Clinicopathological significance and prognostic value of EphA3 and CD133 expression in colorectal carcinoma
- Correspondence to Dr Po Zhao, Department of Pathology, Chinese People's Liberation Army General Hospital, 28 Fuxing Road, Beijing 100853, China;
Contributors QX and PZ performed the research and wrote the manuscript; XQ and ZP designed the research and was also involved in editing the manuscript.
- Accepted 22 February 2011
- Published Online First 17 March 2011
Aims To investigate clinicopathological significance and prognostic implications of EphA3, CD133 and Ki-67 expression in colorectal cancer.
Methods EphA3, CD133 and Ki-67 expression was assessed in 201 cases of paraffin-embedded colorectal carcinoma and 60 cases of distal normal mucosal tissue by immunohistochemistry. Medical records were reviewed and clinicopathological analysis was performed. The differential expression of EphA3 and CD133 protein was detected in 20 cases of fresh resected colorectal carcinoma and 20 cases of matched normal mucosal tissue adjacent to the carcinoma by western blot.
Results The expression of EphA3 and CD133 in carcinoma was significantly higher than that in normal mucosal tissue (p=0.008; p=0.004). EphA3 and CD133 were positively correlated with tumour size (p=0.029; p=0.017), histological grade (all p=0.001), infiltrative depth (all p=0.00), lymph node metastasis (all p=0.00), distant metastasis (p=0.017; p=0.030) and TNM stage (all p=0.001). Patients with high expression of EphA3 and CD133 had the lowest survival (all p=0.001) (median survival time of EphA3 positive and negative cases: 34.0 and 72.0 months; median survival time of CD133 positive and negative cases: 34.0 and 77.0 months). Multivariate survival analysis showed that EphA3 and CD133 expression was correlated significantly with shortened survival in patients with colorectal cancer (Cox regression: p=0.001, HR=4.722, 95% CI 2.667 to 8.361; p=0.001, HR=5.224, 95% CI 2.622 to 10.405). EphA3, CD133 and Ki-67 expression in colorectal cancer had positive correlations with each other (all p=0.001).
Conclusions EphA3 and CD133 may play an important role in the development and progression of tumours, and thus become useful indicators for clinical assessment of tumour biological behaviour and prognosis in patients with colorectal carcinoma.
Eph receptors (Ephs) are the largest family of receptor tyrosine kinases (RTK).1 2 They are divided into two groups, EphAs and EphBs.3 Ephs plays an important role in the development of nervous and vascular systems, and may be involved in carcinogenesis, and progression and prognosis of certain cancers. EphA3 is a member of the Eph family of receptors.4 Some studies have shown that the expression of EphA3 is associated with B and T cell malignant neoplasms.5–7 Some recent studies have indicated that EphA3 is overexpressed in renal carcinoma, hepatic carcinoma, melanoma and sarcoma.8 However, the expression or function of EphA3 in solid tumours has not been widely studied.
CD133 (also called prominin-1) is a transmembrane glycoprotein, with a molecular weight of 120 kDa.9 10 It was initially considered to be a marker of haematopoietic stem cells. Resent research suggests that CD133 is one of the most important cancer stem cell markers.11–14 It is overexpressed in various solid tumours,13 15–18 including retinoblastoma,9 teratocarcinoma,9 leukaemia,9 glioblastoma,11 colorectal carcinoma12 and gastric carcinoma.19 CD133 expression has been found in colorectal cancer as an independent prognostic marker for overall survival.20 21 Ki-67 is a well recognised nuclear antigen-specific marker of cellular proliferation and is mainly used to evaluate the activity of proliferation. The relationship between EphA3, CD133 and Ki-67 in colorectal cancer has not yet been investigated.
Colorectal cancer is a major cause of cancer-related morbidity and mortality.9 22 In the western world it is the second leading cause of death from cancer.22 The incidence of colorectal cancer has recently increased in China.23 Therefore, it is important assess its prognosis according to the expression of some new molecular markers. In this study, we analysed the association of EphA3, CD133 and Ki-67 expression in colorectal cancer with clinicopathological features. Their inter-relationships were also evaluated. It is hoped that this study will provide further understanding of the pathogenesis of this disease.
