IDH-wildtype Gliomas Research Articles

Somatic POLE Mutation and Ultra-Hypermutated Genotype in a De Novo High-Grade, Isocitrate Dehydrogenase Wild-Type Glioma: Treatment Implications.

Pembrolizumab leads to a durable response in ultra-hypermutated, high-grade, glioma.

Diagnostic utility of genetic alterations in distinguishing IDH-wildtype glioblastoma from lower-grade gliomas: Insight from next-generation sequencing analysis of 479 cases.

The accurate diagnosis and classification of gliomas are essential for appropriate treatment planning and prognosis prediction. This study aimed to investigate the molecular diagnostics of IDH-wildtype diffuse astrocytic gliomas and identify potential genetic variants that could differentiate glioblastoma (GBM) from lower-grade gliomas when DNA methylation analysis is not feasible. In total, 479 H3-and IDH-wildtype diffuse astrocytic gliomas were included in this study. All the cases were diagnosed according to the 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumors. Panel sequencing data were collected, and clinicopathological information was retrieved from medical records. Genetic alterations and histological findings were analyzed to determine their diagnostic utility and prognostic implications. Out of 479 cases, 439 (91.6%) were diagnosed with GBM, including 28 cases that were molecularly diagnosed as GBM. However, 40 (8.4%) cases could not be classified according to the 2021 WHO classification and were diagnosed as lower-grade diffuse astrocytic glioma, IDH-wildtype, not elsewhere classified (LGNEC). In addition to the three genetic alterations included in the diagnostic criteria of GBM, PTEN and EGFR mutations were found to be enriched in GBM. Patients harboring mTOR pathway mutations demonstrated a more favorable prognosis and often exhibited morphology resembling subependymal giant cell astrocytoma, along with a high tumor mutational burden. Among patients with mTOR pathway mutations, those lacking molecular diagnostic features of GBM exhibited outstanding survival outcomes, even in the presence of grade 4 histology. Integration of molecular features enhanced the diagnostic accuracy of IDH-wildtype gliomas. Some molecular alterations enriched in GBM offer valuable insights for molecular diagnosis and glioma classification. Furthermore, high-grade diffuse astrocytic gliomas featuring mTOR pathway mutations in the absence of molecular diagnostic features of GBM could represent more favorable tumor types distinct from GBM.

Role of Histogram Features on Arterial Spin Labeling Perfusion Magnetic Resonance Imaging in Identifying Isocitrate Dehydrogenase Genotypes and Glioma Malignancies.

To explore the use of histogram features on noninvasive arterial spin labeling (ASL) perfusion magnetic resonance imaging (MRI) in differentiating isocitrate dehydrogenase mutant-type (IDH-mut) from isocitrate dehydrogenase wild-type (IDH-wt) gliomas, and lower-grade gliomas (LGGs) from glioblastomas. This retrospective study included 131 patients who underwent ASL MRI and anatomic MRI. Cerebral blood flow (CBF) maps were calculated, from which 10 histogram features describing the CBF distribution were extracted within the tumor region. Correlation analysis was performed to determine the correlations between histogram features as well as tumor grades and IDH genotypes. The independent t-test and Fisher's exact test were used to determine differences in the extracted histogram features, age at diagnosis, and sex in different glioma subtypes. Multivariate binary logistic regression analysis was performed, and diagnostic performances were evaluated with the receiver operating characteristic curves. CBF histogram features were significantly correlated with tumor grades and IDH genotypes. These features can effectively differentiate LGGs from glioblastomas, and IDH-mut from IDH-wt gliomas. The area under the receiving operating characteristic curve of the model calculated using combined CBF 30th percentile and age at diagnosis in differentiating LGGs from glioblastomas was 0.73. Integrating age at diagnosis and CBF 10th percentile could be more effective in differentiating IDH-mut from IDH-wt gliomas. Furthermore, the combined model had a better area under the receiving operating characteristic curve at 0.856 (sensitivity: 84.4%, specificity: 82.9%). The histogram features on ASL were significantly correlated with tumor grade and IDH genotypes. Moreover, the use of these features could effectively differentiate glioma subtypes. The combined application of age at diagnosis and perfusion histogram features resulted in a more comprehensive identification of tumor subtypes. Therefore, ASL can be a noninvasive tool for the pre-surgical evaluation of gliomas.

Unraveling glioblastoma diversity: Insights into methylation subtypes and spatial relationships.

