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Immunohistochemical expression of cyclooxygenase-2 and CD44 (stem cell marker) in urinary bladder cancer

Ibrahim, Taiseer R.; Abdel Raouf, Samar M.

doi: 10.1097/01.XEJ.0000455921.18656.71
ORIGINAL ARTICLES
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Background Bladder cancer is the most common urologic cancer and schistosomiasis is responsible for the high incidence of bladder cancer in Egypt. Inflammation may activate stem cells by prostaglandin E2 production mediated by cyclooxygenase-2 (COX-2) expression. Recently, evidence has been accumulating to show that stem cells are involved in inflammation-related carcinogenesis. The cell adhesion molecule CD44 standard (CD44s) has been identified as a cell surface marker associated with cancer stem cell in several types of tumors including urinary bladder cancer.

Aim This study aimed to evaluate the expression of COX-2 and stem cell marker CD44s in schistosomal and nonschistosomal bladder tissues obtained from cystitis and cancer patients.

Materials and methods Fifty formalin-fixed and paraffin-embedded bladder tissues were obtained from four patients with chronic cystitis, 28 patients with transitional cell carcinoma (TCC) (18 schistosomal and 10 nonschistosomal), and 18 patients with squamous cell carcinoma (SCC) (12 schistosomal and six nonschistosomal), and stained by COX-2 and CD44 immunohistochemical markers.

Results There was a significant relation between COX-2 expression and schistosomal infection in TCC (P=0.003), whereas there was no significant difference in the immunoexpression of COX-2 in different grades of TCC. There was no significant relation between CD44 immunostaining and schistosomal infection in TCC and SCC, whereas there was significant difference in the immunoexpression of CD44 and different grades of TCC (P=0.041). There was a significant difference in the expression of both COX-2 and CD44 among different grades of SCC (P=0.003 and 0.007, respectively).

Conclusion There was a strong association between urinary bladder schistosomiasis infection and increased risks of developing TCC and SCC of the urinary bladder. We found that there was a significant positive association between CD44 (stem cell marker) and COX-2 expression in schistosomal-associated lesions of the urinary bladder (P<0.0001).

Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Correspondence to Taiseer R. Ibrahim, MD, Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt Tel: +20 109 426 2229; fax: +20 551 238 7825; e-mail: dr.tisserreeat@yahoo.com

Received May 12, 2014

Accepted May 27, 2014

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Introduction

Bladder cancer represents a significant health problem as it is one of the most common cancers. It is the fifth most common cancer in the west (Jemal et al., 2005; Bryan, 2011). In Egypt, bladder cancer accounts for about 30% of all cancers, and it is the most common malignancy in men and the second most common malignancy in women after breast cancer (Hammam et al., 2008). Most investigators have accepted the association between cigarette smoking and transitional cell carcinoma (TCC), which is most prevalent in western and industrialized countries. In these countries, over 90% of the bladder cancers diagnosed are TCCs (Bulbul et al., 2005; Van Rhijn et al., 2009).

Whereas in developing countries, particularly in the Middle East and Africa, the majority of bladder cancers are squamous cell carcinoma (SCC), the highest incidence has been in Schistosoma-endemic areas, where SCC ranges from two-thirds to three-quarters of all malignant tumors of the bladder (Felix et al., 2008). The high frequency of SCC is because of schistosomiasis-infected bladders that frequently show squamous metaplasia and dysplasia of the transitional epithelium. A relative increase in the frequency of TCC-associated cancer has been noted (Hammam et al., 2008).

Chronic inflammation, in which large amounts of reactive oxygen/nitrogen species are produced, is a well-recognized cause of cancer (Grivennikov et al., 2010). Epidemiological and animal studies have indicated chronic inflammation including urinary tract infection to be involved in the development of bladder cancer (Michaud, 2007).

Infestation by parasites such as Schistosoma haematobium that act as inflammatory agents is associated with urinary bladder cancer; one mechanism of carcinogenesis involves the eggs of parasites (Rollinson, 2009). The deposition of parasite eggs in the host bladder results in irritation, eventual fibrosis, and chronic cystitis, leading to carcinogenesis (Vennervald and Polman, 2009).

