2-Hydroxyglutarate in IDH mutant AML: Predicting Patient Responses, Minimal Residual Disease and Correlations with Methylcytosine and Hydroxymethylcytosine Levels
Authors: Pollyea Daniel A , Kohrt Holbrook E , Zhang Bing , Zehnder James L. , Schenkein David P. , Fantin Valeria , Straley Kimberly , Vasanthakumar Aparna , Abdel-Wahab Omar , Levine Ross L , Godley Lucy , Medeiros Bruno C. 
Published: October 2019

Abstract
Abstract 2509
Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes have recently been shown to be recurrent, frequent and prognostic in acute myeloid leukemia (AML) patients. IDH1 and IDH2 encode NADP+ dependent enzymes responsible for oxidative decarboxylation of isocitrate to α-ketoglutarate (AKG). 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. The ten-eleven translocation 2 (TET2) gene converts 5-methylcytosine (5MC) to 5-hydroxymethylcytosine (5HMC), leading to DNA demethylation. TET2 encodes a dioxygenase that is dependent on AKG, and therefore, increased levels of 2HG may disrupt TET2 function. We explored the potential of 2HG to serve as a clinical biomarker of response to therapy in AML patients with IDH mutations, and correlated fluctuations of 2HG with changes in 5MC and 5HMC levels in a clinical setting.
In a Phase I/II clinical trial of 44 elderly, previously untreated AML patients who received sequential azacitidine and lenalidomide, we identified 12 patients with IDH1/2 mutations (27%). Five had IDH1 mutations (all R132), and seven had IDH2 mutations (R140 N=5, R172 N=2). 2HG levels were measured by mass spectrometry from plasma obtained from bone marrow aspirations. Six patients had pre- and post-treatment samples available for comparison. Clinical responses were determined per LeukemiaNet criteria.
Two of the six patients achieved a complete remission (CR), one with an IDH1 R132 and the other with an IDH2 R140. 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 (Figure 1). A non-parametric comparison (Mann-Whitney U test) in this small sample set showed a trend toward a significant change in pre- vs. post-treatment 2HG levels between patients who experienced a CR and those who did not (p=0.067), suggesting 2HG levels may correlate with leukemic blast clearance.
Both patients who achieved a CR continued to have detectable 2HG levels. Furthermore, patient 8 ultimately relapsed, and at the time of this relapse, which was unexpected based on clinical indicators or routine peripheral blood laboratory findings, there was a 1.8-fold increase in 2HG levels. Therefore, for AML patients with IDH1 and IDH2 mutations, 2HG may provide a useful, non-invasive marker of minimal residual disease.
We hypothesized that decreases in 2HG would allow functional TET2 activity, resulting in increased 5HMC and decreased 5MC. Therefore, we correlated changes in 2HG levels with changes in global 5MC and 5HMC levels, as measured by mass spectrometry, in the patients who achieved a CR.
Patient 8 was wild type for TET2. As predicted, after one cycle of therapy, 2HG decreased 6.1 fold, 5HMC increased 7.8 fold and 5MC decreased 1.4 fold. At relapse, when 2HG rose 1.8 fold, 5HMC decreased 5.4 fold; the expected increase in 5MC was not observed (Figure 2). Although concomitant mutations in IDH and TET2 are exceedingly rare, patient 22 did have this genotype, which allowed for an instructive negative control. After one cycle of therapy, 2HG decreased 2.6 fold. However, in contrast to patient 8, in which it appeared TET2 became functional as shown by increased 5HMC after 2HG decreased, patient 22's mutant TET2 resulted in marginal changes in 5HMC and 5MC levels (1.3-fold increase and 1.1-fold decrease, respectively) (Figure 3). These observations provide in vivo evidence for the interaction between IDH and TET2, and suggest that these interactions are dynamic and can change with therapy.

Disclosures:
Off Label Use: Lenalidomide in AML. Schenkein:Agios: Employment. Fantin:Agios: Employment. Straley:Agios: Employment. Medeiros:Celgene: Honoraria, Research Funding.