Isocitrate Dehydrogenase Mutant Gliomas Research Articles

Isocitrate Dehydrogenase, Patient-Reported Outcomes, and Cognitive Functioning of Glioma Patients: a Systematic Review

Isocitrate dehydrogenase (IDH) mutation status has important prognostic implications in glioma patients, with IDH wild-type (IDH-WT) gliomas being associated with worse prognosis and shorter survival when compared with IDH mutant (IDH-mut) gliomas. Optimization of quality of life is a priority in the management of glioma patients. The goal of this systematic review was to identify studies that explored the association of IDH mutation status with patient-reported outcomes (PROs) and cognitive functioning of glioma patients. Studies that evaluated the association of IDH mutation status with PROs and/or cognitive functioning of glioma patients were identified from the Pubmed/MEDLINE, Clarivate analytics, and Google Scholar databases. Eight studies (7 journal articles and 2 conference abstracts) with a total of 658 low-grade glioma and high-grade glioma patients investigated the association of cognitive functioning and/or QoL with IDH status. IDH-WT status was associated with greater cognitive impairment relative to IDH-Mut status in three studies, while one study did not find the association between IDH status and perioperative cognitive functioning. One study reported worse postoperative cognitive functioning patients with IDH-WT vs. IDH-mut gliomas. In one study, IDH-WT status was linked to greater impairment on physical and communication functioning after surgery. IDH-WT gliomas are associated with greater cognitive burden than IDH-Mut tumors. The association of IDH status with QoL remains less clear. Assessment of IDH status should be considered when evaluating QoL and cognitive complaints of glioma patients. Further studies linking glioma molecular phenotypes with PROs and cognitive functioning are encouraged.

Preoperative Determination of Isocitrate Dehydrogenase Mutation in Gliomas Using Spectral Editing MRS: A Prospective Study.

The edited magnetic resonance spectroscopy (MRS) technique has not yet been formally evaluated for the in vivo detection of 2-hydroxyglutarate (2-HG) in patients with gliomas of various grades. To evaluate the diagnostic accuracy of edited MRS in the preoperative identification of the isocitrate dehydrogenase (IDH) mutation status in patients with gliomas. Prospective. Fifty-eight subjects (31 glioblastomas, 27 grade II and III gliomas). Mescher-Garwood (MEGA)-PRESS and routine clinical brain tumor MR sequences were used at 3T. Data were analyzed using an advanced method for accurate, robust, and efficient spectral fitting (AMARES) from jMRUI software. The amplitudes of the 2-HG, N-acetyl-aspartate (NAA), choline (Cho), and creatine/phosphocreatine (Cr) resonances were calculated with their associated Cramer-Rao lower bound (CRLB). The IDH1 R132H mutation status was assessed by immunohistochemistry for all patients. Patients with grades II and III gliomas with negative immunohistochemistry underwent DNA sequencing to further interrogate IDH mutation status. The differences in 2-HG amplitudes, 2-HG/NAA, 2-HG/Cho, and 2-HG/Cr between IDH-mutant and IDH-wildtype gliomas were assessed using Mann-Whitney U-tests. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic accuracy of each parameter. The 2-HG amplitudes, 2-HG/NAA, and 2-HG/Cho were higher for IDH-mutant gliomas than IDH-wildtype gliomas (P < 0.007). Using a CRLB threshold <30%, a 2-HG cutoff greater than 0 had a sensitivity of 80% (95% confidence interval [CI]: 52-96%) and a specificity of 81% (95% CI: 54-96%) in identifying IDH-mutant gliomas. In the subset of patients with grades II and III gliomas, the sensitivity was 80% (95% CI: 52-96%) and specificity was 100% (95% CI: 40-100%). Among 2-HG ratios, the highest AUC for the identification of IDH mutant status was achieved using the 2-HG/NAA (AUC = 0.8, 95% CI 0.67-.89). Preoperative edited MRS appears to be able to help identify IDH-mutant gliomas with high specificity. Level of Evidence 1 Technical Efficacy Stage 2 J. MAGN. RESON. IMAGING 2021;53:416-426.

3129 – ROLE OF MICROGLIA IN TP53/IDH1 MUTANT GLIOMAS DEVELOPMENT.

