Abstract
BackgroundEarly detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation.MethodsIn this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers.ResultsOf the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity.ConclusionBased on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect.
Highlights
Detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring
DNA methylation profiling was compared between 3 groups of tissue samples (Figure 2): normal urothelial tissue (N, n = 12), corresponding normal-appearing tissue adjacent to the tumor in UC patients (CN, n = 34), and tumor samples saved during transurethral resection (TUR) procedure on UC patients (T, n = 91)
PSQ was performed on DNA samples allocated to the tissue validation set (Table 1: 21Ns, 25 CNs and 53 Ts) and urine validation sets (Table 1: 18 urine sediments from healthy volunteers (NUs) and 73 urine sediments from UC patients (TUs))
Summary
Detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. According to the American Cancer Society estimates for 2013, bladder cancer will account for 72,570 newly diagnosed cases and 15,210 deaths [1]. Bladder cancers can be classified into two groups based on histopathology and clinical behavior: non-muscle-invasive urothelial cancer (NMIUC: pTa-pT1) and muscle-invasive urothelial cancer (MIUC: pT2-pT4). NMIUCs represent approximately 80% of newly diagnosed bladder cancer cases and are treated by transurethral resection (TUR). Several DNA-based urinary markers have been evaluated with the aim of reducing the need for cystoscopy and improving the accuracy of tumor detection. None have been proven to be sufficiently reliable in detecting the entire spectrum of bladder cancers in the clinic [5]
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