IDH2 R172 Mutations Research Articles

Ivosidenib (Tibsovo)

We recommend that Tibsovo in combination with azacitidine be reimbursed by public drug plans for the treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) with an IDH1 R132 mutation who are not eligible to receive intensive induction chemotherapy, if certain conditions are met. Tibsovo in combination with azacitidine should only be covered to treat adult patients with newly diagnosed AML with an IDH1 R132 mutation who are considered ineligible for standard intensive induction chemotherapy and are aged at least 75 years; have an Eastern Cooperative Oncology Group (ECOG) performance status of 2; have severe organ dysfunction in the heart, lungs, kidneys, or liver; and/or have any other comorbidity judged to be incompatible with intensive induction chemotherapy. Tibsovo in combination with azacitidine should only be reimbursed if prescribed by clinicians with expertise in managing patients with AML in a specialized hematology or oncology clinic, and the treatment should be supervised and delivered in institutions with expertise in systemic therapy delivery. The total drug cost of ivosidenib plus azacitidine should not exceed that of venetoclax plus azacitidine. Lastly, it must be feasible to test patients for IDH1 R132

Abstract SY16-01: What IDH mouse models have taught us about hematological diseases, from leukemia to T cell lymphoma

Abstract SY16-01: What IDH mouse models have taught us about hematological diseases, from leukemia to T cell lymphoma

CORR Insights®: Are IDH1 R132 Mutations Associated With Poor Prognosis in Patients With Chondrosarcoma of the Bone?

CORR Insights®: Are IDH1 R132 Mutations Associated With Poor Prognosis in Patients With Chondrosarcoma of the Bone?

IDH1 and IDH2 Gene Mutations in Omani Patients with Acute Myeloid Leukemia: Prognostic Significance and Clinic-pathologic Features.

We sought to define the prevalence of isocitrate dehydrogenase (IDH) mutations, evaluate the clinicopathologic impact of IDH mutations, assess the effect of IDH mutations on the response to the currently offered treatment for acute myeloid leukemia (AML) cases, and determine the impact of other common concurrent mutations with IDH. A single-center retrospective cohort study was conducted at Sultan Qaboos University Hospital (SQUH) from October 2009 to October 2019. We included all Omani patients (pediatric and adult) treated at SQUH with the standard therapy, for whom DNA extraction was performed at diagnosis. The target mutations in both IDH1 and IDH2 genes were screened using the direct polymerase chain reaction product sequencing method. Statistical analysis was conducted using SPSS software. Survival differences were estimated using the log-rank test. Continuous variables were presented as median (IQRs), while categorical variables were presented as frequency. A total of 61 patients treated, for whom DNA extraction was performed at diagnosis were evaluated. The median age was 40 (range = 25.5-65.5). The prevalence of IDH1 R132, IDH2 R140, and IDH2 R172 mutations among the study group was 6.6%, 3.3%, and 1.6%, respectively. Clinicopathologic characteristics associated with IDH mutations at diagnosis included older age, lower white blood cell count, higher median platelet counts, normal karyotype AML, and cytogenetics intermediate-risk group. The overall survival (OS) in patients harboring IDH mutations was poor, with a median OS of nine months. This analysis confirms that the response rate and OS for both IDH-mutated and IDH wild-type AML patients were comparable. This will provide contemporary data to be used for comparison with the results of novel investigational (e.g., selective IDH inhibitor) strategies. The current study results were consistent with the other international studies of IDH mutations in AML and demonstrate the poor prognosis associated with IDH mutations. Clinicopathologic features associated with IDH mutations included older age, lower white blood cell count, higher median platelet counts, normal karyotype AML, and cytogenetics intermediate-risk group.

Clinical and histological study of follicular helper T-cell lymphomas with indolent evolution

IntroductionFollicular helper T-cell lymphomas (TFHL) have an aggressive course with a poor outcome. European and US guidelines recommend anthracycline-based chemotherapy as a first-line treatment, but the 5-year overall survival rate is still approximately 30%. We describe here the features of a cohort of TFHL patients who experienced prolonged survival despite the absence of specific treatment or the initiation of steroid-based therapy. Patients and methodsIn our study, we describe 15 adult patients who suffered from TFHL and had not received intensive chemotherapy at diagnosis for any reason. Biopsies of these cases were centrally reviewed, and the mutational pattern was determined using next-generation sequencing. ResultsThese 15 patients had the classic clinical, biological and pathological features of TFHL, angioimmunoblastic-type. TET2 mutations were found in 83% of patients; RHOA G17V, IDH2 R172 and DNMT3A mutations were found in 67%, 42% and 33% of the patients, respectively. Among the 15 patients, 8 did not receive any treatment, and 7 received steroid-based treatment. Ten patients had progression (5 in each group). Four patients died (3 of them from the progression of their lymphoma). The median follow-up in our cohort was 53 months. The 5-year OS was 66%, 100% for untreated patients and 29% for the others. In those 2 groups, the median time to treatment initiation was 22 months from diagnosis. ConclusionWe described a series of 15 well-characterized TFHL patients with an indolent outcome, suggesting that a watch-and-wait approach can be proposed in selected patients. Identifying factors predicting such evolution is warranted.

