IDH1 Mutations Research Articles

CLIC4 Is a New Biomarker for Glioma Prognosis

Background: Chloride Intracellular Channel 4 (CLIC4) plays a versatile role in cellular functions beyond its role in primary chloride ion transport. Notably, many studies found an association between CLIC4 expression and cancers. However, the correlation between CLIC4 and glioma remains to be uncovered. Methods: A total of 3162 samples from nine public datasets were analyzed to reveal the relationship between CLIC4 expression and glioma malignancy or prognosis. Immunohistochemistry (IHC) staining was performed to examine the results in an in-house cohort. A nomogram model was constructed to predict the prognosis. Functional enrichment analysis was employed to find CLIC4-associated differentially expressed genes in glioma. Immune infiltration analysis, correlation analysis, and IHC staining were employed, aiming to examine the correlation between CLIC4 expression, immune cell infiltration, and ECM (extracellular matrix)-related genes. Results: The expression level of CLIC4 was correlated with the malignancy of glioma and the prognosis of patients. More aggressive gliomas and mesenchymal GBM are associated with a high expression of CLIC4. Gliomas with IDH mutation or 1p19q codeletion express a low level of CLIC4, and a high expression of CLIC4 correlates with poor prognosis. The nomogram model shows a good predictive performance. The DEGs (differentially expressed genes) in gliomas with high and low CLIC4 expression are enriched in extracellular matrix and immune functions. On the one hand, gliomas with high CLIC4 expression have a greater presence of macrophages, neutrophils, and eosinophils; on the other hand, a high CLIC4 expression in gliomas is positively associated with ECM-related genes. Conclusions: Compared to glioma cells with low CLIC4 expression, gliomas with high CLIC4 expression exhibit greater malignancy and poorer prognosis. Our findings indicate that a high level of CLIC4 correlates with high expression of ECM-related genes and the infiltration of macrophages, neutrophils, and eosinophils within glioma tissues.

BIOM-01. CDKN2A HOMOZYGOUS DELETION HAS STRONGER PROGNOSTIC POWER THANIDH MUTATION IN CNS WHO GRADE 4 GLIOMAS

Abstract We conducted retrospective analysis of glioma cohorts in our institute. Medical records were reviewed for 142 glioblastoma patients for 15 years, and pathological slides were examined again for the updated diagnosis according to 2021 WHO classification of CNS tumors. The IDH mutation and CDKN2A deletion were examined by NGS analysis using ONCOaccuPanel®. Traditional prognostic factors were also examined. Mean follow-up duration was 27.5 months (ranged from 4.1 to 43.5 months) and mean overall survival (OS) was 19.4 months (95% CI 16.3 – 20.9 months). After the exclusion of 4 patients with poor status of pathologic samples, total 109 glioblastoma which were diagnosed by previous WHO criteria were changed into 26 (23.9%) astrocytoma, IDH-mutant, CNS WHO grade 4 and 83 (76.1%) glioblastoma, IDH-wildtype, CNS WHO grade 4. Among them, 61 patients (56.0%) had CDKN2A deletion. Group A was classified for 59 patients with IDH-wildtype and present CDKN2A deletion, group B was classified for 16 patients with IDH-mutant and present CDKN2A deletion, group C was classified for 48 patients with IDH-wildtype and absent CDKN2A deletion, and group D was classified for 13 patients with IDH-mutant and absent CDKN2A deletion. Group A had a mean OS of 15.70 months (95% CI 13.86 – 17.54 months), group B had a mean OS of 19.37 months (95% CI 13.43 – 25.30 months), group C had a mean OS of 22.63 months (95% CI 20.10 – 25.17 months), and group D had a mean OS of 33.38 months (95% CI 29.35 – 37.40 months). Multivariate analysis showed IDH mutation (mutant vs. wildtype; HR 6.358), and CDKN2A deletion (absence vs. presence; HR 13.452). The presenting study suggest that CDKN2A deletion should play a powerful prognostic role in CNS WHO grade 4 gliomas as well as low grade glioma. Even if CNS WHO grade 4 gliomas had mutant IDH, they can have poor clinical outcome due to CDKN2A deletion.

