IDH1 R132H Research Articles

Abstract 4134: Characterization of ATRX loss combined with IDH1 R132H mutation in gliomas

Abstract Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and is a potential therapeutic target. ATRX functions as a histone chaperone that helps incorporate histone variant H3.3 into the genome. There is some evidence of its involvement in both nonhomologous-end-joining (NHEJ) and replication stress, but a distinct role for ATRX in DNA repair has not been determined. Our lab has found that another common mutation in gliomas in the isocitrate dehydrogenase (IDH1) gene, leads to deficiency in homologous recombination (HR). We hypothesize that modelling these common mutations in glioma cell lines will to confer sensitivity to combinations of DNA repair inhibitor and damaging agents. To test this, we have created multiple cell lines with a CRISPR knockout of ATRX as well as lines that overexpress the IDH1 R132H in the presence of doxycycline. This will allow comparison of the double mutation and each mutation individually in different glioma cell lines for their DNA repair capacity. Currently, we have found a retention of H2AX foci in these cells after hydroxyurea treatment. A luciferase reporter based DNA repair assay has shown a decrease in HR capacity and no change in NHEJ in ATRX KO cells compared to WT. Additionally, we have begun screening DNA repair inhibitors and DNA damaging agents to identify compounds that specifically target ATRX KO cells or the double mutant cell lines. These preliminary screens have shown sensitivity to Chk1 inhibitors and temozolomide, which is currently being validated. We have also performed a focused DNA repair CRISPR screen to assess which DNA repair genes cause sensitivity or resistance ATRX KO cells. We infected both WT and ATRX KO cells with our pooled CRISPR library and harvested genomic DNA at multiple time points. Next generation sequencing was then used to identify sgRNAs that dropped out of the population compared to control guides. Sensitive hits from these screens were underrepresented in the ATRX KO population compared to WT and resistant hits were overrepresented in ATRX KO. These hits are the in the process of being validated. This work is continuing to expand on the hypothesis that ATRX is an important targetable mutation in low and high grade gliomas. Further understanding its mechanism of action as well as its action in combination with IDH1 R132H will help understand these cancers and lead to more targeted treatments for patients. Citation Format: Jennifer Garbarino, Ranjit Bindra, Ryan Jensen. Characterization of ATRX loss combined with IDH1 R132H mutation in gliomas [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 4134.

Abstract 1389: Exploiting inherent DNA damage repair defects in IDH1/2 mutated gliomas with the CNS penetrant PARP inhibitor, pamiparib

Abstract Our group recently demonstrated that IDH1/2 mutated gliomas exhibit intrinsic homologous recombination (HR) defects mediated by the oncometabolite, 2HG. These defects render IDH1/2 mutated cells exquisitely sensitive to PARP inhibitors (PARPi's), which suggests a novel therapeutic strategy. Here, we studied the potential in vitro and in vivo efficacy of a CNS penetrant PARPi, pamiparib, in combination with radiation therapy (RT) and temozolomide (TMZ) against IDH1/2 mutated gliomas. Our central hypothesis is that multi-modality therapy using agents that are active against 2HG producing cells will increase efficacy, while allowing lower doses of RT and TMZ as a means to mitigate normal tissue toxicity. We performed a comprehensive series of DNA repair functional studies, short and long term viability assays, as well as in vivo studies, using engineered and patient-derived IDH1/2 mutant glioma models. We tested unique combinations of pamiparib, TMZ and RT in these experiments. In vitro short term viability assays and long-term clonogenic survival assays in model IDH1 wild-type (WT) and R132H mutated glioma cells demonstrated that the mutation confers enhanced TMZ sensitivity (IC50 602 vs. 9.5 uM, respectively). A similar differential sensitivity was observed with RT. We also demonstrated significant, mutant IDH1 selective pamiparib sensitivity in vitro (IC50 0.67 vs. 141 uM). Western blot analysis of PARylation, a functional readout of PARPi activity, confirmed PARylation inhibition by pamiparib in a dose dependent manner. Subsequently, we demonstrated synergistic interactions between TMZ and RT combined with pamiparib, particularly at low doses of both DNA damaging agents, in an IDH1 selective manner. Finally, we demonstrated the activity of PARPi's against IDH1 mutant tumors in vivo, including pharmacodynamic and pharmacokinetic properties of pamiparib on target activity within the brain. Pamiparib is a promising, CNS penetrant PARP inhibitor that may selectively target IDH1/2 mutated gliomas, based on intrinsic HR defects associated with these tumors. Pamiparib appears to act synergistically with TMZ and RT, which are the backbone of GBM therapies. These data lay the groundwork for a future trial testing “trimodality therapy” (i.e., pamiparib, RT and TMZ) against IDH1/2 mutant gliomas. Citation Format: Christopher Hong, Amrita Sule, Jason Beckta, Ranjini Sundaram, Ranjit Bindra. Exploiting inherent DNA damage repair defects in IDH1/2 mutated gliomas with the CNS penetrant PARP inhibitor, pamiparib [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 1389.

