Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.为开发区分惰性与侵袭性前列腺癌（prostate cancer, PCa）的新型方法，本研究采用基于芯片的综合高通量相对甲基化（comprehensive high-throughput array-based relative methylation, CHARM）检测技术，依据格里森分级（Gleason grade）对前列腺癌患者全基因组范围内的差异甲基化区域（differentially methylated regions, DMRs）进行筛选，覆盖CpG岛（CpG island, CGI）及非CpG岛区域。初始队列共纳入26份样本，其中低分级[格里森评分（Gleason score, GS）6]、高分级（GS≥7）前列腺癌样本各8份，另纳入10份匹配正常前列腺组织作为对照。本研究共鉴定出正常组织与癌组织间的3567个差异甲基化区域，以及区分低、高分级前列腺癌的913个差异甲基化区域。此类差异甲基化区域大多定位于CpG岛岸（CGI shores）。针对低分级与高分级前列腺癌的比较分析，筛选出排名前五的候选差异甲基化区域（涵盖OPCML、ELAVL2、EXT1、IRX5及FLRT2），并通过焦磷酸测序（pyrosequencing）对初始队列进行验证。其中OPCML与FLRT2的差异甲基化区域进一步在独立队列中得到验证，该队列包含20份低分级、33份高分级前列腺癌组织。在第三队列中，对比发生生化复发（n=70）与未发生生化复发（n=86）的患者，上述差异甲基化区域位点的甲基化水平未呈现显著差异。当分别比较GS 3+4与GS 4+3病例时，复发组（n=30）的OPCML-DMR甲基化水平呈现低于无复发组（n=52）的趋势。本研究表明，采用CHARM技术开展全基因组甲基化谱分析，可揭示正常前列腺组织与前列腺癌组织间，以及依据格里森评分划分的不同风险层级前列腺癌间差异化的DNA甲基化模式。筛选得到的一组差异甲基化区域可作为前列腺癌格里森评分的新型替代生物标志物。