PATH-49. INTRA-OPERATIVE GENETIC ANALYSES FOR DECISION-MAKING DURING TUMOR REMOVAL OF ADULT-TYPE DIFFUSE GLIOMA USING RAPID QUANTITATIVE PCR DEVICE

Abstract BACKGROUND Recent comprehensive molecular analyses of central nervous system (CNS) tumors identified several diagnostic or prognostic markers. Especially, for adult-type diffuse glioma, IDH mutation, TERT promoter (pTERT) mutation and CDKN2A homozygous deletion are quite important markers for diagnosis and prognosis of this type of glioma. Intra-operative identification of these molecular alterations could be effective for decision-making of tumor removal strategy. METHODS In this study, we established intra-operative gene analysis method using rapid quantitative PCR device, GeneSoC, by which we could obtain genotype of these important genes for 20 minutes in an operating room. Using GeneSoC, we could perform 50 cycles of quantitative PCR for approximately 12 minutes. Results; We established rapid gene analyses of mutations of IDH1 R132H, pTERT C225T, pTERT C250T and homozygous deletion of CDKN2A for adult-type diffuse gliomas. For elution of DNA, we incubated at least 1mg of tumor tissue at 95°for 5 minutes. Combined this boiling method with GeneSoC, we could obtain those genetic alterations for 20 minutes after collection of tumor tissue. In analyses of 111 adult-type diffuse glioma cases, sensitivity and specificity for detection of IDH1 p.R132H are 98% and 98%, respectively, and those of pTERT (C225T or C250T) are 100% and 100%, respectively. In analyses of 50 cases, sensitivity and specificity for detection of CDKN2A homozygous deletion are 100% and 83%, respectively. Conclusion; In this study, we could obtain the genotype of important molecular markers intra-operatively, not only during tumor removal but even during tumor biopsy, using GeneSoC device. This rapid genetic analysis might be effective not only for intra-operative diagnosis but also for detection of boundary between tumor and normal tissue.

DDDR-08. PIM1 INHIBITION: A NOVEL THERAPEUTIC APPROACH FOR IDH-MUTANT GLIOMAS

Abstract BACKGROUND PIM1, a constitutively active serine/threonine kinase has emerged as a relevant target in cancer treatment. Previously, we discovered that IDH-mutant glioma exhibited high kinase activity of PIM1, suggesting an increased sensitivity to PIM1 inhibition. Here we sought to investigate PIM1 inhibition as a therapeutic approach for IDH-mutant gliomas. METHODS Patient-derived IDH-mutant and wildtype glioma cell lines were used to evaluate the impact of PIM1inhibition on cell viability, proliferation, and colony formation. mRNA and protein expression of PIM1 were determined by qRT-PCR, Western-blot, and immunohistochemistry. m6A dot-blot assay was conducted to detect m6A RNA levels in glioma cells. m6A modification status and its location in PIM1 transcript were evaluated by m6A RNA immunoprecipitation array. RNA-binding protein immunoprecipitation was performed using anti-YTHDF1 antibody to identify PIM1 RNA-YTHDF1 protein interactions. RESULTS An upregulation of PIM1 protein is demonstrated in IDH-mutant patient tumor tissues and cells. A pan-PIM inhibitor, AZD1208 significantly suppressed cell viability, cell proliferation, and colony formation in IDH-mutant cells but had a lesser effect in IDH-wildtype cells. Interestingly, a lower mRNA expression of PIM1 is observed, while the protein level is significantly elevated in IDH-mutant cells compared to wildtype cells, suggesting post-transcriptional and translational regulation of PIM1 associated with IDH mutation. Mechanistically, 2-hydroxyglutarate, unique to IDH-mutant gliomas, competitively inhibits α-KG-dependent RNA demethylase FTO. We demonstrated suppressed FTO activity and an increased m6A RNA level in IDH-mutant glioma cells. An enrichment of m6A modification at motif “GG (m6A) CU” located in the 3’UTR is found in the PIM1 transcript. Furthermore, we demonstrated that the m6A modification site is recognized by the m6A reader YTHDF1 to promote protein translation, leading to an elevated PIM1 protein expression. CONCLUSION m6A RNA modification contributes to an enhanced PIM1 activity and thereby a selective sensitivity to PIM1 inhibition in IDH-mutant gliomas.

DDEL-05. IMPRESSIVE RESPONSE TO IDH-INHIBITOR THERAPY IN IDH-MUTANT, H3K27M INTACT BRAINSTEM GLIOMAS

Abstract IDH-mutated brainstem gliomas in adults are a rare entity, with only a handful of cases reported in the literature. Diagnosis and treatment of these gliomas remain challenging. There is significant morbidity associated with a surgical biopsy, thus biopsies are often deferred, and tumors of the brainstem are treated presumptively with radiation and temozolomide. However, in the presence of new targeted therapies, confirming pathology for these patients is becoming increasingly important. We present two cases of IDH-mutant grade 2 astrocytomas of the brainstem that revealed both clinical and radiographic responses to IDH-inhibitor therapy. Interestingly, both patients had the same non-canonical IDH mutation, IDH-R132C, towards which ivosedinib is known to have activity. One patient had tumor progression following radiation therapy and 8 cycles of temozolomide and had subsequently exhibited a dramatic radiographic improvement, including resolution of enhancing disease following initiation of ivosidenib therapy. Clinically, this patient improved from a KPS 50 to KPS 100 after 2.5 years of ivosedinib. The other patient also had an impressive clinical improvement after only 3 months of ivosedinib treatment, with radiographic stability, improvement in balance, and resolution of his previous refractory nausea. These case reports suggest that consideration should be given to brainstem biopsy in adults specifically to evaluate for the presence of a targetable IDH mutation.