The origin of glial macrophages and their role in pathology is debated (Gutmann and Kettenmann 2019). In gliomas, paired TP53/IDH1 mutations are associated with lower immune infiltration and better outcomes (Cancer Genome Atlas Research Network et al. 2015) (Amankulor et al. 2017). In contrast, within isocitrate dehydrogenase 1 (IDH1) non-mutant gliomas, higher amounts of glioma associated macrophages (GAMs) in the tumor core correlated with longer patient survival (Zeiner et al. 2019). Mutant IDH1 produces the 2-hydroxyglutarate, inhibiting activation of the microglia in BV-2 model (Han et al. 2019). In addition, IDH1 mutation induces CpG island methylator phenotype and, together with TP53 mutation, changes the gene expression to repress the immune cells infiltration in a mouse model (Amankulor et al. 2017). However, the relevant models to investigate the effects of IDH1-R132H and p53-R248Q mutations on the activation of human microglia of different origin are not yet established. We aim to establish the primary TP53 or/and IDH1 mutant glioma culture in 3D co-aggregate with subpopulation of human adult CX3CR1/CCR2/CD68/PD-L1 monocytes from umbilical cord blood. Role of GAMs will be assessed by using inhibitors of CSF1R (e.g. GW2850), mTOR (Rapamycin), IGF-1/AKT, AMPK/SIRT, NFkB, and SIRPα-CD47 signalling axis. Single-cell RNAseq analysis will reveal the role of microglia in tumour development. We will focus on the pathology of glioma and its interaction with microglia/macrophages in the mammalian central nervous system pathology in order to facilitate the fundamental understanding of these processes, find targets for anti-cancer therapy, and facilitate drug development. Funding: Russian Science Foundation № 20-15-00378.

Metabolic Landscape of a Genetically Engineered Mouse Model of IDH1 Mutant Glioma.

Understanding the metabolic reprogramming of aggressive brain tumors has potential applications for therapeutics as well as imaging biomarkers. However, little is known about the nutrient requirements of isocitrate dehydrogenase 1 (IDH1) mutant gliomas. The IDH1 mutation involves the acquisition of a neomorphic enzymatic activity which generates D-2-hydroxyglutarate from α-ketoglutarate. In order to gain insight into the metabolism of these malignant brain tumors, we conducted metabolic profiling of the orthotopic tumor and the contralateral regions for the mouse model of IDH1 mutant glioma; as well as to examine the utilization of glucose and glutamine in supplying major metabolic pathways such as glycolysis and tricarboxylic acid (TCA). We also revealed that the main substrate of 2-hydroxyglutarate is glutamine in this model, and how this re-routing impairs its utilization in the TCA. Our 13C tracing analysis, along with hyperpolarized magnetic resonance experiments, revealed an active glycolytic pathway similar in both regions (tumor and contralateral) of the brain. Therefore, we describe the reprogramming of the central carbon metabolism associated with the IDH1 mutation in a genetically engineered mouse model which reflects the tumor biology encountered in glioma patients.

Large-Scale Drug Screening in Patient-Derived IDHmut Glioma Stem Cells Identifies Several Efficient Drugs among FDA-Approved Antineoplastic Agents.

The discovery of the isocitrate dehydrogenase (IDH) mutation in glioma led to a paradigm shift on how we see glioma biology. Difficulties in cultivating IDH mutant glioma stem cells (IDHmut GSCs) resulted in a paucity of preclinical models in IDHmut glioma, limiting the discovery of new effective chemotherapeutic agents. To fill this gap, we used six recently developed patient-derived IDHmut GSC lines and performed a large-scale drug screening with 147 Food and Drug Administration (FDA)-approved anticancer drugs. GSCs were subjected to the test compounds for 72 h in concentrations ranging from 0.0001 to 1 µM. Cell viability was assessed by CellTiterGlo and the induction of apoptosis by flow cytometry with Annexin V/propidium iodide staining. The initial screen was performed with two IDHmut GSC lines and identified seven drugs (bortezomib, carfilzomib, daunorubicin, doxorubicin, epirubicin, omacetaxine, plicamycin) with a substantial antiproliferative activity, as reflected by half maximal inhibitory concentrations (IC50) below 1 µM and maximum inhibitory effects (Emax) below 25%. These findings were validated in an additional four IDHmut GSC lines. The candidate drugs, of which plicamycin and omacetaxine are known to cross the blood brain barrier, were used for subsequent cell death analyses. A significant induction of apoptosis was observed at IC50 values of the respective drugs. In summary, we were able to identify seven FDA-approved drugs that should be further taken into clinical investigations for the treatment of IDHmut gliomas.

