Increased Sensitivity of Detection of RASSF1A and GSTP1 DNA Fragments in Serum of Prostate Cancer Patients: Optimisation of Diagnostics Using OBBPA-ddPCR

Abstract

Simple Summary Prostate carcinoma (PCa) incidence rates have continued to increase over the last 3 decades worldwide and PCa is the most common cancer in men in Germany. Classical tumour markers exhibit unsatisfactory sensitivity and specificity, leading to unnecessary prostate biopsies and associated clinical complications. New, minimally invasive biomarkers with high sensitivity and specificity are required for an early diagnosis and a better prognosis. In the present study, we evaluated the performance of a previously developed analytical method to detect trace amounts of aberrant methylated tumour DNA. The optimised method which was developed in this study evaluates DNA methylation changes of RASSF1A and GSTP1 in the blood of PCa patients at different stages compared to benign prostatic hyperplasia patients and healthy individuals. RASSF1A methylation analysis was found to be beneficial as a complementary biomarker for PCa diagnosis in combination with classical PCa markers. Both RASSF1A and GSTP1 exhibited pathological DNA methylation levels in all metastatic PCa patients, which may improve early detection of PCa metastases and therapeutic outcomes. Abstract Identification of aberrant DNA methylation is a promising tool in prostate cancer (PCa) diagnosis and treatment. In this study, we evaluated a two-step method named optimised bias-based preamplification followed by digital PCR (OBBPA-dPCR). The method was used to identify promoter hypermethylation of 2 tumour suppressor genes RASSF1A and GSTP1 in the circulating cell-free DNA (cfDNA) from serum samples of PCa patients (n = 75), benign prostatic hyperplasia (BPH, n = 58), and healthy individuals (controls, n = 155). The PCa cohort was further subdivided into subgroups comprising (I) patients with Gleason Scores (GS) ≤ 7 (n = 55), (II) GS ≥ 8 (n = 10), and (III) patients with metastatic PCa diagnosis (n = 10). We found that RASSF1A methylation levels were significantly increased in all 3 PCa subgroups compared to the controls and BPH cohorts (p < 0.01 for all comparisons). Fractional abundances of methylated GSTP1 DNA fragments were significantly increased in subgroup III of metastatic PCa patients (p < 0.001). RASSF1A methylation analysis was found to be beneficial as a complementary biomarker where further diagnostic validation is most crucial. In combination with free PSA, RASSF1A methylation status helps to identify PCa patients with GS ≥ 8 and grey-zone total PSA values between 2–10 ng/mL. In our study, PCR biases between 80–90% were sufficient to detect minute amounts of tumour DNA with high signal-to-noise ratios as well as high analytical sensitivity and specificity. Both RASSF1A and GSTP1 exhibited strongly increased DNA methylation levels in all metastatic PCa patients. Our data indicates a superior sensitivity of epigenetic biomarker analyses in early detection of PCa metastases that should also help to improve PCa therapy.