Asmaa F.doc J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 36 Immunohistochemical expression of E-cadherin and CD44 adhesion molecules in oral squamous cell carcinoma Asmaa S. Al janabi, B.D.S. (1) Seta A. Sarkis, B.D.S, M.Sc., Ph.D. (2) ABSTRACT Background: Head and neck squamous cell carcinoma is the sixth most common cancer world wide. Despite greater emphasis on multi-modality therapy including surgery, radiation and chemotherapy, advanced stage head and neck squamous cell carcinoma continues to have poor 5-year survival rates (0-40%) that have not significantly improved in the last (30) years. To improve outcomes for this deadly disease , It is required a better understanding of the mechanisms underlying head and neck squamous cell carcinoma tumor growth, metastasis, and treatment resistance. This study evaluates the Immunohistochemical expression of E-cadherin and CD44 adhesion molecules in OSCC and to correlate the expression of either marker with each other, with lymph node metastasis and with tumor grade. Materials and methods: Thirty blocks of OSCC were included in this study. An immunohistochemical staining was performed using anti E-cadherinand anti CD44 monoclonal antibodies. Results: Negative immunohistochemical expression of E-cadherin was found in(66.7%)of the cases and only (33.3%)revealed positive immunoexpression. Positive CD44 immunoreaction was seen in(86.7%)of the cases. There was no statistically significant correlation regarding either marker with respect to the tumor stage, grade and lymph node matastasis. Moreover anon-significant correlation was found between the expression of both markers. Conclusions: this study revealed negative E-cadherin expression in two thirds of the cases, while positive CD44 was illustrated in most of them. Non- significant correlation was found regarding the expression of both markers with tumor stage, grade and lymph node status. Inverse significant correlation was found regarding CD44 expression with the clinical presentation of the study sample. In addition, non significant correlation was found between the E-cadherin and CD44 immunoexpression. Key words: Oral squamous cell carcinoma, Adhesionmolecules, E-cadherin, CD44, Immunohistochemistry. (J Bagh Coll Dentistry 2013; 25(Special Issue 1):36-42). الخالصة بالرغم من التاكیدالشدیدعلى العالج المتعدد االشكال الذي یشمل الجراحة،االشعاع والعالج الكیمیائي،مراحل متقدمة من . سرطان الخالیا الحرشفیة للراس والرقبة ھو السادس االكثرشیوعا في كل العالم:الخلفیة ولتحسین النتائج لھذا المرض الممیت یحتاج الى فھم . والذي لم یتقدم بشكل مفید في السنوات الثالثین االخیرة%) 40-0(سنوات للبقاء ھزیل) 5(سرطان الخالیا الحرشفیة للراس والرقبة یواصل امتالكھ مستوى .افضل لاللیة وراء نمو ورم سرطان الخالیا الحرشفیة للراس والرقبة،االنبثاث ومقاومة العالج موقعھ على سطح الخالیا الطالئیة في مناطق تماس خلیة مع خلیة تعرف 0من اھم الجزیئات اللتصاق خلیة مع خلیة في االنسجة الظھاریة على الظھاروھو واحدمحافظة ولللتكوین ي لكادھرین ضرور -االي .بالرباط االلتصاقي .امكانیة االنبثاث من قبل الخالیا السرطانیةین الذي یكون التصاق الخالیا یرتبط بفقدان تشكل الظھار مع اكتساب ركادھ -في اورام االنسان، فقدان االي مولد المضاد في معظم انسجة االنسان وعرف بامتالكھ وظائف متعددة منذ وجد . ھوجزیئة في غشاء الخلیة الذي وجد اوال في الخلیة اللمفیة وعرف ابتدائیا بامتالكھ لوظائف لصق وایواء الخلیة) 44( - السي دي ربطت بتقدم الورم ) 44( -سي دي -اشارة حامض الھیالورونیك. ا یتعلق بدوره في االسھام بتقدم الورم في مختلف االورام الصلبة بما في ذلك سرطان الخالیا الحرشفیة للراس والعنقوقد درس فیم. اكتشافھ االول .تراق واالنبثاثخبما في ذلك عملیة اال الالصقة في سرطان الخالیا الحرشفیة للفم وربط ظھور كل منھما بمرتبة الورم واالنبثاث للعقد ) 44(- كادھرین والسي دي-ي المناعي لجزیئات االيیم وربط الظھور الكیمیائي النسیجیھذه الدراسة الى تقتھدف .