Molecular alterations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) metabolic genes and additional genetic mutations in newly diagnosed acute myeloid leukemia patients

Leukemic samples from 230 newly diagnosed AML cases were consecutively recruited into the study. Clinical and biological characteristics were collected including clinical history, complete blood counts, peripheral blood (PB) smear, bone marrow (BM) studies, flow cytometric immunophenotyping, and chromosome analysis. Mononuclear cells (MNC) were isolated from the leukemic samples by Ficoll-Hypaque density-gradient centrifugation and subsequently used for molecular analysis. Twenty consented normal individuals were used as controls. Patients were treated according to the standard AML regimen which included idarubicin and cytarabine induction therapy followed by high-dose cytarabine-based consolidation phase. This study was approved by the Ethical Committee for Human Research, Faculty of Medicine Siriraj Hospital, Mahidol University.

Genomic DNA was extracted using standard phenol-chloroform method or Gentra Puregene Blood Kit (Qiagen, Hidden, Germany) according to the manufacturer's protocol. DNA amplicons harbouring exon 4 of IDH1 and IDH2 were amplified by polymerase chain reaction (PCR) using the primer pair; IDHIf (5'-AGCTCTATATGCCATCACTGC-3'), IDH1r (5'-AACATGCAAAATCACATTATTGCC-3'), IDH2f(5'- AATTTTAGGACCCCCGTCTG-3'), and IDH2r (5'-CTGCAGAGACAAGAGGATGG-3') [13]. PCR reactions were performed in a total volume of 20 μL containing 50 ng of genomic DNA, PCR master mixture consisting of 1x Phusion^®HF Buffer (F-520), 200 μM dNTPs, 0.5 μM of each primer, 0.02 U/μL Phusion^® DNA polymerase, and Milli-Q water. The PCR was carried out in a Perkins Elmer PCR2400 thermal cycler (Applied Biosystems, Foster City, CA) using the following steps: initial denaturation at 98°C for 30 seconds (sec), 35 cycles at 98°C for 10 sec, 60°C for 30 sec. and 72°C for 30 sec, and final extension at 72°C for 5 minutes (min). Both amplicons were screened for heterozygous mutations by denaturing high-performance liquid chromatography (DHPLC) on a WAVE 3500HT with DNASep^® HT cartridge technology (Transgenomic Inc, Omaha, NE, USA). The optimized condition and temperature were predicted by the Navigator™ software to determine chromatographic peak pattern. PCR crude sample was injected into DHPLC column and the optimal temperature for IDH1 was 58.5°C and IDH2 was 64°C. Each DHPLC chromatogram was compared to a wild-type reference. The sensitivity of our assay was determined by performing a dilution series containing a different percentage (%) of mutant and wild-type IDH concentrations. Abnormal DHPLC peaks could be clearly detected in 50%, 20%, 10%, 5%, and 3.33% dilutions. The mutational chromatograms were re-amplified in an independent PCR reaction and further subjected to direct sequencing. The sequences were compared to the wild-type IDH1 and IDH2 cDNA (GenBank Accession number, NM_005896.2 and NM_002168.2, respectively) [25].

Mutational analyses of FLT3, PML-RARA, RAS, AML1, and NPM1 were performed according to our previously described method [35‐39]. Briefly, the DNA or RNA was extracted, then the genes of interest were amplified and detected by gel electrophoresis (FLT3), denaturing high performance liquid chromatography (DHPLC) (NPM1), single-strand conformational polymorphism (SSCP) (RAS and AML1). For PML-RARA, the cDNA was synthesized and reverse transcriptase-polymerase chain reaction (RT-PCR) performed.

The relationship between IDH mutations and various patient characteristics such as age, Hb count, WBC count, platelet count, and percentages of blasts was determined by the student t-test, equal variances not assumed for continuous variables. Categorical variables such as FAB classification, cytogenetics, and test. The Kaplan-Meier method and the log-rank test were utilized to estimate the distribution of OS [40]. For all analyses, a p-value of less than 0.05 was considered statistically significant. All reported p-values were 2-sided.

