Isocitrate Dehydrogenase 1 Or 2 Research Articles

TMIC-72. ALL-TRANS RETINOIC ACID INDUCES DURABLE TUMOR IMMUNITY IN IDH MUTANT GLIOMAS AND CIRCUMVENTS REPRESSION OF CRBP1-RETINOIC ACID AXIS

Abstract Diffuse gliomas are fatal, immunologically cold tumors characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) genes. IDH mutations have been shown to result in global genomic hypermethylation and drastically altered transcriptomic landscape. We and others have shown that IDH mutation is direct responsible for the immunological cold landscape observed in IDH mutated glioma. Using a combination of animal models and patient-derived glioma stem cells (GSCs), we identify retinoic acid signaling as a primary regulator of innate and adaptive immune activation in IDHm gliomas. Using the Sleeping Beauty (SB) orthotopic murine model of IDH glioma, which faithfully recapitulates the TME seen in patients, we show that intracellular accumulation of oncometabolite 2-HG leads to hypermethylation and transcriptional repression of Retinol Binding Protein-1 (RBP1), which encode CRBP1, a key chaperone protein required for intracellular all-trans-retinoic-acid biosynthesis in glial cells. Restoration retinoic acid signaling through the addition of all-trans retinoic acid (ATRA) resulted in spontaneous tumor regression and prolonged survival. ATRA administration is further correlated with increased CD8, NK, M1 macrophages and decreased Treg and MDSC populations within the TME. Single-cell sequencing of the TME shows increased type I interferon signaling, myeloid cell polarization, T cell activation, and TCR diversity. Using knockdown and overexpression techniques in patient-derived GSCs, we show that CRBP1 activity is critical for NK mediated immune surveillance and cytotoxicity through regulation of NKG2D ligand expression and immune cell migration through differential regulation of CXCL12 and CCL2. In summary, epigenetic silencing of RBP1 by 2-HG induced hypermethylation leads to a global immune dysregulation resulting in an immunologically cold landscape. Restoration of retinoic acid signaling is a promising immunotherapeutic approach for the treatment of IDHm gliomas.

Ferroptosis in Intrahepatic Cholangiocarcinoma: IDH1105GGT Single Nucleotide Polymorphism Is Associated With Its Activation and Better Prognosis.

ObjectivesIntrahepatic cholangiocarcinoma (ICC) has a dismal prognosis and often demonstrates an anti-apoptotic landscape, which is a key step to chemotherapy resistance. Isocitrate dehydrogenase 1 or 2 (IDH1-2)-mutated ICCs have been described and associated with better prognosis. Ferroptosis is a regulated iron-mediated cell death induced by glutathione peroxidase 4 (GPX4) inhibition, and may be triggered pharmacologically. GPX4 is overexpressed in aggressive cancers, while its expression is inhibited by IDH1R132C mutation in cell lines. We investigated tissue expression of ferroptosis activation markers in ICC and its correlation with clinical-pathological features and IDH1-2 status.Materials and MethodsWe enrolled 112 patients who underwent hepatic resection or diagnostic liver biopsy for ICC. Immunostaining for transferrin-receptor 1 and GPX4, and Pearls’ stain for iron deposits were performed to evaluate ferroptosis activation. Immunostaining for STAT3 was performed to study pro-inflammatory and anti-apoptotic landscape. Main IDH1-2 mutations were investigated in 90 cases by real-time polymerase chain reaction.ResultsGPX4 overexpression was seen in 79.5% of cases and related to poor histological prognostic factors (grading and perineural and vascular invasion; p < 0.005 for all) and worse prognosis (OS p = 0.03; DFS p = 0.01). STAT3 was expressed in 95.5% of cases, confirming the inflammation-related anti-apoptotic milieu in ICC, and directly related to GPX4 expression (p < 0.0001). A high STAT3 expression correlated to a worse prognosis (OS p = 0.02; DFS p = 0.001). Nearly 12% of cases showed IDH1105GGT single nucleotide polymorphism, which was never described in ICC up to now, and was related to lower tumor grade (p < 0.0001), longer overall survival (p = 0.04), and lower GPX4 levels (p = 0.001).ConclusionOur study demonstrates for the first time that in most inflammatory ICCs ferroptosis is not active, and its triggering is related to IDH1-2 status. This supports the possible therapeutic role of ferroptosis-inducer drugs in ICC patients, especially in drug-resistant cases.

