title: Quantitative methylation analysis of BCL2, hTERT, and DAPK promoters in urine sediment for the detection of non-muscle-invasive urothelial carcinoma of the bladder: a prospective, two-center validation study.
abstract: OBJECTIVE
  Urinary hypermethylation of BCL2, hTERT, and DAPK promoters has been previously demonstrated as an accurate biomarker for the detection of urothelial carcinoma of the bladder (UCB) in patients undergoing radical cystectomy. In the present study, we investigated with a validation intent the frequency and levels of methylation of the same 3 genes in tumor tissue and urine sediment of patients undergoing transurethral resection (TUR) for non-muscle-invasive (NMI) UCB.
  
  
  METHODS
  A total of 108 consecutive patients with NMI UCB and 105 controls with no genitourinary malignancies were enrolled in this prospective study conducted in 2 tertiary referral academic urological departments with an advanced molecular laboratory. The frequency and levels of methylated BCL2, hTERT, and DAPK promoters were evaluated with quantitative methylation-specific real-time polymerase chain reaction in DNA extracted from tumor tissue and paired normal bladder mucosa retrieved at the time of TUR in patients, and from urine in patients and controls.
  
  
  RESULTS
  In tumor tissue, at least 1 gene was hypermethylated in 91% patients (BCL2 in 62%, hTERT in 53%, DAPK in 48%). Methylation of hTERT was significantly correlated with tumor grade (P = 0.026). In urine sediment sensitivity and specificity were 76% and 98%, respectively, using BCL2 and hTERT. The number of methylated genes was highly correlated with tumor grade (P = 0.005). Methylated BCL2 and hTERT in urine sediment were highly correlated with those of the corresponding bladder tumor qualitatively (P < 0.001), and only BCL2 also quantitatively (P = 0.005). Methylation levels of BCL2 and hTERT were variably associated with tumor grade and stage, but were significantly correlated with patient age (P = 0.004 and P = 0.027, respectively).
  
  
  CONCLUSIONS
  These findings suggest that quantitative methylation analysis of BCL2 and hTERT, but not DAPK, in urine sediment may be a useful tool in the diagnosis of NMI UCB, deserving future applicability studies.
authors.affiliation: Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Vinci
authors.forename: Gianluca
authors.initials: G
authors.lastname: Giannarini
authors.forename: Cesare
authors.initials: C
authors.lastname: Selli
authors.forename: Jitka
authors.initials: J
authors.lastname: Kuncova
authors.forename: Donata
authors.initials: D
authors.lastname: Villari
authors.forename: Francesca
authors.initials: F
authors.lastname: Valent
authors.forename: Claudio
authors.initials: C
authors.lastname: Orlando
mesh_terms: D000367:Age Factors; D000368:Aged; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000652:urine; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics*; D002295:Carcinoma, Transitional Cell / Q000235:genetics* / Q000473:pathology / Q000652:urine; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000652:urine; D064550:Death-Associated Protein Kinases; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic / Q000235:genetics; D011446:Prospective Studies; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D019098:Telomerase / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology / Q000652:urine
chemical_list: D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D019253:Proto-Oncogene Proteins c-bcl-2; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C509186:TERT protein, human; D019098:Telomerase