Regularly methylated novel pro-apoptotic genes associated with recurrence in transitional cell carcinoma of the bladder.

Epigenetic silencing of tumor suppressor genes by promoter hypermethylation has been shown for a variety of genes in bladder cancer. Various p53 target genes have been investigated, but only few demonstrated promoter hypermethylation when semiquantitative detection methods were applied. To address to the question whether promoter methylation of novel p53 effector genes is a common event in transitional cell carcinoma of the bladder, we selected the p53 target genes apoptotic protein-activating factor (APAF-1), Caspase 8 (CASP-8), death-associated protein kinase, (DAPK-1) and insulin-like growth-factor-binding protein-3 (IGFBP-3), performing quantitative methylation-specific real-time PCR. The individual level of methylation (normalized index of methylation) was correlated with clinicopathological features as well as the biological behavior of the superficial and muscleinvasive tumors. Tissue was obtained from 110 tumor patients and 20 patients without urological malignancy. The median follow-up of the tumor patients was 52 months. Hypermethylation of the promoter region in tumor specimens was common for APAF-1 (100%), DAPK-1 (74%) and IGFBP-3 (66%), but not for CASP-8 (3.6%). It was seen less frequently and with undetectable or low methylation levels in the normal urothelium group. The APAF-1 methylation levels significantly correlated with tumor stage and tumor grade. The APAF-1 and IGFBP-3 methylation levels were able to separate tumors with higher recurrence risk from low-risk tumors in nonmuscleinvasive and muscleinvasive tumors. In multivariate analysis, APAF-1 and IGFBP-3 methylation levels were independent prognostic markers for recurrence in superficial bladder tumors. This study provides new insights into the role of promoter methylation of selected p53 target genes. The extent of promoter methylation of specific genes offers additional prognostical information and is associated with the outcome in patients with nonmuscleinvasive and muscleinvasive bladder cancer.