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An Immunohistochemical Study of E-cadherin and β-catenin in Different Grade of Oral Squamous Cell Carcinoma in a Tertiary Care Centre
Saba Shakil1, Veena Maheshwari1, Nishat Afroz1 and Dr. Murad1
1Dept. of Pathology, Jawaharlal Nehru Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
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*Corresponding Author Dr. Saba Shakil
Article History Received: 30.07.2022 Accepted: 10.08.2022 Published: 20.08.2022
Abstract: Background: The purpose of the present study was to evaluate the expression of cell adhesion molecules, E-cadherin and β-catenin in different grades of oral squamous cell carcinoma and to assess their role as biomarkers and potential prognosticators in oral carcinogenesis. In the present study, the expression of E-cadherin and β-catenin is evaluated with the help of immunohistochemical examination. Materials and Methods: Biopsies/specimens of all suspected cases of oral squamous cell carcinoma were fixed in 10% neutral buffered formalin solution. Histopathological diagnosis was made on hematoxylin and eosin (H&E) stained sections and typed into different grades of oral squamous cell carcinoma.The expression of E-cadherin and β-catenin is assessed by immunohistochemistry. Results: In the present study, a total of 71 cases (35 cases of well differentiated squamous cell carcinoma, 25 cases of moderately differentiated squamous cell carcinoma and 11 cases of poorly differentiated squamous cell carcinoma) were subjected to E-cadherin and β-catenin immunostaining. E-cadherin immunohistochemistry showed high immunoreactivity in 91.43 % cases, and low immunoreactivity in only 8.57% cases. β-catenin immunoreactivity was high in 77.14% cases of well differentiated carcinoma and low in 22.86% cases. Conclusion: The suppression of E-cadherin and β-catenin expression is considered as one of the main molecular events responsible for dysfunction in cell-cell adhesion, which leads to local invasion and ultimately tumor development.
Keywords: Oral squamous cell carcinoma, E-cadherin, β-catenin, Immunohistochemistry. |
INTRODUCTION
Oral cancer ranks the sixth most common cancer among all types of cancers worldwide. India has the largest number of oral cancer cases and contributes one-third of the total burden of oral cancer globally (Borse, V. et al., 2020). Around 77,000 new cases and 52,000 deaths has been reported annually in India, which constitutes approximately one-fourth of global incidences (Laprise, C. et al., 2016). The increasing number of oral cancer cases are the most important concern for community health as it is one of the common types of cancers in India (Sharma, S. et al., 2018).
Oral squamous cell carcinoma belongs to a larger subgroup of tumours termed as head and neck squamous cell carcinomas (HNSCC) and they constitute about 90% of that category among all head and neck cancers. Oral malignancy mostly affects middle and elderly people with male predominance (Kaur, G. et al., 2009).
Oral malignancies gradually progress from normal epithelium, via precursor stages, to invasive and metastatic cancers .Oral cancers often develop from precancerous lesions such as Leukoplakias, erythroplakias and submucous fibrosis (Leemans, C. R. et al., 2011). These often present as identifiable red (erythroplakia) or white (leukoplakia) patches, where development of leukoplakias in the tongue and floor of the mouth are associated with higher risk of malignant transformation (Neville, B. W., & Day, T. A. 2002).
E-cadherin:
It is a calcium dependent transmembrane glycoprotein which is encoded by the CDH1 gene located on chromosome 16q21. It is expressed in most epithelial cells (Gall, T. M., & Frampton, A. E. 2013). E-cadherin play a major role in establishing cell polarity and in maintaining normal tissue architecture. It has a long cytoplasmic and extracellular domains which create interactions between adjacent cells to allow adhesion (Scanlon, C. S. et al., 2013). The intracellular domain of E-cadherin molecules are linked to the actin cytoskeleton via α-catenin and β-catenin (Attramadal, C. G. et al., 2015). This protein complex leads to the formation of cell to cell adhesion in epithelial tissues and prevents the cell dissociation which is required for tumor invasion and metastasis.
β-catenin:
It is a dual function protein that regulates the coordination of cell-cell adhesion and gene transcription. It is a protien that in humans is encoded by the CTNNB1 gene (MacDonald, B. T. et al., 2009). It helps in the cell-cell adhesion by controlling cadherin-mediated cell adhesion at the plasma membrane and by mediating the interplay of adherens junction molecules with the actin cytoskeleton (Brembeck, F. H. et al., 2006).
