Document Type : Original Research

Authors

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

Abstract

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.

Keywords

 

Introduction


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).

Histology

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

Antibody

Immunized Animal

Clone

Dilution

Expression pattern

AR

Rabbit

Polyclone

1:200

Nucleus

р53

Mouse

SP5

1:100

Nucleus

MMP-1

Rabbit

Polyclone

1:50

Cytoplasm

VEGF

Rabbit

Polyclone

1:200

Cytoplasm and membrane

Hsp90

Rabbit

Polyclone

1:100

Nucleus and cytoplasm

Hsp70

Rabbit

Polyclone

1:100

Nucleus and cytoplasm

Bax

Rabbit

Polyclone

1:100

Cytoplasm

CD68

Mouse

KP1

1:100

Cytoplasm

OSN

Rabbit

Polyclone

1:50

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.

Results
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).

  $('.collapse').on('shown.bs.collapse', function(){ $(this).parent().find(".glyphicon-plus").removeClass("glyphicon-plus").addClass("glyphicon-minus"); }).on('hidden.bs.collapse', function(){ $(this).parent().find(".glyphicon-minus").removeClass("glyphicon-minus").addClass("glyphicon-plus"); });

  1. Cronin KA, Lake AJ, Scott S, Sherman RL, Noone AM, Howlader N et al. Annual Report to the Nation on the Status of Cancer, part I: National cancer statistics. Cancer 2018; 124(13): 2785-2800. [DOI:10.1002/cncr.31551] [PMID] [PMCID]
  2. Sfanos KS, De Marzo AM. Prostate cancer and inflammation: the evidence. Histopathology 2012; 60: 199-215. [DOI:10.1111/j.1365-2559.2011.04033.x] [PMID] [PMCID]
  3. Shoskes DA, Lee CT, Murphy D, Kefer J, Wood HM. Incidence and Significance of Prostatic Stones in Men with Chronic Prostatitis/Chronic Pelvic Pain Syndrome. Urology 2007 Aug; 70(2): 235-8. [DOI:10.1016/j.urology.2007.04.008] [PMID]
  4. Torkko KC, Wilson RS, Smith EE, Kusek JW, van Bokhoven A, Lucia MS. Prostate Biopsy Markers of Inflammation are Associated with Risk of Clinical Progression of Benign Prostatic Hyperplasia: Findings from the MTOPS Study. J Urol 2015 Aug; 194(2): 454-61. [DOI:10.1016/j.juro.2015.03.103] [PMID]
  5. Roberts E, Cossigny DAF, Quan GMY. The role of vascular endothelial growth factor in metastatic prostate cancer to the skeleton. Prostate cancer 2013; 2013: 418340. [DOI:10.1155/2013/418340] [PMID] [PMCID]
  6. Chatterjee S, Burns TF. Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach. Int J Mol Sci 2017 Sep 15; 18(9): E1978. [DOI:10.3390/ijms18091978] [PMID] [PMCID]
  7. Pulukuri SM, Rao JS. Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis. Int J oncol 2008 Apr; 32(4): 757-65.
  8. Johnson MI, Robinson MC, Marsh C, Robson CN, Neal DE, Hamdy FC. Expression of Bcl-2, Bax, and p53 in high-grade prostatic intraepithelial neoplasia and localized prostate cancer: relationship with apoptosis and proliferation. Prostate 1998 Dec 1; 37(4): 223-9. https://doi.org/10.1002/(SICI)1097-0045(19981201)37:43.0.CO;2-O [DOI:10.1002/(SICI)1097-0045(19981201)37:43.0.CO;2-O]
  9. Fujita K, Nonomura N. Role of Androgen Receptor in Prostate Cancer: A Review. World J Mens Health 2018 Sep10; 36: e32
  10. Epstein JI, Zelefsky MJ, Sjoberg DD, Nelson JB, Egevad L, Magi-Galluzzi C et al. A contemporary prostate cancer grading system: a validated alternative to the Gleason score. Eur Urol 2016; 69(3): 428-35. [DOI:10.1016/j.eururo.2015.06.046] [PMID] [PMCID]
  11. Guo X, Zhang C, Guo Q, Xu Y, Feng G, Li L et al. The homogeneous and heterogeneous risk factors for the morbidity and prognosis of bone metastasis in patients with prostate cancer. Cancer Manag Res 2018; 10: 1639-1646. [DOI:10.2147/CMAR.S168579] [PMID] [PMCID]
  12. Moskalenko R, Romanyuk А, Danilchenko S, Stanislavov O, Piddubniy A, Zakorko I-М et al. Morphogenetic aspects of biomineralization on the background of benign prostatic hyperplasia. Georgian medical news 2013; 1(214): 54-61.
  13. Sfanos KS, Wilson BA, De Marzo AM, Isaacs WB. Acute inflammatory proteins constitute the organic matrix of prostatic corpora amylacea and calculi in men with prostate cancer. PNAS 2009; 106(9): 3443-8. [DOI:10.1073/pnas.0810473106] [PMID] [PMCID]
  14. Banerjee P, Banerjee S, Brown T, Zirkin B. Androgen action in prostate function and disease. Am J Clin Exp Urol 2018; 6(2): 62-77.
  15. Mohler JL. A role for the androgen-receptor in clinically localized and advanced prostate cancer. Best Pract Res Clin Endocrinol Metab 2008 Apr; 22(2): 357-72. [DOI:10.1016/j.beem.2008.01.009] [PMID] [PMCID]
