Abstract
OBJECTIVE To investigate the correlation of E-cadherin expression with clinicopathological parameters and prognosis of patients with oral squamous cell carcinoma.
METHODS We examined the expression of the protein E-cadherin in 43 oral squamous cell carcinoma (SCC) surgical speciments by SABC immunohistochemistry.
RESULTS There was a significant correlation between the level of E-cadherin expression and tumor stage (P=0.024), invasive pattern (P= 0.009) and lymph node metastasis (P=0.023). No relation was found with age (P=0.084), sex (P= 1.356) and differentiation (P=0.877). Using the Kaplan-Meier method we demonstrated that those cases which showed E-cadherin expression (-) or (+) had a significantly poorer prognosis compared those cases which showed expression (++) or (+++) (P= 0.0146).
CONCLUSION E-cadherin, is an important indicator of clinical diagnoses and prognositic marker for oral SCC patients.
keywords
Ecadherin is a member of the cadherin superfamily, which are calcium-dependent intercellular adhesion molecules believed to play a role in cell recognition and segregation, morphogenetic regulation, and tumor suppression,[1] Reduced expression of E-cadherin is associated with invasion and metastasis in different human cancers, such as primary ovarian carcinomas,[2] esophageal carcinoma,[3] cervical carcinoma,[4] human laryngeal cancer,[5] and rectal cancer[6] etc.
Squamous cell carcinoma (SCC), the most frequent malignancy of the oral cavity, is associated with a poor clinical outcome. Investigation of the expression of proteins associated with clinical characteristics and pathology in SCC would be useful in assessment of tumor behavior, accurately predicting prognosis, and improving the ability to determine the most appropriate therapies for each patient.[7]
In this study, we examined the expression of E-cadherin in surgical resected specimens of oral SCC, and investigated the correlation of E-cadherin expression and clinicopathological parameters and prognosis of patients with oral SCC.
MATERIALS AND METHODS
Patients
We collected 43 surgically treated cases of oral SCC from September 1991 to July 1997 in the Kobe University Hospital. The cases consisted of 32 males and 11 females, ranging in age from 41 to 81 years (average age of 59.6 years). Tumors were classified as well differentiated (n=22), moderately differentiated (n=18), or poorly differentiated (n=3), according to the International Histological Classification of Tumors.[8] Based on the TNM tumor stage classification,[9] orneases were classified as Stage I (5 cases), Stage II (14 cases), Stage III (9 cases), and Stage IV (15 cases).
All patients were treated initially with surgery, after which 17 received postoperative radiotherapy or chemotherapy. Resected specimens were examined in our study.
Carcinoma invasion was classified according to the mode of invasion by Yamamoto et al.[10] : mode 1, well defined margin; mode 2, cords, less obvious margin; mode 3, groups of cells, no distinct margin; mode 4c, diffuse invasion of cord-like type; mode 4d, diffuse invasion of diffuse type. The 43 cases in this study were divided into mode 1 (n=0), mode 2 (n=6), mode 3 (n=18), mode 4c (n=15), and mode 4d (n=4).
Immunohistochemical analysis
Immunohistochemical studies were performed using an indirect streptavidin-biotin immunoperoxidase technique. Sections were dewaxed and rehydrated in concentrated sodium citrate buffer (20x), then heated for 10 min at 750 W output in a microwave oven. Sections were treated with methanol for 20 min to inhibit endogenous peroxide activity, then blocked with serum blocking solution to reduce nonspecific labeling. Sections were incubated with 1:400 diluted HECD-1 (MAb to human E-cadherin, Takara Shuzo, Japan) for 1 h at room temperature. The sections were also treated with irrelevant primary antibody as a negative control. The sections were treated with diluted biotinylated anti-mouse and anti-rabbit Ig antibody for 15 min, then reacted with horseradish peroxidase-conjugated streptavidin (Dako Japan, Kyoto, Japan) for 15 min. After each step, the sections were rinsed in phosphate-buffered saline (PBS) for 15 min. To visualize immunoreactivity, the sections were treated with DAB. Following hematoxylin counterstaining, the slides were permanently mounted. Immunostaining scores were determined by a researcher with no knowledge of the patient clinical status.
