Aberrant promoter methylation is a hallmark of cancer. In this study, we analyzed the methylation profiles of ten tumor suppressors that are frequently methylated in various human cancers [11, 20–25] in bladder UC from different Chinese sub-populations. Our results showed that 93.3%, 95.2% and 100% of patients from Taiwan, Hong Kong and China, respectively, had at least one gene methylated thus suggesting that epigenetic event of gene methylation is frequent in bladder cancer. Although none of these genes showed any aberrant hypermethylation in primary culture of normal urothelium from two individuals, more control samples may be needed for further validation. On the other hand, methylation of the analyzed genes in patients from Hong Kong and/or China showed an obvious diversity as compared to that from Taiwan; for example, IRF8 showed higher frequency of methylation in samples from Taiwan (46.6%) than from Hong Kong (27.6%) and China (25.0%). However, the result from China patients may need to further validate by increasing the sample size. Nevertheless, these differences may be attributed to genetic or environmental differences in these localities as differences in sex, age, stage and grade cannot explain this methylation diversity.
Exposure to environmental carcinogen and uptake of different diets has been shown to be the major reasons causing such distinct DNA methylation epigenotypes [34, 35]. In Taiwan, bladder cancer is particularly common in endemic areas of arsenic-induced Blackfoot disease. Previous studies have demonstrated that arsenic pollution is associated with DAPK and RASSF1A methylation in bladder cancer [36, 37]. It may be one of the factors that contribute to this distinct methylation epigenotype. However, our result demonstrated a similar methylation level of DAPK and RASSF1A in samples from different Chinese sub-populations. It may result from the fact that arsenic exposure from artesian well water has decreased in arsenious-endemic area due to the improvement of drinking water system . However, similar effect from different carcinogens in different localities cannot be excluded. Besides, it has also been reported that influences of dietary factors interact with DNA methylation in colorectal cancer . Thus, different environmental factors together with genetic factors may contribute to these distinct methylation profiles.
In the current study, samples from Taiwan and China displayed a strongly bimodal distribution by the number of methylated genes, which implied that CIMP may exist in bladder UC . However samples from Hong Kong did not exhibit such typical CIMP methylation pattern. This may be due to the fact that genes that we analyzed are not suitable for CIMP analysis in samples from Hong Kong, thus suggesting that the carcinogenesis in bladder UC in Hong Kong may be different from Taiwan and China fundamentally.
In keeping with previous observation [19, 29], our study also demonstrated that methylation of several genes such as APC and RASSF1A were associated with tumor progression. Methylation of RASSF1A was also associated with tumor recurrence. However, the inverse correlation between methylation of p14 and APC with tumor recurrence needs to be further validated.
DNA methylation has been previously demonstrated to be able to predict patient's survival and recurrence [19, 29, 40]. In the current study, patients from Taiwan with methylation in APC or RASSF1A tended to have a shorter RFS. The absence of such correlations in samples from other Chinese sub-populations may be due to similar reasons as discussed above. Previous studies have indicated that bladder cancer patients with APC or RASSF1A methylation show a trend toward poor survival [19, 29]. Furthermore, loss of E-cadherin expression had been reported to be associated with increased risk of recurrence in bladder cancer . Although Muramaki et al did not investigate the role of DNA methylation in their study, loss of E-cadherin expression may result from aberrant promoter methylation. Our results also demonstrated a similar trend that the primary bladder UC patients with E-cadherin methylation had a shorter time of RFS (median = 6.1 months versus 10.9 months, P = 0.07).
Due to the high recurrence rate of bladder UC, patients usually need to have repeated cystoscopy for disease monitoring thus promoting the development of non-invasive strategies. With the advances of cancer epigenetics, detection of methylated genes in voided urine becomes feasible as previously demonstrated [11, 14, 15, 23, 42]. In this study, we utilized a more sensitive quantitative real-time MSP assay (qMSP) for cancer detection in voided urine samples using a combination of methylated markers. Methylation can be detected in low grade samples with high sensitivity. Importantly, methylation can be detected in all of the urine samples from patients with recurrent tumors. However, more samples and specific methylation markers should be included for further validation and improve the diagnostic accuracy. By combining urine cytology with methylation markers and other protein biomarkers such as NMP22 , the sensitivity of cancer detection can also be dramatically increased.