The Ethics Commission of the Faculty Hospital Nitra (Nitra, Slovak Republic) approved the present study. All patients provided written informed consent.

Prostate tissue samples were collected between November 2016 and June 2017 during transurethral resection from 22 patients with bladder outlet obstruction and histologically confirmed benign prostatic hyperplasia (BPH) and acinar adenocarcinoma prostate cancer at the Faculty Hospital Nitra (Nitra, Slovak Republic). At the time of surgery, the mean age of the patients was 70.7 years (age range, 58–85 years). Based on the Tumor-Node-Metastasis (7th edition) clinical stage (30) and the type of treatment, samples were divided into four groups as follows: i) Group 1, samples from patients with BPH (adenocarcinoma-negative); ii) group 2, samples from patients with metastatic hormone naïve acinar prostatic adenocarcinoma (mHNPC); iii) group 3, samples from patients with metastatic acinar prostatic adenocarcinoma (mPC) and receiving ADT; and iv) group 4, samples from patients with metastatic castration-resistant acinar prostatic adenocarcinoma (mCRPC) and receiving ADT and Abi therapy (Table I). The patient clinicopathological characteristics and the number of patients in each group are presented in Table I. Patients in group 3 were treated with leuprorelin acetate (5 mg subcutaneously every 3 months) ADT drug for an average duration of 36 months (duration range, 6–80 months). Patients in group 4 were treated with leuprorelin acetate (5 mg subcutaneously every 3 months) ADT drug for an average duration of 50 months (duration range, 47–60 months). In groups 3 and 4 there was no statistically significant differences in the length of ADT treatment (P=0.40). Patients from group 4 also received abiraterone acetate, a standard treatment for mCRPC, orally at a dose of 1,000 mg/day fasting for an average treatment duration of 15 months (duration range, 3–27 months). At the time of tissue collection, patients in groups 3 and 4 had castrated testosterone levels (<50 ng/dl) (31) due to being treated with ADT. Additionally, patients in group 3 did not have clinical or biochemical signs of cancer progression, and patients in group 4 with CRPC continuing with ADT and Abi therapy, as is standard treatment (31), did not have signs of clinical progression.

The histological diagnosis was determined following conventional biopsy. Fresh tissue samples following collection were fixed in 10% neutrally-buffered formaldehyde solution for 24 h at room temperature, dehydrated in an ascending ethanol series (80, 90 and 100%) at room temperature, and embedded in paraffin. Paraffin sections of 5-µm thickness were prepared using a microtome. Sections for histological analysis were stained with Mayer's hematoxylin and eosin in Dako CoverStainer (Dako; Agilent Technologies, Inc.) at room temperature for 20 min. Sections for IHC analysis were deparaffinized and rehydrated in the automatic pre-treatment module PT Link (Dako; Agilent Technologies, Inc.) at 97°C for 40 min. Sections were inserted into DakoAutostainer 48 Link, and the required protocols were selected according to the manufacturer's instructions. Sections were incubated with primary atibodies against HMW-CK (cat. no. IR051), AMACR (cat. no. IR060), PSA (cat. no. IR514), CK7 (cat. no. IR619) and CK20 (cat. no. IR777) at room temperature for 20 min, and subsequently with EnVision™ Flex/HRP secondary antibody (cat. no. K8000) at room temperature for 20 min (all Dako; Agilent Technologies, Inc.). All primary and secondary antibodies were ready to use (undiluted). The visualization step was performed with EnVision™ Flex/DAB and Chromogen (cat. no. K8000) for 10 min, subsequently washed with water for 5 min. Sections were stained with Mayer's hematoxylin for 5 min at room temperature. Finally, sections were rehydrated in a descending ethanol series (100, 90 and 80%), cleared with 99% xylene and mounted with DAKO Toluene-free mounting medium (Dako; Agilent Technologies, Inc.). Sections were observed using a Nikon Eclipse Ci-L light microscope (magnification, ×200; Nikon Corporation), and images were digitally recorded using an Imaging Source camera (The Imaging Source Europe GmbH). Samples from group 2 (patients with mHNPC) were evaluated using the Gleason grading of prostatic carcinoma (9).