PATH-16. MOLECULAR ALTERATIONS IN IDH-MUTANT ASTROCYTOMA: A MULTI-INSTITUTIONAL RETROSPECTIVE STUDY

Abstract BACKGROUND Many molecular alterations are being identified for diagnosis and prognosis in patients with isocitrate dehydrogenase (IDH)-mutant astrocytoma. However, contemporary survival data for these tumors with respect to their molecular alterations are lacking. Moreover, the prognostic value of histologic grading in IDH-mutant astrocytomas remains controversial. METHODS This was a retrospective study of adult IDH-mutant diffuse astrocytoma patients in whom clinical and molecular alteration data were collected. Overall survival (OS) was estimated using the Kaplan-Meier method. Group differences in OS were assessed by the log-rank test and associations between OS and measures of interest were evaluated using Cox proportional hazards regression models. RESULTS We identified 241 eligible patients with median age of 33 years (range, 16-73 years). The most frequent mutations were in IDH1 (98%), TP53 (91%), ATRX (70%), ARID1A (8%), BRCA2 (6%), TSC2 (6%), CDKN2A (6%), and CREBBP (6%). IDH2 mutations were identified in 2% of cases. By univariate analysis, age >40 years (hazard ratio [HR], 2.02; 95% CI, 1.19-3.43; P =.009) was associated with worse OS. BRCA2 wild-type status compared with mutated BRCA2 (HR, 0.42; 95% CI, 0.20-0.90; P =.024) and histology-based grade 2 compared with grade 3 astrocytoma (HR, 0.41; 95% CI, 0.23-0.74; P =.003) were associated with improved OS. In multivariable analysis, age>40 years (HR, 2.14; 95% CI, 1.22-3.75; P =.008) was associated with worse OS. Patients with WHO grade 2 astrocytoma remained associated with improved OS compared to those with grade 3 disease (HR, 0.45; 95% CI, 0.24-0.84; P =.013). CONCLUSIONS Age and histological grade remain essentials for risk stratification among IDH-mutant astrocytoma patients. Further studies are warranted to determine the role of BRCA2 mutation in prognosis.

EXTH-30. GENETIC ALTERATIONS THAT DEREGULATE RB AND PDGFRA SIGNALING PATHWAYS DRIVE TUMOR PROGRESSION IN IDH2-MUTANT ASTROCYTOMA

Abstract In IDH-mutant astrocytoma, IDH2 mutation is quite rare and biological mechanisms underlying tumor progression in IDH2-mutant astrocytoma remain unclear. In this presentation, we report a unique case of IDH2 mutant astrocytoma, CNS WHO grade 3 that developed tumor progression. We performed a comprehensive genomic and epigenomic analysis for primary and recurrent tumors and found that both tumors harbored recurrent IDH2R172K and TP53R248W mutation with CDKN2A/B hemizygous deletion. We also found amplifications of CDK4 and MDM2 with PDGFRA gain in the recur- rent tumor and upregulated protein expressions of these genes. We further developed, for the first time, a xenograft mouse model of IDH2R172K and TP53R248W mutant astrocytoma from the recurrent tumor, but not from the primary tumor. Consistent with parent recurrent tumor cells, amplifications of CDK4 and MDM2 and PDGFRA gain were found, while CDKN2A/B was identified as homozygous deletion in the xenografts, qualifying for integrated diagnosis of astro- cytoma, IDH2-mutant, CNS WHO grade 4. Cell viability assay found that CDK4/6 inhibitor and PDGFR inhibitor potently decreased cell viability in recurrent tumor cells, as compared to primary tumor cells. On the other hand, in accord with our previous study that IDH1 inhibitor was not sufficient to induce anti-tumor effects in IDH1-mutant high-grade gliomas (Tateishi K et al. Cancer Cell. 2015), we did not find decreased cell viability nor histone methylation status changes after mutation specific IDH2 inhibitor treatment of recurrent cells. These findings underlie that gene alterations that activate retinoblastoma (RB) signaling pathways and PDGFR may drive tumor progression and xenograft formation in IDH2-mutant astrocytoma, which is equivalent to progressive IDH1-mutant astrocytoma. Also, our findings suggest that these genomic alterations may represent therapeutic targets in IDH2-mutant astrocytoma.