Personalized patient care with aggressive hematological malignancies in non-responders to first-line treatment

ABSTRACT Introduction Whereas most patients with aggressive hematological malignancies can achieve disease remission after receiving current standard first-line chemotherapies, the prognosis of those with primary refractory disease remains poor. Conventional salvage chemotherapies are usually unsuccessful, but significant advances have been achieved in precision medicine for these patients in recent years. Areas covered This article presents a comprehensive review of current personalized care for patients with primary refractory aggressive hematological malignancies, focusing on acute leukemias and aggressive non-Hodgkin lymphomas. Expert opinions Salvage treatment for refractory acute myeloid leukemia (AML) should be personalized by the FMS-like tyrosine kinase 3 (FLT3) mutation status. Additionally, treatment for isocitrate dehydrogenase 1 or 2 (IDH1/2)-mutated refractory AML can be specified. Inotuzumab ozogamicin and blinatumomab are essential drugs for refractory B-cell acute lymphoblastic leukemia. These antibodies can be selected based on the disease status, such as measurable residual disease. For refractory diffuse large B-cell lymphoma, treatment options are different according to the cell-of-origin. Finally, accurate pathological phenotyping is an essential first step to optimize refractory peripheral T-cell lymphoma treatment. Although novel personalized approaches have improved the clinical outcomes of these patients, consolidation with hematopoietic stem cell transplantation has a vital role in most cases.

FSMP-07. CYSTATHIONINE-Γ-LYASE DRIVES ANTIOXIDANT DEFENSE IN CYSTEINE-RESTRICTED IDH1 MUTANT ASTROCYTOMAS

Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzymes are crucial for the generation of reducing potential, yet the impact of the mutation on the cellular antioxidant system is not understood. Here, we investigate how glutathione (GSH) levels are maintained in IDH1 mutant gliomas, despite an altered NADPH/NADP balance. We find that IDH1 mutant astrocytomas specifically upregulate cystathionine γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis. Genetic and chemical interference with CSE in patient-derived glioma cells carrying the endogenous IDH1 mutation, sensitized tumor cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with reduced GSH synthesis as shown by in vitro and in vivo serine tracing and led to delayed tumor growth in mice. Thus we show that IDH1 mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis to maintain the antioxidant defense, which uncovers a novel metabolic vulnerability in this dismal disease.

Cystathionine-γ-lyase drives antioxidant defense in cysteine-restricted IDH1-mutant astrocytomas.

BackgroundMutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1-mutant gliomas, despite an altered NADPH/NADP balance.MethodsProteomics, metabolomics, metabolic tracer studies, genetic silencing, and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice.ResultsWe find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1-mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice.Conclusions We show that IDH1-mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited.

