5-Hydroxymethylcytosine Is Correlated with TET2 or IDH1/2 Mutations However, May Not be a Prognostic Value to Predict the Survivals in Normal Karyotype AML

Background: Stem cells display remarkably high levels of 5-hydroxymethylcytosine (5hmC) and, both TET2 or IDH1/2 mutations can impair 5hmC generation. However, the implications of 5hmC have not been evaluated comprehensively in patients with normal karyotype (NK)-AML, especially in aspect of prognostic value in survivals.

Methods: A total of 407 patients were included in the present study, and all met the following eligibility criteria: 1) age ≥ 15 years; 2) a diagnosis of NK-AML confirmed by conventional cytogenetic analysis; and, 3) treatment with induction chemotherapy using a standard protocol (a 3-day course of anthracycline with a 7-day course of cytosine arabinoside). NK-AML patients were diagnosed from October 1998 to September 2012 in seven participating institutes. Among 407 patients with NK-AML who received induction therapy, we selected the 376 patients available the analysis of 5-hmC, retrospectively. For quantitation of 5hmC, Quest 5-hmC DNA ELISA kits (Zymo Research) were used following the manufacturer's protocol. TET2 or IDH1/2 mutation analysis were performed using direct sequencing. We analyzed for 5hmC levels in patients with TET2 or IDH1/2 mutations and, to know the correlation of 5hmC levels with mutant alleles.

Results: The prevalence rates for the mutations were 13.0% in TET2^mut, 7.2% in IDH1 and,14.1% in IDH2^mut. Mutation rates of TET2 or IDH1/2 was 34.6% (130/376). We examined whether the range of 5hmC values correlated with each mutations. TET2, IDH1/2 mutated patients had significant lower levels of 5hmC compared with patients without any TET2 or IDH1/2 mutations (all, p<0.001). The median value of 5hmC level were: TET2^mut (median: 0.051%, range: 0.002%-0.120%), IDH1^mut (median: 0.044%, range: 0.004%-0.641%), IDH2^mut (median: 0.050%, range: 0.001%-0.457%), any mutation of TET2 or IDH1/2 (median: 0.048% , range: 0.001%-0.641%) and, TET2 wild-type and IDH1/2 wild type (median: 0.084%, range: 0.0003%-0.999%). In control group (TET2 wild-type and IDH1/2 wild type), 5hmC levels distributed with broad range but, 5hmC levels were tightly clustered in patients with TET2, IDH1 or IDH2 mutations. With a median follow-up duration of 55.5 months (range, 0.7-179.8 months), there was no significant difference in overall survival (OS), event free survival (EFS) and relapse risk according to TET2^mut or IDH1/2^mut (all, p >0.05). To identify the role of 5hmC levels in clinical significances, we sub-classified this group with tertile category for 5hmC values. However, we could not find the clinical significant in OS, EFS and relapse risk according to the 5hmC levels (all, p>0.05).

Conclusion: TET2 or IDH1/2 mutated patients had lower levels of 5hmC. 5hmC levels distributed with wide range in patients with TET2 and IDH1/2 wild type and tightly clustered in patients with TET2, IDH1 or IDH2 mutations. Apart from affecting the methylation status of the DNA, other processes may be influenced by 5hmC levels in patients with NK-AML with TET2 wild-type and IDH1/2 wild type. In addition, 5hmC may not be a prognostic value to predict the survivals of relapse risk in NK-AML.