Functional mapping with direct electrical stimulation (DES) is widely used during awake neurosurgery to generate causal evidence about person-specific neuroanatomical organization. According to a long-standing clinical and scientific paradigm, if the application of DES to a given brain region does not result in performance errors, that site is considered to be uninvolved in the task. Here, we show that both error rates and performance speed on correct trials are parametrically modulated by when DES starts and stops relative to the timeline of task-driven processing in stimulated brain areas and networks. We propose a framework, causal parametric mapping, which moves beyond the classic approach of binarizing the effects of DES on behavior into "positive" and "negative" mapping trials. Causal parametric mapping offers a method to functionally dissect separable processing stages in the human brain, in real time, with reversible causal evidence during invasive neurosurgical procedures.

PURPOSE:Because of tumor heterogeneity and sampling error, next-generation sequencing (NGS) of glioblastoma (GBM) tumors may provide an incomplete picture of the somatic mutational landscape. We hypothesized that simultaneous targeted NGS of matched tumor tissue and cerebrospinal fluid (CSF), obtained during craniotomy for resection of GBM, would lead to identification of clinically relevant variants not detected by tissue NGS alone. METHODS:We enrolled 50 patients undergoing resection of newly diagnosed (n = 15) or recurrent (n = 35) GBM. CSF was collected intraoperatively via the subarachnoid space (n = 25) or lateral ventricle (n = 25) and assayed by NGS using a hybrid capture liquid biopsy panel. Matched tumor tissue also underwent large panel hybrid capture NGS testing. RESULTS:CSF samples from 28 of 50 patients (56%) passed quality control metrics. At least one CSF variant was detected in 25 of 28 patients (89%), and 22 of 28 patients had matched tissue sequencing results available. In these 22 patients (primary analysis cohort), the median number of variants detected in CSF was higher than in tissue (3 v 2 variants, respectively; P = .0035), and 15 of 22 patients (68%) had ≥1 CSF variant not detected in matched tissue, including clinically relevant alterations in EGFR, PMS2, PIK3CA, and TP53. CONCLUSION:The addition of intraoperatively acquired CSF liquid biopsy to tissue NGS in patients with GBM may improve detection of clinically relevant variants, potentially improving selection of patients for clinical trials.

Immune suppression in glioma is marked by systemic reprogramming of bone marrow-derived cells (BDMCs). Tumor-derived exosomes (TEX) exhibit the requisite biodistribution, genomic contents, and specificity for short and long-range reprogramming of BDMCs. However, in vivo studies have emerged as the major obstacle for discovery of TEX cell-specific glioma immunotherapy. We report a syngeneic murine glioma model capable of tracking endogenously released TEX in real-time. We used the RCAS retroviral system develop IDH-mutant and wild-type glioma mouse models with concurrent induction of spontaneous glioma and reporting of GFP-tagged TEX. Tumor and blood samples were analyzed using spectral flow cytometry, multi-plex immunofluorescence and scRNA sequencing to characterize specific TEX-targeted immune cells and their corresponding spatial topography. Our multimodal analyses reveal unique genotype-specificity of TEX-targeted immune ontogenies. In IDHm gliomas, myeloid cells were ubiquitously endowed with TEX and exhibited central topography within the tumor. Conversely, IDHwt TEX-containing myeloid cells were over-represented along the tumor periphery. These cells exhibited a dichotomous and polarized pattern of TEX uptake with dorsal (CD68/F4/80) and ventral (CD11b/F480) localization. Over 90% of CD4 T cells engulfed TEX and were highly enriched within the medial leading edge of the tumor. CD8 T cells contained the fewest proportion of TEX and were restrained to the meninges and peri-tumoral white matter. Remarkably, TEX were identified in non-neoplastic contralateral hemispheres and the meninges of tumor-bearing mice. Finally, flow analyses of blood samples detected an increase of TEX in circulating myeloid cells of IDHm tumor-bearing mice, emerging at 2 weeks and peaking at 6 weeks after tumor injection. We have developed a novel mouse model that allows unprecedented endogenous tracking of glioma tumor exosomes. Tumor exosomes engage peripheral macrophages and enable recruitment of these cells into the local tumor microenvironment. Our data supports engineering exosomes for glioma immunotherapy.

