Karawan F.docx J Bagh College Dentistry Vol. 28(2), June 2016 A comparative study Oral Diagnosis 52 A Comparative Study of Immunohistochemical Expression of Moesin, Cytokeratin 14 andMMP7 in Oral Squamous Cell Carcinoma and Oral Verrucous Carcinoma Karawan Khaleel Jubair, B.D.S., M.Sc. (1) Wasan Hamdi Younis, B.D.S., M.Sc., Ph.D. (2) Bashar Hamid Abdulla, B.D.S., M.Sc., Ph.D. (2) ABSTRACT Background: Squamous cell carcinoma (SCC) is the most prevalent malignant neoplasm of the oral cavity that exhibits certain histological variations. Verrucous carcinoma (VC) is an uncommon exophytic low-grade well- differentiated variant of SCC. Cellular differentiation and morphology play important roles in cell functions and maintenance of structural integrity .As the cancer is a malignant process in which disorder of the cell growth and behavior occurs, such changes may differ in different tumor types and within different grades of the same tumor. Materials and Methods:Forty two formalin – fixed, paraffin – embedded tissue blocks were included in this study (30 blocks were diagnosed as OSCC and 12 blocks were diagnosed as OVC). An immunohistochemical staining was performed using anti moesin, anti CK14 and anti MMP7 monoclonal antibodies. Results: Moesinimmunoreactivity was recognized in all the studied groups with predominant cytoplasmic expression in OSCC & membranous expression in OVC. No difference was noticed between 2 studied groups &between different grades of OSCC. Cytokeratin 14 positivity was noticed in all studied groups with significant difference between OSCC&OVC (p=0.012) & there was a significant difference between the different grade of OSCC (p=0.047). MMP7 expression was observed in the all studied groups with predominant cytoplasmic pattern in OSCC & nuclear pattern in OVC. No difference was found between the 2studied groups &between the different grades of SCC. A Strong positive linear correlation between MMP7&CK14 was noticed. Conclusion: Verrucous carcinoma has a specific pattern for moesin and MMP7 that differs from OSCC, however; the difference is not significant. CK14 immunoreactivity indicated a significant difference in the degree of cellular differentiation between OSCC &OVC. Keywords: Oral squamous cell carcinoma,oral verrucous carcinoma, moesin,CK14,MMP7. (J Bagh Coll Dentistry 2016; 28(2):52-57). INTRODUCTION The most common oral cancer (OC) of epithelial origin is oral squamous cell carcinoma (OSCC) which is the most frequent malignant neoplasm of the oral cavity, corresponding to almost 95% of all lesions and to about 38% of malignant tumors of the head and neck (1). Oral verrucous carcinoma (OVC) is an uncommon exophytic low-grade well-differentiated variant of squamous cell carcinoma. It is well known for its locally aggressiveness and for its clinically slow- growing behavior with minimal metastatic potential and represent 0.3% to 10% of all oral cavity SCC (1). OSCC is different from OVC in its clinical and histopathological aspects, as well as in its prognosis (2). Several physiologic functions including cell shape, adhesion and motility require a connection between cell membrane proteins and the cortical cytoskeleton (3). A subgroup of superfamily, ezrin-radixin- moesin (ERM), is known to function as a link between the cell membrane and actin cytoskeleton (4). Altered expression of particular ERM proteins is believed to contribute to carcinogenesis and metastasis (5,6). (1) Master student, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. (2)Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. A well controlled balance of cellular proliferation and differentiation is necessary for the development and maintenance of normal epithelium throughout the body. Keratins are cytoplasmic intermediate filament proteins (7), that regulate/modulate different signaling pathways associated with various cellular processes such as protein synthesis, cell growth, and cell differentiation (8,9). Several cellular alterations occur in the cytoskeleton during oncogenic development that can be assessed through the expression of these proteins (10). Degradation of the basement membrane and invasion of the underlying connective tissue by neoplastic cells are recognized as fundamental steps in the progression of many epithelial cancers. MMP-7 (matrilysin) is among the smallest members of the MMP family, has the capacity to start an activation cascade