Between July 2015 and April 2016, a total of 18 male patients with PC were recruited at Alan Walker Cancer Care Centre (Darwin, Australia) for this prospective clinical study. Eligible patients were ≥18 years old, had histologically confirmed prostate adenocarcinoma and Eastern Cooperative Oncology Group performance status of 0 to 1 and had not received prior prostate surgery. Exclusion criteria included metastatic disease at presentation, prior history of malignancy (excluding non-melanoma skin cancer) and serious illness precluding safe administration of RT. These patients were naïve to RT or androgen deprivation therapy (ADT) before transrectal ultrasound (TRUS) biopsy and clinical data were collected. All PC cases were classified into as follows: Low-[clinical (c)T stage ≤2a; PSA<10 ng/ml; GS≤6]; intermediate-(cT=2b; PSA, 10-20 ng/ml; GS=7) and high-risk (cT≥2c; PSA>20 ng/ml; GS=8-10) (2). The present study was approved by the Human Research and Ethics Committee of the Northern Territory (approval no. 2015-2385) Department of Health and Menzies School of Health Research. Written informed consent was obtained from all participants to provide access to prostate tissue biopsies, blood samples collected at various time intervals before, during and after therapy and medical and pathology records from Royal Darwin Hospital and Alan Walker Cancer Care Centre.

Tissue samples were fixed in 10% formalin overnight at room temperature before being embedded in paraffin. The tissue was sectioned to 4 µm and mounted on poly-lysine-coated slides (Dako; Agilent Technologies, Inc.). All tissue sections were stored in a 50˚C water bath. Slides were dried for 30 min in a thermostat at 60˚C. All sections were deparaffinised using xylene and subsequently rehydrated with a series of graded ethanol dilutions. Then, antigen retrieval was performed by placing slides in a Coplin jar with target retrieval solution (Dako; Agilent Technologies, Inc.; pH, 9.0) for 20 min at 90-95˚C in a hot water bath. All sections were marked using a Dako PEP pen (Agilent Technologies, Inc.) for accuracy.

Sections were incubated in methanol containing 3% hydrogen peroxide for 30 min at room temperature and washed twice (3 min/wash) with TBS washing buffer. Goat serum (Dako; Agilent Technologies, Inc.) was applied to all sections and incubated at room temperature for 10 min. Primary antibodies (Novus Biologicals, LLC) were used to determine expression levels of pro-inflammatory TNF-α and IL-6 and TGF-β1 in tumour biopsy samples from patients with PC. All tissues were incubated at room temperature for 1 h using the following primary antibodies: Anti-mouse monoclonal TNF-α (1:50; cat. no. NB600-1422) and TGF-β1 (1:100; cat. no. NBP2-22114SS) and anti-rabbit polyclonal IL-6 (1:100; cat. no. NB600-1131SS). Antibody diluent was substituted with primary antibody for negative control sections (Dako; Agilent Technologies, Inc.). All tissue sections were rinsed in TBS as aforementioned. Then, 3-4 drops of secondary antibody (REAL Link-biotinylated secondary Ab2; cat. no. K5001; Dako; Agilent Technologies, Inc.) were applied to all tissue sections and incubated for 10 min at room temperature. All tissue sections were rinsed twice with TBS then incubated at room temperature with horseradish peroxidase-conjugated streptavidin for 10 min by adding 3-4 drops to the slides (Dako; Agilent Technologies, Inc.). Finally, all sections were developed with 3'-diaminobenzidine for 5 min at room temperature and counterstained with Mayer's haematoxylin at room temperature for another 2 min. All tissue sections were dehydrated via a graded series of ethanol dilutions and washed with xylene. After staining, coverslips were applied and sealed using permanent mounting medium.

IHC-stained slides were evaluated for expression of pro-inflammatory TNF-α and IL-6 and TGF-β1 by light microscopy (magnification, x20) in a blinded manner by two clinical pathology consultants. Expression levels of pro-inflammatory TNF-α and IL-6 and TGF-β1 were evaluated using a semi-quantitative scale based on the proportion of positive-stained cells as follows: -, <10; +, 10-50; ++, 51-80; +++, >80% (6,19,20).

