In this study we demonstrate that the global level of LINE-1 methylation of short-term tumor cell cultures grown from patients with nodal disease is a significant predictor of OS in stage IIIC CM patients. This finding is of remarkable clinical relevance, since, to the best of our knowledge, it provides the first evidence of a molecular marker capable of differentiating the prognosis of CM patients in this high-risk substage. These results are of particular emphasis given the conduct of this study in subjects within a single clinically well-defined clinico-pathological staging sub-group, which has become the focus of several ongoing clinical trials in the US and Europe (i.e., ECOG intergroup trial E4697, EORTC trial 18071, GSK trial 111482 "DERMA").
Genomic DNA hypomethylation has been proposed to have an important impact on tumor biology through the generation of chromosomal instability, reactivation of transposable elements, and loss of imprinting . Thus, a negative correlation between genomic hypomethylation and survival of CM patients could have been expected. Instead, we found that hypomethylation of LINE-1 elements at CpG2 or CpG3 sites was associated with a significantly better OS, as demonstrated by Kaplan-Meier analysis and log-rank test. The positive prognostic value of LINE-1 hypomethylation we have identified in CM is in sharp contrast with data most recently obtained in colon and ovarian cancer patients, in which LINE-1 hypomethylation in neoplastic tissues was associated with a poorer prognosis [10, 11]. This discrepancy, however, is not completely surprising. Indeed, data generated on hematologic malignancies showed that LINE-1 hypomethylation can be either a poor or a good prognostic factor, depending on the patient being affected by chronic myeloid leukemia or acute lymphoblastic leukemia, respectively [19, 20]. Thus, the different behavior of CM, with respect to the other solid tumors so far investigated, might further suggest that the underlying biological effect(s) of LINE-1 hypomethylation on patients' outcome could depend on the tumor histotype. Nevertheless, it should be emphasized that our findings are generated from patients in the same clinico-pathological stage of disease, while the studies on ovarian and colon cancer were conducted on the heterogeneous patients population as a whole, and did not investigate the prognostic potential of LINE-1 methylation in specific clinically defined stages of disease. Thus, it remains to be demonstrated whether this different study approach might contribute to the observed discrepancy. Furthermore, it cannot be ruled out that in the different sources of neoplastic material analyzed, the presence of varying proportions of contaminating normal cells in neoplastic tissues, as well as the different methodological approaches employed might contribute to conclusions that may differ from those we have reached in these studies. In this context, our use of short-term CM cultures has the advantage of eliminating contaminating normal cells, yet representing the methylation status of neoplastic cells of the fresh autologous lesion. In fact, similar levels of LINE-1 methylation were observed between short-term cultures and autologous uncultured CM cells that were purified by anti-HMW-MAA immunomagnetic beads from tumor cell suspensions that were available from 10 patients (data not shown).
The mechanism(s) through which LINE-1 hypomethylation affects survival of CM patients remains to be fully explored; however, some speculation can be made, based on recent data in the literature. Tellez et al  have demonstrated that higher levels of LINE-1 methylation correlate with an increased number of aberrantly hypermethylated tumor suppressor genes (TSG) in cultured melanoma cell lines. This notion has gained further support from our most recent observation showing a direct correlation between higher LINE-1 methylation and increased genome-wide gene methylation, measured through CpG island microarrays (Sigalotti and Maio, manuscript in preparation). Thus, epigenetic inactivation of TSG might account for more aggressive disease we have observed in patients with elevated LINE-1 methylation in their neoplastic cells. This hypothesis is in accordance with initial studies reporting a negative association between survival and the presence of hypermethylated ER-α, RASSF1A, RAR-β2, or MINT31 DNA in neoplastic tissues or sera of stage III/IV CM patients [22–24]. On the other hand, hypomethylation, and consequent transcriptional activation, of LINE-1 elements might per se reduce the tumorigenic potential of neoplastic cells by triggering apoptosis and a senescence-like state through the activity of the second open reading frame of LINE-1 . In our findings, this seems not to be the case, since the lack of correlation between methylation and mRNA expression of LINE-1 elements, suggests that LINE-1 products may not be the driving force for the observed increased OS of LINE-1 hypomethylated patients. Genomic DNA hypomethylation has also been associated with the de novo expression of tumor associated antigens belonging to the Cancer Testis Antigen (CTA) class by neoplastic cells of different histotype, including melanoma stem cells [26–29], and we have recently identified a significant correlation between a hypomethylated status of LINE-1 elements and increased levels and total number of CTA concomitantly expressed in short-term cultures of CM cells (Sigalotti and Maio, unpublished). Besides, pharmacologic DNA hypomethylation has been consistently demonstrated to increase immunogenicity and immune recognition of cancer cells through the up-regulation of different molecules involved in antigen processing and presentation, including HLA class I antigens and co-stimulatory molecules [30, 31]. Thus, it is intriguing to speculate that a better immune recognition of LINE-1 hypomethylated CM cells might contribute to the improved survival of these patients. This hypothesis may find indirect support from most recent gene expression profiling studies that identified the expression of "immune-related" genes in the tumor as a marker of good prognosis in stage III-IV CM [32–34].