of MMPs and is able to degrade a variety of extracellular matrix (ECM) substrates (11). Tumor growth and progression depends critically on the ability of tumor cells to proliferate, cell motility, receptors mediated cell adhesions, production of proteolytic enzymes, and certain extracellular matrix proteins. Moesin, cytokeratin 14 and MMP7 will be used in this study to show the degree of cellular changes, J BagJ Bagh College Dentistry Oral Diagnosis assessing tumor behav comparing these biological parameters in OSCC and OVC. This study aimed to immunohistochemical expression of CK14, moesin and MMP7 in oral squamous cell carcinoma and verrucous carcinoma correlate their histopathological grading. MATERIALS AND METHODS Forty two formalin embedded tissue blocks (30 OSCC and 12 OVC) were collected from labrotaries archives and included in this study. reassessment of sections for each block Four mounted on positively charged slides and stained immunohistochemically with monoclonal antibodies MMP7 monoclonal Abcam expose mouse and rabbit HRP/DAB immunohistochemical detection kit ab80436, Cambridge, UK) RESULTS The age of the studied samples ranged between 24 15.3 years in SCC whi in OVC. Male predominance was found with 60% of SCC group and 75% of VC group. No statistically significant differences in age and sex distribution were observed between the 2 studied groups (Table 1). The most affected site 26.7% (8 cases) while buccal mucosa and alveolar ridge 33.3% (4 cases for each) were the common sites in OVC. Histopathological examination showed that 14 cases (46.7%) were well differentiated SCC, 12 cases (40%) moderately differentiated and 4 cases (13.3%) were poorly differentiated Moesinimmunoreactivity was recognized in all the studied groups with predominant cytoplasmic expression in SCC and membranous expression in OVC(Table 3and figure1). h College Dentistry Oral Diagnosis assessing tumor behav comparing these biological parameters in OSCC and OVC. This study aimed to immunohistochemical expression of CK14, moesin and MMP7 in oral squamous cell carcinoma and verrucous carcinoma correlate their histopathological grading. MATERIALS AND METHODS Forty two formalin embedded tissue blocks (30 OSCC and 12 OVC) were collected from labrotaries archives and included in this study. reassessment of haematoxylin& eosin stained sections for each block Four micrometer thick sections were cut and mounted on positively charged slides and stained immunohistochemically with monoclonal antibodies using anti moesin, anti CK14 and anti MMP7 monoclonal Abcam expose mouse and rabbit HRP/DAB immunohistochemical detection kit ab80436, Cambridge, UK) RESULTS The age of the studied samples ranged between 24-99 years old. The mean age was 59+/ 15.3 years in SCC whi in OVC. Male predominance was found with 60% of SCC group and 75% of VC group. No statistically significant differences in age and sex distribution were observed between the 2 studied groups (Table 1). The most affected site 26.7% (8 cases) while buccal mucosa and alveolar ridge 33.3% (4 cases for each) were the common sites in OVC. Histopathological examination showed that 14 cases (46.7%) were well differentiated SCC, 12 cases (40%) moderately tiated and 4 cases (13.3%) were poorly differentiated (Table 2) Moesinimmunoreactivity was recognized in all the studied groups with predominant cytoplasmic expression in SCC and membranous expression in Table 3and figure1). h College Dentistry assessing tumor behavior and invasiveness; comparing these biological parameters in OSCC This study aimed to immunohistochemical expression of CK14, moesin and MMP7 in oral squamous cell carcinoma and verrucous carcinoma correlate their expression histopathological grading. MATERIALS AND METHODS Forty two formalin – fixed, paraffin embedded tissue blocks (30 OSCC and 12 OVC) were collected from labrotaries archives and included in this study.After histopathological haematoxylin& eosin stained sections for each block. micrometer thick sections were cut and mounted on positively charged slides and stained immunohistochemically with monoclonal using anti moesin, anti CK14 and anti MMP7 monoclonal antibodies Abcam expose mouse and rabbit HRP/DAB immunohistochemical detection kit ab80436, Cambridge, UK) was used. The age of the studied samples ranged 99 years old. The mean age was 59+/ 15.3 years in SCC while it was 61.3 +/ in OVC. Male