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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 39  |  Issue : 2  |  Page : 249-256

Expression of inflammatory molecules cyclooxygenase-2 and forkhead box protein 3 in Wilms’ tumor microenvironment and their clinicopathological significance


Department of Pathology, Faculty of Medicine, Egypt

Date of Submission15-Jun-2019
Date of Acceptance06-Nov-2019
Date of Web Publication30-Sep-2020

Correspondence Address:
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/EGJP.EGJP_32_19

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  Abstract 


Background and purpose The role of inflammation in cancer has been reported in various adult malignant neoplasms. Cyclooxygenase-2 (COX-2) is overexpressed in invasive breast carcinoma, colonic adenocarcinomas, as well as other tumors, and inhibitors of COX-2 have increasingly become therapeutic alternatives in many tumors. Forkhead box protein 3 (FOXP-3) transcription factor is important for the development of Tregs and hence maintenance of immunologic self-tolerance. Activity of Tregs is increased in lung cancer, gastric, and several adult-onset tumors, and they play a role in suppressing the antitumor immune responses. However, the exact role of these inflammatory molecules in pediatric malignancies especially Wilms’ tumor (WT) has not yet been fully addressed.
Aim The aim of the present study was to investigate the expression of the inflammatory molecules COX-2 and FOXP-3 in WTs and their possible association with the different clinicopathological parameters.
Patients and methods In this study, 21 formalin-fixed paraffin-embedded specimens of WTs were evaluated. Anaplasia [unfavorable histology (UFH)] was found in 31.6% of the studied tumors. Protein expression of COX-2 and FOXP-3 was investigated using immunohistochemistry. The degree of expression of both COX-2 and FOXP-3 expression was semiquantitatively assessed for each of the studied tumors using modified H-scoring.
Results The present study revealed that COX-2 was expressed in the cytoplasm of all studied cases (100%). Its expression was higher in anaplastic WT (UFH) (median H-score, 200) compared with tumors with favorable histology (median H-score, 160). However, this difference was not statistically significant (P=0.167). On the contrary, FOXP-3 expression in WT was mostly low. Its expression varied between an H-score of 10 and 130 (median, 20). No significant difference in expression was noted between favorable histology versus UFH tumors. The present study also demonstrated a significant positive association between expression of COX-2 and FOX-P3 in WTs (P=0.033).
Conclusion Our results show that COX-2 is strongly expressed in all WTs with a trend toward higher expression in UFH ones. Furthermore, the significant association between COX-2 expression and FOXP-3 expression in the studied cases suggests that inhibition of COX-2 may lead to an associated suppression of FOXP-3 activity and thereby enhancing the antitumor immune responses in these patient, which is an interesting finding that needs to be further explored.

Keywords: antitumor immune response, cyclooxygenase-2, favorable histology, forkhead box protein 3, tumor microenvironment, unfavorable histology, Wilms’, tumor


How to cite this article:
Guimei MM. Expression of inflammatory molecules cyclooxygenase-2 and forkhead box protein 3 in Wilms’ tumor microenvironment and their clinicopathological significance. Egypt J Pathol 2019;39:249-56

How to cite this URL:
Guimei MM. Expression of inflammatory molecules cyclooxygenase-2 and forkhead box protein 3 in Wilms’ tumor microenvironment and their clinicopathological significance. Egypt J Pathol [serial online] 2019 [cited 2021 Apr 15];39:249-56. Available from: http://www.xep.eg.net/text.asp?2019/39/2/249/296053




  Introduction and review of literature Top


Wilms’ tumor (WT), or nephroblastoma, is the most common genitourinary malignancy in children (Dénes et al., 2013). It is characterized by the presence of persistent blastema, primitive glomeruli and tubules, together with supporting mesenchyme or stroma (Ritchey et al., 1994). It accounts for ∼6% of all childhood tumors, and its incidence corresponds to 1 in 10 000 children (SEER Cancer Statistics Review, 1975-2003, Ries et al. n.d., 2019). The majority of WTs are unilateral and sporadic, and only 1% of the cases are considered hereditary (Breslow et al., 2006). In spite of the great advances achieved in diagnosis and treatment of WT, the most important predictors of outcome in these children remain to be tumor histology and stage of the disease, according to which, treatment is determined (Ghanem et al., 2013).

