Bladder cancer (BC) is the most common malignancy affecting the urinary tract (Jemal et al., 2010; Ferlay et al., 2015). It ranks ninth in worldwide cancer incidence (Ferlay et al., 2015). Globally, the incidence of BC varies significantly, with Egypt, Western Europe, and North America having the highest incidence rates and Asian countries the lowest rates (Ferlay et al., 2010; Siegel et al., 2013). Eissa et al. (2015) found that, in Egypt, BC constitutes about 30% of all malignancies, wherein Schistosomal-associated BC represents the most common malignant tumor among male patients.
Two important common features of bladder tumors are tumor recurrence and multifocality. Moreover, several previous reports suggested that these tumors originated from a primary transformed progenitor cell (Denzinger et al., 2006; Hatefi et al., 2012).
Cancer stem cells (CSCs) are characterized by self-renewal, heterogeneity, resistance to apoptosis, and resistance to conventional therapies. Therefore, the CSC concept fundamentally changed the understanding of tumor development and progression as well as the therapeutic approach (Hatefi et al., 2012). Several studies indicated a rising evidence that urinary bladder carcinogenesis may be possibly associated with the CSC hypothesis (Bryan, 2011). The most important members of CSCs regulatory core are transcription factors such as OCT4, SOX-2, and Nanog. They are defined as key players in the regulatory network for maintaining the ‘stemness’ state of stem cells (Atlasi et al., 2007).
Octamer 4 (OCT4), a member of the POU-domain transcription factor family, is a multifunctional factor in cancer and stem cell biology (Madjd et al., 2009). It is a key regulator of self-renewal and differentiation in embryonic stem cells (Atlasi et al., 2007). It is believed that OCT4 enhances the malignant potential of embryonic stem cells in a dose-dependent way (Madjd et al., 2009).
CD44 is a transmembrane glycoprotein expressed on cancer cell surface and it normally takes part in cell–cell and cell–matrix adhesion interactions. It is also involved in numerous complex-signaling cascades enhancing tumor initiation by interacting with neighboring receptors such as tyrosine kinase (Jaggupilli and Elkord, 2012). Alterations of CD44 glycoproteins have been shown to play an important role in the progression of various malignancies, including urothelial cancer (Kuncova´ et al., 2007).
Inflammation may also activate normal or CSCs through prostaglandin E2 (PGE2) signaling. Therefore, it is accepted that cyclooxygenase-2 (COX2), the PGE2-generating enzyme, is important to inflammation-related carcinogenesis. It is also involved in immune response suppression, apoptosis inhibition, angiogenesis, tumor cell invasion, and metastasis (Shih et al., 2013). It is overexpressed in a variety of premalignant and malignant conditions, including urinary BC (Hammam et al., 2008). Therefore, it has been assumed that COX2 plays important roles not only in tumor initiation and promotion but also in the regulation of stem cell proliferation and differentiation in inflammation-related urinary bladder carcinogenesis (Thanan et al., 2012).
The main objective of this work was to study the expression of CSC markers OCT4 and CD44 in urinary bladder carcinoma and to correlate their expression with the available clinicopathological parameters. This work also aimed to detect the relationship between the expression of both CSC markers (OCT4 and CD44) and COX2 in Schistosomal and non-Schistosomal urinary bladder carcinoma.
Materials and methods
This study was carried out on 77 cases of bladder carcinomas selected from the archives of the Pathology Department of the Faculty of Medicine, Tanta University, and private laboratories during the period of the study from June 2013 to June 2015. Inclusion criteria included all available cases of primary bladder carcinoma with full clinicopathological sheet and the presence of tissue blocks in good condition. Approval from the research ethics committee, Faculty of Medicine, Tanta University, was obtained antecedent to conducting the study. Tissue specimens were in the form of radical cystectomy (20 specimens) and transurethral resection (TUR) (57 specimens). Cases were categorized according to the histopathological types into 60 cases of urothelial carcinoma and 17 cases of squamous cell carcinoma (SCC).
