EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissuespecific methylation

Background: Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression. Methods: EFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients. Results: Analysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples. Conclusions: Biallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.