Locus-Specific Methylation of GSTP1, RNF219, and KIAA1539 Genes with Single Molecule Resolution in Cell-Free DNA from Healthy Donors and Prostate Tumor Patients: Application in Diagnostics

Simple Summary Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, which is constantly accompanied by benign prostate hyperplasia (BPH). To reach a 100% 5-year survival rate in PCa, which is characteristic for PCa if it is diagnosed in early stages, efficient PCa diagnostics against the background of BPH are demanded. The article describes a liquid biopsy approach to differential PCa diagnostics based on the data on locus-specific methylation of the three genes (GSTP1, RNF219, and KIAA1539) obtained with NGS of cell-free DNA from blood plasma of PCa, BPH, and healthy individuals. We offered a diagnostic approach including the analysis of simultaneous methylation status of two CpGs in one cell-free DNA molecule, allowing the discrimination of all patients with absolute sensitivity and specificity. The locus-specific methylation of three genes (GSTP1, RNF219, and KIAA1539 (also known as FAM214B)) in the blood plasma cell-free DNA (cfDNA) of 20 patients with prostate cancer (PCa), 18 healthy donors (HDs), and 17 patients with benign prostatic hyperplasia (BPH) was studied via the MiSeq platform. The methylation status of two CpGs within the same loci were used as the diagnostic feature for discriminating the patient groups. Many variables had good diagnostic characteristics, e.g., each of the variables GSTP1.C3.C9, GSTP1.C9, and GSTP1.C9.T17 demonstrated an 80% sensitivity at a 100% specificity for PCa patients vs. the others comparison. The analysis of RNF219 gene loci methylation allowed discriminating BPH patients with absolute sensitivity and specificity. The data on the methylation of the genes GSTP1 and RNF219 allowed discriminating PCa patients, as well as HDs, with absolute sensitivity and specificity. Thus, the data on the locus-specific methylation of cfDNA (with single-molecule resolution) combined with a diagnostic approach considering the simultaneous methylation of several CpGs in one locus enabled the discrimination of HD, BPH, and PCa patients.