The purpose of this study was to elucidate the relationship between distinct brain regions and molecular subtypes in glioblastoma (GB), focusing on integrating modern statistical tools and molecular profiling to better understand the heterogeneity of Isocitrate Dehydrogenase wild-type (IDH-wt) gliomas. This retrospective study comprised 441 patients diagnosed with new IDH-wt glioma between 2009 and 2020 at Heidelberg University Hospital. The diagnostic process included preoperative magnetic resonance imaging and molecular characterization, encompassing IDH-status determination and subclassification, through DNA-methylation profiling. To discern and map distinct brain regions associated with specific methylation subtypes, a support-vector regression-based lesion-symptom mapping (SVR-LSM) was employed. Lesion maps were adjusted to 2 mm³ resolution. Significance was assessed with beta maps, using a threshold of P < .005, with 10 000 permutations and a cluster size minimum of 100 voxels. Of 441 initially screened glioma patients, 423 (95.9%) met the inclusion criteria. Following DNA-methylation profiling, patients were classified into RTK II (40.7%), MES (33.8%), RTK I (18%), and other methylation subclasses (7.6%). Between molecular subtypes, there was no difference in tumor volume. Using SVR-LSM, distinct brain regions correlated with each subclass were identified: MES subtypes were associated with left-hemispheric regions involving the superior temporal gyrus and insula cortex, RTK I with right frontal regions, and RTK II with 3 clusters in the left hemisphere. This study linked molecular diversity and spatial features in glioblastomas using SVR-LSM. Future studies should validate these findings in larger, independent cohorts to confirm the observed patterns.

Malignant glioma in L-2-Hydroxyglutaric Aciduria: thorough molecular characterization of a case and literature review.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the inactivating homozygous germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

Integrative analysis of the cuproptosis-related gene ATP7B in the prognosis and immune infiltration of IDH1 wild-type glioma

Glioma is the most common malignant tumor in the brain and the central nervous system with a poor prognosis, and wild-type isocitrate dehydrogenase (IDH) glioma indicates a worse prognosis. Cuproptosis is a recently discovered form of cell death regulated by copper-dependent mitochondrial respiration. However, the effect of cuproptosis on tumor prognosis and immune infiltration is not clear. In this research, we analyzed of public databases to show the correlation between cuproptosis-related genes and the prognosis of IDH1 wild-type glioma. Nine out of 12 genes were upregulated in IDH1 wild-type glioma patients, and 6 genes were significantly associated with overall survival (OS), while 5 genes were associated with progression-free survival (PFS). Then, we constructed a prognostic cuproptosis‐related gene signature for IDH1 wild-type glioma patients. ATP7B was considered an independent prognostic indicator, and a low expression level of ATP7B was related to a shorter period of OS and PFS. Moreover, downregulation of ATP7B was correlated not only with the infiltration of activated NK cells, CD8 + T cells and M2 macrophages; but also with high expression of immune checkpoint genes and tumor mutation burden (TMB). In the IDH1 wild-type glioma tissues we collected, our data also confirmed that high tumor grade was accompanied by low expression of ATP7B and high expression of PD-L1, which was associated with increasing infiltration of CD8 + immune cells. In conclusion, our research constructed a prognostic cuproptosis-related gene signature model to predict the prognosis of IDH1 wild-type glioma. ATP7B is deemed to be a potential prognostic indicator and novel immunotherapy biomarker for IDH1 wild-type glioma patients.

Tumor Multiregional Mean Apparent Propagator (MAP) Features in Evaluating Gliomas-A Comparative Study With Diffusion Kurtosis Imaging (DKI).

Glioma classification affects treatment and prognosis. Reliable imaging methods for preoperatively evaluating gliomas are essential. To evaluate tumor multiregional mean apparent propagator (MAP) features in glioma diagnosis and to compare those with diffusion-kurtosis imaging (DKI). Retrospective study. 70 untreated glioma patients (31 LGGs (low-grade gliomas), 34 women; mean age, 47 ± 12 years, training (60%, n = 42) and testing cohorts (40%, n = 28)). 3-T, diffusion-MRI using q-space Cartesian grid sampling with 11 different b-values. Tumor multiregional MAP (mean squared displacement (MSD); q-space inverse variance (QIV); non-Gaussianity (NG); axial/radial non-Gaussianity (NGAx, NGRad); return-to-origin/axis/plane probability (RTOP, RTAP, and RTPP)); and DKI metrics (axial/mean/radial kurtosis (AK, MK, and RK)) on tumor parenchyma (TP) and peritumoral areas (PT) in histopathologically gliomas grading and genotyping were assessed. Mann-Whitney U; Kruskal-Wallis; Benjamini-Hochberg; Bonferroni-correction; receiver operating curve (ROC) and area under curve (AUC); DeLong's test; Random Forest (RF). P value<0.05 was considered statistically significant after multiple comparisons correction. Compared with LGGs, MSD, and QIV were significantly lower in TP, whereas NG, NGAx, NGRad, RTOP, RTAP, RTPP, and DKI metrics were significantly higher in HGGs (high-grade gliomas) (P ≤ 0.007), as well as in isocitrate-dehydrogenase (IDH)-mutated than IDH-wildtype gliomas (P ≤ 0.039). These trends were reversed for PT (tumor grades, P ≤ 0.011; IDH-mutation status, P ≤ 0.012). ROC analysis showed that, in TP, DKI metrics performed best in TP (AUC 0.83), whereas in PT, RTPP performed best (AUC 0.77) in glioma grading. AK performed best in TP (AUC 0.77), whereas MSD and RTPP performed best in PT (AUC 0.73) in IDH genotyping. Further RF analysis with DKI and MAP demonstrated good performance in grading (AUC 0.91, Accuracy 82%) and IDH genotyping (AUC 0.87, Accuracy 79%). Tumor multiregional MAP features could effectively evaluate gliomas. The performance of MAP may be similar to DKI in TP, while in PT, MAP may outperform DKI. 4 TECHNICAL EFFICACY: Stage 2.