PATH-19. DIAGNOSTIC UTILITY OF MOLECULAR PROFILING IN ADULT DIFFUSE GLIOMA

Abstract INTRODUCTION With the publication of World Health Organization Classification of the Central Nervous System Tumors (WHO CNS5), molecular profiling has become essential for the diagnosis of CNS tumors. To test for the diagnostic molecular alterations, we developed a targeted DNA sequencing panel (Nakashima et al. 2024) to analyze 113 genes, chromosomal abnormalities, and MGMT promoter methylation. This study aimed to evaluate our diagnostic approach using the targeted sequencing panel. PATIENTS AND METHODS We included 72 cases of diffuse adult gliomas diagnosed at our institute between June 2023 and May 2024. We analyzed the tumor diagnoses based on the pathological and immunohistochemical findings as well as the targeted sequencing panel findings. RESULTS The tumor diagnoses were as follows: glioblastoma, IDH-wildtype: 32 cases, astrocytoma, IDH-mutant: 17 cases, oligodendroglioma, IDH-mutant and 1p/19q-codeleted: 15 cases, others: 8 cases. Among 32 cases of IDH1/2 mutations, 28 cases (87.5%) with IDH1 R132H mutation were diagnosed by immunohistochemistry using anti-IDH1 R132H antibody, whereas 4 (12.5%) (3 with IDH2 mutation, and 1 with IDH1 R132S) were diagnosed by the targeted sequencing panel. Of the 32 glioblastoma cases, 6 (18.8%) did not show necrosis or microvascular proliferation and were diagnosed based on either TERT promoter mutation, EGFR amplification, or chromosome 7/chromosome 10 abnormalities. Moreover, 24 out of 26 resected cases (92.3%) were diagnosed morphologically, whereas 4 of 6 biopsy cases (66.7%) were diagnosed molecularly (p=0.0056). Among the 17 cases of astrocytoma, IDH-mutant, 2 (11.8%) showed homozygous deletion of CDKN2A/B. There were 4 cases (5.6%) out of 72 that did not satisfy the diagnostic criteria for glioblastoma, astrocytoma, oligodendroglioma. CONCLUSION Approximately 95% of cases were diagnosed based on WHO CNS5 criteria with molecular profiling. We also demonstrate molecular profiling is useful in glioblastoma diagnostics, especially in biopsy cases where morphological diagnostic evidence is not recognized.

Vorasidenib (Voranigo) for low-grade glioma.

The FDA has approved vorasidenib (Voranigo – Servier), an oral isocitrate dehydrogenase (IDH)inhibitor, for treatment of grade 2 astrocytoma or oligodendroglioma in patients ≥12 years old with an IDH1 or IDH2 mutation. It is the first systemic treatment to be approved in the US for this indication.

EPCO-29. PATHOMICS ANALYSIS IDENTIFIES POTENTIAL SUBTYPES OF DIFFUSE GLIOMAS IN H&E-STAINED WHOLE-SLIDE IMAGES

Abstract IDH status is the first criteria of diagnostic definition for diffuse gliomas in the 2021 WHO classification. While sufficient number of molecular tools are available to identify IDH1 and/or IDH2 mutations, they impose a time and cost burden on patients. We previously trained deep learning models visually differentiate IDH wild-type (IDHwt) and IDH mutant (IDHmut) gliomas in a small set of H&E-stained whole image slides (WSIs) from TCGA using an Uncertainty-Aware Convolutional Neural Network (CNN) at 10x magnification. In this follow-up study, we aimed to improve our models by expanding our annotations of a more representative subset of the TCGA public data, increasing the sampling magnification to 20x, redesigning the preprocessing filters, adjusting the training pipeline to reduce overfitting, and assessing the confidence in model prediction via uncertainty metrics. METHODS By modifying the pre-processing methods from our pilot study, we increased the number of expert-annotated WSIs. Using a 2:1:2 train/validation/test split, we fine-tuned a ResNet18 deep learning model with a pre-trained feature extractor and Monte Carlo dropouts. To identify image features critical for separating by IDH status, we extracted the learned embeddings and performed post-hoc analyses (PCA, t-SNE, and UMAP). RESULTS The new set of annotations resulted in ~15-fold increase in the number of WSIs (i.e.,>2.5 million tiles), without any data augmentation. Our prototype model demonstrated clear separation of cases according to IDH status (sensitivity = 0.63, specificity = 0.91, weighted F1 = 0.81). CONCLUSION We successfully improved the prediction of IDH status in H&E-stained WSIs and identified potential subtypes of IDHwt and IDHmut diffuse gliomas after significantly increasing the dataset size with appropriate expert annotations. We are currently addressing the data imbalance by annotating additional IDHmut glioma WSIs. Furthermore, we will integrate other biomarkers (1p19q, EGFR etc.) to increase the robustness of histo-pathomics as a surrogate for molecular tests.