اللمفیة جرى صبغ . لمقاطع النسیجیة المثبتة بالفورمالین والمطمورة بشمع البارافینالشخاص مصابین بسرطان الخالیا الحرشفیة للفم والتي استخرجت من ا استرجاعیة تضمنت ھذه الدراسة ثالثین عینة:المواد والطرق على شرائح نسیجیة ) 44( -كادھرین ومضاد السي دي -اجریت الصبغات الكیمیائیة النسیجیة المناعیة باستخدام مضاد االي. كل عینة بالھیماتوكسلین واالیوسین العادة تقییمھا لغرض الفحص النسیجي المرضي .یقة من العیناتدق :عینة من سرطان الخالیا الحرشفیة للفم كمایلي) 30(نتائج ظھرت :النتائج .1:2مع نسبة الذكور الى االناث %) 70(سنة ومعظم الحاالت ھم من الذكور) 50(سجلت اكثرالحاالت في االعمارمافوق • .%)50(ومعظمھا ظھرت سریریا بشكل تقرح %) 7,36(وجدت معظم الحاالت في اللسان • .واضحة التماییز%) 30(متوسطة التماییز وفقط من الحاالت %) 70(الفحوصات النسیجیة المرضیة اظھرت ان • .كادھرین-اظھرت ایجابیة الظھورالمناعي لالي%) 3,33(وفقط %) 7,66(في ظھرت كادھرین -سلبیة الظھورالكیمیائي النسیجي المناعي لالي • وفقط ) 1(اظھرت الدرجة %) 7,6(، )2(اظھرت الدرجة %) 7,26(، ) 3(اظھرت الدرجة %) 7,46(من بینھا من الحاالت %)7,86(في )44(نسیجي المناعي للسي دي لوحظ ایجابیة الظھورالكیمیائي ال • .)44( -من ایجابیة االصطباغ المناعي للسي دي) 4(اظھرت الدرجة %) 7,6( عالوة على ذلك لم یوجد أي ارتباط معنوي بین الظھورلكال المؤشرتین الحیویتین مع . مرحلة الورم واالنبثاث للعقد اللمفیةو شرتین الحیویتین ودرجةي معنوي بین أي من المؤلم یكن ھناك اي ارتباط احصائ • .بعضھما البعض ارتباط وجد . في معظم الحاالت لسرطان الخالیا الحرشفیة للفم) 44( -المناعي للسي دي ایجابیة الظھور وجدتكادھرین في ثلثي الحاالت بینما -سلبیة الظھورالمناعي لالي ھذه الدراسة اظھرت :االستنتاجات الضافة با. مع الظھور السریري لعینات الدراسة) 44( -عالقة معنویة عكسیة فیما یخص ظھور السي ديوجدت . وحالة العقداللمفیةمرحلة الورم وة المعنوي فیما یخص ظھوركال المؤشرتین الحیویتین مع درج .في الحاالت المدروسة) 44( - كادھرین لم یؤثر معنویا في الظھور المناعي للسي دي-الظھور المناعي لالي فأن الى ذلك INTRODUCTION Over 90% of oral cancers (OCs) are squamous cell carcinomas (SCCs). They constitute a major health problem in developing countries, representing a leading cause of death. The survival index continues to be small (50%), as compared to the progress in diagnosis and treatment of other malignant tumors. (1) Master student. Department of Oral Diagnosis, College of Dentistry, University of Baghdad (2) Assistant Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad This is because patients continue to die from metastatic diseases at regional and distant sites (1). Neoplasia or cancer is viewed as a cell cycle disease. Although this concept implies that every tumor is defective in one or more aspects of the cell cycle control, it clearly does not mean that oncogenesis targets only oncogenes and the cell cycle clock. Development of malignancy appears to require also aberrations in the cell death machinery and cell-cell and/or cell-matrix interactions that cooperate with cell cycle defects. J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 37 Many of the processes in which adhesion molecules play central role – anchorage dependent growth, apoptosis, differentiation, and migration are those that are characteristically dysregulated in malignancy (2). Adhesion molecules (AM) are transmembrane glycoproteins acting as a molecular link between the outside and inside of the cell. The adhesion molecules are involved in the cell differentiation, migration and sorting. Broadly, these proteins can be classified into five families including immunoglobulin superfamily, integrins, cadherins, selectins, and CD44(3). Alterations of these cell adhesion molecules are a common event in cancer. The disrupted cell-cell or cell-ECM adhesion significantly contributes to uncontrolled cell proliferation and progressive distortion of normal tissue architecture. More importantly, changes in cell adhesion molecules play a causal role in tumor dissemination. Loss of cell