Promoter hypermethylation: a common cause of reduced p16(INK4a) expression in uveal melanoma.

Tumors often display unrestricted cell cycling attributable to a dysfunctional G(1)-S checkpoint. One of the mechanisms leading to such a defect is the inactivation of the cyclin-dependent kinase inhibitor p16(INK4a). Although inactivation of p16(INK4a) is observed in a wide range of tumors, including cutaneous melanoma, genetic alteration of p16(INK4a) is reportedly uncommon in uveal melanoma. Here we show that the p16(INK4a) promoter is hypermethylated in 6 of 12 uveal melanoma cell lines and in 7 of 22 primary uveal melanomas analyzed. Five of seven patients with a methylated primary tumor died of metastatic disease compared with 2 of 15 patients with a nonmethylated primary tumor. We also show that all uveal melanoma cell lines with a hypermethylated p16(INK4a) promoter have lost p16(INK4a) expression but have maintained the expression of p14(ARF). Treatment of uveal melanoma cell lines with 5-aza-2'-deoxycytidine results in demethylation of p16(INK4a) and in reexpression of p16(INK4a) mRNA, which is maintained upon withdrawal of the 5-aza-2'-deoxycytidine. In conclusion, p16(INK4a) promoter methylation appears to be a common event in uveal melanoma and is accompanied by the loss of p16(INK4a) expression.

Normal HLA class I, II, and MICA gene distribution in uveal melanoma.

PURPOSE: The molecules of the HLA class I and II molecules as well as the MHC class I chain-related gene A (MICA), a polymorphic and stress-induced cell surface molecule, are involved in T-cell and natural killer-cell (NK-cell) mediated immune responses. In this study we looked for any genetic susceptibility contributed by HLA class I, class II, or MICA genes with regard to the development of uveal melanoma. METHODS: Between 1998 and 2001, 159 uveal melanoma patients were typed for HLA class I and II, and 168 uveal melanoma patients were evaluated for MICA by microsatellite typing. The HLA antigen and MICA allele frequencies were compared with control groups of, respectively, 2,440 and 247 healthy Dutch individuals. RESULTS: HLA class I, HLA class II, and MICA gene frequencies in uveal melanoma patients and healthy Dutch controls showed no significant deviations after correction for the number of comparisons. CONCLUSIONS: We conclude that there is no genetic susceptibility or increased risk attributed to any HLA class I, class II, and MICA polymorphism with regard to the development of uveal melanoma.

Characterization of melanocortin-1 receptor gene variants in uveal melanoma patients.

PURPOSE: Allelic variations of the melanocortin-1 receptor (MC1R) gene have been linked to red hair and sun-sensitive skin types and may play a role in the susceptibility to develop cutaneous malignant melanoma (CMM). To define the role of MC1R gene in uveal melanoma, a case control study was performed, in which the presence of MC1R gene variations in uveal melanoma patients was compared with that of healthy controls. METHODS: MC1R gene variants were analyzed in 162 uveal melanoma patients and 255 healthy controls. After genomic DNA was isolated from venous blood, the MC1R gene was amplified by polymerase chain reaction (PCR) and examined for the presence of variants by single-strand conformation polymorphism (SSCP) analysis. Participants were asked to complete a questionnaire regarding skin type, eye color, and hair color. RESULTS: No disparity was found between the distribution of the MC1R gene variants in both groups. Furthermore, no associations between MC1R genotype and pigment phenotype were found. In contrast to CMM, uveal melanoma patients did not show specific MC1R gene variants. Compared with controls, most uveal melanoma patients had blue eyes (65%, P = 0.060) and skin type III (56%); however, in the uveal melanoma group the presence of dark blond hair was significantly elevated (46%, P = 0.030). These findings are in contrast with studies on CMM, where most patients have skin type II and red/fair hair. CONCLUSIONS: These data suggest that MC1R variants do not play a role in the susceptibility to develop uveal melanoma. Furthermore, most uveal melanoma patients share phenotypic characteristics that differ from findings in CMM patients.