TMET-12. TARGETING AMINO ACID METABOLIC VULNERABILITIES IN GLIOMAS

Abstract Isocitrate dehydrogenase (IDH) wild-type gliomas and IDH mutant gliomas are similar in histologic appearance but differ in their genetic and metabolic backgrounds. This study aimed 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). Using capillary electrophoresis- and ion chromatography–coupled mass spectrometry, the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells, whereas the amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells was lower than NHAE6E7hTERTRas cells. L-asparaginase, which converts asparagine into aspartate, was more effective in NHAE6E7hTERTRas cells than NHAE6E7hTERTIDHmut 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. 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. The experiments using 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 vivo experiments, L-asparaginase suppressed the growth of xenografted glioma cells without mutant IDH, whereas GLUD1 inhibitor inhibited the proliferation 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.

EPCO-10. PROTEIN GENERATOR ENABLES PROTEOMICS-BASED SUBTYPING OF IDH MUTANT ASTROCYTOMA

Abstract The understanding of the proteomic landscape of IDH mutant astrocytoma has been limited in previous studies. To address this knowledge gap, we developed an innovative approach using artificial intelligence (AI) to generate proteomic profiles of tumors based on their transcriptome data. Particularly, we employed a combination of Variational Auto-Encoder and Contrastive Language Image Pretraining techniques to develop an AI model. The model was trained on publicly available databases containing matched RNA and protein data. Leveraging this extensive dataset, we applied the trained model to infer proteomic profiles for over one thousand adult IDH mutant astrocytoma specimens using their transcriptome sequencing data. This novel approach allowed us to explore and identify previously undiscovered subtypes based on the predicted proteomic profiles. As a result, we identified seven distinct clusters: IDHmut-noncodel-Proliferative, IDHmut-noncodel-Mesenchymal, IDHmut-noncodel-Mitochondrial, IDHmut-NFkB-signal, IDHmut-codel-Proliferative, IDHmut-Metabolism, and IDHmut-Neuronal. Of particular clinical significance, the IDHmut-noncodel-Proliferative and IDHmut-noncodel-Immune-Mesenchymal subtypes displayed the worst prognoses, while the IDHmut-Neuronal, IDHmut-NFkB-signal, and IDHmut-Metabolism subtypes exhibited relatively better survival outcomes. Remarkably, within the IDHmut-noncodel-Proliferative cluster, we observed a high prevalence of CDKN2A homozygous deletion, providing a potential explanation for its poor survival prognosis. Conversely, the IDHmut-noncodel-Mesenchymal cluster did not demonstrate any significant genetic events but exhibited elevated expression of mesenchymal marker genes. Furthermore, this cluster displayed notable characteristics, including the presence of gemistocytic differentiated tumor cells and infiltration of activated CD8 T lymphocytes. Moreover, we encompassed an evolutionary analysis of 189 initial-recurrent IDH mutant astrocytoma pairs, and discovered that gemistocytic differentiation represents an early event in tumor progression. In summary, we introduced a novel method utilizing an AI model to generate proteomic profiles, thus enabling the discovery of previously unknown subtypes of IDH mutant glioma with clinical relevance. These findings contribute to a deeper understanding of the molecular landscape of IDH mutant astrocytoma, potentially leading to improved prognostication and personalized treatment strategies.