Magnetic Resonance–Guided Laser Interstitial Thermal Therapy for Recurrent Glioblastoma and Radiation Necrosis: A Single-Surgeon Case Series

To evaluate long-term clinical outcomes among patients treated with laser interstitial thermal therapy (LITT) for predicted recurrent glioblastoma (rGBM). Patients with rGBM treated by LITT by a single surgeon (2013-2020) were evaluated for progression-free survival (PFS), overall survival (OS), and OS after LITT. Forty-nine patients (33 men, 16 women; mean [SD] age at diagnosis, 58.7 [12.5] years) were evaluated. Among patients with genetic data, 6 of 34 (18%) had IDH-1 R132 mutations, and 7 of 21 (33%) had MGMT methylation. Patients underwent LITT at a mean (SD) of 23.8 (23.8) months after original diagnosis. Twenty of 49 (40%) had previously undergone stereotactic radiosurgery, 37 (75%) had undergone intensity-modulated radiation therapy, and 49 (100%) had undergone chemotherapy. Patients had undergone a mean of 1.2 (0.7) previous resections before LITT. Mean preoperative enhancing and T2 FLAIR volumes were 13.1 (12.8) cm3 and 35.0 (32.8) cm3, respectively. Intraoperative biopsies confirmed rGBM in 31 patients (63%) and radiation necrosis in 18 patients (37%). Six perioperative complications occurred: 3 (6%) cases of worsening aphasia, 1 (2%) seizure, 1 (2%) epidural hematoma, and 1 (2%) intraparenchymal hemorrhage. For the rGBM group, median PFS was 2.0 (IQR, 4.0) months, median OS was 20.0 (IQR, 29.5) months, and median OS after LITT was 6.0 (IQR, 10.5) months. For the radiation necrosis group, median PFS was 4.0 (IQR, 4.5) months, median OS was 37.0 (IQR, 58.0) months, and median OS after LITT was 8.0 (IQR, 23.5) months. In a diverse rGBM cohort, LITT was associated with a short duration of posttreatment PFS.

BIOM-26. RAPID IDENTIFICATION OF IDH1-R132 VARIANTS IN GLIOMA WITH LOCKED NUCLEIC ACID LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LNA-LAMP)

Abstract Point mutations that impact the catalytic site of isocitrate dehydrogenase 1 (IDH1) are associated with an extended overall survival of approximately 3.8 years for glioma patients compared to 1 year for those with wild-type IDH1. The difference in survival benefit is associated with the maximal surgical resection of IDH1 mutant tumors, making intraoperative knowledge of the mutational status valuable. Unfortunately, current methods used to determine IDH1-R132 mutational status are incompatible with obtaining real-time results. We have developed a panel of assays that use LNA-LAMP to identify commonly encountered IDH1-R132 variants (R132H, R132C, R132G, R132S, R132L) within 40 minutes. These assays require little preanalytical processing, no complex equipment, and yield results which are easily interpreted by the naked eye. Tumor samples are initially subjected to a 5-minute alkaline digest, then added directly to LNA-LAMP reactions containing primers unique to each IDH1-R132 variant. Discrimination between the wild-type and mutant allele is achieved with the integration of locked nucleic acids within primers. A reaction with an IDH1-R132R specific primer serves as the sample positive control, as all tumor lysates are expected to contain wild-type IDH1 DNA. Lysates are incubated for 35 minutes in a hot water bath, followed by visual interpretation using a pH-based colorimetric indicator, where samples positive for the mutation appear a vibrant yellow while those lacking the mutation remain pink. This method has been rigorously tested with patient-derived tumor lysates, yielding reproducible and accurate results, and has an analytical sensitivity of about 20% mutant DNA in a background of wild-type DNA. This sensitivity is comparable to that of Sanger Sequencing while simultaneously offering a more cost-effective and efficient workflow. LNA-LAMP is amenable for use in the intraoperative window to allow patients with an IDH1-R132 mutation the benefit of a maximal surgical resection.