Materials and methods
A total of 201 consecutive colorectal carcinoma patients who underwent curative surgical resection at the Chinese People's Liberation Army (PLA) General Hospital (Beijing, China) between August 1999 and September 2003, and 60 cases with distal normal mucosal tissue were recruited. Specimens obtained were routinely fixed in 10% neutral formalin and embedded in paraffin. They were obtained from 127 men and 74 women, aged 20–81 years (median 62 years; mean 57.8 years). Of these patients, 24 were grade 1, 110 grade 2 and 67 grade 3, according to histological grading; and 48 were stage I, 72 were stage II, 70 were stage III and 11 were stage IV, according to clinical TNM stage revised by the International Union Against Cancer (UICC) in 2009. All patients were followed up for survival. The follow-up period was defined from the date of surgery to 30 April 2009, during which 126 patients were dead and 75 were alive (median survival time 51 months, range 0.10–115 months).
Twenty cases of fresh resected colorectal carcinoma and 20 cases of matched normal mucosal tissue adjacent to the carcinoma were collected from patients who were treated surgically at the PLA General Hospital in May 2009, snap frozen and stored at −80°C for further use.
All samples were fixed in 10% neutral formalin. Sections (4 μM thick) were cut from wax blocks mounted on to APES-coated glass slides. Slides were dewaxed in xylene twice for 10 min and rehydrated in a graded ethanol series. Antigen retrieval was performed in 0.01 mol/l citrate buffer (pH 6.0) by microwave oven for 2 min and 30 s at 100°C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. After washing with phosphate-buffered saline (PBS), sections with primary polyclonal rabbit antibody to human EphA3 (Santa Cruz, California, USA) diluted 1:400 in blocking solution, polyclonal rabbit antibody to human CD133 (Abcam, Cambridge, Massachusetts, USA) diluted 1:200 in blocking solution, and mouse monoclonal antibody to human Ki-67 (Zymed Laboratories, San Francisco, California, USA) diluted 1:100 in blocking solution were incubated at 4°C overnight, in a humidified chamber. After washing three times with PBS, sections were incubated for 30 min with biotinylated secondary antibody (polyperoxidase-anti-mouse/rabbit IgG, Zymed). After washing in PBS, 3,3′-diaminobenzidine (DAB) was used as the chromogen. Slides were counterstained with haematoxylin for 1 min. Normal human kidney was used as a positive control; the primary antibody was replaced by PBS as a negative control.
Evaluation of score
Immunohistochemically stained slides were reviewed by two investigators independently who were blinded to all clinical data. Slides were analysed under light microscopy using manual methods. In scoring expression of EphA3, CD133 and Ki-67 protein, both the extent and intensity of immunopositivity were considered, according to Zhao et al19 and Hao et al.24 The intensity of positivity was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong. The extent of positivity was scored according to the percentage of cells showing positive staining: 0, <5%; 1, >5–25%; 2, >25–50%; 3, >50–75%; 4, >75% of the cells in the respective lesions. The final score was determined by multiplying the intensity of positivity and the extent of positivity scores, yielding a range from 0 to 12. The expression for EphA3, CD133 and Ki-67 was considered positive when the score was ≥5.
Protein extraction was carried out by lysing colorectal carcinoma and normal mucosal tissue in 1 ml homogenisation buffer (NaCl 11.7 g/l, MgCl26H2O 1 g/l, EDTA 0.76 g/l, KCl 0.37 g/l, Tris 24.2 g/l, pH 7.4) using homogeniser on ice. The homogenates underwent 20 s sonication three times and centrifugation at 12 000 r/min for 10 min at 4°C. The protein content of the supernatant was measured using a standard method. The protein samples were loaded as 50 μg/lane in 12% SDS-PAGE, separated by electrophoresis, and transferred to a nitrocellulose membrane (Amersham Biosciences). Membranes were blocked with 5% non-fat milk in TBST (50 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl, 0.1% Tween 20) for 1 h at room temperature and then incubated with primary antibody (anti-EphA3, 1:200, β-actin, 1:1000, Santa Cruz Biotechnology, USA), anti-CD133, 1:200 (Abcam) in blocking buffer at 4°C overnight. After washing three times with TBST, membranes were incubated with horseradish peroxidase-coupled goat anti-rabbit secondary antibody (1:2000; Santa Cruz Biotechnology, USA) for 2 h at room temperature. Enhanced chemiluminescence was used for visualisation, according to the manufacturer's protocol. The β-actin band was used as loading control. Protein quantity was analysed by LabWork software. The target protein expression was evaluated by the relative intensity ratio of target protein/loading control.
SPSS V.13.0 was used for statistical analysis. The χ2 test was used to examine the various clinicopathological characteristics with the expression of EphA3, CD133 and Ki-67. The Spearman correlation coefficient test was used to assess the association between EphA3, CD133 and Ki-67 expression. Cumulative survival curves were drawn by the Kaplan–Meier method. The difference between the curves was analysed by the log-rank test. Multivariate survival analysis was based on the Cox proportional hazard model. A value of p<0.05 was considered statistically significant.