Fresh prostatic tissue samples immediately after collection were fixed in an aldehyde mixture containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.2–7.4 at 4°C for 1 h and subsequently post-fixed in 1% osmium tetroxide dissolved in 0.1 M sodium cacodylate for 1 h at room temperature. Following dehydration in an ascending acetone series (30, 50, 70, 80, 90, 95 and 100%), samples were embedded into Poly/Bed Embedding Media (Polysciences, Inc.). Ultrathin sections of 70-nm thickness were cut using a Leica EM UC6 ultra microtome (Leica Microsystems GmbH) and contrasted with 10% uranyl acetate solution dissolved in absolute methanol and lead citrate according to methods by Reynolds (32), in order to visualize cellular organelles. The contrasted sections were imaged using a JEM100 CXII transmission electron microscope (JEOL, Ltd.) at an accelerating voltage of 80 kV and a magnification range between ×1,900 and ×7,200.

Paraffin-embedded prostatic tissue sections (5 µm) previously fixed in 10% neutrally-buffered formalin were analyzed using TUNEL assay, using In Situ Cell Death Detection kit, Fluorescein (Roche Diagnostics GmbH) according to the manufacturer's instructions. The samples were deparaffinized using pure xylene (≥99%), rehydrated in a descending ethanol series (100, 90 and 80%) and permeabilized using proteinase K (Roche Diagnostics GmbH). Sections were labelled with TdT-reagent for 60 min in a wet chamber at 37°C in a thermostat. Sections were covered with a Vectashield anti-fade medium (Vector Laboratories, Inc.) and mounted into a sandwich between a microslide and a coverslip. Presence of apoptotic cells was analyzed under a Leica fluorescence microscope (Leica Microsystems GmbH) equipped with a digital camera DFC-480 with a total magnification of ×220. For the positive control, sections were treated with 1,500 U/ml recombinant DNase I (Roche Diagnostics GmbH) prior to TdT labelling and incubated in a humidified atmosphere for 60 min at 37°C in the dark. For the negative control, sections were incubated only with a fluorescein isothiocyanate-labelling solution, according to the manufacturer's protocol (In Situ Cell Death Detection kit; Roche Diagnostics GmbH), in the absence of TdT-reagent for 60 min at 37°C in the dark (data not shown).

Following TUNEL assay, apoptotic cells and fragments exhibited green fluorescence on a dark background. The distribution of apoptosis in the examined samples was different in all 4 groups. In each sample, in areas where apoptosis occurred, images were taken from 10 fields of view at ×200 magnification (ocular, ×10; objective, ×20) under a fluorescence microscope. The number of apoptotic cells was manually counted and statistically analyzed.

ANOVA followed by least significant difference post hoc test was used to analyze the difference in the number of apoptotic cells between the 4 groups. Data were analyzed using SPSS Statistics software (version 20; IBM Corp.). The level of α significance was determined at 0.05, and P<0.05 was considered to indicate a statistically significant difference. One-way ANOVA followed by Tukey's post hoc test was used for comparisons among multiple groups. The differences in the number of apoptotic cells between all groups were evaluated. The calculated values were plotted in a dot graph.

Histologically, BPH tissue from group 1 was formed by dilated hyperplastic glands with two layers, epithelial and myoepithelial. This was verified in one case (sample 5) following IHC examination with HMW-CK and AMACR. The basal cell layer was positive for the HMW-CK antibody, whereas the epithelial layer was negative for the AMACR antibody. In two samples (no. 13 and 14), in addition to gland hyperplasia, chronic active prostatitis with periglandular chronic inflammatory infiltrate and presence of neutrophil leukocytes in the lumen of the glands was detected (data not shown).

Ultrastructurally, in the case of BPH (group 1), well-developed microvilli were visible on the luminal surface of the cells. The epithelial secretory cells were cylindrical in shape and were attached to the basal membrane along with the small basal cells (data not shown). The cytoplasm of the secretory cells contained supranuclearly positioned secretion granules, oval mitochondria, prominent and numerous vesicles of granular endoplasmic reticulum, which were often expanded in the supranuclear area and surface parts. Heterochromatic nuclei had an oval shape and the nucleoli were of a reticular form. Some nuclei were hyperchromatic and had numerous invaginations and signs of degeneration. The incidence of compact nucleoli, swollen mitochondria and lysosomes was low (Fig. 1A). The stroma was sporadically occupied with macrophages (Fig. 1B).

