|Year : 2016 | Volume
| Issue : 2 | Page : 118-122
Expression of calretinin and cytokeratin 19 in radicular cyst, dentigerous cyst, odontogenickerato cyst, and ameloblastoma
Aesha Imran1, Kannan Ranganathan2, Uma Devi K Rao2, Elzibeth Joshua2, Rooban Thavarajah2
1 Department of Oral Pathology, Chettinad Dental College and Research Institute, Kelambakam, India
2 Department of Oral Pathology, Ragas Dental College and Hospital, Uthandi, ECR, Chennai, Tamil Nadu, India
|Date of Web Publication||5-Jul-2016|
Department of Oral Pathology, Chettinad Dental College and Research Institute, Kelambakam, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Context: Calretinin is a protein associated with cellular differentiation and proliferation and is an inhibitor of apoptosis. Cytokeratin is a protein associated with odontogenic epithelial cell, and ameloblasts being epithelial derivative also express some cytokeratin. This study was done to ascertain if calretinin and cytokeratin could be responsible for the differences between aggressiveness of certain odontogenic cysts and tumors.
Aims: To evaluate the expression of calretinin and cytokeratin 19 (CK19) in odontogenic cysts and ameloblastoma.
Materials and Methods: Sixty samples of formalin-fixed paraffin embedded tissue specimens [15 radicular cysts, dentigerous, odontogenic keratocysts (OKCs), and ameloblastoma] were evaluated for the expression of CK19 and calretinin using immunohistochemistry.
Statistical Analysis: Data entry and statistical analysis using Statistical Package for the Social Sciences (SPSS) TM software (version 10.05) were done. Chi-square test was done to compare tissue localization of stain, nature of stain, intensity of stain, and the percentage of cells stained among the study groups. P value less than 0.05 was considered to be statistically significant.
Results: Calretinin showed positive expression in all cases of ameloblastoma. Among the samples of OKC, 13% of the cases showed positivity. All the cases of radicular and dentigerous cysts were completely negative for calretinin. CK19 was negative in all cases of radicular cyst and OKC, whereas among the dentigerous cyst two cases showed mild expression and one case showed moderate staining intensity for CK19. Only one case of ameloblastoma showed moderate staining for CK19.
Conclusion: The results of this study showed that calretinin can be an immunohistochemical marker for neoplastic ameloblastic epithelium and the difference of expression among the lesions is probably due to their diverse histopathological characteristics and their developmental origin. CK19 a marker of simple epithelia and its absence could probably be due to absence of CK19 epitope, superimposition of other cytokeratins, or masking of the epitopes.
Keywords: Calretinin, Cytokeratin 19 (CK19), immunohistochemistry, odontogenic lesions
|How to cite this article:|
Imran A, Ranganathan K, Rao UK, Joshua E, Thavarajah R. Expression of calretinin and cytokeratin 19 in radicular cyst, dentigerous cyst, odontogenickerato cyst, and ameloblastoma. J NTR Univ Health Sci 2016;5:118-22
|How to cite this URL:|
Imran A, Ranganathan K, Rao UK, Joshua E, Thavarajah R. Expression of calretinin and cytokeratin 19 in radicular cyst, dentigerous cyst, odontogenickerato cyst, and ameloblastoma. J NTR Univ Health Sci [serial online] 2016 [cited 2020 Mar 31];5:118-22. Available from: http://www.jdrntruhs.org/text.asp?2016/5/2/118/185442
| Introduction|| |
The odontogenic lesions originate from odontogenic epithelium or ectomesenchyme with varying degrees of inductive tissue interaction.  The most commonly occurring odontogenic cysts are radicular, dentigerous, and odontogenic keratocyst (OKC). , The biological behavior of few OKCs is as aggressive as benign neoplasm such as ameloblastoma. OKC is now designated by the World Health Organization (WHO) as a keratocystic odontogenic tumor (KCOT). The clinically aggressive behavior is a result of the properties of lining epithelial cells and connective tissue capsule.  Calretinin, a calcium binding protein with molecular weight of 29 kDa (KiloDalton), is a member of the large family EF-hand proteins. This protein is expressed primarily in certain subtypes of neurons in the retina and in the central and peripheral nervous system. Its precise behavior is still unknown but possible roles are calcium buffer, calcium sensor, and regulator of apoptosis.  Studies in rats have demonstrated that calretinin is expressed in neural element of tooth pulp, periodontal ligament, viscerosensory nerve fibers of oral tissues and also during odontogenesis in molar tooth germs. Various studies have shown positivity of calretinin in ameloblastoma as well as in aggressive areas of OKC. 
