|Year : 2017 | Volume
| Issue : 1 | Page : 174-183
Evaluation of the role of CD44 as a cancer stem cell marker in colorectal carcinoma: immunohistochemical study
Nanis S Holah1, Hayam A Aiad1, Nancy Y Asaad1, Enas A. B. Elkhouly2, Ayat G Lasheen MSc 1
1 Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El Kom, Egypt
2 Department of Oncology, Faculty of Medicine, Menoufia University, Shebin El Kom, Egypt
|Date of Submission||07-Mar-2016|
|Date of Acceptance||13-Apr-2016|
|Date of Web Publication||25-Jul-2017|
Ayat G Lasheen
Betket El-Sabae, Menoufia, 32651
Source of Support: None, Conflict of Interest: None
The aim of this work was to study the immunohistochemical expression of CD44 in colorectal carcinoma (CRC) cases.
Cancer stem cells (CSCs) have a role in cancer initiation, progression, and metastasis of CRC through their ability of self-renewal and unlimited proliferation, and they seem to be responsible for local relapse and metastasis. Several markers for CSCs have been investigated in CRC, and CD44 was the most likely marker for colorectal CSCs.
Materials and methods
This retrospective study included 71 colorectal specimens (49 CRC, 13 adenoma, and nine normal cases). The studied cases were collected from the Pathology Department, Faculty of Medicine, Menoufia University, during the period from 2011 to 2015. All cases were stained with CD44 antibody. Survival data were available for 31 of 49 studied CRC cases.
All cases of normal colonic epithelium and normal colon adjacent to CRC showed no membranous CD44 immunoreactivity; 84.6% of the studied adenoma cases showed no expression of CD44, whereas 57.1% of CRC cases showed positive expression. There was a statistically significant association between positive epithelial expression of CD44 and left-sided tumors (P = 0.01). There was a statistically significant difference between CRC and colorectal adenoma (P = 0.007) and between CRC and normal cases (P = 0.000) as regards CD44 expression. There was a highly statistically significant association between stromal expression of CD44 and absence of lymph node invasion (P = 0.002) as well as early Dukes' stage (P = 0.01).
CD44 may be involved in the pathogenesis of CRC. Stromal expression only of CD44 is associated with better prognostic factors.
Keywords: CD44 immunostaining, colorectal carcinoma, prognosis, stem cells
|How to cite this article:|
Holah NS, Aiad HA, Asaad NY, Elkhouly EA, Lasheen AG. Evaluation of the role of CD44 as a cancer stem cell marker in colorectal carcinoma: immunohistochemical study. Menoufia Med J 2017;30:174-83
|How to cite this URL:|
Holah NS, Aiad HA, Asaad NY, Elkhouly EA, Lasheen AG. Evaluation of the role of CD44 as a cancer stem cell marker in colorectal carcinoma: immunohistochemical study. Menoufia Med J [serial online] 2017 [cited 2019 Aug 20];30:174-83. Available from: http://www.mmj.eg.net/text.asp?2017/30/1/174/211483
| Introduction|| |
Colorectal carcinoma (CRC) is the third most prevalent type of cancer in the world with the disease incidence rising with advanced age . Each year, CRC is diagnosed in more than 940 000 individuals, and 500 000 individuals die from the disease .
The overall mortality from CRC is 60%, which represents the second leading cause of cancer-related death in western societies .
In Egypt, the incidence of CRC is 2.27% for colon carcinoma and 2.08% for rectal carcinoma .
CD44 is a cell adhesion molecule and a hyaluronic acid receptor, which was considered as a cancer stem cell (CSC) marker. CD44 is known to be involved in cell growth, differentiation, and survival . As an important adhesion molecule, CD44 plays a major role in cancer cell migration, being associated with tumor initiation in xenografts and colony formation, as well as tumor stage, lymph node infiltration, prognosis, and survival. CD44+ cells had CSC properties, meaning that a single cell could self-renew, differentiate, and form a xenograft tumor similar to the original lesion .
There is accumulating evidence that CD44 is involved in the initiation and progression of intestinal tumors and the development of metastasis . In addition, prominent expression of CD44 is a hallmark of highly tumorigenic CRC cells . Accordingly, it was demonstrated that CD44 is a part of an intestinal stem cell gene signature that predicts disease relapse in CRC patients . Several isoforms of CD44 were detected in CRC both using Western blotting and reverse transcription-PCR. CD44 is important for maintaining the properties of CSC and cancer initiation using RNA interference experiments . Downregulation of CD44 inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells . More recently, Dallas et al.  reported that downregulation of CD44 leads to an increase in the metastatic and migratory potential of CRC cells. This is due to alternative splicing of the CD44 pre-RNA . It was observed that high-grade CRC has higher CD44 expression levels compared with low-grade tumors, and this overexpression was associated with reduced patient survival . In contrast, Ylagan et al.  reported that the loss, rather than an increased expression of CD44, is associated with increased tumor aggressiveness. Fernández et al.  demonstrated that CD44 expression levels were related to proliferation in CRC, but not to patient outcome.