In addition to the neoplastic cells, the complex and dynamic tumor microenvironment is composed of extracellular matrix, endothelial cells, immune cells, and a plethora of cytokines and growth factors (Pang et al., 2016). Importantly, inflammatory cells and inflammatory mediators are prominent constituents of the microenvironment in all tumors. The interplay between them and the different components in the tumor microenvironment is now being recognized as a key player in tumor progression, influencing growth, invasiveness, and metastatic potential (Ghanem et al., 2013; Zhu et al., 2012). The inflammation in the microenvironment has been identified as an oncogenic factor leading to tissue remodeling, angiogenesis, cancer cell survival, metastasis as well as immune evasion.

The role of an inflammatory microenvironment in tumor development has been investigated in many adult-onset malignancies, especially those for which inflammation is a known risk factor (Zhu et al., 2012). However, little is known about the role of an inflammatory microenvironment in the development and progression of childhood tumors. Targeting the immune system, either by potentiating immune responses or by inhibiting cancer cell-elicited immunosuppressive mechanisms (immunoediting), is currently at the forefront of cancer research.

Cyclooxygenase-2 (COX-2), also referred to as prostaglandin endoperoxide synthase 2 (PTGS2), metabolizes arachidonic acid and produces a number of biologically active prostaglandins (PGD2, PGE2, PGF2α, and PGI2) and thromboxane A2 (TXA2). There are three isoforms of COX enzymes: COX-1, COX-2, and COX-3 (Sharma et al., 2003); of which, COX-2 is known to be an inducible enzyme that is associated with inflammatory diseases as well as carcinogenesis. It is also suspected to promote angiogenesis, tissue invasion, and resistance to apoptosis (Nzeako et al., 2002; DeNardo et al., 2010). COX-2 protein expression was reported to be increased in several tumors including colorectal cancers (Fujino et al., 2002) as well as ovarian cancers, where higher COX-2 expression was associated with poorer survival rate, thus making COX-2 expression a potential prognostic marker of ovarian cancer (Sun et al., 2017).

In addition to its oncogenic role, COX-2/PGE2 is also suggested to play a role in modulating the immune response against tumors via several mechanisms; it inhibits proliferation of B and T lymphocytes (Harris et al., 2007), augments protumorigenic type 2 lymphocytes (Eruslanov et al., 2010) and promotes M2 macrophage differentiation (Li et al., 2014), induces Tregs, promotes CD4+ and CD8+ T cells differentiation in Tregs, and inhibits effector T cells in a COX-2-dependent manner (Yuan et al., 2010). COX-2/PGE(2) induction of Treg cells supports the cancer-mediated immune suppression, and the extent of COX-2 expression was found to be significantly associated with Tregs prevalence in the tumor microenvironment (Mahic et al., 2006).

The immune-suppressive activity of T-regulatory cells is driven by expression of the forkhead/winged helix (FOXP-3) gene (Rudensky, 2011, p. 3). The forkhead box protein 3 (FOXP-3) is a transcription factor that has a fundamental role in the regulation and development of the immune system as well as maintenance of immunological self-tolerance by suppressing self-reactive T cells (Hori et al., 2003). Considering that most tumor-associated antigens identified to date are antigenically normal self-constituents, it is likely that naturally occurring FOXP-3+ Treg cells hamper the antitumor immune responses in patients with cancer and represents an important cellular target to evoke and augment antitumor immunity (Liu et al., 2017). Although it was first described as restricted to hematopoietic lineages, recent studies have demonstrated FOXP-3 expression in several tissues, including tumor cells. This has been shown in non-small cell lung cancer, pancreatic cancer, melanoma, and breast tumors (Takenaka et al., 2013). Moreover, FOXP-3 expression in tumor cells could be an independent strong prognostic factor for distant metastasis in BC.