Levels of pro-inflammatory TNF-α and IL-6 and TGF-β1 in pre-RT plasma were assessed. For plasma cytokine analysis, ELISA kits were used including human TNF-α (cat. no. KAC1751), TGF-β1 (cat. no. EHTGFBI), IL-6 (cat. no. KAC1261) and IL-8 (cat. no. KAC1301; all Thermo Fisher Scientific, Inc.). Assay kits were chromogen-based and cytokine concentration (colour) was quantified using a Titertek Multiskan MCC/340 plate reader according to the manufacturer's instructions. Each assay was calibrated against a standard curve with a full range predetermined for each cytokine and sample source.

Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc.). Established clinical variables included in the study were age, pre-operative PSA, risk stratification, c and pathological (p)TNM stage and GS. Data are presented as the mean ± SD (n=18). Spearman's correlation test was performed to assess the correlation between pro-inflammatory TNF-α and IL-6 and anti-inflammatory TGF-β1 expression levels and the aforementioned variables. Spearman's correlation test was also used to determine the linear correlation between cytokine expression levels in pre-RT plasma and corresponding prostatic needle biopsy specimens. P<0.05 was considered to indicate a statistically significant difference.

The clinicopathological information of participants, such as age, pre-operative PSA levels, risk stratification, c and pTNM stage, and GS, are summarised in Table I.

The mean age of patients with PC at the time of diagnosis was 66.83±7.93 years (range, 53.00-80.00 years). The mean pre-operative serum PSA levels were 16.03±15.81 ng/ml (range, 4.00-71.00 ng/ml); 33% of patients (6/18) exhibited PSA<10.00, 50% (9/18) exhibited 10.00-20.00 ng/ml PSA and 17% (3/18) exhibited PSA>20.00 ng/ml. The mean GS was 7.88±1.14 (range, 6.00-10.00); 6% of patients (1/18) exhibited GS=6.00, 44% (8/19) exhibited GS=7.00 and 50% (9/18) exhibited GS=8.00-10.00. Tumour staging was divided into c and pT stage. For cT stage, 23% of patients (4/18) were T1a-c, 33% (6/18) were T2a-c and 44% (8/18) were T3a-c. For pT stage, 0% of patients (0/18) were T1a-c, 89% (16/18) were T2a-c and 11% (2/18) were T3a-c. Only one patient (6%) exhibited one ipsilateral pelvic node involved before therapy.

IHC staining for pro-inflammatory TNF-α, IL-6 and anti-inflamatory TGF-β1 revealed elevated levels of these cytokines in most tumour tissue samples compared with healthy tissue (Figs. 1 and 2). Malignant prostate cells exhibited brown cytoplasmic staining, indicating expression of pro-inflammatory TNF-α and IL-6 and TGF-β1 in prostatic needle biopsy specimens from patients with PC.

Serum PSA is used as a guide to initiate prostatic biopsies and to monitor men older than 50 years for PC (21). Serum PSA level is the most commonly used tumour biomarker for PC There was no correlation between expression levels of pro-inflammatory TNF-α and IL-6 and anti-inflammatory TGF-β1 and pre-operative serum PSA levels (data not shown).

Spearman's correlation test was performed to assess the association between cytokine expression levels and GS. GS ranges from 1-5 and describes how much cancer from a biopsy resembles healthy (lower score) or abnormal tissue (higher score). Most cancers score ≥3 in anatomical pathology practice depending on aggressiveness (22). Figs. 1 and 2 show H&E and IHC staining in biopsy samples with GS as follows: 3+3=6, 3+4=7, 4+3=7, 4+4=8, 4+5=9, 5+4=9 and 5+5=10. Lower expression levels of pro-inflammatory TNF-α and IL-6 were associated with high GS; however, no statistically significant association was found (TNF-α, ρ=-0.5096; IL-6, ρ=-0.3169; Fig. 3). Anti-inflammatory TGF-β did not show any association with GS (ρ=-0.3241).

The present study evaluated the potential impact of tumour-derived cytokine production on circulating plasma levels. IHC expression levels of pro-inflammatory TNF-α and IL-6 and anti-inflammatory TGF-β1 were increased in prostatic needle-biopsy specimens with increased pre-RT plasma cytokines levels detected by ELISA (Fig. 4). A statistically significant association was found between staining intensity of proinflammatory TNF-α and IL-6 and anti-inflammatory TGF-β1 in prostatic needle biopsy specimens and concentration in pre-RT plasma (TNF-α, ρ=0.7629; TGF-β1, ρ=0.5742; IL-6, ρ=0.5294; Fig. 4).