A novel and sensitive DNA methylation marker for the urine-based liquid biopsies to detect bladder cancer.

ABSTRACT

BACKGROUND: Better prognostic outcome is closely correlated with early detection of bladder cancer. Current non-invasive urianalysis relies on simultaneously testing multiple methylation markers to achieve relatively high accuracy. Therefore, we have developed an easy-to-use, convenient, and accurate single-target urine-based DNA methylation test for the malignancy. METHODS: By analyzing TCGA data, 344 candidate markers with 424 primer pairs and probe sets synthesized were systematically screened in cancer cell lines, paired tissue specimens, and urine sediments from bladder cancer patients and normal controls. The identified marker was further validated in large case-control cohorts. Wilcoxon rank sum tests and c2 tests were performed to compare methylation levels between case-control groups and correlate methylation levels with demographic and clinical characteristics. In addition, MSP, qMSP, RT-PCR, western blot analysis, and immunohistochemistry were performed to measure levels of DNA methylation, mRNA transcription, and protein expression in cancer cell lines and tissues. RESULTS: A top-performing DMRTA2 marker identified was tested in both discovery and validation sets, showing similar sensitivity and specificity for bladder cancer detection. Overall sensitivity in the aggregate set was 82.9%(179/216). The specificity, from a control group consisting of patients with lithangiuria, prostatoplasia, and prostatitis, is 92.5%(468/506). Notably, the methylation assay had the highest sensitivities for tumors at stages of T1(90.4%) and T2(95.0%) compared with Ta (63.0%), T3(81.8%), and T4(81.8%). Furthermore, the test showed admirable detection rate of 80.0%(24/30) for recurring cancers. While methylation was observed in 39/54(72.2%) urine samples from patients with carcinomas of renal pelvis and ureter, it was detected at extremely low rate of 6.0%(8/133) in kidney and prostate cancers. Compared with SV-HUC-1, the normal bladder epithelial cell line, DMRTA2 was hypermethylated in 8/9 bladder cancer cell lines, consistent with the results of MSP and qMSP, but not correlated with mRNA and protein expression levels in these cell lines. Similarly, DMRTA2 immunostaining was moderate in some tissues but weak in others. Further studies are needed to address functional implications of DMRTA2 hypermethylation. CONCLUSIONS: Our data demonstrated that a single-target DNA methylation signature, mDMRTA2, could be highly effective to detect both primary and recurring bladder cancer via urine samples.