Aberrant promoter methylation of multiple genes during pathogenesis of bladder cancer

Abstract

Purpose: The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features.

Experimental design: We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and chi(2) or Fisher's exact tests as appropriate. All statistical tests were two-sided.

Results: Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion.

Conclusion: Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon.

Figure 1

Promoter methylation levels for the seven markers in the bladder cancer patient samples (CA; n = 93) and normal bladder tissues (N; n = 26). The quantity of each methylated gene promoter was expressed as the ratio of the amount of PCR products amplified from the methylated gene to the amount amplified with the reference gene β-actin multiplied by 1,000. Boxplots show the middle 50% of data, the line is the median, and the bars extend the median by 1.5 times the interquartile range.

Figure 2

Mean methylation index levels markers in bladder cancer samples compared across noninvasive stages and muscle-invasive stage. Methylation index was calculated as the total number of genes having any methylation divided by the total number of genes examined. The median of methylation index in noninvasive stages and muscle-invasive stage is 3.1 and 5.0, respectively. Boxplots show the middle 50% of data, the line is the median, and the bars extend the median by 1.5 times the interquartile range.

Figure 3

Promoter methylation levels for four representative markers in bladder cancer samples for noninvasive stages (1 + 2) and muscle-invasive stage (3). The quantity of methylated allele of each gene was expressed as the ratio of the amount of PCR products amplified from the methylated gene to the amount amplified with the reference gene β-actin multiplied by 1,000. Boxplots show the middle 50% of data, the line is the median, and the bars extend the median by 1.5 times the interquartile range.