|Year : 2015 | Volume
| Issue : 2 | Page : 96-100
Immunohistochemical analysis of cytokeratin 10, cytokeratin 14, epidermal growth factor receptor, tenascin, and Ki-67 in selected odontogenic cysts
Ramya Ramadoss1, Rajkumar Krishnan1, Sudheerkanth Kondamari Peddanna2, Eapen Cherian3, Nandhini Gunasekaran1, Dineshkumar Thayalan1
1 Department of Oral Pathology, SRM Dental College, Ramapuram, Chennai, Tamil Nadu, India
2 Department of Oral Pathology, Mamata Dental College, Khammam, Telangana, India
3 Department of Oral Pathology, Saint Gregorius Dental College, Chelad, Kerala, India
|Date of Web Publication||20-Apr-2015|
Department of Oral Pathology, SRM Dental College, Ramapuram, Chennai - 600 089, Tamil Nadu
Background: Odontogenic cysts are distinct entities and a quite common occurrence in the jaw bones. These are individual lesions which arise from the same odontogenic apparatus but with varying pathogenesis. Various molecules have been implicated to play a role in the pathogenesis of these lesions ranging from cell cycle perturbations to mutations in oncogenes and tumor suppressor genes. Materials and Methods: The role of cytokeratin (CK) 10 and 14, epidermal growth factor receptor (EGFR), tenascin and Ki-67 was studied in 5 cases of dentigerous cysts, 3 cases of odontogenic keratocysts (OKCs) (keratocystic odontogenic tumor), 3 cases of radicular cyst, 2 cases of orthokeratinized odontogenic cyst (OOC), and 2 cases of calcifying odontogenic cyst. Results: The study revealed that CK10 was significantly higher in OOC, CK14 expressed mild to intense positivity in all the odontogenic cysts. EGFR showed significant expression in OKC and tenascin expression was significantly higher in OKC. Conclusion: The study has observed the behavioral differences in the most commonly occurring odontogenic cysts. Further molecular characterization would help in better understanding.
Keywords: Cytokeratin 10 and 14, dentigerous cysts, epidermal growth factor receptor, Ki-67, odontogeni keratocyst radicular cyst, orthokeratinized odontogenic cyst calcifying odontogenic cyst, tenascin
|How to cite this article:|
Ramadoss R, Krishnan R, Peddanna SK, Cherian E, Gunasekaran N, Thayalan D. Immunohistochemical analysis of cytokeratin 10, cytokeratin 14, epidermal growth factor receptor, tenascin, and Ki-67 in selected odontogenic cysts. SRM J Res Dent Sci 2015;6:96-100
|How to cite this URL:|
Ramadoss R, Krishnan R, Peddanna SK, Cherian E, Gunasekaran N, Thayalan D. Immunohistochemical analysis of cytokeratin 10, cytokeratin 14, epidermal growth factor receptor, tenascin, and Ki-67 in selected odontogenic cysts. SRM J Res Dent Sci [serial online] 2015 [cited 2020 Aug 10];6:96-100. Available from: http://www.srmjrds.in/text.asp?2015/6/2/96/155465
| Introduction|| |
The embryonic development of orofacial complex comprises a sequence of highly coordinated patterns of growth and morphogenetic events such as cell migration, fusion of epithelia, tissue invaginations and evaginations, cell polarization, tissue interactions/cell signaling and directed extracellular matrix secretion. This morphogenic complexity of the orofacial region makes it more susceptible for perturbation by a genetic mutation or environmental teratogens. 
Odontogenic cysts and tumors constitute an important aspect of oral and maxillofacial pathology. Odontogenic cysts are encountered relatively commonly in dental practice. The presence of dentition in the jaws and the development due to fusion of various embryonic process increases the incidence of cysts in the jaw bones than the other bones in the body. With rare exceptions, epithelium-lined cysts in the bone are seen only in the jaws. Other than a few cysts that may result from the inclusion of epithelium along embryonic lines of fusion, most jaw cysts are lined by epithelium that is derived from odontogenic epithelium. 