adhesion contacts allows malignant cells to detach and to escape from the primary mass (4,5). E-cadherin, a calcium-dependent cell adhesion molecule,is a cell membrane-associated protein involved in cell–cell adhesion, and loss of expression of the cadherin/catenin complex has been described in various human malignancies (6). Changes or alterations in the function and expression of this cell to cell adhesion molecule have been postulated to be an early event in the multiple step process of tumour metastasis and an important factor in tumour progression(7). The loss of cadherines expression was observed in many types of carcinomas and usually it is associated to late stages of the disease and to the progression of malignant epithelial neoplasias (8). CD44 was first described by Dalchau et al. as a molecule present on the surface of T- lymphocytes, granulocytes, and cortical thymocytes (9). Human CD44 is a transmembrane hyaluronan-binding glycoprotein that can bind to hyaluronic acid, an extracellular matrix, and regulate a variety of cellular functions, such as cell migration, proliferation, cell–cell interaction, and apoptosis (10). These cellular functions of CD44 imply that a disorder of CD44expression plays a crucial role in the behavior of a malignant tumor (11). CD44 plays an important rolein metastases. In OSCC, decreased immnoexpression is associated with increased invasive potential of tumors and the presence of metastases (12, 13). Since (AM) are involved in many fundamental processes of the cell involving normal physiological growth and development as well certain pathological conditions (wound heals, inflammation and neoplasia) and the loss of their expression or disordered expression plays important roles in the behavior of malignant tumors, therefore, this study concerned E- cadherin and CD44 adhesion molecules to elucidate their role in OSCC development and progression. MATERIALS AND METHODS Thirty formalin-fixed paraffin-embedded tissue blocks of OSCC were collected the archieves of the Department of Oral Diagnosis ⁄ College of Dentistry ⁄ Baghdad University;Al- Shaheed Ghazi hospital/Medical city/Baghdad; and private laboratories in Baghdad and Najaf, dated from(2000-2012). Four-micrometer-thick sections were cut from each paraffin tissue block and stained with hematoxylin(Mayer′s) and eosin for diagnostic confirmation and histological grading. Another two 4-µm section was cut from each tissue block and mounted on positively charged slides (Fisher super frost,USA) to be stained with monoclonal antibodies to E-cadherin and CD44 (ABCAM). Negative and positive tissue controls were included into each immunohistochemical run (according to the manufacturer). Immunostaining Five micrometer thick sections were cut and mounted on (Fisher super frost, USA) positively charged slides, then deparaffinized and rehydrated for immunohistochemical staining by E-cadherin and CD44(ABCAM) monoclonal antibodies;Heat mediating Antigene Retrieval was done for CD44 using phosphate buffer PH(6) then the sections were immersed in hydrogen peroxide (H2O2) to block the endogenous peroxidase activity, washed in phosphate-buffered saline (PBS), and then protein blocking reagent and incubated for 20 minutes at 37 c within humid chamber to reduce non specific staining. The tissue sections were incubated with mouse monoclonal [5H9] anti- human E-cadherin antibody (diluted 1:10) and CD44 (diluted 1:50) antibodies for one hour at 37 c.After that the slides were kept in the refrigerator at 4 c over night in humid chamber. The bounded antibodies were detected by the streptavidin-biotin complex method, after an immunoreaction, the sections were counterstained with Hematoxylin (Mayer’s). Scoring system