Diagnostic accuracy and potential covariates for machine learning to identify IDH mutations in glioma patients: evidence from a meta-analysis.

To assess the diagnostic accuracy of machine learning (ML) in predicting isocitrate dehydrogenase (IDH) mutations in patients with glioma and to identify potential covariates that could influence the diagnostic performance of ML. A systematic search of PubMed, Web of Science, and the Cochrane library up to 1 August 2019 was conducted to collect all the articles investigating the diagnostic performance of ML for prediction of IDH mutation in glioma. The search strategy combined synonyms for 'machine learning', 'glioma', and 'IDH'. Pooled sensitivity, specificity, and their 95% confidence intervals (CIs) were calculated, and the area under the receiver operating characteristic curve (AUC) was obtained. Nine original articles assessing a total of 996 patients with glioma were included. Among these studies, five divided the participants into training and validation sets, while the remaining four studies only had a training set. The AUC of ML for predicting IDH mutation in the training and validation sets was 93% (95% CI 91-95%) and 89% (95% CI 86-92%), respectively. The pooled sensitivity and specificity were, respectively, 87% (95% CI 82-91%) and 88% (95% CI 83-92%) in the training set and 87% (95% CI 76-93%) and 90% (95% CI 72-97%) in the validation set. In subgroup analyses in the training set, the combined use of clinical and imaging features with ML yielded higher sensitivity (90% vs. 83%) and specificity (90% vs. 82%) than the use of imaging features alone. In addition, ML performed better for high-grade gliomas than for low-grade gliomas, and ML that used conventional MRI sequences demonstrated higher specificity for predicting IDH mutation than ML using conventional and advanced MRI sequences. ML demonstrated an excellent diagnostic performance in predicting IDH mutation of glioma. Clinical information, MRI sequences, and glioma grade were the main factors influencing diagnostic specificity. • Machine learning demonstrated an excellent diagnostic performance for prediction of IDH mutation in glioma (the pooled sensitivity and specificity were 88% and 87%, respectively). • Machine learning that used conventional MRI sequences demonstrated higher specificity in predicting IDH mutation than that based on conventional and advanced MRI sequences (89% vs. 85%). • Integration of clinical and imaging features in machine learning yielded a higher sensitivity (90% vs. 83%) and specificity (90% vs. 82%) than that achieved by using imaging features alone.

A Potent Blood-Brain Barrier-Permeable Mutant IDH1 Inhibitor Suppresses the Growth of Glioblastoma with IDH1 Mutation in a Patient-Derived Orthotopic Xenograft Model.

Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of α-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood-brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.

The Association between Whole-Brain MR Spectroscopy and IDH Mutation Status in Gliomas.

Mutations in isocitrate dehydrogenase (IDH) have a direct effect on gliomagenesis. The purpose of this study is to quantify differences in brain metabolites due to IDH mutations. Magnetic Resonance Spectroscopic Imaging (MRSI) was performed in 35 patients with gliomas of different grade and varied IDH mutation status. Volumes of interest (VOIs) for active tumor (tVOI), peritumoral area (pVOI), and contralateral normal-appearing white matter (cVOI) were created. Metabolite ratios of Choline (Cho) to both N-acetylaspartate (NAA) and Creatine (Cr) were estimated. Ratios of Glutamate/Glutamine complex (Glx) and myoinositol (mIno) to Cr were also quantified. General linear models (GLMs) were used to estimate the effects of IDH mutation on metabolite measures, with age, gender, and tumor grade used as covariates. GLM analysis showed that maximum Cho/NAA and Cho/Cr in the tVOI were significantly (P < .05) higher in IDH mutant lesions as compared to wild-type. In the pVOI, mean Cho/Cr was found to be significantly different among IDH mutant and wild-type gliomas. Mean Cho/NAA (P = .306) and Cho/Cr (P = .292) within the tVOI were not significantly different. Ratios of Glx/Cr and mIno/Cr in any region showed no significant differences between IDH mutant and wild-type gliomas. No significant differences in metabolite ratios were seen in the cVOI between IDH mutants and wild-types. IDH mutation's effect in gliomas show an increase in Cho in the tumor and perilesional regions as compared to wild-type lesions but do not show widespread changes across the brain.

