AbstractEpigenetic deregulation is a hallmark of cancer characterized by frequent acquisition of new DNA methylation in CpG islands. To gain insight into the methylation changes of canine DLBCL, we investigated the DNA methylome in primary DLBCLs in comparison with control lymph nodes by genome-wide CpG microarray. We identified 1,194 target loci showing different methylation levels in tumors compared with controls. The hypermethylated CpG loci included promoter, 5′-UTRs, upstream and exonic regions. Interestingly, targets of polycomb repressive complex in stem cells were mostly affected suggesting that DLBCL shares a stem cell-like epigenetic pattern. Functional analysis highlighted biological processes strongly related to embryonic development, tissue morphogenesis and cellular differentiation, including HOX, BMP and WNT. In addition, the analysis of epigenetic patterns and genome-wide methylation variability identified cDLBCL subgroups. Some of these epigenetic subtypes showed a concordance with the clinical outcome supporting the hypothesis that the accumulation of aberrant epigenetic changes results in a more aggressive behavior of the tumor. Collectively, our results suggest an important role of DNA methylation in DLBCL where aberrancies in transcription factors were frequently observed, suggesting an involvement during tumorigenesis. These findings warrant further investigation to improve cDLBCL prognostic classification and provide new insights on tumor aggressiveness.