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A gene promoter hypermethylation panel to detect transitional cell carcinoma of the bladder

Introduction:  Promoter methylation has been established as a mechanism for silencing of tumour suppressor genes (TSGs) in TCC. Detection of aberrant methylation has been proposed as a novel diagnostic test, but the desired sensitivity and specificity has not been proven. The gene specific nature of methylation necessitates investigators to identify a panel of TSG's, that when analysed for aberrant methylation in tumour DNA, demonstrate a high sensitivity, specificity and diagnostic accuracy for clinical use. Here, we analyse a panel of 16 genes in the DNA of 96 TCCs and 30 samples of normal urothelium. Methods:  DNA was obtained from 96 TCCs and 30 normal bladders. The DNA was bisulphite modified and quantitative methylation specific PCR (QMSP) was performed for 16 gene promoters. Results:  Methylation was detected for all 16 genes in the 96 TCCs. This ranged from 3% to 75%. The genes with the highest frequency of methylation were RASSF1a (54%), E-cadherin (41%), BCL2 (45%), TNFRS25 (75%), EDNRB (66%), IGFBP3 (43%) and WIF1 (56%). High levels of methylation were detected in the normal controls for RASSF1a (50%) and E-cadherin (20%), with 11 other genes having low levels of methylation. Using a panel of 5 heavily methylated genes, a sensitivity of 57%, specificity of 89% and a diagnostic accuracy of 80% resulted. Conclusion:  The frequency of methylation varied widely for different genes, confirming the gene specific nature of aberrant methylation in TCC. Using our panel of gene loci, the sensitivity and specificity of methylation DNA analysis was not adequate. However, our study does demonstrate the feasibility of TCC DNA methylation analysis and with the identification of further cancer specific gene loci, it may prove a useful early detection strategy.