Noninvasive diagnosis of urothelial cancer in urine using DNA hypermethylation signatures—Gender matters

Abstract

Urothelial cancer (UCa) is the most predominant cancer of the urinary tract and noninvasive diagnosis using hypermethylation signatures in urinary cells is promising. Here, we assess gender differences in a newly identified set of methylation biomarkers. UCa‐associated hypermethylated sites were identified in urine of a male screening cohort (n = 24) applying Infinium‐450K‐methylation arrays and verified in two separate mixed‐gender study groups (n = 617 in total) using mass spectrometry as an independent technique. Additionally, tissue samples (n = 56) of mixed‐gender UCa and urological controls (UCt) were analyzed. The hypermethylation signature of UCa in urine was specific and sensitive across all stages and grades of UCa and independent on hematuria. Individual CpG sensitivities reached up to 81.3% at 95% specificity. Albeit similar methylation differences in tissue of both genders, differences were less pronounced in urine from women, most likely due to the frequent presence of squamous epithelial cells and leukocytes. Increased repression of methylation levels was observed at leukocyte counts ≥500/μl urine which was apparent in 30% of female and 7% of male UCa cases, further confirming the significance of the relative amounts of cancerous and noncancerous cells in urine. Our study shows that gender difference is a most relevant issue when evaluating the performance of urinary biomarkers in cancer diagnostics. In case of UCa, the clinical benefits of methylation signatures to male patients may outweigh those in females due to the general composition of women's urine. Accordingly, these markers offer a diagnostic option specifically in males to decrease the number of invasive cystoscopies.

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer

“…For this purpose, we applied a genome-wide, array-based screening to various urological tumor cell lines of urothelial, prostate and renal origin and compared the results to those obtained from the respective non-tumorous primary cells. To select targets of actual relevance for clinical UC detection, we further compared our in vitro results to those previously obtained in vivo from a genome-wide screening of a small set of urine samples from UC patients and controls [15]. Finally, candidate CpG-sites were evaluated in vivo in a larger set of urine specimens from UC patients, patients with prostate (PC) and renal cancer (RC), and non-cancer controls (population (PCt) and urological hospital controls (UCt)).…”

“…To check whether potentially bladder-specific sites identified in cell culture arrays were also differentially regulated between urine specimens from UC patients and controls and thus potentially suitable for the clinical practice, we compared the results from the cell culture screening with our previously published in vivo array results of a differential analysis derived from 12 urine samples from UC patients and 12 age, gender and smoking-status matched non-UC controls [15], GEO public depository: Accession GSE 120288).…”

“…Final statistical evaluation was carried out in a subset of the aforementioned collective due to our previously published observations that a strong contamination with leukocytes suppresses UC-dependent DNA methylation signals and UC history (de-novo, recurrent) is also an important influencing factor of DNA methylation in cancer cases [15]. Consequently, urine specimens with ≥500 leukocytes/µL and those cancer cases with lacking information on UC history were excluded.…”

“…The preparation of DNA has been described in our previous study in detail [15]. In brief, the urinary sediment was prepared by centrifugation of the voided urine, washing and resolving the pellet with PBS.…”

“…The amplified region was CHR6:28911328-28911620 (genome build GRCh37/hg19). All analyses were carried out according to the protocol of Agena Bioscience GmbH and have been previously described in detail [15].…”