Abstract 6579: Clinical utility of comprehensive genomic testing with artificial-intelligence-based analysis to identify targetable sub-clonal events in relapsed acute myeloid leukemia (AML)

Abstract Next-generation sequencing (NGS) is increasingly used to inform diagnostic, therapeutic, and prognostic decisions in AML at the time of first presentation. We highlight the utility of NGS combined with Watson™ for Genomics (WfG), an artificial-intelligence-based decision-support system, in identifying new clinically actionable alterations as a result of clonal evolution in the relapsed disease setting. In less than 3 minutes, WfG identified an IDH1 R132H pathogenic mutation in the relapsed sample sequenced with the Illumina TruSight Tumor 170-gene panel leading to the compassionate use of ivosidenib. In addition, mutations in two genes resulting in increased sensitivity to PARP inhibitors and mutations in PTEN resulting in activation of the MTOR/PI3K signaling pathway were detected by WfG. In February 2018, a previously healthy 23-year old Caucasian female presented with AML consisting of 80% blasts with positive FLT3 mutation. She received induction cytarabine plus daunorubicin (7+3) followed by multikinase inhibitor therapy with midostaurin on days 8 to 21. A 28-day bone marrow biopsy showed persistent disease with 40% blasts. In March 2018, the patient underwent re-induction chemotherapy with mitoxantrone, etoposide, and cytarabine (MEC) followed by midostaurin on days 8 to 21. A bone marrow biopsy after completion of re-induction therapy showed complete remission with < 5% blasts (CR1). In May 2018, a matched donor was not found on a bone marrow registry, and the patient underwent a post-remission dual unrelated umbilical cord blood (UCB) hematopoietic stem cell transplantation (HSCT). Engraftment was not achieved. In July 2018, the patient had disease relapse with the presence of circulating blasts and 7% of blasts in the bone marrow. While NGS is typically not performed on relapsed samples, WfG identified IDH1 R132H and PTEN C78T pathogenic mutations using the 170-gene panel. Based on these results, the patient initiated azacytidine plus the IDH1 inhibitor ivosidenib. In August 2018, a bone marrow biopsy showed less than 5% blasts positive for a FLT3-ITD mutation. In September 2018, the patient underwent reduced-intensity conditioning with fludarabine, cyclophosphamide, and total body irradiation followed by haploidentical allogeneic HSCT from her mother. Maintenance therapy with azacytidine plus ivosidenib was continued until the present time. Currently, the patient is in remission for over 15 months without evidence of AML minimal residual disease. She has developed mild skin chronic graft-versus-host disease that is controlled with standard treatment. She works on a full-time basis and has excellent functional status. The combination of CGP, artificial intelligence, and expert care has resulted in an excellent outcome in a patient with relapsed AML. In conclusion, our experience suggests that CGP testing should be considered at different time points, at least in the relapsed setting, to help treating physicians alter or help improve clinical outcome. CGP testing in a relapsed setting is precluded because it is not covered by payers. In support of optimal care, we have initiated a new program for compassionate use of genomic testing, where such testing is medically necessary, but not covered by insurance or payer supported. Citation Format: Ravindra Kolhe, Ashis Mondal, Vamsi Kota, Nkhil Sahajpal, Meenakshi Ahluwalia, Allan Njau, Dilhan Weeraratne, Yull Arriaga, David Brotman, Gretchen Jackson, Jane Snowdon. Clinical utility of comprehensive genomic testing with artificial-intelligence-based analysis to identify targetable sub-clonal events in relapsed acute myeloid leukemia (AML) [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 6579.