Patient’s data on age, sex, clinical presentation, and staging were obtained from files of surgery and oncology reports. Paraffin sections of 4–5 micron in thickness were stained with ordinary H&E to assess the histologic diagnosis, tumor grade as well as the extent of invasion (in TUR specimens), and T-stage (in cystectomy specimens) of bladder carcinomas as none of the studied cases had nodal or distant metastasis.
Diagnosis of Schistosomal infestation was based on the detection of schistosoma eggs in tumor tissues or adjacent non-neoplastic tissue. Cases were classified as Schistosomal and non-Schistosomal-associated urothelial carcinomas. Grading was performed according to the WHO classification of bladder tumors (Lopez-Beltran et al., 2004). Staging was performed according to American Joint Committee on Cancer TNM pathologic staging of urinary bladder carcinomas (Edge et al., 2010).
Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections of 4 µm thickness mounted on positively charged slides. Tissue sections were deparaffinized and rehydrated in graded alcohols to distilled water, and then they were incubated in 3% hydrogen peroxide for 10 min to block the endogenous peroxidase. The slides were immersed in acetic acid and heated in microwave at 95°C for 30 min for antigen retrieval, and then left to cool down at room temperature and rinsed with PBS. The slides were then incubated overnight at room temperature with primary antibodies.
- OCT4 immunostaining: Two to three drops of OCT4 mouse monoclonal ready-to-use antibody (CA 95677; Cell Marquee, USA) were placed on each slide.
- CD44 immunostaining: CD44 mouse monoclonal antibody (MS-668-P0; Labvision, 1 : 100 dilution) was used.
- COX2 immunostaining: COX2 rabbit polyclonal antibody (RB-9072-P0; Labvision, 1 : 100 dilution) was used.
Thereafter, the slides were incubated with secondary antibody (Dako En Vision K4007 detection system) for 30 min and 3,3′ diaminobenzidine as chromogen.
The slides of positive and negative controls were included in each run. Positive controls were as follows: seminoma for OCT4, normal human tonsil sections for CD44, and colonic carcinoma for COX2. Negative controls were prepared by excluding the primary antibody and replacing it with PBS.
Interpretation of immunohistochemical sections
Immunoreactivity was quantified by counting the number of positively stained tumor cells per 10 high-power fields; the results were expressed as the percentage of positively stained tumor cells.
Interpretation of OCT4 and COX2 positivity (Thanan et al., 2012)
OCT4 and COX2 staining reactions were detected as brownish staining, mainly in the nucleus and cell cytoplasm, respectively.
For both OCT4 and COX2, the staining intensity was scored as follows: negative (0), weak (+1), moderate (+2), and strong (+3).
The frequency of positive cells in a section was scored as follows: negative (0), less than 25% (+1), 25–50% (+2), 51–75% (+3), and more than 75% (+4).
IHC scoring was assigned by multiplying the intensity score by the frequency score, and was represented as follows: absent expression (0), weak expression + (1–4), moderate expression ++ (5–8), and strong expression +++ (9–12).
Interpretation of CD44 positivity (Lü et al., 2011)
CD44 staining reaction was predominantly membranous. The extent of positivity was scored according to the percentage of immunopositive cells relative to the total number of neoplastic cells as follows: negative, positive cells were up to 1%; +1, positive cells were more than 1% and up to 10%; +2, positive cells were more than 10 and up to 60%; and +3, positive cells were more than 60%.
Data were fed to the computer and analyzed using SPSS software package, version 20 (IBM). Qualitative data were described using number and percent. Significance of the obtained results was judged at the 5% level. The χ2-test, Fisher’s Exact test, and Spearman’s coefficient test were used.