In areas where S. haematobium is not endemic, sporadic bladder cancer may be caused by environment factors, smoking, and genetic polymorphism in tumor suppressor, drug metabolism, and antioxidant genes (Srivastava et al., 2008).

Recently, evidence has been accumulating to show that stem cells are involved in inflammation-related carcinogenesis. According to the cancer stem cell hypothesis, not all tumor cells can participate in tumor evolution and, instead, this property is limited to a subset of cells termed ‘cancer stem cells’, which is a functional term, and defines a tumor subpopulation with tumor-initiating potential, self-renewal properties, and the ability to generate cellular tumor heterogeneity by differentiation. These cancer stem cells do not necessarily arise from normal stem cells; they are also derived from differentiated progenies that have acquired properties by genetic or epigenetic alteration (Ho et al., 2012).

Homotypic adhesion among malignant cells and heterotypic adhesion between malignant cells and other cells, and blood cells and endothelial cells, are necessary to facilitate migration, survival, and implantation. Adhesion molecules are cell surface glycoproteins mediating cell–cell and cell–matrix interactions. These molecules play vital roles both under physiologic and under pathologic conditions (Naor et al., 2002; Pascal et al., 2007) and are classified into four categories: integrins, selectins, cadherins, and immunoglobulin superfamily. CD44 standard (CD44s) is a member of the immunoglobulin superfamily, involved in cell–cell and cell–matrix interactions. CD44s has both physiologic and pathologic functions, including cellular adhesion, aggregation, migration, hyaluranate degradation, lymphocyte activation, lymph node homing, myelopoiesis, lymphopoiesis, and angiogenesis (Omran and Ata, 2012). CD44s is one of the most prominent stem cell markers; it is also known to be involved in tumorigenesis through stimulation of angiogenesis, binding of growth factors to tumor cells, thus increasing the rate of cell division and inhibiting apoptosis. CD44 positive cells are located in the basal layer of the normal urothelium as well as in urothelial cell carcinoma (Dimov et al., 2010).

Prostaglandin E2 (PGE2) has been reported to regulate vertebrate hematopoietic stem cell homeostasis. Inflammation may also activate normal or cancer stem cells by PGE2 signaling. It is known that cyclooxygenase-2 (COX-2), the PGE2-generating enzyme, is important in inflammation-related carcinogenesis (Thanan et al., 2012). It is not detectable in most normal tissue; however, it is induced at sites of inflammation by cytokines, growth factors, and tumor promoters. It plays an important role in cancer initiation and development by activating carcinogens, decreasing apoptosis, immunosuppression, angiogenesis, and induction of metastasis (Pruthi et al., 2004; Méric et al., 2006). COX-2 has been found to be upregulated and overexpressed in tumors of the colon (Sano et al., 1995; Yoshimura et al., 2005), stomach, pancreas, and lung cancers as well as in bladder cancer, suggesting an important role in their tumorigenesis (Yoshimura et al., 2000; Mohamed and Mohamed, 2012).

The aim of this study is to evaluate the expression of COX-2 and stem cell marker CD44 in bladder tissues obtained from cystitis and cancer patients.

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Materials and methods

Fifty formalin-fixed, paraffin-embedded bladder tissues were obtained from four patients with chronic cystitis, 28 patients with TCC, and 18 patients with SCC, with their related data selected randomly from the archive of the Pathology Department, Faculty of Medicine, Zagazig University, in the period 2011–2013. We assessed the pathological grades of urinary bladder cancer according to the WHO classification (Eble et al., 2004). This study was carried out with full approval from the local ethics committee.

All paraffin blocks were cut into 4 μm thickness and stained with ordinary H&E stain to confirm the diagnosis and to determine the associated schistosomal infection.

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Immunohistochemical procedures

Immunostaining was performed using the avidin–biotin peroxidase technique for localization. Paraffin sections mounted on coated slides were deparaffinized with xylene, and then sections were rehydrated through 100, 90, 70, and 50% ethanol. The sections were then treated with 0.01 mol/l citrate buffer (pH 6.0) for 30 min to unmask antigens before further treatment after a quick rinse in PBS.