Abstract 1917: The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma

Abstract Glioblastoma (GBM) is an incurable form of brain cancer with a median survival of ~15 months. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP) represents a significant clinical discovery as these patients have an enhanced survival, with a median survival of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2) which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases. This mutation occurs early in gliomagenesis and further results in aberrant DNA methylation and widespread transcriptional repression. Histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain (MBD) proteins, so we hypothesized that IDH mutant GBM exhibits enhanced reliance on HDAC activity, which is functionally significant to their cell proliferation and survival. Using a panel of 6 patient-derived cell lines grown in defined growth media, we show that G-CIMP is significantly more sensitive in vitro to the clinically approved pan histone deacetylase (HDAC) inhibitor panobinostat, with IDH mutant GBM cells exhibiting ~4 and ~10 fold lower IC/EC50 values in flow cytometric cell viability and apoptosis assays compared to IDH wild type (WT) GBM cells, respectively. For IDH mutant GBM, the average IC50 value was 13.5 nM for cell viability assays and the average EC50 value was 4.8 nM for apoptosis assays. Induction of cleaved caspase 3, a marker of apoptosis, was observed only in IDH mutant GBM cells when exposed to 10 nM panobinostat over 5 days. Analysis of proliferation via BrdU incorporation assays show that treatment with 10 nM panobinastat for 48 hours results in a 90% reduction in proliferation of IDH mutant GBM compared to only 25% reduction in IDH WT GBM cells. Molecular analysis via western blot shows that various acetylated chromatin marks, i.e. H3K14/18/27Ac, are preferentially upregulated in our IDH mutant GBM cell lines when exposed to 10 nM panobinostat for 48 hours. This was also supported by ELISA data showing that IDH mutant GBM cells have ~2 fold increase in histone H3 acetylation compared to IDH WT cells in response to 10 nM and 50 nM panobinostat. These data led us to perform a HDAC activity assay with nuclear extracts from our IDH WT and mutant GBM cells, but this assay showed no significant difference in basal HDAC activity. Overall, our results support a hypothesis where IDH mutant GBMs are more sensitive to panobinostat via preferential uptake of the drug. These data ultimately suggest that G-CIMP tumor cells display a profound sensitivity to the cytotoxic and antiproliferative effects mediated by exposure to the HDAC inhibitor panobinostat. These studies provide a strong foundation for future preclinical work evaluating the use of HDAC inhibitors as a personalized cancer therapy to treat G-CIMP. Citation Format: Thomas K. Sears, Kevin D. Woolard. The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1917.

Correlation between IDH, ATRX, and TERT promoter mutations in glioma.

According to the 2016 World Health Organization (WHO) classification of central nervous system tumors, diffuse astrocytic and oligodendroglial tumors are differentiated by the presence of isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation and the combined loss of the short arm of chromosome 1 and the long arm of chromosome 19 (1p/19q co-deletion). IDH-mutant astrocytoma often has p53 and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutation, showing the alternative lengthening of telomeres (ALT) phenotype, while IDH-mutant and 1p/19q-co-deleted oligodendroglioma often have wild-type p53 and telomerase reverse transcriptase (TERT) promoter mutation, showing telomerase activation. This study analyzed IDH, ATRX, and TERT promoter mutations, and the correlation between them. Immortalized cells overcome the telomere-related crisis by activating telomerase or ALT. In glioma, telomerase is mainly activated by TERT promoter mutation, while ALT is usually associated with ATRX mutation. Although the mechanism of how ATRX mutation induces ALT remains unclear, ATRX loss alone is believed to be insufficient to induce ALT. Treatments targeting telomere maintenance are promising.

Abstract 3846: Targeting epigenetic dysregulation in isocitrate dehydrogenase mutant glioblastoma

Abstract Glioblastoma (GBM) is an incurable form of brain cancer with a median survival time (MdST) of ~15 months after treatment with chemoradiotherapy. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP), represents a significant clinical discovery, as these patients have an enhanced survival, with a MdST of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2), which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent demethylases. This results in aberrant DNA methylation, transcriptional repression, and may drive tumorigenesis. Here, we present data showing that G-CIMP is significantly more sensitive in vitro to inhibitors of the two arms of epigenetic regulation of transcription: the transcriptional activation arm and the transcriptional repression arm. Drug targets include the Bromodomain and Extraterminal (BET) and Histone Deacetylase (HDAC) proteins, involved in epigenetic transcriptional activation of oncogenes and repression of tumor suppressors, respectively. G-CIMP cells show a profound loss of cell viability at submicromolar concentrations using the small molecule inhibitors JQ1 and Panobinostat, which target BET and HDAC proteins, respectively. Western blot analysis indicates that BET inhibition elicits preferential loss of histone phosphorylation and acetylation in G-CIMP, notably serine 10 of Histone 3 and lysine 27 of Histone 3 (H3K27Ac), respectively. Both epigenetic marks are associated with positively regulating transcription. H3K27Ac is a superenhancer with known functions in activating oncogene transcription. These data ultimately suggest that G-CIMP tumor cells are reliant on established epigenetic transcriptional regulation to maintain cell viability. The global methylation status of these tumors may make them particularly sensitive to pharmacologic disruption of these epigenetic pathways. These studies provide a strong foundation for future preclinical work evaluating the prospective treatment of G-CIMP with combinatorial therapy using BET and/or HDAC inhibitors. Citation Format: Thomas K. Sears, Sandipan Datta, Gino Cortopassi, James Angelastro, Kevin Woolard. Targeting epigenetic dysregulation in isocitrate dehydrogenase mutant glioblastoma [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 3846.

