Methylation status of CpG Island in the promoters of p15 and p16 genes in urinary bladder cancer; correlation with adjacent mucosa and pathological parameters

Purpose: A Cross sectional case controlled study was carried on to evaluate DNA methylation patterns in CpG islands of the promoters of p 15 and p 16 genes. This is an epigenetic assessment for studying tumor suppressor gene silencing or transcriptional inactivation in malignancies. Methylation specific PCR (MSP), an intrusion methodology is used in our study to assess methylation status in the promoters of pl5 and pl6 genes carcinoma of the urinary bladder. Subjects and Methods: Twenty tumor biopsy samples were evaluated and compared versus another 20 biopsy samples of the adjacent non-neoplastic mucosa as well as 40 control bladder mucosa samples from 40 normal healthy individuals. MSP entails initial modification of DNA using sodium bisulfite converting all un-methylated, but not methylated (protected) cytocine to uracil, and subsequent amplifications with three primers pairs for each gene, p 15 and p16; the 1st primer pair is specific for unmodified DNA, i.e. wild type gene (p15-W p16-W), the 2 n d pair for methylated gene (p15-M p16- M) and the 3 rd pair for un-methylated gene (p15-U p16- U). Results: In neoplastic tissues, p15-W and p16-W were detected in 7, 6 samples p15-M and p16-M were found in 15, 16 samples whereas p15-U and p16-U were expressed in 5, 4 samples respectively. There was a significant elevation of methylated genes versus wild genes as well as un-methylated genes in tumor tissue. On the other hand, the adjacent nonneoplastic mucosa of bladder cancer exhibited p15-W and p16-W in 17 samples, p15-M and p16-M in 6 and 5 samples and p15-U and p16-U in 14 and 15 samples respectively. There was a significant elevation of wild and un-methylated genes versus methylated genes. As regards the 40 control samples, p15-W and p16-W were found in all samples, p15- M and p16-M were not expressed in any sample whereas, p15-U and p16-U were detected in all samples. There was a significant elevation of wild and un-methylated genes versus methylated genes in these samples. As regards correlation to pathological grade and stage, methylated genes were significantly correlated with increasing tumor size as well as with the presence of lymph nodes metastasis. Conclusion: methylated p 15 and pl6 genes predominate in bladder tumor tissue and are found in its adjacent mucosa in certain cases, this gene paradigm have a significant correlation with the pathological stage of the tumor and can predict the clinical outcome in cystectomy with bladder cancer.