Epigenome-wide DNA methylation landscape of melanoma progression to brain metastasis reveals aberrations on homeobox D cluster associated with prognosis.

Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of <1 year. Alteration in DNA methylation is a major hallmark of tumor progression and metastasis; however, it remains largely unexplored in MBM. In this study, we generated a comprehensive DNA methylation landscape through the use of genome-wide copy number, DNA methylation and gene expression data integrative analysis of melanoma progression to MBM. A progressive genome-wide demethylation in low CpG density and an increase in methylation level of CpG islands according to melanoma progression were observed. MBM-specific partially methylated domains (PMDs) affecting key brain developmental processes were identified. Differentially methylated CpG sites between MBM and lymph node metastasis (LNM) from patients with good prognosis were identified. Among the most significantly affected genes were the HOX family members. DNA methylation of HOXD9 gene promoter affected transcript and protein expression and was significantly higher in MBM than that in early stages. A MBM-specific PMD was identified in this region. Low methylation level of this region was associated with active HOXD9 expression, open chromatin and histone modifications associated with active transcription. Demethylating agent induced HOXD9 expression in melanoma cell lines. The clinical relevance of this finding was verified in an independent large cohort of melanomas (n = 145). Patients with HOXD9 hypermethylation in LNM had poorer disease-free and overall survival. This epigenome-wide study identified novel methylated genes with functional and clinical implications for MBM patients.

Genome-wide characterization of circulating tumor cells identifies novel prognostic genomic alterations in systemic melanoma metastasis.

BACKGROUND: Circulating tumor cells (CTC) have been found in patients with metastatic melanoma and are associated with advanced melanoma stage and poor patient outcome. We hypothesize that CTC harbor genomic changes critical in the development of distant systemic metastasis. Here, we present the first genome-wide copy-number aberration (CNA) and loss of heterozygosity (LOH)-based characterization of melanoma CTC. METHODS: CTC were isolated from peripheral blood monocytes of 13 melanoma patients with regional metastasis stage IIIB/C using antibodies against melanoma-associated cell surface gangliosides. RESULTS: We characterized 251 CNA in CTC. Comparative analysis demonstrated >90% concordance in single-nucleotide polymorphism profiles between paired CTC and tumor metastases. In particular, there were notable recurring CNA across patients. In exploratory studies, the presence of several top CTC-associated CNA was verified in distant metastasis (stage IV) from 27 patients, suggesting that certain genomic changes are propagated from regional metastasis to CTC and to distant systemic metastases. Lastly, an exploratory biomarker panel derived from 5 CTC-associated CNA [CSMD2 (CUB and Sushi multiple domains 2), 1p35.1; CNTNAP5 (contactin associated protein-like 5), 2q14.3; NRDE2 (NRDE-2, necessary for RNA interference, domain containing), 14q32.11; ADAM6 (ADAM metallopeptidase domain 6, pseudogene), 14q32.33; and TRPM2 (transient receptor potential cation channel, subfamily m, member 2), 21q22.3] conferred prognostic utility for melanoma recurrence [hazard ratio (HR), 1.14; CI, 1.00-1.44; P = 0.0471] and death (HR, 2.86; CI, 1.23-14.42; P = 0.0014) in 35 patients with stage IIIB/C melanoma, with a 5-year disease-free survival of 13% vs 69% (P = 0.0006) and overall survival of 28% vs 94% between high-risk and low-risk groups defined by the biomarker panel, respectively. CONCLUSIONS: This study provides the first detailed CNA-based profile of melanoma CTC and illustrates how CTC may be used as a novel approach for identification of systemic metastasis.

Epigenetic changes of EGFR have an important role in BRAF inhibitor-resistant cutaneous melanomas.

BRAF mutations are frequent in cutaneous melanomas, and BRAF inhibitors (BRAFi) have shown remarkable clinical efficacy in BRAF mutant melanoma patients. However, acquired drug resistance can occur rapidly and tumor(s) often progresses thereafter. Various mechanisms of BRAFi resistance have recently been described; however, the mechanism of resistance remains controversial. In this study, we developed BRAFi-resistant melanoma cell lines and found that metastasis-related epithelial to mesenchymal transition properties of BRAFi-resistant cells were enhanced significantly. Upregulation of EGFR was observed in BRAFi-resistant cell lines and patient tumors because of demethylation of EGFR regulatory DNA elements. EGFR induced PI3K/AKT pathway activation in BRAFi-resistant cells through epigenetic regulation. Treatment of EGFR inhibitor was effective in BRAFi-resistant melanoma cell lines. The study demonstrates that EGFR epigenetic activation has important implications in BRAFi resistance in melanoma.

Multi-platform Genome-wide Analysis of Melanoma Progression to Brain Metastasis.

Melanoma has a high tendency to metastasize to brain tissue. The understanding about the molecular alterations of early-stage melanoma progression to brain metastasis (MBM) is very limited. Identifying MBM-specific genomic and epigenomic alterations is a key initial step in understanding its aggressive nature and identifying specific novel druggable targets. Here we describe a multi-platform dataset generated with different stages of melanoma progression to MBM. This data includes genome-wide DNA methylation (Illumina HM450K BeadChip), gene expression (Affymetrix HuEx 1.0 ST array), single nucleotide polymorphisms (SNPs) and copy number variation (CNV; Affymetrix SNP 6.0 array) analyses of melanocyte cells (MNCs), primary melanoma tumors (PRMs), lymph node metastases (LNMs) and MBMs. The analysis of this data has been reported in our recently published study (Marzese et al., 2014).