Evolving Treatment Strategies for Elderly Leukemia Patients with IDH Mutations.

Acute myeloid leukemia (AML) is a debilitating and life-threatening condition, especially for elderly patients who account for over 50% of diagnoses. For over four decades, standard induction therapy with intensive cytotoxic chemotherapy for AML had remained unchanged. However, for most patients, standard therapy continues to have its shortcomings, especially for elderly patients who may not be able to tolerate the complications from intensive cytotoxic chemotherapy. New research into the development of targeted and alternative therapies has led to a new era in AML therapy. For the nearly 20% of diagnoses harboring a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2), potential treatment options have undergone a paradigm shift away from intensive cytotoxic chemotherapy and towards targeted therapy alone or in combination with lower intensity chemotherapy. The first FDA approved IDH2 inhibitor was enasidenib in 2017. In addition, IDH1 inhibitors are in ongoing clinical studies, and the oral BCL-2 inhibitor venetoclax shows preliminary efficacy in this subset of patients. These new tools aim to improve outcomes and change the treatment paradigm for elderly patients with IDH mutant AML. However, the challenge of how to best incorporate these agents into standard practice remains.

Non–small cell lung cancers with isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are important metabolic enzymes that convert isocitrate to α-ketoglutarate. IDH1/2 mutations are associated with the development of multiple malignancies. In this study, we examine the prevalence and features of non-small cell lung cancers (NSCLC) with IDH1/2 mutations. From May 2013 to March 2017, 800 lung cancer samples were successfully sequenced for somatic mutations on the Ion Torrent PGM with the 50-gene AmpliSeq Cancer Hotspot Panel v2 on the Ion Torrent PGM (318 chip). Variants were identified using the Ion Torrent Variant Caller Plugin and reference genome hg19. Golden Helix's SVS software was used for variant annotation and prediction of significance. Nine samples (1.1%) from 8 patients harbored an IDH1 (3 p.R132C and 2 p.R132L) or IDH2 mutation (p.R140W, p.R172S, and p.R172T). All patients' tumors had adenocarcinoma histology, and all patients had a smoking history. Eighty-eight percent of patients' tumors had a coexisting KRAS mutation and 6 of 8 were diagnosed at greater than 70 years of age. Five patients presented with stage IV disease and 3 with stage I. After comparison to a cohort of KRAS-mutated NSCLC with a history of smoking, IDH-mutated cases were significantly older but presented with similar rates of advanced-stage disease and sex distribution. Additional studies are needed to understand the role of IDH1/2 mutations in the development of NSCLC, but such patients who have poor prognostic indicators (KRAS mutation and advanced stage at presentation) may benefit from IDH1/2-directed therapies.

Outcome of AML Patients with IDH1 or IDH2 Mutations from Diagnosis and Refractory/Relapse Phase of the Disease in Routine Practice