Integrative Genomic and Transcriptomic Analysis Reveals Targetable Vulnerabilities in Angioimmunoblastic T-Cell Lymphoma

Follicular helper T-cell lymphoma of the angioimmunoblastic type (AITL) is associated with dismal prognosis. We performed functional genomic approaches including whole-exome sequencing (WES; n=119), transcriptomic (n=78) and methylation (n=40) analysis. We identified recurrent mutations in known epigenetic drivers ( TET2, DNMT3A, IDH2 R172), and also identified novel ones (TET3, KMT2D). Somatic mutation of all three epigenetic drivers ( TET2, IDH2, and DNMT3A) was associated with poor prognosis (p<.001). Mutations in genes regulating T-cell receptor (TCR) signaling ( CD28 VAV1, FYN, PLCG1) or activation ( RHOA G17V), and regulators of the PI3K pathway (PIK(3)C members, PTEN,PHLPP-1/-2) were also found. Genome-wide DNA-methylation analysis integrated with mRNA expression profiling also revealed epigenetic alterations in genes regulating TCR-RHOA/B/C or PI3K-signaling. TET2 loss was noted in 85% AITLs and was significantly associated with RHOA G17V, CD28 and IDH2 R172mutations. AITLs lacking RHOA G17V tended to have mutations regulating the JAK-STAT pathway ( JAK2, JAK3, STAT1, STAT3, SOCS1). RNA-seq analysis identified fusion transcripts in genes regulating TCR activation (8%), revealed a restricted TCR repertoire in the majority of cases (a=87%, b=72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated association of B-cells in the tumor-milieu with better prognosis (p=.006), while dendritic cells were associated with worse prognosis (p=.001), which was further validated by immunohistochemistry using CD20, CD68, and CD163 antibodies. RNA-seq and corresponding WES analysis of 12 AITL patient-derived-xenografts (PDX) showed that bi-allelic TET2 mutations, DNMT3A mutations or sub-clonal mutations ( PLCG1 PHLPP2) werepropagated in sequential passages. Gene signatures related to T FH (follicular helper) and T CM (central memory) were also well-maintained in secondary passages in PDX models. Gene signatures of late PDX passages (3 rd-5 th) were enriched with genes related to proliferation and metabolic reprogramming, and in an independent cohort of AITLs, high expression of T3/T5 related signatures was associated with worse outcome (p=0.02/p=0.009). Low mRNA expression of PHLPP2 predicted poor prognosis (p=.03) and engineered PHLPP2 loss showed enhanced PI(3)K activation and FOXO1 inactivation in CD4+ T-cells in-vitro. Thus, we defined the genomic landscape for AITL, which is largely characterized by epigenetic alterations, TCR signaling and PI3K/AKT dysregulation, which may be amenable for therapeutic targeting.

Ivosidenib for Patients with Clonal Cytopenia of Undetermined Significance and Mutations in IDH1

Background Myeloid neoplasms (MN), including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), are hematologic malignancies that are highly resistant to therapy and confer a poor prognosis. Recent studies have revealed that hematopoietic stem cells and progenitor cells carrying preleukemic mutations, defined as clonal hematopoiesis (CH), are the cells of origin in MN. CH can occur many years before the diagnosis of MN. Multiple mutations, mutations with a high variant allele frequency (VAF), and mutations in TP53, spliceosome genes, and isocitrate dehydrogenase 1/2 ( IDH1/2) have been identified as high-risk features for CH progression to MN. Individuals with cytopenias who harbor acquired mutations in their blood yet do not meet criteria for hematologic malignancy, an entity called clonal cytopenia of undetermined significance (CCUS), have an even higher risk of developing MN (estimated at 95% over 10 years of follow-up). Thus, despite being almost inevitably destined for progression to malignancy, there are no established preventative therapies for patients with CCUS. The IDH1 inhibitor ivosidenib has been extensively evaluated in clinical studies and has been shown to effectively inhibit the gain-of-function activity of the mutated protein. Clinical data has shown that ivosidenib is safe and well-tolerated with excellent response rates even in patients with relapsed or refractory AML. Individuals with lower co-mutational burden are more likely to have a clinical response and achieve molecular remission with ivosidenib. Since CCUS is characterized by a lower mutation burden compared to AML or MDS, we hypothesize that the use of ivosidenib in IDH1-mutant CCUS will result in a higher and more prolonged clinical response. Study Design and Methods NCT05030441 is a decentralized open label, multicenter pilot study exploring the efficacy of ivosidenib in patients with IDH1-mutant CCUS. The study is offered as a decentralized trial (DCT) where patients across the US can participate with study procedures occurring in the patient's home using telemedicine, mobile phlebotomy services and with local providers. By reducing barriers to trial participation, we expect that the DCT design will improve accessibility for this rare patient population. This represents an important step toward making personalized medicine available to a socio-economically diverse patient population and enables rare mutations to be studied at scale. Patients with CCUS harboring the IDH1 R132 mutation are considered eligible if they do not have any concurrent active malignancy requiring treatment. Patients must have unexplained cytopenia lasting ≥6 months, defined as hemoglobin <10 g/dL, absolute neutrophil count <1.8 × 10 9/L and platelets <100 × 10 9/L, as well as adequate baseline organ function including renal and liver function, as per the National Institutes of Health Common Terminology Criteria for Adverse Events (NCI CTCAE) grading. Recruitment and data collection will be coordinated with local providers for patients that enroll remotely. A total of 15 patients will be accrued to receive ivosidenib 500 mg daily for up to 18 cycles. The primary endpoint is rate of improvement in hematologic parameters using specific cutoffs for erythroid, platelet and neutrophil responses based on the MDS response criteria. The study will be considered positive if at least 6 out of the 15 patients have a hematologic improvement. Secondary endpoints include change in mutant IDH1 VAF, measured at baseline and every 3 cycles on peripheral blood samples, safety of ivosidenib, and AML/MDS-free survival which will be evaluated by descriptive statistics and Kaplan-Meier methodology. Enrollment opened in one US center and by decentralized structure in April 2022, with 3 patients accrued, and is expected to open in other US centers in 2023. If the study endpoints are met, our aim is to expand to a larger population with the goal of determining whether ivosidenib can prevent the development of hematologic malignancy in patients with IDH1-mutant CCUS.

Prognostic value of IDH2R140 and IDH2R172 mutations in patients with acute myeloid leukemia: a systematic review and meta-analysis

BackgroundWhether isocitrate dehydrogenase 2 (IDH2) R140 and R172 gene mutations affect the prognosis of patients with acute myeloid leukemia (AML) is controversial. Here, we performed a meta-analysis to assess their prognostic value.MethodsEligible studies were systematically searched from PubMed, Embase, the Cochrane Library and Chinese databases up to June 1, 2022. We extracted the hazard ratios (HRs) and their 95% confidence intervals (CIs) of overall survival (OS) and progression-free survival (PFS) to carry out a meta-analysis by a fixed effect model or random effect model according to the heterogeneity between studies.ResultsA total of 12725 AML patients from 11 studies were included in this meta-analysis, of which 1111 (8.7%) and 305 (2.4%) had IDH2R140 and IDH2R172 mutations, respectively. The results revealed that both IDH2R140 and IDH2R172 mutations had no significant effect on OS (IDH2R140: HR = 0.92, 95% CI: 0.77–1.10, P = 0.365; IDH2R172: HR = 0.91, 95% CI: 0.65–1.28, P = 0.590) or PFS (IDH2R140: HR = 1.02, 95% CI: 0.75–1.40, P = 0.881; IDH2R172: HR = 1.31, 95% CI: 0.78–2.22, P = 0.306) in AML patients. Subgroup analysis of AML patients with IDH2R140 mutation revealed that studies from the USA (HR = 0.60, 95% CI: 0.41–0.89, P = 0.010) and ≤ 50 years old (HR = 0.63, 95% CI: 0.50–0.80, P = 0.000) had longer OS. However, studies from Sweden (HR = 1.94, 95% CI: 1.07–3.53, P = 0.030) had shorter OS. Meanwhile, subgroup analysis of AML patients with IDH2R172 mutation showed that studies from Germany/Austria (HR = 0.76, 95% CI: 0.61–0.94, P = 0.012) and from Sweden (HR = 0.22, 95% CI: 0.07–0.74, P = 0.014) had longer OS, whereas studies from the UK (HR = 1.49, 95% CI: 1.13–1.96, P = 0.005) and studies with nonmultivariate analysis of data type (HR = 1.35, 95% CI: 1.06–1.73, P = 0.014) had shorter OS. In addition, our study also found that patients with IDH2R140 mutation had significantly longer OS (HR = 0.61, 95% CI: 0.39–0.96, P = 0.032) and PFS (HR = 0.31, 95% CI: 0.18–0.52, P = 0.021) than patients with IDH2R172 mutation, despite some degree of heterogeneity.ConclusionsThis meta-analysis demonstrates that IDH2R140 mutation improves OS in younger AML patients and that the prognostic value of IDH2R172 mutation is significantly heterogeneous. Differences in region and data type have a significant impact on the prognosis of AML patients with IDH2R140 and/or IDH2R172 mutations. Additionally, AML patients with IDH2R140 mutation have a better prognosis than those with IDH2R172 mutations, albeit with some degree of heterogeneity.

CLONAL ARCHITECTURE OF RELAPSED OR REFRACTORY FOLLICULAR HELPER T‐CELL LYMPHOMA: AN ANCILLARY STUDY OF THE ORACLE TRIAL, A LYSA STUDY

