Introduction

Bladder cancer is the 10th most common cancer worldwide, with an estimated 549,000 new cases and 200,000 deaths. It is more common in men than in women, with respective incidence and mortality rates of 9.6 and 3.2 per 100,000 in men, about four times those of women globally. Thus, the disease ranks higher among men, in whom it is the sixth most common cancer and ninth leading cause of cancer death (Bray et al., 2018).

In Egypt, it is the second most common malignancy after liver cancer among Egyptian males (Ibrahim et al., 2014). Egyptian men with bladder cancer have the greatest mortality rates (16.3%), twice as high as the greatest rate in Europe (8.3% in Spain and 8.0% in Poland) and 4 times greater than in the USA (3.7%) (Salem and Mahfouz, 2012). The main risk factor for bladder cancer in Egypt was urinary schistosomiasis which was more frequent in Upper (South) Egypt, and its prevalence decreased when going north. Despite control of schistosomiasis, its effect on bladder cancer needs time to disappear (Ibrahim et al., 2014).

Cancer stem cell (CSC) theory can help in understanding the cellular and molecular events during cancer progression contributing to therapy resistance, recurrence, and metastasis. The CSC theory of cancer progression presents tumors as hierarchically organized tissue with CSC population at the top rank that then generate the more differentiated bulk of the tumor cells with lower or limited proliferative potentials. CSCs share similar properties with normal stem cells, including the ability of self-renewal and differentiation that give rise to heterogeneous, differentiated cancer cells making up the bulk of the tumor. Due to this similarity, CSCs are commonly characterized by the expression of surface markers associated with stem cells, such as CD133, CD44, and CD90, although no single marker can be used to define the CSC populations (Beck and Blanpain, 2013).

The existence of bladder CSCs has been suggested as a possible reason for the failure of adjuvant treatment and poor oncological control outcomes with tumor recurrence (Hofner et al., 2014).

Cancer stem cell markers including basal urothelial cell cytokeratins (CK5, CK14, CK17) (Ho et al., 2012) or adhesion molecules (CD44) (Immervoll et al., 2011 and Ho et al., 2012), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) (Ho et al., 2012), and tumor protein 63 (p63) (Ho et al., 2012) have been previously used as prognostic factors and applied to identify urothelial carcinoma stem cells (Hatina and Schulz, 2012 and Ho et al., 2012).

Aldehyde dehydrogenase 1(ALDH1) is an enzyme that takes part in the synthesis and regulation of retinoic acid (RA). It plays a crucial role in the differentiation and regulation of the self-renewal ability of either normal or cancer stem cells (Senol et al., 2015).

CD44 is a transmembrane glycoprotein that mediates cell adhesion to the extracellular matrix through its interaction with hyaluronate (Custódio et al., 2018). Also, CD44 presents cytokines and chemokines to their complimentary receptors on the cellular membrane (Naor et al., 2008). CD44 interacts with osteopontin and regulates its cellular functions leading to tumor progression (Rangaswami et al., 2006). It even interacts with collagen, laminin, and fibronectin where their physiological function is unclear (Jaggupilli and Elkord, 2012). CD44 functions involve ligand-binding receptor, coreceptor, and organizer in cortical actin skeleton (Naor et al., 2008).

CD44 is expressed on cancer cell surface and assists hematogenous spread (Napier et al., 2007). It is also involved in numerous complex signaling cascades enhancing tumor initiations by interacting with neighboring receptors like tyrosine kinase (Jaggupilli and Elkord, 2012).

Material and method

Sixty cases of urothelial carcinoma covering different age groups were retrieved from the pathology department, Ahmed Maher teaching hospital, Egypt, during the period from January 2016 to December 2018. Demographic and clinical data of the patients were collected from the hospital files.

Five-micrometer-thick sections were cut from Formalin-fixed paraffin-embedded tissue blocks and stained with hematoxylin and eosin for histopathological examination and determination of tumor type, grade, variant, the extent of invasion, presence or absence of associated carcinoma in situ, bilharziasis, vascular and perineural invasion, and status of dissected lymph nodes in the radical cystectomy specimens.