ACT-14 A FIRST-IN-HUMAN STUDY OF MUTANT IDH1 INHIBITOR DS-1001B IN PATIENTS WITH RECURRENT GLIOMAS

BACKGROUNDWHO grade II/III gliomas frequently harbor isocitrate dehydrogenase 1 (IDH1) mutations, resulting in intratumoral accumulation of oncometabolite D-2-hydroxyglutarate (D-2-HG) and subsequent clonal expansion. DS-1001b is an oral selective inhibitor of mutant IDH1 R132X that was designed to penetrate the blood-brain barrier.METHODSIn this first-in-human, multicenter, phase I study (NCT03030066), eligible patients (pts) with recurrent/progressive IDH1 mutant glioma received DS-1001b twice daily (bid), continuous. A modified continual reassessment method was used for dose escalation. RANO and RANO-LGG criteria were used to assess tumor response. Pts who planned to undergo salvage surgery after developing progressive disease (PD) and who provided informed consent received DS-1001b treatment until surgery. Tumor samples were also obtained from those pts to measure the free form of DS-1001b and D-2-HG levels.RESULTSBetween Jan 2017 and May 2019, DS-1001b (125–1400 mg bid) had administered for 47 pts, and 15 pts were continuing treatment. Maximum tolerated dose (MTD) was not reached. Most AEs were Gr 1–2. Gr 3 AEs were observed in 40% of pts. No Gr 4 or 5 AEs or serious drug-related AEs were reported. One dose limiting toxicity was Gr 3 white blood cell count decreased (1000 mg bid). Of 35 evaluable pts with contrast enhancing gliomas, one, five and 11 achieved complete response, partial response and stable disease (SD), respectively. Of evaluable 12 pts with contrast non-enhancing gliomas, four achieved minor response and eight achieved SD. Peak plasma concentration (Cmax) and area under the curve (AUC) increased dose-dependently.CONCLUSIONSDS-1001b was well tolerated up to 1400 mg bid with favorable brain distribution, and MTD was not reached. Recurrent/progressive IDH1 mutant glioma pts responded to treatment. Investigation is ongoing to determine the recommended Phase II dose. The latest data will be updated. Funding source: This study was funded by Daiichi Sankyo Co., Ltd.

Single-nucleus chromatin accessibility reveals intratumoral epigenetic heterogeneity in IDH1 mutant gliomas

The presence of genome-wide DNA hypermethylation is a hallmark of lower grade gliomas (LGG) with isocitrate dehydrogenase (IDH) mutations. Further molecular classification of IDH mutant gliomas is defined by the presence (IDHmut-codel) or absence (IDHmut-noncodel) of hemizygous codeletion of chromosome arms 1p and 19q. Despite the DNA hypermethylation seen in bulk tumors, intra-tumoral heterogeneity at the epigenetic level has not been thoroughly analyzed. To address this question, we performed the first epigenetic profiling of single cells in a cohort of 5 gliomas with IDH1 mutation using single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq). Using the Fluidigm HT IFC microfluidics platform, we generated chromatin accessibility maps from 336 individual nuclei, and identified variable promoter accessibility of non-coding RNAs in LGGs. Interestingly, local chromatin structures of several non-coding RNAs are significant factors that contribute to heterogeneity, and show increased promoter accessibility in IDHmut-noncodel samples. As an example for clinical significance of this result, we identify CYTOR as a poor prognosis factor in gliomas with IDH mutation. Open chromatin assay points to differential accessibility of non-coding RNAs as an important source of epigenetic heterogeneity within individual tumors and between molecular subgroups. Rare populations of nuclei that resemble either IDH mutant molecular group co-exist within IDHmut-noncodel and IDHmut-codel groups, and along with non-coding RNAs may be an important issue to consider for future studies, as they may help guide predict treatment response and relapse.A web-based explorer for the data is available at shiny.turcanlab.org.

Diffuse gliomas in patients aged 55 years or over: A suggestion for IDH mutation testing.

