Study population

The present case-control study was approved by the Ethical Board of Jessenius Faculty of Medicine, Comenius University (Martin, Slovak Republic) and conducted in accordance with The Declaration of Helsinki. Most of the subjects were also enrolled in our previous studies (26,27).

The inclusion and exclusion criteria for the case group were as follows: i) age ≥50 years; ii) Caucasian; and iii) histologically confirmed prostate cancer. The indication for prostate biopsy was either a suspicious finding on digital rectal examination (DRE) or elevated serum levels of prostate specific antigen (PSA), or both. Patients with prior or present evidence of other cancers or other major pathologies and patients who had a first-degree relative (brother or father) with a confirmed diagnosis of prostate cancer were excluded.

The inclusion criteria for the control group were as follows: i) age ≥50 years; ii) Caucasian; iii) no current or previous diagnoses of cancer; iv) no evidence of family history of prostate cancer; and v) negative DRE and negative serum PSA according to age-specific reference values (28).

A one-to-one case-control study was designed with an estimated total number of 408 patients and controls each. This number provided an 80% power to detect a difference in the proportions of at least 0.1 in any direction at a significance level of 0.05 assuming the most conservative prevalence in controls (0.5). To account for the non-probabilistic nature of the data-generating process and potential drop-out, the sample size was increased by 35% (408 to 551). A total of 1,076 Caucasian men were studied, including 522 patients with prostate cancer and 554 healthy controls, at the Department of Urology, Jessenius Faculty of Medicine and University Hospital Martin, Comenius University in Bratislava. Prostate cancer patients and controls were enrolled from May 2005 to December 2019. All subjects agreed to participate in the study and written informed consent was obtained from all. Venous blood (3 ml) from all participants was withdrawn into Vacutainer tubes containing EDTA as the anticoagulant.

For all of the patients with prostate cancer, histological evaluation was performed on specimens collected using prostate needle biopsy or transurethral resection of the prostate. Biopsy materials were immediately fixed in a 10% solution of buffered formaldehyde for 24 h at 25°C. Fixed material was embedded into paraffin blocks and histological slides of 3–4 µm thickness were cut and stained using a commercial Hematoxylin and Eosin Staining Kit (cat. no. ab245880; Abcam) according to the manufacturer's protocols. The protocol of the International Society of Urological Pathology was used for histological slides analysis and the final biopsy record (29). Histological slides were assessed using a BX45 light microscope (Olympus Corporation). The Gleason score for the histopathological grade (30) was recorded and the patients were divided into two groups as follows: i) low-grade, score ≤7; and ii) high-grade, score >7.

Cases and controls were tested for total serum prostatic specific antigen (PSA) levels using a Beckman Coulter Access^® Hybritech^® assay (cat. no. 37200, Beckman Coulter, Inc.) according to the manufacturer's protocol.