title: A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urine.
abstract: OBJECTIVE
  To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine.
  
  
  METHODS
  Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patients with BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29).
  
  
  RESULTS
  The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC_a, TERT_a, TERT_b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set).
  
  
  CONCLUSIONS
  We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.
authors.affiliation: Department of Pathology, Urology Social Health Erasmus MC, Amsterdam, The Netherlands.
authors.forename: Tahlita C M
authors.initials: TC
authors.lastname: Zuiverloon
authors.forename: Willemien
authors.initials: W
authors.lastname: Beukers
authors.forename: Kirstin A
authors.initials: KA
authors.lastname: van der Keur
authors.forename: Jesrael R
authors.initials: JR
authors.lastname: Munoz
authors.forename: Chris H
authors.initials: CH
authors.lastname: Bangma
authors.forename: Hester F
authors.initials: HF
authors.lastname: Lingsma
authors.forename: Marinus J C
authors.initials: MJ
authors.lastname: Eijkemans
authors.forename: Jan P
authors.initials: JP
authors.lastname: Schouten
authors.forename: Ellen C
authors.initials: EC
authors.lastname: Zwarthoff
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001681:Biological Assay / Q000379:methods*; D006801:Humans; D008745:Methylation; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local / Q000175:diagnosis / Q000235:genetics / Q000652:urine*; D044023:Receptor, Endothelin B / Q000235:genetics; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000652:urine*; D014556:Urine / Q000737:chemistry*
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D044023:Receptor, Endothelin B; C509186:TERT protein, human; D019098:Telomerase