Then, sections were incubated in 0.3% H2O2 for 30 min to abolish endogenous peroxidase activity (Dako Ko411 Kit; Dako, Glostrup, Denmark) before blocking with 5% horse serum for 2 h at room temperature to inhibit the nonspecific immunoreactions.

Primary monoclonal antibodies were incubated overnight in a humidity chamber using the following dilutions: mouse monoclonal anti-CD44s (1 : 100, clone 156-3c11; Thermo Scientific/Lab Vision Corporation, Fremont, California, USA) and rabbit polyclonal anti-COX-2 (1 : 200, RB-9072-R1; Lab Vision Corporation, Neo Markers). After washing in PBS, they were incubated with biotinylated secondary antibodies for 30 min, followed by the avidin–biotin peroxidase complex for another 30 min according to the instructions of the manufacturer (Universal Detection Kit; Dako). Finally, immune reaction was visualized as a brown color with 3,3-diaminobenzidine tetrahydrochloride (DAB, K0114 Kit; Dako) for 5 min, and then washed in distilled water. Then, the slides were counterstained with Mayer’s hematoxylin for 1 min before mounting.

All the procedures were performed at room temperature. In addition, a negative control for both markers in which the primary antibody was omitted and replaced by PBS was used. Colonic mucosa was used as a positive control for COX-2, whereas normal skin served as a positive control for CD44 for processing with the bladder tissue sections in the same run for precision and standardization of the detailed immunohistochemical results of both markers.

The immunostaining was evaluated semiquantitatively by two pathologists (T.R.I & S.M.AR.). CD44 was membranous in distribution, whereas COX-2 was cytoplasmic. The immunostaining was evaluated on the basis of the intensity obtained from the staining results. The staining intensity was scored as negative (0), weak (+1), moderate (+2), or intense (+3) for CD44 (Lipponen et al., 1998; Omran and Ata, 2012). The intensity of COX-2 immunostaining was graded on a scale of 0–3, where 0=no staining, 1=equivocal staining, 2=moderate to intense staining, and 3=highest intensity staining (Mohammed et al., 1999).

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Statistical analysis

Data were represented as number and percentage. The differences were compared for statistical significance using the χ2-test. A difference was considered significant at P-value less than 0.05. The statistical analysis was carried out using SPSS 16.0 (SPSS Inc., Chicago, Illinois, USA) for Windows.

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Results

This study was carried out in the Pathology Department, Faculty of Medicine, Zagazig University. Fifty patients were included in this study: four patients with chronic cystitis, 28 patients with TCC, and 18 patients with SCC.

Demographic data included in the study are summarized in Table 1. About 72% of our patients were older than 60 years of age and 70% of our patients were men. Of the 50 bladder tissue samples, Schistosoma ova was detected in 34/50 (68%), of which 4/34 (11.7%), 18/34(53%), and 12/34 (35.3%) were found in cystitis, TCC, and SCC, respectively.

Table 1

Table 1

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Results of cyclooxygenase-2 immunostaining

Results of COX-2 immunostaining are summarized in Tables 2 and 3. COX-2 expression was mainly cytoplasmic; also, COX-2 positivity was markedly heterogeneous in most of the cases of TCC, whereas homogeneous staining was usually observed in tumors of higher grade. One out of four (25%) patients with chronic cystitis showed a positive expression of COX-2 (Fig. 1a). COX-2 was immunoexpressed in all cases of schistosomal-associated TCC 18/18 (100%); 9/18 (50%) showed moderate intensity and 9/18 (50%) showed strong intensity. COX-2 was immunoexpressed in eight out of 10 cases of nonschistosomal TCC (80%); 40% showed weak intensity. There was a highly statistically significant relationship between COX-2 expression and schistosomal-associated TCC (P=0.003) (Table 2).