The scoring of the markers was done by examining of at least 1000 cells per section in five different representative fields. The membranous or membranous and cytoplasmic was considered positive for E-cadherin and membranous was considered positive for CD44 immunostaining. The percentage of positive cells was scored as J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 38 follows:score (0):<10% positive cells,score(1):10- 25%positive cells,score(2):25-50%positive cells,score(3):50-75%positive cells and score(4):>75%positive cells (14). Statistical analysis The data was compiled into statistical software, statistical package of social sciences (SPSS) version 17. All variables were compared using Chi- square test. While Pearson correlation coefficient was applied to plot a correlation matrix among the different immunohistochemical markers expression values altogether. P values of less than 0.05 were considered statistically significant. Anova test was carried out to compare the numerical values of the study samples, Spearman′s rho was also applied in order to find any possible correlation between the categorical variables of the study sample. RESULTS: The results of (30) oral squamous cell carcinoma cases were designed as follows: most of the cases (62%) aged > 50 years; the majority of the cases were males (70%) with male to female ratio 2:1.The most common site was the tongue (36.7%). Most of the cases presented clinically as ulcer (50%). Histopathological examination showed that (70%) of the cases were moderately differentiated and only (30%) were well differentiated carcinomas. Negative immunohistochemical expression of E-cadherin was found in(66.7%) of the cases and only (33.3%) revealed positive immunoexpression. Positive CD44 immunoreaction was seen in (86.7%) of the cases, of which (46.7%) presented score (3), (26.7%) score (2), (6.7%) score (1) and only (6.7%) presented score (4) CD44 positive immunostaining. Table 1: Age and sex distribution of the study sample Age No. % 50> 18 62 50≤ 11 37 29* 99 sex Male 21 70 female 9 30 30 100 *1 case the age was not recorded. Table 2: E-cadherin IHC expression in OSCC cases E-cadherin Score* Frequency Valid Percent Valid 0 20 66.7 1 7 23.3 2 2 6.7 3 1 3.3 Total 30 100.0 *score 0:<10% positive cells,score 1:10-25% positive cells,score 2:25-50%positive cells,score 3:50-75% positive cells,score 4:>75% positive cells. Figure 1: Positive brown membranous and /or cytoplasmic immunostaining of E- cadherin in well differentiated OSCC- Buccal mucosa(400X). Figure 2: Positive brown membranous immunostaining of E-cadherin in Moderately-differentiated OSCC-Maxilla (400X). Table 3: CD44 IHC expression in OSCC cases CD44 score * Frequency Percent Valid 0 4 13.3 1 2 6.7 2 8 26.7 3 14 46.7 4 2 6.7 Total 30 100.0 *score 0:<10% positive cells,score 1:10-25% positive cells,score 2:25-50% positive cells,score 3:50-75% positive cells,score 4:>75% positive cells. J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 39 Figure 3: Positive brown membranous immunostaining of CD44 in well differentiated OSCC –Buccal mucosa (400X). Figure 4: Positive brown membranous immunostaining of CD44 in Moderately differentiated OSCC –Buccal mucosa (400X). Table 4: The correlation of E-cadherin & CD44 expressions. CD44 scores E- cadherin scores CD44 scores Pearson Correlation 1 0.124 Sig. (2-tailed) *0.513 N 30 30 E- cadherin scores Pearson Correlation 0.124 1 Sig. (2-tailed) *0.513 N 30 30 *p value more than 0.05 is considered non- significant Table 5: Correlation of CD44 and E-adherin scores with the clinical presentation using Spearman's rho. scores Sperman's rho clinical presentation CD44 score r Sig. (2-tailed) N **-.419 0.033* 26 E-Cadherin score r