IDH-wildtype Gliomas Research Articles

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 >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 < 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.

Simultaneous quantification of perfusion, permeability, and leakage effects in brain gliomas using dynamic spin-and-gradient-echo echoplanar imaging MRI

ObjectiveTo determine the feasibility and biologic correlations of dynamic susceptibility contrast (DSC), dynamic contrast enhanced (DCE), and quantitative maps derived from contrast leakage effects obtained simultaneously in gliomas using dynamic spin-and-gradient-echo echoplanar imaging (dynamic SAGE-EPI) during a single contrast injection.Materials and methodsThirty-eight patients with enhancing brain gliomas were prospectively imaged with dynamic SAGE-EPI, which was processed to compute traditional DSC metrics (normalized relative cerebral blood flow [nrCBV], percentage of signal recovery [PSR]), DCE metrics (volume transfer constant [Ktrans], extravascular compartment [ve]), and leakage effect metrics: ΔR2,ss* (reflecting T2*-leakage effects), ΔR1,ss (reflecting T1-leakage effects), and the transverse relaxivity at tracer equilibrium (TRATE, reflecting the balance between ΔR2,ss* and ΔR1,ss). These metrics were compared between patient subgroups (treatment-naïve [TN] vs recurrent [R]) and biological features (IDH status, Ki67 expression).ResultsIn IDH wild-type gliomas (IDHwt—i.e., glioblastomas), previous exposure to treatment determined lower TRATE (p = 0.002), as well as higher PSR (p = 0.006), Ktrans (p = 0.17), ΔR1,ss (p = 0.035), ve (p = 0.006), and ADC (p = 0.016). In IDH-mutant gliomas (IDHm), previous treatment determined higher Ktrans and ΔR1,ss (p = 0.026). In TN-gliomas, dynamic SAGE-EPI metrics tended to be influenced by IDH status (p ranging 0.09–0.14). TRATE values above 142 mM−1s−1 were exclusively seen in TN-IDHwt, and, in TN-gliomas, this cutoff had 89% sensitivity and 80% specificity as a predictor of Ki67 > 10%.ConclusionsDynamic SAGE-EPI enables simultaneous quantification of brain tumor perfusion and permeability, as well as mapping of novel metrics related to cytoarchitecture (TRATE) and blood–brain barrier disruption (ΔR1,ss), with a single contrast injection.Clinical relevance statementSimultaneous DSC and DCE analysis with dynamic SAGE-EPI reduces scanning time and contrast dose, respectively alleviating concerns about imaging protocol length and gadolinium adverse effects and accumulation, while providing novel leakage effect metrics reflecting blood–brain barrier disruption and tumor tissue cytoarchitecture.Key Points• Traditionally, perfusion and permeability imaging for brain tumors requires two separate contrast injections and acquisitions.• Dynamic spin-and-gradient-echo echoplanar imaging enables simultaneous perfusion and permeability imaging.• Dynamic spin-and-gradient-echo echoplanar imaging provides new image contrasts reflecting blood–brain barrier disruption and cytoarchitecture characteristics.Graphical

Exploring the mechanism of 18F-fluorodopa uptake in recurrent high-grade gliomas: A comprehensive histomolecular-positron emission tomography analysis.

Dihydroxy-6-[18F]fluoro-L-phenylalanine (18F-FDOPA) positron emission tomography (PET) is a valuable tool for managing high-grade gliomas (HGGs), but there is a lack of literature on its relationship with glioma subtypes since the 2021 reclassification of brain tumors. There is also debate surrounding the mechanism of 18F-FDOPA uptake, particularly after chemoradiation therapy. This study aimed to investigate the correlation between 18F-FDOPA uptake and histomolecular characteristics, particularly L-amino acid transporter 1 (LAT1) expression, in recurrent gliomas, and examine their impact on survival in HGGs. Thirty-nine patients with recurrent HGGs (14 isocitrate dehydrogenase [IDH]-mutant, 25 IDH-wildtype) who underwent a brain 18F-FDOPA PET/computed tomography (CT) or PET/magnetic resonance imaging (MRI) followed by surgical resection of the 18F-FDOPA-avid lesion within 6 months, were retrospectively reviewed. PET results were compared with histological examination and for SCL7A5/LAT1 immunostaining. The study also examined the relationship between PET parameters, LAT1 expression, and survival outcomes. Astrocytoma IDH-mutant G4 had higher 18F-FDOPA uptake than glioblastoma IDH-wildtype G4 (maximum tumor-to-normal brain ratio [TBRmax] 5 [3.4-9] vs. 3.8 [2.8-5.9], p = 0.02). IDH-mutant gliomas had higher LAT1 expression than IDH-wildtype gliomas (100 [14-273] vs. 15.5 [0-137], p < 0.05) as well as higher TBRmax (5 [2.4-9] vs. 3.8 [2.8-6], p < 0.05). In survival analysis, LAT1 score >100 was a predictor for longer progression-free survival in IDH-mutant HGGs. To our knowledge, our study is the first to suggest a link between LAT1 expression and IDH mutation status. We showed that higher TBRmax was associated with higher LAT1 expression and IDH mutation status. Further studies are needed to better understand the mechanisms underlying amino acid PET tracers uptake, especially in the post-radiation and chemotherapy settings.