Background For newly diagnosed glioblastoma (GBM), a combination of upfront surgical immunotherapy with aglatimagene besadenovec (CAN-2409), followed by chemoradiation and then adjuvant nivolumab has not been tested. The aim of this study was to test the safety of this regimen and determine metrics of immune activation that may correlate with clinical outcomes.Methods Forty-one patients with suspected newly diagnosed GBM by imaging were enrolled in this multi-institutional, open-label, phase 1b clinical trial before surgical resection. Frozen section confirmation of high-grade glioma was required for administration of CAN-2409. This was then followed with chemoradiation and adjuvant nivolumab. Tumor and blood were assayed for genetic and immune markers before and during treatment.Results The regimen was well tolerated and generated measurable immune activation. Factors linked to survival were identified, such as baseline mutated gene pairs (eg, MED15/HRC), tumor immune cell composition, and changes in systemic cytokine, immune cells, and T-cell diversity. The most significant serial systemic immune changes were observed in a long-term survivor subset of patients with gross total resection (GTR)/methylated methylguanine methyltransferase (MGMT) promoter tumors. Median overall survival (mOS) in these patients was 30.6 months, while it was less for patients with unmethylated or subtotal resections.Conclusions These findings suggest the opportunity for patient stratification and the potential for more durable antitumor immune responses in future clinical trials of this multimodal standard of care and combined immunotherapy regimen. ClinicalTrials.gov identifier: NCT03576612.

While ipilimumab and nivolumab has demonstrated promising intracranial activity among patients with melanoma brain metastases (BM), patients with prior systemic therapy exposure may be at higher risk of poor intracranial disease control and benefit from upfront local therapy. We evaluated outcomes of patients with BMs treated with ipilimumab/nivolumab and stereotactic radiosurgery (SRS). Overall survival (OS) and intracranial progression-free survival (PFS) were estimated from SRS using a non-parametric method, and Cox proportional hazards models were used to test clinically relevant factors. A total of 68 consecutive patients with 413 treated BMs between 2015 and 2025 were included with median follow-up of 19.3 months from SRS. At the time of analysis, 34 (53%) patients were alive. The 2-year OS and intracranial PFS for the entire cohort was 49.5% and 36.1%, respectively. 34 patients (50%) had prior exposure to immune checkpoint inhibition (ICI) and 13 patients (19%) had exposure to prior BRAF/MEK inhibition. On univariable analysis, factors significantly associated with worse OS included receipt of prior ICI (HR 2.3, 95% CI 1.24 - 4.26, p = 0.008) and BRAF/MEK inhibition (HR 2.37, CI 1.21-4.67, p = 0.012). Upfront brain metastasis resection was associated with improved OS (HR 0.39, 95% CI 0.21 – 0.73, p = 0.003). Patients with prior ICI exposure trended towards worse intracranial PFS (median intracranial PFS 5.9 months vs 15.1 months, p = 0.12) and had worse survival after SRS (median OS 17.6 months vs 50.5 months, p = 0.007). While ipilimumab/nivolumab demonstrates encouraging intracranial control and survival among patients with melanoma brain metastases, patients with prior ICI or targeted therapy exposure are at higher risk of poor outcomes and may benefit from additional treatment strategies.

Objectives/Goals: We aim to identify how IDH mutant (IDHm) gliomas use exosomes to modulate the local and systemic immune system. We will do so by characterizing differential miRNA expression between IDHm and IDH wild type (IDHwt) exosomes and identifying the specific immune cell population targeted by exosomes in vivo. Methods/Study Population: Exosome RNA will be isolated from cultured patient glioma samples and perform small RNA sequencing to investigate differential expression of miRNA between IDHwt and IDHm exosomes. We will then utilize miRNA target databases in conjunction with bioinformatic pathway analysis to generate potential target regulatory pathways. To identify the in vivo effect of tumor exosomes, we will generate a novel glioma mouse model that has been genetically engineered to release labeled exosomes using the RCAS retroviral system. We will collect peripheral blood and tumor tissue for flow cytometric immune profiling and single-cell RNA sequencing. The transcriptomic data will be analyzed to identify subsets of immune populations that have taken up the labeled exosomes and assess the resulting expression changes in those cells. Results/Anticipated Results: From the small RNA sequencing and bioinformatics analysis, we expect to find several unique miRNA expressed in IDHm exosomes that induce immunosuppressive pathways in local and systemic immune cell populations when compared to IDHwt exosomes. Furthermore, using our novel murine model, we expect to be able to track endogenously released exosomes in the local tumor microenvironment and in the circulating blood. We hypothesize that IDHm exosomes specifically target precursor myeloid cells within the local and peripheral circulating immune populations and induce the expansion of monocytes, M2 macrophages, and mono-MDSCs. Discussion/Significance of Impact: Immunosuppression in IDHm glioma has hindered the development of adequate therapies to treat this fatal disease. Our study will illuminate the mechanism by which tumor exosomes can suppress immune surveillance. These results will help identify new therapeutic targets to sensitize the immune system against glioma cells.

