Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer (PCa). We correlated quantitative promoter methylation levels of APC, TGFb2 and RASSF1A in 219 radical prostatectomies diagnosed between 1998 and 2001 with clinicopathological follow-up data available including Gleason Pattern (GP), Gleason Score (GS) and pathological stage and explored their potential in predicting biochemical recurrence using univariate and multivariate analyses. We observed that the average methylation levels of APC increased significantly from GS 6 to GS7, and pT2 to pT3a, and that of TGFb2 increased from GS 6 to GS7, but not for RASSF1A. PCa samples were also stratified into high methylation (HM) and low methylation (LM) groups based on the PMR scores of all cases analyzed for each marker. The HM frequency of APC was greater in pT3a than pT2, and in GS 8 than GS 6. The HM frequency also increased significantly from GP3 to GP4 for APC, TGFb2 and RASSF1A. APC methylation level was a significant predictor of biochemical recurrence in univariate analysis (p-value 5 0.028). Finally, we combined methylation data of these three genes with the previously reported novel methylation biomarker HOXD3. Quantitative methylation assessment of a multiplex panel of markers, consisting of APC, HOXD3 and TGFb2, outperforms any single marker for the prediction of biochemical recurrence (p-value 5 0.017). Our study demonstrated that quantitative increase in promoter methylation levels of APC, HOXD3 and TGFb2 are associated with PCa progression.

DNA methylation in gene promoters causes gene silencing and is a common event in cancer development and progression. The ability of aberrant methylation events to serve as diagnostic and prognostic markers is being appreciated for many cancers, including prostate cancer. Using quantitative MethyLight technology, we evaluated the relationship between HOXD3 methylation and clinicopathological parameters including biochemical recurrence, pathological stage, Gleason score (GS), and Gleason pattern in a series of 232 radical prostatectomies performed between 1998 and 2001. HOXD3 methylation was significantly greater in GS 7 cancers vs GSr6 cancers (P-value o0.001) as well as pT3/pT4 vs pT2 cancers (P-value o0.001). The proportion of cases with high methylation in GS 7 vs rGS 6 and pT3/pT4 vs pT2 were also significantly different (P-values ¼ 0.002 and 0.005, respectively). There were also significant increases in methylation from Gleason pattern 2-3 and from pattern 3 to 4/5 (paired t-test P-values ¼ 0.01 and o0.001, respectively), whereas methylation from lymph node metastases was decreased when compared with matched tumor tissue (P-value ¼ 0.029). HOXD3 methylation was associated with biochemical recurrence in univariate analysis (P-value ¼ 0.043) and showed evidence for interaction with pathological stage as a predictor variable in Cox regression analysis (P-value ¼ 0.028). The results indicate that HOXD3 methylation distinguishes low-grade prostate cancers from intermediate and high-grade ones and may also have prognostic value when considered together with pathological stage.