Cytokeratin belonging to intermediate family protein that are divided into two subfamilies based on their charges, immunoreactivity, and amino acid sequence: Acidic proteins with low molecular weight and basic proteins with high molecular weight.  The expression pattern of intermediate filaments has been investigated in normal and neoplastic human cells including oral epithelial cells, odontogenic epithelia, tumors, and cysts. These investigators hypothesize that intermediate filaments expression patterns are characteristic for each kind of cells.  Cytokeratin 19 (CK19) is the smallest known acidic type of cytokeratin, having molecular mass of 40 kD (KiloDalton). It is not paired in epithelial cells. It is expressed in most simple epithelia, in various ductal epithelia, intestinal epithelia, gastric foveolar epithelium, and in the mesothelium. It is expressed in developing tooth germs, and also in neoplastic epithelial cells of some odontogenic tumors. It is also detected in cell rests of Malassez, cell rests of Serres, and lining of odontogenic cysts.  Various other studies have demonstrated the presence of cytokeratin in human oral embryonic tissues.  Fukumashi et al. has demonstrated the expression of CK19 in odontogenic epithelium to discuss histogenesis of epithelium in tumors such as ameloblastoma.  The aim of this study is to evaluate the expression of calretinin and CK19 in odontogenic cysts and ameloblastoma.
| Materials and methods|| |
The study was conducted in the Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, Chennai, and was approved by the Institutional Review Board. The study material comprised of 60 formalin fixed, paraffin embedded specimens taken from archival tissue blocks. Clinically, radiographically, and histologically confirmed cases of dentigerous cyst (group I, N = 15), OKC (group II, N = 15), radicular cyst (group III, N = 15), and ameloblastoma (group IV, N = 15) were included in the study.
Sections were made from paraffin embedded blocks and transferred to amino propyl epoxy saline coated (APES) slides. The slides with tissue sections were treated with three changes of xylene to remove paraffin wax, followed by descending grades of alcohol and rehydration with water. The slides were then transferred to citrate buffer and autoclaved for antigen retrieval at 15 lbs pressure for 15 min. The slides were allowed to cool and washed in phosphate buffer solution (PBS) (pH 7.2) for 5 min. Peroxidase and protein blocking were done using 3% hydrogen peroxide and protein block reagent, respectively, for 10 min each. The slides were incubated with primary antibodies (concentrated, rabbit polyclonal anticalretinin antibody, Biogenex super sensitivity detection system) at room temperature for overnight. Anti-human CK19 antibody (concentrated mouse monoclonal antibody; Biogenex super sensitive detection system) was added to the tissues and incubated for 2 h at room temperature. The secondary antibody used was an enhancer followed by streptavidin solution for 20 min from the secondary polymer kit (Biogenex). A drop of freshly prepared DAB (3'-diaminobenzidine tetrahydrochloride-A substrate chromogen) was added. Three changes of PBS washes were performed after every step during the immunostaining procedure. The sections were counterstained with hematoxylin and mounted with DPX® .
Criteria for evaluation of staining
Labeling index (LI) was calculated by dividing the number of positive cells by the total number of cells counted in the slide and expressed as percentage. A minimum of thousand cells was counted for each slide. The cytoplasmic staining intensity was evaluated and graded as mild (+), moderate (++), and intense (+++) as described. Mild staining is denoted by light brown color, moderate by brown color, and intense by dark brown color. The cells that did not take up any brown stain were considered negative.
Data entry and statistical analysis was done using Statistical Package for the Social Sciences (SPSS) TM software (version 10.05) (SPSS-Inc., Chicago, IL). Chi-square test was done to compare tissue localization of stain, nature of stain, intensity of stain, and the percentage of cells stained among the study groups. P value less than 0.05 was considered to be statistically significant.
| Results|| |
Among the odontogenic cysts, all the cases of dentigerous cysts and radicular cysts were negative for calretinin stain, whereas only two cases (6.7%) of OKC exhibited positive staining. Of the two positive cases of OKC, one showed mild stain and the other exhibited moderate staining intensity. All the cases of ameloblastoma expressed positive calretinin stain with 66.6% (N = 10) showing mild staining, 26.6% (N = 4) moderate staining and 6.6% (N = 1) intense staining [Table 1]. When the staining intensity of OKC and ameloblastoma was compared, a significantly higher intensity of calretinin staining was noted in ameloblastoma (P = 0.004). Mean labeling index of the study group that had stained positive for calretinin was calculated as 1.28 ± 3.39 for group II (OKC) and 19.21 ± 13.22 for group IV (ameloblastoma).
Among the odontogenic cysts, all the cases of radicular and OKC were negative for CK19, whereas among the cases of dentigerous cyst, two cases showed mild staining (13.3%) and one case (6.6%) was moderately stained [Table 2]. Among the cases of ameloblastoma, only one case (6.6%) showed moderate staining for CK19 [Table 3]. No significant difference was noted in the staining intensity between odontogenic cysts and ameloblastoma.
| Discussion|| |
Odontogenic tumors originate from successional and accessional dental lamina and can be differentiated into various entities. The mechanisms that trigger proliferation of the odontogenic epithelial rests are unknown, but various subcellular and developmentally related factors may be responsible for their differentiation.  Ameloblastoma is the most frequently encountered tumor arising from enamel organ. , Among the cysts, KCOT deserves a special consideration because of its destructive behavior, in a lesser degree when compared to ameloblastoma.  There are clinical and radiographic similarities between ameloblastoma and OKC that may also be reflected at the histologic level if the tissue sample is small and if neoplastic epithelium displays reactive changes induced by inflammation. Although the histology presentation of both OKC and ameloblastoma are different, both represent an aberration in some stage of odontogenesis. 
