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ORIGINAL ARTICLE
Year : 2012  |  Volume : 3  |  Issue : 4  |  Page : 236-239

Immunohistochemical analysis of dentigerous cyst and ameloblastoma using cytokeratin 19 & 14, p53, p63 and ki-67


1 Department of Oral Pathology, Army College of Dental Sciences, Secunderabad, Andhra Pradesh, India
2 Department of Oral Pathology, SRM Dental College, Ramapuram, Chennai, Tamil Nadu, India

Date of Web Publication12-Jul-2013

Correspondence Address:
K Sudheer Kanth
Army College of Dental Sciences, Secunderabad, Andhra Pradesh
India
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DOI: 10.4103/0976-433X.114967

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  Abstract 

Introduction: Odontogenic cysts and tumours constitute an important aspect of oral and maxillofacial pathology. Although they arise from the same odontogenic apparatus, they are distinct entities with different pathogenesis and differ considerably in their biological behavior in terms of aggression and capacity to spread (metastasis). This could be attributed to the nature of their epithelium and alteration in the cell cycle control. Hence this study is done to compare an odontogenic cyst dentigerous cyst (DC) and odontogenic tumor (Ameloblastoma) with a panel of immunohistochemical markers including cytokeratin (CK) 14 and 19, p53, p63, Ki67 to explain for the differences between the two. Materials and Methods: IHC was performed with the above mentioned markers with five cases each of DC and ameloblastoma. Results: The mean scores of each of the marker used were found to be significantly higher in ameloblastoma in comparison to DC. Conclusion: The study confirms that considerable difference exists between these two lesions in terms of their clinical characters and biologic behavior. Furthermore, immunohistochemical analysis with the combination of the markers used in this study may be useful in differentiating DC and ameloblastoma.

Keywords: Immunohistochemistry, dentigerous cyst, ameloblastoma, cytokeratins, p53, p63, Ki-67


How to cite this article:
Kanth K S, Kumar T D, Kumar A R. Immunohistochemical analysis of dentigerous cyst and ameloblastoma using cytokeratin 19 & 14, p53, p63 and ki-67. SRM J Res Dent Sci 2012;3:236-9

How to cite this URL:
Kanth K S, Kumar T D, Kumar A R. Immunohistochemical analysis of dentigerous cyst and ameloblastoma using cytokeratin 19 & 14, p53, p63 and ki-67. SRM J Res Dent Sci [serial online] 2012 [cited 2018 Sep 22];3:236-9. Available from: http://www.srmjrds.in/text.asp?2012/3/4/236/114967


  Introduction Top


The most common odontogenic cyst in the oral cavity, accounting for 20% of the developmental cysts of the jaws is dentigerous cyst (DC). It is almost always associated with the crown of a tooth attached to the cemento-enamel junction. They are believed to be formed from the fluid accumulated between reduced enamel epithelium and tooth crown, and results in expanded follicle beyond the 3 mm normal diameter, because of which they are usually associated with impacted or un-erupted teeth. [1],[2] These odontogenic cysts are reported to transform to neoplasm and are shown to be the origin for certain rare neoplasm like ameloblastoma. [3],[4]

Though uncommon and benign, ameloblastoma are locally aggressive odontogenic neoplasm that accounts for approximately ten percent of all tumors that arise in the mandible and maxilla. [5] Ameloblastoma is classified as central or peripheral. Central ameloblastomas are classified as multicystic/solid (MCA) or unicystic (UCA). [6] With an unknown etiology, these neoplasms are hypothesized to develop from odontogenic epithelia derived from various sources, like dental follicular lining epithelium. [7] High aggressive nature and recurrence after surgery has been associated with multicystic/solid ameloblastomas compared with unicystic and peripheral ameloblastomas. [8] The neoplastic epithelial proliferation in the form of an ameloblastoma is far more pronounced in the DC than in the other odontogenic cysts. It is well evidenced that the lining of a DC might undergo neoplastic transformation to an ameloblastoma. [3],[4]