In view of these findings, and in order to learn more about the role the inflammatory microenvironment in the development of WT, the aim of the present study was to analyze the immunohistochemical expression of both COX-2 and FOXP-3 in WT in relation to the different clinicopathological characteristics hoping that this may aid the search for new possible molecular markers that could better stratify the patients into high-risk and low-risk groups for better fine tuning of their treatment.


  Patients and methods Top


Patient samples

Twenty-one cases of WT were collected from the archives of the Pathology Department, Faculty of Medicine, Alexandria University, Egypt. Written informed consents were obtained from the parents of the patients included in the study. Formalin-fixed paraffin-embedded specimens were cut in 5-µm thick sections and stained with hematoxylin and eosin to confirm the diagnosis and the grade of tumor. The study included 14 cases of favorable histology (FH) and seven cases with unfavorable histology (UFH). Anaplasia was identified in the latter group according to the COG definition by the presence of a three-fold increase in nuclear size compared with adjacent nuclei, hyperchromasia, and multipolar mitotic figures. Eight (38%) cases had a stage I disease, six (29%) cases were stage II disease, five (24%) were stage III, and two (10%) were stage IV. The study was approved by the ethics committee of the College of Medicine, University of Alexandria.

Histology and immunohistochemistry of inflammatory and immune markers

Immunohistochemistry was performed using anti-COX-2 rabbit polyclonal antibody (Clone ab15191; Abcam, Cambridge, UK) and anti-FOXP-3 mouse monoclonal antibody [clone (2A11G9), sc-53876, Santa Cruz, California, USA] antibody. The Abcam detection kits were used (rabbit specific HRP/DAB detection kit, ab64162, and mouse specific HRP/DAB detection kit, ab64259; Abcam). Sections were deparaffinized and dehydrated. Microwave unmasking of antigens was performed for 20 min in 0.01 mol/l citrate buffer at 98°C (pH 6). The sections were then left to cool for 1 h. Endogenous peroxide was subsequently blocked with 3% hydrogen peroxide for 10 min, followed by washing for 5 min with phosphate buffer saline. The specimens were incubated overnight at 4°C with anti-COX-2 and FOXP-3 antibodies diluted at 1 : 100 and 1 : 50, respectively. They were then washed three times in phosphate buffer saline for 5 min each and incubated for 30 min with labeled-polymer-conjugated secondary antibody. Finally, they were washed and developed with 3,3-diaminobenzidine tetrahydrochloride for 5 min, lightly counterstained with hematoxylin (cas number 517-28-2; Sigma-Aldrich, St. Louis, Missouri, USA), dehydrated, and mounted. Positive and negative controls were included in all the runs. Tonsil tissue and human breast carcinoma tissue were used as positive controls for FOXP-3 and COX-2, respectively

Quantification of immune cells and inflammatory markers

Modified H-scoring

A semiquantitative (modified H-score) method was used to assess the degree of positive staining. This method assigns an immunohistochemical H-score to each patient on a continuous scale of 0–300, based on the percentage of cells at different staining intensities visualized at different magnifications (Pirker et al., 2012). Cytoplasmic staining was scored according to four categories: 0 for ‘no staining,’ 1+ for ‘light staining visible only at high magnification,’ 2+ for ‘intermediate staining,’ and 3+ for ‘strong, dark staining, visible even at low magnification.’ The percentage of cells at different staining intensities was determined by visual assessment, with the score calculated using the formula, 1 Å∼ (% of 1 +cells)+2 Å∼ (% of 2+cells)+3 Å∼ (% of 3+cells), depending on the percentage of stained cells.