Sig. (2-tailed) N -.277 0170 26 * Correlation is Significant at the 0.05 level (2-tailed) . ** negative(-ve) indicates a reverse correlation . There was no statistically significant correlation regarding either markers with respect to the tumor grade, lymph node status and stage .Moreover a statistically non significant correlation was found between the expressions of both markers. DISCUSSION This study is not a large epidemiological one that expressed the incidence and prevalence of different clinicopathological features of OSCC. The clinicopathological information were evaluated and analyzed for only (30) OSCC surgical specimens, however, there is a close correlation between the present data and other published data concering the incidence of OSCC in previous foreign and Iraqi studies records. Assessment of E-cadherin immunohistochemistry The results of this study showed reduction in the immunoexpression of E-cadherin in (66.7%) of the cases, this is in agreement with the findings of Williams et al. who found E-cadherin underexpression in carcinoma in situ cases and infiltrative tumors and Santos et al. who recorded E-cadherin underexpression in (90%) of oral squamous cell carcinoma cases (15,16). Generally E-cadherin is expressed as membranous immunostaining, but cytoplasmic expression (a translocation of this marker into the cytoplasim) was detected in some cases of the study sample such finding was also found by Massarelli et al. and Aguiar et al. who observed higher cytoplasmic expression of E-cadherin in OSCC with nodal metastasis(17,18). In fact, a redistribution of the E-cadherin complex out of tight junctions can affect its functions in cell-cell adhesion and increase its degradation by cytoplasmic endocytosis resulting in cytoplasmic E-cadherin expression(19). This study revealed a non-significant correlation regarding E-cadherin expression with any of the clinicopathological data including the tumor stage, grade and lymph node involvement. Generally, more aggressive oral carcinomas show loss of epithelial cell cohesion, and this is often associated with a reduction in E-cadherin expression. However, loss of cohesion may also be due to reduced E-cadherin function as a result of sequence mutation or by abnormalities in the cadherin-catenin complexes. Thus, E-cadherin may still be detected by immunohistochemistry, even in non-functional form, and this possibly explains some of the apparently conflicting results produced by immunohistochemical studies on oral carcinoma. Furthermore, Several factors such as the sample size, methods of histological grading of J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 40 malignancy, the visual judgment of pathologists ,type of antibody used, immunohistochemical techniques, the choice whether to use frozen or paraffin embedded material, relative subjectivity in interpreting and scoring the staining results and the cellular heterogeneity of OSCC may also be responsible for those conflicting results(20). Assessment of CD44 immunohistochemistry The expression of different CD44 isoforms in HNSCC has been studied, but their role remained controversial. Whereas some studies have found a correlation between increased CD44 expression and HNSCC progression, others have reported no such correlations or negative correlations (21). In the present study isoform (10)epithelial isoform is used, but regardless to its type, the study focused on the tissue specificity of the marker namely epithelial cells in which CD44 expressed in carcinomas was indicated according to the manufacturer data sheet. The results of this study showed that most of the studied cases(86.7%) presented positive CD44 immunostaining ,of which (46.7%) revealed score (3)immuno