Various proliferative studies and immunohistochemical demonstration of proliferative markers suggest that increased cell proliferation plays a role in the development of odontogenic cyst (e.g., DC) and neoplastic tumors (e.g., ameloblastoma). [9],[10] This increased cell proliferation can result from perturbations in cell cycle, mutations in oncogenes or tumor suppressor genes. Although they arise from the same odontogenic apparatus, they are distinct entities with different pathogenesis. Many concepts have been proposed which explain the pathogenesis of different odontogenic cysts and tumors. Hence, it is important to know their cell cycle pattern, biological behavior and neoplastic pattern of DC and ameloblastoma.

With all the above background facts, the study was aimed to compare differences in a panel of markers (cytokeratin [CK] 14, CK 19, p53, p63 and Ki67) between the DC and ameloblastoma using immunohistochemistry technique. This will assess the differences in their nature and behavior and may be of value in distinguishing dentigrous cyst from ameloblastoma.


  Materials and Methods Top


This laboratory based study was carried out on 10 archival specimens, which included five cases of ameloblastoma and five cases of DC [Table 1]. The diagnosis was based on histopathological appearance and the clinical and radiographic features. All the cases were seen by four independent observers to avoid interobserver variability. Formalin fixed paraffin embedded tissues were sectioned at 3 μm thick sections using semiautomatic microtome and placed on slides coated with poly-L-lysine for immunohistochemical staining. Consecutive sections were placed on slides precoated with egg albumin for routine hematoxylin and eosin staining.
Table 1: Clinicopathological data of cases selected

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Immunohistochemistry and interpretation of slides

Immunohistochemical staining was performed for all the above mentioned markers using their corresponding antibody (Biogenex, USA), to assess localization and intensity. Presence of brown colored product of the stain used - Diaminobenzidine at the site of the target antigen was indicative of positive immunoreactivity for the expression of the particular marker used. Positive controls consisted of tissue sections of squamous cell carcinoma for CK 14, colon carcinoma for CK 19, breast carcinoma for p53, skin for p63 and tonsil for Ki67. Brown staining of the target antigen sites was taken as positive staining. Negative control included sections of tissue incubated overnight in tris buffered saline containing bovine serum albumin as a blocking agent without the primary antibody. The evaluation of study case was carried out subsequently in a similar way and was graded as positive or negative.

Intensity of the stain was scored on the following scale: 0 no staining, 1 mild staining, 2 moderate staining, and 3 intense staining. Localization of the stain was categorized as either nuclear or cytoplasmic. In the case with staining heterogeneity, the expression was grouped according to the predominant staining intensity. Four observers evaluated the slides to eliminate interobserver variability.

Statistical analysis was carried out by estimating the mean and standard deviation for each study group. Mean values were compared between different study groups by Mann-Whitney U-test. For all tests, a P ≤ 0.05 was considered as the level of significance.


  Results Top


In DC, positive immunoreactivity was shown in the basal and the suprabasal layers of the lining epithelium and ameloblastoma showed strong positivity in the peripheral columnar cells and weak positivity in the central stellate reticulum type cells.

The mean scores for each marker used were compared between ameloblastoma and DC and was found to be higher in ameloblastoma than DC. Expression of CK19, p53, p63, and Ki67 were found to be statistically highly significant between the groups [Table 2].
Table 2: Comparison of mean scores between two groups

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  Discussion Top


Only, very few comparative studies have been carried out so far between odontogenic cyst and odontogenic tumors with a panel of markers. This study is a comparative study involving DC and ameloblastoma with five different types of markers to compare the expression of these markers in ameloblastoma and DC, which will help to assess in detail the behavior and nature of the odontogenic cyst and odontogenic tumor. In this study, the expression of CKs 19 and 14, p53, p63 and Ki67 was evaluated in a total of 10 cases, which included histopathologically diagnosed cases of DC and follicular type of ameloblastoma. Significant clinical and biological differences were found between the DC and ameloblastic tumors. This observation is crucial because it will affect the treatment and prognosis.

