Dataset Information

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

ABSTRACT: Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30-44.52] %); TERT mutations-in 40/70 (AUC of 0.786; MAF = 28.29 [19.03-38.08] %); ≥1 mutation-in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78-47.49] % vs. 27.13 [17.00-37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

SUBMITTER: Jain M

PROVIDER: S-EPMC9953558 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Jain Mark M Tivtikyan Alexander A Kamalov David D Avdonin Savva S Rakhmatullin Tagir T Pisarev Eduard E Zvereva Maria M Samokhodskaya Larisa L Kamalov Armais A

Biomedicines 20230208 2

Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine ...[more]

PMID: 36831030

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Project description:BackgroundBetter 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.MethodsBy 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.ResultsA 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.ConclusionsOur 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.

Dataset Information

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Publications

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of <i>GPR126</i> Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

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