Multimodal MRI-based radiomic nomogram for predicting telomerase reverse transcriptase promoter mutation in IDH-wildtype histological lower-grade gliomas.

The presence of TERTp mutation in isocitrate dehydrogenase-wildtype (IDHwt) histologically lower-grade glioma (LGA) has been linked to a poor prognosis. In this study, we aimed to develop and validate a radiomic nomogram based on multimodal MRI for predicting TERTp mutations in IDHwt LGA. One hundred and nine IDH wildtype glioma patients (TERTp-mutant, 78; TERTp-wildtype, 31) with clinical, radiomic, and molecular information were collected and randomly divided into training and validation set. Clinical model, fusion radiomic model, and combined radiomic nomogram were constructed for the discrimination. Radiomic features were screened with 3 algorithms (Wilcoxon rank sum test, elastic net, and the recursive feature elimination) and the clinical characteristics of combined radiomic nomogram were screened by the Akaike information criterion. Finally, receiver operating characteristic curve, calibration curve, Hosmer-Lemeshow test, and decision curve analysis were utilized to assess these models. Fusion radiomic model with 4 radiomic features achieved an area under the curve value of 0.876 and 0.845 in the training and validation set. And, the combined radiomic nomogram achieved area under the curve value of 0.897 (training set) and 0.882 (validation set). Above that, calibration curve and Hosmer-Lemeshow test showed that the radiomic model and combined radiomic nomogram had good agreement between observations and predictions in the training set and the validation set. Finally, the decision curve analysis revealed that the 2 models had good clinical usefulness for the prediction of TERTp mutation status in IDHwt LGA. The combined radiomics nomogram performed great performance and high sensitivity in prediction of TERTp mutation status in IDHwt LGA, and has good clinical application.

MGMT promoter methylation in 1p19q-intact gliomas.

Standard-of-care for 1p19q-intact anaplastic gliomas is defined by the international randomized phase III CATNON trial, which found an overall survival (OS) benefit for adjuvant temozolomide (TMZ) when added to radiotherapy. Paradoxically, TMZ did not appear to benefit patients with IDH-wildtype gliomas, regardless of MGMT promoter status. The authors concluded that well-powered prospective study on the clinical efficacy of TMZ for patients with IDH-wildtype anaplastic gliomas (meeting criteria for glioblastoma) is warranted. Given that the prognostic and predictive role of MGMT status for grade 2-3 gliomas is unresolved, we determined the effect of MGMT status on OS in patients with 1p19q-intact gliomas in the National Cancer Database (NCDB). We queried the NCDB from 2018 to 2019 for patients with diffuse (grade 2) and anaplastic (grade 3) IDH-wildtype or -mutant astrocytomas who received chemotherapy with follow-up through 2022. The Kaplan-Meier method and Cox proportional hazards regressions models were used to determine the association of MGMT with OS. We identified 1514 patients who were newly diagnosed with IDH-wildtype (n = 802, 33% methylated) or -mutant astrocytomas (n = 712, 48% methylated) and received chemotherapy during initial management. An unmethylated promoter was associated with poorer survival in patients with IDH-wildtype (3-year OS 34% [95%CI 29-39%] vs. 46% [95%CI 39-54%], p < .001, adjusted HR 1.53 [95%CI 1.24-1.89]) but not IDH-mutant astrocytomas (3-year OS 79% [95%CI 74-84%] vs. 80% [95%CI 75-86%], p =0 .81, HR 1.04 [95%CI 0.73-1.50]). This ancillary analysis supports conclusions from the CATNON trial for adjuvant TMZ as standard-of-care for anaplastic astrocytomas (IDH-mutant and 1p19q-intact), irrespective of MGMT status. Determining the optimal strategy for diffuse gliomas that are IDH-wildtype will be particularly important. MGMT promoter methylation should be considered as a stratification factor in future clinical trials for these patients.