Abstract Somatic mutations of isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) genes are found in 7-14% and 8-19% of AML. IDH1R132, IDH2R140 and IDH2R172mutations are most frequent in cytogenetically normal AML (25-30% of cases) and significantly associated with NPM1 mutations (except for IDH2R172). Their prognostic significance depends on mutational context (NPM1 and FLT3-ITD status) and on type of mutation: IDH1R132 has a possible adverse effect, IDH2R140 is associated with a favorable effect while the IDH2R172 effect remains controversial (Green C, Blood 2011; Papaemmanuil E, NEJM 2016). IDH1/2 mutations induce a neomorphic enzyme that overproduces the 2-hydroxyglutarate oncometabolite and thus have emerged as promising therapeutic targets. Indeed, AG-120 (IDH1 inhibitor) and AG-221 (IDH2 inhibitor) have shown encouraging activity in phase I trials including Rel/Ref AML pts though median OS are not yet reported. The prognosis of IDH mutated pts at this phase of the disease is not well described in routine practice outside clinical trials. The primary objective of this study was to describe characteristics and outcome of AML pts with IDH1/2 mutations treated in routine practice by intensive chemotherapy from both diagnosis and Rel/Ref phase of the disease. This study included 1603 pts admitted at the Toulouse University Hospital and/or registered in the Oncomip regional Network from January, 1st, 2000 to December, 31st, 2014. This database included all cases of AML pts treated by intensive chemotherapy since 2000 then, starting from 2007, all consecutive patients whatever their treatment (Bories P, Am J Hematol 2014; Bertoli S, Blood 2013). Molecular analyses were performed at diagnosis or retrospectively from stored samples. Only first relapses after standard intensive chemotherapy were considered for this analysis and refractory disease was defined as failure following one course of induction chemotherapy including or not a second course for patients with more than 5% bone marrow blasts at day 15. Treatment distributions were as follows: 984 pts received intensive chemotherapy, 224 azacitidine and 312 best supportive care. Mutational status of IDH1 and IDH2 was available for 465 pts of whom, 422 received intensive chemotherapy as first line therapy, 21 azacitidine and 20 BSC. The study focalized on pts treated by intensive chemotherapy: 349 IDH1/2wt (82%), 32 IDH1R132 (7.5%), 31 IDH2R140 (7.3%) and 11 IDH2R172 (2.6%). IDH2R140 (59y, IQR, 54-66.5) and IDH2R172 (60y, 42.5-64) pts were older than IDH1R132 (53y, 43.8-59.3) and IDHwt (52y, 39-62) pts. IDH2R172 pts had lower WBC count (2.1 G/l, 1.35-4.6) as compared to IDHwt (18 G/l, 4.3-66.9), IDH1R132 (14.7 G/l, 2.1-47.5) and IDH2R140 (21.5 G/l, 4.1-43). De novo AML was found in 85%, 94%, 74% and 73% of IDHwt, IDH1R132, IDH2R140 and IDH2R172 pts, respectively. Only 3 IDH1R132 and 3 IDH2R140 pts had unfavorable karyotype whereas 2 IDH2R140 pts had CBF-AML. Complete response was achieved in 80%, 91%, 74% and 100% of IDHwt, IDH1R132, IDH2R140 and IDH2R172 pts, respectively. Median overall survival (OS) from diagnosis was 23.6, 20.9, 35.8 and 41.1 months in IDHwt, IDH1R132, IDH2R140 and IDH2R172 pts, respectively. 185 Rel/Ref patients with IDH1/2-defined mutational status received salvage therapy with intensive chemotherapy (68%); azacitidine (18%) or other treatments (14%): 144 IDHwt (36 Ref/108 Rel), 18 IDH1R132 (3 Ref/15 Rel) and 23 IDH2R140/R172 (8 Ref/15 Rel). Complete response was achieved in 62 IDHwt (43%), 9 IDH1R132 (50%) and 12 IDH2R140/R172 (52%) pts, respectively. Median OS and 3-year OS from failure or relapse were: IDHwt: 7.6 months/19%; IDH1R132: 5.9 months/8% and IDH2R140/R172: 11 months/23%. Observational data should be considered complementary to that provided by randomized clinical trials. They provide data collected in a non-selected general population, while participants in clinical trials are generally under very restrictive eligibility criteria. In refractory or relapsed patients who received salvage therapy, substantial differences may be observed according to the subtype of mutations, IDH1R132 having the poorest prognosis. IDH2R172 appears to confer a more favorable outcome (3-year OS from diagnosis: 61%). Disclosures Tavitian: Novartis: Membership on an entity's Board of Directors or advisory committees. Huguet:Pfizer, Novartis, BMS, Ariad, Jazz, Amgen: Membership on an entity's Board of Directors or advisory committees. Récher:Celgene, Sunesis, Amgen, Novartis, Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding.

