Abstract
Background: Recently, two meta-analysis reports have suggested that CD133 expression in the primary tumor is significantly associated with shorter survival in colorectal cancer (CRC), and that CD133 may play an important role in CRC progression. However, the expression of CD133 in lymph node metastases as well as in primary tumors in CRC remains to be elucidated. Materials and Methods: We analyzed CD133 expression in both primary tumors and lymph node metastases in stage III CRC by immunohistochemistry, and its correlation with clinicopathological factors and outcomes. Results: Through immunohistochemistry we demonstrated that 69.6% of CRC primary tumors and 62.3% of lymph node metastases were CD133-positive. High CD133 expression in lymph node metastases was significantly associated with the number of lymph node metastases. Moreover, patients with CD133-negative staining of either primary tumor or lymph node metastases had a higher overall survival rate than those with CD133-positive staining, although this finding was not statistically significant. Conclusion: CD133-positive cancers may be more aggressive than CD133-negative ones during the process of lymph node metastasis. Further investigation of the role of CD133-positive cells in lymph node metastases in CRC is required.
Colorectal cancer (CRC) is the third most common type of cancer in men and the second most common in women. Globally, it is the fourth most common cause of cancer-related death after lung, stomach, and liver cancer (1). Besides surgery, adjuvant chemotherapy is often administered to improve survival, especially in advanced stages of the disease. Therefore, identification of risk factors for resistance to adjuvant chemotherapy may benefit patient prognosis.
The cancer stem cell (CSC) theory suggests that cancers may be hierarchically organized, with only a small population of cancer cells maintained as CSCs that can undergo both self-renewal and differentiation (2). CSCs have been purified using cell surface markers such as CD133, CD44 (3), ALDH1 (4), EpCam (5), and Lgr5 (6). Among them, CD133 is now thought to be the most robust cell surface marker for colorectal CSCs (7, 8).
The cell surface marker CD133 (also known as prominin-1) is a glycoprotein with five transmembrane loops and a molecular weight of 120 kDa. It is localized in membrane protrusions (9). In 2007, two studies revealed CD133 as a candidate marker for colorectal CSCs, though its cellular function was not clear (7, 8). CD133-positive tumor-initiating cells were able to maintain themselves, as well as differentiate and re-establish tumor heterogeneity upon serial transplantation (7). Moreover, CD133-positive cells, but not CD133-negative cells, were shown to form tumors when injected into immunodeficient mice (8). These results suggested that CD133-positive cells have a much higher capacity for initiating tumors than their CD133-negative counterparts. Following these initial studies, in 2012 and 2013, two meta-analysis reports suggested that CD133 expression is to a high degree related to shorter patient survival, and may play an important role in the progression of CRC (10, 11). Moreover, several studies have reported that CD133-positive rectal cancers might be more resistant to preoperative chemoradiotherapy (12-14). These results implied that CD133-positive cells, in not only the primary tumor, but also at the metastatic sites could contribute to CSC-induced tumor progression and chemoradio-resistance. However, to date, there have been only a few studies examining CD133 expression in metastastic sites. Therefore, in the present study, we analyzed the CD133 expression in both primary and metastatic sites for stage III CRC, as well as its correlation with clinicopathological factors and outcomes. To the best of our knowledge, this is the first study to examine CD133 expression in lymph node metastases as well as primary tumors by immunohistochemistry.
Materials and Methods
Patients and specimen. We examined 138 surgically-resected stage III colorectal adenocarcinomas collected at the Department of Surgical Oncology, University of Tokyo, from March 2000 to December 2007. Patients with the following criteria were excluded from study inclusion: receipt of radiotherapy or chemoradiotherapy before the operation, a diagnosis of multiple CRCs, a history of cancer in another organ, familial CRC, and inflammatory bowel disease. All patients were followed-up for overall survival (OS). OS was defined as the time from surgery to death or the point when the patient was last recorded to be alive. Moreover, in all cases, regular follow-up examinations were performed; abdominal and chest computed tomography was performed every 6 months and the presence of tumor markers was checked every 3 months. Each patient underwent a total colonoscopy every year to investigate colonic recurrence of cancer. All fresh specimens were fixed in 10% formalin and embedded in paraffin. The histomorphology of primary tumors and lymph nodes was confirmed by the Department of Pathology, University of Tokyo. The research had approval from the appropriate ethics committee (The University of Tokyo Hospital). Written informed consent was obtained for use of the samples in the research.
CD133 immunohistochemical staining. CD133 expression was examined both in primary tumors (pCD133) and lymph node metastases (lyCD133). A single 4-μm-thick section was used for immunohistochemical staining, as described below. Tissues were treated with xylene and ethanol, and then washed with phosphate-buffered saline (PBS). Endogenous peroxidase was blocked with 3% hydrogen peroxidase solution in methanol for 15 min. After washing with PBS, heat-induced antigen retrieval was performed in 0.01 M sodium citrate buffer at pH 6.0. Tissues were washed with PBS and incubated with 5% bovine serum albumin for 30 min to block non-specific proteins from binding to the antibody. Each slide was incubated overnight at 4°C with primary anti-CD133 antibody (AC133; Miltenyi Biotec, Auburn, CA, USA) at a dilution of 1:100. After three PBS washes, the slides were incubated with a Dako Envision Kit (Dako, Carpinteria, CA, USA) following the manufacturer's recommended protocol. Following a further three PBS washes, each slide was incubated for 3 min in 2% 3, 3’-diaminobenzidine tetrahydrochloride and 50 mM tris-buffer (pH 7.6) containing 0.3% hydrogen peroxidase as a chromogen. Mayer's/Lillie-Mayer's hematoxylin was used for counterstaining. Renal tubules were used as a positive control; as a negative control, the antibody was replaced with PBS.