Diffuse gliomas are defined on the isocitrate dehydrogenase (IDH) gene (IDH) mutational mutational status. The most frequent IDH mutation is IDH1 R132H, which is detectable by immunohistochemistry; other IDH mutations are rare (10%). IDH mutant gliomas have better prognosis. Further, IDH wild-type low-grade (II/III) gliomas have clinical behaviors similar to those of glioblastoma (GBM) and it was suggested that they are submitted to similar post-surgical treatment. The incidence of IDH mutant gliomas (2%) and that of GBMs with non-canonical IDH mutations (< 1%) are very low in patients ≥ 55 years. For this reason, it was suggested that immunohistochemistry against IDH1 R132H is sufficient to classify GBM as IDH wild-type in this age group. However, no indication was provided for IDH mutational testing in low-grade diffuse gliomas. To address this issue, 273 diffuse gliomas were tested for IDH1 R132H immunohistochemistry. 2/4 diffuse astrocytomas (DAs), 4/9 anaplastic astrocytomas (AAs), 2/256 GBMs, and 4/4 oligodendrogliomas had positive staining. No other IDH mutations were found in immuno-negative low-grade cases by DNA sequencing. To validate our findings, we considered 311 diffuse gliomas in patients ≥ 55 years in The Cancer Genome Atlas database. Fifty-five out of 311 gliomas had IDH R132H mutations (9/16 DAs; 8/48 AAs; 3/211 GBMs; 35/36 oligodendrogliomas), one DA, and one oligodendroglioma had other IDH mutations. IDH mutant gliomas had significantly higher frequency of O-6-methylguanine-DNA methyltransferase promoter methylation (P = 0.0008) and longer overall survival (P < 0.0001). In conclusion, low-grade gliomas are a minor part of gliomas (117/584) in patients ≥ 55 years, albeit they represent most IDH mutant gliomas in this age group (64/69 cases). IDH non-canonical mutations can be found in immunonegative low-grade gliomas (2/54). In view of its significance for prognosis and therapeutic management, our results suggest that IDH mutational status is assessed in all diffuse gliomas in patients ≥ 55 years by immunohistochemistry, followed by IDH sequencing in low-grade immunonegative cases.

Measurement of 2-hydroxyglutarate enantiomers in serum by chiral gas chromatography-tandem mass spectrometry and its application as a biomarker for IDH mutant gliomas

(R)-2-hydroxyglutarate (R-2-hg) is a metabolite produced under physiologic conditions but also by tumors harboring isocitrate dehydrogenase (IDH) mutations. Detection is challenging as it must be distinguished from its enantiomer (S)-2-hydroxyglutarate (S-2-hg), which is also produced in the body but can increase under hypoxic conditions. A chiral gas chromatography-tandem mass spectrometry (GC–MS/MS) assay was developed, which separated enantiomers using a chiral column and quantified levels using a stable-isotope internal standard. The assay improves upon current methods by avoiding chiral derivatization and implementing a simplified sample extraction procedure. The assay was validated and serum 2-hg levels from healthy patients were measured, establishing a new, comprehensive reference range for normal levels of each enantiomer. Differences in basal levels were observed between races, but not sex. Age also correlated with S-2-hg levels, but not R-2-hg levels. Finally, serum levels of 2-hg enantiomers were measured in a pilot study of 11 patients with and without IDH mutant gliomas. An increase in R-2-hg levels was observed in 2/3 patients with actively growing IDH mutant gliomas. S-2-hg levels were increased in 4/11 patients, irrespective of IDH status. The results presented demonstrate the feasibility of a GC–MS/MS assay for measurement of 2-hg enantiomer levels for clinical use.

EXTH-45. THERAPEUTIC EFFICACY OF MUTANT ISOCITRATE DEHYDROGENASE 1 (IDH1) INHIBITOR SYC-435 WITH STANDARD THERAPY IN PATIENT-DERIVED IDH1 MUTANT GLIOMA XENOGRAFT MOUSE MODELS

