title: Promoter hypermethylation of tumor suppressor genes correlates with tumor grade and invasiveness in patients with urothelial bladder cancer.
abstract: OBJECTIVE
  To investigate the promoter methylation status at selected loci which encode for key proteins involved in apoptosis, DNA repair, cell cycle control and progression in urothelial cell carcinoma of bladder and compare the findings from tissue samples with that of plasma.
  
  
  METHODS
  Total genomic DNA was isolated from 43 non-muscle invasive (low grade) and 33 muscle invasive (high grade) urothelial bladder cancer samples along with 10 control cases of normal bladder mucosa. Promoter methylation status was investigated for RASSF1A, APC, MGMT, CDKN2A and CDKN2B genes using real-time methylation-specific PCR with SYBR® green. Plasma samples from 16 patients with muscle invasive high grade bladder cancer were also subjected to similar analyses.
  
  
  RESULTS
  Promoter hypermethylation was frequently observed in RASSF1A, APC and MGMT gene promoters (p-value < 0.001). The methylation was more prominent in the muscle invasive high grade bladder cancer when compared to non-muscle invasive low grade group (p-value < 0.001) and normal bladder mucosa (p-value < 0.05). The RNA expression of RASSF1A, APC and MGMT was also found to be decreased in the muscle-invasive high grade bladder cancer when compared to the non muscle invasive low grade group (p-value < 0.05). RASSF1A, MGMT and CDKN2A showed comparable results when data from 16 plasma samples was compared to the corresponding tissue samples.
  
  
  CONCLUSIONS
  Our results suggest that epigenetic silencing of RASSF1A, APC and MGMT genes is strongly associated with invasive high grade urothelial bladder cancer. Thus, status of promoter methylation has the potential to serve as valuable tool for assessing aggressiveness of urothelial cell carcinoma of bladder.
authors.affiliation: Office of Research and Graduate Studies, Aga Khan University, Stadium Road, Karachi, 74800 Pakistan.
authors.forename: Shumaila M
authors.initials: SM
authors.lastname: Bilgrami
authors.affiliation: Department of Biology, Syed Babar Ali School of Science and Engineering, Lahore University of Management Sciences, Sector-U, D.H.A., Lahore, 54792 Pakistan.
authors.forename: Sohail A
authors.initials: SA
authors.lastname: Qureshi
authors.affiliation: Department of Microbiology and Pathology, Aga Khan University, Stadium Road, Karachi, 74800 Pakistan.
authors.forename: Shahid
authors.initials: S
authors.lastname: Pervez
authors.affiliation: Department of Surgery, Aga Khan University, Stadium Road, Karachi, 74800 Pakistan.
authors.forename: Farhat
authors.initials: F
authors.lastname: Abbas
keywords: Paraffin embedded tissue; Plasma; Promoter methylation; Tumor suppressor genes; Urothelial cell carcinoma of bladder