IDH1/2 gene hotspot mutations in central nervous system tumours: analysis of 922 Chinese patients

Mutations of isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) genes have been identified as early molecular events in the development of astrocytomas and oligodendrogliomas. Data regarding the status and prevalence of IDH1/2 mutations in Chinese patients are limited. Herein we report our data from West China Hospital, a major Chinese medical centre. IDH1(R132H) mutation was analysed by immunohistochemistry with the mutation-specific IDH1(R132H) antibody in 1011 patients, including 922 central nervous system (CNS) tumours and 89 non-neoplastic CNS lesions, and PCR-based direct sequencing of IDH1/2 gene mutation in 570 of these samples. Correlation with clinicopathological features and immunohistochemical expression of p53, EGFR, PTEN and Ki-67 was examined. Our data showed that IDH1/2 mutation was present in oligodendrogliomas, anaplastic oligodendrogliomas, diffuse or anaplastic astrocytomas, and glioblastomas, with decreasing frequency, but not in other types of CNS tumours or non-neoplastic lesions examined. IDH1(R132) mutation was most frequent in oligodendrogliomas (57/62, 91.9%), with IDH1(R132H) mutation as the most frequent mutation form. Only one case for each of the rare mutations (R132C, R132G, R132L, and R132S) was identified in the 570 samples analysed by sequencing. Younger age, low expression of p53 and low Ki-67 index were significantly correlated with IDH1 mutation status (p=0.000). All tumours with IDH1(R132) mutations were supratentorial, with frontal lobe as the most frequent site for IDH-mutated gliomas. Only three IDH2(R172) mutation cases were detected in this series. Univariate survival analysis in 459 glioma patients with diffusely infiltrating gliomas showed that IDH1 mutations as well as the more classical prognosticators (age, WHO grade, p53 and Ki-67 index) were of prognostic significance. Multivariate analysis by Cox proportional hazard regression model demonstrated that lack of IDH1 mutation was an independent prognostic factor for both progression-free survival [relative risk (RR)=2.450, 95% confidence interval (CI)=1.351-4.444] and disease-specific survival (RR=2.489, 95%CI=1.155-5.363).

IDH1/2 Mutation and MGMT Promoter Methylation – the Relevant Survival Predictors in Czech Patients with Brain Gliomas

Gliomas are a heterogeneous group of tumours varying in prognosis, treatment approach, and overall survival. Recently, novel markers have been identified which are linked to patient prognosis and therapeutic response. Especially the mutation of the enzyme isocitrate dehydrogenase 1 or 2 (IDH1/2) gene and the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status seem to be the most important predictors of survival. From 2012 to 2015, 94 Czech patients with primary brain tumours were enrolled into the study. The IDH1/2 mutation was detected by denaturing capillary electrophoresis. The methylation status of the MGMT gene and other 46 genes was revealed by MS-MLPA. In all 94 patients, the clinical data were correlated with molecular markers by Kaplan-Meier analyses and Cox regression model. The MGMT promoter methylation status was established and compared to clinical data. In our study eight different probes were used to elucidate the MGMT methylation status; hypermethylation was proclaimed if four and more probes were positive. This 3 : 5 ratio was tested and confirmed by Kaplan-Meier and Cox analyses. The study confirmed the importance of the IDH1/2 mutation and hypermethylation of the MGMT gene promoter being present in tumour tissue. Both markers are independent positive survival predictors; in the Cox model the IDH hazard ratio was 0.10 and in the case of MGMT methylation it reached 0.32. The methylation analysis of the panel of additional 46 genes did not reveal any other significant epigenetic markers; none of the candidate genes have been confirmed in the Cox regression analyses as an independent prognostic factor.

Abstract SY43-01: Stereochemistry matters: L-2HG as a tumor response to hypoxia

Abstract Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3). Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that can influence gene expression and cellular differentiation. Citation Format: Craig B. Thompson, Andrew M. Intlekofer, Raymond G. Dematteo, Sriram Venneti, Lydia W. S. Finley, Chao Lu, Ariën S. Rustenburg, Patrick B. Grinaway, John D. Chodera, Justin R. Cross, Alexander R. Judkins. Stereochemistry matters: L-2HG as a tumor response to hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY43-01. doi:10.1158/1538-7445.AM2015-SY43-01

Hypoxia Induces Production of L-2-Hydroxyglutarate

Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the "oncometabolite" D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of α-ketoglutarate (α-KG)-dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D-enantiomer or the L-enantiomer. Although cancer-associated IDH1/2 mutants produce D-2HG, biochemical studies have demonstrated that L-2HG also functions as a potent inhibitor of α-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of α-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage primarily by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3).

