Abstract
Objective
To evaluate the value of E-cadherin and epithelial cell adhesion molecule (Ep-CAM) expression in laryngeal biopsy materials for predicting cervical lymph node metastasis in patients with supraglottic laryngeal carcinoma.
Methods
All patients participating in the study were selected from among the surgically treated patients at the department of Otolaryngology, Head and Neck Surgery, Erciyes University School of Medicine between 1991 and 2005. The study consisted of thirty patients who had pathologically metastatic lymph nodes (pN + group) and 30 age-, sex-, T value- and differentiation matched patients without pathologically metastatic lymph nodes (pN0 group). Immunohistochemical studies were performed with E-cadherin and Ep-CAM antibodies on representative tumor sections collected from paraffin sections of laryngeal biopsy materials. The expression of E-cadherin and Ep-CAM was compared between the pN0 and pN + groups. The association between immunostaining of E-cadherin and Ep-CAM was also evaluated.
Results
There was no significant difference between the two groups in terms of E-cadherin and Ep-CAM expression. There was also a very poor agreement between the expression of E-cadherin and Ep-CAM.
Conclusion
Multi-institutional and multidisciplinary immunohistochemical studies conducted with standardized methodology and also with more patient participation may help to obtain more specific results.
1
Introduction
The management of cervical lymph node metastasis is a significant part of the treatment of head and neck squamous cell carcinoma (SCC), which is directly related to decreased survival and regional recurrence. Ipsilateral single node metastasis reduces survival by 50% whereas contralateral or bilateral metastasis contributes an additional 50% decrease . However, management of N0 neck is still controversial because of the existing limitations in ascertaining the neck disease precisely. A definitive diagnostic tool that reflects the true status of N0 neck has not emerged until today . Immunohistochemical studies concerning the different steps in the metastasis process are currently being intensively investigated to find an immunohistochemical parameter predictive of lymph node involvement .
E-cadherin is a calcium-dependent intercellular glycoprotein in epithelial cells that has a critical role in cell adhesion and suppression of tumor invasion . A weakness or loss of E-cadherin expression was reported to be relevant for increased neck metastasis in head and neck SCC .
Epithelial cell adhesion molecule (Ep-CAM) is a calcium-independent type I glycoprotein that mediates cell signaling, migration, proliferation, differentiation and also cell-cycle regulation . Ep-CAM also modulates cell adhesion interactions mediated by E-cadherin . Studies concerning the expression of Ep-CAM in head and neck SCC have shown contradictory results in the literature .
In the present study, we aimed to evaluate the value of E-cadherin and Ep-CAM expression in laryngeal biopsy materials in predicting cervical lymph node metastasis in patients with supraglottic laryngeal carcinoma. We also aimed to evaluate the association between E-cadherin and Ep-CAM expression in tumor cells to determine a possible concordance.
2
Materials and methods
The study protocol was approved by the Ethics Committee of the Kayseri Erciyes University School of Medicine. Two hundred ninety-four patients with supraglottic laryngeal carcinoma were included in the present study. All patients were treated surgically at the department of Otolaryngology, Head and Neck Surgery, Erciyes University School of Medicine between 1991 and 2005. All patients were staged according to the tumor, node and metastasis staging system of the American Joint Committee on Cancer . Tumor sections were also graded according to the World Health Organization classification . All patients underwent different types of laryngectomies with unilateral or bilateral neck dissection. Laryngectomy types were determined by the status of the primary tumor, the patient’s condition and pulmonary function. Tumors not crossing the midline were treated with unilateral neck dissection, otherwise bilateral dissection was performed. Medical records of the patients were retrospectively evaluated and pathologically metastatic lymph nodes were found in 94 of the 294 patients. Among the 94 patients, 30 were selected via random sampling (pN + group). Thirty age-, sex-, T value- and differentiation matched controls were also selected from among the 200 patients without pathologically metastatic lymph nodes (pN0 group). None of the pN0 patients had developed any neck recurrence during the three years following surgery.
Representative tumor sections were collected from paraffin sections of the laryngeal biopsy materials of patients for immunohistochemical study. E-cadherin (Lab Vision, Fremont, CA) and Ep-CAM (Lab Vision, Fremont, CA) antibodies were applied using the streptavidin–biotin method . Tumors were analyzed using full tissue sections ( n = 60). All cases were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Tissue sections (3 or 4 μm thick) were deparaffinized in xylene and rehydrated in a graded series of ethanol. Heat-induced epitope retrieval was performed by placing sections in plastic Coplin jars containing a preheated target retrieval solution (DAKO, Carpinteria, CA) boiled for 2 × 10 minutes in a microwave oven in an EDTA buffer. Sections were then allowed to cool at room temperature for at least 15 minutes. Mouse monoclonal antibodies specific for E-cadherin and Ep-CAM were applied at a dilution of 1:20 and 1:100, respectively. Subsequent steps of the immunostaining procedure were performed in a humidity chamber as previously described. Formalin-fixed and paraffin-embedded samples of breast carcinoma were used as a positive control for Ep-CAM. Tonsillar carcinoma was the positive control for E-cadherin. Negative controls were obtained by the substitution of phosphate buffered saline for the primary antibodies.