Expression of epidermal growth factor receptor: risk factor in uveal melanoma.

PURPOSE: To investigate the prognostic significance of the expression of epidermal growth factor receptor (EGFR) in uveal melanoma. EGFR is a transmembrane glycoprotein, and its expression has been correlated with the development of metastases in various malignancies. METHODS: Frozen sections from 22 primary uveal melanomas were examined for EGFR expression by a three-step immunoperoxidase staining, using a mouse anti-human EGFR IgG2b monoclonal antibody. The results were compared with patient survival and clinical and histopathologic parameters. RESULTS: EGFR expression could not be determined on one tumor due to excessive pigmentation. Two patients died of causes unrelated to melanoma, and two patients were lost to follow-up. Out of 21 tumors, six tumors showed immunoreactivity for EGFR. Five of these six patients (83%) died due to metastases, compared with 2 (17%) of 12 patients with no EGFR expression (Kaplan-Meier analysis P = 0.0004). EGFR-positive tumors tended to have a greater tumor prominence and a higher mitotic rate. CONCLUSIONS: The expression of EGFR was significantly correlated with death due to metastatic disease and therefore can be regarded as an important prognostic factor in human uveal melanoma.

Frequent loss of heterozygosity on chromosome 6p in uveal melanoma.

Lack of expression of HLA class I antigens is frequently observed on primary uveal melanoma, and is correlated with improved patient survival. Several mechanisms may contribute to the observed loss of HLA class I expression, including changes at the DNA level. In this study, we used microsatellite analysis as a molecular genetic approach to examine loci on chromosome 6p for loss of heterozygosity (LOH). Three pairs of microsatellite markers were used to screen 20 formalin-fixed, paraffin-embedded uveal melanomas for LOH on the short arm of chromosome 6. In all cases, normal adjacent scleral tissue was used as a control. We identified LOH in eleven cases from microsatellite locus D6S105 to the telomere, in eight cases from microsatellite locus D6STNFa to the telomere (area includes D6S105), and in seven cases from microsatellite locus D6S291 to the end of chromosome 6p (includes D6STNFa and D6S105). In seven cases, retention of heterozygosity was found at all three loci using these primers. Our results suggest that loss of heterozygosity on chromosome 6p is a common feature in uveal melanoma. We did not find a correlation between the presence of LOH and locus-specific HLA-A and -B expression.

High frequency of allele-specific down-regulation of HLA class I expression in uveal melanoma cell lines.

Uveal melanoma is the most common primary intra-ocular tumor in adults and has a high mortality rate due to liver metastases, for which no effective treatment is available. To investigate whether immunotherapy might be feasible in uveal melanoma, the HLA class I surface expression of 6 uveal melanoma cell lines was analyzed by flow cytometry using a broad panel of allele-specific monoclonal antibodies. To up-regulate HLA expression, cells were also cultured with IFN-alpha or -gamma. In general, expression of HLA-A alleles was high (except for cell line EOM-3) and could be further up-regulated by both IFN-alpha and -gamma. In cell line EOM-3, IFN-gamma treatment resulted in significant HLA-A expression while IFN-alpha treatment did not. Expression of HLA-B alleles was low or even negative. Variable effects were observed after IFN treatment. In 3 cell lines, expression of some HLA-B alleles could not be induced by IFN-alpha or -gamma: HLA-B44 in cell line 92-1, HLA-B15 in cell line OCM-1 and HLA-B5 in cell line MEL-202. The other B alleles of these cell lines showed enhanced expression levels upon IFN stimulation. In OMM-1 cells, IFN-alpha and -gamma increased the expression of HLA-A but did not induce expression of the 2 B alleles, indicating an HLA-B locus-specific loss. We thus found a high frequency of allele-specific and locus-specific down-regulation of HLA expression in uveal melanoma cell lines. Some of these defects were not restored by IFN-alpha or -gamma treatment. The lack of HLA expression may explain why uveal melanoma cells escape immune surveillance by cytotoxic T cells and complicate the development of immunotherapy in uveal melanoma.