Indian Journal of Dental Research

: 2009  |  Volume : 20  |  Issue : 1  |  Page : 71--76

Expression of E-cadherin in primary oral squamous cell carcinoma and metastatic lymph nodes: An immunohistochemical study

Gurkiran Kaur1, Sunitha Carnelio1, Nirmala Rao1, Laxmi Rao2,  
1 Department of Oral and Maxillofacial, Manipal College of Dental Sciences and Kasturba Medical College, Manipal, Karnataka, India
2 Departments of General Pathology, Manipal College of Dental Sciences and Kasturba Medical College, Manipal, Karnataka, India

Correspondence Address:
Sunitha Carnelio
Department of Oral and Maxillofacial, Manipal College of Dental Sciences and Kasturba Medical College, Manipal, Karnataka


Background: E-cadherin plays an important role in cell-to-cell adhesion and cell motility and its loss is associated with oral squamous cell carcinoma (OSCC) progression. The aim of this study was to determine the expression of E-cadherin in various grades of OSCC and to correlate changes in the expression between these various grades and metastatic lymph nodes. Materials and Methods: Immunohistochemistry (IHC) was used to detect E-cadherin expression in normal oral mucosa, primary OSCC ( n = 37), and metastatic lymph nodes ( n = 10). E-cadherin immunoreactivity was correlated with grades of differentiation and with clinicopathological features. Results: E-cadherin immunoreactivity was found to inversely correlate with the loss of cell differentiation. The expression of E-cadherin decreased significantly in advanced cases of OSCC. However, increase in E-cadherin immunoreactivity was seen in early lesions, that is, in well differentiated ( n = 9) and moderately differentiated OSCC ( n = 13). Furthermore, E-cadherin was negative in majority of metastatic lymph nodes (7/10). Conclusions: Loss of the cell adhesion and E-cadherin plays an important role in progression of OSCC, that is, down regulation of its expression is associated with de-differentiation and metastasis.

How to cite this article:
Kaur G, Carnelio S, Rao N, Rao L. Expression of E-cadherin in primary oral squamous cell carcinoma and metastatic lymph nodes: An immunohistochemical study.Indian J Dent Res 2009;20:71-76

How to cite this URL:
Kaur G, Carnelio S, Rao N, Rao L. Expression of E-cadherin in primary oral squamous cell carcinoma and metastatic lymph nodes: An immunohistochemical study. Indian J Dent Res [serial online] 2009 [cited 2022 Jan 19 ];20:71-76
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Full Text

Head and neck carcinoma constitutes the fifth most frequent human tumor diagnosed worldwide and accounts for approximately 400,000 new cases annually. [1] In India, squamous cell cancer constitutes more than 90% of the total of head and neck carcinoma with an incidence rate of 12.6 per lakh of the population. [2] Staging and grading of the disease, are two best parameters that predict survival. [3] The success of clinical therapy for head and neck tumors depends upon the status of lymph node metastasis. [4]

Many risk factors have been attributed to the development of cancer, and over the past few decades there has been a sharp focus on genetic factors. [5] However, defined genetic alterations have not been ascribed to the distinct stages of tumor development. An understanding into the mechanism of metastatic behavior of carcinoma and recognizing cases at higher risk of metastases might help to design new strategies for the diagnosis and treatment. [5],[6],[7],[8] Therefore, efforts to identify novel molecular predictors of behavior and therapeutic targets for these remain a high priority.

Recent investigations have led to an insight in the mechanism of cellular adhesion. It has been found that the formation of tight tissue sheets depends on strong intercellular adhesion and represents a prerequisite for the generation of characteristic properties of epithelia. To achieve intercellular adhesion, epithelial cells possess various cell junctions, the composition and function of which must be tightly regulated. There has been an increasing interest in a large family of transmembrane glycoproteins called 'cadherins' of which the 'epithelial cadherin' (E-cadherin) plays a pivotal role in epithelial cell-cell adhesion. E-cadherin, a calcium dependant transmembrane glycoprotein of the type-1 cadherin super-family is an invasion/tumor suppressor gene, which belongs to the family of cell-cell adhesion molecules essential for maintaining the structural integrity and organization of stratified squamous epithelia. It is encoded by the CDH1 gene that is located on chromosome 16q-22.1 and is a classical cadherin that forms the key functional component of adherens junction between epithelial cells. [5],[6],[9],[10] Both in vitro and in vivo studies have shown that absence of membranous E-cadherin in carcinoma cells contribute toward increased invasiveness and metastatic ability. [11],[12],[13],[14],[15],[16],[17] Also, it is shown that decreased E-cadherin expression in cancerous tissue correlates with the poor prognosis of OSCC patients. Detection of E-cadherin expression is useful to confirm the cervical lymph node metastasis. [18]

The purpose of this study was to investigate the expression of E-cadherin in paired primary and metastatic OSCC and to elucidate its role as a reliable and potential marker in determining the biological behavior of the disease.

