Document Type : Original Research


1 Department of Pathology, Medical Institute, Sumy State University, Sumy, Ukraine

2 Department of Pathology, Medical Institute, Sumy State University, Sumy, Ukraine.

3 Department of Surgery and Oncology, Medical Institute, Sumy State University, Sumy, Ukraine


Background & Objective: To study the immunophenotype of prostate cancer (PC) with the presence and absence of intraluminal inclusions (IIn), depending on the grade score.
Methods: A total of 30 PC samples with IIn (group E) and 30 PC samples without them (group C) were studied. These groups were divided into 2 subgroups, depending on the grade of malignancy, which was determined according to the Gleason score as moderate and high-grade tumors. Macroscopic analysis, hematoxylin-eosin staining, immunohistochemistry (androgen receptors, p53 and Bax proteins, Hsp70 and Hsp90, CD68, VEGF, OSN, MMP-1) were used.
Results: The expression level of VEGF was higher in the more differentiated tumors of the control group (P<0.01). Increased expression of prognostic-adverse markers p53 (in the presence of IIn, P<0.01) and MMP-1 (P<0.05) was observed. Also, a higher level of OSN expression was found in PC tissue with IIn (P<0.01) due to its participation in the processes of biomineralization. The expression level of CD68 and Bax protein was higher in the PC group with IIn (both P<0.01). Furthermore, Hsp90 had a significantly lower expression level in the PC of group E (P<0.05).
Conclusion: the presence of IIn in the PC samples of group E promotes tissue remodeling with mechanical trauma, chronic inflammation, and fibrosis development. The presence of IIn in PC leads to the increase of OSN, CD68 and Bax expression and decrease of Hsp90 and VEGF expression. High expression of p53 and MMP-1 and low expression of OSN and VEGF was identified as a characteristic of high-grade tumors.




Prostate cancer (PC) is one of the most common causes of cancer-related deaths all over the world. According to the American Cancer Society, in 2010-2014 PC incidence and mortality rates were 118.2 and 19.5 cases per 100.000, respectively (1). The PC development and progression are closely related to the presence of chronic inflammation, which is associated with the presence of intraluminal inclusions (IIn) (prostatic calculi and amyloid corpuscles, or corpora amylacea) (2). The presence of IIn is also associated with an increased number of CD68 positive activated macrophages, the development of chronic pelvic pain syndrome and PC (3-4). Formation of IIn is a complex process, which involves the interaction of both tumor cells and tumor stroma. The remodeling of stromal components is manifested by the angiogenesis (the appearance of VEGF-positive cells) and the expression of stress tissue factors (Hsp70 and Hsp90), which have a tumorigenic effect and promote the development of bone metastases (5-6). Destruction of connective tissue (due to increased expression of MMP-1) causes migration and invasion of cancer cells, and this results in the development of the metastatic PC (7). Progression of PC is accompanied by increased levels of apoptosis (expression of the mutant p53 protein and Bax protein) and changes in sensitivity to androgens (decreased expression of receptors to androgens) (8,9).

The course and prognosis of the malignant process is directly related to the grade score of the tumor tissue. The Gleason grading system is generally accepted to estimate the morphological status of PC (10). It has been shown that prognosis of the disease deteriorates and probability of metastasis (predominantly to the bones) increases significantly with increasing of PC grade score (11).

The aim of this study is to evaluate the immune-histochemical phenotype of prostate cancer with the presence and absence of intraluminal inclusions, depending on the grade score.


Materials and Methods

Samples of Prostate Cancer

The study was conducted on the biopsy material obtained during surgeries at the Sumy Regional Clinical Hospital and Sumy City Clinical Hospital 1. The selected PC samples were divided into two groups according to the presence/absence of IIn. In total, 30 PC samples with inclusions (experimental group, E) and 30 PC samples without inclusions (control group, C) were studied. The control and experimental groups were divided into 2 subgroups, depending on the grade score, which was determined according to the Gleason grading system: tumors with a moderate (C2 and E2) and high (C3 and E3) grade score. The groups C2 and E2 included tumors with 7-8 Gleason scores (class 2-4) and the groups C3 and E3 included tumors with 9-10 Gleason scores (class 5) (10).

The Ethics Commission

This study was approved by the ethics committee of the Medical Institute of Sumy State University (Proceedings 3/6; June 7, 2016).