MATERIALS AND METHODS:
Study Period:
The present study includes 71 cases of oral squamous cell carcinoma diagnosed histopathologically in the department of Pathology, JNMCH, AMU, Aligarh. Normal buccal mucosa was taken as positive control and tonsil lymphoid cells as negative control.
Histopathological Evaluation:
Biopsies/specimens of all suspected cases of oral squamous cell carcinoma were fixed in 10% neutral buffered formalin. Paraffin embedded tissue blocks were prepared. Histopathological diagnosis was made on hematoxylin and eosin (H&E) stained sections and typed into different grades of oral squamous cell carcinoma.The selected paraffin embedded tissue blocks were subjected for immunohistochemistry (IHC) procedure.
Immunohistochemistry Procedure:
Immunohistochemistry was performed on paraffin embedded tissue sections (3-4µ in thickness) on poly l lysine coated slides. Sections were deparaffinized in xylene and rehydrated by descending grades of ethyl alcohol (95%, 80%, 70% and 50%). Antigen retrieval was done by immersing the slides in pre-warmed citrate buffer (pH-6.0) and heating in microwave oven at 95-98ºC for 20 minutes. Blocking of endogenous peroxidase was done by dipping slides (kept in moist chamber) in mixture of 50ml methanol with 1.5 ml hydrogen peroxide. Primary ready to use antibody (Thermo Scientific monoclonal rabbit anti-human E-cadherin antibody; clone:EP700Y) and Thermo Scientific polyclonal rabbit antihuman β-catenin antibody; clone:E247) in dilution of 1:50-500 was added to the sections and slides were incubated for 60 minutes at room temperature for E-cadherin and for 30 minutes at room temperature for β-catenin. Secondary antibody (Horse radish peroxidase) was added to the sections and incubated for 30 minutes. After washing in wash buffer, Diaminobenzidine (DAB) chromogen was added to the sections for 10 minutes and slides were counterstained with hematoxylin. Slides were then dehydrated by dipping in ascending grades of ethyl alcohol and finally mounted in DPX.
Evaluation of Immunohistochemistry:
The evaluation of the expression of E-cadherin and β-catenin were visually estimated under microscope using IHC immunoreactivity score (IRS), which is a semiquantitative method based on the proportion of positive stained tumor cells and the intensity of staining. IRS= proportion positive score x intensity score. The proportion of positive staining was scored as 0 if < 20% of cells were positive, 1 if 21-40 % of cells were positive, 2 if 41-60% were positive, 3 if 61-80% were positive and 4 if > 80% cells were positive. The intensity was scored as 0 for absence of staining, 1+ for weak intensity staining, 2 + for moderate intensity staining, 3+ for strong intensity staining. The total score ranges between 0-12. Immunoreactivity was divided into three groups on the basis of final score: a total score of 0 is considered as negative immunoreactivity score, 1-4 score as low immunoreactivity score and >4 as high immunoreactivity score.
Statistical Analysis:
All the qualitative variables were analysed using Pearson Chi Square and Fisher Exact Test and all the quantitative variables were analysed using Kruskal Wallis one way ANOVA test. Along with these, Pearson product moment Correlation analysis was also used to see the correlation between E-cadherin and β-catenin expression in OSCC. A p value of <0.05 was considered significant.
RESULTS:
Clinicopathological Characteristics:
A total of 71 cases of OSCC were analysed, of which 35 cases were of well differentiated squamous cell carcinoma (WDSCC), 25 cases of moderately differentiated squamous cell carcinoma (MDSCC) and 11 cases of poorly differentiated squamous cell carcinoma (PDSCC). Males were most commonly affected (86%) than females (14%). Majority of the patient belongs to 4th and 5th decade with mean age group of 47.49 ± 12.89. Tongue was the most common site of involvement occuring in 35 (49.2%) cases followed by buccal mucosa with 25 cases (35%).