predominance was found with 60% of SCC group and 75% of VC group. No statistically significant differences in age and sex distribution were observed between the 2 studied The most affected site in SCC was the tongue 26.7% (8 cases) while buccal mucosa and alveolar ridge 33.3% (4 cases for each) were the common sites in OVC. Histopathological examination showed that 14 cases (46.7%) were well differentiated SCC, 12 cases (40%) moderately tiated and 4 cases (13.3%) were poorly (Table 2). Moesinimmunoreactivity was recognized in all the studied groups with predominant cytoplasmic expression in SCC and membranous expression in Table 3and figure1). h College Dentistry Vol. 2 ior and invasiveness; comparing these biological parameters in OSCC compare immunohistochemical expression of CK14, moesin and MMP7 in oral squamous cell carcinoma and verrucous carcinoma and to expression with the MATERIALS AND METHODS fixed, paraffin embedded tissue blocks (30 OSCC and 12 OVC) were collected from labrotaries archives and After histopathological haematoxylin& eosin stained micrometer thick sections were cut and mounted on positively charged slides and stained immunohistochemically with monoclonal using anti moesin, anti CK14 and anti antibodies(Abcam UK). Abcam expose mouse and rabbit HRP/DAB immunohistochemical detection kit (Catalog No. was used. The age of the studied samples ranged 99 years old. The mean age was 59+/ le it was 61.3 +/- 14.4 years in OVC. Male predominance was found with 60% of SCC group and 75% of VC group. No statistically significant differences in age and sex distribution were observed between the 2 studied in SCC was the tongue 26.7% (8 cases) while buccal mucosa and alveolar ridge 33.3% (4 cases for each) were the common sites in OVC. Histopathological examination showed that 14 cases (46.7%) were well differentiated SCC, 12 cases (40%) moderately tiated and 4 cases (13.3%) were poorly Moesinimmunoreactivity was recognized in all the studied groups with predominant cytoplasmic expression in SCC and membranous expression in Vol. 28(2), June 53 ior and invasiveness; comparing these biological parameters in OSCC compare the immunohistochemical expression of CK14, moesin and MMP7 in oral squamous cell and to with the fixed, paraffin – embedded tissue blocks (30 OSCC and 12 OVC) were collected from labrotaries archives and After histopathological haematoxylin& eosin stained micrometer thick sections were cut and mounted on positively charged slides and stained immunohistochemically with monoclonal using anti moesin, anti CK14 and anti (Abcam UK). Abcam expose mouse and rabbit HRP/DAB (Catalog No. The age of the studied samples ranged 99 years old. The mean age was 59+/- 14.4 years in OVC. Male predominance was found with 60% of SCC group and 75% of VC group. No statistically significant differences in age and sex distribution were observed between the 2 studied in SCC was the tongue 26.7% (8 cases) while buccal mucosa and alveolar ridge 33.3% (4 cases for each) were the common sites in OVC. Histopathological examination showed that 14 cases (46.7%) were well differentiated SCC, 12 cases (40%) moderately tiated and 4 cases (13.3%) were poorly Moesinimmunoreactivity was recognized in all the studied groups with predominant cytoplasmic expression in SCC and membranous expression in groups and between different grades of SCC. Cytokeratin 14 positivity was noticed in all studied groups with significant SCC and OVC p=0.012 and there was a significant difference between the different grade of OSCC (p= June 2016 Figure1: cytoplasmic expression in SCC, B: membranous &cytoplasmic in VC No difference was noticed between 2 studied groups and between different grades of SCC. Cytokeratin 14 positivity was noticed in all studied groups with significant SCC and OVC p=0.012 and there was a significant difference between the different grade of OSCC (p=0.047) (figure2). Figure2: cytoplasmic expression in SCC,B: cytoplasmic expression inVC A B A B Figure1:Moesinimmunostainig,A: cytoplasmic expression in SCC, B: membranous &cytoplasmic in VC No difference was noticed between 2 studied groups and between different grades of SCC. Cytokeratin 14 positivity was noticed in all studied groups with significant SCC and OVC p=0.012 and there was a significant difference between the different grade 0.047) (figure2). CK14 immunostaining, A: cytoplasmic expression in SCC,B: cytoplasmic