Follicular helper T-cell lymphoma (TFHL) results from the oncogenic transformation of a TFH cell, driven by mutations in genes involved in epigenetic regulation (TET2, DNMT3A, IDH2) and T-cell signaling (RHOA). Demonstration of TET2 and DNMT3A mutations in B cells, myeloid cells, or hematopoietic progenitor cells have suggested that TFHL can emerge from clonal hematopoiesis (CH) in a multi-step process. However, the frequency, the extension, and the relevance of such CH in TFHL oncogenesis are unclear. To describe and characterize the clonal architecture of refractory/relapsed (R/R) TFHL, we collected bone marrow samples of patients included in the ORACLE trial (NCT03593018), a phase 3 trial evaluating the oral 5-azacitidine (CC-486) compared to single-agent chemotherapy in patients with R/R TFHL. Bone marrow (BM) cells from 32 patients at inclusion were cultured in methylcellulose supplemented with standard growth factors. BFU-E, CFU-GM, and CFU-GEMM were counted and individually harvested. A total of 517 colonies derived from the 32 patients were then individually genotyped by next-generation sequencing (NGS) using a capture-based panel (limit of detection: 1%) covering 43 genes recurrently mutated in CH and/or TFHL, including TET2, DNMT3A, IDH2, and RHOA. Meanwhile, bulk BM cells from 29 patients were sequenced with the same NGS panel and then compared to the mutations found in cfDNA (31 patients) and tumor biopsies (28 patients). Overall, the frequency of CH and the phylogenetic link between the myeloid progenitors and the TFHL was evaluable in 30 TFHL patients. We successfully genotyped 471/517 (91%) colonies. CH defined by at least one somatic mutation detected in at least one myeloid colony or total BM cells was found in 29/30 (97%) patients. Among the 26/29 patients with mutated myeloid colonies, the median percentage of mutated colonies was 21% (IQR 7%–53%) and 7 had subclonal architecture with a median of 3 different clones. The 2 most frequent mutations were DNMT3A and TET2, detected in 24/30 (80%) and 18/30 (60%) patients' BM, respectively. We confirmed the absence of RHOAG17V and IDH2R172 mutations in BM cells of TFHL patients. While 17/18 patients with a detectable TET2 mutation in CH had a TET2 mutated TFHL, only 13/23 patients with a DNMT3A mutated CH had a DNMT3A mutated TFHL. We thus identified 3 groups of patients according to the distribution of mutations found in the BM and the LN lymph nodes: 16/30 (53.3%) patients had a TFHL clonally derived from CH (CH-related TFHL), 13 (43.3%) had TFHL, and clonally distinct CH (CH-unrelated TFHL), and one patient (3.3%) had undetectable CH. No significant difference in patient outcome was observed regarding the presence of CH-related or unrelated TFHL, but our results are limited by the small number of patients and treatment heterogeneity. Encore Abstract—previously submitted to EHA 2023 Keywords: aggressive T-cell non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Molecular Alterations in IDH 1/2 Genes among Iraqi Adult Acute Myeloid Leukemia Patients

BACKGROUND: The recurrent somatic variations in IDH1/2 genes in AML play imperative roles in epigenetic dysregulation and the pathogenesis of AML, which could be useful prognostic markers for risk stratification. AIM: The aim of the study was to detect the frequency of R132 mutations in the IDH1 gene and R140Q mutation in the IDH2 gene with their treatment outcomes. PATIENTS, MATERIALS AND METHODS: IDH molecular alterations were detected by high-resolution-melting (HRM)-based real-time PCR assay in 56 newly diagnosed AML patients. RESULTS: IDH molecular alterations were identified in 39.3% of AML patients; IDH1 R132 and IDH2 R140Q mutations were present in 32.1% and 12.5% of patients, respectively. The mean age of patients with mutant IDH (52±14.87 years) is higher than in wild type (41.68±20.4 years), P = 0.041. Females were seen in 53% of mutant IDH patients while in the wild-type 73.3% were males (P = 0.038). There were significantly lower mean levels of hemoglobin, absolute neutrophil count, and platelet count in mutant IDH than in wild-type (P = 0.015, 0,.03 and 0.01, respectively). After induction remission therapy, 68.2% of mutated IDH and 64.7% of unmutated IDH patients didn't achieve complete remission (P > 0.05). After 6 months; 59.1% of mutated IDH and 64.7% of unmutated IDH had unfavorable outcomes (P > 0.05). CONCLUSIONS: IDH mutations are common in Iraqi adult AML patients and present in older age and females predominance with lower Hb level, WBC count, absolute neutrophil count, platelet count, and less extramedullary involvement. There is an insignificant association with treatment outcomes.

Clinicopathologic Features of IDH2 R172-Mutated Myeloid Neoplasms.

IDH1 and IDH2 are among the most commonly mutated genes in myeloid neoplasms (MNs). It has been proposed that IDH2 R172 mutations (mR172) define a molecular subtype of acute myeloid leukemia (AML), but the clinicopathologic features of AML with mR172 have not been fully described. We retrospectively identified and characterized all mR172 MNs with increased blasts in our archive for comparison to a similar number of MNs with IDH2 R140 (mR140) and IDH1 R132 (mR132) mutations (n = 39). mR172 cases had lower leukocyte counts and bone marrow cellularity than did non-mR172 cases. mR172 MNs often displayed blasts with highly invaginated, cleaved nuclei and typically expressed CD34, HLA-DR, CD117, and CD13 but often with diminished CD33. mR172 cases often had co-occurring mutations in myelodysplasia-associated genes and/or an adverse karyotype. Despite frequent adverse-risk genetic changes, in our cohort mR172 cases had significantly improved overall survival vs non-mR172 cases (P = .01), and we validated that mR172 was associated with improved survival in an independent large data set. We show that MNs with mR172 represent a morphologically and phenotypically distinct subtype, which in our cohort exhibited relatively favorable survival that is not captured in current AML risk assignment.

