Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) were recently demonstrated in acute myeloid leukemia (AML), but their prevalence and prognostic impact remain to be explored in large extensively characterized AML series, and also in various other hematologic malignancies. Here, we demonstrate in 893 newly diagnosed cases of AML mutations in the IDH1 (6%) and IDH2 (11%) genes. Moreover, we identified IDH mutations in 2 JAK2 V617F myeloproliferative neoplasias (n = 96), a single case of acute lymphoblastic leukemia (n = 96), and none in chronic myeloid leukemias (n = 81). In AML, IDH1 and IDH2 mutations are more common among AML with normal karyotype and NPM1mutant genotypes. IDH1 mutation status is an unfavorable prognostic factor as regards survival in a composite genotypic subset lacking FLT3ITD and NPM1mutant. Thus, IDH1 and IDH2 mutations are common genetic aberrations in AML, and IDH1 mutations may carry prognostic value in distinct subtypes of AML.
Somatic mutations in the genes encoding the isocitrate dehydrogenases IDH1 and IDH2 were revealed in more than 70% of World Health Organization grade 2 and 3 astrocytomas, oligodendrogliomas, and glioblastomas.1-3 Mutations in IDH1 and IDH2 were mutually exclusive and affected the arginines on position 132 of IDH1 and position 172 of IDH2.3 Patients with malignant gliomas with IDH1 or IDH2 mutations showed a better response to therapy than those with wild-type IDH genes.3 Mutations in these residues of IDH significantly disturb the function of both isocitrate dehydrogenases, as demonstrated by impaired production of nicotinamide adenine dinucleotide phosphate.3,4 In acute myeloid leukemia (AML), mutant IDH enzyme activity results in accumulation of the cancer-associated metabolite 2-hydroxyglutarate.5,6
Recently, acquired mutations in the gene encoding IDH1 were identified in 8%7 and 5.5%8 of newly diagnosed AML cases. IDH1 mutations were significantly associated with normal karyotype and NPM1 mutations.7,8 Overall, the IDH1 mutation status did not suggest a relationship with overall survival (OS), but the sample sizes were limited in these studies.7,8 However, a trend for an adverse effect on OS was suggested in normal karyotype AML with NPM1wild-type.7
The prevalence and prognostic value of IDH mutations in AML, as well as other hematologic malignancies, remain to be further established. In this study, we determined the frequencies of both IDH1 and IDH2 mutations in cohorts of AML, acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and JAK2 V617F myeloproliferative neoplasia (MPN). In a cohort of 893 cases of AML, we investigated their distribution in relationship with cytogenetic and molecular risk categories as well as recurrent gene mutations commonly apparent in AML, and we evaluated the impact of IDH mutations on treatment outcome.
Bone marrow aspirates or peripheral blood samples of cohorts of patients with various hematologic malignancies were collected after written informed consent in accordance with the Declaration of Helsinki. All experiments described were approved by the Erasmus University Medical Center Institutional Review Board. AML, ALL, and CML patients were treated according to the HOVON (Dutch-Belgian Hematology-Oncology Cooperative Group) AML protocols HO04, HO04A, HO29, HO42, HO42A, and HO43, ALL protocols HO18, HO37, HO70, and HO71, and CML protocol HO51 (http://www.hovon.nl). The MPN samples were collected, and the JAK2 V617F mutation was determined in our routine molecular diagnostics facility.