expression inVC A comparative study Moesinimmunostainig,A: cytoplasmic expression in SCC, B: membranous &cytoplasmic in VC No difference was noticed between 2 studied groups and between different grades of SCC. Cytokeratin 14 positivity was noticed in all studied groups with significant difference between SCC and OVC p=0.012 and there was a significant difference between the different grade 0.047) (figure2). CK14 immunostaining, A: cytoplasmic expression in SCC,B: cytoplasmic expression inVC omparative study Moesinimmunostainig,A: cytoplasmic expression in SCC, B: membranous &cytoplasmic in VC No difference was noticed between 2 studied groups and between different grades of SCC. Cytokeratin 14 positivity was noticed in all difference between SCC and OVC p=0.012 and there was a significant difference between the different grade CK14 immunostaining, A: cytoplasmic expression in SCC,B: cytoplasmic expression inVC omparative study J Bagh College Dentistry Vol. 28(2), June 2016 A comparative study Oral Diagnosis 54 Collectively, MMP7 expression was observed in the all studied groups with predominant cytoplasmic pattern in SCC and nuclear pattern in OVC(figure3). Figure 3:MMP7 immunostaining, A:cytolasmic expression in SCC,B: nuclear expression in VC No difference was found between the 2studied groups and between the different grades of SCC. A Strong positive linear correlation between MMP7and CK14 was noticed (Tables 4 and 5) DISCUSSION Assessment of moesinimmunohistochemical expression: All the studied cases showed positive expression for moesin, interestingly, moesin cytoplasmic expression was detected in 86.7% of SCC group which is higher than that of VC group (66.7%), while VC showed membranous expression(16.7%) compared to SCC group (3.3%). This would corroborate the finding of (12), however in this studied series the difference in this protein expression failed to reach the level of statistical significance which may be attributed to the small sample size. There are several explanations for the shift in localization: Firstly, conformational and functional changes of moesin results in redistribution of this molecule in tumor cells (13). Secondly, CD44(a cell surface receptor) has been cleaved by MMP-1 in carcinoma cells at a membrane proximal domain, thereby suggesting that functional moesin migrates with CD44 degraded from the cell surface to the cytoplasm (14). Thirdly, according to carcinogenesis, it is possible that increased membranous degredation in more aggressive neoplasms and mutation of moesin gene cannot cross-link between plasma membrane and actin filament (15). The last reason may explain the present work results regarding the grades in which proportional decrease in the moesin score with increasing histopathologic grade was observed, however no statistical significance in median moesin score was noticed between the different grades of SCC. Assessment of cytokeratin 14 immunohistochemical expression: The results showed that SCC cases showed CK14 immunostaining in all the neoplastic cells, irrespective of the grade. These findings are in contrast to those ofSu et al.(16). WhileChu et al. (17)found that over 90% of cases of SCC of various origins were CK14 positive. In previous studies performed by Morgan and Lane(18), CK 14 expression was detected regardless of the differentiation compartment. In the current study, all oral epithelial layers in VC exhibited strong immunostaining for CK 14 and that in accordance withOliveira et al.(10). The difference was significant regarding the CK 14 profile between OSCC and OVC, p=0.012,which emphasized the biological behavior of the studied lesions, especially pattern of OVC as the CKs profile was similar to the CKs profile in well differentiated oral squamous cell carcinoma. On the other hand there was significance among the different grades of OSCC(p=0.047). Moreover, it confirms the use of this protein as a marker of cellular differentiation. Assessment of MMP7 immunohistochemical expression: In the present work, all the studied cases showed positive reaction to MMP7. In VC, nuclear immunostaining was the main staining pattern of the cells, which also reported in other studies (19). The explanation of nuclear expression is based on findings of a recent study carried out by (20) which focused on epithelial cell adhesion molecule (EpCAM, a single-transmembrane protein), MMP7 is a target of EpCAM, which is induced at the transcriptional level upon nuclear translocation of EpICD. Expression patterns and levels of EpCAM and MMP7 correlated closely in vivo in tumors suggesting that there is biological relevance of this association during malignancy (20).While the absence of MMP-7 from epithelial cells of OVC was found in a study conducted byImpola et al.