expression, and only(13.3%) of the studied cases were negative.Similar results reported in other studies in different cancers including head and neck, breast ,lung ,gastrointestinal, bladder, cervical carcinomas by employing immunohistochemical staining,RT- PCR and Northern blotting techniques and using different isoforms of CD44(22,23,24). In the present study no correlation was found between the histological grade of the tumors and the CD44 expression,this finding is in agreement with Herold-Mende et al and Van Hale who also found no correlation regarding CD44splice variants expression and any clinicopathological variables (25,26). However, Ue et al. found that the reduction in the expression of certain variants of CD44 was correlated with tumor cell differentiation in primary OSCC cases(27). Concerning the relation of CD44 expression with the tumor stage, A non –significant correlation was found . Similar results recorded by Van Hal et al. and Kanke et al. who found (96%) CD44v6 immunoexpression in HNSCC, with no correlation to the tumor stage (26,28). While other investigators revealed that CD44 play crucial role in tumor progression and its expression is correlated well to the tumor stage (29-31). Regarding the relation of CD44 immunoexpression with lymph node involvement, this study showed a non-significant correlation, As mentioned previously, this finding may be due to the small size of the node positive cases enrolled in this study (14 out of 30). Another investigators found direct correlation between increased or decreased expression of CD44variant isoforms with lymph node involvement and development of metastasis within different kinds of tumors in different organs(32,33) . The present study revealed an inverse significant correlation between CD44 immunostianing and the clinical presentation of the studied cases. No previous studies highlighted such correlation to compare with; however, this inverse correlation suggests that reduced CD44expression is associated with increased tumor aggrasiveness (ulcer), while increased expression of CD44 is associated with decreased tumor aggressiveness (mass). There are some possible explanations for the discrepant results among different studies regarding the correlation between CD44expression and the clinicopathological presentation, these could be, the employment of different antibodies (CD44 different isoforms), which makes comparison between research groups difficult.Moreover, certian CD44 variant domain epitopes may become hidden and not regognized by some antibodies due to post -translational changes which alter the three-dimensional conformation of the protein.In addition, assessment of immunostaining positivity is dependant on what region of the tumor is examined, size of the study sample, method used for assessment of CD44 expression (RT-PCR, FISH, Immunohistochemistry ….etc), techniques of immunohistochemistry (manual or automated) (34,35,36). Assessment of the correlation between E- cadherin and CD44 immunohistochemical expression: Regarding the correlation between both markers, the results revealed a non-significant correlation between them, i.e,each marker acts independently(each marker works alone) . Furthermore, either markers showed no correlation to tumors stage, grade and lymph node status.Similar results revealed by (Carmen et al. and Vazifeh et al., IVSL) (36,37).However,different findings revealed by Simionescu et al.