Immunohistochemical Analysis:
Immunostaining by E-cadherin and β-catenin showed positivity of different intensity in different grades of squamous cell carcinoma. The normal stratified squamous epithelium of the oral mucosa showed strong, continuous E-cadherin membranous positivity (Fig. 1 A) and strong β-catenin membranous and cytoplasmic positivity (Fig. 2 A).
Table 1: E-cadherin Expression in different grades of OSCC
|
Grade of OSCC |
E-cadherin Immunoreactivity Score |
||||||||
|
Total Cases No. |
Negative (0) |
Low (1-4) |
High (5-12) |
||||||
|
Cases |
(%) |
Cases |
(%) |
Cases |
(%) |
||||
|
WDSCC |
35 |
0 |
0.00 |
3 |
8.57 |
32 |
91.43 |
||
|
MDSCC |
25 |
2 |
8.00 |
19 |
76.00 |
4 |
16.00 |
||
|
PDSCC |
11 |
3 |
27.27 |
8 |
72.73 |
0 |
0.00 |
||
|
Total |
71 |
5 |
7.04 |
30 |
42.25 |
36 |
50.70 |
||
p-values: a:b < 0.001 ; a:c < 0.001 ; b:c > 0.05
E-cadherin immunohistochemistry showed high immunoreactivity in 91.43% cases of WDSCC (Fig. 1 B) and low immunoreactivity in only 8.57% cases. 76% cases was showing low immunoreactivity in MDSCC (Fig. 1 C). In PDSCC, 72.73 % cases showed low staining for E-cadherin (Fig. 1 D). Besides 27.27 % cases were totally negative for E-cadherin staining, thereby stating the loss of E-cadherin with de-differentiation or lower grade of tumor morphology. A statistical significant difference in E-cadherin expression was noted on comparing well differentiated carcinoma with moderately differentiated carcinoma, p-value < 0.001; and well differentiated carcinoma with poorly differentiated carcinoma, p-value < 0.001. However, no statistical significance (p value > 0.05) was found in E-cadherin expression amongst moderately differentiated carcinoma and poorly differentiated carcinoma. (Table 1)
Figure Image are available at PDF file
In WDSCC, β-catenin immunoreactivity was high in 77.14% cases (Fig. 2 B) and low in 22.86% cases, but none of the case showed negative immunoreactivity. 56% cases of MDSCC showed low immunoreactivity (Fig. 2 C) While in PDSCC, 81.82% casesshowed low immunoreactivity and 9% cases were showing absent staining for β-catenin (Fig. 2 D). Thereby stating the fact that as the tumor progresses to a lower grade, there is loss of β-catenin expression. A statistical significant difference in β-catenin expression was noted on comparing well differentiated carcinoma with moderately differentiated carcinoma, p-value < 0.01; and well differentiated carcinoma with poorly differentiated carcinoma, p-value < 0.001. However, no statistical significance (p value > 0.05) was found in β-catenin expression amongst moderately differentiated carcinoma and poorly differentiated carcinoma. (Table 2)
Figure Image are available at PDF file
Table 3: E-cadherin and β-catenin Mean IHC score
|
Grades of OSCC |
(E-Cadherin) |
(β-catenin) |
P-Value |
||
|
Mean IHC Score |
Standard Deviation |
Mean IHC Score |
Standard Deviation |
||
|
WDSCC |
8.57 |
±2.77 |
7.62 |
±3.03 |
<0.001 |
|
MDSCC |
3.76 |
±2.02 |
3.88 |
±2.22 |
|
|
PDSCC |
1.27 |
±1.27 |
2.27 |
±1.48 |
|
This table showed that E-cadherin and β-catenin Mean IHC score in well differentiated OSCC was 8.57±2.77 and 7.62±3.03, in moderately differentiated OSCC was 3.76±2.02 and 3.88±2.22 and in poorly differentiated OSCC was 1.27±1.27 and 2.27±1.48 respectively, showing a significant reduction in the mean IHC score from 8.57 to 1.27 and 7.62 to 2.27 as the tumor progresses from well differentiated to poorly differentiated OSCC. This difference in the expression of epithelial E-cadherin and β-catenin was statistically significant (p<0.001) between all the histopathological grades of oral squamous cell carcinoma. (Table 3)
Table 4: Comparision of OSCC with and without lymph node metastases
|
Groups |
E-Cadherin |
P-Value |
β-catenin |
P-Value |
||||
|
<=4 (%) |
5 to 12 (%) |
<=4 (%) |
5 to 12 (%) |
|||||
|
OSCC without Lymph node metastasis [n= 42] |
16 (22.5) |
26 (36.6) |
0.031 |
20 (28.2) |
22 (31.0) |
0.733 |
||
|
OSCC with Lymph node metastasis [n= 29] |
19 (26.8) |
10 (14.1) |
15 (21.1) |
14 (19.7) |
||||