Comparison of DNA Sequencing and Multispecific Monoclonal Antibody (Msmab-1) based Immunohistochemical Detection of IDH1/2 Mutation in Acute Myeloid Leukemia Cases.

Background: The IDH1/2 mutation is an important epigenetic modifier involved in the pathogenesis of AML. It is associated with variable prognosis in AML cases. Lack of proper molecular diagnostic infrastructure and high cost limits the routine use of PCR with sequencing as routine diagnostic methodology. The aim of this study was to find the prevalence of IDH mutation in AML cases using both PCR with sequencing and Immunohistochemistry method.
 Methods: We evaluated 60 patients registered at KGMU, Lucknow for diagnosis and treatment of AML. PCR followed by sequencing was done. IHC staining of the IDH1/2 mutation was performed on all cases using bone biopsy or clot section (in cases of pediatric AML cases).
 Results: Out of the total 60 patients of AML 4(6.7%) patients had IDH1R312 mutation and 5(8.3%) Patients had IDH2R172 mutation. IDH2 R140 mutation was not detected in any sample. On immunohistochemistry analysis 10 cases showed positive staining against anti IDH1/2 mutant (R132/R172) antibody, clone MsMab-1 with a sensitivity of 77.8% and specificity of 94.1%.
 Conclusion: IHC could be an alternative method to direct Sanger sequencing for IDH1/2 mutation detection in AML cases. However, the antibody used in the study is not effective for individual assessment of IDH1 and IDH2 mutation.
 Keywords: Immunohistochemistry, Sanger sequencing, IDH1/2 Mutation, Acute Myeloid Leukemia

Mutation Analysis and Clinicopathological Implications of Isocitrate Dehydrogenase 1/2 Mutation in Acute Myeloid Leukemia Patients in North India: A Tertiary Centre Based Study

Acute myeloid leukemia has been classified on the basis of morphology, cytochemistry and genetic profile. IDH gene is an epigenetic modifier whose mutation is involved in pathogenesis of various malignancies. This study was planned to study the prevalence of IDH1/2 mutation in denovo AML cases at a tertiary care Centre in North India and also to study its clinicopathological effect in these cases. We evaluated 60 patients registered at our Centre for diagnosis and treatment of AML. Routine investigations, bone marrow examination, flow cytometry were done followed by PCR and sequencing. Out of the total 60 patients of AML 4(6.7%) patients had IDH1R312 mutation and 5(8.3%) Patients had IDH2R172 mutation. IDH2 R140 mutation was not detected in any sample. IDH mutation was significantly associated with high risk AML group. No significant clinicopathological correlation was seen. In this study significant association was observed between IDH mutation and high risk AML cases. There was no paediatric case with IDH mutation. IDH mutation and AML might have an age related association.

Molecular Features Associated with Response to Combination Therapy with Enasidenib (ENA) Plus Azacitidine (AZA) in Newly Diagnosed IDH2-Mutated Acute Myeloid Leukemia (AML)