Evaluation of CD133 immunostaining. The evaluation of CD133 immunostaining was performed according to the method reported by Maeda et al. (15). Slides were examined under a microscope at low power (from ×40 to ×200 magnification) to identify the region containing the highest percentage of CD133-positive cells (hot spot) in the cancer nest. Ten hot-spot fields inside the tumor tissue were selected, and CD133 expression was evaluated in 1,000 tumor cells (100 cells per field) under high-power (magnification ×400). Expression of CD133 was defined as positive when CD133 staining was found in more than 5% of the entire tumor. CD133 expression was independently evaluated by two observers who had received training in pathological diagnosis (S.K. and J.K.) and who were unaware of the clinical findings. Discrepancies between their findings were resolved by discussion. Their inter-observer agreement was calculated using κ-statistics (κ-score=0.8). The patients were classified as lyCD133-positive (lyCD133+), when at least 1 of the lymph node metastases was positive, and lyCD133-negative (lyCD133−), when all lymph node metastases were negative. The correlation between CD133 expression and clinicopathological features and overall survival was analyzed.
Clinicopathological features in colorectal cancer cases used in the study.
Immunohistochemical detection of CD133 in colorectal cancer (CRC) lymph node metastasis and primary lesion. (A) Immunohistochemical staining of CD133 in lymph node metastasis (original magnification, ×100). CD133 expression was detected on the luminal cell surface of the colorectal tumor gland and in intra-glandular cellular debris. (B) Hematoxylin and eosin staining of a serial section of a metastatic lymph node (original magnification ×100). (C) Immunohistochemical staining of CD133 in a primary lesion (original magnification ×100). (D) Hematoxylin and eosin staining of a serial section of a primary lesion (original magnification ×100).
Statistical analysis. All statistical calculations were performed using the JMP Pro 11.0.0 statistical software (SAS Institute Japan, Tokyo, Japan). The association between CD133 expression and clinicopathological features was analyzed using the Chi-square test or the Student's t-test. Overall survival curves were calculated using the Kaplan-Meier method, and differences were evaluated using the log-rank test. Multivariate analysis was performed with all variables that had yielded a p-value of <0.05 in univariate analysis using Cox's proportional-hazard modeling to identify the factors with a significant influence on death. Differences with a p-value of <0.05 were considered statistically significant.
Results
Clinicopathological findings. Clinicopathological findings are listed in Table I. Clinicopathological features were analyzed according to the tumor-node-metastasis classification of malignant tumors (7th edition) developed by the International Union Against Cancer (16). The median patient age was 67.1 years old (range=32-92 years); 62% patients were male and 38% were female. The mean numbers of examined lymph nodes and lymph node metastases were 23.2 (range=1-75) and 3.0 (range=1-34), respectively. The median follow-up period was 10.7 years. Seventy-nine patients (57.2%) received adjuvant chemotherapy, as shown in Table I; 19 patients received 5-fluorouracil and leucovorin, 16 patients received S-1 (combination drug with tegafur, gimeracil, oteracil potassium), and 44 patients received a 5-fluorouracil-based regimen.
CD133 expression in primary tumors and lymph node metastases. CD133 expression was detected on the luminal cell surface of the colorectal tumor glands, but not in the cytoplasm. In some tumor glands, the intra-glandular cellular debris was also CD133-positive. CD133 was not detected in the normal colonic mucosa adjacent to cancerous lesions (Figure 1). The number of patients with pCD133+ and lyCD133+ staining results was 96 (69.6%) and 86 (62.3%), respectively (Table II). Out of the 96 pCD133+ patients, 73 (76.0%) were also positive for lyCD133; this rate was significantly higher than in pCD133-patients (p<0.001).
Expression of CD133 in CRC and its relationship with overall survival (OS). (A) Association of pCD133 expression with OS, evaluation was performed by Kaplan–Meier curves (log-rank test, p=0.1104). (B) Association of lyCD133 expression with OS, evaluation was performed by Kaplan–Meier curves (log-rank test, p=0.0644).
Relationship between CD133 expression and clinicopathological features. The relationship between CD133 expression, including both pCD133 and lyCD133, and clinicipathological features is shown in Table III. pCD133 expression had no correlation with any clinicopathological factors. However, the number of lymph node metastases in lyCD133+ patients was significantly higher than in lyCD133− patients (3.5±4.2 vs. 2.1±1.6; p=0.004), even though the number of examined lymph nodes was not significantly different. Moreover, the number of lyCD133+ patients who received adjuvant chemotherapy was significantly higher than the number of lyCD133− patients (55/86 vs. 24/52; p=0.041).