Table 2

Table 2

Table 3

Table 3

Fig. 1

Fig. 1

In SCC, COX-2 was immunoexpressed in 11/12 (91.6%) of schistosomal-associated SCC; 3/11 showed weak staining intensity, 5/11 showed moderate staining intensity, and 3/11 showed marked staining intensity. COX-2 was immuoexpressed in all cases of nonschistosomal-associated SCC; 50% showed weak staining intensity. There was no significant relationship between COX-2 expression and schistosomal SCC (P=0.695).

There was no significant difference in COX-2 expression among different grades of TCC (P=0.127) (Figs 2a, 3a, and 4a and Table 3).

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

A significant difference was observed in COX-2 expression among different grades of urinary bladder SCC (P=0.003) (Figs 5a and 6a).

Fig. 5

Fig. 5

Fig. 6

Fig. 6

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Results of CD44 immunostaining

Results of CD44 immunostaining are summarized in Tables 4 and 5. One (25%) case of cystitis was negative for CD44, whereas three (75%) cases showed moderate staining intensity (Fig. 1b). In schistosomal TCC, CD44 was immunoexpressed in 17/18 (nearly 94%) and 55.5% showed marked staining intensity. CD44 immunoreactivity was detected in 80% of nonschistosomal-associated TCC. There was no significant relationship between CD44 immunostaining and schistosomal infection in TCC (P=0.284).

Table 4

Table 4

Table 5

Table 5

Among 25 specimens of TCC that were CD44 positive, four had a weak staining intensity (three cases grade 1 and 1 case grade 2), nine had moderate staining intensity, and 12 cases showed strong immunoexpression (50% were grade 1). A significant difference was detected in CD44 immunoexpression among individual grades of TCC (P=0.041) (Figs 2, 3b, and 4b).

In SCC, CD44 was expressed in all cases of nonschistosomal SCC [6/6 (100%)] and immunoexpressed in 10/12 (83%) of schistosomal SCC. There was no significant relationship between CD44 immunoexpression and schistosomal infection in SCC (P=0.058).

As shown in Table 5, all cases of well-differentiated SCC were CD44 positive (50% showed strong staining), whereas cases of poorly differentiated SCC showed CD44 negative staining in 33% (2/6) and weak staining in 57% (4/6) of cases. A statistically significant difference was observed in CD44 expression among different grades of SCC (P=0.007) (Figs 5b and 6b).

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Coexpression of CD44 and cyclooxygenase-2 in urinary bladder tissues

There was a significant positive association between CD44 (stem cell marker) and COX-2 expression in schistosomal-associated lesions of the urinary bladder (P<0.001) (Table 6).

Table 6

Table 6

In contrast, no significant association was detected between CD44 and COX-2 expression in nonschistosomal-associated lesions of the urinary bladder (P=0.283).

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Discussion

Infection and inflammation have been recognized as important risk factors for carcinogenesis and malignancies. The International Agency for Research on Cancer (IARC) has estimated that ∼18% of cancer cases worldwide are attributable to infectious diseases (IARC, 2008). However, cancer itself can cause inflammation through the production of proinflammatory factors (Coussens and Werb, 2002). Inflammation-associated tissue injury may activate stem cells. PGE2 has been reported to function as a potent regulator of human mesenchymal stem cell migration and proliferation, and plays a crucial role in the regulation of several types of stem cells. Ma et al. (2011) and Ohnishi et al. (2013) considered that oxidative and nitrative DNA damage in stem cell may play a key role in inflammation-related carcinogenesis. Many cancers including bladder cancer show high PGE2 levels because of upregulated expression of COX-2, a key enzyme in PGE2 biosynthesis (Hammam et al., 2008).

It is known that COX-2 is important to inflammation-related carcinogenesis. Therefore, inflammation may also activate normal or cancer stem cells by PGE2 signaling (Logan et al., 2007). Recently, the COX-2 polymorphism has been considered a risk factor for the development and invasion of urinary carcinoma (Gangwar et al., 2011).