Immunohistochemical staining was performed using immunostainer (Shandon Sequenza) using the labeled streptavidin biotin method with the following reagents: Citrate buffer: 10X concentrate, pretreatment antigen–retrieval (Spring Bioscience, Catalog number: DCB-125), Hydrogen peroxide block (Lab vision, USA, Catalog number: TA-060-HP), Ultravision large volume detection system (Lab vision, USA, Catalog number: TP-060- HL) including Ultra V block, Biotinylated goat anti-polyvalent plus (link) & Streptavidin peroxidase plus (label), and DAB plus substrate system (Lab vision, USA, Catalog number: TA-060-HDX) including DAB plus chromogen & DAB plus substrate. The primary antibodies were ALDH1A1: rabbit polyclonal antibody (Gene Tex Catalog number: GTX123973), supplied as a vial (0.1 ml), concentrated to be diluted with a diluent (phosphate buffered saline) at a concentration of 1:700 and CD44: Mouse monoclonal antibody (Thermo Scientific Catalog number: MS-668-R7), supplied as a vial (7 ml), ready to use.

Sections of normal liver and breast carcinoma were used as positive control for ALDH1A1 and for CD44, respectively (Keymoosi et al., 2014; Schlossman et al., 1995). Sections of the same tissue were used following the same procedure, but the PBS was used instead of the primary antibody used as internal negative controls.

ALDH1A1 positivity was defined as cytoplasmic staining of tumor cells, while CD44 positivity was defined as membranous staining of tumor cells.

The staining intensity of antibodies were evaluated applying a semi-quantitative system, ranging from negative to strong: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong staining). The percentage of positive cells for each antibody was graded as follows: 1 (< 25% positive cells), 2 (25–50% positive cells), 3 (50–75% positive cells), and 4 (> 75% positive cells). The overall score was calculated by H-score (histochemical score) for each case by multiplying the intensity of staining by the percentage of positive cells, and a final score of 0 to 300 was given. The mean of H-scores was chosen as cut-off value to classify the samples as high or low expression (Keymoosi et al., 2014).

Data collected was revised, coded, tabulated, and introduced to a PC using statistical package for social sciences (IBM SPSS 20.0). Chi-square, Fisher’s exact tests, and Pearson correlation coefficient (r) were used. P value is significant when ≤ 0.05.

Results

Fifty-four out of our sixty cases were derived from transurethral resection of bladder tumors (TURBT) and six from radical cystectomies.

The mean age of patients was 58.53, and the age ranged between 24 and 83 years with a standard deviation of 12.62 years. Fifty patients were males (83.3%), and ten patients were females (16.7%).

Regarding immunohistochemical results, there was only a statistically significant relationship between the presence of bilharziasis and expression of ALDH1 (P = 0.020). Nine cases (81.8%) out of 11 bilharziasis-associated urothelial carcinomas showed ALDH1 high expression (Fig. 1a). There was no statistically significant relationship between CD44 expression and presence of bilharziasis.

Fig. 1
figure 1

(a) High ALDH1expression in a case of infiltrating urothelial carcinoma associated with bilharzias ova (arrow) (ALDH IHC, × 200). (b) A focus of carcinoma in situ in a case of infiltrating urothelial carcinoma showing high ALDH1 expression (ALDH IHC, × 400). (c) High ALDH1expression in a case of infiltrating urothelial carcinoma with focal squamous differentiation (arrow) (ALDH IHC, × 400). (d) Low ALDH1 expression in a case of nested variant of urothelial carcinoma (ALDH IHC, × 200)

Full size image

There was no statistically significant relationship between ALDH1 or CD44 expression and age, gender, tumor grade, histological variants of urothelial carcinoma, depth of invasion (T), presence of carcinoma in situ in adjacent mucosa, lymphovascular invasion, lymph node involvement, and tumor recurrence.

Also, there was no statistically significant relationship between ALDH1 and CD44 expression or between combined ALDH1 and CD44 expression and all clinicopathological parameters (Table 1).

Table 1 Clinicopathological parameter among cases
Full size table

Tumor recurrence was detected in 14 cases (23.3%). Four of them had the same grade and stage of the primary tumor, while the remaining ten cases showed progression in grade and/or depth of invasion (T).