  16. Levesque A, Eastman A. p53-based cancer therapies: Is defective p53 the Achilles heel of the tumor? Carcinogenesis 2007; 28(1): 13-20. [DOI:10.1093/carcin/bgl214] [PMID]
  17. Thomas DJ, Robinson M, King P, Hasan T, Charlton R, Martin J, et al. p53 expression and clinical outcome in prostate cancer. Br J Urol 1993 Nov; 72(5 Pt2): 778-81. [DOI:10.1111/j.1464-410X.1993.tb16267.x] [PMID]
  18. Khor LY, Desilvio M, Li R, McDonnell TJ, Hammond ME, Sause WT, et al. Bcl-2 and bax expression and prostate cancer outcome in men treated with radiotherapy in Radiation Therapy Oncology Group protocol 86-10. Int J Radiat Oncol Biol Phys 2006 Sep 1; 66(1): 25-30. [DOI:10.1016/j.ijrobp.2006.03.056] [PMID] [PMCID]
  19. Khor LY, Moughan J, Al-Saleem T, Hammond EH, Venkatesan V, Rosenthal SA, et al. Bcl-2 and Bax expression predict prostate cancer outcome in men treated with androgen deprivation and radiotherapy on radiation therapy oncology group protocol 92-02. Clin Cancer Res 2007 Jun 15; 13(12): 3585-90. [DOI:10.1158/1078-0432.CCR-06-2972] [PMID] [PMCID]
  20. Jindala DG, Jindalb V, Joshia S, Bhojiac I, Chawdhryc A. Heat shock proteins in pathology: a review. Journal of Pierre Fauchard Academy (India Section) 2016; 30(3-4): 84-7. [DOI:10.1016/j.jpfa.2016.11.002]
  21. Kurahashi T, Miyake H, Hara I, Fujisawa M. Expression of major heat shock proteins in prostate cancer: correlation with clinicopathological outcomes in patients undergoing radical prostatectomy. J Urol 2007 Feb; 177(2): 757-61. [DOI:10.1016/j.juro.2006.09.073] [PMID]
  22. Cornford PA, Dodson AR, Parsons KF, Desmond AD, Woolfenden A, Fordham M, et al. Heat shock protein expression independently predicts clinical outcome in prostate cancer. Cancer Res 2000 Dec 15; 60(24): 7099-105.
  23. Lebret T, Watson RW, Fitzpatrick JM. Heat shock proteins: their role in urological tumors. J Urol 2003 Jan; 169(1): 338-46. [DOI:10.1016/S0022-5347(05)64123-7]
  24. Nolan KD, Kaur J, Isaacs JS. Secreted heat shock protein 90 promotes prostate cancer stem cell heterogeneity. Oncotarget 2017 Mar 21; 8(12): 19323-41. [DOI:10.18632/oncotarget.14252]
  25. Zhang C, Song X, Zhu M, Shi S, Li M, Jin L et al. Association between MMP1-1607 1G>2G polymorphism and head and neck cancer risk: a meta-analysis. PloS One 2013; 8(2): 285-93. [DOI:10.1371/journal.pone.0056294] [PMID] [PMCID]
  26. Zhong WD, Han ZD, He HC, Bi XC, Dai QS, Zhu G, et al. CD147, MMP-1, MMP-2 and MMP-9 protein expression as significant prognostic factors in human prostate cancer. Oncology 2008; 75(3-4): 230-6. [DOI:10.1159/000163852] [PMID]
  27. Pulukuri SM, Rao JS. Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis. Int J Oncol 2008 Apr; 32(4): 757-65.
  28. Kervancioglu E, Kosan M, Erinanc H, Gonulalan U, Oguzulgen AI, Coskun EZ et al. Predictive values of vascular endothelial growth factor and microvessel-density levels in initial biopsy for prostate cancer. Med Sci 2016; 32(2): 74-9. [DOI:10.1016/j.kjms.2015.12.001] [PMID]
  29. Gautam KA, Singh AN, Srivastav AN, Sankhwar SN. Angiogenesis in prostate cancer and benign prostatic hyperplasia assessed by VEGF and CD-34 IHC: A comparative clinico-pathological study. Afr J Urol 2018 June; 24(2): 98-103. [DOI:10.1016/j.afju.2018.01.009]
  30. Weber DC, Tille JC, Combescure C, Egger JF, Laouiti M, Hammad K, et al. The prognostic value of expression of HIF1α, EGFR and VEGF-A, in localized prostate cancer for intermediate- and high-risk patients treated with radiation therapy with or without androgen deprivation therapy. Radiat Oncol 2012; 7: 66. [DOI:10.1186/1748-717X-7-66] [PMID] [PMCID]
  31. Lanciotti M, Masieri L, Raspollini MR, Minervini A, Mari A, Comito G, et al. The Role of M1 and M2 Macrophages in Prostate Cancer in relation to Extracapsular Tumor Extension and Biochemical Recurrence after Radical Prostatectomy. Biomed Res Int 2014; 2014: 486798. [DOI:10.1155/2014/486798] [PMID] [PMCID]
  32. Chang DK, Jamiesonaf NB, Grimmondag SM. Stratified Medicine for Pancreatic Cancer. In: Padmanabhan S, editor. Handbook of Pharmacogenomics and Stratified Medicine. London, UK: Academic Press; 2014. p. 807-14. [DOI:10.1016/B978-0-12-386882-4.00034-7] [PMCID]
  33. Jacob K, Webber M, Benayahu D, Kleinman HK. Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone. Cancer Res 1999 Sep 1; 59(17): 4453-7.