Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In addition, changes in DNA methylation have been implicated in the pathogenesis of AML. Recently it was discovered that TET proteins are able to convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), which is an important intermediate in the demethylation pathway. In this study, we measured 5-hydroxymethylcytosine levels in AML patients, and correlated these with mutational status and overall survival (OS).
Samples from 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years), included in the EORTC/GIMEMA AML-12 06991 clinical trial, were analyzed for mutations in DNMT3A, TET2, IDH1 and IDH2. 5-hydroxymethylcytosine levels were measured using HPLC-MS/MS.
In healthy control cells, 5hmC levels were confined to a narrow range (1.5 fold difference), whereas in AML cells, a much wider range was detected (15 fold difference). In remission, 5hmC values were normalized to levels comparable to healthy bone marrow and peripheral blood, indicating that the aberrant 5hmC levels at diagnosis are intrinsic to the leukemic cells. Patients with mutations in TET2 and patients with mutations in IDH1/2 had significantly lower levels of 5hmC compared to patients without mutated TET2 and IDH1/2 (both P<.001), whereas mutations in DNMT3A did not influence 5hmC levels. Patients with bi-allelic TET2 inactivation displayed lower 5hmC levels than patients with one affected allele (P=.003). Among the patients that did not harbor TET2 or IDH mutations, still a wide variation in 5hmC levels was observed, and importantly, both low and very high 5hmC levels correlated with inferior OS (P=.02, HR=1.80 and P=.04, HR=1.97, respectively). Multivariate analysis revealed that abnormal levels of 5hmC were independent prognostic indicators for OS. The difference in OS could not be explained by an initial inferior response to therapy, however, the relapse rate was considerably higher in patients with low and very high 5hmC levels compared to patients with intermediate 5hmC.
Both low and very high levels of 5hmC are markers of poor prognosis in AML, lending further support for testing therapies targeting the DNA hydroxymethylation pathway.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.