IDH-Mutation Is a Weak Predictor of Long-Term Survival in Glioblastoma Patients.

BackgroundA very small proportion of patients diagnosed with glioblastoma (GBM) survive more than 3 years. Isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations define a small subgroup of GBM patients with favourable prognosis. However, it remains controversial whether long-term survivors (LTS) are found among those IDH1/2 mutated patients.MethodsWe retrospectively analyzed 207 GBM patients followed at Lariboisière Hospital (Paris) between 2005 and 2010. Clinical parameters were obtained from medical records. Mutations of IDH1/2 were analyzed in these patients, by immunohistochemistry for the R132H mutation of IDH1 and by high-resolution melting-curve analysis, followed by Sanger sequencing for IDH1 and IDH2 exon 4 mutations. Mutation rates in LTS and non-LTS groups were compared by Chi square Pearson test.ResultsSeventeen patients with survival >3 years were identified (8.2% of the total series). The median overall survival in long-term survivors was 4.6 years. Subgroup analysis found that the median age at diagnosis was significantly higher for non long-term survivors (non-LTS) compared to LTS (60 versus 51 years, p <0.03). The difference in the rate of IDH mutation between non-LTS and LTS was statistically not significant (1.16% versus 5.9%, p = 0.144). Among LTS, 10 out of 16 tumors presented a methylation of MGMT promoter.ConclusionsThis study confirms that long-term survival in GBM patients is if at all only weakly correlated to IDH-mutation.

Leukemia-Associated Oncometabolite 2-Hydroxyglutarate Regulates Epigenetic Modifications in Response to Oxygen Availability in Cells without IDH1/2 Mutations

Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) result in production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). D-2HG blocks differentiation of leukemia cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases and TET family DNA methylcytosine dioxygenases, which function as important regulators of chromatin structure and gene expression. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although leukemia-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. However, biologic sources and/or activities of L-2HG remain unknown. Possible physiologic roles for both 2-hydroxyglutarate enantiomers are suggested by the existence of evolutionarily conserved ‘waste disposal’ enzymes, known as D2HGDH and L2HGDH, whose sole function is to eliminate D-2HG or L-2HG, respectively. Indeed, children born with genetic deficiency of either enzyme develop severe developmental disorders known as D- and L-2HG acidurias, characterized by systemic elevation of 2HG. We sought to investigate physiologic sources and functions of D-2HG and L-2HG by manipulating D2HGDH and L2HGDH in cells without IDH1/2 mutations. Using this approach, we found that under conditions of oxygen limitation, cells potently and selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG production is not mediated by IDH1 or IDH2, but instead appears to occur via promiscuous substrate usage by lactate dehydrogenase A (LDHA), and to a lesser extent, malate dehydrogenase 2 (MDH2). Hypoxic induction of L-2HG increases repressive histone modifications, including H3K9me3 and H3K27me3, chromatin marks associated with an undifferentiated cellular state. Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that might prevent a cell from undergoing differentiation when it lacks the capacity for oxidative metabolism. As hematopoietic stem cells (HSCs) reside in hypoxic niches, oxygen-regulated L-2HG might influence self-renewal of HSCs and represent a physiologic system exploited by IDH1/2 mutant-derived D-2HG in leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MOLECULAR GENETIC DETERMINANTS OF LONG-TERM SURVIVAL WITH GLIOBLASTOMA

