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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 2  |  Page : 230-234
Immunohistochemical expression of interlukin10 (IL10) and heat shock protein-90 (HSP-90) in prostatic carcinoma


1 Department of Microbiology, Mustansiriyah University, College of Medicine, Baghdad, Iraq
2 Department of Pathology, Mustansiriyah University, College of Medicine, Baghdad, Iraq

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Date of Web Publication18-Apr-2020
 

   Abstract 


Background: Specific cytokines are related to pathologically changed prostate, propose that the balance in cytokine differs in normal and pathological prostate. Of these cytokines the interleukins 10, due to its “pleiotropic” actions in inflammation and angiogenesis, and HSP-90 due to its expression in tumor cells at high levels, suggesting that it has an important role for growth and/or survival of tumor cells. Aims: Evaluation of HSP-90 and IL10 immunoreactivity in benign prostatic hyperplasia (BPH) and prostatic carcinoma and to correlate this expression with clinicopathological parameters. Settings and Design: A retrospective study in which 83 Paraffin-embedded tissue specimens including (43) BPH, (40) prostatic carcinoma and (20) normal prostate as control were included between the period of January 2015 and January 2017. Patients, Material and Methods: All the cases were evaluated histopathologically and stained immunohistochemically for IL10 and HSP-90. Only cytoplasmic staining was considered as positive. Immunoreactivity scoring for both markers expression was calculated based on both staining intensity and percentage. Statistical Analysis: Was done using SPSS Version 21 statistical analysis software. P value of <0.05 was considered statistically significant. Result: Statistical analysis of HSP-90 and IL10 expression revealed a highly significant correlation of expression of these two markers in advanced Gleason grading and tumor, node, and metastasis (TNM) staging cases of prostatic carcinoma. Conclusion: High expression of IL10 and HSP-90 is associated with high grade and stage of prostatic carcinoma. This provides a base for further studies and researches on the role of these investigated proteins as prognostic markers immunotherapy targets for carcinoma of the prostate.

Keywords: Heat shock protein -90, immunohistochemical expression, interlukin10, prostatic carcinoma

How to cite this article:
Bakir WA, Gaidan HA, Al-kaabi MM. Immunohistochemical expression of interlukin10 (IL10) and heat shock protein-90 (HSP-90) in prostatic carcinoma. Indian J Pathol Microbiol 2020;63:230-4

How to cite this URL:
Bakir WA, Gaidan HA, Al-kaabi MM. Immunohistochemical expression of interlukin10 (IL10) and heat shock protein-90 (HSP-90) in prostatic carcinoma. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 May 31];63:230-4. Available from: http://www.ijpmonline.org/text.asp?2020/63/2/230/282694





   Introduction Top


Carcinoma of the prostate is an expanding risk throughout the world. It has the highest incidence of cancer among men and is the second cause of cancer-related death in men in the United States. However, it has variable mortality rates and incidence in the world.[1]

In Iraq, the incidence of prostatic carcinoma was 3.5% of all newly diagnosed cases of cancer in males.[2]

In spite of researches over the years, the exact mechanisms underlying the development and progression of prostatic carcinoma are not obvious. The growth, development, and differentiation of the prostate are regulated by growth factors, steroid hormones particularly androgen, and cytokines.[3]

Cytokines (including interleukin IL10) have an important role in the progression and development of prostate carcinoma, affecting the tumor bulk and host-tumor interaction. Heat shock proteins (HSP) (in particular HSP-90) have a role in cytokines biosynthesis regulation according to some studies.[4],[5]

The observation that specific cytokines are related to pathologically changed prostate, propose that the balance in cytokine differs in normal and pathological prostate.[6],[7] Increasing researches concentrate on the interleukins 10, due to its “pleiotropic” actions; it has an important role in regulating the inflammation, immune responses, and pathogenesis of many tumors[8],[9] including carcinoma of the prostate.[10]

Interleukin10 is a cytokine with potent anti-inflammatory properties and has multiple effects on inflammation and immunoregulation.[11] It is produced by many inflammatory cells like B cells, T cells, macrophages, mast cells, dendritic cells, and keratinocyte and it regulates their differentiation. The main function of IL10 is terminating inflammatory responses.[12]