NCOG-37. SINGLE CENTER EXPERIENCE OF IDH INHIBITORS IN THE TREATMENT OF RECURRENT HIGH GRADE GLIOMAS

Abstract BACKGROUND The antitumor effect of IDH inhibitors in high grade gliomas (HGG) is not well known, with conflicting views regarding the role of IDH status and the potential for resistance to DNA damage treatments. We present the outcomes of patients with HGG that were treated with IDH inhibitors at our institution. METHODS We performed a retrospective review of patients with IDH mutant gliomas treated with FDA approved IDH inhibitors (ivosidenib and enasidenib) in the past 5 years at our institution. One cycle of IDH inhibitor was defined as treatment for 28 days. RESULTS We identified 10 HGG patients (both oligodendrogliomas and astrocytomas). Median age at diagnosis was 31 years and 7 were male. Seven patients were diagnosed as WHO grade 3. Six patients were WHO grade 3 oligodendrogliomas, three WHO grade 4 astrocytomas, and one patient WHO grade 3 astrocytoma. Only one patient received enasidenib and the rest were treated with ivosidenib. Eight patients were started on IDH inhibitors after 3 recurrences. Median number of cycles was 5. One patient received 23 cycles, and one patient received 11 cycles. All patients underwent resection, 4 received chemotherapy (including temozolomide, bevacizumab, and CCNU), and 4 radiation prior to starting IDH inhibitors. One patient was treated with Tumor Treating Fields. Five patients progressed during the study follow up, and 3 patients died. Four patients are still on treatment. No serious adverse events. Median PFS after starting an IDH inhibitor was 6 months. DISCUSSION IDH inhibitors were well tolerated in IDH mutant HGG patients previously treated with DNA-damaging agents. It may have a role in the treatment of HGG with multiple recurrences with 3 patients showing stable disease for more than 18 months. More studies are needed to clarify the role of IDH inhibitors in patients with IDH mutant HGG.

CTNI-15. A PERIOPERATIVE STUDY OF SAFUSIDENIB IN PATIENTS WITH IDH1 MUTATED GLIOMA

Abstract BACKGROUND IDH mutations cause aberrant accumulation of (R)-2-hydroxyglutarate (2-HG), an oncometabolite that promotes tumorigenesis through abnormal hypermethylation of histones and DNA as well as suppression of normal cellular differentiation. IDH inhibition significantly improves progression free survival in patients with IDH mutant WHO grade 2 glioma, however most patients progress, and there is a lack of understanding on mechanistic reasons for failure. METHODS We tested Safusidenib, an oral, blood brain barrier penetrant IDH1 R132X enzyme inhibitor in a single arm, open label perioperative study. Patients with IDH1 mutated glioma, treatment naïve to radiation or chemotherapy, received Safusidenib (250 mg BID) for four weeks following surgical biopsy and prior to definitive resection (Part A). After resection patients continued Safusidenib until toxicity or progression (Part B). Translational endpoints include paired whole genome transcriptome sequencing (WGTS), single nuclei RNA sequencing (snRNAseq), spatial transcriptomics and spatial metabolomics as well as longitudinal sampling of blood and CSF for circulating tumour DNA (ctDNA). Here we are reporting Part A of the study. RESULTS As of May/27/2024 10 patients (mean age 37, 40% female) have been enrolled: seven with astrocytoma, three with oligodendroglioma; six had prior surgical resection. Pharmacokinetic sampling showed a mean tumor concentration 2457 ng/mL (range 957.0 – 4810) with tumor:plasma ratio 0.33. Consistent with mechanism, tumor 2-HG reduced 86% from biopsy (mean 75.0μM, range 7.8-203) to surgery (10.3μM, range 2.3 – 30.8), as well as identification of novel biomarkers of response. Paired tumour snRNAseq reveals transcriptional shift with changing cell-to-cell communication, and increased immune infiltration post-treatment, validated by spatial transcriptomics. Additionally, we find reduced heterogeneity consistent with a more differentiated tumour state. Treatment was well tolerated with ongoing follow-up. CONCLUSIONS This innovative trial facilitates intra-patient comparisons and confirmation of the on-target effects of Safusidenib, enhancing understanding of the mechanisms of response and resistance. The approach serves as a proof of concept for advancing drug development in glioma through perioperative trials.

CTIM-09. INB-200: FULLY ENROLLED PHASE 1 STUDY OF GENE-MODIFIED AUTOLOGOUS GAMMA-DELTA (ΓΔ) T CELLS IN NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME (GBM) PATIENTS RECEIVING MAINTENANCE TEMOZOLOMIDE (TMZ)

