with overlaps with Noonan syndrome. While nearly 75% of CFC cases have germline BRAF mutations, the frequency of BRAF mutations is inferior in Noonan and LEOPARD syndromes. BRAF mutations in CFC are often found in the cysteine-rich domain of the BRAF protein (exons 6, 11, 12, 13, 14, 15 and 16). Conversely, somatic BRAF mutations are more common, grouped into classes according to their kinase activity level, and have been associated with a variety of malignancies, including pediatric and adult brain tumors (low- and high-grade gliomas, glioblastomas and low-grade astrocytomas). BRAF V600E is the most common somatic mutation, but other mutations are currently being identified. Many of these mutations are within the kinase domain, while others are located across the gene and have less well-understood functional consequences. Germinal V600E mutation has been described once, in a CFC patient having a severe phenotype. Nevertheless, it seems that patients with germline BRAF mutations and gliomas have not yet been reported. Given the rarity of CFC, it is unclear whether there is a high risk of brain tumors. Further molecular studies will enable us to refine recommendations for the prevention, monitoring and care of people with germline BRAF mutations, given the success of BRAF inhibitors in certain cancers, with encouraging results in gliomas.

TMOD-21. MOUSE MODEL TO CONDITIONALLY EXPRESS A DIFFUSE MIDLINE GLIOMA DERIVED MUTATION IN THE ONCOGENIC PHOSPHATASE PPM1D

Abstract Diffuse Midline Gliomas (DMGs) are incurable brain tumors of children and adults, and there is an urgent need for new therapeutic approaches. Their location within critical areas of the brain precludes complete surgical removal. While current treatment mainly relies on radiation therapy, its efficacy remains palliative. DMGs commonly harbor truncating mutations in the Mn2+/Mg2+-dependent protein phosphatase 1D (PPM1D), leading to a gain-of-function in PPM1D by enhancing protein stability and activity. Thus, investigating PPM1D protein expression and its regulatory mechanisms is crucial for understanding its oncogenic role and identifying therapeutic targets. To address this, we developed a Ppm1d-loxP-exon6-loxP-exon6-E518X-tag mouse allele (Ppm1d-FL). This allele allows conditional expression of DMG-derived truncating mutations from the endogenous Ppm1d locus when the loxP sites are recombined in the presence of Cre recombinase. Tosimulate primary gliomas, we utilized the RCAS/tv-a retrovirus system and Cre/loxP recombination platforms. Mouse pups were injected with RCAS retroviruses bearing oncogenic platelet-derived growth factor B, Cre recombinase, and luciferase reporter genes into the brainstem. We confirmed that Cre leads to expected recombination of Ppm1d-FL, leading to expression of DMG-derived truncated Ppm1d-E518X-Tag mRNA. We examined tumor-free survival data, which indicates accelerated tumor formation with Ppm1d-FL compared to littermate controls with wildtype Ppm1d. The tumors demonstrated expression of molecular biomarkers commonly found in DMGs. Single-cell RNA sequencing has identified pathways activated or inactivated upon Ppm1d mutation. These data shed light on the complex interplay between PPM1D mutations and glioma pathogenesis and may guide the design of therapies that target PPM1D molecular pathways.

NCOG-13. ASSESSING CAREGIVER OUTCOMES OF PRIMARY BRAIN TUMOR PATIENTS: A SYSTEMATIC REVIEW OF THE LITERATURE

Abstract BACKGROUND Family caregivers in neuro-oncology are known to have high levels of unmet support needs. Intervention trials and effective support options are scarce. The Response Assessment in Neuro-Oncology (RANO-) Cares working group has been established to determine standards of caregiver outcomes (CO) research reporting in neuro-oncology. We present the current state of methodological quality of reporting on neuro-oncology caregiver outcomes in randomized controlled trials (RCTs). METHODS A systematic literature review (PubMed/Medline, Embase, Web of Science, Emcare, Cochrane Library, PsycINFO; July 2023) was performed to assess to what degree RCTs assessing outcomes of family caregivers of adult primary brain tumor patients adhere to established reporting standards. Using Covidence, screening and data extraction was independently performed by two researchers, with a third guiding consensus. A 33-item checklist (23 applicable to secondary analysis reports) based on the International Society for Quality of Life (ISOQOL) Research criteria for patient-reported outcome reporting was utilized to assess reporting standards. Risk of bias was assessed per RCT. RESULTS Fifteen publications (12 unique RCTs) included 684 neuro-oncology caregivers, with low overall risk of bias. Ten publications (66%) reported on COs as a primary aim and eight (80%) of these satisfied ≥2/3 of the key methodological criteria. Of the five secondary analysis reports (33%) two met ≥2/3 of applicable key criteria. Criteria often missed are related to sample size calculations, details on statistical approaches, discussion of limitations and clinical significance of studies. Only three of the 12 RCTs were definitive trials aimed at improving COs and five should be considered as pilot or feasibility studies. CONCLUSION RCTs investigating neuro-oncology COs have high reporting standards and risk of bias was low. Future studies should focus on specific caregiver interventions to reduce high caregiver burden and with that patients’ quality of life.

EPCO-56. INHERITED PREDISPOSITION TO ADULT GLIOMAS: OVERVIEW OF THE CURRENT CLINICAL PRACTICE AND FUTURE RECOMMENDATIONS

Abstract Gliomas are the most common adult primary malignant brain tumor and occur in ages 45-65 years old. While most gliomas are sporadic in nature, a small portion is related to cancer predisposition syndromes (CPS), such as Li Fraumeni or Lynch syndromes. Given that 5–10% of patients will have a family history of glioma, there is currently an effort in identifying germline variants associated with gliomas. Additionally, with the advent of somatic tumor testing, it is possible to pick up suspected germline mutations in genes that cause CPS. Our experience includes a 32-year-old patient diagnosed with astrocytoma IDH mutant, Grade 2 who was found to have a BRAC1 mutation on somatic brain tumor testing. 10 years later, the patient was found with metastatic ovarian cancer and a germline BRAC1 mutation. Early identification of CPS can lead to early detection or prevention of malignancies in affected patients and relatives. However, gliomas are rarely the primary or presenting tumor of CPS, and guidance of CPS screening for these individuals is still lacking. Due to the increasing interest and importance of CPS testing in patients with glioma we have developed a collaborative protocol between our neuro-oncologists and genetic counselors aso that patients have access to germline testing. We propose that all patients with glioma at age of 50 or under, patients with strong family history of cancer including gliomas, and patients with suspected germline mutations based on tumoral somatic mutational profile should be referred to the genetic counselors. Our preliminary findings include 8 patients who completed germline genetic testing. Of these, one patient tested positive for a BRCA1 pathogenic variant. We are planning to continue to collect this data to better inform clinical practice.