Abstract 1059: Targeting IDH1/2 mutant cancers with unique combinations of DNA repair inhibitors

Abstract Mutations in the Isocitrate Dehydrogenase-1 and -2 (IDH1/2) genes occur in the vast majority of solid tumors including low-grade and secondary high-grade gliomas. These neomorphic mutations occur early on in gliomagenesis leading to the production of 2-Hydroxyglutarate (2HG). 2HG has been implicated in tumorigenesis via inhibiting α-ketoglutarate (αKG)-dependent dioxygenases. Our group recently demonstrated that the production of 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state of “BRCAness”. We initially found that mutant IDH1/2-induced BRCAness confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, a finding which now has been replicated by multiple independent laboratories. Although IDH1/2 mutations were first identified in gliomas and acute myeloid leukemia (AML) cells, multiple other tumor types have subsequently been shown to harbor these mutations. Current clinical trials are testing the efficacy of PARP inhibitors as a monotherapy, as well as in combination with other DNA repair inhibitors. Here, we demonstrate that novel combinations of DNA repair inhibitors can be utilized to synergistically target IDH1/2-mutant glioma cells. In particular, we demonstrate potent synergy with ATRi and PARPis, a finding which was validated in multiple structurally unique drugs within these classes. We have shown that in an in vivo flank model bearing IDH1 R132H mutation, AZD6738 (ATRi) and Olaparib (PARPi) combination treatment reduces the tumor burden significantly relative to monotherapy and vehicle. As this combination is active in BRCA1/2-mutant cancers, in particular after the emergence of PARPi resistance, these data suggest are consistent with an underlying HR defect in IDH1/2-mutant gliomas. These preclinical investigations will provide a blueprint for future clinical trials combining PARP and ATR inhibitors in the treatment of IDH1/2 mutant cancers. Citation Format: Amrita D. Sule, Ranjini Sundaram, Ranjit Bindra. Targeting IDH1/2 mutant cancers with unique combinations of DNA repair inhibitors [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 1059.

High Throughput Molecular Characterization of Normal Karyotype Acute Myeloid Leukemia in the Context of the Prospective Trial 02/06 of the Northern Italy Leukemia Group (NILG).

By way of a Next-Generation Sequencing NGS high throughput approach, we defined the mutational profile in a cohort of 221 normal karyotype acute myeloid leukemia (NK-AML) enrolled into a prospective randomized clinical trial, designed to evaluate an intensified chemotherapy program for remission induction. NPM1, DNMT3A, and FLT3-ITD were the most frequently mutated genes while DNMT3A, FLT3, IDH1, PTPN11, and RAD21 mutations were more common in the NPM1 mutated patients (p < 0.05). IDH1 R132H mutation was strictly associated with NPM1 mutation and mutually exclusive with RUNX1 and ASXL1. In the whole cohort of NK-AML, no matter the induction chemotherapy used, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 (FLT3-ITD) and U2AF1 were associated with a worse overall and disease-free survival (p < 0.05). FLT3-ITD positive patients who proceeded to alloHSCT had a survival probability similar to FLT3-ITD negative patients and the transplant outcome was no different when comparing high and low-AR-FLT3-ITD subgroups in terms of both OS and DFS. In conclusion, a comprehensive molecular profile for NK-AML allows for the identification of genetic lesions associated to different clinical outcomes and the selection of the most appropriate and effective treatment strategies, including stem cell transplantation and targeted therapies.

A Nomogram Modeling 11C-MET PET/CT and Clinical Features in Glioma Helps Predict IDH Mutation.