From the above data, the expression of CD44 in CRC is still controversial.
The aim of the present study was to evaluate the role of immunohistochemical expression of CD44 in CRC cases and its relationship with clinicopathological parameters and patients' outcome.
| Materials and Methods|| |
This retrospective study included 71 cases divided into 49 CRC, 13 adenoma, and nine normal cases (obtained from cases diagnosed as Hirschsprung disease). The cases were collected from the Pathology Department, Faculty of Medicine, Menoufia University, during the period from 2011 to 2015. The clinicopathological data were collected from patients' records, and survival data were available for 31 of 49 of the studied CRC cases. Ethical approval was obtained from Institutional Review Board.
The hematoxylin and eosin-stained sections were evaluated for histological type, according to Hamilton et al. . Tumor grade was determined based on gland formation, either low grade (≥50% gland formation) or high grade (<50% gland formation) . The mitotic figures in tumor cells were counted in 10 randomly selected high-power fields . Similarly, apoptosis was counted in 10 high-power fields . Lymphovascular invasion was assessed . Pathologic staging of the tumor was performed according to TNM American Joint Committee on Cancer-Union International Center , as well as according to modified Dukes' staging system .
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded material that was sectioned at 4 μm thickness and placed onto positively charged slides. The primary antibody was Rabbit monoclonal antibody against CD44 (anti-CD44 antibody with a dilution of 1: 200, overnight at 4°C) (Thermo Fisher Scientific, Fremont, California, USA). Negative control slide was included in each run by omitting the primary antibody. Positive control slide of normal tonsil was also included in each run. All slides were deparaffinized using xylene and then rehydrated in decreasing concentrations of ethanol. Inhibition of endogenous peroxidase activity, using 3% hydrogen peroxidase for 15 min, was carried out. Antigen retrieval was carried out using microwave heating (20 min; 10 mmol/citrate buffer, pH 6.0). The primary antibody was applied to the slides. The slides were incubated overnight with the primary antibody at room temperature, washed using PBS, and then incubated with secondary antibody for 15 min followed by PBS wash. Finally, the detection of bound antibody was accomplished using a modified labeled avidin–biotin reagent for 20 min, and then PBS wash was carried out. A 0.1% solution of diaminobenzidine was use for 5 min as a chromogen. The slides were counterstained with Mayer's hematoxylin for 5–10 min. The method used for immunostaining was streptavidin–biotin amplified system.
Interpretation of immunohistochemical staining
Positive or negative expression was evaluated according to the presence or absence of membranous expression of CD44 .
H score was used. The intensity (I) of the staining was scored as follows: '0', no reaction; '1', weak reaction; '2', moderate reaction; and '3', strong reaction. The extent of the signal was scored as percentage of positive cells (P). Overall staining score was calculated by multiplying the intensity with the percentage of positive cells (score=P × I; maximum=300) .
Stromal expression of CD44 was positive if more than one-third of the stroma showed membranous CD44 expression and considered negative if less than one-third of the stroma showed membranous CD44 expression .
On revision of patients' files for CRC cases from 2011 to 2015, overall survival and distant recurrence-free survival times were available for 31 of 49 (63.3%) patients.
Data were collected, tabulated, and statistically analyzed using statistical package for the social sciences program for Windows, version 21 (SPSS; SPSS Inc., Chicago, Illinois, USA). Data were presented as mean ± SD and percentages. The χ2-test, Fisher's exact test (F), Kruskal–Wallis test (K), Mann–Whitney test (U), and Kaplan–Meier test were used. All P values were two-sided; P values of 0.05 or less were considered statistically significant and highly significant when P value was 0.01 or less .
| Results|| |
The clinicopathological data of the studied colorectal adenoma and carcinoma cases are demonstrated in [Table 1] and [Table 2], respectively.
|Table 2 Clinicopathological data of the studied colorectal carcinoma cases|
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The present study revealed that all cases of normal colonic epithelium [Figure 1] and the normal mucosa adjacent to CRC cases showed negative CD44 immunoreactivity. There was a highly statistically significant difference between studied CRC cases and both normal colonic mucosa and normal mucosa adjacent to CRC cases as regards CD44 expression (P = 0.002 and <0.000, respectively) [Table 3] and [Table 4], respectively).