IL10 is produced by tumor cells also exerting their immunosuppressive effect on macrophage and dendritic cell leading to inhibition of cell differentiation, maturation and antigen presentation allowing tumor cell evasion from immune surveillance.[13],[14]

HSP-90 is a common heat-related protein. The “90” represents its weight (90 kilo Daltons). It is a chaperone protein that shows expression during stress and participates with many other proteins in perfect folding, maintain stabilization of proteins against heat stress, and play a role in protein degradation.[15],[16]

The expression of HSP-90 in tumor cells at high levels, suggesting that it has an important role in their development and progression.[17]

Therefore, using HSP targeting drugs has emerged as a novel, promising and potential anticancer agent in cancer therapy especially in prostatic carcinoma as the function of the androgen receptor that plays a major role in prostatic carcinoma development and progression depends on HSP activity.[15],[17],[18],[19],[20],[21]

In this study, we evaluate the immunohistochemical expression of IL10 and HSP-90 in benign and malignant prostate and investigate the association with pathological grading and clinical staging in Iraqi patients with prostatic carcinoma.


   Patients, Material and Methods Top


This study is a retrospective study, formalin-fixed paraffin-embedded tissue blocks were collected from archived materials with the permission of patients between the period of January 2015 and January 2017.

The paraffin blocks represented cases of surgically removed transurethral prostatectomy or open prostatectomy as single therapy (no radiation or hormonal therapy) from 83 male patients (age range 52–68 years, mean age 62.4 years) in at that period. Clinicopathological parameters such as age, the gender of the patient, and histological/morphological types were obtained from the available histopathologic reports.

All the histopathological diagnosis for the cases had been revised.

Forty-three (43) cases were diagnosed as benign prostatic hyperplasia (BPH), while 40 cases were diagnosed as prostatic carcinoma depending on histopathological examination.

Gleason grading system was used for grading of prostatic carcinoma: Nine tumors were Gleason score (3–5); 21 were scoring (6–7); 10 score (8–10).

Tumor, node, and metastasis (TNM) staging system was used to determine the pathological stage in prostatic carcinoma: 7 tumors were stage II (pT2), 17 were stage III (pT3), and 16 were stage IV (pT4).

Twenty (20) samples of normal prostate taken from autopsies from the Institute of Forensic Medicine were used as a control in the study.

Material and method of immune staining

Each paraffin block was sectioned into 5 μm. From each block three sections were taken, one for hematoxylin and eosin (H and E stain) for histopathology revision, two were used on positively charged slides (Fisherbrand) for immunohistochemical staining with IL10 and HSP-90 monoclonal antibody.

The hematoxylin and eosin (H and E-stained sections) had been reassessed for the morphological types of the tumor, grade, and other parameters by two independent pathologists.

For IL10 immunohistochemical detection, mouse antihuman IL10 antibody (F-8) (SC-8438, IgG 2b) was used, while for HSP-90 detection we used mouse antihuman-HSP-90 QA antibody SC 13119, IgG29 (supplied by Santa Cruz Biotechnology, Inc.), at 1:50 dilution with an overnight incubation at 4°C for both.

The primary antibody was reacting with antigen in the tissue, and after that, a biotin-labeled secondary antibody binds to the primary antibody.

After that, the conjugate is added and the biotinylated secondary anti-body will form a complex with the peroxidase-conjugated streptavidin.

The substrate then added, which contains 3, 3 diaminobenzidines (DAB) in a chromogen solution which results in the formation of a brown-colored precipitate at the antigen site.

The appearance of the reaction of brown products at the site of the target antigen is associated with a positive reaction in the peroxidase secondary detection system. Counterstain was then used as blue staining of the cell.

Appropriate positive control slide (human tonsil for IL10 and human placenta tissue for HSP-90 as indicated by manufacturer instructions) and negative control slide (technically negative by omitting the primary antibody) were included in each run of immunohistochemical staining.

Immunostaining evaluation was done by two independent histopathologists who were blinded to the clinical diagnosis of the tissues at assessment time.