The present study was performed on 77 biopsies of urinary bladder carcinoma. They included 60 cases of urothelial carcinoma and 17 cases of SCC. Schistosoma ova were evidenced in 36 (46.7%) of the total studied cases, 25 (41.7%) cases of urothelial carcinoma and 11 (64.7%) cases of SCC. The clinicopathological features of the studied cases of urinary bladder carcinoma are summarized in Table 1.
Immunohistochemical results of OCT4
OCT4 expression was detected as brown nuclear staining; it was positive in 57/77 (74%) studied cases, 46 of which were cases of urothelial carcinoma and 11 cases of SCC (Fig. 1). The adjacent apparently normal urothelium expressed negative immunohistochemical reaction for OCT4. The relations between OCT4 expression and the studied clinicopathological parameters are summarized in Table 2.
It was found that OCT4 expression was significantly correlated with tumor grade in urothelial carcinoma (P=0.026). Its expression also increased with increasing grade in SCC cases, yet not reaching statistical significance. OCT4 expression was also significantly correlated with Schistosomal association (P<0.001) among both urothelial and SCC cases. OCT4 immunoreactivity was significantly correlated with the tumor stage (P=0.006) in the studied cases of cystectomy specimens as well as with the degree of muscle invasion (P=0.003) in the studied cases of TUR.
Immunohistochemical results of CD44
A positive membranous reaction of CD44 was noted in 63/77 (81.8%) cases of the study group. These positive cases included 46/60 cases of urothelial carcinoma and all 17 cases of SCC (Fig. 2). The adjacent, apparently normal urothelium, expressed positive basal immunohistochemical reaction for CD44. The relations between CD44 expression and the studied clinicopathological parameters are shown in Table 3.
CD44 expression was inversely correlated with the tumor grade (P<0.001). There was no correlation detected between CD44 expression and urothelial carcinoma associated with schistosomiasis. However, CD44 expression significantly correlated neither with the tumor grade nor with Schistosomal association among SCC cases. As regards the tumor stage, it was noticed that CD44 expression decreased in tumors with high stage but the values did not reach statistically significant levels. Moreover, CD44 expression significantly decreased with muscle invasion in TUR cases (P<0.001).
Immunohistochemical results of COX2
A positive, brown, cytoplasmic staining reaction for COX2 was detected in 73/77 (94.8%) studied cases, 56 of which were of urothelial carcinoma and all 17 cases of SCC (Fig. 3). The adjacent, apparently normal, urothelium expressed negative immunohistochemical reaction for COX2. A summary of the relation between COX2 expression and the studied clinicopathological parameters is presented in Table 4.
COX2 expression significantly increased with increasing tumor grade and was also significantly correlated with Schistosomal association among the urothelial carcinoma cases. In contrast, it was correlated neither with tumor grade nor with Schistosomal association among SCC cases. Moreover, COX2 expression did not significantly correlate with the tumor stage in the studied cystectomy specimens or with the degree of muscle invasion in TUR specimens.
Correlation between the markers studied
A correlation between OCT4, CD44, and COX2 immunoreactivity in cases of urinary bladder carcinoma negative and positive for Schistosomal infection was made (Tables 5 and 6):
- There was a highly significant positive association between OCT4 and COX2 expression in Schistosomal-associated cases (P=0.006) when compared with non-Schistosomal-associated (P=0.023) cases of the urinary bladder carcinoma.
- There was a significant association between CD44 and COX2 expression in non-Schistosomal-associated carcinoma. However, no significant correlation was found between the two markers in Schistosomal-associated cases.
BC is the most common malignancy affecting the urinary tract (Jemal et al., 2010; Ferlay et al., 2015). In Egypt, BC constitutes about 30% of all malignancies, and it is still the most common malignant tumor in male population (Eissa et al., 2015); schistosomiasis is detected in 39.7% of urinary bladder carcinomas (Mokhtar et al., 2007).