Abstract Purpose: Noninvasive prognostic biomarkers to inform clinical decision-making are an urgent unmet need for the management of patients with glioblastoma (GBM). We previously showed that higher circulating cell-free DNA (ccfDNA) concentration is associated with worse survival in GBM. However, the biology underlying this is unknown. Experimental Design: We prospectively enrolled 129 patients with treatment-naïve GBM with blood drawn prior to initial resection (baseline) and at the time of the first postradiotherapy MRI. We performed ccfDNA methylation deconvolution to determine cellular sources of ccfDNA. ELISA was performed to detect citrullinated histone 3 (citH3), a marker of neutrophil extracellular traps (NET). Multiplex proteomic analysis was used to measure soluble inflammatory proteins. Results: We found that neutrophils contributed the highest proportion of prognostic ccfDNA. The percentage of ccfDNA derived from neutrophils was correlated with total [ccfDNA] but only in patients receiving preoperative corticosteroids. At baseline and on therapy, [citH3] was significantly higher in the plasma of patients with GBM receiving corticosteroids compared with corticosteroid-naïve GBM or no-cancer controls. Unsupervised hierarchical clustering of ccfDNA methylation patterns yielded two clusters, with one enriched for patients with the NETosis phenotype and who received corticosteroids. Unsupervised clustering of circulating inflammatory proteins yielded similar results. Conclusions: These data suggest neutrophil-mediated NETosis is the dominant source of prognostic ccfDNA in patients with GBM and may be associated with glucocorticoid exposure. If further studies show that pharmacological inhibition of NETosis can mitigate the deleterious effects of corticosteroids, these plasma markers will have important clinical utility as noninvasive correlative biomarkers.

Supplementary Table S1. Representativeness of study participants Supplementary Table S2. Analysis of the intracranial response duration in the COMBI-MB trial. Supplementary Table S3. Analysis of overall survival in the COMBI-MB trial. Supplementary Table S4. Baseline clinical features of the COMBI-BRV trial. Supplementary Table S5. Gene expression score (SingScore) and multiplex immunohistochemical results. Supplementary Table S6. Summary statistics of the linear mixed models utilized to assess the association between protein/pathway expression/score and biopsy sites. Supplementary Table S7. Mutation annotated file of exome sequencing from melanoma biopsies.

Gene expression of major signalling pathways and multiplex immunohistochemical comparison of markers between timepoints and tumor sites. A) Gene expression of related oncogenic signalling pathways between treatment timepoints and site, B) Estimated mean difference of proteins and pathways between biopsy categories (95% confidence interval for the estimated mean difference is depicted as a segment), C) Multiplex immunohistochemical staining for key intracellular signalling proteins, D) Changes in immune intratumoral immune cell densities between treatment timepoints and site, E and F) Multiplex immunohistochemical staining depicted marked decrease in immune cell densities EDT in PT4 (steroid treated). PRE, collected prior to treatment; EDT, collected early during treatment (i.e., after 10-14 days of dabrafenib). MBM, melanoma brain metastasis; ECM, extracranial metastasis. PT, patient.

