Multiple Highly Methylated CpG Sites as Potential Epigenetic Markers for the Diagnosis of Prostate Cancer

Prostate cancer represents the second most frequently diagnosed malignant tumour in males. Serum PSA is used as a screening tool for prostate cancer. However, there are limitations to this approach due to the number of false positives it generates, which can result in overdiagnosis and over-treatment. The objective of this study was to identify accurate DNA methylation markers for the diagnosis of prostate cancer. In order to achieve this objective, whole-methylome sequencing was performed on tumour tissue samples, and the identified epi-genetic markers were validated using multiplex methylation-specific PCR. Abstract: Background/Objectives: Prostate cancer (PCa) remains the leading cause of cancer deaths in men. Serum Prostate-Specific Antigen (PSA) test is widely used for PCa screening. This method is controversial, as it can lead to over-diagnosis and over-treatment. Using DNA methyl-ation sequencing, our aim was to identify new sensitive and specific epigenetic markers in PCa tissue. Methods: DNA methylome analysis was performed from 15 paired tumors (T) and non-tumour adjacent (NT) prostate tissues by Enzymatic Methyl-seq Kit (EM-seq). Results: 66 CpGs sites representing eight genes were identified as hypermethylated in T versus NT, and were confirmed by multiplex methylation-specific PCR (MM-SPCR). A very good correlation between EM-Seq and MM-SPCR results was observed (Pearson's correlation of 0.93). As an indicator of the overall methylation status, the percentage of methylated reference (PMR) was measured for each marker. Overall, a significant difference was found in the mean PMR values of T vs. NT (86.2  30.3% vs. 5.0  3.2%, p < 0.0001). Area under the ROC curve (AUC) was calculated from PMRs for each marker, with AUC values ranging from 0.987 to 1.0. This is an indication of the very high diagnostic accuracy of these markers in the tissue of prostate cancer patients. Conclusions: A total of 66 hypermethylated CpG sites were identified in paired prostate tumour tissue in comparison to non-tumour adjacent tissue by EM-seq. They represent a set of eight genes, including CLDN5, GSTP1, NBEAL2, PRICKLE2, SALL3, TAMALIN, TJP2 and TMEM106A, which could be used as diagnostic markers for prostate cancer.