Abstract Mutation in isocitrate dehydrogenase 1 (IDH1) occurs in &gt;70% of WHO grade II/III astrocytomas, oligodendrogliomas and secondary glioblastoma. The mutant enzyme catalyzes the reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid, leading to cancer initiation. In this study, we examined therapeutic efficacy of SYC-435 (1-hydroxypyridin-2-one), a newly developed mutant IDH1inhibitor, in IDH1 mutant gliomas. IDH1 R132H mutation (homozygous) was detected in BT142 anaplastic oligoastrocytoma (AOA) and R132C mutation (mutant allele frequency 39–50%) in V0914AOA by pyrosequencing. Suppression of cell growth by SYC-435 was observed with more sensitive of mutant over wild-type IDH1. Patient-derived orthotopic xenograft mouse models of BT142AOA and V0914AOA were treated with vehicle, SYC-435 (15 mg/kg/day x 28 days), temozolomide (50 mg/kg/day x 5 days)/fractionated radiation (2 Gy/day x 5 days) (standard therapy), and SYC-435/standard therapy starting 2 weeks after intracranial tumor implantation. Log rank analysis showed SYC-435 alone did not alter survival times. Although standard therapy significantly prolonged survival times in both models (P&lt; 0.0005), SYC-435/standard therapy further extended survival times (P&lt; 0.05) in V0914AOA and exhibited similar trend in BT142AOA. Elevation of 2-HG/α-KG ratio and methylation of H3K4/H3K9 in V0914AOA tumor compared to wild-type model was detected at the end of treatments. SYC-435 with/without standard therapy tended to reduce 2-HG/α-KG ratio and dramatically reduced methylation of H3K4/H3K9. RNA-seq analysis showed sirtuin signaling pathway, mitochondrial dysfunction and oxidative phosphorylation pathways with mitochondrial DNA (mtDNA) encoded molecules were highly affected by all treatments. However, mtDNA regulation did not correlate to survival benefits. In conclusion, SYC-435 possesses anti-tumor effects that are more sensitive in IDH1 mutant gliomas and generated strong synergistic activities with standard therapies for survival benefits with reduced methylation of H3K4/H3K9. Treatments significantly affected mtDNA but significance to survival benefits remains to be elucidated. Our data support the clinical testing of SYC-435 in patients with IDH1 mutant glioma.

CBMT-07. BIOMARKERS OF AGGRESSIVENESS IN IDH MUTANT GLIOMA

Abstract BACKGROUND Gliomas with isocitrate dehydrogenase (IDH) mutations in adults evolve from lower-grade gliomas to secondary glioblastomas (GBM), a fatal disease with fast progression. IDH mutation occurs early in tumorigenesis, and persistently contribute to the reprograming of glucose, lipid and amino acid metabolism. This offer a plethora of potential biomarkers of progression. However, because it is extremely difficult to detect the distribution and transfer of metabolites changing in every moment in a single cell, the involvement of metabolites produced by mutant IDH in malignant progression remains understudied. MATERIALS AND METHODS Raman imaging spectroscopy, which can image chemical bonds and concentration of molecules at submicron spatial resolution, enables detection of spatiotemporal changes of metabolomes in live cells. We developed the software called Biomolecular Component Analysis (BCAbox) to deconvolute the recorded raw Raman spectra, leading to detection of unique spectral features of different classes of biomolecules. RESULTS AND CONCLUSIONS We applied Raman imaging spectroscopy to GBM cell lines that were transfected with IDH1 mutant gene. Our results indicated that lipid metabolism has a unique profile in IDH1 mutant gliomas. Subsequent mass spectrometry analysis of extracted organelle revealed the exact classes of lipids altered in the IDH mutant glioma and suggested biomarkers unique to IDH1 mutant. We will report our validation studies of the biomarkers in patient-derived IDH mutant glioma cell lines and patients derived-orthotopic xenograft mouse models with different degrees of aggressiveness and in matched primary versus recurrent gliomas. The results of the present study may provide novel insights into the discovery of metabolic biomarkers for the malignant progression in IDH mutant gliomas.

Magnetic Resonance Spectroscopy for Identification of Isocitrate Dehydrogenase Mutation in Gliomas

Magnetic Resonance Spectroscopy for Identification of Isocitrate Dehydrogenase Mutation in Gliomas

Magnetic Resonance Spectrobiopsy for Prediction of Isocitrate Dehydrogenase Mutation in Glioma

Magnetic Resonance Spectrobiopsy for Prediction of Isocitrate Dehydrogenase Mutation in Glioma

Identification of IDH and TERTp mutation status using 1 H-MRS in 112 hemispheric diffuse gliomas.

