EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation

Abstract

BackgroundUveal 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.MethodsEFS 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.ResultsAnalysis 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.ConclusionsBiallelic 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.

Molecular pathology of uveal melanoma

“…Upregulation of E-cadherin and Wnt/beta-catenin signalling pathways 42,43 Downregulation of the helix-loop-helix inhibitor ID2 39,40 Increased expression of matrix metalloproteinases (MMPs) and downregulation of their tissue inhibitors (TIMPs) by UM [44][45][46][47] ALCAM expression 48 NOTCH pathway activation 49 Biallelic methylation of EFS 50 …”

Decreased expression of EFS is correlated with the advanced prostate cancer

“…Although there are several reports on the relationship between other CAS family members and cancer, there is only one report on the EFS expression in carcinogenesis. In that report, Neumann et al have shown that bi-allelic methylation of EFS promoter is a common event in melanoma and which has been associated with poor prognosis [16].…”

Differentially methylated embryonal Fyn-associated substrate (EFS) gene as a blood-specific epigenetic marker and its potential application in forensic casework

“…Neumann et al [28] detected differential methylation patterns of the EFS gene when analysing various human tissues including blood, sperm and buccal cells. It is known that EFS encodes for a protein playing a role in coordinating cell adhesion via tyrosine-kinasebased signalling.…”

“…As mentioned above, Neumann et al [28] detected differential methylation patterns of the EFS gene when analysing various human tissues including the forensically relevant tissues:…”

“…Neumann et al analysed the methylation status of the EFS gene, and more specifically the region Chr14: 23,835,859-23,835,970 (GrCh37/hg19) including a total of 11 CpG sites, in various tissues including blood, buccal cells, sperm, brain and reported tissue-specific methylation patterns [28]. They found that EFS is highly methylated in blood, completely unmethylated in sperm and partially methylated in buccal cells.…”