Among recurrent cases, eight cases showed high ALDH1 expression, and six showed low ALDH1 expression. Seven recurrences showed high CD44 expression, and the other seven showed low CD44 expression.

Five of seven of primary tumors with low ALDH1 expression showed high ALDH1 expression within the recurrent tumor, and the other two cases remained of low expression. Four out of these five cases (80%) had progression in grade and/or depth of invasion in the recurrent tumor. Regarding CD44, four of eight of primary tumors with low CD44 expression showed high CD44 expression within the recurrent tumor, and the other four cases remained of low expression. Three out of these four cases (75%) had progression in grade and/or depth of invasion in the recurrent tumor.

Discussion

Bladder cancer is the 10th most common form of cancer worldwide (Bray et al., 2018). In Egypt, it is the second most common malignancy after liver cancer among Egyptian males (Ibrahim et al., 2014).

Cancer stem cells (CSCs) are subpopulation of cancer cells that are distinct from normal stem cells and bulk cancer cells in terms of tumorigenic and self-renewal capacities and are responsible for tumor initiation, differentiation, recurrence, metastasis, and drug resistance (Jinesh et al., 2014).

The target of conventional anticancer treatment is mature cancer cells, which account for most cancer cells. Although CSCs only constitute a small proportion of cancer cells, they have strong vitality and natural resistance to conventional therapies, which often leads to treatment failure. The discovery of therapies targeting CSCs will achieve better treatment effects at a lower cost (Liu et al., 2015).

The expression of ALDH genes, especially ALDH1, has been reported to be associated with UCSCs with a worse prognosis in a large number of previous studies (Kitamura et al., 2013 and Shen et al., 2015).

CD44 has been applied individually or in combination with other putative CSC markers to isolate CSCs in various solid tumors (Jaggupilli and Elkord, 2012).

In the current study, the expression of ALDH1 among UC cases was low in 30 cases (50%) and high in 30 cases (50%). These findings are in contrary to those of Su et al. (2010), of Kitamura et al. (2013), and of Liu et al. (2015) who revealed higher number of low than high ALDH1 expression cases, but all these studies had larger sample size and used monoclonal antibody, while we used polyclonal one. Moreover, all these studies used different scoring systems; both Kitamura et al. and Liu et al. considered at least one ALDH1-positive cell as being positive. Su et al. used a cutoff value of 10% to determine whether a tumor had low or high ALDH1A1 expression.

There was only a statistically significant relationship between the presence of bilharziasis and expression of ALDH1 (P = 0.020). This suggests that ALDH1 may play a role in carcinogenesis of urinary bladder urothelial carcinoma as it is related to the presence of bilharziasis which is a known risk factor. Bilharziasis or schistosomiasis is a snail-borne poverty-related disease caused by blood-dwelling trematode worms. It is Schistosoma haematobium, causing urogenital schistosomiasis that with continuous infection and reinfection through many years can lead to a state of chronic inflammation which results in nitric-oxide-mediated DNA alterations which may lead to genetic instabilities and potential malignant transformation. Inflammation and passage of parasite eggs through the tissue result in repeated tissue damage which lead to restorative hyperplasia of the damaged tissue. This may promote the propagation of cells in which genotoxic DNA damage has been completed.

Although there was no relationship between ALDH1 expression and other clinicopathological parameters, it is worth to be mentioned that 75% of urothelial carcinoma with differentiation or special variant show low ALDH1 expression (Fig. 1 c and d); this suggests relationship between urothelial carcinoma with differentiation or special variant and low ALDH1 expression but not at significant level, perhaps due to small number of cases.

Su et al. (2010) found that ALDH1 expression was significantly associated with tumor recurrence and high-grade carcinoma, while Keymoosi et al. (2014) found significant correlation between ALDH1expression and high-grade carcinoma, depth of tumor invasion, lymph node metastasis, and lymphovascular invasion. Liu et al. (2015) also found significant correlation between ALDH1expression and high-grade carcinoma and depth of tumor invasion. El-Dien et al. (2017) detected significant relation with depth of invasion.

Senol et al. (2015) detected that ALDH1 expression was significantly associated with presence of concomitant carcinoma in situ (Fig. 1b) and tumor recurrence.