In a total cohort of 230 consecutive AML patients (36 acute promyelocytic leukemia (APL) and 194 non-APL), 44 patients with IDH mutation were identified (19.13%) by DHPLC showing abnormal chromatogram patterns that were different from the wild-type profiles. These mutations were further confirmed by sequencing analysis (Figure 1). Twenty IDH1 mutations (8.7%) included six missense mutations leading to amino acid (AA) substitution with different frequencies: c.G395A; p.R132H in 8 cases (40.0%), c.C394T; p.R132C in 6 cases (30.0%), c.C394A; p.R132S in 2 cases (10.0%), c.C394G; p.R132G in 2 cases (10.0%), c.G395T; p.R132L in 1 cases (5.0%), and c.A297G; p.I99M in 1 cases (5.0%) (Table 1). In addition, one silent polymorphism (c.315 G > T; IDH1 ^G105G ) was observed in 3 patients (1.30%). Twenty-four IDH2 mutations (10.4%) included two missense mutations with different frequencies: c.G419A; p.R140Q in 20 cases (83.3%) and c.G515A; p.R172K in 4 cases (16.7%). All patients with the mutations were heterozygous and retained a wild-type allele (Figure 1). No mutated patients harbored dual mutations of both genes, indicating that these mutations are mutually exclusive.

Twenty IDH1-mutated patients showed no significant differences in age, hemoglobin level, WBC counts, platelet counts or percentages of blasts as compared to the IDH1 wild-type group, although a trend towards more females was observed (15 females vs 5 males; P = .058). Twenty-four IDH2- mutated patients also showed no significant differences in sex, WBC counts or percentages of blasts but an older age (49.5-year vs. 43-year; P = .001), a higher platelet count (59 vs. 45 × 10⁹/L; P = .048), and a trend towards higher hemoglobin level (9.25 g/dL vs. 7.7 g/dL; P = .059) were observed in the IDH2-mutated group as compared to the wild-type group (Tables 2 and 3). Among IDH1-mutated cases, the majority was classified as AML with maturation (AML M2) (13/20 cases, 65%) followed by APL (5/20 cases, 25%). None of the cases with APL, acute monoblastic or monocytic leukemia, acute erythroid leukemia, and acute megakaryoblastic leukemia was found among IDH1-mutated group. Similarly, AML with maturation (AML M2) was the most common subtype among the IDH2-mutated group (11/24 cases, 46%) (Table 3). The second most common subtypes were APL (6/24 cases, 25%) and acute myelomonocytic leukemia (6/24 cases, 25%). The OS of patients with or without IDH1 or IDH2 mutations did not differ among the entire series of AML patients and AML patients with normal cytogenetics (P = 0.200 and 0.272, respectively). No significant difference was demonstrated in subgroups of younger patients (age < 60) and patients with/without additional mutations as compared to the wild-type patients (P = 0.471 and 0.812, respectively).

Cytogenetic information was available in 226 of 230 patients, 126 patients (55.75%) of whom had normal karyotype and 100 patients (44.25%) had an aberrant karyotype. Of 20 AML cases with IDH1 mutation, 11 cases had normal karyotype (55%). In the aberrant karyotype, we found 5 cases with t(15;17) chromosome translocation, 2 cases with del(9q), and 1 case with trisomy 8 (Table 2). Of 24 cases with IDH2 mutation, 12 cases had normal karyotype (50%). In the aberrant karyotype, we observed 6 cases with t(15;17), 2 cases were IDH2 R172K harboring trisomy 11, 1 case with trisomy 8, 1 case with t(8;21), and 1 case with del(12)(p12.1p13.1) (Table 3).

IDH1 mutations were significantly associated with NPM1 mutation as compared with wild-type cases (14/19, 74% vs. 45/185, 24%; P < 0.001)(Table 4). Some cases had additional mutations including FLT3-ITD, FLT3-TKD, NRAS, and PML-RARA. IDH2 mutations were also significantly associated with NPM1 mutations when compared with the respective wild-type cases (12/20, 60% vs. 47/184, 26%; P < 0.001)(Table 4). AML1 mutations were not observed in any mutated IDH1 or IDH2 patients.