(14). Indeed, the cellular and molecular processes involved in malignant neoplasms are complex. Further studies are required to clarify the role of E- cadherin and CD44 AMs in the development and progression of OSCCs. Fortunately, the progress in the area of adhesion molecules is expected to be rapid in the following years. This may result in novel prognostic and therapeutic tools in the problematic field of head and neck cancer. It J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 41 seems that this interesting journey is long and we are just at the beginning. REFERENCES 1. Pantel K, Brankenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004; (6):448-456. 2. Lukas Z, Dvofiak K. Adhesion Molecules in Biology and Oncology. Acta Vet Brno 2004; 73: 93-104. 3. Nair KS, Naidoo R, Chetty R. Expression of cell adhesion molecules in oesophageal carcinoma and its prognostic value. J Clin Pathol 2005; 58:343-51. 4. Okegawa T, LiY, Pong RC, Hsieh JT. Cell adhesion proteins as tumor suppressors. J Urol 2002; 167:1836- 43. 5. Keleg S, Büchler P, Ludwig R, Büchler MW, Friess H. Invasion and metastasis in pancreatic cancer. Mol Cancer 2003; 2:14. 6. Kurtz KA, Hoffman HT, Zimmerman MB. Decreased E-cadherin but not beta-catenin expression is associated with vascular invasion and decreased survival in head and neck squamous carcinomas. Head Neck Surg 2006; (1): 142–146. 7. Weinberg RA. The Biology of Cancer. Garland Science; 2009. 8. Zhong LP, LI J, Zhang CP, Zhu HG, Sun J, Zhang ZY. Expression of E-cadherin in cervical lymph nodes from primary oral squamous cell carcinoma patients. Arch Oral Biol 2007; 52(8):740-747. 9. Dalchau RJ. Kirkley JW, Fabre. Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat. Eur J Immunol 1980; 10:737– 744 (IVSL) 10. Sato S, Miyauchi M, Takekoshi T. Reduced expression of CD44 variant 9 is related to lymph node metastasis and poor survival in squamous cell carcinoma of tongue. Oral Oncol 2000; 36:545–549. 11. Gonzalez-Moles MA, Gil-Montoya JA, Ruiz-Avila I, Esteban F, Delgado-Rodriguez M, Bascones-Martinez A. Prognostic significance of p21WAF1/CIP1, p16INK4a and CD44s in tongue cancer. Oncol Rep 2007; 18:389–396. 12. Carinci F, Stabellini G, Calvitti M, Pelucchi S, Targa L, Farina A, Pezzetti F, Pastore A. CD44 as prognostic factor in oral and oropharyngeal squamous cell carcinoma. J Craniofac Surg 2002; 13: 85–89. 13. Sato S, Miyauchi M, Kato M, Kitajima S, KitagawaS, Hiraoka M, Kudo Y, Ogawa I, Tata T. Upregulated CD44v9 expression inhibits the invasion of oral squamous cell carcinoma cells. Pathobiology 2004; 71(4):171–175. 14. Simionescu CL, Margaritescu M, Surpateanu L, Mogoanta R, Zavoi R, Ciurea P, Surlin A, Stepan. The study of E-cadherin and CD44 immunoexpression in Oral Squamous Cell Carcinoma. Romanian Journal of Morphology and Embryology 2008; 49(2):189-193. 15. Williams HK, Sanders DS, Jankowski JA, Landini G, Brown AM. Expression of cadherins and catenins in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med 1998; 27:308-17. 16. Santos-Garcia A, Abad-Hernandezm M, Fonsecasanchez E, Julian-Gonzalez R, Galindo- Villardon P, Cruz-Hernandezj J, Bullon-Sopelana A. E-cadherin, laminin and collagen IV expression in the evolution from dysplasia to oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal 2006; 11(2): 100–105. 17. Lin YC, Wu MY, Li DR, Wu XY, Zheng RM. Prognostic and clinicopathological features of E- cadherin, alpha-catenin, beta-catenin, gamma-catenin and cyclin D1 expression in human esophageal squamous cell carcinoma, World J Gastroenterol 2004; 10(22): 3235–3239. 18. Massarelli E, Erika B, Ngoc KT, Diane D Liu , Julie GI, Lee JJ, EL-Naggar A K, Waun KH, Vassiliki AP. Loss of E-cadherin and P27 Expression Is Associated with Head and Neck Squamous Tumorigenesis. American Cancer Society, Wiley Inter Science, January; 2005. 19. Aguiar Júnior FCA, Kowalski LP, Almeida OP. Clinicopathological and immunohistochemical evaluation of oral squamous cell carcinoma in patients with early local recurrence. Oral Oncol 2007; 43:593- 601. 