10091-COT-12 ELUCIDATING THE PATHOPHYSIOLOGY OF POST-OPERATIVE VENOUS THROMBOEMBOLISM IN IDH-WILDTYPE MALIGNANT GLIOMAS

Abstract BACKGROUND We have previously reported elevated expression of podoplanin and high incidence of post-operative venous thromboembolism (VTE) in IDH-wildtype gliomas (Watanabe et al., World Neurosurgery, 2019). Podoplanin binds to C-type lectin-like receptor-2 (CLEC-2) on the surface of platelets and induces platelet aggregation, potentially leading to the development of VTE. In the present report, we monitor serum podoplanin and CLEC-2 after surgery in malignant glioma patients to directly determine if this is the cause of post-operative VTE. METHODS Fourty-four malignant glioma patients undergoing surgery at the Department of Neurosurgery, Niigata University Hospital between April, 2018 and August, 2020 were assessed. Platelet counts as well as serum D-dimer, podoplanin, soluble CLEC-2, and C2PAC index (soluble CLEC-2 divided by platelet count) were measured within 10 days after surgery. Serum podoplanin and soluble CLEC-2 were assessed by enzyme-linked immunosorbent assay (ELISA). Values for IDH-wildtype glioma patients and IDH-mutant glioma patients were compared to patients undergoing microvascular decompression (MVD) and healthy volunteers (HV). RESULTS Post-operative VTE was observed in 8 out of 35 (23%) IDH-wildtype glioma patients, compared to 1 out of 9 (11%) IDH-mutant glioma patients. Serum soluble CLEC-2 levels (p = 0.0004) and C2PAC index (p = 0.0002) were significantly elevated in IDH-wildtype glioma patients compared to IDH-mutant glioma, MVD and HV groups. A C2PAC index threshold of 3.7 provide an 87.5% sensitivity and 51.9% specificity to predict post-operative VTE in IDH-wildtype gliomas. We serially monitored podoplanin and soluble CLEC-2 in select patients, and found that podoplanin is transiently elevated, followed by soluble CLEC-2 after surgery. CONCLUSION Platelet aggregation due to the activation of podoplanin-CLEC2 axis leads to post-operative VTE in IDH-wildtype malignant glioma patients. The C2PAC index is a potential biomarker for the early detection of VTE after surgery in these patients.

Druggable genomic landscapes of high-grade gliomas.

Despite the putatively targetable genomic landscape of high-grade gliomas, the long-term survival benefit of genomically-tailored targeted therapies remains discouraging. Using glioblastoma (GBM) as a representative example of high-grade gliomas, we evaluated the clonal architecture and distribution of hotspot mutations in 388 GBMs from the Cancer Genome Atlas (TCGA). Mutations were matched with 54 targeted therapies, followed by a comprehensive evaluation of drug biochemical properties in reference to the drug's clinical efficacy in high-grade gliomas. We then assessed clinical outcomes of a cohort of patients with high-grade gliomas with targetable mutations reviewed at the Johns Hopkins Molecular Tumor Board (JH MTB; n = 50). Among 1,156 sequence alterations evaluated, 28.6% represented hotspots. While the frequency of hotspot mutations in GBM was comparable to cancer types with actionable hotspot alterations, GBMs harbored a higher fraction of subclonal mutations that affected hotspots (7.0%), compared to breast cancer (4.9%), lung cancer (4.4%), and melanoma (1.4%). In investigating the biochemical features of targeted therapies paired with recurring alterations, we identified a trend toward higher lipid solubility and lower IC50 in GBM cell lines among drugs with clinical efficacy. The drugs' half-life, molecular weight, surface area and binding to efflux transporters were not associated with clinical efficacy. Among the JH MTB cohort of patients with IDH1 wild-type high-grade gliomas who received targeted therapies, trametinib monotherapy or in combination with dabrafenib conferred radiographic partial response in 75% of patients harboring BRAF or NF1 actionable mutations. Cabozantinib conferred radiographic partial response in two patients harboring a MET and a PDGFRA/KDR amplification. Patients with IDH1 wild-type gliomas that harbored actionable alterations who received genotype-matched targeted therapy had longer progression-free (PFS) and overall survival (OS; 7.37 and 14.72 respectively) than patients whose actionable alterations were not targeted (2.83 and 4.2 months respectively). While multiple host, tumor and drug-related features may limit the delivery and efficacy of targeted therapies for patients with high-grade gliomas, genotype-matched targeted therapies confer favorable clinical outcomes. Further studies are needed to generate more data on the impact of biochemical features of targeted therapies on their clinical efficacy for high-grade gliomas.