Scoring

Estimating the number of cells that give brown cytoplasmic staining (positive) under a light microscope. The intensity of immunohistochemical staining was estimated in 10 fields (X40 magnification). The total number of cells in each field was counted. The total score for staining was divided by the number of all cells per field in 10 fields, so the positivity stained cells percentage (in the 10 fields) was estimated for each case by holding the mean of the percentage of the cells with a positive stain in the 10 fields.

The expression of HSP-90 and IL10 <10% of positive neoplastic cells was used to define the low expression, 10–50% of neoplastic cells to define the moderate expression and >% of neoplastic cells to define the extensive or high expression.

Secondary staining kit is goat antiserum IgG CAT #SC-2050 (Santa Cruiz Biotechnology Inc.).

Statistical analysis

Statistical analysis was obtained using the Statistical Package for Social Sciences (SPSS) version 21 program. The quantitative data was handled with a Student test (t-test), that is, IL10+, IL10–, HSP-90+, HSP-90 -and controls. Absolute variables were evaluated by Pearson Chi-squared analysis test with ANOVA test if required.

P value was considered significant if it is <0.05 and highly significant if it is less than 0.001.


   Results Top


The immunohistochemical expression for both markers was cytoplasmic with different intensity as demonstrated in the [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d.
Figure 1: Photomicrograph of Immunohistochemical expression of IL10 and HSP 90 in prostatic carcinoma. (a and b) IL-10 staining in the cytoplasm of epithelial luminal cells of prostatic carcinoma with high and moderate expression respectively (X400). (c) HSP 90 staining in the cytoplasm of epithelial luminal cells of a focus of prostatic carcinoma with high expression (X40). (d) the same focus of figure C at high power (X400) demonstrate cytoplasmic high expression of the marker

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In 83 investigated tissue samples of benign and malignant prostate (43 benign prostatic hyperplasias and 40 prostatic carcinomas), there was a positive expression of HSP-90 in 33.5% of cases of benign prostatic hyperplasia) and in 76.2% of cases of prostatic carcinoma as summarized in [Table 1].
Table 1: Comparison between the mean percent of the expression of heat shock protein-90 (HSP-90) and interleukin10 (IL10) in the studied groups

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Regarding IL10 cytokine expression, it was positive in 23.7% of cases of benign prostate and in 62.3% of prostatic carcinoma as summarized in [Table 1].

Regarding HSP-90 immunohistochemical expression and Gleason scoring, the expression was highest (75.93%) in prostatic carcinoma with high Gleason score Gs (8–10) and lowest (52.3%) in low Gleason score Gs (3–5) as shown in [Table 2].
Table 2: HSP-90 protein expression in prostatic carcinoma samples according to Gleason histological grade and TNM stage

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Regarding HSP-90 immunohistochemical expression and TNM staging, the expression was highest (72.81%) in prostatic carcinoma with high TNM staging (stage pT4) and lowest (46.47%) in low TNM staging (stage pT2) as shown in [Table 2].

There was a significant association between HSP-90 expressions and Gleason scoring or TNM staging, P value < 0.05, significant at the 0.01 levels as shown in [Table 2]

Regarding IL10 immunohistochemical expression and Gleason scoring, the expression was highest (62.25%) in prostatic carcinoma with high Gleason score Gs (8–10) and lowest (43.69%) in low Gleason score Gs (3–5) as shown in [Table 3].
Table 3: IL10 protein expression in prostatic carcinoma samples according to Gleason histological grade and TNM stage

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Regarding IL10 immunohistochemical expression and TNM staging the expression was highest (60.54%) in prostatic carcinoma with high TNM staging (stage pT4) and lowest (40.53%) in low TNM staging (stage P T2) as shown in [Table 3].

There was a significant association between IL10 expression and Gleason scoring and TNM staging, P value < 0.05, significant at the 0.01 levels as shown in [Table 3].

There was a highly significant correlation of HSP-90 and IL10 expression in prostatic carcinoma while there was a significant correlation in benign prostatic samples as shown in [Table 4].
Table 4: Correlation of HSP-90 and IL-10 protein expression in study group

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   Discussion Top


This is the first study in Iraq to evaluate the immunohistochemical expression of IL10 and HSP-90 in prostatic cancer.

There were previous studies that discuss the role of these markers in cancer biology, development, prognosis and treatment.[22],[23],[24] However, a few kinds of research that investigate the immunohistochemical expression of these two markers that have been used to compare our results with them.