Purpose: We developed a 11C-Methionine positron emission tomography/computed tomography (11C-MET PET/CT)-based nomogram model that uses easy-accessible imaging and clinical features to achieve reliable non-invasive isocitrate dehydrogenase (IDH)-mutant prediction with strong clinical translational capability.Methods: One hundred and ten patients with pathologically proven glioma who underwent pretreatment 11C-MET PET/CT were retrospectively reviewed. IDH genotype was determined by IDH1 R132H immunohistochemistry staining. Maximum, mean and peak tumor-to-normal brain tissue (TNRmax, TNRmean, TNRpeak), metabolic tumor volume (MTV), total lesion methionine uptake (TLMU), and standard deviation of SUV (SUVSD) of the lesions on MET PET images were obtained via a dedicated workstation (Siemens. syngo.via). Univariate and multivariate logistic regression models were used to identify the predictive factors for IDH mutation. Nomogram and calibration plots were further performed.Results: In the entire population, TNRmean, TNRmax, TNRpeak, and SUVSD of IDH-mutant glioma patients were significantly lower than these values of IDH wildtype. Receiver operating characteristic (ROC) analysis suggested SUVSD had the best performance for IDH-mutant discrimination (AUC = 0.731, cut-off ≤ 0.29, p < 0.001). All pairs of the 11C-MET PET metrics showed linear associations by Pearson correlation coefficients between 0.228 and 0.986. Multivariate analyses demonstrated that SUVSD (>0.29 vs. ≤ 0.29 OR: 0.053, p = 0.010), dichotomized brain midline structure involvement (no vs. yes OR: 26.52, p = 0.000) and age (≤ 45 vs. >45 years OR: 3.23, p = 0.023), were associated with a higher incidence of IDH mutation. The nomogram modeling showed good discrimination, with a C-statistics of 0.866 (95% CI: 0.796–0.937) and was well-calibrated.Conclusions: 11C-Methionine PET/CT imaging features (SUVSD and the involvement of brain midline structure) can be conveniently used to facilitate the pre-operative prediction of IDH genotype. The nomogram model based on 11C-Methionine PET/CT and clinical age features might be clinically useful in non-invasive IDH mutation status prediction for untreated glioma patients.

A phase I clinical trial on intratumoral and intracavitary administration of ipilimumab and nivolumab in patients with recurrent glioblastoma.

2534 Background: Intravenous (IV) administration of ipilimumab (IPI) and nivolumab (NIVO) has low activity in recurrent glioblastoma (rGB). Intratumoral (IT) and intracavitary (IC) administration of IPI and NIVO is under evaluation in the GlITIpNi phase I clinical trial. Methods: Patients (pts) with resectable rGB were recruited to cohorts C1, C2 and C4; pts with non-resectable rGB were recruited in C3 (biopsy only). IT administration (brain tissue lining the resection cavity during surgery) of IPI (10 mg)(C1), or IPI (5 mg) plus NIVO (10 mg)(C2, C3 and C4), was followed by IC administration of NIVO at escalating doses of 1, 5 or 10 mg Q2w in both C3 and C4 (via an Ommaya reservoir). In all cohorts, pts received 10 mg NIVO IV Q2w (6x in C1/C2, and 12x in C3/C4). Corticosteroids were contraindicated. Results: Forty-six pts (31 male; median age 56y (38-74); IDH1 R132H mutation in 2 pts in C1/C2; NGS somatic mutation analysis for C3/C4 ongoing) with rGB following resection, RT and temozolomide were enrolled (3, 24, 13 and 6 pts in C1, C2, C3 and C4, respectively). All pts received IT administrations. Pts in C1/C2 received a median of 5 IV NIVO administrations. Study treatment has been completed in all pts in C1/C2, in 9 pts in C3, and in 3 pts in C4; pts received a median of 4 (0-10) and 3 (0-7) postoperative IC/IV administrations in C3 and C4, respectively. Two pts in C2 and 1 pt in C3 had an increased perilesional cerebral edema (G3) with neurological deterioration after surgery/IT-injection, that was reversible with steroids. Most frequent AE were fatigue (32 pts, 64%), fever (20 pts, 44%), and headache (25 pts, 50%). In 4 pts from C3, the Ommaya was removed because of bacterial colonization (asymptomatic). There were no G5 AE. There was no dose/AE correlation with increasing IC NIVO doses in C3/C4. Repetitive CSV analysis during therapy (C3/C4) revealed increased lymphocyte counts in 4 pts; scRNA- and TCR-sequencing is ongoing. Gene expression profiling for C1/C2, and pharmacokinetic analysis of NIVO and IPI in CSV for C3/C4 are ongoing. After a median FU of 62w (16-165) for pts in C1/C2, 16 pts have died; median OS is 71w (95% CI 8-134), 1- and 2y-OS% are respectively 51% (95% CI 31-71), and 34% (95% CI 10-59). OS compares favorably to a historical cohort of Belgian rGB pts (n = 469; Log-Rank p .001). After a median FU of 10w (1-37) for pts in C3/C4, 2 pts have died; median OS has not been reached. One pt in C3 achieved a PR that is ongoing at 12m. Conclusions: IT/IC administration of NIVO and IPI is feasible and sufficiently safe to warrant further investigation in pts with rGB. Clinical trial information: NCT03233152 .