Expression of EphA3 and CD133 in colorectal carcinoma and normal mucosa
EphA3 protein was expressed positively in 51.2% (103/201) of cancer tissues and 21.7% (13/60) of normal mucosal tissues. Expression of EphA3 protein was found in the cytoplasm (figure 1). CD133 protein was expressed positively in 54.7% (110/201) of cancer tissues and 25% (15/60) of normal mucosal tissues. Expression of CD133 protein was found in the membrane and cytoplasm of cancer cells (figure 2). In well differentiated carcinoma, CD133 protein was found predominantly around the lumens of cancerous glands. A significant difference was found in the expression of EphA3 or CD133 protein between colorectal cancer and normal mucosal tissues (p=0.008; p=0.004).
Association of EphA3 and CD133 expression with clinicopathological features
EphA3 and CD133 expression were present more in larger tumours (p=0.029, p=0.017). Significant positive correlations were found between EphA3 expression and depth of invasion (p=0.001) and lymph node metastasis (p=0.001). EphA3 and CD133 expression positively correlated with histological grade (p=0.001), depth of invasion (p=0.001), lymph node metastasis (p=0.001) and distant metastasis (p=0.017, p=0.003). An apparent relationship of EphA3 and CD133 with TNM stage was observed; their expression was gradually increased with the progression of TNM stage (χ2=60.763, p=0.001; χ2=52.944, p=0.001) (table 1).
Prognostic implication of EphA3 and CD133 expression in colorectal cancer
All 201 patients were followed up for survival to assess EphA3 and CD133 expression. Kaplan–Meier survival curves and the log-rank test showed that EphA3 positive cases had a significantly shorter median survival time (34.0 months) than EphA3 negative cases (72.0 months) (log rank=107.796, p=0.001) (figure 3). CD133 positive cases had a significantly shorter median survival time (34.0 months) than CD133 negative cases (77.0 months) (log rank=83.731, p=0.001) (figure 4). The overall 5-year survival rate of EphA3 negative patients (84.7%) was higher than that of the EphA3 positive group (16.5%), and that of the CD133 negative patients (82.4%) was higher than that of the CD133 positive group (22.7%). By the Cox proportional hazard model, EphA3 expression and CD133 expression were found to be significant independent prognostic factors (all p=0.001). Other independent prognostic factors included histological grade, distant metastasis, TNM stage and Ki-67 (table 2).
EphA3 and CD133 protein were positive by western blot in all specimens. Furthermore, the expression of EphA3 and CD133 in all colorectal carcinoma specimens was higher than that in matched normal mucosal tissue adjacent to the carcinoma by western blot (figure 5).
Correlations between EphA3, CD133 and Ki-67
Ki-67 was positive in 59.7% (120/201) of cases of colorectal carcinoma and was found in the nuclei of cancer cells (figure 6). It was associated with clinicopathological features such as tumour size, histological grade, invasive depth, metastasis in regional lymph nodes, distant metastasis, TNM stage and prognosis. Positive correlations between EphA3 and CD133, EphA3 and Ki-67, and CD133 and Ki-67 were found (r=0.653, p=0.001; r=0.700, p=0.001; r=0.801, p=0.001) (table 3).
The incidence of colorectal cancer has increased recently and emerged as an important public health problem in China. It has been suggested that the 5-year survival rate for colorectal cancer patients is very low.23 25–28 Countless treatment protocols, including chemotherapy and radiation, have been applied to colorectal cancer. However, complete cure of colorectal cancer has not been accomplished. Therefore, effective screening, preventive strategies and new molecular markers of prognosis for colorectal cancer are necessary to enhance our capability to predict clinical outcome of the disease.
