Identification and Validation of the Methylated TWIST1 and NIDI Genes through Real-Time Methylation-Specific Polymerase Chain Reaction Assays for the Noninvasive Detection of Primary Bladder Cancer in Urine Samples

摘要

Background: Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers.Objective: To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples.Design, setting, and participants: A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methyla-tion-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls. Measurements: A urine sample was classified as valid when >10 copies of the gene encoding fs-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared.Results and limitations: MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively.Conclusions: Detection of the methylated TWIST1 and N1D2 genes in urine sediments using MSP provides a highly (>90%) sensitive and specific, noninvasive approach for detecting primary BCa. Trial registration: BlCa-001 study - EudraCt 2006-003303-40.