Abstract IN8bio’s DeltEx drug resistant immunotherapy (DRI), comprising TMZ-resistant γδ T cells engineered to express methylguanine-DNA methyltransferase (MGMT), enables γδ T cells to target upregulated NKG2D ligands on tumor cells in real time during alkylating chemotherapy exposure. Updated results from the fully enrolled Phase 1 trial evaluating DRI in adult newly diagnosed GBM subjects with IDH wild type and mutant tumors, adequate organ function and KPS ≥ 70%. Cohorts (C) 1, 2 and 3 received 1, 3 or 6 doses (1 x 107 DRI cells/dose) into the resection cavity with 150 mg/m2 of IV TMZ on Day 1 of each of 6 Stupp maintenance cycles. The primary endpoint is safety and secondary endpoints include survival; immunologic correlative analyses are included. Dose limiting toxicities (DLTs) are defined as treatment related ≥ grade (G) 3 cardiopulmonary or hepatic toxicity, G4 toxicity exceeding 72 hours or neurologic deterioration that exceeds 2 weeks. A total of 23 subjects were enrolled and 13 dosed (61% male; median age 68 (range: 21-74); 92% IDH-WT, 54% MGMT unmethylated). No DLTs, cytokine release syndrome (CRS), neurotoxicity (ICANS) or treatment related deaths were reported. The most common adverse events were decreased WBC/platelet count, asthenia, fatigue, hydrocephalus, headache, decreased appetite, urinary tract infection, thrombosis and balance disorder with ≥G3 platelet count decreased in 23% and WBC/ neutrophil/ lymphocyte count decreased in 7.7% each. Peripheral TMZ-based lymphodepletion was maintained for up to 1 year evidenced by near or below normal range T, B, and NK subsets. With median follow-up of 10.8 months, 83% of subjects exceeded the median 7 month Progression Free Survival (PFS) of the Stupp regimen alone, had manageable toxicity with a continued encouraging trend in PFS. Subject with IDH mutant tumor remains progression free for > 35 months. Long-term follow-up for durability of PFS and OS continues.

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.

EXTH-70. PRECLINICAL TRIALS OF THE ANTIBODY-DRUG CONJUGATE TISOTUMAB VEDOTIN IN ADULT-TYPE DIFFUSE GLIOMAS

Abstract The gene F3, encoding Tissue Factor (TF), is expressed in many cancers and contributes to their malignancy. Our previous work showed that, among adult-type diffuse gliomas, IDH wild-type (IDHwt) glioblastomas (GBM) express more TF than IDH mutant (IDHmut) gliomas (PMID: 27664011, 30266764, 31222125). Tisotumab vedotin (TisVed), an anti-TF antibody conjugated to monomethyl auristatin E, is a therapeutic designed to target cells expressing TF. We therefore sought to determine the therapeutic potential of TisVed in IDHwt vs. IDHmut gliomas. TisVed was more active against cultured IDHwt GBM cells than IDHmut glioma cells (P<0.0001). This activity was increased by 55% with daily washout of the soluble TF secreted by IDHwt GBM cells. TisVed extended the survival of mice intracranially engrafted with IDHwt GBM by 34% (37.5 days vs. 28 days for IgG control, P=0.006), but not mice with IDHmut glioma (30.5 days, P=0.88). TisVed also reduced the growth of IDHwt GBM flank xenografts by 58% (P=0.045). Unconjugated Tis had no antitumor effect intracranially or in the flank. All 12 flank-engrafted mice treated with Tis, and 9/12 mice treated with TisVed, developed large hematomas in and around the flank xenografts, compared to 0/12 IgG-treated mice (P<0.001). Similarly, a semiquantitative analysis showed that intracerebral hemorrhage was more extensive in Tis/TisVed- treated GBM12 mice than in IgG-treated GBM12 mice (P=0.046). These data indicate that TisVed targets high TF-expressing IDHwt GBM, but not low TF-expressing IDHmut glioma, that its activity is predominately through the vedotin conjugate rather than inhibition of TF signaling, and that it may have some effect against TF-expressing IDHwt GBM, but intracerebral hemorrhage may be a concern.