Biological material was fixed in 10% neutral buffer formalin solution for 24 hours. Subsequently, the material was dehydrated and paraffin embedded. Paraffin series were sliced at a thickness of 4 μm on a rotational microtome Shandon Finnesse 325 (Thermo Scientific, USA). Deparaffinized and rehydrated sections were stained with hematoxylin and eosin.

Immunohistochemistry (IHC)

In summary, 4 µm-thick serial sections made from prepared paraffin blocks were applied to SuperFrost adhesive slides (Thermo Scientific, USA). The deparaffinized sections were subjected to demasking of the antigens by thermal treatment in citrate buffer (pH 6.0) at a temperature of 95-98°C. The UltraVision Quanto Detection System HRP Polymer (Thermo Scientific, USA) detection system was used for visualization of results. It includes reduction of the endogenous peroxidase activity with 3% hydrogen peroxide, blocking of non-specific background reaction with the "Ultra V Block", and enhancing with the "Primary Antibody Amplifier Quanto". Diaminobenzidine (DAB) was used as a chromogen. The following antibody panel was used (Thermo Scientific, USA): androgen receptors (AR), pro-apoptotic protein Bax and protein p53 (p53), matrix metalloproteinase 1 (MMP-1), vascular endothelial growth factor (VEGF), heat shock protein 86 kDa (Hsp90), heat shock protein of 70 kDa (Hsp70), CD68, and osteonectin (OSN) (Table 1).



Table1. Antibody panel for IHC


Immunized Animal



Expression pattern




















Cytoplasm and membrane





Nucleus and cytoplasm





Nucleus and cytoplasm


















Morphometric study was conducted using the morphometric programs "SEO Scan ICH 285 AK-F IEE-1394" (Ukraine) and "Zen 2.0" (Carl Zeiss, Germany). The number of positive tumor cells was counted in fields with a diameter of 1000 μm. Photographing and storage of images were conducted using the digital imaging systems "SEO Scan ICH 285 AK-F IEE-1394" (Ukraine) and "ZEN" for Carl Zeiss microscopes (Germany). We used active (tissue with established positive and negative reactions) and passive control of results.

Statistical Analysis

The normality of all data sets was assessed by the Shapiro-Wilk test. In case of an abnormal distribution, a nonparametric method, the Mann-Whitney test, was used. In case of correct distribution, the data were compared using parametric Student’s t-test to determine the reliability of the difference. The results were considered statistically significant with a probability of more than 95% (P<0.05). The graphical representation of statistical analysis results was performed using the GraphPad Prism 7.04.

Histological Structure of Tumor Tissue

Histological analysis of the PC tissue of the experimental group with a moderate grade score (E2) showed the presence of glands, which were formed by atypical cells (Figs. 1a and 1b). These cells had hyperchromic nuclei, and the crybroid and pseudotrabecular structures were present. Well-developed stromal component was observed between tumor glands. Most glands had a lumen and were located close to each other.

The PCs of group E3 were characterized by a significant violation of histological structure and simplification of tumor glands. These glands formed multiple chains and nests. Tissue of the tumors had a small amount of the stromal components (Figs. 2a and 2b). Most of the glands did not have a lumen. In addition, in majority of cases, the samples of this group were represented only by the tumor field.

Samples of the experimental group (E2 and E3) were characterized by the presence of IIn in the tumor glands. These inclusions were round-formed and repeated the shape of gland lumen. Prostatic calculi were characterized by dark brown color and a more homogeneous structure. Corpora amylacea had a layered structure and a dark pink color.

The tissue samples of both experimental and control groups had the inflammatory infiltration around tumor glands. However, the severity of the inflammatory process was higher in the experimental group. Inflammatory infiltration consisted mainly of macrophage cells, lymphocytes, and neutrophils (Figs. 1a, 1b, 2a, 2b, 3a, 3b, 4a and 4b).

IHC Characteristic of Tumor Tissue

IHC examination of AR expression in the PC tissue of subgroups E2 and E3 showed a clear nuclear reaction in tumor cells and in single cells of the peripheral tumor stroma (Figs. 1c, 1d, 2c, 2d and 6). The number of positive cells for the subgroup E2 corresponded to 396.79±26.02, and this number was 360.91±36.87 tumor cells for the subgroup E3 in the view field. The number of positive nuclei was 417.17±39.61 and 424.88±53.76 cells in the view field for samples of subgroups C2 and C3, respectively (Figs. 3c, 3d, 4c, 4d and 6).


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