IDH1 and IDH2 mutations in AML, refractory anemia with excess blasts, ALL, CML, and JAK2 V617F MPN were determined by cDNA amplifications using FW1-IDH1 cDNA WAVE 5′-CTTCAGAGAAGCCATTATCTG-3′ and REV2-IDH1 cDNA WAVE 5′-TCACTTGGTGTGTAGGTTATC-3′ (IDH1 R132), FW1-IDH2 cDNA WAVE 5′-GAACTATCCGGAACATCCTG-3′ and REV2-IDH2 cDNA WAVE 5′-CTTGACA- CCACTGCCATC-3′ (IDH2 R172), or FW-IDH2-Ex4 5′-GTTCAAGCTGAAGAAGATGTG-3′ and REV-IDH2-Ex5-6 cDNA WAVE 5′-TGAGATGGACTCGTCGGTG-3′ (IDH2 R140). All polymerase chain reaction (PCR) reactions were carried out at an annealing temperature of 60°C in the presence of 25mM deoxynucleoside triphosphate, 15 pmol primers, 2mM MgCl2, Taq polymerase, and 1 times buffer (Invitrogen). Cycling conditions were as follows: 1 cycle 5 minutes at 94°C, 30 cycles 1 minute at 94°C, 1 minute at annealing temperature, 1 minute at 72°C, and 1 cycle 7 minutes at 72°C. All IDH1 and IDH2 reverse-transcribed PCR products were subjected to denaturing high performance liquid chromatography (dHPLC) analyses using a Transgenomic WAVE system. Samples were run at 61.4°C (IDH1 R132), 57.7°C (IDH2 R172), or 6.1°C (IDH2 R140). PCR products showing aberrant dHPLC profiles were purified using the Multiscreen-PCR 96-well system (Millipore) followed by direct sequencing with the appropriate forward and reversed primers using an ABI-PRISM3100 genetic analyzer (Applied Biosystems). PCR products were sequenced with FW-IDH1 cDNA WAVE (IDH1 R132), FW-IDH2 cDNA WAVE (R172 IDH2), or FW-IDH2-Ex4 (R140 IDH2). We validated this strategy using 350 cases of de novo AML that were previously analyzed using PCR on genomic DNA followed by direct sequencing.
Information on the IDH1 and IDH2 mutation status of all AML cases is available as supplemental 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article) and of all AML cases that were previously gene expression–profiled at the Gene Expression Omnibus (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov/geo, accession no. GSE6891).
The relation between IDH mutations and various patient characteristics was determined by the Student t test, equal variances not assumed (continuous variables) and the Fisher exact test (categorical variables).
We distinguished the following cytogenetic risk categories: (1) favorable: t(8;21), inv(16) or t(15;17); (2) unfavorable: inv(3)/t,3,3 t(6;9), 11q23 abnormalities other than t(9;11), −5, 5q−, −7, 7q−, or t(9;22) (cytogenetically abnormal [CA] unfavorable); (3) very unfavorable: monosomal karyotypes9 ; (4) intermediate-risk I: cytogenetically normal (CN) and (V); intermediate-risk II: the remaining AML cases (CA rest).
OS endpoints were death (failure) and alive at last follow-up (censored), as measured from entry onto trial. Event-free survival (EFS) endpoints were remission induction failure, disease relapse, or death from any cause, measured from entry onto trial. Distribution estimations and survival distributions of OS and EFS were calculated by the Kaplan-Meier method and the log-rank test.
Results and discussion
To determine the frequencies of IDH1 and IDH2 mutations in AML, we screened cDNA of 893 newly diagnosed AMLs by reverse-transcribed PCR/dHPLC followed by direct sequencing (1). IDH1 mutations were identified in 55 AML cases (6%) and IDH2 mutations in 97 cases (11%). A total of 152 (17%) mutations in either IDH1 or IDH2 were apparent in 150 cases. IDH1 and IDH2 mutations were mutually exclusive except in 2 cases of AML (nos. 7272 and 10400) with dual mutations in IDH1 and IDH2. The R132H mutation was the most prevalent mutation in IDH1 (n = 31, 56%). In addition, various other IDH1 protein mutations were identified (R132C, n = 15, 28%; R132G, n = 6, 11%; R132L, n = 3, 6%). We identified 74 IDH2 R140Q mutations,6,14 22 cases with an IDH2 R172K mutation, and a single case with a R172M substitution (no. 7309).
In addition to AML, we investigated the prevalence of IDH1 and IDH2 mutations in JAK2 V617F MPN (n = 96), ALL (n = 96), including cases with BCR-ABL (n = 21), MLL fusions (MLL-AF4, MLL-AF9, or MLL-ENL) (n = 6), SIL-TAL (n = 2), E2A-PBX (n = 2), and SET-NUP (n = 1) and CML in chronic phase (n = 81). We identified a mutation in IDH1 (R132C) and IDH2 (R140Q) in 2 independent cases of JAK2 V617F MPN, indicating that these mutations can be present before leukemic transformation.14 In addition, we identified an IDH2 R140Q mutation in a single case of ALL. No IDH mutations were present in CML.
AML with IDH1mut and IDH2mut are more prevalent at older age and present with significantly higher average platelet counts at diagnosis compared with AML with IDHwild-type (1). IDH1 and IDH2 mutations were significantly more frequently present among cytogenetically normal AML (P < .001, CN; 1). In addition, IDH mutations appear to be significantly associated with NPM1mutant (P < .001; 1). The specificity of the pathogenetic involvement of IDH gene mutations in AML is also suggested by the observations that they did not significantly associate with various other recurrent mutations (ie, FLT3ITD [internal tandem duplication] FLT3TKD [tyrosine kinase domain], N-RAS, K-RAS, or CEBPA gene mutations).
