Department of Molecular Medicine, Aarhus University Hospital, Denmark.
Department of Histopathology, Aarhus University Hospital, Denmark.
Mol Oncol. 2018 Apr;12(4):545-560. doi: 10.1002/1878-0261.12183. Epub 2018 Mar 13.
Current diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, leading to overdiagnosis and overtreatment. Aberrant promoter hypermethylation of specific genes has been suggested as novel candidate biomarkers for PC that may improve diagnosis and prognosis. We here analyzed ST6GALNAC3 and ZNF660 promoter methylation in prostate tissues, and ST6GALNAC3, ZNF660, CCDC181, and HAPLN3 promoter methylation in liquid biopsies. First, using four independent patient sample sets, including a total of 110 nonmalignant (NM) and 705 PC tissue samples, analyzed by methylation-specific qPCR or methylation array, we found that hypermethylation of ST6GALNAC3 and ZNF660 was highly cancer-specific with areas under the curve (AUC) of receiver operating characteristic (ROC) curve analysis of 0.917-0.995 and 0.846-0.903, respectively. Furthermore, ZNF660 hypermethylation was significantly associated with biochemical recurrence in two radical prostatectomy (RP) cohorts of 158 and 392 patients and remained significant also in the subsets of patients with Gleason score ≤7 (univariate Cox regression and log-rank tests, P < 0.05), suggesting that ZNF660 methylation analysis can potentially help to stratify low-/intermediate-grade PCs into indolent vs. more aggressive subtypes. Notably, ZNF660 hypermethylation was also significantly associated with poor overall and PC-specific survival in the RP cohort (n = 158) with long clinical follow-up available. Moreover, as proof of principle, we successfully detected highly PC-specific hypermethylated circulating tumor DNA (ctDNA) for ST6GALNAC3, ZNF660, HAPLN3, and CCDC181 in liquid biopsies (serum) from 27 patients with PC vs. 10 patients with BPH, using droplet digital methylation-specific PCR analysis. Finally, we generated a three-gene (ST6GALNAC3/CCDC181/HAPLN3) ctDNA hypermethylation model, which detected PC with 100% specificity and 67% sensitivity. In conclusion, we here for the first time demonstrate diagnostic biomarker potential of ST6GALNAC3 and ZNF660 methylation, as well as prognostic biomarker potential of ZNF660. Furthermore, we show that hypermethylation of four genes can be detected in ctDNA in liquid biopsies (serum) from patients with PC.
当前用于前列腺癌 (PC) 的诊断和预后工具并不理想，导致过度诊断和过度治疗。异常启动子超甲基化的特定基因被认为是 PC 的新型候选生物标志物，可能改善诊断和预后。我们在这里分析了前列腺组织中的 ST6GALNAC3 和 ZNF660 启动子甲基化，以及液体活检中的 ST6GALNAC3、ZNF660、CCDC181 和 HAPLN3 启动子甲基化。首先，我们使用包括 110 例非恶性 (NM) 和 705 例 PC 组织样本在内的四个独立患者样本集，通过甲基化特异性 qPCR 或甲基化阵列进行分析，发现 ST6GALNAC3 和 ZNF660 的高甲基化具有高度的癌症特异性，ROC 曲线分析的 AUC 为 0.917-0.995 和 0.846-0.903。此外，ZNF660 高甲基化与两个根治性前列腺切除术 (RP) 队列的 158 例和 392 例患者的生化复发显著相关，在 Gleason 评分≤7 的患者亚组中也具有显著相关性（单变量 Cox 回归和对数秩检验，P<0.05），提示 ZNF660 甲基化分析可能有助于将低/中分级 PC 分为惰性和更具侵袭性的亚型。值得注意的是，ZNF660 高甲基化与 RP 队列（n=158）的总生存和 PC 特异性生存不良也显著相关，该队列具有可获得的长期临床随访。此外，作为原理证明，我们使用液滴数字甲基化特异性 PCR 分析，成功检测到 27 例 PC 患者和 10 例 BPH 患者血清中 ST6GALNAC3、ZNF660、HAPLN3 和 CCDC181 的高度 PC 特异性高甲基化循环肿瘤 DNA (ctDNA)。最后，我们生成了一个三基因（ST6GALNAC3/CCDC181/HAPLN3）ctDNA 高甲基化模型，该模型对 PC 的特异性为 100%，敏感性为 67%。总之，我们首次证明了 ST6GALNAC3 和 ZNF660 甲基化的诊断生物标志物潜力，以及 ZNF660 的预后生物标志物潜力。此外，我们表明，PC 患者液体活检（血清）中的 4 个基因的高甲基化可以被检测到。
