Background
Cutaneous melanoma (CM) is a very aggressive neoplasm of growing incidence and mortality in industrialized countries, and the leading cause of skin cancer-related deaths worldwide [
1]. Surgery, in early phases of disease has curative potential for patients; for advanced CM conventional therapies have failed to prolong survival [
2]. At present, the best predictor of 5-year survival is the clinico-pathological stage of disease, which defines overall survival (OS) rates ranging from 95% to 7% for stage I to IV patients, respectively [
3]. However, within the same clinico-pathological stage category, patients often behave radically differently, and the current lack of prognostic molecular markers impairs our ability to identify CM patients with highly aggressive as opposed to more indolent courses of disease [
4].
In mammals, DNA methylation of cytosine at the 5C-position in the context of CpG dinucleotides represents a major epigenetic mechanism controlling gene expression, chromosome X inactivation, imprinting and repression of endogenous parasitic sequences (for review see [
5]). Global genomic DNA hypomethylation (i.e., overall reduction of the 5-methylcytosine content) is a frequent molecular event in cancer and has been observed in neoplastic cells of different histotypes [
6]. Genomic hypomethylation might contribute to cancer development and progression through various mechanisms including generation of chromosomal instability, reactivation of transposable elements, and loss of imprinting [
5]. Substantial decreases in the 5-methylcytosine content in the genome mainly reflect the hypomethylation of repetitive genomic sequences. Among these, methylation levels of the
Long Interspersed Nucleotide Element-1 (
LINE-1) may represent a surrogate marker for the overall level of genomic DNA methylation [
7]. Preliminary investigations of
LINE-1 methylation in solid tumors have identified increasingly greater hypomethylation of these sequences with progression of gastric and prostatic cancer [
8,
9]. Furthermore, decreased methylation of
LINE-1 correlated with higher FIGO stage and advanced tumor grade of ovarian cancer [
10]. Of interest, a increased hypomethylation of
LINE-1 elements has been associated with poorer prognosis in colon and ovarian cancers [
10,
11]; however, these studies did not investigate the role of
LINE-1 methylation as a prognostic factor in patients at identical stages of disease.
Despite these promising initial data, to the best of our knowledge no studies have investigated the influence of the overall level of genomic DNA methylation on CM prognosis. Accordingly, we investigated whether the extent of methylation of the LINE-1 repetitive elements may account for the differing survival patterns of CM patients of identical clinico-pathological stage of disease. The study was conducted on a series of 42 consecutive stage IIIC CM patients for whom the autologous short-term cell cultures were available. The latter were analyzed early during in vitro passage, and utilized instead of tumor tissues to overcome possible alterations in the evaluation of levels of LINE-1 methylation due to the unavoidable presence of contaminating normal cells. Results demonstrated that LINE-1 hypomethylation identifies CM patients with a significantly better prognosis as compared to those with hypermethylated LINE-1 sequences. These findings demonstrate that evaluation of LINE-1 methylation levels may greatly help in guiding the daily clinical management of CM patients, and provide a strong rationale for the development of a large prospective validation study.
Discussion
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 [
5]. 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[
21] 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[
25]. 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].
Conclusion
Irrespective of the underlying biological mechanism(s) triggered by
LINE-1 hypomethylation, the prognostic value of
LINE-1 methylation here identified for stage IIIC CM patients bears several important practical clinical implications. Among these, the goal to provide CM patients with improved clinico-pathological sub-stage and/or follow-up-procedures would be enhanced using
LINE-1 methylation status, and these findings might be used to select and/or stratify patients for adjuvant treatment based on the methylation level of
LINE-1 in their tumors. In addition, the significant positive prognosis of
LINE-1 hypomethylated patients should prompt the incorporation of this in new studies aimed at understanding whether pharmacologic DNA hypomethylation [
35] could be regarded as a feasible chemoprevention approach in the initial phases of disease and/or in patients at high-risk of disease recurrence.
Our present findings will be further investigated in prospective multicenter studies in which the prognostic significance and the predictive value for different treatments of CM will be validated. Providing further support to our initial data will finally allow to establish the appropriateness of adding the evaluation of LINE-1 methylation into the routine clinico-pathological ascertainment of CM patients, in order to help personalizing their comprehensive clinical management.
Competing interests
LS and MM have applied for a patent based on the findings reported in this manuscript. All other authors declare no competing interests.
Authors' contributions
LS participated in acquiring laboratory data, data analysis and interpretation, study coordination, and drafted the manuscript. EF performed the pyrosequencing analyses, and contributed in data acquisition and analysis. EB performed the statistical analyses. AC, GP, FC contributed in cellular biology procedures, molecular assays and data acquisition. SC, contributed in data interpretation. SM participated in acquisition of clinical data and data interpretation. JMK participated in data interpretation and manuscript drafting. MM conceived of the study, participated in its design and coordination, and contributed in producing the final draft of the manuscript. All authors read and approved the final manuscript.