Diagnostic accuracy of anti-3-[18F]-FACBC PET/MRI in gliomas

PurposeThe primary aim was to evaluate whether anti-3-[18F]FACBC PET combined with conventional MRI correlated better with histomolecular diagnosis (reference standard) than MRI alone in glioma diagnostics. The ability of anti-3-[18F]FACBC to differentiate between molecular and histopathological entities in gliomas was also evaluated.MethodsIn this prospective study, patients with suspected primary or recurrent gliomas were recruited from two sites in Norway and examined with PET/MRI prior to surgery. Anti-3-[18F]FACBC uptake (TBRpeak) was compared to histomolecular features in 36 patients. PET results were then added to clinical MRI readings (performed by two neuroradiologists, blinded for histomolecular results and PET data) to assess the predicted tumor characteristics with and without PET.ResultsHistomolecular analyses revealed two CNS WHO grade 1, nine grade 2, eight grade 3, and 17 grade 4 gliomas. All tumors were visible on MRI FLAIR. The sensitivity of contrast-enhanced MRI and anti-3-[18F]FACBC PET was 61% (95%CI [45, 77]) and 72% (95%CI [58, 87]), respectively, in the detection of gliomas. Median TBRpeak was 7.1 (range: 1.4–19.2) for PET positive tumors. All CNS WHO grade 1 pilocytic astrocytomas/gangliogliomas, grade 3 oligodendrogliomas, and grade 4 glioblastomas/astrocytomas were PET positive, while 25% of grade 2–3 astrocytomas and 56% of grade 2–3 oligodendrogliomas were PET positive. Generally, TBRpeak increased with malignancy grade for diffuse gliomas. A significant difference in PET uptake between CNS WHO grade 2 and 4 gliomas (p < 0.001) and between grade 3 and 4 gliomas (p = 0.002) was observed. Diffuse IDH wildtype gliomas had significantly higher TBRpeak compared to IDH1/2 mutated gliomas (p < 0.001). Adding anti-3-[18F]FACBC PET to MRI improved the accuracy of predicted glioma grades, types, and IDH status, and yielded 13.9 and 16.7 percentage point improvement in the overall diagnoses for both readers, respectively.ConclusionAnti-3-[18F]FACBC PET demonstrated high uptake in the majority of gliomas, especially in IDH wildtype gliomas, and improved the accuracy of preoperatively predicted glioma diagnoses.Clinical trial registration ClinicalTrials.gov ID: NCT04111588, URL: https://clinicaltrials.gov/study/NCT04111588

Proteomic landscape of primary and metastatic brain tumors for heterogeneity discovery.

Despite recent advancements in our understanding of driver gene mutations and heterogeneity within brain tumors, whether primary or metastatic (also known as secondary), our comprehension of proteomic changes remains inadequate. The aim of this study is to provide an informative source for brain tumor researches, and distinguish primary brain tumors and secondary brain tumors from extracranial origins based on proteomic analysis. We assembled the most frequent brain tumors as follows: gliomas from WHO grade 2 to 4, with IDH1 mutations and wildtypes; brain metastases (BrMs) originating from lung cancer (LC), breast cancer (BC), ovarian cancer (OC), and colorectal cancer (CC). A total of 29 tissue samples were analyzed by label free quantitative mass spectrometry-based proteomics. In total, 8165 protein groups were quantified, of which 4383 proteins were filtered at 50% valid intensity values for downstream analysis. Proteomic analysis of BrMs reveals conserved features shared among multiple origins. While proteomic heterogeneities were found for discriminating different grades of gliomas, as well as IDH1 mutant and wildtype gliomas. In addition, notable distinctions were observed at the pathway level between BrMs and gliomas. Specifically, BrMs exhibited characteristic pathways focused on proliferation and immunomodulation after colonizing the brain, whereas gliomas primarily engaged in invasion processes. We characterized an extensive proteomic landscape of BrMs and gliomas. These findings have promising implications for the development of targeted therapies for BrMs and gliomas.

