In this study we demonstrate that the genome-wide DNA methylation profile of tumor cells from CM patients with nodal metastases is a significant predictor of OS within stage IIIC. This finding provides the first evidence that genome-wide methylation profiles can serve as molecular markers of prognosis for CM patients in this high-risk group, which has been the focus of multiple adjuvant trials.
Promoter hypermethylation has been proposed to have an important impact on tumor biology through the silencing of TSG and the alteration of virtually every cellular pathway relevant to CM development and progression
. Accordingly, initial studies evaluated the status of specific genes known to be methylated in cancer, and showed an association of promoter hypermethylation and advancing tumor stage, reduced disease-related survival and/or OS in CM patients
[7, 8]. These studies relied on the a priori selection of few genes and generally enrolled CM patients from highly heterogeneous disease stage-groupings.
The results of the present study demonstrate the adverse prognostic impact of genome-wide hypermethylated profiles in relation to OS, and more importantly, for patients within a single stage sub-grouping of disease, have shown remarkable prognostic significance for the methylation profile. This observation strongly supports the notion that the constitutive methylation profile of cancer cells is intimately linked to the behavior of the tumor, and drives differing outcomes of disease. This concept is further strengthened by the discovery that, among all clinico-pathological and molecular factors examined, only those linked to genome-wide methylation were significantly associated with OS among CM patients analyzed. In this context, the multivariate Cox model identified whole-methylome-profile-defined classification as the most robust prognostic marker, suggesting its superior ability in the identification of biologically relevant methylation backgrounds as compared to LINE-1 methylation
Genome-wide methylation profiles could be recapitulated using a 17-gene signature, which was sufficient to correctly assign CM patients to the identified prognostic groups. Intriguingly, none of the genes composing the signature has been previously reported to be methylated in CM, demonstrating that “unbiased” methylation profiling represents an appropriate and possibly more effective tool for the identification of novel prognostic/predictive epigenetic markers in human cancer.
The mechanisms through which the different methylome profiles affect the survival of CM patients remain to be defined. A direct contribution of the products of the 17 signature genes is unlikely since no meaningful association was observed between methylation and expression of analyzed genes (Figure
4). The methylation signature, thus, appears as an effective bona fide prognostic marker, accounting for the overall methylation profile of tumor cells, without a direct impact on tumor biology that can presently be defined. A similar observation was recently reported by Tanemura et al.
 who evidenced a significant positive association of MINT31 hypermethylation and improved disease-free survival and OS in stage III CM patients. Since no established product is known for MINT31 locus, the authors suggested that the methylation status of the locus could be linked to the activation status of additional genes yet to be identified
. This appears to be true, more in general, for the widely described phenomenon of the CpG island methylator phenotype (CIMP), which has been described in several tumor types, and refers to a high frequency of concomitant aberrant hypermethylation of different genes and/or chromosomal loci
. Indeed, presence of CIMP, rather than accounting for the transcriptional suppression of the specific genes tested, identifies tumors that have a higher propensity to manifest genome-wide hypermethylation, and thus are more likely to inactivate genes critical for tumor progression and response to therapy, leading to a worst prognosis
. In line with this notion, functional enrichment analysis of the genes that were significantly differentially methylated between LM and HM groups revealed a perturbation of several biological pathways, including cytokine signaling, cell adhesion, drug and retinol metabolism, and natural killer cell mediated cytotoxicity (Additional file
1). Thus, the global alteration of these pathways could account for the different OS of CM patients bearing different methylomes. Intriguingly, biological processes involved in immune response are highly represented in the genes differentially methylated between HM and LM patients (Additional file
2), suggesting that an improved immune recognition of CM cells with a LM profile might contribute to the better survival of these patients. Though this hypothesis has still to be demonstrated and is currently under investigation, an initial support may come from the well known involvement of promoter methylation in regulating the expression of different molecules involved in the immune recognition of cancer cells, including: i) the de novo expression of the Cancer Testis Antigens (CTA) tumor associated antigens by neoplastic cells of different histotype and melanoma stem cells following promoter hypomethylation; ii) the direct correlation between levels and total number of CTA expressed in short-term cultures of CM cells and LINE-1 hypomethylation (Sigalotti and Maio, unpublished); iii) the ability of pharmacologic DNA hypomethylation to increase immunogenicity and immune recognition of cancer cells through the up-regulation of HLA class I and co-stimulatory/accessory molecules
Irrespective of the underlying biological features associated with the different whole-genome methylation profiles, the prognostic value of methylome classification here identified for stage IIIC CM patients bears several important practical clinical implications. Among these are: i) providing an improved clinico-pathological sub-staging; ii) modulating post-surgery follow-up-procedures; iii) selecting patients at higher risk of recurrence for adjuvant treatment(s); iv) stratifying patients in clinical trials based on their differential prognosis. This latter aspect is of particular relevance also in view of multiple studies that have explored new adjuvant regimens in stage III CM patients, in the US and European cooperative groups. These considerations may not be restricted to CM. Indeed, a recent work, investigating whole-genome methylation profiles in breast cancer, demonstrated that the group of patients with high-methylation tumors disproportionately included individuals with a poor prognosis defined by the 70-gene expression prognostic signature of van’t Veer et al.