20. Fujita Y, Krause G, Scheffner M. Hakai. A c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 2002; 4:222–231. 21. Georgolios A, Batistatou A, Manolopoulos L, Charalabopoulos K. Role and Expression patterns of E-cadherin in Head and Neck Squamous Cell Carcinoma (HNSCC). J Exp Clin Cancer Res 2006; 25(1). 22. Reategui EP, de Mayolo AA, Das PM, Astor FC, Singal R, Hamilton KL, Goodwin WJ, Carraway KL, Franzmann EJ: Characterization of CD44v3- containing isoforms in head and neck cancer. Cancer Biol Ther 2006; 5:1163–1168 23. Stoll C, Baretton G, Soost F, Terpe HJ, Domide P, Lohrs U. Prognostic importance of the expression of CD44 splice variants in oral squamous cell carcinomas. Oral Oncol1999; 35: 484-489. (IVSL) 24. Kahara N, Ozaki T, Doi T, Nishida K, Kawai A, Shibahara M, Inoue H. CD44 in soft tissue sarcomas. Virchows Arch 2000; 436: 574-578. 25. Ylagan LR, Scholes J, Demopoulos R. CD44: a marker of squamous differentiation in adenosquamous neoplasms. Arch Pathol Lab Med 2000; 124: 212-215. 26. Herold-Mende C, Seiter S, Born AI, Patzelt E, Schupp M, Zöller J, Bosch FX, Zöller M. Expression of CD44 splice variants in squamous epithelia and squamous cell carcinomas of the head and neck. J Pathol 1996;179:66–73 27. Van Hal NL, Van Dongen GA, Stigter-van Walsum M, Snow GB, Brakenhoff RB. Characterization of CD44v6 isoforms in head and neck squamous cell carcinoma. Int J Cancer 1999; 82: 837- 845. 28. Ue T, Yokozaki H, Kagai K, Higashikawa K, Yasui W, Sugiyama M, Tahara E, Ishikawa T. Reduced expression of the CD44 variant exons in oral squamous cell carcinoma and its relationship to metastasis. J Oral Pathol Med 1998; 27: 197-201. 29. Kanke M, Fujii M, Kameyama K, Kanzaki J, Tokumaru Y, Imanishi Y, Tomita T, Matsumura Y: Clinicopathological significance of expression of CD44 variants in head and neck squamous cell carcinoma. Jpn J Cancer Res 2000; 91:410–415 30. Sato S, Miyauchi M, Kato M, Kitajima S, KitagawaS, Hiraoka M, Kudo Y, Ogawa I, Tata T. Upregulated CD44v9 expression inhibits the invasion of oral squamous cell carcinoma cells. Pathobiology 2004; 71(4):171–175. J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Immunohistochemical Oral Diagnosis 42 31. García-Montesinos-Perea B, Val-Bernal JF, Saiz- Bustillo R. Epidermoid carcinoma of the lip: an immunohistochemical study. Med Oral Patol Oral Cir Bucal 2005; 10:454-461. 32. Kosunen R, Pirinen K, Ropponen M, Pukkila J, Kellokoski J, Virtaniemi R, Sironen M, Juhola E, Kumpulainen R, Johansson J, Nuutinen, Kosma. CD44 expression and its relationship with MMP-9, clinicopathological factors and survival in oral squamous cell carcinoma. Oral Oncol 2007; 43:51– 59.(IVSL) 33. Massano J, Regateiro FS, Januario G, Ferreira A. Oral squamous cell carcinoma: review of prognostic and predictive factors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102:67–76. 34. Lyons AJ& Jones J. Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma. Int JOral Maxillofac Surg (2007); 36 :671–679. 35. Mack B &Gires O. CD44s and CD44v6 expression in head and neck epithelia. PLoS One 2008; 3: 3360. 36. Carmen MF, Antonio LA, Fernanda F, Cassiano FW, Raimunda R, Roseana A, Lelia B, Leao PP. Immunohistochemical expression of E-cadherin and CD44v6 in Squamous Cell Carcinomas of the Lower Lip and Tongue. Braz Dent J 2009; 20(1):64-69. 37. Vazifeh L, Mostaan M, Khorsandi T, Shahriar- Mohammed RS, Fatemeh HS, Fatemeh M, Homa S, Reza B, Hasti B, Nasrin Y. Correlation between E- cadherin and CD44 adhesion molecules expression and cervical lymph node metastasis in oral tongue SCC:Predictivre significance or not. Cancer Research Centre, Mashhad University of Medical Sciences, Iran, Pathology –Research and Practice 2011; 207: 448-451.(IVSL)