P11.53.B MOLECULAR PROFILING AND ACTIONABLE MUTATIONS IN ADULT PATIENTS WITH IDH WILD TYPE GLIOMAS AND GLIONEURONAL AND NEURONAL TUMORS

Abstract BACKGROUND Next generation sequencing (NGS) assays enable the identification of genetic alterations that may guide a personalized treatment in glioma patients. MATERIAL AND METHODS From July 2021 to March 2023 a NGS analysis (Myriapod® NGS Cancer DNA and RNA panels) was performed in a large prospective cohort of adults with an histological diagnosis of IDH wild type glioma or circumscribed astrocytoma or glioneuronal and neuronal tumor at the Division of Neuro-Oncology, University and City of Health and Science Hospital, Turin, Italy. The aim was to identify potential actionable molecular alterations and provide targeted therapy at recurrence accordingly. RESULTS We collected tumor samples from 221 patients. NGS analysis was available in 211 patients (95.5%), while NGS was not feasible in 10 patients (4.5%). 188 patients out of 211 (89.1%) had an IDH wild type glioblastoma (GBM) (median age 61 years, range 24-84; F/M: 97/91), 6 patients (2.8%) had IDH wild type lower grade gliomas (LGG) (median age 63 years, range 43-78; F/M: 3/3), and 17 patients (8.1%) had circumscribed astrocytoma or glioneuronal and neuronal tumor (median age 37 years, range 18-67; F/M: 13/4). Overall, actionable molecular alterations were found in 13/211 patients (6.2%). In this regard, 7/211 patients (3.3%) had BRAF V600E mutation (3 IDH wild type GBM, 1 IDH wild type LGG, and 3 circumscribed/glioneuronal and neuronal tumor), 4/211 patients (1.9%) had FGFR3-TACC3 fusion (all GBM), 1/211 patients (0.5%) had PIK3CA mutation (GBM), and 1/211 patients (0.5%) had NTRK1 fusion (glioneuronal tumor). Other molecular mutations, including ALK, ERBB2, MET, KIT, KRAS, PDGFRA, and RET have not been detected in our cohort. Three patients with druggable mutations recurred after first-line therapy: 1 patient with BRAF mutated GBM, 1 patient with GBM harboring FGFR3-TACC3 fusion, and 1 patient with NTRK1 fusion positive glioneuronal tumor received specific BRAF/MEK inhibitor (dabrafenib/trametinib), FGFR inhibitor (pemigatinib), and NTRK inhibitor (larotrectinib), respectively. A remarkable activity of BRAF/MEK and NTRK inhibitors was observed (8 months and 5 months, respectively *), while FGFR inhibitor did not display a significant activity to control disease (9 weeks). * ongoing CONCLUSION The incidence of actionable molecular alterations in IDH wild type gliomas and glioneuronal and neuronal tumors was low. However, the possibility of using targeted therapies may lead to an important impact on the outcome.