However, during the past decade, certain changes have been observed in the features of BC in Egypt, with a significant increase in the incidence of urothelial carcinoma and a decrease in SCC cases. A notable increase in patients’ mean age with a dramatic decline in the incidence of associated schistosomiasis was also reported (Salem and Mahfouz, 2012; Gayyed and Tawfiek, 2015).
The present study included 77 cases of urinary bladder carcinoma, with a mean age of 61.9 (range: 25–80 years) years. This finding also correlated with the changing pattern of BC in Egypt in the last decade, as mentioned by Salem and Mahfouz (2012).
Male population was more commonly affected compared with female population, and the male-to-female ratio was 4 : 1. The male predominance was probably attributed to the fact that male population is more exposed to the established risk factors, including bilharzial infestation, cigarette smoking, and occupational exposure to the chemical carcinogens, as explained by other researchers (Damjanov and Golubović, 2011; Salem and Mahfouz, 2012).
The relapsing nature of BC makes it one of the most expensive human malignancies to manage (Kurtova et al., 2015). CSCs have some distinct properties among the tumor populations as they are capable of self-renewal and differentiation, and their presence is one of the major factors believed to be involved in making BC resistant to conventional drug therapies (Ojha et al., 2016).
OCT4 is a transcription factor that regulates the expression of several target genes, including NANOG, SOX-2, REX-1, and CDX-2 (Jóźwicki et al., 2014). One of the possible mechanisms responsible for the aggressive behavior of cancers and the worse clinical outcomes of tumors expressing OCT4, is the presence of the stem cell phenotype, which is responsible for dedifferentiation and chemoresistance (Kumar et al., 2012).
In the current study, OCT4 was expressed by carcinoma cells in 74% of the studied cases. These results were higher than those reported by Abdou et al. (2013), who stated that 56.6% of their malignant cases expressed OCT4. However, Hatefi et al. (2012) reported higher results using a semiquantitative RT-PCR approach to detect OCT4 expression in BC.
The results of the current study showed that OCT4 expression was significantly increased with advanced tumor grade, stage, and with evident muscle invasion. Intense OCT4 expression by carcinoma cells is associated, to a great extent, with poor differentiation states, as OCT4 is reported to maintain cells in an undifferentiated state by binding to SOX-2 and regulating the transcription factor called Nanog (Papapetrou et al., 2009). These results are in agreement with those of Hatefi et al. (2012), who reported a significant correlation between the expression of OCT4 and the tumor grade and stage, suggesting that OCT4 could be used as a molecular marker for the prognosis of bladder tumors Jóźwicki et al. (2014) also found that positive OCT4 expression was significantly associated with intrabladder tumor recurrence after operation. They recommended that a high percentage of OCT4-positive cells should support more radical therapy.
As regards the association with Schistosomal infection, the current study showed that OCT4 expression was significantly increased in schistosomiasis-associated BC. This indicates that Schistosomal infection increased the number of OCT4-positive cells, and these results were similar to those reported by Ma et al. (2011) and Thanan et al. (2012).
CD44 has been identified as a cell surface marker associated with CSCs in several types of tumors, including urinary BC (Klatte et al., 2010). It was expressed in 81.1% of the studied cases of urinary bladder carcinoma. Among urothelial carcinoma cases, a highly significant inverse statistical correlation was detected between CD44 expression and the tumor grade. Similarly, Omran and Ata (2012) and Abd El-Fattah et al. (2014) found a statistically significant decrease in CD44 expression with the increase in the tumor grade in cases of urothelial carcinoma. This could be explained by the more aggressive behavior usually seen with high-grade tumors with loss of adhesion molecules such as CD44.
There was no statistically significant relation found between CD44 expression and Schistosomal infection. These results are in accordance with those of Omran and Ata (2012) and Ibrahim and Abdel Raouf (2014) also found no association between the expression of CD44 and bilharzias ova in both urothelial carcinoma and SCC cases. They stated that the absence of a statistically significant relation between CD44 expression and schistosomiasis may be attributed to the inhibition of its expression by cytokines released from the inflammatory cells in bilharzial granuloma.
