Isocitrate dehydrogenase (IDH1 and IDH2) mutations can be detected in brain tumors1, 2, 3 and acute myeloid leukemia (AML)4, 5, 6, 7 and are associated with distinct clinical and biological features. The mutations in these metabolic enzymes result in increase of 2-hydroxyglutarate (2HG) and decrease of α-ketoglutarate (α-KG), a cofactor of TET2, leading to disturbance of DNA and histone demethylation.8, 9 Consistent with these pathophysiological effects, IDH mutations are virtually mutually exclusive with loss-of-function mutations of TET2, which converts methylcytosine to hydroxymethylcytosine, a process probably related to DNA demethylation.8, 9 Taking the advantage of relative depletion of α-KG in IDH-mutated cells, which makes the cells more dependent on glutaminolysis pathway to supply α-KG, inhibition of this pathway by bis-2-5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a glutaminase inhibitor, has been shown to preferential kill IDH-mutated cells.10 Thus, the IDH mutations show a linkage between metabolism and cancers, demonstrate crosstalk between genetic and epigenetic dysregulation, and provide a well known pathway for possible targeted therapy. However, the role of IDH mutation in leukemogenesis remains to be defined. Here we show that IDH1 R132 and IDH2 R140 mutations can retain in myeloid but not lymphoid cells or skin tissue from three IDH-mutated AML patients in long-termed complete remission (CR), whereas the concomitant NPM1 or FLT3-ITD mutations are not detectable anymore.
We analyzed paired bone marrow samples collected at diagnosis, in CR, and at relapse in 19, 18 and 9 patients who harbored mutations at IDH1 R132, IDH2 R140 and IDH2 R172, respectively, using polymerase chain reaction followed by direct sequencing. A total of 114, 114 and 72 samples from these patients, respectively, were studied. The patients and materials were derived from our previous studies.4, 11 Screening for mutations of NPM1, IDH1/2 and FLT3-ITD was described previously.4, 11, 12 This study has been approved by the Institutional Review Board of the National Taiwan University Hospital.
We found a nice correlation between the mutation and disease status in all samples from these patients, except for one patient with IDH1 R132H, and two patients with IDH2 R140Q mutation, in whom the mutation persistently retained in CR samples during the follow-up period of 4, 4 and 10 years, respectively, after the last chemotherapy (Figure 1). Patient A, a 75-year-old woman, had AML M2 subtype with a normal karyotype and concomitant NPM1 and IDH1 R132H mutations. She has remained in continuous CR till the time of this study for 50 months after cessation of the treatment. However, the IDH1 mutation signal, though weakened at the time when CR was first achieved, was enhanced later while the patient was still in CR and the NPM1 mutation was no longer detectable (Figure 1a). Patient B, a 46-year-old woman, had AML M4 subtype with a normal karyotype and concurrent IDH2 R140Q and NPM1 mutation. She obtained a continuous CR and the NPM1 mutation disappeared in the following 46.6 months, but IDH2 R140Q retained persistently. Patient C, a 68-year-old man, had AML M2 subtype with a normal karyotype and concurrent IDH2 R140Q and FLT3-ITD. There was no detectable IDH2 R140Q or FLT3-ITD in the first CR. However, throughout his second CR that lasted for 124 months till his death of an accident unrelated to AML, IDH2 R140Q could be detected repeatedly, while FLT3-ITD remained undetectable (Figure 1c). The absence of the original NPM1 mutant in patients A and B and the FLT3-ITD in patient C were confirmed by sensitive real-time PCR assays (Figures 1a–c), which were performed as described previously.12, 13 DNA from the skin of patient C and the sorted CD3+ lymphocytes of peripheral blood (PB) from patients A and B in CR did not reveal the IDH mutation (Figures 1a–c), indicating that the IDH mutation was acquired in these patients. In contrast, the sorted CD15+ PB myeloid cells from patients A and B still obviously harbored the IDH1 R132H and IDH2 R140Q mutations, respectively.
We further measured the 2HG, an oncometabolite resulting from IDH mutations,14 in the PB white blood cells from patients A and B collected in the recent CR stage. The 2HG in white blood cells collected from a healthy volunteer and patients A and B were extracted by 1000 μl of ice-cold methanol and distilled water (1:1, v/v), followed by evaporation to dryness and reconstituted in 1000 μl of 70% acetonitrile for ultra high performance liquid chromatography -MS/MS analysis. The 2HG concentration was quantified by an Agilent 1290UHPLC system coupled with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A volume of 7 μl of the extract was injected into an Atlantis HILIC column (2.1 × 100 mm, 3 μm; Waters, Milford, MA, USA). The column was thermostated at 40 °C. The chromatographic system was operated using a gradient of water and acetonitrile (both solvents were modified by the addition of 0.1% trifluoroacetic acid) at a rate of 0.5 ml/min. A Jet Stream electrospray ion source with capillary spray voltage of 4000 V and sheath gas temperature of 325 °C was used for sample ionization. Two ion pairs were used for quantification (147>129) and qualification (147>57.1) in multiple reaction monitoring (MRM) mode, and the collision energy was at 5 and 17 eV, respectively. We found that PB white blood cells from patients A and B contained >55-fold higher 2HG than that in a normal control (Figure 1d).