P10.17.A GLUTAMINE METABOLISM AS A PROGNOSTIC FACTOR IN GBM AND OLIGODENDROGLIOMA

Abstract BACKGROUND Increased uptake of principal nutrients is one of the attributes of cancer cells. A recent study has shown that glutamine, but not glucose, consumption differed markedly between gliomas and adjacent healthy tissue. However, the prognostic role of glutaminolysis or glutaminolysis-related genes (GRGs) in gliomas remains unclear. Here we sought to determine if the GRG signature correlates with glioma patient survival and if it could identify patients with different outcomes. MATERIAL AND METHODS The RNA-seq and clinical data for The Cancer Genome Atlas (TCGA) was obtained from the GlioVis webpage. We compared the expression of the GRGs (GLUD1, GLUL, GLS1, GLS2, GOT1, GOT2, GPT, GPT2, SLC1A5, and SLC7A5) between 4 non-tumor, 220 IDH wild-type (IDHwt) and 389 IDH mutant (IDHmut) gliomas, of which 154 harbored 1p/19q codeletion. We further performed unsupervised hierarchical clustering for all glioma patients and compared survival between clusters. RESULTS Our results revealed that most GRGs were differentially expressed between non-tumor, IDHwt, and IDHmut gliomas. We found that gliomas grouped into 3 clusters based on the varying GRGs expression. Eighty-eight percent of glioblastomas (GBM, IDHwt) and 19% of all IDHmut tumors were characterized by low GRGs expression. These were denoted as cluster 1, and the remaining samples belonged to clusters 2 or 3, corresponding to intermediate and high GRGs expression groups, respectively. We performed survival analysis separately for IDHwt and IDHmut gliomas and found that cluster 1 patients had the worst prognosis, regardless of IDH status. We further separated IDHmut gliomas according to 1p/19q codeletion status. Oligodendroglioma (1p/19q codeleted) patients had a median survival of 33.2 months, 134.3 months, and 169.8 months in clusters 1, 2, and 3, respectively. No cluster-dependent differences in survival were seen for astrocytoma. For IDHwt (GBM) we found that patients from clusters 1 and 2 had survival of 14 months and 25 months, respectively. Due to the small number of deceased GBM patients in cluster 3, we could not estimate the median survival. The univariate analysis confirmed that cluster membership was an independent predictor of survival in oligodendroglioma (cluster 2 and 3 vs. 1, HR 0.14; 95% CI, 0.05-0.4; p=0.0005) and GBM (cluster 1 vs. 2 and 3, HR 3.05; 95% CI, 1.5-5.9; p=0.0012). CONCLUSION The GRG signature could define metabolic groups with distinct survivals in oligodendroglioma and GBM and constitute a new prognostic factor. We are planning further investigations to confirm our findings.

P12.05.B THE ROLE OF ONCO-METABOLITE IN THE IDH MUTANT GLIOBLASTOMA MICROENVIRONMENT

Abstract BACKGROUND Recently, we found that the reactive astrocytes in the IDH wild type glioblastoma contribute to anti-tumor immunity and support pro-oncogenic signaling. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients due to an onco-metabolite R-2-hydroxyglutarate using Next generation sequencing and human ex-vivo slice model. MATERIAL AND METHODS We purified and transcriptionally profiled astrocytes from 9 patients with confirmed IDH1-R132H mutation, by means of RNA-sequencing and the data were analyzed using the established pipelines. We also used spatial transcriptomics to evidently show the spatial distribution of astrocytes in IDH-mutated/wildtype glioma samples. We validated our findings using human organotypic slice model inoculated with IDH-mutant cell line or treated with oncometabolite 2-hydroxy glutarate. Additionally, LC-MS was further used to give us a chart of neurotransmitters due to altered microenvironment. RESULTS Our results from RNA sequencing showed a transcriptional transformation of reactive astrocytes within the microenvironment of IDH-mutated tumors compared to wildtype glioma by means of RNA-sequencing of purified astrocytes. And, using our established human neocortical GBM model inoculated with IDH mutant tumor and R-2HG treatment, we showed that we were able to activate inflammatory transcriptional programs in astrocytes, mediated by the presence of microglia. Further, by spatially mapping the transcriptomic profiles of purified microglia, we were able to confirm that microglia also demonstrate inflammatory activation in IDH mutated glioma. This inflammatory astrocyte transformation is associated with a loss of neurotransmitter homeostasis (disrupted levels of glutamate) in the treated sections, as has been previously reported in IDH mutated tumors. Additionally, R-H2G increased neuronal spiking rate in, pointing to potential excitotoxicity. CONCLUSION Our work provides a crucial contribution towards understanding the role of R-2HG in the IDH mutant glioma microenvironment and sheds light on the significant microenvironmental differences to IDH wild-type glioma.