BACKGROUND:Glioblastoma (GBM) is the most aggressive adult primary brain cancer, characterized by significant heterogeneity, posing challenges for patient management, treatment planning, and clinical trial stratification. METHODS:We developed a highly reproducible, personalized prognostication, and clinical subgrouping system using machine learning (ML) on routine clinical data, magnetic resonance imaging (MRI), and molecular measures from 2838 demographically diverse patients across 22 institutions and 3 continents. Patients were stratified into favorable, intermediate, and poor prognostic subgroups (I, II, and III) using Kaplan-Meier analysis (Cox proportional model and hazard ratios [HR]). RESULTS:The ML model stratified patients into distinct prognostic subgroups with HRs between subgroups I-II and I-III of 1.62 (95% CI: 1.43-1.84, P < .001) and 3.48 (95% CI: 2.94-4.11, P < .001), respectively. Analysis of imaging features revealed several tumor properties contributing unique prognostic value, supporting the feasibility of a generalizable prognostic classification system in a diverse cohort. CONCLUSIONS:Our ML model demonstrates extensive reproducibility and online accessibility, utilizing routine imaging data rather than complex imaging protocols. This platform offers a unique approach to personalized patient management and clinical trial stratification in GBM.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months, despite treatment, with most patients experiencing recurrence within 9 months of resection. The propensity of recurrence in GBM exemplifies the fatal course of the disease and remains an underlying area of study as novel instances of recurrence are encountered. The authors present a unique case of a 31-year-old male patient with a history of cerebellomedullary junction astrocytoma who later developed a supratentorial GBM followed by recurrence centered around a preexisting ventriculoperitoneal catheter and located in the hemisphere contralateral to his first GBM. Each of these lesions was initially thought to represent de novo glial neoplasms because of the absence of intervening T2 fluid-attenuated inversion recovery signal change between each lesion. However, next-generation sequencing using the GlioSeq™ platform revealed similar mutational profiles in both GBMs, suggesting an alternative method of migration of tumor cells to the shunt catheter site, and a local inflammatory environment likely triggering recurrence. This study concludes that in rare instances, in the presence of dormant glioma cells, intracranial foreign bodies may promote an inflammatory microenvironment that may activate tumorigenesis.

SummaryPediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Throughin vitropharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

ABSTRACT Diffuse gliomas are epigenetically dysregulated, immunologically cold, and fatal tumors characterized by mutations in isocitrate dehydrogenase (IDH). Although IDH mutations yield a uniquely immunosuppressive tumor microenvironment, the regulatory mechanisms that drive the immune landscape of IDH mutant (IDHm) gliomas remain unknown. Here, we reveal that transcriptional repression of retinoic acid (RA) pathway signaling impairs both innate and adaptive immune surveillance in IDHm glioma through epigenetic silencing of retinol binding protein 1 (RBP1) and induces a profound anti-inflammatory landscape marked by loss of inflammatory cell states and infiltration of suppressive myeloid phenotypes. Restorative retinoic acid therapy in murine glioma models promotes clonal CD4 + T cell expansion and induces tumor regression in IDHm, but not IDH wildtype (IDHwt), gliomas. Our findings provide a mechanistic rationale for RA immunotherapy in IDHm glioma and is the basis for an ongoing investigator-initiated, single-center clinical trial investigating all-trans retinoic acid (ATRA) in recurrent IDHm human subjects.

Low-grade glioma (LGG) is the most common brain tumor affecting pediatric patients (pLGG) and BRAF mutations constitute the most frequent genetic alterations. Within the spectrum of pLGGs, approximately 70%-80% - 80% of pediatric patients diagnosed with transforming pleomorphic xanthoastrocytoma (PXA) harbor the BRAF V600E mutation. However, the impact of glioma BRAF V600E cell regulation of tumor infiltrating fi ltrating immune cells and their contribution to tumor progression remains unclear. Moreover, the efficacy fi cacy of BRAF inhibitors in treating pLGGs is limited compared with their impact on BRAF-mutated melanoma. Here we report a novel immunocompetent RCAS-BRAF V600E murine glioma model. Pathological assessment indicates this model seems to be consistent with diffuse gliomas and morphological features of PXA. Our investigations revealed distinct immune cell signatures associated with increased trafficking fi cking and activation within the tumor microenvironment (TME). Intriguingly, immune system activation within the TME also generated pronounced inflammatory fl ammatory response associated with dysfunctional CD8+ + T cells, increased presence of immunosuppressive myeloid cells and regulatory T cells. Further, our data suggests tumor-induced inflammatory fl ammatory processes, such as cytokine storm. These fi ndings suggest a complex interplay between tumor progression and the robust inflammatory fl ammatory response within the TME in preclinical BRAF V600E LGGs, which may significantly influence fl uence animal survival.

