6-gene promoter methylation assay is potentially applicable for prostate cancer clinical staging based on urine collection following prostatic massage.

The detection of prostate cancer (PCa) biomarkers in bodily fluids, a process known as liquid biopsy, is a promising approach and particularly beneficial when performed in urine samples due to their maximal non-invasiveness requirement of collection. A number of gene panels proposed for this purpose have allowed discrimination between disease-free prostate and PCa; however, they bear no significant prognostic value. With the purpose to develop a gene panel for PCa diagnosis and prognosis, the methylation status of 17 cancer-associated genes were analyzed in urine cell-free DNA obtained from 31 patients with PCa and 33 control individuals using methylation-specific polymerase chain reaction (MSP). Among these, 13 genes indicated the increase in methylation frequency in patients with PCa compared with controls. No prior association has been reported between adenomatosis polyposis coli 2 (APC2), homeobox A9, Wnt family member 7A (WNT7A) and N-Myc downstream-regulated gene 4 protein genes with PCa. The 6-gene panel consisting of APC2, cadherin 1, forkhead box P1, leucine rich repeat containing 3B, WNT7A and zinc family protein of the cerebellum 4 was subsequently developed providing PCa detection with 78% sensitivity and 100% specificity. The number of genes methylated (NGM) value introduced for this panel was indicated to rise monotonically from 0.27 in control individuals to 4.6 and 4.25 in patients with highly developed and metastatic T2/T3 stage cancer, respectively. Therefore, the approach of defining the NGM value may not only allow for the detection of PCa, but also provide a rough evaluation of tumor malignancy and metastatic potential by non-invasive MSP analysis of urine samples.