Supervised cluster analysis of bladder samples at 1,370 loci (784 genes) using the Illumina GoldenGate methylation assay. N (n=12) represents normal tissue from patients without UC, CN (n=34) represents corresponding normal-appearing tissue from UC patients, Ta-T1 (n=49) represents non-invasive urothelial tumors, and T2-T4 (n=38) represents invasive tumors. No methylation is shown in blue with increasing DNA methylation is shown in yellow. Indicated on the right is whether each loci is located within a CpG island (CGI) or is a Polycomb PRC2 target.

A) Venn diagram of the overlap of hypermethylated and hypomethylated loci. The hypermethylated and hypomethyated cancer-specific loci were determined using three separate comparisons of tissue from UC patients (CN, Ta-T1, and T2-T4) to 12 N samples (5% FDR, Wilcoxon test). B) The majority of loci hypermethylated in corresponding normal-appearing tissues and tumor tissues are CpG islands, in contrast to the hypomethylated loci. C) Starburst plots of transcriptome and epigenetic differences between normal urothelia and urothelial tumors (26). Each data point in the Starburst plot represents the FDR value calculated for gene expression and DNA methylation independently. Red vertical and horizontal lines indicate FDR level at 0.05. FDR values as log₁₀ transformed data are plotted for normalized gene expression (y-axis) and DNA methylation (x-axis) for each gene. The sign (negative or positive log₁₀(FDR)) depends on the direction of fold normalized gene expression and DNA methylation β value difference. If the fold gene expression is up in tumors compared to normal, −1 is multiplied to log₁₀(FDR), providing positive values. If the fold gene expression levels are lower in tumors compared to normal, 1 is multiplied to log₁₀(FDR), providing negative values. Concurrently, if the DNA methylation β value difference indicates hypermethylation in tumors compared to normal, −1 is multiplied to log₁₀(FDR), providing positive values. If the DNA methylation β value difference indicates hypomethylation in tumors compared to normal, 1 is multiplied to log₁₀(FDR), providing negative values. FDR values decrease (becoming more statistically significant) as the log₁₀(FDR) proceed in either direction from the p=0.05 cutoff. Hypermethylated genes with decreased expression levels in non-invasive (Ta-T1, 31%) and invasive (T2-T4, 27%) urothelial tumors are labeled with red data points, while hypomethylated genes having increased expression levels in Ta-T1 (26%) and T2-T4 (25%) are labeled with green data points.

Aberrant DNA hypermethylation at ZO2 in corresponding normal-appearing tissues reveals the presence of a field defect in bladders with cancer. A) DNA methylation data from the Illumina GoldenGate assay performed on normal urothelium from patients without UC (N, green), normal appearing urothelium taken from bladders with cancer (CN, dark blue), and urothelial tumors (T, red). Expression of ZO2 and GAPDH were measured by RT-PCR. Horizontal lines represent the median value. Red arrows indicate the CpG sites queried. *** represents p<0.001, ** represents p<0.01, as determined by unpaired t-tests (N&CN, N&T) or paired t-tests (CN&T). B) Bisulfite sequencing of ZO2 was performed on one N, one CN and the matched T using primers indicated by the black arrows in order to confirm the Illumina methylation results. C–D) Tissue samples were taken from five patients of their tumors (red, T) and at increasing distances from the tumor (0.5 to 2 cm) in the surrounding normal-appearing tissue in multiple directions (light blue, a to d). Additionally, distant normal-appearing samples were taken at least 5 cm from the tumor (dark blue, C). DNA methylation was measured by Pyrosequencing. The green line represents the median methylation value of normal samples from cancer-free patients.

X-inactivation patterns reveal that the urothelium of two female patients with UC remains polyclonal. A) Bisulfite sequencing was performed on a region located on the X-chromosome with an A/T SNP (rs4826507). This SNP is located within an exonic region that is methylated on the active X chromosome and unmethylated on the inactive X chromosome (23). Pink and blue boxes represent the portion of sequences from each clonal population. The tumors are monoclonal while the samples of corresponding normal-appearing urothelium taken at various distances from the site of the tumors are polyclonal, indicating that a single clone has not grown across the urothelium. B) Two of the possible explanations for the presence of abnormal DNA methylation in normal-appearing urothelium from bladders with cancer are that either aberrant DNA methylation accumulates in one cell, followed by the clonal expansion of that cell population across the urothelium and subsequent transformation, or that there is a generalized epigenetic defect that occurs independently in many different cells, which does not result in a growth advantage but does predispose cells to undergo transformation. The X-inactivation results support the latter.

Hypermethylated loci are enriched in Polycomb (PcG) targets. The majority of loci hypermethylated in corresponding normal-appearing tissues and tumor tissues are PcG targets, in contrast to the hypomethylated loci.