|Figure 1: (a) A case of normal non-neoplastic colonic epithelium showed negative CD44 immunoreactivity. (IHC. × 200). (b) A case of adenoma showed weak expression of CD44. (IHC. ×400). (c) A case of colorectal carcinoma showed mild membranous expression of CD44. (IHC. ×400). (d) A case of colorectal carcinoma showed moderate membranous expression of CD44. (IHC. ×400). (e) A case of colorectal carcinoma showed strong membranous expression of CD44. (IHC. ×400), and. (f) A case of colorectal carcinoma showed positive membranous expression of CD44 in stroma, whereas negative membranous expression in tumor cells. (IHC. ×400). IHC, immunohistochemical staining.|
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|Table 3 Comparison between immunohistochemical expression of CD44 in studied normal non-neoplastic and colorectal carcinoma cases|
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|Table 4 Relationship between immunohistochemical expression of CD44 in studied colorectal carcinoma cases and adjacent normal mucosa|
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Furthermore, 84.6% of colorectal adenoma cases were negative for CD44 and only two (15.4%) cases showed positive membranous expression of CD44 [Figure 1]. There was a highly statistically significant difference between studied adenoma and CRC cases as regards CD44 expression (P = 0.007) [Table 5].
|Table 5 Comparison between immunohistochemical expression of CD44 in studied adenoma and colorectal carcinoma cases|
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The current study revealed that 57.1% of the studied CRC cases showed positive epithelial expression of membranous CD44 in tumor cells [Figure 1]. Total of 63.27% of cases showed positive expression in the stroma [Figure 1]. There was a statistically significant association between positive epithelial expression of CD44 and left-sided tumors (P = 0.013) [Table 6]. Moreover, there was a highly statistically significant association between stromal expression of CD44 and absence of lymph node invasion (P = 0.002) as well as early Dukes' stage (P = 0.02) [Table 7].
|Table 6 Relationship between immunohistochemical expression of CD44 and clinicopathologic factors in studied colorectal carcinoma cases|
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|Table 7 Relationship between stromal expression of CD44 and clinicopathologic factors in studied colorectal carcinoma cases|
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On revision of patients' files, survival data were available for 31 of 49 studied CRC cases. The range of survival time was 2–40 months with a mean ± SD of 15.8 + 10.1 months and a median of 14 months. Nine of 31 CRC cases died because of the disease. Univariate survival analysis for studied CRC cases revealed no statistical significance between overall survival and CD44 expression, nor any of the clinicopathological data. There was a statistically significant association between distant recurrence-free survival and male sex (P = 0.02) [Figure 2] as well as lower grade (P = 0.03) [Figure 2] and [Table 8].
|Table 8 Univariate distant recurrence-free survival analysis for studied colorectal carcinoma cases|
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|Figure 2: (a) Kaplan–Meier distant recurrent free survival for CRC patients with different sexes (P = 0.02 'significant', log rank = 5.12). (b) Hazard function curve of distant recurrent free survival for CRC patients with different sexes indicating that female patients were more hazardous. (c) Kaplan–Meier distant recurrent free survival for CRC patients with different grades (P = 0.03 'significant', log rank = 6.83). and (d) hazard function curve of distant recurrent free survival for CRC patients with different grades indicating that patients had high grade were more hazardous. CRC, colorectal carcinoma.|
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However, multivariate Cox regression analysis revealed that sex was the most independent prognostic factor affecting patients' distant recurrence-free survival (P = 0.04) [Table 9].
|Table 9 Multivariate Cox regression analysis for the detection of the independent prognostic factor affecting patients' distant recurrence free survival|
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| Discussion|| |
CSCs constitute a small percentage of the tumor population and can be defined by their characteristics. Therefore, separating these CSCs from the entire tumor population is an essential step for studying them . Several markers for CSCs have been investigated and CD44 has been thought to be the most likely marker in CRC . CD44 is a transmembrane glycoprotein, which was used for the isolation of mesenchymal stem cells from bone marrow  and is associated with cell–matrix and cell–cell interactions . CD44 expression was shown to be relevant to tumor progression in various types of human cancers . CD44 had long been thought of as a marker of tumor invasiveness and metastasis and had also been described as a putative colorectal CSC marker . CD44 upregulation had been shown to be an earlier marker of neoplastic transformation of colonic epithelium . However, CD44 downregulation in the metastatic phase of CRC had been demonstrated . Some previous studies in the colon found that CD44 pattern of staining was mainly membranous ,, and this was similar to that reported in the present study.