Abstract B108: NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects

Abstract Adult-type diffuse gliomas are aggressive brain cancers. The widespread standard treatment for these high-grade gliomas (HGG) is the Stupp Protocol, consisting of maximal safe surgical resection followed by concurrent chemoradiotherapy. However, since the Stupp Protocol, no significant therapeutic advances have been as widely and easily adopted, halting improvements in HGG patient survival. Therefore, identifying new drug targets or treatments that enhance the Stupp Protocol would result in therapeutic benefit. HGG are classified by distinct genetic and metabolic alterations; the most well-known being somatic gain of function mutations in isocitrate dehydrogenase (IDH) 1/2 genes. These mutations distinguish the two most severe HGGs, namely Grade 4 IDH-Mutant Astrocytoma and IDH-Wildtype Glioblastoma (GBM). IDH-mutant tumors accumulate the oncometabolite D-2-Hydroxyglutarate, which alters metabolic pathways and DNA damage responses. One metabolic alteration is decreased nicotinamide adenine dinucleotide (NAD+) levels, a metabolite essential for cellular homeostasis, and crucial in IDH-mutants as targeting NAD+ homeostasis is effective against IDH-mutant tumors. Here, we investigated if additional metabolic vulnerabilities within NAD+ and purine biosynthetic pathways could be identified in HGG cells. For this, a panel of cell lines, including an isogenic IDH1-mutant cell line, was screened for their sensitivity to a range of metabolic inhibitors in combination or not with chemo- and radiotherapy. This screen identified NUDT5, a Nudix hydrolase that metabolizes ADP-Ribose into Ribose-5-Phosphate (R-5-P) to replenish NAD+ pools, and synthesizes nuclear ATP for hormone driven chromatin remodeling, as an important determinant in glioma cell survival. Cell viability assays determined NUDT5 inhibition (NUDT5i) sensitized IDH1-wildtype HGG cells to different radiotherapy modalities, specifically X-rays and alpha particles, whilst IDH1-mutant HGG were sensitive to NUDT5i alone. Live-cell imaging analysis indicated NUDT5i induced cytostasis followed by cytotoxicity in the IDH1-mutant, suggesting IDH1-mutations confer exploitable metabolic dependencies on NUDT5 for proliferation and survival. NUDT5i did not significantly deplete NAD+ levels and NAD+ precursor supplementation failed to rescue cell survival and proliferation, indicating effects of NUDT5i are NAD+ independent. Our results indicate NUDT5 plays a crucial role in HGG proliferation and survival. Given that NUDT5 produces R-5-P for downstream nucleotide synthesis, we hypothesize that NUDT5i impairs nucleotide homeostasis and leads to replication stress. Current investigations include nucleoside supplementation and characterization of replication stress signaling by western blotting. We anticipate our results to prelude further investigations into NUDT5 as a potential therapeutic target in HGG. We suggest NUDT5i could enhance the effectiveness of the Stupp Protocol regardless of IDH-Status and advance HGG therapies. Citation Format: Thomas J.R Cox, Eren Ozcagli, Giuseppe Schettino, Lisiane B Meira. NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B108.

BIOM-49. FGFR ALTERATIONS IN GLIOMAS; CLINICAL, RADIOGRAPHIC AND MOLECULAR CHARACTERIZATION

Abstract BACKGROUND FGFR-TACC is the most prevalent type of gene fusion in adult gliomas. It is present in 3-6% of IDH WT (wild-type) gliomas. Due to their histopathological and molecular features, it has been suggested that FGFR3-TACC3 fusions (F3T3) gliomas represent a unique entity among IDH WT gliomas. F3T3 are potent oncogenes that confer sensitivity to FGFR inhibitors. Preliminary data showed promising results of these alterations as actionable targets by FGFR inhibitors. METHODS We retrospectively analyzed molecular data on 912 glioma patients treated at our institution from 2014 to 2023. RESULTS We identified 22 gliomas (2.5%) with FGFR alterations. Median age was 60 years, 14 were female. 14 patients (64%) self-identified as Hispanic. 18 had a diagnosis of glioblastoma, 3 had other gliomas, and one had a low-grade neuroepithelial neoplasm. Ten patients had tumor location in the temporal lobe, 6 frontal, 5 parietal, 3 occipital, 1 multifocal, and 1 basal ganglia. 19 patients had enhancing tumors, while three were non-enhancing. The most common FGFR alteration was FGFR3-TACC3 fusion in 14/22 patients, 6 had FGFR mutations (either single base or amino acid substitutions), one had FGFR-LMNB2 fusion, and one had FGFR3 amplification. 86.4% of our patient cohort (19/22) had a confirmed TERT promoter mutation, while 100% of GBM patients with known TERT promoter status had an associated TERT promoter mutation. 10/22 had CDKN2A/B alterations. One patient had pathology-proven systemic metastases. CONCLUSIONS FGFR3-TACC3 fusions (F3T3) gliomas represent a unique entity among IDH WT gliomas. In our cohort, 100% of the patients with F3T3-positive glioblastomas also had an associated TERT promoter mutation. To our knowledge, this is one of the larger cohorts showing the association between FGFR3 and TERT promoter alterations in glioblastomas. RESULTS will be validated through the upcoming clinical trial ETCTN 10559, including tumor evolution and mechanisms of resistance.