The CKs represent a group of structurally related intermediate filament proteins. CKs are identified either by molecular weight or by numerical designation, 1 through 19. [11] In this study, DC showed mild staining in the basal layer and intense positivity in the peripheral columnar/cuboidal cells and central stellate cells in ameloblastoma. Our results were consistent with the study carried out by Christian Stoll et al., 2005 [12] who observed a positive staining for CK 19 mainly in the basal cell layers in DC. Kumamoto et al., 2001 [13] observed the expression of CK 19 which was diffusely present in neoplastic cells in ameloblastomas, suggesting that these tumors have odontogenic epithelial properties.

CK 14 stains the cytoplasm of epithelial cells. CK expression patterns differ according to cell type, development stage, differentiation status and anatomical site. CK 14 is considered as the main intermediate filament of the odontogenic epithelium as it is observed in dental lamina, reduced enamel epithelium and all the cells of the enamel organ. Thus, the presence of CK 14 at this stage might have a role in preserving and supporting the epithelial-mesenchymal interactions. In our study, ameloblastoma showed intense positive staining in the peripheral columnar cells and stellate cells while DC showed mild positivity in the basal layer cells. Our results were consistent with the results of Crivelini et al., [14] who observed that CK 14 expression was seen in most of the tumoral cells, except in part of the central stellate cells, peripheral columnar cells with vacuolated cytoplasm, or metaplastic squamous cells and keratinizing cells.

Ki67 staining was observed in the nucleus of proliferating cells. A more specific marker of proliferating cells, maximally expressed during S phase, is Ki67 antigen, which is rapidly degraded after mitosis. It is a non-histone protein initially expressed in mid G1, increasing in level through S and G2 and peaking in M. In our study, it stained mildly positive in basal cells in the DC, intensely in the peripheral columnar cells and central stellate reticulum like cells in ameloblastoma. Our results were consistent with the study done by Müller and Slootweg, [15] who observed proliferative activity for Ki67 antigen and p53 and found that proliferation rates were more in ameloblastomas than odontogenic cysts.

p53 gene is well recognized as a tumor suppressor gene situated on chromosome 17p 13 and is one of the most frequently altered genes in tumors. p53 stains positive in the nucleus of a variety of tumor cells. Mutations and loss of heterozygosity of p53 gene and/or accumulation of p53 product protein have been associated with increased cellular proliferation and malignant potential. In our study, it stained mild positive in the basal layer cells of DC, and ameloblastomas stained intensely positive in the peripheral columnar cells and central stellate cells indicating that the negative growth regulation of normal p53 is suppressed, at least to some extent, resulting in an increase in proliferation.

Kumamoto et al., 2004 [16] has observed that expression of p53 was significantly higher in plexiform cases than in follicular cases. Elevated expression of p53 in benign and malignant ameloblastomas suggest that alterations p53 cascade is involved in oncogenesis and/or malignant transformation of odontogenic epithelium. The results indicated that the ameloblastomas has greater proliferative potential, which can contribute to explain its more aggressive and invasive characteristics.

p63 plays an essential role in epithelial development and the proliferation of limb and craniofacial structure. p63 stains the nuclei of basal or progenitor cells in a variety of epithelia. In recent studies, up-regulated expression and/or activity of p63 have been demonstrated in malignancies. In the present study, the expression of p63 in DC is mild in the basal layer, and ameloblastoma showed intense staining in the peripheral columnar/cuboidal cells and central stellate cells. The expression of p63 suggests that these p53 homologs play a role in differentiation and proliferation of odontogenic epithelial cells. Variations of predominantly expressed isoforms suggest that p63 might differentially function in odontogenic tissues.