Abstract Standard of care (SOC) for ndGBM begins with maximal safe resection followed by adjuvant radiotherapy and temozolomide, and maintenance temozolomide. IGV-001 is an autologous biologic-device combination immunotherapy for the treatment of ndGBM that consists of autologous GBM tumor cells and an antisense oligonucleotide against IGF-1R mRNA, irradiated and administered via biodiffusion chambers implanted in the abdomen. In a phase 1b study, IGV-001 was well tolerated without unexpected adverse events in subjects with ndGBM. Multiple efficacy signals were observed, including significant improvements in progression-free survival (PFS), overall survival (OS), radiographic evidence of tumor response, and changes in immune response biomarkers. Here, we present early safety data from the phase 2b randomized, multicenter, double-blind, placebo-controlled study (NCT04485949) designed to assess efficacy and safety of IGV-001 in subjects with ndGBM across 20 sites in the United States. After surgical resection, subjects were randomized 2:1 and treated with IGV-001 or placebo followed by SOC. The primary outcome is PFS, defined as the time from randomization to first progression, as determined by blinded central radiology review, or death. Secondary outcomes include OS, defined as the time from randomization to death due to any cause, and safety. As of May 22, 2024, 99 subjects were randomized and 95 implanted with IGV-001 or placebo plus SOC. A total of 39/45 (86.7%) subjects had sufficient follow-up time after initiated treatment with concurrent radiation and temozolomide. Nine of 72 randomized (12.5%) discontinued treatment, including 7 who stopped during the SOC treatment period. None ceased treatment for adverse events, protocol deviations, or death. A total of 11/72 (15.3%) randomized subjects discontinued the study after randomization. A review of blinded safety data did not show any emerging risk and supports no change to the benefit-risk profile of IGV-001 versus placebo. Updated data will be presented.

Abstract BACKGROUND Glioblastoma (GBM) is the most common primary brain malignancy in adults. Standard of care (SOC) for suspected GBM begins with maximal safe resection followed by adjuvant radiotherapy and temozolomide, and maintenance temozolomide. Imvax has utilized its Goldspire™ platform to create IGV-001, an autologous biologic-device combination product for the treatment of newly diagnosed GBM (ndGBM). IGV-001 consists of autologous GBM tumor cells and an antisense oligonucleotide against IGF-1R mRNA (IMV-001), irradiated and administered via biodiffusion chambers implanted in the abdomen. Together, these components stimulate immunogenic cell death and antigen release. In a phase 1b study in patients with ndGBM, IGV-001 was well tolerated without unexpected adverse events. Multiple efficacy signals were also observed, including significant improvements in progression-free survival, overall survival, radiographic evidence of tumor response, and changes in immune response biomarkers. A phase 2b randomized, multicenter, double-blind, placebo-controlled study to assess the safety and efficacy of IGV-001 in patients with ndGBM (ClinicalTrials.gov identifier: NCT04485949) is ongoing. Here, we present the first safety data from the study. MATERIAL AND METHODS After surgical resection, patients are treated with IGV-001 or placebo followed by SOC. The study will enroll approximately 93 patients at 20 sites in the United States, with a 2:1 randomization favoring IGV-001. The primary outcome is progression-free survival, defined as the time from randomization to first progression, as determined by blinded central radiology review, or death. Secondary outcomes include overall survival, defined as the time from randomization to death due to any cause, and safety. RESULTS As of February 2, 2024, a total of 72 patients have been randomized and 69 have been implanted with IGV-001 or placebo plus SOC. The median age was 60.0 years (range, 24-70 years). Most randomized patients (76.4%) were 51-70 years of age, and the male:female ratio was 2.27. A total of 39/45 (86.7%) patients with enough follow-up time, initiated treatment with concurrent radiation and temozolomide. A total of 9/72 (12.5%) randomized patients discontinued treatment, including 7 patients who discontinued during the SOC treatment period. No patients discontinued treatment for adverse events, protocol deviations, or death. A total of 11/72 (15.3%) randomized patients discontinued the study after randomization. CONCLUSION To date, the review of blinded safety data did not show any emerging risk and supports that there is no change to the benefit-risk profile of treatment with IGV-001 versus placebo. The data will be updated before presentation.