Cysts of the maxilla, mandible and the perioral regions, vary markedly in histogenesis, incidence, behavior, and treatment.  The importance of this fact necessitates the need to differentiate each cyst and to study their characteristics in an elaborate manner. Immunohistochemical studies have been widely carried out to reason out better molecular level of understanding of the odontogenic cysts. Immunohistochemistry can give sensitive results even at low levels of antigen expression. This will aid in better treatment modalities as to achieve a better prognosis.  The study aims to assess the behavioral patterns and histogenesis in selected odontogenic cysts.
| Materials and methods|| |
Formalin-fixed paraffin-embedded tissue blocks of 5 cases of dentigerous cysts, 3 cases of odontogenic keratocyst (OKC), 3 cases of radicular cyst, 2 cases of orthokeratinized odontogenic cyst (OOC), and 2 cases of calcifying odontogenic cyst were retrieved from the archives of Department of Oral and Maxillofacial Pathology, SRM Dental College, Chennai.
Immunohistochemical analysis was performed on 3 μ tissue sections on poly-L-Lysine coated slides (Biogenix Life Sciences Limited, CA, USA). Prediluted primary monoclonal mouse anti-cytokeratin (CK) 10 and 14, epidermal growth factor receptor (EGFR), tenascin, and Ki-67 (Biogenix Life Sciences Limited, CA, USA) were used followed by the secondary super sensitive polymer horseradish peroxidase detection system (Biogenix Life Sciences Limited, CA, USA). Diaminobenzidine was used as the chromogen and counterstained with Harris hematoxylin. Presence of brown colored end product at the site of target antigen was indicative of positive immunoreactivity. Evaluation of immunoreactivity was based on the staining intensity and was classified as nonreactive, weak, moderate, and intense. The grades were given a score of 0 for nonreactive, 1 for weak, 2 for moderate, and 3 for intense staining.
Statistical analysis was done by estimating the mean standard deviation for each study group. Mean values were compared between different study groups by Kruskal-Wallis one-way ANOVA followed by Mann-Whitney U-test. P < 0.05 was considered as the level of significance.
| Results|| |
The mean scores of immunoreactivity of the markers used in the study are listed in the [Table 1]. The mean score of CK10 in OOC (2.0 ± 0.0) was significantly higher than the mean scores in OKC (1.0 ± 0.0), dentigerous cyst (0.2 ± 0.4), and in radicular cyst (0.0 ± 0.0) (P < 0.05). Further, the mean value in OOC was significantly higher than the mean values in DC, COC, RC (P < 0.05). However, there was no significant difference in mean values between other cysts (P > 0.05).
The mean score of CK14 in COC (3.0 ± 0.0) was significantly higher than the mean scores of other cysts, OKC (1.7 ± 0.6), OOC (2.0 ± 0.0), DC (1.0 ± 0.0), and RC (2.0 ± 0.0). COC exhibited a significant difference than other cysts (P < 0.05).
The mean score of EGFR in OKC (2.3 ± 0.6) was significantly higher than the other cysts, DC (2.0 ± 0.0), OOC (1.0 ± 0.0), COC (1.0 ± 0.0), and RC (2.0 ± 0.0), hence a significant difference was seen (P < 0.05). The mean score of tenascin in OKC (2.7+-0.6) was significantly higher than the other cysts, DC (1.0 ± 0.0), OOC (0.0 ± 0.0), COC (2.0 ± 1.4), and RC (0.0 ± 0.0), hence a significant difference was seen (P < 0.05). The mean score of Ki-67 in OKC (2.3 ± 0.6) was significantly higher than the other cysts, DC (1.0 ± 0.0), OOC (1.0 ± 0.0), COC (1.5 ± 0.7), and RC (0.0 ± 0.0), hence a significant difference was seen (P < 0.05).
| Discussion|| |
Odontogenic cysts are unique to the jaws and originate from the tissue associated with tooth development. The abnormal tissue in each of these tumors can often be correlated with similar tissue in normal odontogenesis from inception to tooth eruption. Cysts of the maxilla, mandible and perioral regions vary markedly in histogenesis, incidence, behavior, and treatment.  The distribution of jaw cyst in a general population has been reported as - radicular cyst (55%), dentigerous cyst (17%), nasopalatine cyst 12%, odontogenic keratocyst (globulomaxillary cyst (1-3%), traumatic cyst (0.7%), and eruption cyst (0.7%). 