Samples from patients with acinar adenocarcinoma and who did not receive treatment (group 2) presented with small glands in a random arrangement, hyperchromatic nuclei and prominent nucleoli, amphophilic cytoplasm without basal cell layer, according to IHC staining for AMACR-positivity and HMW-CK negativity (data not shown). The Gleason score was determined according to this architectural pattern (3+3, 3+4, 5+5). In the samples from group 3 (patients who received ADT), groups of individually arranged cells with round-shaped nuclei and light cytoplasm were observed as a treatment effect (data not shown). Since the Gleason score is not recommended to be applied in patients subjected to ADT (2,33), the Gleason score was not used as a criteria in the present study.

The ultrastructural images of samples from the groups 2 and 3 were similar and were characterized by the presence of secretory cells without basal cells. In both groups, disintegrated acinus and hyperchromatic nuclei were identified; the nuclei presented with an oval shape with numerous invaginations and signs of degeneration. In addition, the hypertrophic nuclei were of compact form and high in number. The granular endoplasmic reticulum and Golgi complex presented with similar structures in the two groups. In group 2, some apoptotic bodies were identified (Fig. 1C). Furthermore, the number of swollen mitochondria with altered cristae morphology in secretory cells of samples from the group 3 was high (Fig. 1D). Impaired integrity of the basal membrane was observed in samples from the 4 groups.

Histologically mCRPC (group 4) was characterized by solid and solid-alveolar arranged foci (33) of malignant cells with hyperchromatic nuclei and prominent nucleoli, small to moderate amount of amphophilic cytoplasm, a shift of the nucleus/cytoplasm ratio and numerous mitoses, including atypical mitoses (Fig. 2A). Locally, foci of glandular formation were noted. IHC staining of malignant cells revealed positivity for PSA and AMACR (Fig. 2B and C, respectively). IHC staining of the myoepithelial layer with HMW-CK and staining for CK7 and CK20 was negative (data not shown). A portion of the malignant cells were arranged in clusters, rows, or as single cells (34), and presented with a lightly stained eosinophilic cytoplasm and small hyperchromatic nuclei as a sign of the therapeutic effect (Fig. 2D). As aforementioned, the Gleason score was not determined.

Benign glands with basal cell hyperplasia were also identified in group 4, as confirmed by the IHC positivity for HMW-CK (Fig. 2E). Basal cells in the benign glands manifested enlargement and clearing of the cytoplasm following treatment compared with the untreated BPH (Fig. 2E and F).

Ultrastructurally, mCRPC, in comparison with naïve prostate cancer (group 2), was characterized by the presence of a large amount of connective tissue, smooth muscle bundles and solitary, malignant cells arranged into small groups located in the prostate stroma with prominent fibrils of collagen (Fig. 1E). The nuclei of these malignant cells were small, oval in shape and their membrane formed numerous invaginations on the inside. Chromatin in the nucleus was evenly distributed and formed hyperchromatic clusters. The nuclei contained numerous, large, predominantly compact nucleoli. The cytoplasm contained a great number of electron-dense mitochondria with altered cristae, lysosomes and autophagic vacuoles (Fig. 1F).

Apoptosis is the most common form of death in eukaryotic cells. In each group, apoptosis was observed in the glands but rarely in the stroma (Fig. 2G-I; data not shown for group 1). Apoptotic fragments also occurred in the lumen of the gland.

The results from the statistical analysis revealed that the number of apoptotic cells in the assessed fields of view was significantly increased in hormonally naïve, high-risk prostate cancer tissues compared with benign tissue samples. The one-way ANOVA test revealed significant differences among the groups (P=0.014); the post-hoc Tukey's test confirmed the significant difference in the apoptosis frequency was between group 1 (12.40±15.08) and group 2 (49.33±14.40) (P=0.014). Furthermore, the number of apoptotic cells in samples from patients who received ADT treatment (groups 3 and 4) was mainly decreased compared with patients who did not receive any treatment (group 2). Although there was no significant difference in the number of apoptotic cells between groups 2 and 3 (P=0.215; group 3 mean ± SD, 28.67±26.39), the number of apoptotic cells in group 4 was significantly decreased compared with that in group 2 (P=0.049; group 4 mean ± SD, 19.00±6.71) (Fig. 3).