TMET-19. TARGETING AMINO ACID METABOLIC VULNERABILITIES IN IDH-MUTANT AND IDH-WILDTYPE GLIOMAS

Abstract IDH-wildtype and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. The aim of the study is to reveal the difference in metabolites between IDH-wildtype glioma and IDH-mutant glioma, and to find the effective treatment according to the status of IDH in gliomas. Two artificial cell lines made from human astrocyte were used: NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant). Capillary electrophoresis- and ion chromatography–coupled mass spectrometry revealed that the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells. L-asparaginase, which converts asparagine into aspartate, was more effective in former cells than latter cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect, which meant that the antitumor effect was at least partially due to the L-asparaginase-induced autophagy. Metabolic assay also showed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. Pharmacological or genetical inhibition of GLUD1 which converts glutamate to 2-oxoglutarate, suppressed proliferation of the cells by inducing ROS and apoptosis in NHAE6E7hTERTIDHmut cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells, revealing that cytotoxicity by GLUD1 inhibitor was at least partially due to the inhibitor-induced ROS. Mutant IDH1 overexpressed glioma cell line showed similar sensitivity to GLUD1 inhibitor to NHAE6E7hTERTIDHmut, which suggested that the difference of sensitivity to GLUD1 inhibitor was due to the status of mutant IDH. In addition, GLUD1 inhibitor suppressed the growth of xenografted glioma cells with overexpressed mutant IDH1. In conclusion, L-asparaginase and Glud1 inhibitor will be new therapeutic option for IDH-wildtype glioma and IDH-mutant glioma, respectively.

NIMG-57. PERFORMANCE OF DEEP LEARNING IN MGMT PROMOTER METHYLATION STATUS PREDICTION USING BRAIN MRI: RESULTS FROM A LARGE COHORT OF IDH-WILDTYPE GLIOMAS TESTED BY A SINGLE METHYLATION ASSAY

Abstract There have been several attempts to predict the MGMT promoter methylation status of gliomas using deep learning (DL), with mixed results. These studies did not account for IDH mutation status and did not utilize a uniform methylation assay, introducing potential confounders in the experimental design. We assessed the performance of DL applied to brain MRI to predict the MGMT promoter methylation status of IDH-wildtype gliomas tested with a standardized assay. We included 539 IDH-wildtype gliomas tested with real-time methylation-specific PCR from 2017-2022; 205 (38%) were MGMT promoter methylated. The assay targets 8 downstream MGMT CpG sites using ACTB as control; methylation status is determined based on the difference of PCR crossing-point values: methylated if difference &amp;lt; 5, unmethylated if difference &amp;gt; 7, indeterminate otherwise. We used post-contrast T1 and T2 MRIs as inputs for our models trained using stratified 5-fold cross-validation, 400 epochs, batch size of 16, a learning rate scheduler starting at 10-3, weighted binary cross-entropy loss, and AdamW optimizer. We repeated the experiments using two 3D DL architectures (DenseNet121 and EfficientNetB0) and two image preprocessing pipelines: the BraTS protocol and one that uses padding instead of registration to an MRI atlas, keeping constant other experimental variables. For Densenet121, the best cross-validation AUROCC and F1-score (median, median absolute deviation) were 0.67 (0.06), 0.59 (0.02) using the BraTS protocol, and 0.67 (0.06), 0.58 (0.01) using padding. For EfficientNetB0, were 0.67 (0.02), 0.66 (0.01) using the BraTS protocol and 0.67 (0.01), 0.68 (0.01) using padding. Our findings suggest that it is unlikely that DL models trained on brain MRI can predict the MGMT promoter methylation of IDH-wildtype gliomas. Importantly, our study is the first to focus on an IDH-wildtype adult diffuse glioma cohort tested with a single, standardized MGMT methylation assay, eliminating the variability introduced by different assays and IDH status.