(21), who consideredthat matrilysin expression A B J Bagh College Dentistry Vol. 28(2), June 2016 A comparative study Oral Diagnosis 55 correlates with the aggressive phenotype of many cancers. While the cytoplasmic stain was the predominant one in the SCC; this is accordance withWeber et al.(22), who found that cytoplasmic MMP-7 is mainly produced by tumor cells themselves and is associated with short survival times in head and neck cancer(23), also demonstrated that MMP-7cytoplasmic expression is significantly correlated with lymph node metastasis in oral cavity cancers. As OSCC &OVC are carcinoma,both of them have the ability to induce angiogenesis and local invasion of the basement membrane so,both of them expressed MMP7 positive immunoreactions, no difference was observed between both tumors and among the 3 grades of SCC concerning the degree of expression. As conclusion; in this project moesin and CK14 are present normally in squamous cells. They changed their cellular localization and expression pattern in neoplastic epithelia. As VC is a very well differentiated variant of SCC, it has specific pattern for moesin, CK14 and MMP7 that differed from SCC, however, the current work not reach the level of significance in moesin and MMP7. CK14 immunoreactivity indicated a significant difference in the degree of cellular differentiation between OSCC &OVC. Table 1: The difference in age and sex between (SCC) group and VC group Variables SCC Verrucous P N % N % Age group (years) 0.67 [NS] <50 9 30.0 2 16.7 50-69 13 43.3 6 50.0 70+ 8 26.7 4 33.3 Total 30 100.0 12 100.0 Range (24-99) (32-83) Mean 59 61.3 SD 15.3 14.4 Sex 0.36 [NS] Female 12 40.0 3 25.0 Male 18 60.0 9 75.0 Total 30 100.0 12 100.0 Table 2: Frequency distribution of SCC group by grading Grading for SCC N % Well differentiated 14 46.7 Moderately differentiated 12 40.0 Poorly differentiated 4 13.3 Total 30 100 Table 3: The difference in type of moesin tissue stain expression between SCC and VC groups Moesin tissue stain expression Verrucous Ca (Vs Squamous cell Ca) P SCC Verrucous N % N % Mainly cytoplasmic 26 86.7 8 66.7 0.24 NS Mainly membranous 1 3.3 2 16.7 Mainly mixed 3 10.0 2 16.7 Total 30 100.0 12 100.0 Table 4: The difference in median score of the 3 markers staining between SCC and VC groups. Verrucous Ca (Vs Squamous cell Ca) SCC Verrucous N % N % P Moesin score 0.18[NS] Score-I 0 0.0 0 0.0 Score-II 9 30.0 7 58.3 Score-III 15 50.0 3 25.0 Score-IV 6 20.0 2 16.7 J Bagh College Dentistry Vol. 28(2), June 2016 A comparative study Oral Diagnosis 56 Total 30 100.0 12 100.0 Median Score-III Score-II Mean rank 23 17.8 MMP7-score 0.09[NS] Score-I 1 3.3 0 0.0 Score-II 15 50.0 3 25.0 Score-III 14 46.7 9 75.0 Score-IV 0 0.0 0 0.0 Total 30 100.0 12 100.0 Median Score-II Score-III Mean rank 19.8 25.9 Cytokeratin score 0.012 Score-I 1 3.3 0 0.0 Score-II 17 56.7 2 16.7 Score-III 12 40.0 10 83.3 Score-IV 0 0.0 0 0.0 Total 30 100.0 12 100.0 Median Score-II Score-III Mean rank 18.9 28.1 Table 5: The difference in average score of 3 types of markers staining between the three grades of SCC Grading Well differentiated Moderately differentiated Poorly differentiated N % N % N % P Moesin score 0.12[NS] Score-I 0 0.0 0 0.0 0 0.0 Score-II 3 21.4 3 25.0 3 75.0 Score-III 7 50.0 7 58.3 1 25.0 Score-IV 4 28.6 2 16.7 0 0.0 Total 14 100.0 12 100.0 4 100.0 Median Score-III Score-III Score-II Mean rank 17.4 15.8 8 r=-0.314 P=0.09[NS] MMP7-score 0.18[NS] Score-I 0 0.0 0 0.0 1 25.0 Score-II 10 71.4 4 33.3 1 25.0 Score-III 4 28.6 8 66.7 2 50.0 Score-IV 0 0.0 0 0.0 0 0.0 Total 14 100.0 12 100.0 4 100.0 Median Score-II Score-III Score-III Mean rank 13.1 18.7 14.3 r=0.216 P=0.25[NS] Cytokeratin score 0.047 Score-I 0 0.0 0 0.0 1 25.0 Score-II 4 28.5 8 66.7 2 50.0 Score-III 10 71.5 4 33.3 1 25.0 Score-IV 0 0.0 0 0.0 0 0.0 Total 14 100.0 12 100.0 4 100.0 Median Score-III Score-II Score-II Mean rank 19.7 13.1 11.4 r=0.17 P=0.37[NS] J Bagh College Dentistry Vol. 28(2), June 2016 A comparative study Oral Diagnosis 57 REFERENCES 1. 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