This table showed that there was a significant difference in E- cadherin expression at the primary site of OSCC, with or without lymph node metastases but no significant difference observed in β-catenin expression. (Table 4)
Table 5: The Relation between the Expression of E-cadherin and beta-catenin
|
Pearson Correlation- Coefficient |
E-cadherin (IRS Score) |
E-cadherin (IRS Score) |
Β-catenin (IRS Score) |
|
1 |
.559** |
||
|
P-Value |
P < 0.01 |
When correlation test was performed it was found that E -cadherin (IRS Score) was positively corelated with β-catenin (IRS Score) means E -cadherin (IRS Score) was found directly proportional to β-catenin (IRS Score) with significant results (r = 0.559, P < 0.01) (Table 5)
DISCUSSION:
Oral cancer is the most prevalent cancer in the world, accounting for over 30% of all cancers in India (Jetley, S. et al., 2021). Despite improvement in diagnostic and therapeutic methods, morbidity and mortality rates of oral squamous cell carcinoma have not improved much. Adhesion molecules play a central role in pathogenesis and progression of malignant tumours (Fillies, T. et al., 2005). Therefore, it is important to evaluate the role of cell adhesion molecules like E-cadherin and β-catenin in tumour metastasis of oral squamous cell carcinoma.
In the present study, we found well differentiated SCC constituted the maximum number of cases (49.3%) followed by moderately differentiated SCC (35.2%); similar to the study done by Iype et al., 2001 who found 52.6% well-differentiated tumors, 34.2% moderately differentiated and 4.2% poorly differentiated tumors (Iype, E. M. et al., 2001). Jetley et al., 2021 in their study also found 72% patients had well differentiated, 24% had moderately differentiated, and only 2% had poorly differentiated oral squamous cell carcinoma (Jetley, S. et al., 2021).
The present study revealed increase prevalence of oral OSCC in the males with 61 cases (85.9%) as compared to females with only 10 cases (14.1%) with male to female ratio of 6.1:1. Sharma et al., 2010 have also observed a male: female ratio of 2.2:1 in OSCC cases in their study (Sharma, P. et al., 2010).
Amongst the 180 cases of OSCC, maximum numbers of patients were found in the age group of 41-50 years with mean age of 47.49 ±12.89 years. Sharma et al., 2010 in their study, have observed an increased prevalence of OSCC cases in the 4th and 5th decade of life (Sharma, P. et al., 2010). Johnson, N. 2005 stated that oral cancer is a disease generally affecting individuals with an average age of 60 years (Johnson, N. 2005).
Tongue (49.3% cases) was found to be the most commonly involved site followed by buccal mucosa (35.2%) in our study, which could be due to increased exposure to various risk factors like tobacco chewing, smoking and betel nuts in the Indian population. These findings are in concordance with those of Mehrotra et al., (2003) who reported the most common sites as tongue (42.57%) followed by cheek (19.14%) (Mehrotra, R. et al., 2003).
In the present study, E-cadherin immunohistochemistry showed high immunoreactivity in 91.43 % cases, and low immunoreactivity in only 8.57% cases. In poorly differentiated carcinoma 72.73 % cases showed low immunoreactivity and 27.27 % cases were totally negative for E-cadherin staining. While none of the case showed high immunoreactivity for E-cadherin. So, we noted that degree of E-cadherin expression decreased as the grade of the tumor was increased.
Kaur et al., 2009 studied E-cadherin expression in different histological grades of oral squamous cell carcinoma and reported strong expression in 90% WDSCC , 92.9% MDSCC and 15.4% PDSCC as compared to weak and homogenous staining showed by 10%, 7.1% and 69.2% cases respectively.WDSCC expressed E-cadherin often as strongly as normal stratified squamous epithelium, while in poorly differentiated squamous cell carcinoma expression of E-cadherin was lost or cytoplasmic, and in moderately differentiated tumors it was expressed in a heterogeneous fashion. E-cadherin immunoreactivity was found to inversely correlate with the loss of cell differentiation (Kaur, G. et al., 2009).