 Materials and Methods

Before the commencement of this study, we considered the ethical aspects and obtained an ethical committee approval. The tissue specimen for the study included 42 formalin fixed, paraffin-embedded tissue blocks retrieved from the departments of Oral and Maxillofacial and General Pathology of a tertiary referral and teaching hospital.

Among these, ten blocks of histologically diagnosed well-differentiated, 14 moderately differentiated and 13 poorly differentiated OSCC were taken as cases. Broders histological grading system was followed. [19] Five blocks of normal mucosa were taken as controls. Ten cases of metastatic lymph nodes were also included in the study. Exclusion criteria included patients who had received neoadjuvant cancer therapy.


The antibodies and reagents used for immunohistochemistry (IHC) technique were obtained from Sigma Aldrich Chemicals Private Limited and Dako Cytomatio, Denmark; and consisted of primary antibody, monoclonal mouse anti E-cadherin antibody (0.2 ml) clone NCH-38; biotinylated secondary antibody, goat-antimouse IgG (0.5 ml); conjugate-streptavidin-peroxidase polymer (ultras 0.25 ml); substrate-diaminobenzidine tetrahydrochloride (DAB) (15 ml); and slide adhesive-poly L-lysine solution.

Sections (3 mm) were deparaffinized in xylene and rehydrated by immersion in a graded series of ethanol dilutions. Microsections were dipped in freshly prepared 3% H 2 O 2 in methanol for 20 min to block endogenous peroxidase activity and rinsed with phosphate buffered saline (PBS) for 5 min. For antigen retrieval, microsections were immersed in 0.01 mM sodium citrate buffer solution in a microwave oven. Sections were further incubated in blocking serum for 30 min and then for 60 min with anti E-cadherin antibody at 37 o C (1:200 dilution) in a humid chamber. For reagent (negative) control, normal mouse immunoglobulin of the same class was used instead of the primary antibody. The sections were washed in three changes of PBS thoroughly for five min each. Incubation with biotinylated secondary antibody (F c conjugated goat-antimouse IgG, 1:200 dilution) was carried out at room temperature for 30 min, followed by washing in three changes of PBS for 5 min each. Sections were incubated with streptavidin-peroxidase conjugate, diluted at a concentration of 1:200 for 30 min with subsequent washing in three changes of PBS for 5 min each. For visualization, sections were incubated with DAB for 10 min. Before mounting, the sections were then lightly counterstained by using Mayer's hematoxylin for 1 min, washed with PBS, and deionised distilled water. Presence of brown colored end product at the site of target antigen was indicative of positive reactivity.

Positive control tissue sections were used to ensure homogenous accurate and reproducible staining and this included normal oral mucosa. Furthermore, normal squamous and glandular epithelia within the oral tissue were used as internal positive controls for E-cadherin staining.

Assessment of E-cadherin Immunoreactivity

Immunostaining was assessed in each case in three fields that were randomly selected and its average was taken for evaluation for cells expressing E-cadherin. The distribution and intensity of E-cadherin immunostaining were assessed semi quantitatively by two independent observers to eliminate interobserver bias. An analysis of the hematoxylin and eosin (H and E) stained slides of these patients, allowed regrading of lesions whose grade had been altered after recutting of archival blocks. A scale of 0 to ++ was used (0, absence of staining; +/0, heterogeneous staining; +, weak staining; and ++, strong staining).

Pattern of expression of E-cadherin by tumor cells was also graded by these observers on a scale of 1 to 4 (1, membranous staining; 2, both membranous and cytoplasmic staining; 3, cytoplasmic staining; and 4, absence of staining).

Statistical Analysis

The Chi-square test was performed to examine the significance of E-cadherin expression in terms of intensity, location in tumor cells and within tumor islands, and to correlate with histopathological grading. Intergroup comparison was also done using the same test. Kendall's tau-b test was performed to estimate the interobserver bias.


Among the five controls of normal oral mucosa, the E-cadherin expression yielded positive signals in the surface epithelia, but not in the connective tissue. The immunostaining expression was seen as a strong membranous homogenous staining of the basal/parabasal and superficial cell layers of the epithelium, but excluding the upper most epithelial layer [Figure 1].

Following the evaluation of normal oral mucosa, 37 cases of OSCC were tested for the expression of E-cadherin. A decrease of membranous staining and an increase in cytoplasmic staining of E-cadherin was seen more frequently in poorly differentiated tumors [Figure 2], than in well and moderately differentiated tumors [Table 1]. In cases of well-differentiated OSCC, expression of E-cadherin was intense [Figure 3] and [Figure 4] as that of the normal stratified squamous epithelium [Table 2]. A moderately differentiated OSCC showed heterogeneous staining, where in the central cell mass of tumor islands was differentiated and expressed membranous E-cadherin staining, while the cells at the periphery were dedifferentiated and expressed cytoplasmic staining only [Figure 5]. Staining intensity and staining location showed significant difference in poorly differentiated OSCC when compared to that of well-differentiated and moderately differentiated OSCC [Table 3].

