Epigenetic Inactivation of Tumor Suppressor Genes in Serum of Patients with Cutaneous Melanoma

Abstract

Small amounts of cell-free DNA circulate in both healthy and diseased human blood, while increased concentrations of DNA are present in the serum of cancer patients. Tumor-specific mutations or epigenetic modifications have predominantly been detected in tissue specimens. The purpose of this study was to investigate methylation of five different genes involved in tumor suppression and DNA repair (suppressors of cytokine signaling 1 and 2 (SOCS1, SOCS2)), Ras-association domain family protein 1A (RASSF1a), D-type p16(INK4a) cyclin-dependent kinase inhibitor (CDKN), and O6-methylguanine DNA-methyltransferase (MGMT)) in the serum of 100 patients using methylation-specific PCR. In all, 41 melanoma patients (stage I = 18; stage II = 10; stage III/IV = 13), 13 healthy controls without nevi, and 10 individuals with more than 15 nevi of >5 mm in size were investigated. For comparison, sera from patients with other skin tumors (nine basal cell cancers, five Kaposi's sarcoma), different metastasized cancers (five breast cancers, five colon cancers), and several chronic inflammatory diseases (n = 12) were also analyzed. In addition, we examined if methylation was involved in silencing transcription of these genes in 12 melanoma specimens. SOCS1, SOCS2, RASSF1a, CDKN2a, and MGMT were methylated in 75, 43, 64, 75, and 64% of melanoma samples, respectively. Of the 41 melanoma patients, 83% had one hypermethylated gene, while 66, 51, and 41% had two, three, or four hypermethylated genes, respectively. Also, 20% of these patients showed hypermethylation for all genes, while only 17% showed no methylation. Importantly, the methylation profile of the selected genes from melanoma patients was distinct from the other analyzed tumors. Transcription of SOCS1, SOCS2, CDKN2a, and RASSF1a genes was significantly reduced in fresh melanoma samples, while MGMT showed a 12-fold upregulation at the messenger ribonucleic acid level (P < 0.001). Our findings suggest that epigenetic silencing of the studied tumor suppressor genes is a common and probably important mechanism for melanoma formation. This convenient method using a simple blood sample may contribute to classification of melanoma and awaits clinical validation.

Genomics of Human Malignant Melanoma

“…In spite of the existence of a large dataset that has revealed more than 80 genes downregulated by promoter methylation, to allow their clinical utilisation, the detected elements must be distinguished based on the number of primary tumour samples involved in the study and the frequency of positive results as eligibility criteria for diagnosis or to determine whether they are a candidate therapeutic target. Promoter hypermethylation of two molecules involved in the cell cycle, Ras association domain-containing protein 1 (RASSF1A) and the cyclin-dependent kinase inhibitor 2A-coding gene (CDKN2A), has been confirmed by multiple, substantive experiments; these findings have also been confirmed in melanoma cell lines [Furuta et al, 2004;Marini et al, 2006;Sigalotti et al, 2010]. A higher level of methylation of oestrogen receptor alpha (ERα) compared to normal tissues has also been revealed in both tumour specimens and cell lines [Furuta et al, 2004;Mori et al, 2006].…”

SOCS2: inhibitor of JAK2V617F-mediated signal transduction

“…[1][2][3] Aberrant methylation of SOCS genes, correlated with transcriptional silencing, has been described in solid tumors and hematopoetic diseases. [4][5][6][7][8][9][10] Silencing of SOCS genes may lead to both cytokine hypersensitivity and independency, thereby contributing to tumorigenesis. 4,10 SOCS gene hypermethylation and deletion has also been described in cases of myeloproliferative disorders (MPDs), carrying the Janus kinase 2 (JAK2)V617F point mutation.…”

Detection of mutated BRAFV600E variant in circulating DNA of stage III–IV melanoma patients

“…16 Circulating cell free tumor DNA has been detected in plasma and serum by assessing allelic imbalance at microsatellite markers [17][18][19] and tumor-related gene methylation. [20][21][22] Recent studies indicate that these markers may show a predictive value of the response to biochemotherapy in stage IV patients. 23,24 Here, we report the results of a pilot study aimed at evaluating the BRAFV600E mutation as a molecular marker in circulating DNA from cutaneous melanoma patients.…”