Statistical analysis

Data were entered using statistical package for the social science program for statistical analysis (SPSS version 21, Armonk, NY, USA). Data were entered as numerical or categorical as appropriate. Data were described using minimum, maximum, mean, SD, and 95% confidence interval of the mean for the normally distributed area. Categorical variables were described using frequency and percentage of total. Comparisons were carried out between two studied independent not normally distributed subgroups using Mann–Whitney U test. Comparisons were carried between more than two studied independent not normally distributed subgroups using Kruskal–Wallis test. χ2 test was used to test the association between qualitative variables. Fisher’s exact test and Monte Carlo correlation were carried out when indicated. Results were considered statistically significant when the P value was less than 0.05.


  Results Top


Patient data

The present study is a retrospective study of 21 formalin-fixed paraffin-embedded tissue specimens of WT. All patients had been treated by surgery followed with postoperative chemotherapy/radiotherapy in Alexandria University Hospital, Egypt, according to the NWTSG/COG guidelines. All data regarding patient age, tumor grade, tumor stage, as well as follow-up were retrieved from the archives of the pathology and oncology departments.

The patients’ ages ranged between 3 and 132 months, with a median age of 24 months. Ten (47.62%) out of the 21 cases showed the typical triphasic pattern on histology, whereas six (28.57%) had a biphasic pattern, and only five (23.81%) tumors had a monophasic pattern (blastemal only). Fourteen (66.67%) out of the 21 cases had FH, and seven (33.33%) cases showed UFH. Data concerning patients’ follow-up were retrieved from the patients’ records. The average follow-up was 2 years after the start of treatment. After those 2 years, only three patients developed recurrence and died from the disease, whereas the remaining 18 patients were alive and free of the disease.

Cyclooxygenase-2 expression in Wilms’ tumors

In the present study, COX-2 expression was noted in the cytoplasm of the tumor cells in all studied cases (100%). COX-2 expression score (as measured by modified H-scoring) varied between 60 and 300, with a median value of 170 in the different tumors. COX-2 expression was stronger and more evident in the epithelial compartment of the tumors (primitive tubules) compared with the stromal and blastemal areas, which both showed less expression ([Figure 1]).
Figure 1 (a) Cytoplasmic expression of COX-2 in the primitive tubules of WT. (immunoperoxidase, ×200). (b) Strong cytoplasmic expression of COX-2 in the primitive tubules (thick arrows) compared with blastema in which less number of cells show positive staining (thin arrows) (immunoperoxidase, ×400). COX-2, cyclooxygenase-2; WT, Wilms’ tumor.

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Modified H-score for COX-2 expression was higher in UFH WTs (median H-score, 202) compared with tumors with low-grade tumors (FH), where the median H-score value was 160. Yet this difference was not statistically significant (P=0.167, Mann–Whitney U test). Similarly, COX-2 expression scores were higher in high-stage tumors (stages III and IV) compared with low-stage tumors (stages I and II); however, this difference was also not statistically significant (P=0.178, Mann–Whitney U test) ([Figure 2]).
Figure 2 (a) COX-2 expression H-scores were higher in anaplastic WT (median H-score, 202) compared with favorable histology tumors (median H-score, 160) (P=0.167). (b) COX-2 expression scores was higher in high-stage tumors (median H-score, 208) compared with low-stage tumors (median H-score, 166) (P=0.178). COX-2, cyclooxygenase-2; WT, Wilms’ tumor.

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Forkhead box protein 3 expression in Wilms’ tumors

FOXP-3 expression was noted in all studied tumors. Its expression was noted in the nuclei of not only the tumor-associated immune cells but also in some tumor cells themselves. The FOXP-3 H-scores varied between a minimum of two, where only very few cells showed positive staining, and a maximum of only 130 in others. The median modified H-score was only 20, thus denoting that FOXP-3 expression was significantly less than COX-2 expression in the examined tumors ([Figure 3]).
Figure 3 (a) FOXP-3 expression is noted in the nuclei of few tumor cells mostly in the primitive tubular and glomerular structures (arrows), whereas the majority of the tumor cells are negative for FOXP-3 (H-score of this case was 20) (immunoperoxidase, ×400). (b) Few tumor-infiltrating immune cells show positive nuclear expression of FOXP-3 (immunoperoxidase, ×200). FOX-3, forkhead box protein 3.