Calretinin is a calcium binding protein of EF-hand family and has a role as a calcium buffer, sensor and also been demonstrated as an antiapoptotic factor. , In odontogenesis calretinin was expressed focally in dental lamina, outer enamel epithelium, stellate reticulum, and stratum intermedium at different stages. In contrast, it was intensely expressed in the inner enamel epithelium and presecretory ameloblasts. This distribution suggests a possible role of this protein in enamel formation.  Calretinin is known to be a distinctive and specific immunohistochemical marker for ameloblastoma. 
The expression of calretinin in our studies was in accordance with earlier studies by Devilliers, Sundaragiri, Chitra, and Desliva ,,, in which ameloblastomas showed 100% positive staining. None of these studies showed staining in OKC except for studies done by Desilva,  in which 40 % of OKC were positive for calretinin. In our study, 13% of OKC cases showed positive staining for calretinin. The localization of the protein in ameloblastoma was intense in areas of stellate reticulum such as epithelium, whereas in peripheral areas its expression was weak. , In OKC, the positive cells were seen in the basal and parabasal layers of the liningepithelium.  These findings suggest that the role of calretinin in ameloblastoma could be a calcium ferry in the path of transition toward enamel matrix, thus making calretinin a definitive marker for neoplastic ameloblastic epithelium.  The location of positivity (the stellate reticulum like epithelium was strongly reactive for calretinin) explains the role of calretinin as a antiapoptotic proliferating protein in the outer layer and apoptotic differentiating factor in the stellate reticulum like areas. In OKC, the positivity in suprabasal layers similar to other markers, such as PCNA and p53, could point to increased activity of calcium binding protein and abnormal cell cycle control. ,
In our study, 80% of dentigerous cysts did not show any staining for CK19, while 3.3% cases showed mild staining and 6.6% cases showed moderate staining intensity. Stoll et al. reported that 48.3% of dentigerous cyst showed positive staining of CK19 in the superficial layers.  In our study, staining was not detected for CK19 in any of the layers in the epithelial lining of OKC. Recent studies done by Stoll et al. on OKCs also showed a complete negative staining. 
CK19 is a marker of simple epithelia and is an obligatory constituent of all normal epithelium, junctional epithelium, and odontogenic epithelium. Although OKC is lined by odontogenic epithelium, all the cases in our study showed negative staining for CK19. A reason for negative staining could be due to the superimposition of the cornification markers, such as cytokeratin1 and 10, as suggested by Morgan et al.  Shear et al. suggested that there could be change in the pattern of cytokeratin profiles in odontogenic epithelial cells during odontogenesis and also when quiescent cells proliferate in certain pathological situations such as odontogenic cysts and tumors. Hence, negative staining could also be attributed to the epithelial characteristics, modification, or absence of epitope. 
In our study, none of the cases of radicular cysts showed any staining characteristics for CK19. However, 48.3% of the radicular cysts showed positive immunoreactivity in the suprabasal layers in study conducted by Stoll et al.  In our study, 6.6% of ameloblastoma showed positive staining characteristics for CK19, while 93.3% of cases did not show immunoexpression. In the study conducted by Fukumashi et al., all the cases of follicular and desmoplastic ameloblastomas showed positivity for CK19, whereas 95% of plexiform and 67% of granular ameloblastomas exhibited positive staining.  Kumamoto et al. conducted a study on all histological types of ameloblastoma and found decreased expression in keratinizing cells of acanthomatous type suggesting the dedifferentiation from odontogenic epithelial characteristics.  The reasons for the absence of staining in our study could be due to the dedifferentiation of cells in ameloblastoma that is similar to the results of abovementioned authors.  Further, keratin profile of an epithelium is linked to several factors such as differentiation, proliferation, and histogenesis. The biological rules of keratin expression are yet to be defined fully and often there may be subtle differences between keratin at the molecular levels that may be reflected as absence of immunoreactivity with antibodies that have generated to known epitopes. 
| Conclusion|| |
In conclusion, the results of the current study show that calretinin was shown to be a specific immunohistochemical marker for neoplastic ameloblastic epithelium and the discrepancy among the selected odontogenic lesions (higher immunoreactivity for ameloblastoma, less reactivity for OKC, and nonreactivity for dentigerous and radicular cysts) is probably interconnected with diverse clinical and histopathological characteristics. The variable expression of calretinin in ameloblastoma and OKC may reveal a role of this protein as a second messenger in the control of abnormal cell cycle proliferation and also indicates that higher the expression of the protein the least differentiated will be odontogenic epithelium and hence the calretinin expression in stronger in ameloblastoma and weaker in OKC. CK19 is a marker of simple epithelia and its absence could probably be due to absence of CK19 epitope, superimposition of other cytokeratins, or masking of the epitopes.
Ragas Dental College and Hospital, Uthandi, ECR, Chennai - 603103.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]