Introduction: Isocitrate dehydrogenase 2 (IDH2) mutations (mIDH2), which typically occur at arginine residues R140 and R172, induce production of the oncometabolite 2-hydroxyglutarate (2-HG), causing aberrant hypermethylation of DNA and histones and blockade of differentiation. In vitro, combining ENA, an mIDH2 inhibitor, plus AZA showed synergistic effects, leading to greater-than-additive reductions in DNA methylation and enhanced differentiation. In a phase 2 trial (NCT02677922), combination treatment (Tx) with ENA + AZA significantly improved morphologic response rate compared with AZA monotherapy in adult patients (pts) with mIDH2 newly diagnosed (ND) AML. We investigated the association between molecular characteristics and clinical outcomes with ENA + AZA and AZA only in that trial. Methods: Total 2-HG in peripheral blood was assessed by liquid chromatography-mass spectrometry (Covance) at baseline (BL), cycle (C) 1 day (D) 15, C2D1, C2D15, and on D1 of every other cycle from C3 to C19. IDH2 variant allele frequency (VAF) was quantified in DNA from bone marrow mononuclear cells by digital PCR (Sysmex) and co-occurring mutations were identified by targeted next-generation sequencing using a 37-gene myeloid panel (ArcherDx). Gene VAFs (including IDH2) were assessed at BL and on D1 of C2, C3, C5, C11, and C17. Clinical response categories included complete remission (CR), incomplete response (IR; ie, non-CR response), or no response (NR; ie, stable or progressive disease), per IWG 2003 response criteria for AML. Statistical analyses were performed using GraphPad Prism v.8.0.0 and R v.4.1.0. Results: In all, 101 pts were randomized to ENA + AZA (n = 68) or AZA only (n = 33). At BL, most pts (74%) had an IDH2-R140 mutation. SRSF2, RUNX1, FLT3-ITD, NPM1, and SF3B1 were significantly (P ≤ 0.05) preferentially co-mutated with IDH2-R140 (vs R172), and DNMT3A and ETV6 were preferentially co-mutated with IDH2-R172 (vs R140). BL 2-HG levels and IDH2 VAFs were similar between treatment arms and IDH2 variants, and were not significantly different between clinical response categories (CR/IR/NR). In the ENA + AZA arm, median 2-HG was significantly reduced from BL by C1D15 (n = 60; -91%; P ≤ 0.001) with maximal reduction at C5 (n = 43; -95%; P ≤ 0.001). IDH2 VAF was significantly reduced from BL by C3 (n = 39; -23%; P ≤ 0.01) with maximal reduction at C17 (n = 7; -99%; P ≤ 0.05). In the AZA-only arm, 2-HG was not significantly reduced from BL at any time and IDH2 VAF was significantly reduced only at C11 (n = 7; P < 0.05). Marked IDH2 VAF reductions were observed primarily in complete responders, reaching undetectable levels (ie, VAF < 1%) in 50% (18/36) of pts who achieved CR with ENA + AZA (P = 0.0005 vs IR/NR), and the rate of undetectable IDH2 was higher in the IDH2-R140 subgroup vs R172 (59% vs 22%, respectively). In the AZA-only arm, 2/4 CR pts (both IDH2-R140) had undetectable IDH2 mutations (Figure A). In both Tx arms, achievement of clinical response was associated with VAF reductions in the combined DNA methylation gene pathway (IDH1/2, DNMT3A, TET2) and receptor tyrosine kinase (RTK) pathway (KIT, CSF3R, FLT3, JAK2), with maximal reductions from BL of ~85% and 100%, respectively, by C11 with ENA + AZA (n = 19 and n = 15; both P ≤ 0.001), and maximal reductions in the AZA-only arm of 50% at C17 (n = 2) for DNA methylation and 100% at C5 (n = 4) for RTK (statistical comparisons not reported due to small numbers). Pts who achieved CR with ENA + AZA also showed a significant VAF reduction in the RAS gene pathway (CBL, PTPN11, N/KRAS), with maximal reduction from BL of 100% by C5 (P ≤ 0.001); the AZA-only arm was not evaluable because very few pts responded. Among all pts with RAS pathway mutations, ENA + AZA significantly prolonged event-free survival vs AZA-only (P = 0.002) (Figure B). Conclusions: Outcomes in this mIDH2 ND-AML cohort demonstrate robust clinical validation of the synergistic effects of ENA and AZA seen in vitro. BL co-mutational patterns differed between the IDH2-R140 and R172 variants. Rapid reductions in 2-HG and IDH2 VAF were observed during combination Tx with ENA + AZA. Significant differences in undetectable IDH2 VAF were observed between complete and non-complete responders and were more common in pts with IDH2-R140 mutations than with R172. Additional research is needed to further elucidate the benefits of ENA + AZA in pts with RAS pathway mutations, which are associated with resistance to ENA and AZA monotherapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Atypical cartilage in type II germ cell tumors of the mediastinum show significantly different patterns of IDH1/2 mutations from conventional chondrosarcoma

AbstractNeoplastic cartilage is a common component of teratomas in type II germ cell tumors. Although IDH1/2 mutations have been well-described in somatic cartilaginous tumors, ranging from benign enchondromas to highly aggressive dedifferentiated chondrosarcomas, the presence of IDH1/2 mutations in cartilaginous neoplasms arising from germ cell tumors has not been previously investigated. To better understand the relationship between these tumors and their bone/soft tissue counterpart, we studied the IDH1/2 mutational status of 20 cases of primary mediastinal mixed germ cell tumors with areas of readily identifiable cartilaginous differentiation. Our study found that cartilaginous lesions arising in germ cell tumors have a different frequency and distribution of IDH1/2 mutations compared to those at somatic sites. We identified IDH1/2 mutations in only 15% (3/20) of cases, compared to a frequency in the literature among differentiated chondroid tumors of bone and soft tissue of 54%, a highly significant decreased frequency (p = 0.0011; chi-square test). Furthermore, they were exclusively IDH2 R172 mutations that occurred at a non-significant, increased frequency in the germ cell tumor group compared to conventional chondrosarcoma (15% vs. 5%, respectively, p > 0.05, chi-square test). The unexpected finding, therefore, was entirely attributable to the absence of IDH1 R132 mutation in chondroid neoplasia of germ cell origin (p < 0.00001, Fisher exact test). Our results suggest that a subset of cartilaginous lesions arising within type II germ cell tumors have a similar oncogenic mechanism to their bone/soft tissue counterpart but that the majority form using different oncogenic mechanisms compared to their somatic counterparts.

Evaluation of two new highly multiplexed PCR assays as an alternative to next-generation sequencing for IDH1/2 mutation detection.

