Methylation of Pten As A Prognostic Factor in Malignant Melanoma of the Skin

TO THE EDITOR The molecular mechanisms of malignant melanoma, especially epigenetic silencing, are little understood. In particular, RAS/RAF kinases and changes in p16 and the PTEN state have attracted interest (see the introduction to the Supplementary material). In our study, epigenetic silencing of the tumor suppressor genes PTEN, p16, and RASSF1A and mutations of BRAF and NRAS were analyzed in 619 samples of 230 cutaneous melanomas (median tumor thickness, 1.90mm) from 230 patients. All sections were reevaluated to confirm diagnoses and known histological prognosis parameters. In addition, local dermal actinic aging was scored semi-quantitatively as previously described (Helmbold et al., 2006Helmbold P. Lautenschlager C. Marsch W. Nayak R.C. Detection of a physiological juvenile phase and the central role of pericytes in human dermal microvascular aging.J Invest Dermatol. 2006; 126: 1419-1421Google Scholar). Mutations of codon 600 of BRAF and codon 61 of NRAS were analyzed. Promoter methylation of PTEN, p16, and RASSF1A was investigated by methylation-specific PCR (Schagdarsurengin et al., 2006Schagdarsurengin U. Gimm O. Dralle H. Hoang-Vu C. Dammann R. CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma.Thyroid. 2006; 16: 633-642Google Scholar). Mutation and methylation analyses were confirmed in different regions of the same tumor (mean: 2.59±0.98 Figure 1). Expression of PTEN was studied immunohistologically in selected corresponding paraffin sections. Intercorrelations between the investigated molecular and clinicopathological features were studied. Prospective follow-up data were used for survival analysis. Details of all methods are provided in the Supplementary material (Material and Methods, Supplementary Tables S1 and S2). Download .pdf (.38 MB) Help with pdf files Supplementary material In 147 of 221 (67%) melanomas, a BRAF mutation was detected. Single-nucleotide substitutions at position 1,799 (T>A) were detected more frequently compared with two nucleotide changes (GT>AA) at positions 1,798 and 1,799 (52 and 22%, respectively). There was a negative correlation between the types of BRAF mutation (P=0.001) as well as between BRAF (T>A) and NRAS (A>T) mutations (P=0.037). Ulcerated tumors exhibited a higher frequency of BRAF mutations compared with nonulcerated melanomas (75 and 58%, respectively; P=0.006). We found a strong negative correlation between BRAF (T>A) mutations and dermal actinic aging (P=0.004). (For further details see Supplementary materials: Results, Supplementary Figure S1 and Supplementary Table S3). Methylation of RASSF1A, p16, or PTEN was detected in 25 of 217 (12%), 46 of 213 (22%), or 120 of 200 (60%) melanomas, respectively. There was no correlation between the investigated mutations and/or promoter hypermethylations. Positive correlations were found between PTEN hypermethylation and the degree of actinic aging as well as the TANS (trunk, upper arm, neck, and scalp) location of the tumor (P=0.011 and P=0.043, respectively). Twelve selected samples were investigated for correlation between PTEN methylation and immunohistological PTEN expression. In the subgroup of melanomas with an unmethylated PTEN promoter, +++ (strong), ++ (equivalent to endothelial cells), + (low), or - (no) PTEN expression was found in 3 of 6, 2 of 6, 0 of 6, or 1 of 6 tumors, respectively. In contrast, in the hypermethylated subgroup, the corresponding frequencies were 0 of 6 (+++ PTEN), 0 of 6 (++ PTEN), 1 of 6 (low PTEN), or 5 of 6 (- PTEN). Further details are given in the Supplementary material: Results, Supplementary Figures S2 and S3 and Supplementary Table S3. Kaplan–Meier estimations showed that patients with tumors that are methylated for PTEN have an increased risk of dying compared with patients without such methylation (P=0.035) (Figure 1). This was confirmed by multivariate Cox regression analysis (Table 1). Survival analyses showed an additional prognostic influence of tumor thickness, tumor surface diameter, mitotic index, and age on survival, and tumor thickness and ulceration on recurrence-free survival (Figure 1, Table 1; Supplementary material: Results, Supplementary Figure S4, Supplementary Tables S4 and S5).Table 1Multivariate Cox regression analysis of overall survival and recurrence-free survivalOdds ratio95% CIP-valuenOverall survival176 Tumor diameter2.541.56–4.130.000 Tumor thickness2.011.22–3.290.006 PTEN methylation1.751.12–2.730.014 Mitotic index1.681.08–2.600.020 Age1.541.00–2.390.052Recurrence-free survival223 Tumor thickness4.292.43–7.580.000 Ulceration1.841.09–3.090.023CI, confidence interval; n, number of cases; P, significance level. Open table in a new tab CI, confidence interval; n, number of cases; P, significance level. The most interesting result of our study is that PTEN methylation acts as an independent prognostic parameter, and to our knowledge this has not previously been reported. However, PTEN methylation as a prognostic marker was not superior to the strongest traditional markers (tumor thickness and ulceration). Thus, it cannot act as a single survival parameter for clinical purposes. The observed frequency of PTEN methylation was consistent with other epigenetic data and PTEN expression analyses (Zhou et al., 2000Zhou X.P. Gimm O. Hampel H. Niemann T. Walker M.J. Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation.Am J Pathol. 2000; 157: 1123-1128Google Scholar; Mikhail et al., 2005Mikhail M. Velazquez E. Shapiro R. Berman R. Pavlick A. Sorhaindo L. et al.PTEN expression in melanoma: relationship with patient survival, Bcl-2 expression, and proliferation.Clin Cancer Res. 2005; 11: 5153-5157Google Scholar; Mirmohammadsadegh et al., 2006Mirmohammadsadegh A. Marini A. Nambiar S. Hassan M. Tannapfel A. Ruzicka T. et al.Epigenetic silencing of the PTEN gene in melanoma.Cancer Res. 2006; 66: 6546-6552Google Scholar). Our data show a direct relationship between PTEN hypermethylation and its decreased expression. The lack of expression in an unmethylated tumor could be attributed to loss of heterozygosity of PTEN, which is frequently observed in melanoma (Zhou et al., 2000Zhou X.P. Gimm O. Hampel H. Niemann T. Walker M.J. Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation.Am J Pathol. 2000; 157: 1123-1128Google Scholar). PTEN methylation is lower in the melanomas of Japanese individuals (Furuta et al., 2004Furuta J. Umebayashi Y. Miyamoto K. Kikuchi K. Otsuka F. Sugimura T. et al.Promoter methylation profiling of 30 genes in human malignant melanoma.Cancer Sci. 2004; 95: 962-968Google Scholar). This could be related to the detection of a pseudogene located on chromosome 9 (Zysman et al., 2002Zysman M.A. Chapman W.B. Bapat B. Considerations when analyzing the methylation status of PTEN tumor suppressor gene.Am J Pathol. 2002; 160: 795-800Google Scholar). However, in silico analysis with our utilized primers revealed only homology with the PTEN CpG island promoter on chromosome 10q23 (Schagdarsurengin et al., 2006Schagdarsurengin U. Gimm O. Dralle H. Hoang-Vu C. Dammann R. CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma.Thyroid. 2006; 16: 633-642Google Scholar). It has been reported that decreased PTEN expression correlated significantly with ulceration, but not with survival (Mikhail et al., 2005Mikhail M. Velazquez E. Shapiro R. Berman R. Pavlick A. Sorhaindo L. et al.PTEN expression in melanoma: relationship with patient survival, Bcl-2 expression, and proliferation.Clin Cancer Res. 2005; 11: 5153-5157Google Scholar). Interestingly, the frequency of PTEN methylation significantly increased with the degree of actinic aging and TANS location in our study. This suggests an influence of chronic UV light exposure. The frequency of BRAF mutations in our study (67%) and the absence of a link between BRAF mutation and survival are consistent with previous findings (Akslen et al., 2005Akslen L.A. Angelini S. Straume O. Bachmann I.M. Molven A. Hemminki K. et al.BRAF and NRAS mutations are frequent in nodular melanoma but are not associated with tumor cell proliferation or patient survival.J Invest Dermatol. 2005; 125: 312-317Google Scholar). In contrast to a previously postulated influence of UV light on BRAF mutagenesis (Besaratinia and Pfeifer, 2008Besaratinia A. Pfeifer G.P. Sunlight ultraviolet irradiation and BRAF V600 mutagenesis in human melanoma.Hum Mutat. 2008; 29: 983-991Google Scholar), we observed an inverse correlation between BRAF (T>A) mutation and dermal actinic aging, and no correlation between BRAF mutation and TANS tumor location. Thus, chronic UV exposition, at least, seems not to be a direct predictor of BRAF (T>A) mutation. Another difference from previous studies was the absence of synergy between the BRAF mutation and RASSF1A hypermethylation in our study. Such a synergy was previously suggested on the basis of only 17 cases (Reifenberger et al., 2004Reifenberger J. Knobbe C.B. Sterzinger A.A. Blaschke B. Schulte K.W. Ruzicka T. et al.Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas.Int J Cancer. 2004; 109: 377-384Google Scholar). In summary, our results show that mutation of BRAF and methylation of PTEN occur frequently in melanoma. PTEN methylation correlates with tumor localization or dermal actinic aging and is an independent predictor of impaired patient survival. Reinhard Dammann and Peter Helmbold received grant support from Deutsche Krebshilfe (107742). Reinhard Dammann received grant support from BMBF (FKZ 01ZZ0104) and DFG (DA552). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid