Abstract
We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this “bladder-specific” region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.
Highlights
Urothelial cancer (UC) is the most frequent neoplasm of the urothelial tract
We aimed to identify urinary DNA methylation biomarkers that are capable of distinguishing cancerous urothelial cells from those of the prostate and kidney
According to the results from our screening in cell culture, we studied the methylation differences of the bladder specific (BLSP) amplicon between all groups in all available urine samples from urothelial cancer (UC) patients and compared the results to those of PC and renal cancer (RC) patients as well as controls with non-malignant urological diseases (UCt) and population controls (PCt) (Tables S1 and S4, Figure 2)
Summary
Urothelial cancer (UC) is the most frequent neoplasm of the urothelial tract. Advanced age, smoking, male gender, and exposure to certain chemicals at the workplace or via the environment such as aromatic amines or arsenic are well-accepted risk factors [1]. Current guidelines suggest invasive urethrocystoscopy accompanied by cytology as the standard of care in symptomatic persons for diagnosing de-novo and recurrent UC and independent on stage, grade or risk of recurrence. Many patients, men, experience significant discomfort, secondary hematuria, and infections associated with de-novo or recurrent UC diagnosis due to invasive cystoscopy [3,4]. To minimize the number of cystoscopies, numerous approaches to non-invasively diagnose UC have been developed in urine. Genetic approaches such as analyzing DNA mutations and DNA methylation signatures, among others, have been shown to be most promising [5,6,7]
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