OS04.4.A LONG- AND SHORT-TERM SURVIVORS AMONG ADULT PATIENTS WITH DIFFUSE WHO GRADE 2-3 GLIOMA: CLINICAL FEATURES, MOLECULAR RECLASSIFICATION AND DNA METHYLATION PATTERNS

Abstract BACKGROUND Diffuse gliomas show considerable heterogeneity in terms of survival. The WHO classifications of 2016 and 2021 improved prognostic stratification due to the definition of biologically and clinically homogeneous tumor entities. However, further prognostic models considering clinical and molecular factors are needed to balance survival benefit with adverse treatment-related long-term effects in therapeutic decisions. MATERIALS AND METHODS Patients with diffuse gliomas of WHO grades 2 and 3 diagnosed in 2000 - 2019 have been included. Long term survivors (LTS) were defined as patients with an overall survival (OS) of &amp;gt;10 years after radiological diagnosis, whereas short-term survivors (STS) had an OS of &amp;lt;1 year. Histological diagnosis was performed by a board-certified neuropathologist. DNA methylation analysis of formalin-fixed, paraffin-embedded tumor tissue was conducted using the Illumina EPIC 850k platform. The Heidelberg Methylation Classifier was applied to obtain methylation-based diagnoses. RESULTS Of 697 patients with WHO grade 2 and 3 glioma, 182 (26.1%) were LTS, and 42 (6.0%) were STS. LTS were younger than STS (median age: 36 vs. 59.5; p&amp;lt;0.001) and had a higher Karnofsky Performance Score at diagnosis (median: 90% vs. 80%; p&amp;lt;0.001). LTS more frequently presented with epileptic seizures (p&amp;lt;0.001), while more STS had motoric (p&amp;lt;0.001), sensory (p=0.021), and visual deficits (p=0.027) as well as aphasia (p&amp;lt;0.001) and behavioral changes (p=0.009). Histological diagnoses (including pre-2016) showed astrocytic histology in 117 (70.9% LTS, 29.1% STS), oligodendroglial in 61 (93.4% LTS, 6.6% STS), oligo-astroglial in 43 (97.7% LTS, 2.3% STS) and not otherwise specified in 3 (all STS; p&amp;lt;0.001). Molecular reclassification of pre-2016 diagnoses (n = 143) yielded divergent entities in 46 (32.2%) cases. Overall, integrated molecular diagnoses included astrocytoma (isocitrate dehydrogenase [IDH]-mutant] in 65 patients (95.4% LTS and 4.6% STS), oligodendroglioma (IDH-mutant, 1p/19q-codeleted) in 64 patients (98.4% LTS and 1.6% STS), IDH-wildtype gliomas in 24 patients (100% STS), anaplastic pilocytic astrocytoma in 2 STS, diffuse midline glioma (H3K27-altered) in 1 STS, and dysembryoplastic neuroepithelial tumor in 1 LTS, while not otherwise specified diagnoses were found in 4 patients (75% LTS, 25% STS; p&amp;lt;0.001). Unsupervised clustering based on DNA methylation profiling revealed two major clusters consisting of IDH-wildtype STS (cluster A) and IDH-mutant patients (cluster B; 94.6% LTS, 5.4% STS). In cluster B, one subcluster (n = 8) contained 3/4 IDH-mt STS. Further analyses of these cases including validation with publicly available DNA methylation data are ongoing. CONCLUSION LTS and STS show distinct clinical and molecular features. Further profiling of cases with divergent survival could provide more refined prognostic stratification.