Editing of IDH1 R132H Mutation in Human Induced Pluripotent Stem Cells to Investigate Tumor Genesis in Glioma (2207)

Editing of IDH1 R132H Mutation in Human Induced Pluripotent Stem Cells to Investigate Tumor Genesis in Glioma (2207)

Design and synthesis of novel 2-arylbenzimidazoles as selective mutant isocitrate dehydrogenase 2 R140Q inhibitors

A series of novel 2-arylbenzimidazoles have been designed, synthesized and evaluated for their inhibitory activity against IDH2 R140Q mutant. The preliminary results indicated that four compounds 7b, 7c, 7m and 7r displayed the potent inhibitory activity against IDH2 R140Q mutant. Among them, compound 7c showed the highest inhibitory activity, with the IC50 value of 0.26 μM, which was more active than positive control enasidenib. The exquisite selectivity of 7c for IDH2 R140Q mutant isoform was demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.

The Landscape of the Anti-Kinase Activity of the IDH1 Inhibitors.

Isocitrate dehydrogenases constitute a class of enzymes that are crucial for cellular metabolism. The overexpression or mutation of isocitrate dehydrogenases are often found in leukemias, glioblastomas, lung cancers, and ductal pancreatic cancer among others. Mutation R132H, which changes the functionality of an enzyme to produce mutagenic 2-hydroxyglutarate instead of a normal product, is particularly important in this field. A series of inhibitors were described for these enzymes of which ivosidenib was the first to be approved for treating leukemia and bile duct cancers in 2018. Here, we investigated the polypharmacological landscape of the activity for known sulfamoyl derivatives that are inhibitors, which are selective towards IDH1 R132H. These compounds appeared to be effective inhibitors of several non-receptor kinases at a similar level as imatinib and axitinib. The antiproliferative activity of these compounds against a panel of cancer cells was tested and is explained based on the relative expression levels of the investigated proteins. The multitargeted activity of these compounds makes them valuable agents against a wide range of cancers, regardless of the status of IDH1.

IDH1 Expression via the R132H Mutation-Specific Antibody in Adrenocortical Neoplasias-Prognostic Impact in Carcinomas.

ContextMutations to isocitrate dehydrogenase (IDH) appear to play a prognostic or predictive role in several neoplasias. Immunohistochemical staining designed to detect a specific R132H mutation to IDH1 showed expression in the normal adrenal cortex, raising interest to study the potential role of IDH1 in the pathogenesis of adrenocortical tumors.ObjectiveThe objective of this work is to study the role of IDH1 and its mutations in adrenocortical tumors.Design and patients IDH1 R132H immunohistological staining was performed on a cohort of 197 adrenocortical tumors. The exon of the IDH1 gene was sequenced in 16 tumors.ResultsPositive IDH1 R132H immunohistochemical staining correlated with a better prognosis among patients with a malignant adrenocortical tumor. However, IDH1 R132H immunohistochemistry did not distinguish between local and metastasized tumors. We were unable to identify IDH1 mutations among our adrenocortical tumors using a targeted next-generation sequencing panel or via exon sequencing.ConclusionsAmong adrenocortical carcinomas, IDH1 R132H immunopositivity correlated with a better prognosis. Thus, IDH1 R132H immunohistochemical staining could serve as a prognostic or as a potential predictive marker in adrenocortical carcinomas. Further research is needed to identify the possible alterations in IDH1 that could explain our findings, because we identified no known mutations to the IDH1 gene.

In vivo MRS measurement of 2-hydroxyglutarate in patient-derived IDH-mutant xenograft mouse models versus glioma patients.

To generate a preclinical model of isocitrate dehydrogenase (IDH) mutant gliomas from glioma patients and design a MRS method to test the compatibility of 2-hydroxyglutarate (2HG) production between the preclinical model and patients. Five patient-derived xenograft (PDX) mice were generated from two glioma patients with IDH1 R132H mutation. A PRESS sequence was tailored at 9.4 T, with computer simulation and phantom analyses, for improving 2HG detection in mice. 2HG and other metabolites in the PDX mice were measured using the optimized MRS at 9.4 T and compared with 3 T MRS measurements of the metabolites in the parental-tumor patients. Spectral fitting was performed with LCModel using in-house basis spectra. Metabolite levels were quantified with reference to water. The PRESS TE was optimized to be 96 ms, at which the 2HG 2.25 ppm signal was narrow and inverted, thereby leading to unequivocal separation of the 2HG resonance from adjacent signals from other metabolites. The optimized MRS provided precise detection of 2HG in mice compared to short-TE MRS at 9.4 T. The 2HG estimates in PDX mice were in excellent agreement with the 2HG measurements in the patients. The similarity of 2HG production between PDX models and parental-tumor patients indicates that PDX tumors retain the parental IDH metabolic fingerprint and can serve as a preclinical model for improving our understanding of the IDH-mutation associated metabolic reprogramming.