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Among the 14 cases that showed FH, FOXP-3 scores ranged between 2 and 130, with a median score of 20, compared with a median of 30 for cases with UFH. Therefore, there was no statistically significant difference between FH and UFH cases regarding FOXP-3 expression. Furthermore, FOXP-3 expression did not show any statistically significant correlation with the age, sex, or tumor stage ([Figure 4]).
Figure 4 (a) FOXP-3 expression (H-scores) in FH (low grade) and UFH (high grade) WTs, (P=0.470). (b) FOXP-3 expression scores in WTs with low-stage and high-stage tumors (P=0.518). FH, favorable histology; FOX-3, forkhead box protein 3; UFH, unfavorable histology; WT, Wilms’ tumor.

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All cases that showed a high COX-2 expression showed a simultaneously high expression of FOXP-3. Thus, a statistically significant positive correlation was found between COX-2 and FOXP-3 expression in the studied cases (t=0.361, P=0.033) ([Figure 5]).
Figure 5 A statistically significant positive correlation between COX-2 (H-scores) and FOXP-3 (H-scores) in the studied cases (t=0.361, P=0.033). COX-2, cyclooxygenase-2; FOX-3, forkhead box protein 3.

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Diagnostic test accuracy analysis showed that COX-2 H-score was a statistically insignificant discriminator of occurrence of death, with area under the receiver operating characteristic curve (area under the curve) of 0.500 (95% confidence interval, 0.277–0.723) (Z=0.000, P=1.000). Similarly, FOXP-3 H-score was not found to be a statistically significant discriminator of occurrence of death, with area under the receiver operating characteristic curve (area under the curve) of 0.639 (95% confidence interval, 0.403–0.834) (Z=0.560, P=0.5754).


  Discussion Top


In the past decade, several studies have pointed to a significant role of an inflammatory tumor microenvironment in adult-onset cancers. It has been suggested to play a role in the establishment as well as the progression of many tumors (Dénes et al., 2013). However, the contribution of the inflammatory microenvironment in childhood malignancies has not yet been fully addressed. We have investigated the expression of two important inflammatory molecules, COX-2 and FOXP-3, in 21 cases of WT.

In the present study, the expression of COX-2 was noted in tumor cells of all the studied cases, as well as in all compartments of the tumor: epithelial, stromal, and blastema. These results are in accordance with a previous comparative study that had investigated COX-2 expression in five cases of WT in comparison with adult tumors and had reported that expression of COX-2) was more localized in the stroma and was associated with infiltration of tumor-associated macrophages (TAM) (Maturu et al., 2014). TAM infiltration is known to be induced by COX-2 in the tumor microenvironment, especially in the tumor stroma, and TAMs can also induce expression of COX-2 (Hou et al., 2011). The abundant COX-2 expression in WTs could be attributed to several factors; one of these factors is the presence of infiltrating immune cells within the tumor stroma that express COX-2. Moreover, the abundant tumor fibroblasts in the stromal compartment could be generating COX-2 in response to macrophage infiltration or the inflammatory tumor microenvironment.

The present study also identified a trend toward a higher expression of COX-2 in higher grade tumors (UFH) as shown by higher H-scores of COX-2 expression in UFH tumors compared with FH tumors. This is a relatively novel finding that places COX-2 expression as an important prognostic marker in WT. In spite of the great advances in the diagnostic and therapeutic techniques, the presence of histological anaplasia still remains the most important determinant of prognosis and response to therapy in patients with WT, and specific disease biomarkers that could help stratify high-risk from low-risk patients, and therefore fine-tune management, are in great demand.

