This study utilized methylation profiles to define subtypes of bladder cancer and associated these subtypes with clinical disease presentation and carcinogen exposure histories. As expected, our initial analyses demonstrate that the profiles identified in tumors are significantly distinct from those identified in non-diseased bladder epithelium. Among tumors, we demonstrated that the mean methylation level differs among methylation profile classes, suggesting that there are distinct phenotypes associated with the methylation profiles, and that membership in the most methylated classes is associated with ORs for invasive bladder cancer of ≥4. This is consistent with our initial analyses using a highly selected reduced number of loci in a smaller series of tumors, which demonstrated that a greater propensity for DNA methylation was associated with more aggressive forms of bladder cancer (15
). Our previous work also suggested that the propensity identified by a small number of genes may in fact have been exemplifying a more widespread process of epigenetic dysregulation across the genome (15
Again, consistent with this previous report and previously published work (14
), we also have demonstrated associations between male gender, age and former smoking status with specific subgroups of bladder tumors defined by methylation profile. Compared with non-smokers and controls, current-smokers demonstrated relatively similar odds of membership in all methylation-based subgroups of bladder cancer. This suggests that the specificity of class membership is based on additional exposures, beyond current smoking. For example, high water arsenic levels were associated with cases that had a class 3 methylation profile, suggesting that arsenic exposure has a distinct phenotype represented by a highly specific epigenetic profile. Arsenic exposure has been associated with epigenetic effects in animal models (36
) and we have demonstrated that specific gene methylation events are associated with arsenic exposure in bladder cancer (14
). This class was also almost four times more probably to be an invasive tumor compared with class 1, consistent with our findings that arsenic exposure is associated with more aggressive disease and poorer patient survival (28
). Although there is controversy over the levels at which arsenic exposure is carcinogenic in humans, our data suggest that levels found commonly in the USA (40
) give rise to a detectable specific molecular subgroup of this disease. Former smoking cases were more probably to be class 2 or 3 molecular subtypes, similarly suggesting that this exposure leads to an intermediate overall hypermethylation status. We cannot tell from this type of cross-sectional analysis if this represents a state attained by smoking and then quitting or a return to a lesser state following quitting, but model-system studies should be initiated to better understand the mechanisms by which smoking leads to these effects. Finally, we also observed that males were more probably to be in the class 3 molecular subtype as compared with females. Men generally are three to four times more at risk of developing bladder cancer as compared with females and previous authors have shown that after accounting for exposures such as cigarette smoking, urinary infections and occupational hazards, men still had an excessive risk of bladder cancer as compared with women (42
). This excessive risk may be related to anatomical differences between men and women, and may be related, especially in older men, with an inability to completely void their bladder (due to prostate enlargement or other conditions) allowing exposures present in residual urine to persist for a longer time. As greater frequency of urination decreases the risk of bladder cancer, this persistence may provide different selective pressures by gender driving these methylation profiles (44
). However, further research is needed to further examine this potential mechanism.
Strengths of this study included the large size and population-based nature of the study, as well as the use of the Illumina GoldenGate Methylation Bead Array for methylation profiling and detailed exposure assessment including the use of average water arsenic concentrations measured by an inductively coupled plasma mass spectrometer. Limitations of this study include the retrospective nature of the study and thus, the inability to determine true causality to the associations described. Another limitation is the use of only five non-diseased bladder epithelium obtained from individuals without cancer as these individuals may not be representative of normal bladder tissue. The methylation of these normal tissues is relatively homogenous and is significantly distinct from those of the bladder tumors (supplementary Figure 1
is available at Carcinogenesis
Online). Therefore, we believe that these are relatively representative and can serve as an appropriate comparator. Larger examinations of non-diseased tissue would be necessary to determine how representative these samples are and how much the demographics of the individual and their exposure history can influence the pattern of DNA methylation in non-diseased bladder epithelium. A final limitation is the time reference for arsenic exposure as the measures of water arsenic are taken at the time of enrollment, which for cases, is following diagnosis, and thus it is possible that the exposure levels at that time do not reflect those which may have been related to bladder tumorigenesis. At the same time, long-term reproducibility of these measures is particularly probably for stable populations, and those for which remediation efforts have not occurred, such as the New Hampshire population under study, and thus these measures are probably reflective of exposures over some period of time (47
). We have previously demonstrated reproducible measures of arsenic in tap-water over a 3–5 year period in this population and found that our population used their tap water system for >15 years on average (9
In summary, this study demonstrates that profiles of DNA methylation can be used to distinguish phenotypically and clinically important subgroups of bladder cancer. Smoking history as well as arsenic exposure, age and gender are not only risks of bladder cancer in general but also predispose individuals to specific molecular subtypes of disease. The novelty of these results lies in the use of array-based methodologies to examine CpG methylation of a large number of CpG loci instead of examining only specific promoter regions of certain genes, thereby allowing for a more comprehensive understanding of the epigenetic landscape of bladder tumors. These findings indicate that the methylation profiles of CpG loci can be used as a potential diagnostic marker of bladder cancer and can help further identify novel molecular subtypes of bladder cancer. Future work should examine if these subtypes can be used to create more individualized, targeted regiments of therapy for bladder cancer and aid in the prognosis of this disease.