Interaction between IDH1 WT and calmodulin and its implications for glioblastoma cell growth and migration

Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas.

Metabolic changes related to the IDH1 mutation in gliomas preserve TCA-cycle activity: An investigation at the protein level.

The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA-cycle activity in IDH1 mut gliomas.

Malignant transformation of pleomorphic xanthoastrocytoma and differential diagnosis: case report

BackgroundPleomorphic xanthoastrocytoma (PXA) is a rare astrocytic glioma, characterized by large pleomorphic and frequently multinucleated cells, spindle and lipidized cells, a dense pericellular reticulin network, and numerous eosinophilic granular bodies according to the grade II glial tumor standards of the World Health Organization’s (WHO) 2016 guidelines. PXA rarely transforms into anaplastic PXA or glioblastoma (GBM) and anaplastic PXA, classified as WHO grade III, has a more aggressive clinical behavior with poorer prognosis than PXA.Case presentationHere we describe an unusual case of PXA in a 19-year-old woman, first admitted with headache and a mass in the left temporal lobe in 2005 that was removed. Twelve years later, she returned with left temporal headache, diplopia and tinnitus. A local tumor recurrence was found, and a second resection was performed. The specimen showed highly malignant findings, such as necrosis, microvascular proliferation, and multiple mitoses. The integrated diagnosis was made as high grade glioma, probably derived from PXA. Immunohistochemical (IHC) stains were positive for oligo2, and approximately 21% positive for Ki-67, while negative for CD34, IDH1 R132H. INI1 and ATRX were retained. As the histological classification was glioblastoma, the patient received GBM-appropriate chemotherapy and radiation therapy and outpatient follow-ups have demonstrated no obvious symptoms for 1 year after surgery. Additional molecular analyses found BRAF V600E mutations in both resections, supporting the idea that the recurrent tumor had derived from PXA.ConclusionsThis case highlights the complexities of differential diagnosis based on the World Health Organization’s 2016 guidelines. More integrated criteria to differentiate anaplastic PXA from GBM and epithelioid GBM, combined with genetic screening results, might be needed.

Clinicopathological associations and prognostic values of IDH1 gene mutation, MGMT gene promoter methylation, and PD-L1 expressions in high-grade glioma treated with standard treatment

Introductionthe objective was to evaluate the impact of IDH1 R132H mutation, MGMT methylation and PD-L1 expression in high grade glioma that received standard therapy (surgery, radiation and chemotherapy) to overall survival (OS).Methodsthis is a retrospective study of 35 high grade glioma cases. Genotyping of IDH1 gene alteration on the mutation hotspot R132 (Sanger sequencing method with Applied Biosystems 3500 Genetic Analyzer), EZ DNA Methylation-Gold kit (Zymo Research) is used to study the methylation, Cell line BT549 (ATCC HTB-122) and HCT-116 (ATCC CCL-247) were used as unmethylated control and partially methylated control respectively. Anti-human PD-L1 antibody clone E1L3N®from Cell Signalling Technology (USA) and Rabbit XP®were used to see PDL-1 expression.Resultsanaplastic astrocytoma cases had more MGMT promoter methylation (50%) than glioblastoma multiforme (GBM) (20%), more IDH1 R132H mutation (42%) than GBM (4.3%). Immunohistochemistry tumor proportion score method (TPS) identified 17% and 8.7% were PD-L1 positive in AA and GBM groups, respectively. Cases with IDH1 R132H mutation and MGMT methylation still showed better OS although with high PD-L1 expression.ConclusionIDH1 R132H mutation and MGMT methylation were good prognostic markers. High expression of PD-L1 apparently might not indicate poor overall survival in the presence of IDH1 R132 mutation and MGMT methylation.

Novel IDH1-Targeted Glioma Therapies.