The degree of staining for E-cadherin was scored as described by Hiraki et al.[11] (+++): extensive staining comparable to control epithelium at the invasion front of SCC. (++): staining reduced from control levels, but greater than 50 % of the level of positive staining. (+): positive staining, but reduced to less than 50 % of control levels. (-): very little or no staining. Normal epithelium proximal to the tumor within the specimen was used as a control.
Statistical analysis
The χ2-test was used to assess the statistical significance of E-cadherin expression in relation to clinicopathological parameters. Survival curves were obtained using the Kaplan-Meier method. P values < 0.05 were considered significant.
RESULTS
Under light microscopy, E-cadherin immunostaining in tumor cells was observed not only in the membrane but also in the cytoplasm (Fig.1).
E-cadherin immunohistochemical staining of oral SCC. A: E-cadherin (+++) expression (original magnification, * 200); B: E-cadherin (++) expression (original magnification, * 200); C: E-cadherin (+) expression (original magnification, * 200); D: E-cadherin (-) expression (original magnification, * 200).
E-cadherin expression (+++) was observed in 10 cases (23.3%), expression (++) in 11 (25.6%), expression (+) in 13 (30.2%), and no expression in 9 cases (20.9%). E-cadherin expression (+) and (-) was significantly associated with more advanced stage tumors (P=0.024), demonstrating a diffusely invasive pattem (P=0.009) and lymph node metastasis (P=0.023). No relationship was observed between E-cadherin expression and age (P=0.084), sex (P=1.356) or tumor differentiation (P=0.877, Table 1). The survival curve demonstrated a significant difference between the cases with expression (-) or (+) of E-cadherin from the expression (++) or (+++) population (P=0.0146, Fig.2).
Relationship between the expression status of E- cadherin and clinicopathological parameters of oral SCC
Kaplan-Meier survival curves according to the tissue status of E-cadherin in oral SCC.The cases with E-cadherin expression(-,+) were significantly poorer in prognosis compared to those with E-cadherin expression(++,+++)(P=0.0146).
DISCUSSION
E-cadherin is an important suppressor molecule of tumor development. E-cadherin functions by enhancing adhesion between the cells and thereby inhibits cellular proliferation and growth of tumors. The human E-cadherin gene is located at chromosome 16q22.1. The expression of E-cadherin in human tumors has been reported by multiple researchers. Though the data from these reports are not very consistent, E-cadherin has been shown to be related to tumor biological behavior. The relationship is that down-regulation of E-cadherin expression weakens adhesion between cells, followed by the infiltration, dissemination and metastasis. This event contributes to the poorer prognosis of the patients.[2-7]
In our study, the down-regulation of E-cadherin expression in oral SCC correlated with increased lymph node metastasis and poorer prognosis, as seen in previous reports. Furthermore, reduced expression of E-cadherin correlated with tumor stage and invasive pattern, but not with age, sex and differentiation, results similar to those of Shinohara et al. (12) who reported no significant relationship between down-regulation of E-cadherin and differentiation in oral SCC.
Recently, Bosch et al.[13] showed that E-cadherin, is an important indicator of clinical diagnoses and prognostic marker of head and neck SCC patients. The prognostic strength of E-cadherin was independent of, and stronger than histological grading, N stage, tumor site, and even stronger than the TNM stage. Based on these results, evaluation of E-cadherin in SCC by immunostaining is recommended as a significant prognostic marker. In our studies of 43 cases of OSCC after surgery over a period of 8 years, we evaluated E-cadherin expression using an immunohistochemical staining technique. Our findings support the belief that assessment of E-cadherin expression, only by immunostaining, has prognostic importance in patients with oral SCC.
Studies relating to how E-cadherin may influence tumor behavior, have shown that E-cadherin gene mutations were not identified in human oral SCC cells,[14] and that the interaction between α-, β-and γ-catenin,[15] S100A4,[9] dysadherin[16] and E-cadherin may affect the metastatic potential of tumor cells and, consequently, the prognosis of the cancer patients. Thus, to elucidate the mechanism underying the effect of E-cardhrin on tumor prognosis will be the next focus of our research.
In conclusion, we believe E-cadherin is an independent important indicator for clinical diagnoses, a prognositic marker and a useful factor for the prediction of metastatic potential of oral SCC patients. E-cadherin status will be of great value to clinical oncologists in determining the best therapeutic methods for patients to obtain improved outcomes.
- Received May 9, 2006.
- Accepted June 7, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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