Analysis of OS associated with CD133 expression in stage III CRC. Patients with pCD133− tumors and lyCD133− tumors had higher OS rates than those with pCD133+ and lyCD133+ tumors, although no statistically significant association was found (p=0.1104, p=0.0644, respectively) (Figure 2). Multivariate analysis of significant prognostic factors from univariate analysis was performed (Table IV). Neither pCD133 nor lyCD133 expression was found to be an independent risk factor for OS.
Discussion
In the present study, we examined CD133 expression of both primary cancerous lesions and lymph node metastases in stage III CRC by immunohistochemistry and demonstrated that, in our patient cohort, 69.6% of primary tumors and 62.3% of lymph node metastases were positive for CD133 expression. Although pCD133 expression was not associated with any clinicopathological features, high lyCD133 expression, but not low lyCD133 expression, was significantly associated with the number of lymph node metastases, although the number of examined lymph nodes was not significantly different. Moreover, 76.0% of pCD133-positive patients were also lyCD133-positive, and patients with CD133-negative primary tumor or lymph node metastases had a higher overall survival rate than those with CD133-positive tumors, but this was not statistically significant.
Correlation between pCD133 and lyCD133 expression.
From recent studies on the biological importance of CSCs, CD133 is considered to be a representative marker for CSCs in CRC. Two meta-analyses evaluating the association between the expression of CD133 and the clinicopathological features and outcome of CRC patients suggested that CD133 is an efficient prognostic factor in CRC. This has led to immunohistochemical studies of CD133 expression using primary cancerous lesions (10, 11). However, to the best of our knowledge, to date there have been no reported studies examining CD133 expression in lymph node metastases in CRC. Therefore, in the present study, we further investigated CD133 expression of lymph node metastases as well as primary tumors in stage III CRC, and analyzed the correlation between CD133 expression and clinicopathological features or survival time.
Comparison of the clinicopathological features between CD133+ and CD133− patients.
Univariate and Multivariate analysis of prognostic valuables for overall survival in colorectal cancer.
In our series, we showed that CD133 expression of lymph node metastases correlated with the number of lymph node metastases. Recent studies have reported that CD133 expression was associated with lymph node metastasis (12, 17-19). Moreover, there have been three reports showing that CD133+/CXCR4+ cancer cells correlated with the presence of lymph node metastasis (20-22). According to the meta-analysis by Chen et al., who examined CD133 expression in primary tumors, high CD133 expression was associated with greater T3-T4 tumor invasion, N-positive cases, and vascular invasion cases, but it was not associated with histologal type, lymphatic invasion, and distant metastasis (11). In this study, lyCD133 expression was significantly associated with the number of lymph node metastases, but pCD133 expression was not. This result indicates that CD133-positive cancer might be more aggressive during the process of lymph node metastasis. A larger number of cases is required, however, to clarify the correlation between lyCD133 expression and lymph node metastasis. Moreover, to reveal this mechanism, a more detailed biological study would be required.
In our study, we showed that the rate of lyCD133+ staining was significantly higher in pCD133+ patients than in pCD133− patients. Moreover, patients with CD133-negative staining of either the primary tumor or the lymph node metastases had a higher OS rate than those with CD133-positive staining, but this was not statistically significant. Until now, it may have been assumed that the majority of pCD133+ patients were also positive for CD133 expression at the metastatic sites, especially lymph node metastases, and this was demonstrated in our study. Our results regarding the correlation between CD133 expression and patient OS did not show a significant difference. The first reason for this might be the sample size. The study was limited by the relatively small number of examined cases. Second, the rate of lyCD133+ patients who received adjuvant chemotherapy was significantly higher than that of lyCD133− patients. Therefore, it is apparent from our results that a larger, case-matched control study is needed.
Our results should be interpreted cautiously due to existense of certain limitations. Study methodology is an important problem in studies of CD133 expression. In particular, the different anti-CD133 antibodies used by different researchers and the different cut-off scores for the definition of positive staining have been pointed out (23). Different CD133 antibodies may result in different immunohistochemical staining patterns (cytoplasmic versus membranous). Moreover, the different cut-off scores for the definition of positive staining may result in contradicting results. In the present study, we used an anti-CD133 antibody from Miltenyi Biotec (AC133) to detect CD133 expression by immunohistochemistry. We have used the same antibody and the same cut-off scores since we began our immunohistochemistry studies of CD133 expression in CRC (14). However, further prospective large-scale studies using the same antibody, immunohistochemical staining pattern evaluation, and cut-off value with respect to scores for the definition of positive staining, are required.
In conclusion, we demonstrated that high CD133 expression in lymph node metastases is significantly associated with the actual number of lymph node metastases. From our results, it may be suggested that CD133-positive cancer is more aggressive during the process of lymph node metastasis than CD133-negative cancer. Further investigation on the role of CD133-positive cells in lymph node metastases in CRC is warranted.
Footnotes
Coflicts of Interests
No conflicts of interest exist for the authors.
- Received August 31, 2015.
- Revision received October 28, 2015.
- Accepted October 29, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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