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DNA methylation abnormalities comprise a common phenomenon in human lung cancer and thus it is suggested that DNA methylation based biomarkers may assist in diagnosis, prognosis and targeted therapeutics. Although a lot of data has already been produced, the spectrum of DNA methylation markers with the high potential in clinical use is not yet clear. In this study we have developed pyrosequencing-based assays to scan the proximal promoters of p16, RARb, MGMT, p57, CYGB, MAGEA3 and GAS genes in 48 NSCLCs and their counterpart normal tissues. We were able to read up to 12 CpGs per promoter (Figure 1) in a semi-quantitative manner and thus calculate the methylation index (MtI) for each gene. GAS, p57 and MAGEA3 were heavily methylated in normal tissue while their MtI was reduced in tumour tissue. In contrast, p16, RARB, MGMT and CYGB presented very low basal levels of methylation in normal tissue while they were frequently methylated in tumour tissue. In particular, we identified 33/48 (69%) tumour samples carrying at least one methylated gene (MtI>0.1) and only 3/48 (6%) corresponding normals with this degree of methylation. The overall index for these four genes was significanlty higher in tumours (0.15) than normals (0.056) (paired T test, p=1.5×10-4. It is of note that p16 methylation correlated with concordant RARB methylation (p=0.004) and MGMT methylation (p=0.009) suggesting a methylator phenotype in a subgroup of the samples. There was also a trend to associate high methylation index with nodal metastasis (Mann-Whitney, p=0.1). It is also important to note that the genes with measurable levels of methylation in normal tissue presented with very diverse methylation levels at the different CpGs screened (one way ANOVA, CYGB p=3.6×10-17, MAGEA3 p=4.4×10-10, p57 p=2.2×10-14, GAS p=1.3×10-14). This underlines the need for quantitative methods that screen a long stretch of the gene promoters in order to select CpGs with higher sensitivity and specificity for future diagnostic purposes. Our findings indicate that pyrosequencing based assays offer multiple internal quality controls for bisulphite conversion. and provide an excellent platform for quantitatively assessing DNA methylation in human specimens.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]