Mutations in the isocitrate dehydrogenase (IDH) 1 gene are commonly found in human glioma, with the majority of low-grade gliomas harboring a recurrent point mutation (IDH1 R132H). Mutant IDH reveals an altered enzymatic activity leading to the synthesis of 2-hydroxyglutarate, which has been implicated in epigenetic mechanisms of oncogenesis. Nevertheless, it is unclear exactly how IDH mutations drive glioma initiation and progression, and it is also not clear why tumors with this mutation generally have a better prognosis than IDH wild-type tumors. Recognition of the high frequency of IDH mutations in glioma [and also in other malignancies, including acute myeloid leukemia (AML) and cholangiocarcinoma] have led to the development of a number of targeted agents that can inhibit these enzymes. Enasidenib and ivosidenib have both gained regulatory approval for IDH mutant AML. Both agents are still in early clinical phases for glioma therapy, as are a number of additional candidates (including AG-881, BAY1436032, and DS1001). A marked clinical problem in the development of these agents is overcoming the blood-brain barrier. An alternative approach to target the IDH1 mutation is by the induction of synthetic lethality with compounds that target poly (ADP-ribose) polymerase (PARP), glutamine metabolism, and the Bcl-2 family of proteins. We conclude that within the last decade, several approaches have been devised to therapeutically target the IDH1 mutation, and that, potentially, both IDH1 inhibitors and synthetic lethal approaches might be relevant for future therapies.

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.

Synthetic Lethal Interaction between Apto-253 and IDH1/2 Mutations

APTO-253 is a small molecule with a novel mechanism of action and potent antiproliferative activity against cell lines derived from a wide range of human malignancies. A Phase 1 study of APTO-253 was completed in patients with advanced solid tumors. It was well tolerated and produced evidence of antitumor activity in patients but did not cause myelosuppression even at the maximum tested dose. A phase 1a/b trial of APTO-253 in patients with relapsed/refractory acute leukemias (including AML) and high-risk MDS (NCT02267863) is currently underway. We previously reported that APTO-253 was converted intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. Both APTO-253 and Fe(253)3 stabilized DNA quadruplex (G4) structures in the Myc promotor region to reduce the expression of Myc (Figure 1). Stabilization of G4 structures has been reported to stall replication forks and produce DNA strand breaks. Treatment of cancer cells with APTO-253 produced time and concentration-dependent γH2AX foci formation and DNA double strand breaks that have to be repaired by homologous recombination. Loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 whose magnitude was similar to the effects of PARP inhibitors, olaparib. Thus, APTO-253 is a member of the limited repertoire of drugs which can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. The goal of this project was to identify synthetic lethal interactions in addition to BRCA1/2 deficiency that can guide combination drug studies. The normal function of the isocitrate dehydrogenase (IDH) enzymes is to catalyze the conversion of isocitrate to α-ketoglutarate (αKG) in the citric acid cycle. IDH1 mutations occur in more than 70% of low-grade gliomas, 20% of high-grade glioblastomas and with a frequency of about 10% in AML, cholangiocarcinomas, melanomas and chondrosarcomas. Additionally, mutations are also identified in IDH2 in about 4% of gliomas and 10% of AMLs. IDH1/2 alterations are heterozygous missense mutations that confer a neomorphic activity on the encoded enzyme such that they covert αKG to (R)-2-hydroxyglutarate [(R)-2HG]. (R)-2HG is an oncometabolite with pleiotropic effects on cell biology including chromatin methylation and cellular differentiation. It was reported that IDH1/2 mutations induce a homologous recombination defect that renders tumor cells exquisitely sensitive to PARP inhibitors. We used 2 pairs of isogenic IDH1 wild type and mutant cell lines to test the hypothesis that cells carrying IDH1/2 mutations are hypersensitive to APTO-253. Induction of the expression of IDH1 R132H in THP-1 AML cells increased the 2-HG concentration ~150-fold whereas induction of wild-type IDH1 expression increased it only 2-fold (doi: 10.1038/nm.3788). THP-1 cells expressing the mutant IDH1 R132H were hypersensitive the treatment of APTO-253 compared to cells expressing the wild-type IDH1 (Figure 2). Comparison of wild-type and isogenic IDH1 R132H HCT116 cells disclosed a ~100-fold higher level of (R)-2-HG in the mutant cells (doi: 10.1126/scitranslmed.aal2463), and an increase in sensitivity to the cytotoxic effect of APTO-253 (Figure 3). These results are consistent with the observation that both APTO-253 and increased levels of 2-HG cause DNA damage that depends on homologous recombination for repair and that this favors a synthetic lethal interaction. We surmise that the ability of APTO-253 to markedly reduce the expression of Myc further contributes to the favorable interaction. This data further supports the conclusion that defects in homologous DNA repair can enhance the potency of APTO-253 and suggest the use of this non-myelosuppressive drug in both IDH1/2 mutant AML and solid tumors. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Howell:Aptose Biosciences, Inc: Consultancy, Research Funding.

Comparison of Three Assays for Identification of IDH Mutations in AML

Introduction Isocitrate dehydrogenase (IDH) mutations are present in up to 20% of acute myeloid leukemia (AML) patients and lead to production of 2-hydroxyglutarate which promotes impaired differentiation and leukemic cell proliferation. Currently, there are two FDA-approved IDH inhibitors for the treatment of AML: enasidenib and ivosidenib. It is now imperative to establish timely testing so that patients can take advantage of these new therapies. Here we compared three different genotyping strategies to identify the most reliable and cost-effective way of picking up IDH1 and IDH2 mutations in AML. Methodology We have tested 60 AML patient samples across three different methods for detection of IDH1/2 mutations: (i) Capillary Electrophoresis-Single Strand Conformational Analysis (CE-SSCA) with Sanger sequencing, (ii) Next generation sequencing (NGS) myeloid capture panel (SureSeq myPanel, Oxford Gene Technology, OGT; UK) and (iii) multi-cancer NGS amplicon panel (QIAseq Targeted DNA Panel, Qiagen, Germany). The NGS capture assay is a 26 gene panel designed for myeloid-related genes while the NGS amplicon panel includes 33 genes associated with haematological and non-haematological malignancies (Figure 1). We have compared the techniques and assessed the quality of the results by measuring gene coverage, read depth across the panels, sensitivity and specificity per test, and performed a final valuation including some practical considerations: cost analysis, staff timings, ease of assay operation, turnaround times (TATs), ease of analysis and reporting as well as batch size limits. We initially tested 424 AML cases with CE-SSCA and have successfully transferred to amplicon panel testing (to date 200 cases). Results The results from the initial 60 cases were concordant between both NGS technologies and Sanger sequencing. In total across all 3 assays, we have detected IDH mutations in 19% of the 624 samples tested (Figure 2). Two samples showed false positive results by CE-SSCA, but these were identified by Sanger sequencing; therefore CE-SSCA alone is an inadequate test and all positive samples by CE-SSCA have to be confirmed by Sanger sequencing. Two samples showed IDH1 R132H mutation with variant allele frequency less than 10%, which was detected by all methods, however the Sanger sequencing trace was very small and could potentially have been missed. The gene minimum read depth for capture NGS was 682 and 677 for amplicon, and the gene mean depth was 1571 and 934 reads, respectively. We did not identify any new genetic variants in IDH1/2. The cost of reagents was higher for the NGS capture panel at $235 per patient compared to amplicon based at $86 per patient, with CE-SSCA/Sanger sequencing combined costing $50. The amplicon NGS allows processing of up to 96 samples per batch, allowing for high throughput testing, while capture NGS only allows for 16 samples to be processed. The library preparation time (Figure 1) is shorter for NGS amplicon (2 days; 12 hours staff time) compared to capture (3 days; 17 hours staff time), with both techniques taking longer to set-up when compared to CE-SSCA (2 hours) and Sanger sequencing (1 day; 6 hours staff time). Looking at assay operation, CE-SSCA and Sanger are the less complex assays allowing for laboratory accessibility, whereas although NGS amplicon was relatively straight forward, NGS capture was slightly more complex, lending itself to specialist laboratory setup. When looking at analysis, CE-SSCA was straightforward; however certain patterns that are very similar to positive controls can lead to false positives. Both NGS analysis were very straightforward, taking &lt;5min per sample, while CE-SSCA/Sanger can take up to 10min per sample in more difficult cases. Importantly, NGS-based approaches incorporate other clinically-relevant genes whereas CE-SSCA is limited to IDH1/2 evaluation with additional testing being required for other targets. Conclusion: We have established a multi-cancer NGS amplicon assay for the detection of IDH mutations in AML patients. It reduces test costs for patients, improving testing efficiencies, allowing additional clinically-relevant genes to be analysed in parallel It has also helped streamline testing for different cancer types which can now all follow the same workflow and be automated thus improving TAT's contributing to better patient management. Acknowledgements: Celgene provided funding for this study. Disclosures Taussig: Celgene: Research Funding.