P12.08.A CELL-EXTRINSIC D-2-HYDROXYGLUTARATE PROMOTES HYPERMETHYLATION AT LINEAGE-SPECIFIC REGULATORY REGIONS TO PREVENT MICROGLIAL ACTIVATION IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in diffuse gliomas. Their composition and transcriptional programs differ according to the IDH mutation status of glioma cells. The underlying mechanisms remain until now little known. MATERIAL AND METHODS We compared bulk DNA methylome (Methylation EPIC array) and transcriptome of TAMs (CD11b+ purified by magnetic-activated cell sorting) from 25 IDH-mutant and 11 IDH-wildtype gliomas. The contamination of the CD11b+ fraction by tumor cells was determined by the detection of IDH and/or TERT mutations using droplet digital PCR. Only samples with a purity of at least 95% were used for analyses. To determine experimentally direct effects of D-2-hydroxyglutarate (D-2-HG), which is the oncometabolite produced and realeased into the microenvironment by IDH-mutated glioma cells, we used primary cultures of human microglial cells obtained from glioma surgery or epilepsy surgery. We exposed microglia or not to (D-2-HG) during 14 days, and analyzed their DNA methylome and transcriptome. RESULTS TAMs from IDH-mutant gliomas compared with TAMs from IDH-wildtype gliomas exhibit decreased expression of pro-inflammatory pathways (mainly IFN-γ, IFN-α, and TNF-α signaling) and genes related to G2-M checkpoint, glycolysis and Epithelial-Mesenchymal Transition (EMT). We uncovered that TAMs from IDH-mutant gliomas exhibit significant hypermethylation at promoters and enhancers linked to decreased expression of genes involved in inflammatory responses. Notably, these hypermethylated regions were enriched for binding motifs of transcription factors controlling an environment-dependent transcriptional network in microglial cells (such as PU.1, AP-1, C/EBP and RUNX). These findings were confirmed by comparing methylome data of TAMs from IDH-mutant gliomas and methylome of microglia from normal brains available in the literature. A 14 days exposition to D-2-HG of human microglial cells in vitro partially recapitulates the observed methylation changes. Transcriptome analyses of these cells show that this oncometabolite dampens the IFN-α/γ pathways and attenuates the response to LPS while inducing a metabolic shift toward oxidative phosphorylation. CONCLUSION Our findings establish a link between an extrinsic cell-effect of D-2-HG and the hypermethylation of DNA of microglial cells at regulatory regions in IDH-mutant gliomas. Our observations support the concept that oncometabolites may disrupt directly the function of immune cells of the tumor microenvironment.

P02.18.B INTEGRATIVE OMICS ANALYSIS OF IDH1 MUTANT GLIOMA PATIENTS REVEALS ALTERATIONS IN BUTYRATE METABOLISM

Abstract BACKGROUND Mutations in isocitrate dehydrogenase (IDH) 1 or 2 define glioma classification and determine the biology of these tumors. Although IDH is an essential enzyme in the cellular metabolism, powerful genome-wide analyses focus mostly at the genetic and epigenetic levels, while differences between distinct glioma entities at the proteomics, metabolomics, and functional levels are still poorly understood. Here, we provide an integrative multi-omics analysis of glioma patients to reveal metabolomic vulnerabilities of gliomas stratified by IDH mutation. MATERIAL AND METHODS We performed a multi-omics analysis of two cohorts of glioma patients (n=28) stratified based on IDH mutation status. Molecular assessment included LC-MS analysis, EPIC DNA methylation arrays, RNA-seq/HTA array-based transcriptomics and label-free quantitative proteomics. In 13 patients 13C-glucose was injected prior to surgery and tumor and adjacent brain tissue was collected. Gut microbial profiling using 16S ribosomal RNA (rRNA) sequencing was performed in a separate cohort of 128 glioma patients and 24 healthy individuals. Additional validation was performed in isogenic glioma stem-like cultures with CRISPR knock-in of IDH mutation. RESULTS We show that several molecular layers collectively contribute to biological differences between IDH mutant and wild-type gliomas, with strongest differences observed at the DNA methylation level. IDH mutant gliomas present specific molecular features linked to tumor metabolism. We confirm previous findings on 2-HG, cysteine and de novo glutathione synthesis pathways, which are specifically altered in IDH mutant gliomas. We further identify butyrate metabolism as a novel pathway highly active in IDH mutant gliomas. Butyrate is a bacterial metabolite with multiple systemic effects that is normally synthesized in the gut. This is supported by our gut microbiota metagenomics analysis of glioma patients. Yet the detection of enzymes associated with butyrate metabolism in tumor tissue and cell lines suggests a differential/enhanced metabolic rewiring of the butyrate signaling in IDH mutant gliomas CONCLUSION Our data shows the importance of multiple molecular layers in shaping specific features of IDH mutant gliomas. We unravel an activated butyrate metabolism in IDH mutant gliomas that may underly a novel gut-brain axis in glioma patients.