There is a growing interest in noninvasively defining molecular subsets of hemispheric diffuse gliomas based on the isocitrate dehydrogenase (IDH) and telomerase reverse transcriptase gene promoter (TERTp) mutation status, which correspond to distinct tumor entities, and differ in demographics, natural history, treatment response, recurrence, and survival patterns. To investigate whether metabolite levels detected with short echo time (TE) proton MR spectroscopy (1 H-MRS) at 3T can be used for noninvasive molecular classification of IDH and TERTp mutation-based subsets of gliomas. Retrospective. In all, 112 hemispheric diffuse gliomas (70 males/42 females, mean age: 42.1 ± 13.9 years). Short-TE 1 H-MRS (repetition time (TR) = 2000 msec, TE = 30 msec, number of signal averages = 192) and routine clinical brain tumor MR protocols were acquired at 3T. 1 H-MRS data were quantified using LCModel software. TERTp and IDH1 or IDH2 (IDH1/2) mutations in the tissue were determined by either minisequencing or Sanger sequencing. Metabolic differences between IDH mutant and IDH wildtype gliomas were assessed by a Mann-Whitney U-test. A Kruskal-Wallis test followed by a Tukey-Kramer test was used to analyze metabolic differences between IDH and TERTp mutational molecular subsets of gliomas. A Spearman rank correlation coefficient was used to assess the correlations of metabolite intensities with the Ki-67 index. Furthermore, machine learning was employed to classify the IDH and TERTp mutational status of gliomas, and the accuracy, sensitivity, and specificity values were estimated. Short-TE 1 H-MRS classified the presence of an IDH mutation with 88.39% accuracy, 76.92% sensitivity, and 94.52% specificity, and a TERTp mutation within primary IDH wildtype gliomas with 92.59% accuracy, 83.33% sensitivity, and 95.24% specificity. Short-TE 1 H-MRS could be used to identify molecular subsets of hemispheric diffuse gliomas corresponding to IDH and TERTp mutations. 3 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;51:1799-1809.

P11.47 Generation, characterisation and drug screening of patient-derivedIDH1-mutated glioma cell lines

Abstract BACKGROUND Despite considerable scientific efforts, endogenous in vitro isocitrate dehydrogenase (IDH)-mutated glioma models remain scarce. Availability of these models is key to understanding underlying molecular mechanisms and vital for the development of new therapeutic interventions. We established and characterized a set of seven cell lines derived from IDH1-mutated gliomas and utilized them to investigate IDH-mutant glioma drug-response in vitro. MATERIAL AND METHODS Fresh tumor material was collected directly from the operating room, mechanically and enzymatically dissociated and cultured under serum-free culture conditions. IDH mutation status was verified at several passages with Sanger sequencing, as well as with whole exome sequencing. D-2-hydroxyglutarate (D2-HG) levels were determined with mass-spectrometry. Genome-wide methylation profiling was performed using the Infinium MethylationEpic BeadChip array. Cell viability was measured using the ATP-based CellTiter-Glo assay. Cell proliferation was determined through cell counting. Drug screens were performed with an FDA-approved anti-cancer drug set from the NIH as well as two IDH-mutant specific inhibitors. RESULTS Over the last decade our lab processed over 800 glioma samples from all WHO grades and IDH mutational status. Optimisation of culture conditions led to the establishment of seven IDH1-mutant glioma cell lines. The IDH1-mutation was present during all tested passages in these cell lines. Whole exome sequencing showed a high concordance between tumor and cell lines with regard to driver gene mutations. Similarly, copy-number changes based on genome-wide methylation data also show a close resemblance between the parental tumor and resulting cell lines. The Heidelberg methylation profiler classified each cell line and its parental tumor as IDH mutant glioma. When exposed to an IDH1-mutant specific inhibitor, all cell lines showed a concentration-dependent decrease of D2-HG levels. The inhibitors showed little to no effect on viability up to 10uM during 8 days, however, long term treatment up to 4 weeks revealed a decrease in cell doubling time in 2 of 6 cultures. Drug screen results either alone or in combination with an IDH1-mutant specific inhibitor identified several interesting candidates that are currently followed up in additional experiments. CONCLUSION We established a unique set of patient-derived IDH1-mutant glioma cell lines that closely resemble their respective tumors and can be used to identify new therapeutic strategies.

To be Wild or Mutant: Role of Isocitrate Dehydrogenase 1 (IDH1) and 2-Hydroxy Glutarate (2-HG) in Gliomagenesis and Treatment Outcome in Glioma.