In the past decade, COX-2 overexpression has been reported in several human cancers, including breast, lung, skin, colon, bone, cervical, as well as esophageal tumors and was reported to have a poor prognostic significance (Han et al., 2014; Pang et al., 2016). In colon cancer, high COX-2 protein expression was significantly correlated with tumor size, infiltration depth, Duke’s stage, tumor differentiation, distant metastasis, and lymph node metastasis, and both COX-2 and HER-2 were found to be important markers for invasion and metastasis in colorectal cancer (Wu and Sun, 2015, p. 2). COX-2 mediates these oncogenic effects through numerous signaling pathways, including activation of vascular endothelial growth factor, which leads to increased cell proliferation and angiogenesis (Xu et al., 2014). It was also mentioned that there is increased expression of the proto-oncogenes, BCL-2, and the epidermal growth factor receptor, and this occurs through the activation of the mitogen-activated protein kinase and the phosphoinositide 3-kinase/AKT pathway, respectively (Buchanan et al., 2003). In addition, COX-2 increases the transcriptional activity of the antiapoptotic mediator nuclear factor κB, activates matrix metalloproteases (MMP-2 andMMP-9), and suppresses the production of interleukin-12, leading to immunosuppression (Pang et al., 2016).

In the present study, FOXP-3 expression was observed in tumor cells as well as in tumor-infiltrating immune cells in all studied cases of WT. However, the level and the degree of expression was relatively low compared with COX-2 expression in the same tumors, as shown by the relatively low H-scores for FOXP-3. It is well established that FOXP-3 plays a key role in Treg function and is an obligate marker of CD4+CD25+ Tregs. FOXP-3+ Tregs exert an immune suppressive influence by expression of cytokines such as interleukin-10 and transforming growth factor-b (Weiner, 2001). A high density of tumor-infiltrating FOXP-3+ Treg in tumor specimen has been associated with poor outcome in various solid tumors, including pancreatic (Hiraoka et al., 2006) and hepatocellular carcinoma (Kobayashi et al., 2007). Therefore, the relatively low expression of FOXP-3 in WT goes hand in hand with the relatively good prognosis of this neoplasm.

Similar to our results, the expression of FOXP-3 has not been restricted to tumor immune cells; it has been demonstrated in the tumor cells themselves in several tumors, including breast cancers (Ladoire et al., 2011), lung (Tao et al., 2012), and thyroid carcinomas (Cunha et al., 2012). In colorectal cancer, high FOXP-3 expression in tumor cells and not in tumor-infiltrating lymphocytes was associated with poor prognosis compared with patients with low FOXP-3 expression (Kim et al., 2013). The expression of FOXP-3 by cancer cells enabled them to downregulate effector T-cell responses directed against the tumor. This would give clinical evidence for an effective mechanism of a direct tumor-derived evasion from immunological destruction in CRC.The expression of FOXP-3 in the studied cases showed a significant positive association with COX-2 expression levels. In accordance with our findings, it has been previously suggested that COX-2-PGE2 signal pathway suppressed dendritic cells, natural killer, T cells, and type-1 immunity, but promoted type 2 immunity leading to tumor immune evasion and resistance to cancer immunotherapy (Liu et al., 2015). Similarly, a study by Baratelli et al. (2005) has demonstrated that PGE2, a product of COX-2, upregulated FOXP-3 at both mRNA and protein levels and enhanced FOXP-3 promoter activity in human lymphocytes). Another study on gastric carcinoma has demonstrated that an elevated FOXP-3 expression in tumor-infiltrating Treg cells correlated with COX-2 and PGE2 expression and was associated with a higher TNM stage. This effect was reversed by COX inhibitors and PGE2 receptor-specific antagonists (Yuan et al., 2010, p. 3).


  Conclusion Top


Our data therefore demonstrate a strong expression of COX-2 in WT with higher expression levels noted in the more aggressive UFH type. COX-2 expression was also associated with FOXP-3 expression, which, in turn, points to a possible role of COX-2 in upregulating FOXP-3 and thereby inhibiting the immune response against the tumor cells. COX-2 inhibitors may thus be beneficial in the treatment of WT especially those with UFH by overcoming FOXP-3-dependent immune suppression.

However, the results of this study need to be further validated on a large sample of cases as well as address the exact molecular events underlying this association.[37]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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