CNSC-52. OLIGODENDROGLIOMA SPATIAL TRANSCRIPTOMIC SEQUENCING REVEALS THERAPY-DEPENDENT EVOLUTION OVER TIME

Abstract Oligodendroglioma evolution is incompletely understood. Here we analyze serial oligodendroglioma samples to define genomic and cellular mechanisms underlying treatment resistance. RNA sequencing was performed on 53,406 spatial transcriptomes from 31 oligodendrogliomas (IDH mutant, 1p/19q-codeleted) that were resected from 16 patients. There were 15 CNS WHO grade 2 and 16 grade 3 oligodendrogliomas, which were stratified for temporal progression across grades (n=5 grade 2 to 2, n=5 grade 2 to 3, n=6 grade 3 to 3) and treatments between serial resections (n=2 TMZ/RT, n=2 TMZ, n=1 mTOR inhibition, n=1 IDH inhibition, n=10 surgical monotherapy). Spatial transcriptomic clusters were defined using cell signature gene sets, cell cycle analysis, differentially expressed marker genes, immunohistochemistry, spatial copy number alterations (CNAs), and deconvolution of 18,628 oligodendroglioma single-cell transcriptomes from 16 independent patients. Specificity was tested using immunofluorescence on independent oligodendrogliomas that did not recur after surgical monotherapy (n=8 samples, median follow-up 8.4 years) and spatial transcriptomic sequencing on 8 matched-paired astrocytomas from 4 patients. Integrated uniform manifold approximation and projection revealed a proneural/NPC-like cluster that was enriched in grade 3 oligodendroglioma samples and represented the starting point in oligodendroglioma lineage differentiation using RNA velocity, Monocle, and subclonal spatiotemporal CNA evolution. Comparison across treatment regimens showed (1) enrichment of the proneural/NPC-like cluster in recurrent versus primary tumors that were treated with surgical monotherapy, (2) enrichment of a proneural/OPC-like cluster and depletion of an oligodendrocyte-like cluster after TMZ/RT, (3) enrichment of proneural/NPC-like and immediate-early response clusters after TMZ/RT, (4) enrichment of the oligodendrocyte-like cluster following mTOR inhibition, and (5) enrichment of cancer associated fibroblasts (CAFs) following IDH inhibition. These data show the spatiotemporal landscape of oligodendroglioma is associated with branched lineage differentiation that is modified by postoperative therapy, and suggest that therapy-dependent druggable dependencies may exist to treat oligodendrogliomas that are resistant to standard interventions.

EXTH-45. PRECLINICAL EVALUATION OF THE ANTIBODY-DRUG CONJUGATE TISOTUMAB VEDOTIN IN ADULT-TYPE DIFFUSE GLIOMAS

Abstract The gene F3, encoding Tissue Factor (TF), is expressed in many cancers and contributes to their malignancy. Our previous work showed that, among adult-type diffuse gliomas, IDH wild-type (IDHwt) glioblastomas (GBM) express more TF than IDH mutant (IDHmut) gliomas (PMID: 27664011, 30266764, 31222125). Tisotumab vedotin (TV), an anti-TF antibody conjugated to monomethyl auristatin E, is a therapeutic designed to target cells expressing TF. We therefore sought to determine the therapeutic potential of TV in IDHwt vs. IDHmut gliomas. TV was more active against cultured IDHwt GBM cells than IDHmut glioma cells (P<0.0001). This activity was increased by 55% with daily washout of the soluble TF secreted by IDHwt GBM cells. TV extended the survival of mice intracranially engrafted with IDHwt GBM by 34% (37.5 days vs. 28 days for IgG control, P=0.006), but not mice with IDHmut glioma (30.5 days, P=0.88). TV also reduced the growth of IDHwt GBM flank xenografts by 58% (P=0.045). Unconjugated tisotumab had no antitumor effect intracranially or in the flank. All 12 flank-engrafted mice treated with tisotumab, and 9/12 mice treated with TV, developed large hematomas in and around the flank xenografts, compared to 0/12 IgG-treated mice (P<0.001). Similarly, a semiquantitative analysis showed that intracerebral hemorrhage was more extensive in tisotumab/TV- treated GBM12 mice than in IgG-treated GBM12 mice (P=0.046). These data indicate that TV targets high TF-expressing IDHwt GBM, but not low TF-expressing IDHmut glioma, that its activity is predominately through the vedotin conjugate rather than inhibition of TF signaling, and that it may have some effect against TF-expressing IDHwt GBM, but intracerebral hemorrhage may be a concern.

TMIC-19. EXCESSIVE LIPID PRODUCTION SHAPES GLIOMA TUMOR MICROENVIRONMENT

Abstract Disrupted lipid metabolism is a characteristic of gliomas. This study utilizes an ultrastructural approach to characterize the prevalence and distribution of lipids within gliomas. This study made use of tissue from IDH1 wild type (IDH1-wt) glioblastoma (n=18) and IDH1 mutant (IDH1-mt) astrocytoma (n=12) tumors. We uncover a prevalent and intriguing surplus of lipids. The bulk of the lipids manifested as sizable cytoplasmic inclusions and extracellular deposits in the tumor microenvironment (TME); in some tumors the lipids were stored in the classical membraneless spheroidal lipid droplets (LDs). Frequently, lipids accumulated inside mitochondria, suggesting possible dysfunction of the beta-oxidation pathway. Additionally, the tumor vasculature has lipid deposits in their lumen and vessel walls; this lipid could have shifted in from the tumor microenvironment or have been produced by the vessel-invading tumor cells. Lipid excess in gliomas stems from disrupted beta-oxidation and dysfunctional oxidative phosphorylation pathways. The implications of this lipid-driven environment include structural support for the tumor cells and protection against immune responses, non-lipophilic drugs, and free radicals.