The present report is a systematic study of DNA methylation patterns on adult de novo
AML. Our unsupervised clustering analysis of 116 patients revealed that distinct epigenetic signatures could be identified based on a limited number of genes. Recent reports, using different epigenomic approaches with a much larger number of CpGs 
, also defined epigenetic profiles for cases harboring balanced translocations such as t(8;21), inv(16), t(15;17), or MLL rearrangements. Nevertheless, our data showed that most of these cases with chromosome rearrangements were clustered in a large group (Group II) that shows abundant and common epigenetic modifications on CpG islands and hypomethylation of selected non-clustered CpGs. Within Group II, we identified a subset of 20 genes conforming an epigenetic signature common to AML with fusion proteins and a subset of AML cases from the intermediate cytogenetic prognostic subgroup. Furthermore, the pathway analysis of these aberrantly methylated genes showed a significant involvement of the Wnt signaling pathway. These results are consistent with our recent studies and reveal a significant role of the epigenetic modifications of Wnt antagonists in the pathogenesis of AML 
. On the other hand, cases harboring complex karyotypes clustered within Group I AML, which did not show a hypermethylated signature, supporting the hypothesis put forward by Koeger et al. of a negative correlation between epigenetic and chromosomal instability 
Our results, along with previous studies, suggested a link between genetic/chromosome rearrangements and the induction of aberrant DNA methylation 
. We previously showed that the overexpression of the MLL/AF9 fusion protein on HSPC induced acute myeloid, lymphoid, or mixed-lineage leukemia in immunodeficient mice, and that these transformed HSPC could be lineage-directed by altering either the growth factors or the recipient mouse strain 
. Using this leukemic HSPC model, we demonstrated that MLL/AF9
recapitulated the epigenetic profile observed in the MLL-positive patients, as has been shown in mice models with other genetic insults 
, thus supporting a direct role for MLL fusion proteins in the down-regulation of target genes by DNA methylation 
. However, the mechanisms underlying this signature have yet to be explored. Furthermore, the observed ALL MLL methylation profile included a significantly higher number of aberrantly methylated CpGs than the AML cases. These results support the essential role of DNA methylation in the plasticity of the hematopoietic system 
, suggesting interplay between transcription factors downstream of cytokine receptors and the DNA methylation machinery.
overexpression on HSPC failed to reproduce the epigenetic signature observed in the primary patients, suggesting that these two CBF fusion proteins are insufficient to target DNA methylation to specific sites, as they are not capable of inducing a fully transformed phenotype 
. These results suggest that, either longer periods of time (more than 12 weeks) are needed to induce the epigenetic modifications or that these fusion proteins do not have a direct impact on the DNA methylation profile (as is shown to happen in the HSPC model). Further research exploring the mechanisms driving the specific epigenetic signature of the different cytogenetic subgroups seems warranted.
In addition, another major objective of our study was to determine whether patterns of DNA methylation signature could be used to improve patient prognostification, mainly among the abundant normal karyotype group of cases. As other 
we were not able to correlate the presence of a methylation signature with different clinical outcomes among normal karyotype cases, as it has been previously reported using larger number of CpGs 
. However, using specific biomarkers, differences in overall survival in patients with AML have been revealed by analysis of concurrent aberrant methylation of specific genes, such as ESR1
, and IGSF4 
. Using the SignS web tool, we were able to identify the methylation status of the deleted in bladder cancer 1 (DBC1
) and the cyclin-dependent kinase inhibitor 2B (CDKN2B/p15
) as predictors of overall survival in the AML subgroup with a normal karyotype. Only aberrant methylation of the DBC1
promoter was observed to have statistically significant prognostic value among cases with a normal karyotype in an independent case series. DBC1
have been shown to have tumor-suppressor activity through the negative regulation of G1 cell cycle progression, and their loss of function has been associated with an advantage in proliferation, growth, and malignant transformation 
. Our results suggest that the clinical importance of CDKN2B
methylation in AML is still controversial 
Hypermethylation associated with reversible epigenetic silencing of DBC1
has been reported in hematological disorders 
and in solid tumors 
. This silencing mechanism has been postulated as an early and age-independent event in the development of malignancy 
. We identified aberrant methylation of DBC1
, located at 9q33.1, as an adverse prognostic marker in the AML subgroup with a normal karyotype. Furthermore, when this epigenetic event is combined with FLT3
status, it constitutes a unique and powerful predictor of clinical outcome within AML cases with a normal karyotype. However, under current therapeutic regimens, this epigenetic marker does not retain its independence in the multivariate analysis. The identification of patients with aberrant DNA methylation patterns that can predict survival will be essential in future designs of clinical trials with demethylating agents.
In conclusion, comprehensive epigenetic profiling of AML provides relevant biological information and new clinical markers that should be integrated in the design of clinical trials with demethylating agents.