Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes are recurrent, frequent and prognostic in patients with acute myeloid leukemia (AML). IDH1 and IDH2 encode nicotinamide adenine dinucleotide phosphate (NADP) dependent enzymes responsible for the oxidative decarboxylation of isocitrate to α-ketoglutarate (AKG)[1]. Heterozygous point mutations in IDH1 and IDH2 result in the loss of this activity, concomitant with a neomorphic ability to convert AKG to 2-hydroxyglutarate (2HG), resulting in significant elevations in this oncometabolite, which is not detectable in patients with AML with wild type IDH [2, 3]. The ten-eleven translocation (TET) family proteins convert 5-methylcytosine (5MC) to 5-hydroxymethylcytosine (5HMC), leading to DNA demethylation [4]. TET2 encodes a dioxygenase that is dependent on AKG, and therefore, increased levels of 2HG may disrupt TET2 function [5]. We explored the potential of 2HG to serve as a clinical biomarker of response to therapy in patients with AML with IDH mutations, and correlated fluctuations of 2HG with changes in 5MC and 5HMC levels in a clinical setting. A clinical trial of elderly, previously untreated patients with AML who received sequential azacitidine and lenalidomide was approved by the Stanford University Institutional Review Board. Bone marrow aspirate was collected at specified pre-and post-treatment time points with the informed consent of the patients, in accordance with the Declaration of Helsinki. Plasma and mononuclear cells were isolated and preserved as previously described [6]. DNA and RNA were extracted with the use of Qiagen kits (Qiagen, Valencia, CA). Genotyping for IDH1/2, TET2, FMS-like tyrosine kinase 3 internal tandem duplication (FLT-3–ITD) and D835 tyrosine kinase domain mutation (FLT-3–TKD), nucleophosmin (NPM) and CCAAT enhancer binding protein alpha (CEBPA) were performed as previously described [6]. 2HG levels were measured as previously described by liquid chromatography-mass spectrometry from plasma obtained from bone marrow aspirations [2], and normalized according to the total blast count. 5HMC and 5MC levels were measured as previously described by high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry from DNA extracted from bone marrow aspirate mononuclear cells [7]. Six patients with IDH mutations had pre-and posttreatment samples available for comparison. Clinical responses were determined according to European Leukemia Net criteria [8]. The median age was 77, and 3/6 were female. Three had a normal karyotype, and three had other coexisting mutations. Two had IDH1 mutations (R132), and four had IDH2 mutations (R140 n 3, R172 n 1)(Table I). Two of the six patients achieved a complete remission (CR), one with an IDH1 R132 (patient B) and the other with an IDH2 R140 (patient C). A comparison of pre-and post-treatment samples from these patients revealed decreases in 2HG levels of 16.9-and 17.9-fold after three cycles of therapy. Four patients did not achieve a CR, although some did experience decreases in the percentage of blasts. These patients had substantially smaller decreases, or increases, in 2HG levels when measured after one cycle (non-responding patients did not receive more than one cycle of therapy)[Figure 1 (A)]. A non-parametric comparison (Mann–Whitney U-test) in this small sample set showed a trend toward a significant change in preversus post-treatment 2HG levels between patients who experienced a CR and those who did not (p 0.067).