JS05.6.A CONTINUOUS DYNAMIC MAPPING OF LANGUAGE FUNCTION AND COGNITIVE CONTROL AND SPATIAL CORRELATION TO WHITE MATTER TRACTS USING INTRAOPERATIVE MRI TRACTOGRAPHY

Abstract BACKGROUND Subcortical mapping of white matter tracts (WMTs) subserving complex processes as movement, language and cognition is critical in glioma surgery. Continuous dynamic mapping (CDM) is an indispensable tool for mapping of the corticospinal tract (CST). There is, however, no widespread method for CDM of more advanced brain functions. MATERIAL AND METHODS Patients undergoing awake craniotomy for diffuse gliomas at the Oslo University Hospital with the use of neuronavigation, diffusion tensor imaging (DTI) and CDM coupled with structured evaluation as well as intraoperative magnetic resonance imaging (iMRI) were included in a retrospective analysis. Subcortical CDM was conducted when resection became close to the arcuate fasciculus (AF) and the frontal aslant tract (FAT) with clinical testing using language paradigms and Stroop task. The current was delivered through a monopolar suction probe applying a modified Taniguchi technique with short train of pulses. The spatial relationship between the stimulation response and the examined tracts was determined by measuring the shortest distance between the resection cavity and the relevant white matter tracts (WMTs) using iMRI DTI. RESULTS Twenty IDH-mutant and eight IDH wild type gliomas operated 2021-2023 were included in the analysis. Subcortical CDM of the AF and the FAT proved to be amenable as both language function and the ability to perform Stroop test were affected when the stimulation site gradually became close (&amp;lt; 1 cm) to the examined tracts. Positive stimulation response was verified by repeat challenge. The stimulation intensity was thereafter reduced in a stepwise fashion when there was a consistent hesitation in the patient’s response. Resection was continued until the tumor border was reached and not beyond the threshold of 3mA. The threshold of stimulation intensity correlated to the minimal distance from the resection cavity to the delineated WMTs. We did not observe any seizure activity induced by CDM. CONCLUSION We have experienced subcortical CDM to be a safe and reliable method, where distance to identified WMT appear to be correlated to the applied stimulation intensity.

P19.08.B INCREASED CORTICAL EXCITABILITY TO TRANSCRANIAL MAGNETIC STIMULATION CONTRIBUTE TO EPILEPTOGENESIS IN IDH1-MUTANT GLIOMAS

Abstract BACKGROUND In-vitro studies indicate that gliomas with a mutation of the isocitrate dehydrogenase (IDH) increase neuronal excitability of the peritumoral cortex contributing to epileptogenesis in these patients. However, in-vivo evidence is missing. This study evaluates the electric characteristics of the brain-tumor-interface (BTI) relative to the IDH mutation status. MATERIAL AND METHODS To investigate peritumoral cortical excitability (CE), we applied 5258 pulses of transcranial magnetic stimulation (TMS) at the BTI of IDH-mutant (IDH-mt) and IDH-wildtype (IDH-wt) glial tumors in 39 patients. CE was assessed by the resting motor threshold (RMT) and the synchronized electromyographic (EMG) activity (i.e., event-related spectral perturbation, ERSP) after TMS. ERSP values were related to the IDH status, tumor grading, antiepileptic drug intake (AED) and the spatial relationship to the tumor borders. RESULTS There was not significant group difference in RMT. TMS to the BTI triggered an EMG synchronization decreasing linearly with the distance to the functional hotspot. In contrast, IDH-mt gliomas demonstrated an increased cortical output of the peritumoral brain tissue compared to IDH-wt gliomas. This effect was not attributable to AED intake or other histological and molecular characteristics. Notably, cortical hyperexcitability was detectable well beyond the tumor border. CONCLUSION s: This study provides in-vivo evidence of cortical hyperexcitability at the BTI of IDH-mt gliomas which could contribute to the epileptogenesis in these patients.

Non-Invasive Assessment of Isocitrate Dehydrogenase-Mutant Gliomas Using Optimized Proton Magnetic Resonance Spectroscopy on a Routine Clinical 3-Tesla MRI.