Immunohistochemical analysis was performed with the consensus of one pathologist and one head and neck surgeon using a double-headed light microscope. Both were unaware of patient characteristics. The immunostaining of tumor cells was divided into four grades as follows: no reactive cells (−), less than 25% of reactive cells (+), 25% to 50% of reactive cells (++), and 50% or greater expression of reactive cells (+++). Immunoreactivity of more than 25% of the tumor cells was considered as overexpression . The immunohistochemical staining scores were then statistically compared between the pN0 and pN + groups. The association between immunostaining of E-cadherin and Ep-CAM was also evaluated.
Statistical analysis was performed using Statistical Package for the Social Sciences (v. 15; SPSS Inc.; Chicago; IL; USA). A two-tailed unpaired Student’s t test was used to compare the parametric conditions between the two groups and chi-square for comparison of categorical variables. A p value less than 0.05 was considered significant for all comparisons. An agreement of CD44 and MMP-9 immunohistochemistry was assessed with Cohen κ statistic. The agreement was determined with κ values as follows: 0 to 0.20, very poor; 0.2 to − 0.40, poor; 0.41 to 0.60, fair; 0.61 to 0.80, good; and 0.81 to 1.00, excellent.
2
Materials and methods
The study protocol was approved by the Ethics Committee of the Kayseri Erciyes University School of Medicine. Two hundred ninety-four patients with supraglottic laryngeal carcinoma were included in the present study. All patients were treated surgically at the department of Otolaryngology, Head and Neck Surgery, Erciyes University School of Medicine between 1991 and 2005. All patients were staged according to the tumor, node and metastasis staging system of the American Joint Committee on Cancer . Tumor sections were also graded according to the World Health Organization classification . All patients underwent different types of laryngectomies with unilateral or bilateral neck dissection. Laryngectomy types were determined by the status of the primary tumor, the patient’s condition and pulmonary function. Tumors not crossing the midline were treated with unilateral neck dissection, otherwise bilateral dissection was performed. Medical records of the patients were retrospectively evaluated and pathologically metastatic lymph nodes were found in 94 of the 294 patients. Among the 94 patients, 30 were selected via random sampling (pN + group). Thirty age-, sex-, T value- and differentiation matched controls were also selected from among the 200 patients without pathologically metastatic lymph nodes (pN0 group). None of the pN0 patients had developed any neck recurrence during the three years following surgery.
Representative tumor sections were collected from paraffin sections of the laryngeal biopsy materials of patients for immunohistochemical study. E-cadherin (Lab Vision, Fremont, CA) and Ep-CAM (Lab Vision, Fremont, CA) antibodies were applied using the streptavidin–biotin method . Tumors were analyzed using full tissue sections ( n = 60). All cases were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Tissue sections (3 or 4 μm thick) were deparaffinized in xylene and rehydrated in a graded series of ethanol. Heat-induced epitope retrieval was performed by placing sections in plastic Coplin jars containing a preheated target retrieval solution (DAKO, Carpinteria, CA) boiled for 2 × 10 minutes in a microwave oven in an EDTA buffer. Sections were then allowed to cool at room temperature for at least 15 minutes. Mouse monoclonal antibodies specific for E-cadherin and Ep-CAM were applied at a dilution of 1:20 and 1:100, respectively. Subsequent steps of the immunostaining procedure were performed in a humidity chamber as previously described. Formalin-fixed and paraffin-embedded samples of breast carcinoma were used as a positive control for Ep-CAM. Tonsillar carcinoma was the positive control for E-cadherin. Negative controls were obtained by the substitution of phosphate buffered saline for the primary antibodies.
Immunohistochemical analysis was performed with the consensus of one pathologist and one head and neck surgeon using a double-headed light microscope. Both were unaware of patient characteristics. The immunostaining of tumor cells was divided into four grades as follows: no reactive cells (−), less than 25% of reactive cells (+), 25% to 50% of reactive cells (++), and 50% or greater expression of reactive cells (+++). Immunoreactivity of more than 25% of the tumor cells was considered as overexpression . The immunohistochemical staining scores were then statistically compared between the pN0 and pN + groups. The association between immunostaining of E-cadherin and Ep-CAM was also evaluated.
Statistical analysis was performed using Statistical Package for the Social Sciences (v. 15; SPSS Inc.; Chicago; IL; USA). A two-tailed unpaired Student’s t test was used to compare the parametric conditions between the two groups and chi-square for comparison of categorical variables. A p value less than 0.05 was considered significant for all comparisons. An agreement of CD44 and MMP-9 immunohistochemistry was assessed with Cohen κ statistic. The agreement was determined with κ values as follows: 0 to 0.20, very poor; 0.2 to − 0.40, poor; 0.41 to 0.60, fair; 0.61 to 0.80, good; and 0.81 to 1.00, excellent.
3
Results
Both groups consisted of male patients. The mean ages of the pN0 and pN + groups were 55.2 ± 7.9 and 57.2 ± 9 years, respectively. There was no statistically significant difference between the two groups with regard to age and sex ( p > 0.05). Both groups consisted of four T2 (13.3%), 18 T3 (60%) and eight T4 tumors. There were 16 well (53.3%), 12 moderately (40%) and two poorly (6.7%) differentiated tumors in each group. There was no statistical difference between tumor stages or differentiation in relation to expression of E-cadherin ( Table 1 ; Fig. 1 ) or Ep-CAM ( Table 2 ; Fig. 2 ). There was no significant difference between the two groups in terms of E-cadherin and Ep-CAM expression ( Table 3 ). There was also a very poor agreement between expression of E-cadherin and Ep-CAM ( κ value < 0.2).
