Epigenetic signature of differentially methylated genes in cutaneous melanoma

Background: Cutaneous melanoma (CM) is the most aggressive subtype of skin cancer, with increasing incidence over the past several decades. DNA methylation is a key element of several biological processes such as genomic imprinting, cell differentiation and senescence, and deregulation of this mechanism has been implicated in several diseases, including cancer. In order to understand the relationship of DNA methylation in CMs, we searched for an epigenetic signature of cutaneous melanomas by comparing the DNA methylation profiles between tumours and benign melanocytes, the precursor cells of CM. Methods: We used 20 primary CMs and three primary cell cultures of melanocytes as a discovery cohort. The tumours mutational background was collected as previously reported. Methylomes were obtained using the HM450K DNA methylation assay, and differential methylation analysis was performed. DNA methylation data of CMs from TCGA were recovered to validate our findings. Results: A signature of 514 differentially methylated genes (DMGs) was evident in CMs compared to melanocytes, which was independent of the presence of driver mutations. Pathway analysis of this CM signature revealed an enrichment of proteins involved in the binding of DNA regulatory regions (hypermethylated sites), and related to transmembrane signal transducer activities (hypomethylated sites). The methylation signature was validated in an independent dataset of primary CMs, as well as in lymph node and distant metastases (correlation of DNA methylation level: r > 0,95; Pearson's test: p < 2.2e-16). Conclusions: CMs exhibited a DMGs signature, which was independent of the mutational background and possibly established prior to genetic alterations. This signature provides important insights into how epigenetic deregulation contributes to melanomagenesis in general (AU)

LINE-1 hypomethylation and mutational status in cutaneous melanomas.

Epigenetic dysregulation is an important emerging hallmark of cutaneous melanoma development. The global loss of DNA methylation in gene-poor regions and transposable DNA elements of cancer cells contributes to increased genomic instability. Long interspersed element-1 (LINE-1) sequences are the most abundant repetitive sequence of the genome and can be evaluated as a surrogate marker of the global level of DNA methylation. In this work, LINE-1 methylation levels were evaluated in cutaneous melanomas and normal melanocyte primary cell cultures to investigate their possible association with both distinct clinicopathological characteristics and tumor mutational profile. A set of driver mutations frequently identified in cutaneous melanoma was assessed by sequencing (actionable mutations in BRAF, NRAS, and KIT genes, and mutations affecting the TER T promoter) or multiplex ligation-dependent probe amplification (MLPA) (CDKN2A deletions). Pyrosequencing was performed to investigate the methylation level of LINE-1 and CDKN2A promoter sequences. The qualitative analysis showed a trend toward an association between LINE-1 hypomethylation and CDKN2A inactivation (p=0.05). In a quantitative approach, primary tumors, mainly the thicker ones (>4 mm), exhibited a trend toward LINE-1 hypomethylation when compared with control melanocytes. To date, this is the first study reporting in cutaneous melanomas a possible link between the dysregulation of LINE-1 methylation and the presence of driver mutations.

DNA Methylation Levels of Melanoma Risk Genes Are Associated with Clinical Characteristics of Melanoma Patients.

In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples.

Role of immunoexpression of nitric oxide synthases by Hodgkin and Reed-Sternberg cells on apoptosis deregulation and on clinical outcome of classical Hodgkin lymphoma.

Hodgkin and Reed-Sternberg (H-RS) cells of classical Hodgkin lymphoma (cHL) present an impaired expression of immunoglobulin genes, but escape apoptotic death. We investigated whether nitric oxide synthases (NOS) are expressed by H-RS cells, studied their association with EBV status and the expression of apoptotic proteins, and investigated their relationship to the clinical outcome of 171 patients. NOS1 and NOS2 were expressed in a large number of cases, whereas NOS3 expression was not detected. Positive associations were found between NOS1 and p53, bax and NOS2, bcl-2 and NOS2, bax and p53, and between bax and fasL. Inverse correlations were established between EBV and NOS2 and between EBV and bcl-2. A shorter overall survival (OS) was associated with strong expression of NOS2. In conclusion, NOS are expressed by H-RS cells of cHL.