To investigate the prognostic value of IDH1 mutations, 829 AML patients younger than 60 years were considered for survival analysis. The median follow-up of these patients is 33.2 months. The OS of patients with AML with or without IDH1mutant or IDH2mutant genotypes among the entire series of patients with AML did not differ (P = .05; Figure 1A). OS of IDHmutant patients in the subgroups with intermediate-risk cytogenetics (P = .13), normal karyotypes (Figure 1B, P = .11), and intermediate-risk cytogenetics with FLT3wild-type (P = .32), FLT3ITD (P = .09), NPM1wild-type (P = .06), or NPM1mutant (P = .25) genotypes were not significantly different from those with IDHwild-type. Similar results were obtained in analyses as regards EFS. Of note, IDHmutant patients within the AML subtype NPM1wild-type were associated with an inferior EFS (P = .02).
Because there is significant overlap in the occurrence of mutations in NPM1mutant and FLT3ITD, we also assessed the value of IDH gene mutations in each of the 4 composite variants, but no significant prognostic effect of IDH mutations was apparent as regards OS or EFS among FLT3ITD/NPM1mutant (OS, P = .24; EFS, P = .24) and FLT3wild-type/NPM1mutant (OS, P = .77; EFS, P = .75; Figure 1C and 1D, respectively). Only 8 AML patients with IDH mutations were identified among FLT3ITD/NPM1wild-type, which prevents reliable survival analysis. However, among the FLT3wild-type/NPM1wild-type AML subtype, the presence of IDH1 mutations (n = 14 cases) predicted for both significantly reduced OS (Figure 1E, P = .032) and EFS (P = .005). These data suggest an only moderate prognostic effect of IDH1mut because it is not evident in genetically heterogeneous series of AML, but only in intermediate-risk AML in the absence of NPM1mut and FLT3ITD. Apparently, the NPM1mutant and FLT3ITD markers override the prognostic effect of IDH1mut. In this regard, we wish to note that the numbers of the 4 composite subgroups, even though this study was performed in a relatively large series of AML, become obviously increasingly small, which limits the statistical power of these analyses and prohibits the interesting exploratory analysis for IDH1 and IDH2 mutations separately.
Acquired IDH gene mutations (ie, not only in IDH1 but also IDH26 ) are common abnormalities in AML. The results of the current study demonstrate that the frequency of IDH2 mutations exceeds those of IDH1. Together, IDH1 and IDH2 mutations account for a considerable frequency of approximately 17% in adult AML. The presence of IDH gene mutations appears to be associated with normal karyotypes and NPM1 mutations. The observation that IDH1 mutations appear to correlate with significantly inferior outcome in patients FLT3wild-type/NPM1wild-type AML requires confirmation in future studies.
The online version of this article contains a data supplement.
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The authors thank Dr Gert J. Ossenkoppele (Free University Medical Center, Amsterdam, The Netherlands), Dr Jaap Jan Zwaginga (Sanquin, Amsterdam, The Netherlands), Dr Edo Vellenga (University Hospital, Groningen, The Netherlands), Dr Leo F. Verdonck (University Hospital, Utrecht, The Netherlands), Dr Gregor Verhoef (Hospital Gasthuisberg, Leuven, Belgium), and Dr Matthias Theobald (Johannes Gutenberg-University Hospital, Mainz, Germany), who provided AML cell samples; our colleagues from the stem cell transplantation and molecular diagnostics laboratories for storage of the samples and molecular analyses; and H. Berna Beverloo (Erasmus University Medical Center) for cytogenetic analyses.
This work was supported by the Dutch Cancer Society (Koningin Wilhelmina Fonds).
Contribution: S.A. performed research, analyzed data, and wrote the paper; S.L. performed research and analyzed data; F.G.K., A.S., J.E.K., A.Z., and A.W.R. performed research; W.J.L.v.P. analyzed data; B.L. designed research, analyzed data, and wrote the paper; and P.J.M.V. designed and performed research, analyzed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Peter J. M. Valk, Erasmus University Medical Center Rotterdam, Department of Hematology, Ee1391a, Dr Molewaterplein 50, 3015 GE Rotterdam Z-H, The Netherlands; e-mail: email@example.com.