BACKGROUND: The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long-term survival of more than three years after diagnosis. We aimed to elucidate the poorly characterized molecular aberrations in glioblastomas of longterm survivors. METHODS: We performed genome- and/or transcriptome-wide molecular profiling of primary tumor samples from 94 glioblastoma patients of the German Glioma Network, including 28 longterm survivors with >36 months overall survival (OS), 20 short-term survivors with <12 months OS, and 46 patients with intermediate OS. Integrative bioinformatic analyses were performed to characterize molecular aberrations in the distinct survival groups considering established molecular markers such as isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations, and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. RESULTS: Patients with long-term survival were younger and more often had IDH1/2-mutant and MGMT promoter-methylated tumors. Gene expression profiling revealed over-representation of a distinct (proneural-like) expression signature in long-term survivors that was linked to IDH1/2 mutation. However, among the IDH1/2-wildtype patients, tumors from long-term survivors did not show distinct gene expression profiles and included proneural, classical and mesenchymal glioblastoma subtypes. Genomic imbalances also differed between IDH1/2-mutant and -wildtype tumors, but not between survival groups of IDH1/2-wildtype patients. CONCLUSIONS: Our data support an important role for MGMT promoter methylation and IDH1/2 mutation in glioblastoma long-term survival and corroborate the association of IDH1/2 mutation with distinct genomic and transcriptional profiles. However, IDH1/2-wildtype glioblastomas from long-term survivors do not carry distinct DNA copy number changes or gene expression signatures, indicating that host factors play a major role in allowing for long-term survival. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Predicting the likelihood of an isocitrate dehydrogenase 1 or 2 mutation in diagnoses of infiltrative glioma.

Several variables are associated with the likelihood of isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation in gliomas, though no guidelines yet exist for when testing is warranted, especially when an R132H IDH1 immunostain is negative. A cohort of 89 patients was used to build IDH1/2 mutation prediction models in World Health Organization grades II-IV gliomas, and an external cohort of 100 patients was used for validation. Logistic regression and backward model selection with the Akaike information criterion were used to develop prediction models. A multivariable model, incorporating patient age, glioblastoma multiforme diagnosis, and prior history of grade II or III glioma, was developed to predict IDH1/2 mutation probability. This model generated an area under the curve (AUC) of 0.934 (95% CI: 0.878, 0.978) in the external validation cohort and 0.941 (95% CI: 0.918, 0.962) in the cohort of The Cancer Genome Atlas. When R132H IDH1 immunostain information was added, AUC increased to 0.986 (95% CI: 0.967, 0.998). This model had an AUC of 0.947 (95% CI: 0.891, 0.995) in predicting whether an R132H IDH1 immunonegative case harbored a less common IDH1 or IDH2 mutation. The models were also 94% accurate in predicting IDH1/2 mutation status in gliomas from The Cancer Genome Atlas. An interactive web-based application for calculating the probability of an IDH1/2 mutation is now available using these models. We have integrated multiple variables to generate a probability of an IDH1/2 mutation. The associated web-based application can help triage diffuse gliomas that would benefit from mutation testing in both clinical and research settings.

Molecular characterization of long-term survivors of glioblastoma using genome- and transcriptome-wide profiling

The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long-term survival of more than three years after diagnosis. To elucidate the molecular aberrations in glioblastomas of long-term survivors, we performed genome- and/or transcriptome-wide molecular profiling of glioblastoma samples from 94 patients, including 28 long-term survivors with >36 months overall survival (OS), 20 short-term survivors with <12 months OS and 46 patients with intermediate OS. Integrative bioinformatic analyses were used to characterize molecular aberrations in the distinct survival groups considering established molecular markers such as isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations, and O(6) -methylguanine DNA methyltransferase (MGMT) promoter methylation. Patients with long-term survival were younger and more often had IDH1/2-mutant and MGMT-methylated tumors. Gene expression profiling revealed over-representation of a distinct (proneural-like) expression signature in long-term survivors that was linked to IDH1/2 mutation. However, IDH1/2-wildtype glioblastomas from long-term survivors did not show distinct gene expression profiles and included proneural, classical and mesenchymal glioblastoma subtypes. Genomic imbalances also differed between IDH1/2-mutant and IDH1/2-wildtype tumors, but not between survival groups of IDH1/2-wildtype patients. Thus, our data support an important role for MGMT promoter methylation and IDH1/2 mutation in glioblastoma long-term survival and corroborate the association of IDH1/2 mutation with distinct genomic and transcriptional profiles. Importantly, however, IDH1/2-wildtype glioblastomas in our cohort of long-term survivors lacked distinctive DNA copy number changes and gene expression signatures, indicating that other factors might have been responsible for long survival in this particular subgroup of patients.