Primary tumors of patients with lymph node metastases (n = 10) were either heterogeneous or negative. Remarkably, in seven of the ten cases the infiltrated lymph nodes were E-cadherin negative.

To eliminate the subjective bias, two observers independently evaluated the expression of E-cadherin and the P value was found to be nonsignificant for all grades of OSCC. Thus, the E-cadherin expression graded by observer-1 was subjected to further statistical analysis (P = 1).


The main observations of this study were that well-differentiated OSCC expressed E-cadherin often as strongly as normal stratified squamous epithelium, while in poorly differentiated OSCC expression of E-cadherin was lost or cytoplasmic, and in moderately differentiated tumors it was expressed in a heterogeneous fashion. Similar were the results of Tanaka et al, [14] who observed a significant relationship between reduced E-cadherin and invasiveness of OSCC. Bagutti et al. [15] have shown that least differentiated tumors showed a reduced expression of E-cadherin in later stages, and these tumor cells are said to acquire invasive phenotype. [17] The exact mechanism involved in the variable expression of E-cadherin is unclear. Various studies have suggested that abnormalities lie within E-cadherin and its associated mediators, leading to disruption or loss of function. Loss of adhesion or reduction of this protein staining can be caused by deletion or mutation silencing by CpG methylation or otherwise altered gene expression of E-cadherin. [12],[13],[16] But the role of this protein mutated in OSCC is yet to be proved. Reduced expression of this molecule is generally taken as an indicator of malignancy and this is supported by studies which show that the invasive behavior was suppressed by transinfection with E-cadherin; [17],[18],[19] while studies on E-cadherin by Shinohara et al.[16] showed no significant association between degree of differentiation and E-cadherin expression. However, they found that E-cadherin expression was reduced in tumors showing high invasiveness and in patients with cervical lymph node metastasis. Remarkably in normal oral mucosa, membranous E-cadherin staining is localized at the intercellular junctions (basal and parabasal layers) and lost at the upper third of the epithelium, which may be due to normal desquamation process. [20],[21],[22],[23]

Cytoplasmic staining was observed in moderately differentiated (four cases) and poorly differentiated carcinomas (eight cases). Various possibilities related to E-cadherin expression in cytoplasm are either an increased production rate, a failure to translocate, or to anchor onto the cell membrane. Alternatively, it could be related to that seen in noncancerous diseases where in the release of intercellular contact results in the endocytic uptake of desmosomal glycoprotein in membrane vesicles, which remains associated with the plaque component and is seen as clumps. Breakdown of these membranous vesicles results in a diffuse cytoplasmic staining, which have been supported by experimental studies. [24],[25],[26]

There are controversial reports on expression of E-cadherin in metastatic lymph nodes. [4],[21],[27],[28],[29],[30],[31] Though Zhong et al.[18] indicate that detection of E-cadherin expression is useful to confirm the cervical lymph node metastasis but maybe useless to detect the cervical lymph node micrometastasis. They also indicate that further studies are essential to reveal the detail mechanism of E-cadherin expression in formation of lymph node metastatic focus. Studies on breast cancer by Kowalski et al.[32] have shown that E-cadherin immune reactivity was significantly increased in metastatic lesions when compared with their primary sites. Hung et al.[30] found an increased E-cadherin immunoreactivity in metastatic OSCC lesions, which would predict worst prognosis. In our patients, staining was either absent or heterogenous (+/0) for E-cadherin. Further, it was also observed that majority of them were generally E-cadherin absent, irrespective of whether they originated from poorly or moderately differentiated OSCC. However, Bulkholm et al.[33] reported that there was no significant difference between the expression of E-cadherin and the presence of regional metastasis in human breast carcinoma and that the variation in the expression of E-cadherin complex may vary from tumor to tumor. Studies on colorectal cancer by Gofuku et al.[34] showed that reduction of alpha catenin was a more sensitive and useful indicator than the reduction of E-cadherin in evaluating the potential for tumor invasion and metastasis. Cases of moderately differentiated carcinoma revealed that the periphery of tumor islands did show cytoplasmic staining, while central cells showed membranous staining. These features suggest that the peripheral cells fail to differentiate and that this protein plays an important role as a differentiation factor and invasion suppressor in epithelial tissues in vivo. [17],[19]

The sample size of this study though small gives an insight into the expression of E-cadherin in different grades of OSCC and the metastatic lymph nodes. We believe, further larger samples and trials are required to establish whether abnormalities of either E-cadherin or its related molecules such as catenin and actin are the prime cause of disruption of the E-cadherin/catenin complexes or whether these complexes are disrupted via other mechanisms. These might give an insight into the early molecular interaction which is critical in the initiation and progression of the tumor and also help us to understand the mechanism of metastatic behavior, and aid in the design of new strategies for diagnosis and treatment of OSCC.


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