Tyrosine kinases are important regulators of the intracellular signal-transduction pathway. Some studies have evaluated the sequences of protein kinase genes in a number of human tumours.29–32 The Ephs are the largest family of transmembrane receptor tyrosine kinases, with 14 members cloned.1 2 They play important roles in varying biological functions with their ligands. Changed expression of Ephs and their glycosyl phosphatidylinositol-linked ephins are associated with tumour vascular systems and may be involved in carcinogenesis, progression and prognosis of many types of human cancers including breast, lung and prostate cancers, melanoma and leukaemia.33 34 EphA3, which was one of the first Ephs to be cloned, was originally recognised and isolated from membranes of pre-B cell acute lymphoblastic leukaemia cells and presents in numerous haematopoietic tumours.6 Some studies have shown that the expression of EphA3 is associated with B and T cell malignant neoplasms.5–7 EphrinA5 (ligand of EphA3) stimulation decreased adhesion to fibronectin surfaces of Jurket T cells overexpressing EphA3, indicating a potentially interesting role in regulating adherence and motility of malignant T cells.35 Previous studies have indicated that EphA3 is overexpressed in renal carcinoma, hepatic carcinoma, melanoma, sarcoma8 and lung cancer.11 It has been reported that EphA3 can decrease the adhesive and migratory capacity of RMS cells.36 Some authors have identified an EphA3 gene in colorectal tumours.29 However, no report has evaluated the expression of EphA3 in colorectal carcinoma. In this study, we examined the expression of EphA3 in colorectal carcinoma and normal mucosal tissue. The expression of EphA3 positively correlated with tumour size, histological grade, depth of invasion, lymph node metastasis, distant metastasis and TNM stage. EphA3 expression was commonly up-regulated in colorectal carcinoma. EphA3 expression appeared as one of the specific molecular markers in the prognosis of colorectal carcinoma. High expression levels of EphA receptors have usually correlated with more malignant and metastatic tumours.37 38 Therefore, we propose that EphA3, a member of EphA, may display an important function in carcinogenesis and progression of colorectal cancer.
A current focus of cancer research is cancer stem cells that have been proposed to be tumour initiating cells. CD133 has been used as a marker to identify the cancer stem cell population for a variety of malignancies.12 13 15 16 Cancer neural stem cells have been identified and isolated through the CD133 marker.13 It has been reported that CD133-positive cells in colorectal cancer exhibit properties of self-renewal activity and high tumourigenic potential.11 12 However, it has also been reported that CD133 expression is not restrict to stem cells, and both CD133-positive and CD133-negative subsets of human metastatic colon cancer exhibit long-term tumourigenic potential.39 40 In this study a positive expression of CD133 was found in 54.7% patients who had a worse prognosis than CD133-negative patients, suggesting that CD133-positive cells may contain more cancer stem-like cells. Currently, the function of CD133 is unclear. The presence of five tyrosine residues on the cytoplasmic tail may indicate that the protein is phosphorylated in response to ligand binding and initiates a signal transduction cascade. Its presence on early, undifferentiated cells is suggestive of a growth factor receptor.9 Our study demonstrated that CD133 expression was commonly up-regulated in colorectal cancer. Furthermore, it was seen more frequently in advanced colorectal cancer. It is suggested that CD133 may play an important role in the development and progression of the tumour. It was also found that CD133 appeared as one significant independent prognostic factor. Ki-67 is a well recognised nuclear antigen-specific marker of cellular proliferation, and is mainly used to evaluate the activity of proliferation. It is already used in pathological diagnosis. The results concerning Ki-67 in our study were in accordance with previous studies.41 42
This research shows that the expression of EphA3 and CD133 in carcinoma was significantly higher than that in normal mucosal tissues by immunohistochemistry. This result was verified by western blot analysis. Furthermore, the expressions of EphA3, CD133 and Ki-67 were positively correlated with each other, implying higher putative proliferative activity in EphA3 and CD133-positive cancer cells. Although the detailed molecular mechanism involved in this process is unclear, our study has potentially clinical benefits. EphA3 and CD133 expression that could be detected by immunohistochemistry may be a useful molecular marker to predict prognosis. The combined detection of EphA3, CD133 and Ki-67 expression, to some extent, can reflect the biological behaviour of colorectal cancer cells, thus guiding the choice of chemotherapy and molecular targeting therapy in selected colorectal carcinoma patients.
The expression of EphA3 and CD133 in carcinoma was significantly higher than that in normal mucosal tissue. Both were positively correlated with tumour size, histological grade, infiltrative depth, lymph node metastasis, distant metastasis and TNM stage.
EphA3 and CD133 expression appeared as significant independent prognostic factors for clinical outcome in patients with colorectal cancer.
There are positive correlations between EphA3, CD133 and Ki-67.
Detecting the EphA3 and CD133 expression levels of colorectal cancer patients can be used to identify a subgroup of patients with poor outcome. EphA3 and CD133 may be useful targets for therapeutic intervention in selected colorectal carcinoma patients.
We greatly appreciate the excellent technical assistance of all the staff of the molecular pathology laboratory, Chinese People's Liberation Army General Hospital.
Competing interests None.
Ethics approval This study was conducted with the approval of the Chinese People's Liberation Army General Hospital Research Ethics Committee and informed consent was obtained according to the committee's regulations and the Declaration of Helsinki.
Provenance and peer review Not commissioned; externally peer reviewed.