The isocitrate dehydrogenase (IDH) mutation has become one of the most important prognostic biomarkers in glioma management, indicating better treatment response and prognosis. IDH mutations confer neomorphic activity leading to the conversion of alpha-ketoglutarate (α-KG) to 2-hydroxyglutarate (2HG). The purpose of this study was to investigate the clinical potential of proton MR spectroscopy (1H-MRS) in identifying IDH-mutant gliomas by detecting characteristic resonances of 2HG and its complex interplay with other clinically relevant metabolites. Thirty-two patients with suspected infiltrative glioma underwent a single-voxel (SVS, n = 17) and/or single-slice-multivoxel (1H-MRSI, n = 15) proton MR spectroscopy (1H-MRS) sequence with an optimized echo-time (97 ms) on 3T-MRI. Spectroscopy data were analyzed using the linear combination (LC) model. Cramér-Rao lower bound (CRLB) values of <40% were considered acceptable for detecting 2HG and <20% for other metabolites. Immunohistochemical analyses for determining IDH mutational status were subsequently performed from resected tumor specimens and findings were compared with the results from spectral data. Mann-Whitney and chi-squared tests were performed to ascertain differences in metabolite levels between IDH-mutant and IDH-wild-type gliomas. Receiver operating characteristic (ROC) curve analyses were also performed. Data from eight cases were excluded due to poor spectral quality or non-tumor-related etiology, and final data analyses were performed from 24 cases. Of these cases, 9/12 (75%) were correctly identified as IDH-mutant or IDH-wildtype gliomas through SVS and 10/12 (83%) through 1H-MRSI with an overall concordance rate of 79% (19/24). The sensitivity, specificity, positive predictive value, and negative predictive value were 80%, 77%, 86%, and 70%, respectively. The metabolite 2HG was found to be significant in predicting IDH-mutant gliomas through the chi-squared test (p < 0.01). The IDH-mutant gliomas also had a significantly higher NAA/Cr ratio (1.20 ± 0.09 vs. 0.75 ± 0.12 p = 0.016) and lower Glx/Cr ratio (0.86 ± 0.078 vs. 1.88 ± 0.66; p = 0.029) than those with IDH wild-type gliomas. The areas under the ROC curves for NAA/Cr and Glx/Cr were 0.808 and 0.786, respectively. Noninvasive optimized 1H-MRS may be useful in predicting IDH mutational status and 2HG may serve as a valuable diagnostic and prognostic biomarker in patients with gliomas.

Astrocytoma, IDH-Mutant

Astrocytoma, IDH-mutant is defined as infiltrative diffuse glioma harboring IDH1/2 mutation without accompanying 1p/19q codeletion in the current diagnostic system based on the 5th edition of the World Health Organization Classification of Tumors of the Central Nervous System. This revision delineates this neoplasm as a molecularly and clinically relevant tumor type. The evidence for the clinical management of patients with glioma has been largely established based on the results of clinical studies using the diagnostic criteria before the molecular classification. Many clinical studies investigated astrocytoma, IDH-mutant in combination with IDH-wildtype gliomas or oligodendrogliomas. The aim of the present study was to discuss the optimal management of astrocytoma, IDH-mutant based on the growing number of recent clinical studies incorporating molecular analyses. Particularly, the significance of the extent of surgical removal has increased after the definition of this tumor type in comparison with other types of gliomas.

The prognostic power of [11C]methionine PET in IDH-wildtype diffuse gliomas with lower-grade histological features: venturing beyond WHO classification.

IDH-wildtype (IDH-wt) diffuse gliomas with histological features of lower-grade gliomas (LGGs) are rare and heterogeneous primary brain tumours. [11C]Methionine (MET) positron emission tomography (PET) is commonly used to evaluate glial neoplasms at diagnosis. The present study aimed to assess the prognostic value of MET PET in newly diagnosed, treatment naïve IDH-wt gliomas with histological features of LGGs. Patients with a histological diagnosis of IDH-wt LGG who underwent preoperative (< 100days) MET PET/CT and surgery were retrospectively included. Qualitative and semi-quantitative analyses of MET PET images were performed. Progression-free survival (PFS) and overall survival (OS) were analysed by Kaplan-Meier curves. Cox proportional-hazards regression was used to test the association of imaging and clinical data to PFS and OS. We included 48 patients (M:F = 25:23; median age 55). 39 lesions were positive and 9 negative at MET PET. Positive MET PET was significantly associated with shorter median PFS (15.7months vs. not reached, p = 0.0146) and OS time (32.6months vs. not reached, p = 0.0253). Incomplete surgical resection and higher TBRmean values were independent predictors of shorter PFS on multivariate analysis (p < 0.001 for both). Higher tumour grade and incomplete surgical resection were independent predictors of OS at multivariate analysis (p = 0.027 and p = 0.01, respectively). MET PET is useful for the prognostic stratification of patients with IDH-wt glial neoplasms with histological LGGs features. Considering their huge biological heterogeneity, the combination of MET PET and molecular analyses may help to improve the prognostic accuracy in these diffuse gliomas subset and influence therapeutic choices accordingly.