Molecular and clinical research based on isocitrate dehydrogenase (IDH) mutations is much sought after in glioma research since a decade of its discovery in 2008. IDH enzyme normally catalyzes isocitrate to α-keto-glutarate (α-KG), but once the gene is mutated it produces an 'oncometabolite', 2-hydroxyglutarate (2-HG). 2-HG is proposed to inhibit α-KG-dependent dioxygenases and also blocks cellular differentiation. Here, we discuss the role of the IDH1 mutation in gliomagenesis. The review also focuses on the effect of 2-HG on glioma epigenetics, the cellular signaling involved in IDH1 mutant glioma cells and the therapeutic response seen in mutant IDH1(mIDH1) harboring glioma patients in comparison to the patients with wild-type IDH1. The review encompasses the debatable impacts of the mutation on immune microenvironment a propos of various mIDH1 inhibitors in practice or in trials. Recent studies revealing the relation of IDH mutation with the immune microenvironment and inflammatory status in untreated versus treated glioblastoma patients are highlighted with respect to prospective therapeutic targets. Also at the molecular level, the association of mIDH1/2-HG with the intracellular components such as mitochondria and other neighboring cells is discussed.

Abstract 5263: 1H and 13C MRS-based metabolic markers of IDH1 mutant glioma response to temozolomide therapy

Abstract Low-grade gliomas, driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene, are less aggressive than primary glioblastoma (GBM), but nonetheless always recur and ultimately lead to patient death. To improve patient survival, one therapeutic strategy is treatment with the alkylating chemotherapeutic agent Temozolomide (TMZ), previously reserved for the treatment of primary GBM. Though the treatment of IDH1 mutant patients with TMZ improves survival, early detection of treatment-associated effects remains challenging. The goal of this study was, therefore, to determine the value of magnetic resonance spectroscopy (MRS)-based biomarkers for detection of response to treatment. To this end, we examined the global metabolic alterations that occurred following TMZ treatment in a genetically engineered IDH1 mutant immortalized Normal Human Astrocyte (NHAIDHmut)-based cell model using 1H MRS combined with chemometrics and 13C MRS combined with labeling of cells with [1-13C]-glucose and [3-13C]-glutamine. Cells were treated either with the IC50 value of TMZ (100 μM; N=5), or with DMSO (0.2%; N=5) for 72 hours. Then, metabolites were extracted from cells using the dual-phase extraction method. 1H-MRS and proton-decoupled 13C-MRS spectra were acquired using a 500 MHz Bruker Avance spectrometer. 1H MRS data was subjected to multivariate analysis using SIMCA. Specific metabolites were also quantified using Mnova7, integrals normalized to TSP and to cell number and statistical significance of differences determined using unpaired Student’s t-test. Pathway enrichment analysis of dysregulated metabolites was performed using MetPA. principal component analysis score plot showed separation of TMZ-treated from DMSO-treated control cells. Further, improved separation between the groups was obtained by orthogonal-partial least squares discriminant analysis. Most significant metabolites contributing to class separation were identified using correlation ≥0.6 and variable importance in projection score ≥1. Glutamine, glutamate, 2-hydroxyglutarate (2-HG), pyruvate, succinate, glucose, phosphocholine, isoleucine, valine, lysine, phenylalanine, NAD(P)+ and AMP/ADP/ATP were observed to be significantly higher in TMZ-treated cells as compared to controls. Based on pathway impact score, tricarboxylic acid (TCA) cycle was identified to be the significantly altered pathway following treatment Further, there was a significant increases in glucose- and glutamine-derived glutamate and 2-HG, indicating an increase in flux to the TCA cycle following treatment. These findings demonstrate that IDH1 mutant glioma show a clear metabolomic fingerprint in response to TMZ therapy, which, combined with complementary hyperpolarized 13C MRS approaches, may help assess early response to TMZ therapy in mutant IDH1 glioma. Note: This abstract was not presented at the meeting. Citation Format: Elavarasan Subramani, Chloe Najac, Georgios Batsios, Pavithra Viswanath, Marina Radoul, Anne Marie Gillespie, Russell O. Pieper, Sabrina M. Ronen. 1H and 13C MRS-based metabolic markers of IDH1 mutant glioma response to temozolomide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5263.