title: Multiplexed methylation profiles of tumor suppressor genes in bladder cancer.
abstract: Changes in DNA methylation of tumor suppressors can occur early in carcinogenesis and are potentially important early indicators of cancer. The objective of this study was to assess the methylation of 25 tumor suppressor genes in bladder cancer using a methylation-specific (MS) multiplex ligation-dependent probe amplification assay (MLPA). Initial analyses in bladder cancer cell lines (n = 14) and fresh-frozen primary bladder tumor specimens (n = 31) supported the panel of genes selected being altered in bladder cancer. The process of MS-MLPA was optimized for its application in body fluids using two independent training and validation sets of urinary specimens (n = 146), including patients with bladder cancer (n = 96) and controls (n = 50). BRCA1 (71.0%), WT1 (38.7%), and RARB (38.7%) were the most frequently methylated genes in bladder tumors, with WT1 methylation being significantly associated with tumor stage (P = 0.011). WT1 and PAX5A were identified as methylated tumor suppressors. In addition, BRCA1, WT1, and RARB were the most frequently methylated genes in urinary specimens. Receiver operating characteristic curve analyses revealed significant diagnostic accuracies in both urinary sets for BRCA1, RARB, and WT1. The novelty of this report relates to applying MS-MLPA, a multiplexed methylation technique, for tumor suppressors in bladder cancer and body fluids. Methylation profiles of tumor suppressor genes were clinically relevant for histopathological stratification of bladder tumors and offered a noninvasive diagnostic strategy for the clinical management of patients affected with uroepithelial neoplasias.
authors.affiliation: Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain.
authors.forename: Maria José
authors.initials: MJ
authors.lastname: Cabello
authors.forename: Laura
authors.initials: L
authors.lastname: Grau
authors.forename: Noreli
authors.initials: N
authors.lastname: Franco
authors.forename: Esteban
authors.initials: E
authors.lastname: Orenes
authors.forename: Miguel
authors.initials: M
authors.lastname: Alvarez
authors.forename: Ana
authors.initials: A
authors.lastname: Blanca
authors.forename: Oscar
authors.initials: O
authors.lastname: Heredero
authors.forename: Alberto
authors.initials: A
authors.lastname: Palacios
authors.forename: Manuel
authors.initials: M
authors.lastname: Urrutia
authors.forename: Jesus María
authors.initials: JM
authors.lastname: Fernández
authors.forename: Antonio
authors.initials: A
authors.lastname: López-Beltrán
authors.forename: Marta
authors.initials: M
authors.lastname: Sánchez-Carbayo
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D022521:Ligase Chain Reaction; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D009367:Neoplasm Staging; D012372:ROC Curve; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor