Association of tissue promoter methylation levels of APC, TGFβ2, HOXD3 and RASSF1A with prostate cancer progression

Abstract

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.

Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors

“…Aberrations to DNA methylation in certain GP morphologies require further investigation as it may improve personalized patient treatment by utilizing biomarker information. The DNA methylation of APC, CYP26A1, HOXD3, HOXD8, RASSF1, TBX15 and TGFβ2 genes were investigated in the current study, as they have previously been revealed as promising prognostic biomarkers for PCa with the potential for clinical utility in independent patient cohorts (23)(24)(25).…”

“…DNA was extracted from 10 µm sections of previously prepared FFPE blocks matching the selected H&E slides using a QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA), as previously described (23)(24)(25). DNA methylation was analyzed in the same GP4 area used for histological evaluation.…”

“…Briefly, a total of 100-400 ng extracted DNA from the GP4 region for each GS7 case was bisulfite modified using EZ DNA Methylation Gold kit (Zymo Research Corp, Irvine, CA, USA), according to the manufacturer's protocol. Methylated genes were detected using a MethyLight quantitative polymerase chain reaction (qPCR) assay that was conducted using the TaqMan ® Gold with Buffer A pack (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as described previously (23)(24)(25). The primer and probe sequences are presented in Table II.…”

“…Additionally, DNA methylation markers are emerging as promising biomarkers of prostate carcinogenesis, thus complementing, if not altogether avoiding, the requirement for the histopathological examination of biopsy tissue samples (22). The results of our previous studies demonstrated that the DNA hypermethylation of a panel of seven genes [adenomatous polyposis coli (APC), cytochrome P450 family 26 subfamily A member 1 (CYP26A1), homeobox D3 (HOXD3), HOXD8, Ras association domain family member 1 (RASSF1), T-box 15 (TBX15) and transforming growth factor-β2 (TGFβ2)] is associated with PCa disease progression and clinical outcome (23)(24)(25). These markers were initially identified through a genome-wide methylation screen of low-grade and high-grade PCas (23).…”

“…These markers were initially identified through a genome-wide methylation screen of low-grade and high-grade PCas (23). Subsequently, the prognostic potential of a subset of these markers was validated in an independent cohort (24,25). However, to the best of our knowledge, their association with the tumor architectural features of aggressive PCa has not yet been investigated.…”