In the present study, there was no expression of CD44 in normal colonic epithelium, and this is similar to the findings of Liu et al. . Moreover, there was a highly significant difference between carcinoma cases and normal cases as regards CD44 expression (P = 0.002). This result is similar to the findings of Chai et al. , and this might be attributed to the fact that normal colonic tissue did not express variant isoforms of CD44, whereas tumor cells seemed to express a wide variety of CD44 isoforms . However, this result is in disagreement with that of Huang et al. , who found that larger subpopulation within normal colonic tissues showed positive CD44 expression because CD44 is normally expressed in the lower two-thirds of colonic crypts . This difference might be attributed to different techniques used, as that study used flowcytometry and Aldefluor kits, whereas our study used immunohistochemistry only.
In the present study there was a highly significant difference between carcinoma cases and adjacent normal mucosa as regards CD44 expression (P < 0.000). This result is similar to that of Liu et al. , who found that CD44+ cells were not present in the normal intestinal mucosa adjacent to colorectal tumors and this might be attributed to the fact that normal colonic tissue did not express variant isoforms of CD44, whereas tumor cells seemed to express a wide variety of CD44 isoforms .
The current study showed that only two (15.4%) adenoma cases showed positive membranous expression of CD44, and this is in agreement with the findings of Imazeki et al. , who also found that adenomatous colorectal polyps showed membranous expression of CD44 and attributed it to the fact that variant forms of CD44− mRNA might be expressed in the early stage of colorectal carcinogenesis.
In the current study we observed a statistically significant difference between adenoma and carcinoma cases with regard to CD44 epithelial expression. This result is similar to that of Choi et al. , who found a significant difference between adenoma and gall bladder carcinoma cases as regards CD44 expression.
Moreover, the present study showed that 57.1% of CRC cases had positive CD44 expression compared with 51% reported by Lugli et al. , and 70% reported by Chun et al. . This might be attributed to the fact that CD44 is implicated in the carcinogenesis of CRC as it is essential for stemness maintenance of colorectal CSCs as it is involved in the activation of the tyrosine kinase receptor c-Met, which is involved in the regulation of proliferation, motility, invasion, and metastasis .
CD44 showed no significant relationship, neither with grade nor with stage; this result is in agreement with that of Chen et al. . In contrast, Zhao et al.  found a significant association between CD44 expression and stage II and III. This difference might be due to the low number of CRC cases (49 case) compared with 187 cases in the study by Zhao.
Furthermore, the current study showed a statistically significant association between positive expression of CD44 and left-sided tumors (P = 0.013). This result is in agreement with the findings of Michl et al. . The left side localization of CRC was said to carry a better prognosis compared with the right side . The latter authors attributed that to the differences in embryologic origin, genetic and environmental factors, and fecal exposure as well as the difference in time to detection. In contrast, Lugli et al. , found that loss of expression of CD44 was associated with left-sided tumor.
H score of CD44 had a statistically significant association with older age (P = 0.04). This result is in disagreement with that of Hong et al. , Liu et al. , and Chen et al. , who found that there was no significant relationship between CD44 and age of studied CRC patients.
The differences in our results compared with others might be due to the presence of numerous CD44 isoforms, which might have remarkable homology in their antigenic repertoire , increasing the possibility of cross-reactivity between the antibodies and the studied sample size.
These current results suggest the upregulation of CD44 had favorable prognosis. This result is similar to that of Ahmed et al. , who found that CD44 expression was associated with favorable prognostic criteria in studied breast carcinoma cases. Moreover, several previous studies showed that CD44 expression was associated with good prognosis of CRC ,,.
In the current study, there was a highly statistically significant association between stromal expression of CD44 and absence of lymph node invasion (P = 0.002) as well as early Dukes' stage (P = 0.01). Similarly, Furuta et al. , found that the stromal expression was associated with good prognostic factors. The presence of CD44 extracellular molecule that retained the hyaluronate binding domains and occupied the sites to which the cancer cells would attach could facilitate cancer cell attachment and prevent the process of metastasis . A similar effect of CD44 expression in tumor cells and stroma may be attributed to tumor-stroma cross talk.
Survival analysis was performed for CD44 expression and there was no significant association between CD44 and overall survival. This result was similar to that of Galizia et al. . In contrast, Huh et al.  found that CD44 overexpression was associated with poor prognosis.
Increased expression of CD44 in carcinoma compared with adenoma and normal colonic tissue may indicate the involvement of CD44 in the pathogenesis of CRC. Expression of CD44 in stromal expression of CD44 is associated with the absence of lymph node invasion and earlier Dukes' stage. This might suggest that stromal expression only of CD44 is associated with better prognostic factors.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]