title: Multi-institutional external validation of urinary TWIST1 and NID2 methylation as a diagnostic test for bladder cancer.
abstract: OBJECTIVE
  We previously reported a clinical trial in which we were unable to replicate the excellent diagnostic metrics produced in the developmental study of the TWIST1 and NID2 gene methylation assay. In this expanded trial with subjects enrolled from another institution, we reexamine the diagnostic capabilities of the test to externally validate our previous study.
  
  
  METHODS
  TWIST1 and NID2 gene methylation was assessed in DNA isolated from the urine of subjects at risk of bladder cancer undergoing cystoscopy for hematuria or bladder cancer surveillance. The diagnostic gold standard was cystoscopy. Two thresholds of TWIST1 and NID2 gene methylation were used for determining test result positivity, those published by Renard et al. and Abern et al. The sensitivity, specificity, positive and negative predictive values, diagnostic likelihood ratios, and receiver operating characteristic curves were calculated for each gene, as well as their combination. In all, 3 methods were used to combine TWIST1 and NID2 into a single composite test: (1) believe-the-positive decision rule-if either gene is methylated the test result is positive, which maximizes test sensitivity; (2) believe-the-negative decision rule-if either gene is not methylated the test result is negative, which maximizes test specificity; and (3) a likelihood-based logistic regression model approach that balances sensitivity and specificity. Clinical utility was determined using a decision curve analysis.
  
  
  RESULTS
  A total of 209 subjects were evaluated: 40% for hematuria and 60% for bladder cancer surveillance. Approximately 75% were male, most of the prior cancers being low-grade Ta. Using cystoscopy as the gold standard, areas under the curve were 0.67 for TWIST1, 0.64 for NID2, and 0.66 for combined TWIST1 and NID2. Decision rule results revealed optimization of sensitivity at 67% using Renard thresholds and specificity using the Abern thresholds at 69%. We found improved sensitivity (78%) in current smokers. Decision curve analyses revealed that the methylation assay provided only a modest benefit even at high probabilities of missed cancer.
  
  
  CONCLUSIONS
  A urine DNA test measuring TWIST1 and NID2 methylation was externally examined with a larger cohort and its results continue to be poor. These 2 biomarkers are unlikely to replace cystoscopy, but they may be worthy of study in active smokers.
authors.affiliation: Division of Urology, Duke University Medical Center, Durham, NC.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Fantony
authors.affiliation: Department of Urology, University of Illinois Chicago, Chicago, IL.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Abern
authors.affiliation: Division of Urology, Duke University Medical Center, Durham, NC.
authors.forename: Ajay
authors.initials: A
authors.lastname: Gopalakrishna
authors.affiliation: Division of Urology, Duke University Medical Center, Durham, NC.
authors.forename: Richmond
authors.initials: R
authors.lastname: Owusu
authors.affiliation: Division of Urology, Duke University Medical Center, Durham, NC.
authors.forename: Kae
authors.initials: K
authors.lastname: Jack Tay
authors.affiliation: Division of Urology, Urology of Virginia, Virginia Beach, VA.
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Lance
authors.affiliation: Division of Urology, Duke University Medical Center, Durham, NC. Electronic address: brant.inman@duke.edu.
authors.forename: Brant A
authors.initials: BA
authors.lastname: Inman
mesh_terms: D000368:Aged; D019540:Area Under Curve; D014408:Biomarkers, Tumor / Q000032:analysis*; D002135:Calcium-Binding Proteins; D002295:Carcinoma, Transitional Cell / Q000175:diagnosis* / Q000235:genetics / Q000652:urine; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism / Q000652:urine*; D015331:Cohort Studies; D019175:DNA Methylation; D005260:Female; D006801:Humans; D016013:Likelihood Functions; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000652:urine*; D012372:ROC Curve; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000378:metabolism / Q000652:urine*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000652:urine
chemical_list: D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; C114729:NID2 protein, human; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1
keywords: Diagnostic test; Epigenetic; Sensitivity; Smoking; Specificity; Urothelial carcinoma