The aim of the study is to demonstrate their role in tumor progression and to correlate with clinicopathological parameters in prostatic tumors.

In this retrospective study, we evaluate the immunohistochemical expression of IL10 cytokine and HSP-90 in 43 cases of benign prostatic hyperplasia (BPH) and in 40 cases of prostatic carcinoma (PCa).

The expression of IL10 was higher (62.3%) in prostatic carcinoma than in benign prostatic hyperplasia (23.7%) with P value less than 0.001, this result agreed with the previous study by Cardillo and. Ippollti (2006), but the results of cases with positive expression in this study was lower than that of our study (it was 11.99% in BPH and 19.01% in PCa), the explanation of this difference in the result in prostatic carcinoma may due to the fact that larger number of cases of prostatic carcinoma of high Gleason grading (G3) and high TNM staging (pT4) in our study while in Cardillo and Ippollti's study, the largest number of cases were in intermediate Gleason grading (G2) and intermediate TMN staging (pT2) and this will further be explained by the results below.

The expression of IL10 cytokine was higher (62.25%) in high Gleason grade Gs3 (8–10) than in lower grades (43.69% in Gs 1 and 58.3% in Gs 2), this also agreed with finding in the previous study by Cardillo M.R. and F. Ippollti 2006.[5]

The expression of IL10 cytokine was higher (60.54%) in high TNM stages (pT4) than in lower stages (40.53% in pT2 1 and 55.61% in pT3), this also agreed with finding in the previous study by Cardillo and Ippollti.[5] regarding HSP-90, its expression was higher (76.2%) in prostatic carcinoma than in BPH (33.5%), this was agreed with the previous study by Cardillo and Ippollti, but the results of cases with positive expression in this study was lower than that of our study (it was 15.29% in BPH and 27.69% in PCa). The explanation of this difference is the same as in IL10 (due to difference in Gleason staging and TNM staging of cases).

There was a significant correlation between HSP-90 expressions and Gleason Grading and TNM staging.

The expression of HSP-90 was higher (75.93%) in high Gleason grade Gs3 (8–10) than in lower grades (52.3% in Gs 1 and 69.27 in Gs 2) and this is agreed with the above previous study by Cardillo M.R. and F. Ippollti and explain the above difference between in the results of two studies.

There was a significant correlation between HSP-90 expressions and Gleason grading.

The expression of HSP-90 was higher (72.81%) in high TNM stages (pT4) than in lower stages (46.47% in pT2 1 and 67.82% in pT3) also this was agreed with the previous study by Cardillo and Ippollti.[5]

There was a significant correlation between HSP-90 expression and TNM staging.

The higher expression of both proteins (HSP-90 and IL10) in higher Gleason grading and higher TNM staging suggest that these two proteins may be useful as tumor progression markers in prostatic carcinoma.[4],[25]

A highly significant correlation found between the expressions of HSP-90 and IL10 in prostatic carcinoma (correlation coefficient was 0.731 and P value < 0.00), while there was a significant correlation between the expressions of HSP-90 and IL10 in benign prostatic hyperplasia (correlation coefficient was 0.425 and P value < 0.03) as summarized in [Table 4]. This finding supports the known particular role of the different cytokines in benign and neoplastic cells that were proved by various previous studies by Cardillo et al.,[5] Bodmer,[6] and Harashima et al.[4]

The higher immunoreactivity of HSP-90 (76.2%) than that of IL10 (62.3%) in advanced stage and high-grade tumors, give the suggestion that treatment with anti- HSP-90 may be of benefit in advanced cases of prostate carcinoma, this finding was agreed with other previous studies, by Reebye et al.[21] Simone et al.,[26] and Harashima et al.[4]


   Conclusion Top


The results of this study demonstrate that high expression of IL10 and HSP-90 is associated with high grade and stage of prostatic carcinoma and provide a base for further studies and researches on the role of these investigated proteins as prognostic markers immunotherapy targets for carcinoma of the prostate.

Acknowledgment

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad - Iraq for its general support in the present work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Methaq Mueen Al-kaabi
Department of Pathology, Mustansiriyah University, College of Medicine, Baghdad
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_460_19

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