OBJECTIVE:Preoperative stereotactic radiosurgery (SRS) is emerging as a viable alternative to standard postoperative SRS. Studies have suggested that preoperative SRS provides comparable tumor control and overall survival (OS) and may reduce the incidence of leptomeningeal disease (LMD) and adverse radiation effects (AREs). It is unknown, however, if preoperative SRS remains effective in cohorts including large brain metastases (> 14 cm3) or if preoperative SRS affects steroid taper/immunotherapy. Here, the authors report the results of a phase 2 single-arm trial assessing a prospectively acquired series of 26 patients who underwent preoperative SRS, without a volumetric cutoff, compared with a propensity score-matched concurrent cohort of 30 patients who underwent postoperative SRS to address these salient questions. METHODS:Demographics, oncological history, surgical details, and outcomes were collected from the medical records. Coprimary endpoints were local tumor control (LTC) and a composite outcome of LTC, ARE, and LMD. Additional outcomes were OS, steroid taper details, and immunotherapy resumption. For survival analyses, cohorts were propensity score matched. RESULTS:Preoperative and postoperative SRS patients were comparable in terms of age, sex, Karnofsky Performance Status score, oncological history, and operative details. Gross tumor volume (GTV) was significantly higher in the preoperative group (median 12.2 vs 5.3 cm3, p < 0.001). One-year LTC (preoperative SRS: 77.2% vs postoperative SRS: 82.5%, p = 0.61) and composite outcome (68.3% vs 72.7%, p = 0.38) were not significantly different between the groups. In multivariable analysis, preoperative SRS did not have a significant effect on LTC (HR 1.57 [95% CI 0.38-6.49], p = 0.536) or the composite outcome (HR 1.18 [95% CI 0.38-3.72], p = 0.771), although the confidence intervals were large. The median OS (preoperative SRS: 17.0 vs postoperative SRS: 14.0 months, p = 0.61) was not significantly different. Rates of LMD were nonsignificantly lower in the preoperative SRS group (3.8% vs 16.7%, p = 0.200). Greater GTV volume was associated with prolonged (> 10 days) steroid taper (OR 1.24 [95% CI 1.04-1.55], p = 0.032). However, in multivariable analysis, preoperative SRS markedly reduced the steroid taper length (OR 0.13 [95% CI 0.02-0.61], p = 0.016). Time to immunotherapy was shorter in the preoperative SRS group (36 [IQR 26, 76] vs OR 228 [IQR 129, 436] days, p = 0.02). CONCLUSIONS:Compared with postoperative SRS, preoperative SRS is a safe and effective strategy in the management of cerebral metastases of all sizes and provides comparable tumor control without increased adverse effects. Notably, preoperative SRS enabled rapid steroid taper, even in larger tumors. Future studies should specifically examine the interaction of preoperative SRS with steroid usage and resumption of systemic therapies and the subsequent effects on systemic progression and OS.

Abstract INTRODUCTION Increased extent of resection (EOR) is a known predictor of seizure freedom in glioma patients. However, it is unknown how tumor genomic alterations affect EOR-induced seizure control. OBJECTIVE To evaluate the genotype-specific impact of EOR on post-operative seizure control METHODS Records of 553 glioma patients who underwent tumor resection from 2012-2023 were reviewed. All patient tumors were analyzed with next generation sequencing and FISH analysis. The unsupervised machine learning algorithm non-negative matrix factorization (NMF) was used to cluster the genomic data. Clinical variables including extent of resection of contrast enhancing tumor and non-enhancing tumor, temozolomide therapy, and radiation therapy were analyzed using Cox Proportional Hazards models. RESULTS NMF clustering of tumor sequencing data revealed four molecular groups: Group 1 (n=56): CDKN2a loss, EGFR copy number gain or mutation, TERT promoter (TERTp) mutation, and MGMT methylated; Group 2 (n=27): IDH mutation, TP53 mutation, and MGMT methylated; Group 3 (n=60): TP53 mutation, TERTp mutation and MGMT methylated; Group 4 (n=244): MGMT unmethylated. Thresholds in the post-operative residual non-enhancing tumor volume (NETV) were identified which marked the most difference in seizure control. For patients in Group 1, the NETV threshold that led to improved epilepsy outcomes was <94.7 cc (p=0.047), whereas the threshold for Group 4 was <89.1 cc (p=0.016). When analysis was conducted for Group 2, the threshold of 10.2 cc failed to reach significance (p=0.1). Interestingly, the threshold analysis for Group 3 found that having a NETV of > 10.1 cc showed improved seizure outcomes (p=0.016). A multivariable cox proportional hazard analysis found that twelve-month seizure freedom was predictive of better OS in group 1 (HR 0.02, p=0.05) and group 4 (HR 0.21, p=1.63e-9). CONCLUSION Our study reveals clinically distinct molecular groups of glioma that display different seizure responses. This data suggests a tumor-specific approach to aggressiveness of extent of resection in order to achieve optimal seizure control.