High incidence of <i>IDH</i> mutations in acute myeloid leukaemia with cuplike nuclei (2011)

IDH1 and IDH2 encode cytoplasmic/peroxisomal isocitrate dehydrogenase 1 (IDH1) and mitochondrial isocitrate dehydrogenase 2 (IDH2) respectively, which are homodimeric NADP+-dependent enzymes that catalyse the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), generating NADPH from NADP+. Somatic heterozygous mutations of IDH1 and IDH2 have been identified in 70–80% of low-grade diffuse gliomas and glioblastomas, and in a smaller subset of acute myeloid leukaemia (AML) cases (Yan et al, 2009; Marcucci et al, 2010). These mutations are oncogenic and almost exclusively occur at IDH1 codon 132 and IDH2 codons 140 and 172, where they affect the respective arginine residues. These mutations reduce the enzymes’ affinity for isocitrate and increase the affinity for α-KG and NADPH, thereby preventing oxidative decarboxylation of isocitrate to α-KG and facilitating reduction of α-KG to D-2-hydroxyglutarate (D-2-HG) (Ward et al, 2010). While the incidence of IDH mutations in unselected AML is low, these mutations are clustered in AML with normal karyotype and nucleophosmin gene (NPM1) mutations (Marcucci et al, 2010; Ward et al, 2010). We and others have described a rare morphological subset of AML with cuplike nuclei (AML-CN) (Kroschinsky et al, 2008; Chen et al, 2009) that represents approximately 1% of AML cases and is associated with normal karyotype and NPM1 and fms-like tyrosine kinase-3 gene (FLT3) mutations (Kroschinsky et al, 2008; Chen et al, 2009). Therefore, we sought to investigate the incidence of IDH mutations in AML-CN. After approval by the Institutional Review Boards of UT Southwestern Medical Center and MD Anderson Cancer Center, patient information was obtained from a search of records from 2002 to 2010. Available Wright-Giemsa-stained peripheral blood and bone marrow aspirate smears and hematoxylin-eosin-stained bone marrow clot and trephine biopsy sections were reviewed. The selection criterion for inclusion was the presence of > 10% blasts with prominent nuclear invaginations (or ‘cuplike’ nuclei) spanning at least 25% of the nuclear area (Fig 1) (Chen et al, 2009). Twelve patients with sufficient amplifiable DNA were identified. All cases were classified as AML without maturation (WHO classification) or AML M1 (French-American-British classification). All cases had normal cytogenetics, which was probably due to the small sample size, as 86% (19/22) of AML-CN had normal cytogenetics in our previous study (Chen et al, 2009). An age-matched control group of 23 cases of similarly classified (AML M1), cytogenetically normal AML without cuplike nuclei was established. Four-colour flow cytometric immunophenotypic analysis was performed on bone marrow aspirate specimens. Conventional cytogenetic analysis was performed on bone marrow aspirate specimens using standard Giemsa trypsin G-banding procedures. Either all coding exons or exon 4 along with flanking intronic regions/splice sites of IDH1 and IDH2 were sequenced as previously described (van den Bent et al, 2010; Rakheja et al, 2011). Molecular assays for detection of FLT3 and NPM1 mutations were performed as previously described (Chen et al, 2009). In three patients, conditioned flow cytometry medium was available for measurement of D-2-hydroxyglutarate (D-2-HG) and L-2-hydroxyglutarate (L-2-HG). The two stereoisomers were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described (Rakheja et al, 2011). Morphologic features of blasts with cuplike nuclei (arrows). The blasts are medium-sized with prominent nuclear invagination (cuplike nuclei) and scant to moderate cytoplasm containing a few azurophilic granules (Wright-Giemsa bone marrow aspirate smear, ×1000, Olympus, Center Valley, PA, USA). The clinical, immunophenotypic, genetic, and metabolic findings of AML-CN cases and cytogenetically normal AML M1 controls are summarized in Table I. Compared to the control cases, the AML-CN cases showed statistically significant (P < 0·05) associations with presence of IDH mutations (67%), presence of NPM1 mutations (100%), lack of CD34 (100%) expression, and lack of HLA-DR (67%) expression. Furthermore, triple mutations (mutations for IDH, NPMI, and FLT3) were seen exclusively in AML-CN cases (60%). Eight of 12 AML-CN cases (67%) had IDH heterozygous mutations, involving either IDH1 (R132G in two patients, R132C in two patients) or IDH2 (R140Q in four patients). In contrast, only five of 23 (22%) control AML cases harboured these mutations (IDH1 in three cases, IDH2 in two cases) (P = 0·0244). One AML-CN case had a synonymous single nucleotide polymorphism (SNP) of IDH1 (cd.105 GGC>GGT, p.105 G>G). These findings are particularly notable as the reported incidence of IDH mutations is approximately 10% in unselected AML and only 20–30% in cytogenetically normal AML. Our data suggest that cuplike nuclei are the morphological expression of distinct molecular changes in this unique subgroup of AML. IDH mutations facilitate the reduction of α-KG to D-2-HG, a putative ‘oncometabolite’ which may contribute to dysregulated epigenetic programming in cancer cells (Figueroa et al, 2010; Xu et al, 2011). In two cases of AML-CN, we confirmed the high levels of D-2-HG in the presence of IDH mutations (Table I), suggesting that plasma/serum or urine D-2-HG may potentially serve as a useful metabolic biomarker to monitor disease progression and detect minimal residual disease. IDH mutations were found to be associated with inferior outcome in a subset of AML patients with mutated NPM1 without FLT3-internal tandem duplication (Paschka et al, 2010). With a follow-up period up to 7·5 years, there was no significant difference in overall survival between our AML-CN cohort and AML control cohort (35% survival vs. 50% survival). Prospective studies of a larger series of cases would be required to assess for potential clinical significance. In conclusion, AML-CN has distinct genetic features characterized by a high incidence of normal cytogenetics, and IDH, NPM1, and FLT3 mutations. Morphological recognition of cuplike nuclear morphology in AML should prompt appropriate molecular and metabolic workup. The authors thank Midori Mitui and Richard A. Gibbs for their help with sequence analysis, and Richard L. Boriack for performing biochemical measurements. DNA sequencing was performed at UT Southwestern DNA sequencing core facility or MD Anderson Cancer Center DNA analysis facility. The molecular and metabolic work performed at UT Southwestern was supported in part by a grant from The Clinical Development Fund of the Department of Pathology at UT Southwestern (DR). The MD Anderson Cancer Center DNA analysis facility is funded by NCI # CA16672 (DAF). DR, SK, and WC were the principal investigators of this study; they analysed the data and wrote the manuscript; DR, DW, DMM, and VRR provided the sequencing data; MS, RC, and NK provided the clinical or pathologic data; NK helped with study design; MA provided the patient samples; and LJM analysed the data and helped write the manuscript. All authors read and approved the final version of the manuscript. The authors declare no competing financial interests.