BIOS-03. COPY NUMBER VARIATION (CNV) AND CLINICAL OUTCOMES IN IDH-WILDTYPE VERSUS IDH-MUTANT GLIOMA

Abstract Genomic classification of gliomas has the potential for improved prognostication and treatment guidance. However, much work remains to elucidate the full potential of genomics in glioma characterization. IDH mutant gliomas generally carry an improved survival compared to their wild-type counterparts, but their clinical course is significantly more variable and challenging to predict. The goal of this study is to characterize tumor genetic/genomic parameters associated with progression-free survival (PFS) and overall survival (OS) of both IDH-wildtype and IDH-mutant gliomas and compare the two subtypes for further stratification. We collected data for 203 patients diagnosed with IDH-wildtype gliomas (WHO Grade IV) and 88 patients with IDH-mutant gliomas (WHO Grade II-IV) from the Emory Healthcare system. Microarray single nucleotide polymorphism-copy number analysis was performed to detect total CNVs which are associated with tumor mutational burden. We analyzed total CNV based on the median, quartiles, and optimal cut points via the ideal cutpoint calculator, Cutoff Finder. We then estimated the correlation between % total CNV, PFS, and OS using the Pearson correlation coefficient. For IDH-wildtype gliomas, the mean CNV was 18.69 (standard deviation (SD): 9.62), and the median was 16.00 (IQR: 1-57). The optimal cutoff was defined as 39 for PFS analysis and 20.5 for OS analysis. For IDH-mutant gliomas, the mean CNV was 12.8 (SD: 11.36), and the median was 8.5 (IQR: 5 – 17.5). The optimal cutoff was defined as 10.5 for OS analysis and 7.5 for PFS analysis. Overall, higher CNV was associated with lower PFS and OS in both groups. However, the results were only statistically significant for OS at the optimal cutoff (20.5) for IDH-wildtype gliomas (p-value = 0.0379), and for PFS at the optimal cutoff (7.5) for IDH-mutant gliomas (p-value = 0.0420). These findings suggest that CNV may be useful as a potential prognosis factor for both tumor subtypes in different contexts.

RTID-07. A RANDOMIZED WINDOW OF OPPORTUNITY TRIAL WITH DOSE ESCALATION TO EVALUATE FLUOXETINE AND TEMOZOLOMIDE IN GLIOMA

Abstract The blood brain barrier limits the efficacy of many drugs in brain tumors. Fluoxetine (Prozac) is FDA-approved and brain penetrant. Preclinical studies show synergy between fluoxetine and temozolomide (TMZ), resulting in lysosomal stress and enhanced DNA damage in human-derived glioma in vitro and in vivo. We present our randomized surgical window of opportunity trial (NCT05634707) in patients with recurrent IDH wild type glioma. The primary objective is to evaluate if fluoxetine induces tumoral lysosomal stress while exploratory objectives will determine if combination therapy enhances tumor DNA damage. We will randomize 30 patients, ≥ 24yo, with recurrent glioma and who are eligible for re-treatment with TMZ (MGMT methylated, progression &amp;gt;6 months post TMZ). Following informed consent, patients will be randomized 1:2 to control or experimental arms. Control patients will receive 50 mg/m2 TMZ for 7 days pre-resection. The experimental arm will receive 20 mg/day fluoxetine for 5 days before escalating to 40 mg/day. If &amp;lt; 3/10 participants receiving 40mg/day experience dose-limiting toxicity (DLT), subsequent experimental arm patients will escalate to 60 mg/day. All experimental arm participants will receive 50 mg/m2 TMZ for 7 days after starting fluoxetine, starting 21 days pre-resection. The regimen of 50 mg/m2 TMZ for 7 days is based on a similar pre-surgical trial combining TMZ with experimental therapy (NCT05188508). This reduced dose was chosen to limit potential synergistic toxicities like thrombocytopenia or neutropenia. Following resection, patients will continue TMZ, initially at reduced dose for the first post-op cycle, before reverting to standard doses of 150-200mg/m2 (up to 12 cycles or progression). All patients (control and experimental) will be offered fluoxetine post-operatively. The primary study outcome will be quantification of tumoral lysosomal stress via Lysosomal-associated membrane protein 1 (LAMP1) expression while DNA damage will be assessed via γH2A histone family member X (γH2AX) using immunohistochemistry.