We found high β-catenin immunoreactivity in well differentiated carcinoma 77.14% cases and low in 22.86% cases but none of the case showed negative immunoreactivity. While in poorly differentiated carcinoma, 81.82% cases showed low immunoreactivity and only 1 case (9.09%) each showed high and negative immunoreactivity. Thereby, it supports the fact that as the tumor grade increased, there is loss of β-catenin expression and depicts a poor prognosis. Similar study done by Zaid, K. W. 2014 also documented β-catenin expression in different histological grades of oral squamous cell carcinoma and reported the expression of β-catenin in 87.3% of the cells in WDOSCC, 68.7% of MDSCC and 43.4% of PDSCC (Zaid, K. W. 2014).
In the present study, Lymph node metastasis was present in 29 (40.8%) cases of squamous cell carcinoma. Out of these, 19 (26.8%) cases showed low immunoreactivity and 10 (14.1%) cases showed high immunoreactivity for E-cadherin expression which was statistically significant. While, 15 (21.1%) cases showed low immunoreactivity and 14 (19.7%) cases with high immunoreactivity for β-catenin expression,which was statistically non-significant. Rodrigo et al., 2002 in a study on 101 patients with supraglottic laryngeal carcinoma found reduced E-cadherin expression which correlated with the presence of nodal metastatic disease (Rodrigo, J. P. et al., 2002). However, studies by Wang et al., 2009 and Bukholm et al., 1998 did not find any significant relationship between patients of oral squamous cell carcinoma with lymph node metastasis (Wang, X. et al., 2009; Bukholm, I. K. et al., 1998).
The present study showed a statistically significant difference in the expression of E-cadherin in patients who had lymph node metastasis showing decreased expression in these patients. However, there was no statistically significant expression of β-catenin in patients with lymph node metastasis. Study done by Balasundaram et al., 2014 who also analysed the down regulation of molecular markers E-cadherin and β-catenin in oral squamous cell carcinoma. However, they did not show any significant difference in the expression of E- cadherin as well as β-catenin in oral squamous cell carcinoma with and without lymph node metastases (Fadare, O. et al., 2005).
It is likely that these discrepancies may be due to variation in the experimental model or tissue used, clinico-pathological characteristics of the patients, sample size, and methodology.
In the present study, as for the relationship between E-cadherin and β-catenin, the expression of β-catenin was also found to be aberrant when aberrant expression of E-cadherin was present, and the expression of β-catenin was also maintained when the expression of E-cadherin was normal. Hence, the expression pattern of β-catenin depicted a very close relationship with the pattern of E-cadherin expression. Thereby, this finding suggests some connection between the direct down-regulation of the cadherin/catenin complex and metastasis of oral squamous cell carcinoma. The E-cadherin and β-catenin proteins are directly linked to each other so that abnormality in these proteins suggests disruption of cell-cell adhesion that might lead to tumor metastases. Zaid, K. W. 2014 demonstrated that the expression of E-cadherin and β-catenin was significantly correlated with the histological degree of oral squamous cell carcinoma (Zaid, K. W. 2014).
Loss of expression of both the proteins is of prognostic significance with respect to the histologic grades of oral squamous cell carcinoma, revealing the fact that as the tumor progress to a lesser differentiation, loss of expression occurs. We can suggest from our study that invasiveness and metastasis could be analysed by use of immunohistochemical analysis of E-cadherin and β-catenin which can help in predicting the tumor behaviour, prognosis, survival and management of the patient.
We can use these biomolecules as biomarkers for more research on the microinvasion of the tumor for early diagnosis and better treatment modality and survival of the patients.
CONCLUSION:
The suppression of E-cadherin and β-catenin expression is considered as one of the main molecular events responsible for dysfunction in cell-cell adhesion, which leads to local invasion and ultimately tumor development. The E-cadherin–catenin complex plays a central role in cellular adhesion and loss of this function results in increased invasiveness and tumor metastasis.
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