Patients
In this retrospective study, we included 61 patients with prostatic adenocarcinoma, who did not undergo treatment previously. These patients were admitted at the Clinic of Urology from January 2019 to June 2020 with increased prostate-specific antigen (PSA) levels in serum. All tumors were diagnosed to be primary, without previous therapy. In this study, we excluded patients who underwent prostate cancer-related therapies, including radiation therapy, chemotherapy, and androgen deprivation therapy. The Ethics Committee of the First Affiliated Hospital of Guangxi Medical University approved this study (Protocol number:2021KY-E-238), and all patients gave written informed consent before their enrollment in the study. All methods were performed in accordance with the relevant guidelines and regulations.
CE-TRUS imaging
All patients included in this study underwent CE-TRUS before prostate biopsy. The ultrasound equipment used was the LOGIQ E9 system (GE Healthcare, Milwaukee, WI, USA) with a transrectal probe operating at a frequency of 3–9 MHz. During CE-TRUS, 2.4 mL SonoVue (Bracco, Milan, Italy) was administered intravenously as a rapid bolus injection, followed by a 5-mL saline flush. The acoustic power of the equipment was set at a mechanical index of 0.10. The contrast imaging plane was considered the transverse plane of the CE-TRUS abnormality. The results of the imaging examinations were saved in DICOM format.
Image and data analyses
Prominent differences exist in the contrast enhancement of peripheral zone (PZ) lesions and that of transition zone (TZ) lesions. The normal inner gland and coexisting benign prostate hyperplasia often appear to be hypervascular, therefore, regions of interest (ROIs) were drawn only on the PZ biopsy sites on CE-TRUS images. However, in case of systematic biopsies, ROIs with diameters of approximately 5 mm were traced around the biopsy sites. Furthermore, ROIs were drawn as closely as possible to encompass the CE-TRUS abnormalities in case of targeted biopsies. The CE-TRUS image analysis was performed individually by two sonographers with more than 10 years of work experience, who were blinded to all clinical and pathological information. If the conclusions obtained by these sonographers were inconsistent, a consistent conclusion would be reached after discussion. According to the enhancement patterns of prostate lesions, the patients in this study were divided into low-and high-enhancement groups. An abnormal imaging lesion was defined with reference to the surrounding normal prostate tissue or compared to the other side, with asymmetric or asynchronous enhancement areas. Based on the normal internal gland, if the enhancement level was similar to or higher than that of the internal gland, high enhancement was defined. However, an enhancement level lower than that of the internal gland was defined as low enhancement. In the case of uneven enhancement, the enhancement level was based on the enhancement in more than 50% of the lesion area. Time intensity curve (TIC) analysis for each ROI was performed using the TIC analysis software of LOGIQ E9.
Prostate biopsy
Within 1 week after CE-TRUS, 2–3 targeted biopsy cores were taken from areas of abnormal CE-TRUS findings, and a systematic 12-core transrectal ultrasound-guided prostate biopsy was performed by a sonographer with more than 10 years of experience in the field. Core samples were extracted using an automatic biopsy gun (C. R. Bard, Covington, GA, USA) that triggers an 18 G needle with a core length of 25 cm. Specimens were marked based on the site of biopsy.
Pathological analysis and immunostaining
PCa tissue samples were extracted using ultrasound-guided targeted or systematic biopsy. The grading of tumors on needle biopsy samples was undertaken in accordance with the ISUP grades. Each sample were assigned one of the following ISUP grades: grade 1 (GS ≤3 + 3), grade 2 (GS 3 + 4), grade 3 (GS 4 + 3), grade 4 (GS 4 + 4, 3 + 5, and 5 + 3), and grade 5 (GS 9–10).
The biopsy specimens of prostate tissues were immersed in formalin for 5–6 hours in preparation for optimal immunostaining, and then these sample tissues were embedded in paraffin. A pathologist, with more than 10 years of experience in this field, reviewed all the histological slides from ultrasound-guided targeted or systematic biopsies and selected a few slides for quantitative evaluation. The selected samples were stained for CD31 using the “hot spot” method introduced by Weidner et al. (11) for the assessment of MVD. After staining, the most vascularized areas were identified using a fluorescence microscope with a 10× objective, and three fields were selected for counting of the vessels under 20× magnification. The average count was designated as the MVD. None of the luminal structures, single vascular endothelial cell buds, strand-shaped endothelial cells, or clustered endothelial cells was considered as an immature vessel. The vessels that showed an obvious luminal structure were considered relatively mature vessels. The calculations used for mature and immature vessel indices were:
Mature vessel index = Number of relatively mature vessels/MVD
and
Immature vessel index = Number of immature vessels/MVD
Paraffin-embedded PCa tissue sections with a thickness of 4 mm were obtained. The sections were mounted on glass slides coated with silane. These sections then underwent deparaffinization using xylene and rehydration with graded alcohol solutions. The treated sections were pressed with citric acid. Next, they were rinsed with phosphate-buffered saline (PBS) and treated with 50 mL of 3% H2O2 solution for 10 min at room temperature for inactivation of endogenous peroxidase. Non-immune goat serum (50 mL) was added to each section for incubation at room temperature for 10 min. The sections were further incubated with CD31 antibody (MAB-0720) at room temperature for 60 min. After incubation with primary antibody, the sections were washed with PBS three times. Then, the sections were again incubated with 50 mL of immunochromogenic reagent D-3004-15 (secondary antibody, horseradish peroxidase [HRP] labeled) at room temperature for 30 min. After adding 100 mL of freshly prepared DAB chromogenic solution to the sections and washing them thrice, the sections were stained with DAB chromogenic solution (100 mL), incubated for 3–5 minutes, and photographed using an optical microscope (Olympus Corporation, Tokyo, Japan). The sections were rinsed with tap water, counterstained with hematoxylin, rinsed with PBS to return to blue, dehydrated with gradient alcohol, rendered transparent with xylene, and finally sealed with neutral gum. The cytoplasm of vascular endothelial cells of all study sections showed a strong positive reaction.
Statistical analysis
All analyses were conducted using SPSS 25.0 (SPSS Inc., Chicago, IL, USA). The comparisons between sample means of the two groups were performed using independent sample t-test or non-parametric Mann–Whitney test. Correlations were analyzed using Pearson or Spearman correlation analysis. A statistically significant difference was indicated by P <0.05.