IMMU-57. EFFECT OF THE ONCOMETABOLITE D2HG ON MYELOID-DERIVED SUPPRESSOR CELL POPULATIONS

Abstract Mutations in the isocitrate dehydrogenase 1/2 (IDH1/2) enzyme correlate with better prognosis of gliomas, partly due to enhanced infiltration of nonimmunosuppressive granulocytic cells compared to IDH wild-type (IDHwt) tumors. Altered cytokine/chemokine secretion from tumor cells and reprogramming of myeloid cell metabolism are among the factors that drive differences in immune profiles of IDHwt vs. IDHmut tumors. IDH mutant protein produces the oncometabolite D-2-hydroxyglutarate (D2HG) instead of the canonical alpha-ketoglutarate (αKG), a co-factor for histone and DNA demethylases. Since D2HG is known to be secreted into the tumor microenvironment (TME), we interrogated the effect of this metabolite on the epigenetic landscape and behavior of granulocytic and monocytic myeloid-derived suppressor cells (g/mMDSCs). Co-culturing bone marrow cells with either IDHmut or IDHwt glioma cells demonstrated that mMDSCs, which we previously showed to be the abundant tumor infiltrating MDSC subtype in IDHwt tumors, but not gMDSCs specifically experience higher levels of apoptosis when exposed to IDHmut cells. Treatment of bone marrow cells with physiologically relevant concentrations of either D2HG or αKG demonstrated that D2HG re-capitulates the pro-apoptotic effect on mMDSCs. Furthermore, co-cultures of IDHmut glioma cells and bone-marrow cells induced a pronounced increase in global methylation on mMDSCs specifically, and we observed that mMDSCs and gMDSCs differentially express histone demethylases via western blotting. Collectively, these results suggest that D2HG can differentially impact mMDSC vs gMDSC biology by epigenetic reprogramming. Future studies will elucidate the specific targets of D2HG in mMDSCs that renders these cells more susceptible to apoptosis with the goal of identifying the mechanisms that can be modulated to down-regulate mMDSC infiltration and/or survival within the TME.

ANGI-05. CDKN2A/B HOMOZYGOUS DELETION IN IDH-MUTANT ASTROCYTOMA ALTERS TUMOR CELL INVASION AND VASCULAR FORMATION

Abstract High-grade gliomas represent the most common type of primary adult malignant brain tumor historically diagnosed and graded from histologic criteria alone. Gliomas harboring isocitrate dehydrogenase (IDH) 1 or 2 mutations are associated with lower grade tumors and portend a favorable prognosis relative to IDH-wildtype glioma. However, molecular profiling has identified additional alterations in lower grade gliomas that confer an aggressive high-grade phenotype and a worse clinical prognosis. Of these, the loss of CDKN2A/B in IDH-mutant astrocytoma is the strongest implicated alteration and is sufficient to define a grade 4 tumor regardless of histologic grade. However, there remain no effective therapies targeted at molecular subgroups in aggressive gliomas to date. Here, we sought to elucidate the biologic pathways and functional outcomes associated with the acquisition of high-grade behavior under loss of CDKN2A/B in IDH-mutant astrocytoma to inform future therapeutic strategies. We analyzed a cohort of patient IDH-mutant astrocytomas with whole exome and RNA sequencing data and found several candidate genes/pathways regulated concurrent with CDKN2A/B loss, including regulators of embryonic development, extracellular matrix organization and regulation of vascular processes. Moreover, we identified 1 tumor that displayed intratumoral heterogeneity of CDKN2A/B expression across biopsies. Using grade 4 IDH-mutant astrocytoma patient-derived (PDX) and established cell-lines harboring CDKN2A/B homozygous deletion, we then stably re-expressed cell cycle regulators p14, p15, and p16 using a Tet-inducible lentiviral system and confirmed their decreased proliferation as compared to parental lines. IDH-mutant cell lines with re-expression of p14, p15 or p16 were characterized by differential transcription factor activation and distinct vascular formation patterns as compared to parental lines. These lines also displayed an increased cell migration and invasive potential and were associated with enhanced integrin signaling through integrin beta-3. Together, our work supports a hypothesis of regional tumor evolution with distinct biologic phenotypes across IDH-mutant gliomas.

Differential metabolic alterations in IDH1 mutant vs. wildtype glioma cells promote epileptogenesis through distinctive mechanisms.

Glioma-related epilepsy (GRE) is a hallmark clinical presentation of gliomas with significant impacts on patient quality of life. The current standard of care for seizure management is comprised of anti-seizure medications (ASMs) and surgical resection. Seizures in glioma patients are often drug-resistant and can often recur after surgery despite total tumor resection. Therefore, current research is focused on the pro-epileptic pathological changes occurring in tumor cells and the peritumoral environment. One important contribution to seizures in GRE patients is metabolic reprogramming in tumor and surrounding cells. This is most evident by the significantly heightened seizure rate in patients with isocitrate dehydrogenase mutated (IDHmut) tumors compared to patients with IDH wildtype (IDHwt) gliomas. To gain further insight into glioma metabolism in epileptogenesis, this review compares the metabolic changes inherent to IDHmut vs. IDHwt tumors and describes the pro-epileptic effects these changes have on both the tumor cells and the peritumoral environment. Understanding alterations in glioma metabolism can help to uncover novel therapeutic interventions for seizure management in GRE patients.