CD44 adhesion molecules are a family of cell surface transmembrane glycoproteins that serves as receptors for hyaluronate and bind extracellular components such as collagen, laminin, chondroitin sulfate, and fibronectin (Welsh et al., 1995). Interest has been focused on the significance of cell adhesion molecules in the process of neoplastic transformation and on the invasive potential of urothelial carcinomas (Kuncová et al., 2007). CD44 has been identified as one of the cell surface markers associated with cancer stem cells in several types of tumors including urinary bladder cancer (Klatte et al., 2010). Numerous isoforms of CD44 are generated through alternative mRNA splicing. Variant isoforms (CD44v) with insertions in the membrane-proximal extracellular region are abundant in epithelial-type carcinomas and have been found to be associated with the progression of gastrointestinal malignancies. The metastatic potential of the tumor is associated with altered expression of adhesion molecules such as CD44s and its variants (Ponta et al., 2003).

The present work was carried out on bladder tissues of 50 patients including four patients with chronic schistosomal cystitis, 28 patients with TCC (18 schistosomal and 10 nonschistosomal), and 18 patients with SCC (12 schistosomal and six nonschistosomal). Schistosomal ova was detected in 34/50 (68%) of our patients.

In this work, COX-2 was immunoexpressed in all cases of schistosomal-associated TCC 18/18 (100%) and expressed in 11/12 (91.6%) of schistosomal-associated SCC. There was a highly statistically significant relationship between COX-2 expression and schistosomal-associated TCC (P=0.003). These results were consistent with those obtained by Hammam et al. (2008).

Hassan et al. (2013) reported that COX-2 immunoexpression was found in all cases of SCC that were positive for Schistosoma ova, whereas 90% of schistosomal-associated TCC were found to express COX-2.

All cases of the TCC that were positive for Schistosoma ova were found to be positive for the COX-2 marker. These findings suggest that schistosomal infestation stimulates the production of COX-2, resulting in the development of bladder cancer. Similar findings have been reported previously by Youssef et al. (2011); they reported a significant difference in COX-2 alterations between patients with bilharzial-related bladder cancer and those with nonbilharzial bladder cancer (P<0.05).

In the current work, we found that there was no significant difference between COX-2 expression and different grades of TCC (P=0.343). Hammam et al. (2008) found that COX-2 was significantly higher in grade 3 TCC than grades 1 and 2. Matsuzawa et al. (2002) found, in their study, that COX-2 was markedly upregulated in human bladder TCC, and showed that from grade 1 to grade 3, there was a stepwise increase in the COX-2 score; this finding indicates that COX-2 might play an important role in bladder tumor development and progression, and it might be useful as a biomarker in bladder cancer. Tabriz et al. (2013) found a statistically significant relationship between COX-2 expression and different grades of urinary bladder TCC, and found that high-grade tumors showed a higher expression of COX-2 versus other grades.

There was a statistically significant inverse correlation in COX-2 expression among different grades of SCC (P=0.003); these results are not in accordance with those of Youssef et al. (2011), who reported that COX-2 overexpression was associated with increasing tumor grade (P<0.001) in bilharzial bladder cancer.

Several studies showed a significant increase in COX-2 expression with advancing tumor grade and stages of bladder cancer (Wadhwa et al., 2005). In contrast to the role of COX-2 in tumor progression, there are also studies in which COX-2 expression was not associated with the primary tumor stage and histological grading (Eltze et al., 2005).

Overall, the discrepancies found in the findings of several studies from our study may be related to different sample sizes, selection of different cut-off values for marker expression, and application of different antibodies and technical methods.

In the current study, CD44 immunoexpression was localized to the cell membrane; CD44 reactivity was detected in 3/4 chronic schistosomal cystitis. In TCC, CD44 immunoreactivity was detected in about 94% (17/18) of schistosomal TCC and in 80% (8/10) of nonschistosomal TCC. There was no statistically significant association between schistosomal infection and CD44 immunoexpression in TCC (P=0.284).

In SCC, CD44 was expressed in all cases of nonschistosomal SCC [6/6 (100%)] and was immunoexpressed in 10/12 (83%) of schistosomal SCC. There was no significant association between CD44 immunoexpression and schistosomal infection in SCC (P=0.057).

