Study of β-catenin, E-cadherin and vimentin in oral squamous cell carcinoma with and without lymph node metastases

A total of 60 cases of primary OSCC diagnosed over a period of 2 years (2010–2012) in the Department of Pathology, All India Institute of Medical Sciences were included for the study. All the patients had been surgically treated with tumour resection and radical neck dissection. None of the patients did receive any tumour specific therapy (chemotherapy or radiotherapy) before the resection. Thirty cases with a clinical suspicion of malignancy but diagnosed as inflammatory lesions on histology and 30 histologically confirmed normal mucosal margins from the resection specimens were included as control group. This study was approved by the Ethics Committee of All India Institute of Medical Sciences.

Specimens from all the cases were fixed in 10% formaldehyde solution and embedded in paraffin. Histological diagnosis was made on haematoxylin and eosin stained sections according to the revised criteria given by the World Health Organization (2005). OSCCs were classified into well, moderately and poorly differentiated grades. Staging was done based on TNM staging into four categories, stage I-IV.

Tissue sections (4 μm) cut from representative paraffin blocks were deparaffinised in xylene and rehydrated through graded alcohols. Endogenous peroxidase was blocked using 4% hydrogen peroxide. For antigen retrieval, the sections were processed by conventional microwave heating in 10mMol/L sodium citrate retrieval buffer (pH 6.0) for 30 minutes. The sections were then incubated for overnight with primary antibody at 4°C in a humid chamber for β catenin (Spring bioscience), E-cadherin (Spring bioscience) and vimentin (Thermo scientific). The dilutions used for the primary antibodies were 1:250, 1:100 and 1:400 for β catenin, E-cadherin and vimentin respectively. The sections were subsequently incubated with anti-mouse immunoglobulin in phosphate buffered saline (PBS) containing carrier protein and 15 mM Sodium Azide (large volume universal DAKO LSAB kit, Peroxidase, M/s Dakopatts, Denmark) at room temperature for 30 minutes for β-catenin, E-cadherin and vimentin. The sections were then washed three times with PBS (pH 7.2) for 2 min. The reaction product was developed with 3, 30-diaminobenzidine and counterstained with haematoxylin. Immunoreactivity in the tissue was judged independently by two pathologists who were blinded to the clinical data and other immunohistochemical results. Normal oral mucosal tissues were used as positive control. Negative controls were included in each slide run with omission of primary and secondary antibodies.

Immunoreactivity was semi quantitatively evaluated on the basis of staining intensity and distribution using the immunoreactive score[9, 10].

Immunoreactive score = intensity score x proportion score. The intensity score was defined as 0: negative; 1: weak; 2: moderate; or 3: strong, and the proportion score was defined as 0: negative; 1: <10%; 2: 10-50%; 3: >50-80%; or 4: >80% positive cells. The total score ranged from 0 to 12. Immunoreactivity was divided into three groups based on the final score: negative immunoreactivity was defined as a total score of 0, low immunoreactivity was defined as a total score of 1–4, and high immunoreactivity was defined as a total score >4.

The correlation between clinicopathological parameters and β catenin, E-cadherin and vimentin expression were analysed using the chi square test and Fisher’s exact test. A p value <0.05 was considered statistically significant.

A total of 60 cases of OSCC were analyzed, of which 30 cases had lymph node metastasis. Majority of them were males (77%) and females constituted 14 (23%) cases. Age range was 23 years to 72 years with a mean age of 44.79 years. Tongue was the most common site involved (52%). Well differentiated, moderately differentiated and poorly differentiated squamous cell carcinomas included 38, 20 and 2 cases respectively. Of 60 cases, 21(35%) cases were Stage III, 17(28%) Stage IV, 16(27%) Stage I and 6(10%) in Stage II.

Both the control groups revealed a strong membranous staining pattern of E-cadherin with an absence or reduced expression in the superficial layer of mature well differentiated cells. Strong membranous β-catenin expression was observed in the suprabasal to the basal layers with absence of staining in the superficial layer in both the control groups. Vimentin expression was detected in the cytoplasm of the connective tissue mesenchymal cells of normal oral mucosal tissue, but not in the squamous epithelium (Figures 1 and2).

Comparison of expression of β-catenin, E-cadherin and vimentin in OSCC with and without lymph node metastases is enumerated in Table 1. OSCCs showed a weaker expression of both β-catenin and E-cadherin than the control groups (p <0.05). However, there was no significant difference in the degree of β-catenin (p = 1.000) and E-cadherin (p = 0.771) expression in study groups of OSCC with and without lymph node metastases. Vimentin expression was seen in all cases of OSCC. Vimentin was expressed in the cytoplasm of the tumour cells with weak to moderate intensity. However, there was no significant difference in the degree of vimentin (p = 0.360) expression in both the study groups (Figure 3).

Patients without lymph node metastases were nearly equally distributed in age groups of ≤50 years and >50 years where as patients with lymph node metastases predominantly belonged to the ≤50 years group. There were no statistically significant differences between the clinical variables (age, sex, site, size of tumour and histological differentiation) in OSCC with and without lymph node metastases as enumerated in Table 2.

There was no statistically significant difference between the expressions of β-catenin, E-cadherin or vimentin and clinicopathological variables (age, sex, site, size of tumour, histological differentiation and stage) in OSCCs with and without lymph node metastases as listed in Table 3. Thus, all these variables did not affect the expression of adhesion molecules.