CpG methylation in repetitive regions of the genome is becoming widely used to estimate global methylation (reviewed in 10). Yet, few studies have compared %5MeC within repetitive genomic regions in DNA from matched sources. The NEB study provided an opportunity to evaluate these comparisons. In this study of 50 patients, associations were not observed between LINE-1
mean %5MeC measured across DNA types. No correlation was observed between genomic DNA sources and tumor tissues. LINE-1
%5MeC in buffy coat DNA was higher among males compared to females; and a significant inverse association between tumor %5MeC levels and tumor stage and grade was found. This difference between males and females is consistent with several recent studies of LINE-1
in blood DNA (4
), but not all (22
). Similar to published studies, associations between smoking or age and tissue methylation were not observed (4
Our analysis also supports reports that levels of LINE-1
methylation in tumor tissue decreases with stage and grade. One study by Choi et al., (25
) examined different interspersed repeats (LINE-1
) and tandem repeats (Sat-α, NBL-2, and D4Z4) in blood among 10 individually-matched tissue DNAs in 4 cancer-free individuals undergoing autopsy. In contrast to work presented here, the investigators reported LINE-1
levels were consistent between tissues (n=10) and individuals (n=4). An inverse association between LINE-1
%5MeC and matched adjacent normal urothelium and bladder tumor samples from cases was also observed; however correlations were not provided. The study by Choi et al., was one of the first studies demonstrating that tumor tissue methylation levels were lower than normal urothelial tissue and blood cell DNA. This study was one of the first to report substantial variability and lack of comparability in methylation outcome using different DNA sequences to estimate global methylation.
In the current study, the strongest correlation in methylation levels was observed between buffy coat and serum, both originating from blood. A recent study compared different blood cell types among 48 matched DNAs and noted similar moderate correlations within LINE-1
methylation of blood cell subtypes (26
). It has been suggested that a proportion of serum DNA is actually blood cell DNA although the exact proportion remains uncertain in bladder cancer patients. Other literature suggests that serum DNA originates from lysed, malignant, haemopoietic apoptotic, and/or necrotic circulating cells in cancer patients (27
The potential for LINE-1
methylation as a predictive biomarker of bladder cancer risk is an active area of research. Three case-control studies report associations between blood cell methylation and bladder cancer risk using post-diagnostic samples; but it is unclear whether a similar relationship is observed before diagnosis (8
). Such pre-diagnostic studies will elucidate whether hypomethylation in LINE-1
is an independent risk factor for bladder cancer, or a question of reverse causality. Environmental exposures such as smoking may impact LINE-1
methylation and its utility as a biomarker. Smoking modified the association between LINE-1
levels and bladder cancer risk in two studies (4
), but not in a third study (5
). These data suggest the need for researchers to continue modeling environmental exposures (when possible) during development of disease specific biomarkers.
It has been hypothesized that LINE-1
hypomethylation in case blood DNA may reflect levels in bladder tumor tissue. Our study suggests that they are not directly related. Rather, it appears that hypomethylation of LINE
-1 in genomic DNA reflects other factors associated with bladder carcinogenesis such as retropositioning and enhancement of gene expression. Hypomethylation of a specific LINE-1
promoter has been shown to activate an alternate transcript of the MET
oncogene in bladder tumors and normal bladder urothelium (29
). It is unclear whether the LINE-1
methylation levels in genomic DNA actually change expression of specific functional LINE-1
elements and other cancer genes; however these findings provide a mechanism through which LINE-1
methylation could modify cancer susceptibility.
Limitations of this study include lack of inclusion of healthy controls, limiting the analysis of bladder cancer risk factors such as smoking exclusively to cases. Healthy tissue nor exfoliated cells were collected in this cohort, therefore we were unable to compare LINE1 methylation levels across the four types of DNA isolated from healthy controls. This study was sufficiently powered to evaluate the study aims of examining modest correlations between paired DNA samples, however a larger sample of cases would have allowed for additional subgroup analyses. For example, the low proportion of non-smoking cases limited power to estimate differences by smoking status. Strengths of this study include the study design; 50 patients with matched genomic and tumor tissue DNAs for comparison. Isolation of DNA from FFPE tissue did limit our analysis to 34 tissue case samples. Yet even with the smaller sample size, we had sufficient power to compare %5MeC from measured in tissue to %5MeC measured in buffy coat, buccal, and serum. Another strength of the study was the use of Pyrosequencing; an accurate and reproducible method used to measure %5MeC. The role of incomplete bisulfite conversion and high CVs across replicates was evaluated through sensitivity analyses, strengthening the overall interpretation of our results.
To our knowledge, this is the first study to systematically compare LINE-1 methylation across these specific types of matched DNAs. The mean promoter LINE-1 %5MeC measurements were correlated between buffy coat and serum DNA samples but not with tumor or buccal cell DNA. Although genomic LINE-1 levels were not directly related to those observed in bladder tumor tissue, current case-control studies appear to support an association between global hypomethylation in blood and cancer risk; however the possibility of reverse causality cannot be eliminated. Future studies should include case-control and prospective studies of pre-diagnostic blood samples, to elucidate temporal associations between LINE-1 methylation and bladder cancer risk.