In conclusion, on comparing the staining intensities of different panel markers between the odontogenic cyst (DC) and odontogenic tumor (ameloblastoma) it confirms and supports the previous evidence that considerable difference exists between these two lesions in terms of their clinical characters and biologic behavior. Hence, immunohistochemical analysis with the combination of the markers used in this study may be useful in differentiating DC and ameloblastoma.

 
  References Top

1.Shibata Y, Asaumi J, Yanagi Y, Kawai N, Hisatomi M, Matsuzaki H, et al. Radiographic examination of dentigerous cysts in the transitional dentition. Dentomaxillofac Radiol 2004;33:17-20.  Back to cited text no. 1
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2.Masloub SM, Abdel-Azim AM, Elhamid ES. CD10 and osteopontin expression in dentigerous cyst and ameloblastoma. Diagn Pathol 2011;6:44.  Back to cited text no. 2
    
3.Kahn MA. Ameloblastoma in young persons: A clinicopathologic analysis and etiologic investigation. Oral Surg Oral Med Oral Pathol 1989;67:706-15.  Back to cited text no. 3
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4.Leider AS, Eversole LR, Barkin ME. Cystic ameloblastoma. A clinicopathologic analysis. Oral Surg Oral Med Oral Pathol 1985;60:624-30.  Back to cited text no. 4
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5.Zemann W, Feichtinger M, Kowatsch E, Kärcher H. Extensive ameloblastoma of the jaws: Surgical management and immediate reconstruction using microvascular flaps. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:190-6.  Back to cited text no. 5
    
6.Carlson ER, Marx RE. The ameloblastoma: Primary, curative surgical management. J Oral Maxillofac Surg 2006;64:484-94.  Back to cited text no. 6
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7.Kumamoto H, Ooya K. Immunohistochemical detection of retinoblastoma protein and E2 promoter-binding factor-1 in ameloblastomas. J Oral Pathol Med 2006;35:183-9.  Back to cited text no. 7
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8.Lau SL, Samman N. Recurrence related to treatment modalities of unicystic ameloblastoma: A systematic review. Int J Oral Maxillofac Surg 2006;35:681-90.  Back to cited text no. 8
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9.Henriques áC, Vasconcelos MG, Galvão HC, de Souza LB, de Almeida Freitas R. Comparative analysis of the immunohistochemical expression of collagen IV, MMP-9, and TIMP-2 in odontogenic cysts and tumors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:468-75.  Back to cited text no. 9
    
10.da Silva TA, Batista AC, Mendonça EF, Leles CR, Fukada S, Cunha FQ. Comparative expression of RANK, RANKL, and OPG in keratocystic odontogenic tumors, ameloblastomas, and dentigerous cysts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:333-41.  Back to cited text no. 10
    
11.Batsaki JG. Tumors of the Head and Neck: Clinical and Pathological Considerations 2nd ed. 1979. Williams and Wilkins.  Back to cited text no. 11
    
12.Stoll C, Stollenwerk C, Riediger D, Mittermayer C, Alfer J. Cytokeratin expression patterns for distinction of odontogenic keratocysts from dentigerous and radicular cysts. J Oral Pathol Med 2005;34:558-64.  Back to cited text no. 12
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13.Kumamoto H, Kimi K, Ooya K. Detection of cell cycle-related factors in ameloblastomas. J Oral Pathol Med 2001;30:309-15.  Back to cited text no. 13
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14.Crivelini MM, de Araújo VC, de Sousa SO, de Araújo NS. Cytokeratins in epithelia of odontogenic neoplasms. Oral Dis 2003;9:1-6.  Back to cited text no. 14
    
15.Müller H, Slootweg P. Clear cell differentiation in an ameloblastoma. J Maxillofac Surg 1986;14:158-60.  Back to cited text no. 15
    
16.Kumamoto H, Izutsu T, Ohki K, Takahashi N, Ooya K. p53 gene status and expression of p53, MDM2, and p14 proteins in ameloblastomas. J Oral Pathol Med 2004;33:292-9.  Back to cited text no. 16
    



 
 
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