The pathogenesis of odontogenic cysts is still uncertain, the underlying cause and mechanisms of epithelial proliferation are not yet agreed upon in some odontogenic cysts. Significant clinical and biological differences are found between the odontogenic cysts. It is very important to differentiate both since they show different behavior patterns which in turn affect the treatment and prognosis.  The present study aims at immunohistochemical characterization of odontogenic cysts using the markers CKs 10 and 14, EGFR, tenascin, and Ki-67. The markers were chosen in such a way that they represent a particular characteristic, which will help to assess in detail the behavior and nature of the odontogenic cysts.
The CKs represent a group of structurally related intermediate filament proteins. They are divided into 2 types namely basic or type I CKs and acidic or neutral type II CKs. Depending on their molecular weight, it may be classified as low molecular weight CKs (LMCKs) and high molecular weight CKs (HMCKs), which vary from 40,000-68,000. Different sets of CKs are expressed in various epithelia. ,,,, CK10 is an acidic type of CK and represents the degree of keratinization. CK10 clone DEK-10 was used in our study, it presented with significant staining in OOC followed by OKC, dentigerous cyst, and in radicular cyst.
The results of the present study were consistent with the study done by Da Silva et al.  who observed an intense expression of CK10 in suprabasal and superficial layers of the epithelium in the OOC, thus revealing that a constant process of keratinization is taking place, as seen in epidermal tissue, and also in the orthokeratinized epithelium, dermoid, and epidermoid cysts. OKC expressed CK10 only in the superficial layer, indicating the absence or the scarcity of mature keratinocytes. ,
Cytokeratin 14 is the main intermediate filament of odontogenic epithelium. It is expressed in the inner enamel epithelium in early and late stages of odontogenesis.  The absence of CK14 in areas where epithelial lining was separated from underlying connective tissue might indicate a role for CK14 in maintaining epithelial attachment. In the present study, CK14 clones LL002 stained cytoplasm of epithelial cells. The staining pattern was mild to moderate with significant staining in COC followed by OKC, OOC, DC, and RC. The result was in accordance with the results of Da Silva et al. who observed that the CK14 showed a continuous and constant expression in both basal and suprabasal epithelial layers in OOCs and variable expression in the more superficial layers, thus showing that cell differentiation was not occurring abruptly. In OKCs, CK14 showed a continuous and constant expression in the basal layer.
Epidermal growth factor receptor is the receptor for EGF. It is a member of the ErbB family receptors. EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas and are capable of inducing cell transformation. EGFR clone E30 used in the present study stained the cell membrane of positive cells. It presented with intense positivity in OKC, moderate positivity in calcifying odontogenic cyst and mild in OOC, dentigerous cyst, and radicular cyst. 
The above findings were in accordance with Tiejun Li et al. who studied the immunohistochemical pattern of expression of EGF in OKC, dentigerous cyst, and radicular cysts. OKC linings expressed higher levels of EGF than those of dentigerous and radicular cysts. Percentage of positivity was higher in OKC than other cysts in the study by Shrestha et al.  However, the expression of EGFR in OOC and COC was not studied before. Expression of EGFR in OKC and COC suggests an intrinsic growth potential.  Whereas OOC showed mild expression when compared with OKC suggesting a more indolent behavior.
Tenascin is a polymorphic high molecular mass extracellular matrix glycoprotein. It plays an important role in the maintenance of a correct microenvironment for basic cell functions such as cell adhesion and differentiation. Tenascin expression is higher during embryonic development period. Increased expression suggests marked instability in cystic structure and it may influence the epithelial-mesenchymal signalizing relations and propitiate a more favorable microenvironment for epithelial proliferation and migration. Tenascin clone DB7 used in the present study stained fibroblasts and the extracellular matrix of connective tissue. It stained intensely positive in OKC, moderate in calcifying odontogenic cyst, mild in a dentigerous cyst and negative in OOC and radicular cyst. 