As regards the relationship between CD44 immunoreactivity and the tumor stage, it was noticed that CD44 expression decreased within tumors with high stage, but did not reach statistically significant values. This observation might be attributed to the small number of cystectomy specimens in this study. However, among TUR specimens, CD44 expression was inversely correlated with muscle invasion. These results coincided with those reported by Omran and Ata (2012) and Abd El-Fattah et al. (2014), who found that CD44 expression inversely correlated with the tumor stage, which may enable prediction of the progression of this tumor, as loss or decrease in this adhesion molecule (CD44) would stimulate invasiveness of tumor cells. Moreover, according to Hofner et al. (2014), expression of both CD44 as well as its splicing variant CD44v6 showed a tendency to decrease along with tumor dedifferentiation and invasion.
Inflammation also activates normal or CSCs through PEG2 signalling. The latter is mediated by COX2, which is an inflammation-associated enzyme involved in the pathogenesis of many solid tumors (Gangwar et al., 2011).
In the present study, COX2 was immunoexpressed in 94.8% of studied cases. It was positive in 100% of schistosomiasis-associated urothelial carcinoma. There was a high statistically significant relationship between COX2 expression and schistosomiasis-associated urothelial carcinoma. The present results are consistent with those obtained by Hammam et al. (2008) and Ibrahim and Abdel Raouf (2014). These findings suggest that Schistosomal infestation stimulates the production of COX2, resulting in the possible development of bladder carcinoma. Similar findings have been previously reported by Youssef et al. (2011); they observed a significant difference in COX2 alterations between patients with bilharzial-related bladder carcinoma and those with nonbilharzial-related bladder carcinoma.
Among SCC cases, COX2 was immunoexpressed in all cases. However, in schistosomiasis-associated cases, COX2 positivity was higher than that in nonschistosomiasis-associated cases; however, the differences did not reach statistically significant values. Similar results were reported by Hassan et al. (2013) and Ibrahim and Abdel Raouf (2014). The increase in COX2 expression with schistosomiasis-associated bladder carcinoma should be expected, as it may be attributed to inflammatory process induced by schistosomiasis and mediated by COX2, a known inflammatory-associated enzyme.
In the current work, we found that there was a significant association between COX2 expression and the different grades of urothelial carcinoma cases. These results are in accordance with those reported by Tabriz et al. (2013). The current findings might suggest a role played by COX2 in the progression of urothelilal carcinoma, especially in the context of being an important factor in inflammation-induced carcinogenesis. In contrast, however, Ibrahim and Abdel Raouf (2014) found that there was no significant difference between COX2 expression and grades of urothelial carcinoma. The discrepancies in results need to be further evaluated by extending the study on a larger scale, thereby allowing the comparison between Schistosomal and non-Schistosomal cases to be statistically better evaluated.
Concerning grading of SCC cases, no association was found between COX2 expression and tumor grade. These results are in contrast to those of Youssef et al. (2011), who reported that COX2 overexpression was associated with increasing tumor grade of bilharzias squamous bladder carcinoma. This finding may be due to the small number of SCC cases in the current study.
There are different risk factors that induce different levels of expression of stem cell markers in urinary BC; one of them is Schistosomal infection. The present study demonstrated that Schistosomal-induced urinary BC correlated with the expression of OCT4, whereas cancer without Schistosomal infection correlated with the expression of CD44. Moreover, OCT4 expression could be used not only as a poor prognostic marker in BC but also for the development of new therapeutic strategies, especially in cases of early BC. Research should be continued to isolate urothelial CSCs to provide new insights into the biological basis of BC progression. COX2 seems to play a role in the progression of urothelial carcinoma of the urinary bladder and may also be involved in inflammation-mediated stem cell proliferation/differentiation in urinary bladder carcinogenesis; a point that needs further study.
Conflicts of interest
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