These results are in accordance with those of Gadalla et al. (2004), who reported that no difference was found between CD44 expression in bilharzial and nonbilharzial bladder cancer. Omran and Ata (2012) found, in their study, that no association was observed between the expressions of both CD44s and CD44v6 and bilharzial ova in both TCC and SCC. The absence of a statistically significant relation between CD44s and CD44v6 expression and bilharziasis may be attributed to the fact that the bilharzial antigens do not affect the expression of CD44s and also by inhibition of their expression by cytokines released from the inflammatory cells in bilharzial granuloma.

Thanan et al. (2012) found that immunoreactivity of the stem cell marker CD44v6 was significantly higher in urinary bladder cancer tissues without S. haematobium infection than in the S. haematobium-associated cancer tissues (P<0.001).

In this work, there was a statistically significant difference in CD44 expression among different grades of TCC (P=0.041). CD44 immunoexpression was detected in 100% of grade 1 and 2 TCC and in 62.5% of grade 3 TCC. Our current findings on CD44 expression are in agreement with previously reported studies in which higher CD44 expression was observed in low-grade tumors. Gadalla et al. (2004) found that there was a reduction in CD44 expression with increasing tumor grade and stage of TCC, which may additionally enable prediction of the progression of this tumor. Kuncová et al. (2007) reported that progression to higher grades of TCC was associated with a decrease in CD44 expression, higher proliferative activity of tumor cells, and more frequent p53 overexpression. Erdogan et al. (2008) observed that higher expression of CD44 was correlated inversely with the infiltrative potential of urothelial carcinoma. However, Omran and Ata (2012) reported that there was a direct association between the percentage of expression of CD44s and CD44v6 and the grade of TCC (P<0.05).

In the present study, we report a decrease in the intensity of CD44s in poorly differentiated SCC; a highly statistically significant difference was observed in CD44 expression between well-differentiated and poorly SCC (P=0.007). Gadalla et al. (2004) could not find a significant difference in CD44 expression between squamous metaplasia, preinvasive carcinoma, and invasive SCC, and no detectable difference in CD44 expression with tumor grade. Omran and Ata (2012) found an inverse correlation between CD44s expression and progression to SCC, where metaplastic urothelium showed higher expression than invasive carcinoma.

There are different risk factors that induce different levels of expression of stem cell markers in urinary bladder cancer; one of them is S. haematobium infection. In the present study, we examined the coexpression of CD44 and COX-2 in urinary bladder tissues in relation to S. haematobium infection. We found that there was a significant positive association between CD44 (stem cell marker) and COX-2 expression in schistosomal-associated lesions of the urinary bladder (P<0.001). In contrast, no significant association was detected between CD44 and COX-2 expression in nonschistosomal-associated lesions of the urinary bladder (P=0.283).

These results were not in agreement with those of Thanan et al. (2012), who found that there was a significant difference between CD44v6 immunoreactivity and COX-2 expression in urinary bladder cancer without S. haematobium (P=0.002), whereas there was no correlation between them in S. haematobium-induced urinary bladder cancer (P=0.320). They assumed that COX-2 plays important roles not only in tumor initiation/promotion but also in the regulation of stem cell proliferation and differentiation in inflammation-related urinary bladder carcinogenesis.

Other studies on different organs reported that both the increased frequency of CD44 positive cells (tumor stem cell) and upregulation of the expression of COX-2 were involved in breast cancer induction (Wang et al., 2010) and therapy (Kundu et al., 2014). Sun et al. (2013) found that COX-2 was detected in CD44 positive stem-like cells in gastric cancer.

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Conclusion

There was a strong association between urinary bladder schistosomiasis infestation and increased risks of developing TCC and SCC of the bladder. The results of this study suggest a role of CD44s gene activity in the progression of both TCC and SCC of the urinary bladder. We found that there was a significant positive association between CD44 (stem cell marker) and COX-2 expression in schistosomal-associated lesions of the urinary bladder (P<0.0001). Further studies with a large number of cases are needed to confirm our results and to elucidate the role of COX-2 and CD44s in human bladder carcinomas.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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