title: Quantitation of promoter methylation of multiple genes in urine DNA and bladder cancer detection.
abstract: BACKGROUND
  The noninvasive identification of bladder tumors may improve disease control and prevent disease progression. Aberrant promoter methylation (i.e., hypermethylation) is a major mechanism for silencing tumor suppressor genes and other cancer-associated genes in many human cancers, including bladder cancer.
  
  
  METHODS
  A quantitative fluorogenic real-time polymerase chain reaction (PCR) assay was used to examine primary tumor DNA and urine sediment DNA from 15 patients with bladder cancer and 25 control subjects for promoter hypermethylation of nine genes (APC, ARF, CDH1, GSTP1, MGMT, CDKN2A, RARbeta2, RASSF1A, and TIMP3) to identify potential biomarkers for bladder cancer. We then used these markers to examine urine sediment DNA samples from an additional 160 patients with bladder cancers of various stages and grades and from an additional 69 age-matched control subjects. Data were analyzed on the basis of a prediction model and were internally validated using a jacknife procedure. All statistical tests were two-sided.
  
  
  RESULTS
  For all 15 patients with paired DNA samples, the promoter methylation pattern in urine matched that in the primary tumors. Four genes displayed 100% specificity. Of the 175 bladder cancer patients, 121 (69%, 95% confidence interval [CI] = 62% to 76%) displayed promoter methylation in at least one of these genes (CDKN2A, ARF, MGMT, and GSTP1), whereas all control subjects were negative for such methylation (100% specificity, 95% CI = 96% to 100%). A logistic prediction model using the methylation levels of all remaining five genes was developed and internally validated for subjects who were negative on the four-gene panel. This combined, two-stage predictor produced an internally validated ROC curve with an overall sensitivity of 82% (95% CI = 75 % to 87%) and specificity of 96% (95% CI = 90% to 99%).
  
  
  CONCLUSIONS
  Testing a small panel of genes with the quantitative methylation-specific PCR assay in urine sediment DNA is a powerful noninvasive approach for the detection of bladder cancer. Larger independent confirmatory cohorts with longitudinal follow-up will be required in future studies to define the impact of this technology on early detection, prognosis, and disease monitoring before clinical application.
authors.affiliation: Department of Otolaryngology--Head and Neck Surgery, The Johns Hopkins School of Medicine, 818 Ross Research Building, 720 Rutland Ave., Baltimore, MD 21205-2196, USA.
authors.forename: Mohammad Obaidul
authors.initials: MO
authors.lastname: Hoque
authors.forename: Shahnaz
authors.initials: S
authors.lastname: Begum
authors.forename: Ozlem
authors.initials: O
authors.lastname: Topaloglu
authors.forename: Aditi
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Eli
authors.initials: E
authors.lastname: Rosenbaum
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
authors.forename: William H
authors.initials: WH
authors.lastname: Westra
authors.forename: Mark
authors.initials: M
authors.lastname: Schoenberg
authors.forename: Marianna
authors.initials: M
authors.lastname: Zahurak
authors.forename: Steven N
authors.initials: SN
authors.lastname: Goodman
authors.forename: David
authors.initials: D
authors.lastname: Sidransky
mesh_terms: D000368:Aged; D000465:Algorithms; D000704:Analysis of Variance; D014408:Biomarkers, Tumor / Q000235:genetics*; D016022:Case-Control Studies; D019175:DNA Methylation; D005260:Female; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D016015:Logistic Models; D008297:Male; D008875:Middle Aged; D015233:Models, Statistical; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000652:urine
chemical_list: D014408:Biomarkers, Tumor