The fact that these three patients retained the IDH mutations in the bone marrow cells during the long period of CR while they lost the accompanying NPM1 mutation (patient A and B) or FLT3-ITD (patient C) detected at diagnosis suggests that IDH mutation alone is not sufficient for leukemogenesis in vivo, compatible with the two-hit theory. This finding also coincides with a recent report that patients with hereditary D-2-hydroxyglutaric aciduria and germline mutation of IDH2 R140Q were free of cancers.15 Different from minimal residual disease in CR, which can only be detected by sensitive real-time PCR or multi-color flow cytometry, the IDH mutant in the three patients could be readily detected by the direct sequencing, and the mutant loads appeared to step up and finally existed in most myeloid cells based on the high mutation signals in sequence tracings and the high 2HG levels in the PB cells (Figure 1).
Our finding suggests that some bone marrow cells harboring IDH mutation alone can survive chemotherapy and expand, but are not adequate to transform myeloid cells to AML. The facts that the patients with IDH1/2 mutations at diagnosis always retained the original IDH mutation in relapsed bone marrow samples shown previously,4, 11 whereas some patients remained in continuous CR but persistently harboring IDH mutations in myeloid cells, as shown in this study, suggest that IDH mutation is important in maintaining the leukemia phenotype probably through cooperation with other oncogenic mutations, but itself alone is not sufficient for leukemogenesis in vivo. It also suggests that monitoring IDH mutants after treatment in AML patients bearing the mutations at diagnosis can be misleading in assessment of disease responses. Moreover, we provide a new insight into the role of IDH mutations in leukemogenesis.
Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009; 360: 765–773.
Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F et al. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol 2009; 27: 4150–4154.
Weller M, Felsberg J, Hartmann C, Berger H, Steinbach JP, Schramm J et al. Molecular predictors of progression-free and overall survival in patients with newly diagnosed glioblastoma: a prospective translational study of the German Glioma Network. J Clin Oncol 2009; 27: 5743–5750.
Chou WC, Hou HA, Chen CY, Tang JL, Yao M, Tsay W et al. Distinct clinical and biologic characteristics in adult acute myeloid leukemia bearing the isocitrate dehydrogenase 1 mutation. Blood 2010; 115: 2749–2754.
Marcucci G, Maharry K, Wu YZ, Radmacher MD, Mrozek K, Margeson D et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol 2010; 28: 2348–2355.
Paschka P, Schlenk RF, Gaidzik VI, Habdank M, Kronke J, Bullinger L et al. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. J Clin Oncol 2010; 28: 3636–3643.
Ward PS, Patel J, Wise DR, Abdel-Wahab O, Bennett BD, Coller HA et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 2010; 17: 225–234.
Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 2010; 18: 553–567.
Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell 2011; 19: 17–30.
Seltzer MJ, Bennett BD, Joshi AD, Gao P, Thomas AG, Ferraris DV et al. Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res 2010; 70: 8981–8987.
Chou WC, Lei WC, Ko BS, Hou HA, Chen CY, Tang JL et al. The prognostic impact and stability of isocitrate dehydrogenase 2 mutation in adult patients with acute myeloid leukemia. Leukemia 2011; 25: 246–253.
Chou WC, Hou HA, Liu CY, Chen CY, Lin LI, Huang YN et al. Sensitive measurement of quantity dynamics of FLT3 internal tandem duplication at early time points provides prognostic information. Ann Oncol 2011; 22: 696–704.
Chou WC, Tang JL, Wu SJ, Tsay W, Yao M, Huang SY et al. Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin (NPM1) mutations. Leukemia 2007; 21: 998–1004.
Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009; 462: 739–744.
Kranendijk M, Struys EA, van Schaftingen E, Gibson KM, Kanhai WA, van der Knaap MS et al. IDH2 mutations in patients with D-2-hydroxyglutaric aciduria. Science 2010; 330: 336.
This work was supported by grants NSC 96-2628-B002-013-MY2, 97-2314-B002-015-MY3, 97-2628-B-002-002-MY3, 98-2314-B-002-033-MY3, 100-2325-B-002-032, 100-2325-B-002-033- from the National Science Council (Taiwan), NHRI-EX97-9731BI from the National Health Research Institute, DOH100-TD-C-111-001 from the Department of Health (Taiwan), NTUH 98-S1052, NTUH 98-S1383 from the National Taiwan University Hospital and the YongLin Healthcare Foundation.
W-CC and H-FT designed the experiment. W-CC, K-YP, C-HK and H-FT analyzed the data and wrote the paper. W-CL, B-SK and WT provided important materials.
The authors declare no conflict of interest.
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Chou, WC., Peng, KY., Lei, WC. et al. Persistence of mutant isocitrate dehydrogenase in patients with acute myeloid leukemia in remission. Leukemia 26, 527–529 (2012). https://doi.org/10.1038/leu.2011.215
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