In MDS and related conditions, unbalanced chromosomal aberrations, inactivating mutations and epigenetic silencing may have similar pathogenetic consequences. For example, they all can result in decreased or defective expression of tumor suppressor genes. Progressive aberrant hypermethylation is associated with malignant evolution. While various mechanisms have been identified as possible causes of chromosomal instability, to date the pathogenic pathways responsible for epigenetic instability have not been identified. Recent discovery of TET2 mutations in MDS/MPN, sAML and especially CMML provided an intriguing possibility that dysfunction of this gene may result in increased promoter methylation. This hypothesis is based on the description of the function of the highly related TET1 gene: the corresponding protein mediates hydroxylation of methylcytosine to methylhydroxycytosine, which abrogates DNMT1-mediated maintenance methylation during cell division. Consequently, TET2 mutations could lead to loss of epigenetic stability. To investigate our hypothesis, we utilized a high-density bisulfite-based methylation array (27,758 CpG sites; Illumina ®) to analyze whole epigenome methylation patterns, as well as selection of individual candidate genes showing aberrant hypermethylation. Patients with TET2 mutation positive CMML (N=14) and other forms of MDS/sAML (N=13) and CML (N=3) were compared to those with WT TET2 CMML (N=12) and other forms of MDS/sAML (n=7). Controls (N=28) included bone marrow samples from healthy volunteers and elderly, otherwise healthy, individuals undergoing orthopedic procedures. We developed an analytic algorithm that included assessment of methylation expressed either as a continuous variable (where β is proportional to the percentage of cells with methylated status for each individual locus) or as a dichotomous variable (where hypermethylation was defined as a β value greater than the 97th percentile of controls). Comparison of the average methylation level across all genes showed no significant differences between patient groups. However, using the dichotomized method, in mutant TET2 patients 4, 1440 and 2760 CpG sites were concordantly hypermethylated in 75%, 50% and 25%, respectively. In those with WT TET2, 10, 2444 and 5960 genes were concordantly hypermethylated in 75%, 50% and 25% of patients, respectively. There was little overlap between concordantly hypermethylated genes in TET2 mutant and WT cases. When we examined genes that were concordantly methylated in at least 50% of patients with TET2 mutations and less than 25% of TET2 WT patients: a set of 130 genes was identified. Of these, we selected 4 genes that were the most consistently methylated in TET2 mutant cases: ZPBP2, SP140, TRAK1 and PSGF1. When compared with WT cases, each of these genes was significantly methylated as compared to mutant cases (p=.01, .01, .03, .02, respectively). When patients with CMML were selected as a subgroup to match for similar pathomorphologic features between mutant and WT cases 7, 762 and 6341 CpG sites were concordantly hypermethylated in 75%, 50% and 25% of patients with CMML and TET2 mutations, respectively, whereas CMML with WT TET2 showed concordant hypermethylation in 57, 1809 and 8706 genes in 75%, 50% and 25% cases, respectively. There were 104 genes that were methylated in greater than 50% of TET2 mutation positive patients and less than 25% of TET2 WT patients. Examples of hypermethylated markers associated with TET mutations include CRYAB, TUBB3, MT1B and TNFSF12. Of these, TNFSF12 was significantly hypermethylated compared to TET2 WT CMML patients (p=.05). We then focused on p15 (CDKN2B) and p16 (CDKN2A), reported to be frequently hypermethylated in MDS and thereby serving as epigenetic indicator gene. CDKN2A methylation was significantly increased 4/7 CpG sites in CMML patients with TET2 mutation as compared to those with WT TET2. In contrast, 2/7 loci showed opposite methylation pattern. When CDKN2B methylation status was analyzed 2/3 loci were significantly hypermethylated mutant cases and 1/3 was hypermethylated in WT cases. These results were similar when patients with all forms of MDS were included in the analysis. In conclusion, our results indicate that TET mutations are associated with a distinct aberrant hypermethylation profile. It is possible that the defective function of TET2 gene may lead to disturbance of epigenetic stability leading to inactivation of distinct set of tumor suppressor genes.
Advani:Cephalon: Research Funding.
Asterisk with author names denotes non-ASH members.