title: Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer.
abstract: Non-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients.
  
  
  
  We used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time's and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination.
  
  
  
  For Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers' methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82.
  
  
  
  We showed that combined analysis of FGFR3 mutation and DNA methylation markers on urine can be a useful strategy in diagnosis, surveillance and for risk stratification of patients with NMIBC. These results provide the basis for a highly accurate noninvasive test for population screening and allowing to decrease the frequency of cystoscopy, an important feature for both patient quality of life improvement and care cost reduction.
authors: OncoDiag SAS, Agoranov, Paris, France. roperch@oncodiag.fr.
authors: Jean-Pierre
authors: 0000-0002-6745-6067
authors: JP
authors: Roperch
authors: OncoDiag SAS, Agoranov, Paris, France.
authors: Bernard
authors: B
authors: Grandchamp
authors: Departement of Urology, Saint-Louis Hospital, Paris, France.
authors: François
authors: F
authors: Desgrandchamps
authors: Departement of Urology, Saint-Louis Hospital, Paris, France.
authors: Pierre
authors: P
authors: Mongiat-Artus
authors: Departement of Urology, Bichat-Claude Bernard Hospital, Paris, France.
authors: Vincent
authors: V
authors: Ravery
authors: Departement of Urology, Bichat-Claude Bernard Hospital, Paris, France.
authors: Idir
authors: I
authors: Ouzaid
authors: GRC-05, University Institute of Oncology, University Paris-6, Paris, France.|Department of Urology, Pitié Hospital, Paris, France.|Department of Urology, Tenon Hospital, Paris, France.
authors: Morgan
authors: M
authors: Roupret
authors: Department of Urology, Pitié Hospital, Paris, France.
authors: Véronique
authors: V
authors: Phe
authors: Department of Urology, Tenon Hospital, Paris, France.
authors: Calin
authors: C
authors: Ciofu
authors: INSERM, ECEVE, UMR 1123, CIC-EC 11425, Paris, France.|University Paris Diderot, ECEVE, UMR 1123, Sorbonne Paris Cité, Paris, France.|Department of Epidemiology and Clinical Research, Bichat-Claude Bernard Hospital, Paris, France.
authors: Florence
authors: F
authors: Tubach
authors: GRC-05, University Institute of Oncology, University Paris-6, Paris, France.|Department of Urology, Pitié Hospital, Paris, France.|Department of Urology, Tenon Hospital, Paris, France.
authors: Olivier
authors: O
authors: Cussenot
authors: OncoDiag SAS, Agoranov, Paris, France.|Computational Bioscience Research Center, KAUST University, Thuwal, Saudi Arabia.
authors: Roberto
authors: R
authors: Incitti
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000465:Algorithms; D019540:Area Under Curve; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000652:urine*; D002295:Carcinoma, Transitional Cell / Q000175:diagnosis* / Q000235:genetics / Q000652:urine; D019175:DNA Methylation / Q000235:genetics; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D016015:Logistic Models; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012372:ROC Curve; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics*; D058112:Septins / Q000235:genetics; D015238:Sulfotransferases / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000652:urine; D000098552:Slit Homolog 2 Protein
chemical_list: D014408:Biomarkers, Tumor; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3; D015238:Sulfotransferases; D058112:Septins; D000098552:Slit Homolog 2 Protein
keywords: Diagnosis; Genetic and Epigenetic DNA biomarkers; Non-muscle-invasive bladder cancer; Surveillance; Urine-based assay