IDH1 and IDH2 somatic mutations have been identified in solid tumors and blood malignancies. The development of inhibitors of mutant IDH1 and IDH2 in the past few years has prompted the development of a fast and sensitive assay to detect IDH1R132 , IDH2R140 and IDH2R172 mutations to identify patients eligible for these targeted therapies. This study aimed to compare two new multiplexed PCR assays - an automated quantitative PCR (qPCR) on the PGX platform and a droplet digital PCR (ddPCR) with next-generation sequencing (NGS) for IDH1/2 mutation detection. These assays were evaluated on 102 DNA extracted from patient peripheral blood, bone marrow and formalin-fixed paraffin-embedded tissue samples with mutation allelic frequency ranging from 0.6% to 45.6%. The ddPCR assay had better analytical performances than the PGX assay with 100% specificity, 100% sensitivity and a detection limit down to 0.5% on IDH1R132 , IDH2R140 and IDH2R172 codons, and a high correlation with NGS results. Therefore, the new highly multiplexed ddPCR is a fast and cost-effective assay that meets most clinical needs to identify and follow cancer patients in the era of anti-IDH1/2-targeted therapies.

Overall survival by IDH2 mutant allele (R140 or R172) in patients with late-stage mutant-IDH2 relapsed or refractory acute myeloid leukemia treated with enasidenib or conventional care regimens in the phase 3 IDHENTIFY trial.

7005 Background: IDH2 gene mutations (m IDH2) occur in up to ̃20% of patients (pts) with acute myeloid leukemia (AML), most commonly as R140Q (in ̃75% of cases) or R172K (̃25%) point mutations. The functional effects and prognostic relevance of m IDH2-R140 and m IDH2-R172 can vary (Papaemmanuil 2016). In the randomized, phase 3 IDHENTIFY trial, enasidenib (ENA), an oral mIDH2 inhibitor, did not significantly improve overall survival (OS) vs conventional care regimens (CCR) as salvage treatment (Tx) for older pts with m IDH2 relapsed/refractory (R/R) AML in ITT analysis, but a trend for improved OS with ENA was detected in pts with IDH2-R172 mutations. We further investigated OS and correlative biomarkers in IDHENTIFY pt subgroups defined by m IDH2 variant (R140/R172). Methods: This open-label trial (NCT02577406) enrolled pts ≥ 60 years of age who had received 2 or 3 prior AML Tx. Pts were preselected to a CCR (azacitidine, intermediate- or low-dose Ara-C, or supportive care), and were then randomized 1:1 to ENA 100 mg/d or CCR in 28d cycles. Co-occurring gene mutations were identified by targeted next-generation sequencing (37-gene panel) of bone marrow mononuclear cell (BMMC) DNA. Total 2-HG levels were determined by LC/MS. Results: Of 319 pts enrolled, 88 pts (28%; 43 ENA, 45 CCR) had m IDH2-R172 and 229 (72%; 115 ENA, 114 CCR) had m IDH2-R140. Median baseline (BL) 2-HG levels were similar between Tx arms and m IDH2 variant subgroups, as were IDH2 variant allele frequencies. Pts with m IDH2-R172 had fewer median BL co-mutations (4 [range 2–8]) than did pts with m IDH2-R140 (5 [1–11]) ( P &lt; 0.0001). The most frequently co-occurring mutations were SRSF2 and RUNX1 in the R140 cohort (59% each) and DNMT3A in the R172 cohort (57%). Compared with the R172 cohort, the R140 group was enriched with SRSF2, FLT3 (-ITD/-TKD), NPM1, RUNX1, and JAK2 mutations, whereas DNMT3A and TP53 mutations were more common in the R172 group. In Cox multivariate analysis including m IDH2 variant (R140/R172), DNMT3A mutation status, and number of gene mutations at BL, m IDH2-R172 was significantly ( P = 0.04) correlated with improved OS (vs. R140) in the ENA arm, whereas the number of BL gene mutations was significantly ( P &lt; 0.01) associated with OS in the CCR arm. Median OS in the R172 subgroup was 14.6 mo with ENA vs 7.8 mo with CCR (HR, 0.59 [95%CI 0.35-0.98]; P = 0.039) and 1-yr survival rates were 62% and 30%, respectively. In m IDH2-R140 pts, median OS was 5.7 mo in both Tx arms (0.93 [0.70-1.24]; P = 0.61), and 1-year survival rates were 29% and 25% with ENA and CCR, respectively. Conclusions: Mutational burden and co-mutational profiles differed between pts with m IDH2-R140 and m IDH2-R172 R/R AML. ENA improved survival outcomes for pts with IDH2-R172 mutations, with median OS and 1-year survival rate approximately double those in the CCR arm. Clinical trial information: NCT02577406.

Monitoring of Dynamic Changes and Clonal Evolution in Circulating Tumor DNA From Patients With IDH-Mutated Cholangiocarcinoma Treated With Isocitrate Dehydrogenase Inhibitors.

IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.