Da Silva et al. observed that the reaction with antitenascin antibody was negative in OOC and positive in OKC, in the basement membrane zone and in the extracellular matrix next to the basement membrane, independent of the presence of inflammation or not. This finding evidences an important distinction between the 2 cysts because tenascin is an adhesion-modulating protein, expressed in unstable environments as in neoplasms and healing areas. Few studies have shown the expression of tenascin in the stroma of odontogenic neoplasms. Tenascin immunostaining was mainly intense as a thick band deep to the epithelial-mesenchymal interface in both RCs and OKCs. The intense tenascin immunoexpression observed in the RCs was usually associated with inflammation. An intense fibronectin reactivity was observed in the basement membrane region and at the cystic wall of OKCs. The higher tenascin and fibronectin expression in the capsule of OKCs suggests marked instability in the cystic structure and may contribute to its aggressive behavior.
In our study out of the two cases of calcifying odontogenic cyst one case showed intense positivity in the connective tissue, whereas the other case showed mild positive staining. Unlike the study done by Lukinmaa et al., no staining was appreciated in the epithelial elements. They also observed immunostaining with monoclonal antibodies which showed reactivity of both LMCK and HMCK, the degree of coexpression decreasing with the increasing morphological diversity of the cyst/tumour epithelium.  Staining for the matrix glycoprotein tenascin-C was seen not only in the connective tissue, where its distribution patterns corresponded to the stage of hard tissue formation, but also in epithelial elements. The staining patterns were analogous to those described during normal tooth formation. Both the morphological characteristics and expression patterns of the various CK types and tenascin-C implied that COC represents a pathological counterpart of normal odontogenesis. Our findings of tenascin in COC were consistent with Lukinmaa et al., who observed immunostaining for the matrix glycoprotein tenascin-C in the connective tissue of COC. The expression of dentigerous cyst and OKC was consistent with De Oliveira et al.  Negativity in tenascin expression by OOC was consistent with Da Silva et al.
Ki-67 is one of the most widely studied proliferating cell antigens. The expression of Ki-67 antigen is limited to cells on phase G1, S, and G2 with the highest levels present in the M phase. The Ki-67 expression is not only of diagnostic use but can also be used as a prognostic indicator. , High Ki-67 labeling index or growth fractions in one factor that predicts a poor prognosis. Ki-67 clone BGX-297 stains nucleus of proliferating cells.  It stained moderate to intense positive in OKC (keratocystic odontogenic tumor [KCOT]), mildly positive in OOC dentigerous cyst, calcifying odontogenic cyst and negative in radicular cyst.  Kim et al. observed increased proliferation rates analyzed with the help of Ki-67 marker and also increased apoptosis in OKC (KCOT) than dentigerous cysts.  De Paula et al. found that proliferation markers showed increased rates in inflamed OKC than noninflamed keratocysts.  Slootweg observed proliferative activity by positivity for Ki-67 antigen and p53 and found that proliferation rates were more in ameloblastomas than odontogenic cysts. 
On statistical comparison of staining intensities between the odontogenic cysts taken for study CK10 was significantly higher in OKC and in OOC. CK14 was significantly higher in calcifying odontogenic cyst. EGFR was significantly higher in OKC than in other cysts. Tenascin was significantly higher in OKC. Ki-67 was significantly higher in OKC.
The study revealed that CK10 was significantly higher in OOC suggesting the presence of mature keratinocytes, CK14 is predominate keratin type found in odontogenic epithelia and it was expressed mild to intense in all the odontogenic cysts. EGFR showed significant expression in OKC suggesting an intrinsic growth potential. Tenascin expression was significantly higher in OKC, tenascin is present in neoplasms and it is an adhesion modulating protein. The markers used for the study CK 10 and 14, EGFR, tenascin and Ki-67 can be used to elucidate the clinical importance of odontogenic cysts as there was significant difference in their expression and each marker is helpful to assess different characters of odontogenic cysts.
| Conclusion|| |
The findings from this study substantiate the recent categorization of OKC as KCOT, which falls under the category of benign odontogenic tumor arising from odontogenic epithelium without the mesenchyme. The study has observed the behavioral differences in the most commonly occurring odontogenic cysts. Further molecular characterization would help in better understanding.
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