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Deregulation of the apoptotic pathway, one of the hallmarks of tumor growth and -progression, has been shown to have prognostic value for tumor recurrence in rectal cancer. In order to develop clinically relevant biomarkers, we studied the methylation status of promoter regions of key apoptosis genes in rectal cancer patients, using methylation-sensitive restriction enzymes. DNA was extracted from fresh-frozen tumor tissues of 49 stage I-III rectal cancer patients and 10 normal rectal tissues. The results of this pilot study were validated in 88 stage III tumor tissues and 18 normal rectal tissues. We found that methylation of the intrinsic apoptotic pathway genes Apaf1, Bcl2 and p53 correlated with the apoptotic status (M30) of the tumor. Combined survival analyses of these three genes, based on the number of genes showing high methylation (all low, 1 high, 2 high or all high), showed shorter patient survival and recurrence-free periods with an increasing number of methylated markers. Multivariate analyses showed significant differences for overall survival (p = 0.01; HR = 0.28 (0.09-0.83)), cancer-specific survival (p = 0.004; HR = 0.13 (0.03-0.67)) and distant recurrence-free survival (p = 0.001; HR = 0.22(0.05-0.94)). The shortest survival was observed for patients showing low methylation of all markers, which-as was expected-correlated with high apoptosis (M30), but also with high proliferation (Ki-67). The study of epigenetic regulation of apoptosis genes provides more insight in the tumorigenic process in rectal cancer and might be helpful in further refining treatment regimens for individual patients.

DNA methylation of apoptosis genes in rectal cancer predicts patient survival and tumor recurrence