Contrasting results may be explained by several factors: (1) different antibodies used for the analysis, (2) different criteria used to classify positive staining and different cutoff used to discriminate high and low expression tumors, (3) different treatment modalities done before sample collection, and (4) non-homogeneity of patient selection criteria.

Keymoosi et al. (2014), Su et al. (2010), Kim et al. (2013), and Senol et al. (2015) reported that overexpression of ALDH1 was positively associated with prognostic factors of bladder cancers indicating that ALDH1 could be applied as a prognostic marker in urothelial carcinomas.

Regarding the expression of CD44 in UC cases, the present study revealed low expression in 32 cases (53.3%) and high expression in 28 cases (46.7%) (Fig. 2). These findings are similar to that of Keymoosi et al. (2014) but in contrary to that of El-Dien et al. (2017).

Fig. 2
figure 2

(a) Low CD44 expression in a case of low-grade urothelial carcinoma, T1 (CD44 IHC, × 400). (b) High CD44 expression in a case of infiltrating high-grade urothelial carcinoma, T2 (CD44 IHC, × 400)

Full size image

There was no relationship between CD44 expression and any of the clinicopathological parameters.

Omran and Ata (2012) found that CD44 expression is significantly associated with depth of invasion. Omar et al. (2015) reported significant relationship with tumor grade, depth of invasion, and vascular invasion.

El-Dien et al. (2017) reported that higher CD44 expression was associated with bilharziasis, while Wasfy and El-Guindy (2017) reported significant relationship with both tumor grade and depth of invasion.

It is to be noted that the antibody used in this study detects the standard isoform of CD44 (CD44s). Many isoforms are generated out of alternative splicing of CD44 during oncogenic signaling. The presence of many isoforms of CD44 undoubtedly contributes to some of the variability in research (Basakran, 2015).

In the current study, the pattern of CD44s staining in normal urothelium was membranous and slightly cytoplasmic, with the maximum intensity in the basal cell layers. The extent of staining decreased in a step-wise manner towards the surface, leaving the upper part of the intermediate and the most superficial layers of urothelium with highly differentiated umbrella cells entirely negative for CD44; this agrees with Kuncova et al. (2007) and Salih et al. (2016).

Gadalla et al. (2004) reported that there was markedly less expression of CD44 in invasive UC than in normal and pre-invasive carcinomas. Erdogan et al. (2008) reported that higher CD44 expression was revealed in low-grade and noninvasive tumors. And Salih et al. (2016) documented that higher CD44 expression was revealed in low-grade and noninvasive tumors. All of these findings are supporting evidence indicating involvement of CD44 in the tumorogenesis of UC.

There was no significant relationship between combined expression of ALDH1 and CD44 proteins among studied cases (P = 0.796). This is in contrary to the significant correlation found by Keymoosi et al. (2014) from combined analysis of both ALDH1 and CD44 that showed 30% of urothelial carcinomas displayed the ALDH1+/CD44+ phenotype, whereas 58% of cases were ALDH1−/CD44+ phenotype, and only 3% tumors expressed ALDH1+/CD44− phenotype. This finding suggests that the ALDH1+ population is a subset of the CD44+ bladder cancer cells. Liu et al. (2015) concluded that only a proportion of the CD44-positive cells express ALDH1, while most ALDH1-positive cells express CD44, thereby indicating that ALDH1 has higher specificity as a CSC marker as compared to CD44.

There was no correlation between combined expression of ALDH1 and CD44 and the clinicopathological parameters of UC cases. No data was detected in literature discussing this point to compare with.

It is worth to be mentioned that the findings among the fourteen recurrent cases suggest that increased expression of either ALDH1 or CD44 within the recurrent tumor may be associated with progression in grade or pathological T stage of tumor or both.

Referring to the current study and the related previous researches, diverse conclusions have been achieved, but many related results were detected. Most conclusions documented that in a way or another, ALDH1 and CD44 stem cell marker expression is related to at least one clinicopathological parameter of urinary bladder urothelial carcinoma, and so they could be considered as prognostic markers. This topic is worthy to be further evaluated and studied on a larger number of patients as it has a very important role in introducing therapies targeting CSCs and that will achieve better treatment effects at a lower cost and may prevent morbidities and mortalities caused by invasive treatment.