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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 2  |  Page : 168-171
Immunohistochemical analysis of CK 18 in dental follicles of impacted third molars: A pilot study


1 Department of Oral Pathology, Coorg Institute of Dental Sciences, Virajpet, India
2 Department of Oral and Maxillofacial Surgery, A. B. Shetty Memorial Institute of Dental Sciences, Dheralakatte, Mangalore, India
3 Department of Pathology, Kidwai Memorial Institute of Oncology, M. H. Marigowda Road, Bangalore, India

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Date of Submission16-Mar-2012
Date of Decision12-Aug-2012
Date of Acceptance22-Nov-2012
Date of Web Publication20-Aug-2013
 

   Abstract 

Objective: Odontogenic tumors are lesions derived from epithelial, ectomesenchymal, and/or mesenchymal elements that still are, or have been, part of the tooth-forming apparatus. Approximately 80% of odontogenic tumors occur in the mandible, with a marked predilection for the posterior region, and are often associated with an unerupted tooth. The aim of this study was to determine whether cytokeratin (CK) 18 immunostaining decorated the follicular tissue removed at the time of prophylactic extraction of impacted mandibular third molars, which might suggest oncofetal transformation.
Materials and Methods : Fifty-four impactions met the study inclusion criteria, of which 24 cases showed the presence of reduced enamel epithelium and/or connective tissue with odontogenic epithelium, which were subjected to CK 18 immunostaining.
Results: All 24 cases with adequate epithelium were CK 18 immunonegative.
Conclusion: There was no oncofetal transformation in the odontogenic epithelia of the dental follicles studied. Thus, although we reaffirm that evaluation of follicular tissue is imperative since disease conditions may be found in minute follicular spaces, development of odontogenic cysts and tumors is unlikely.

Keywords: CK 18 immunohistochemistry, dental follicle, impacted mandibular third molar, pathology

How to cite this article:
Tegginamani AS, Prasad R, Kumar RV. Immunohistochemical analysis of CK 18 in dental follicles of impacted third molars: A pilot study. Indian J Dent Res 2013;24:168-71

How to cite this URL:
Tegginamani AS, Prasad R, Kumar RV. Immunohistochemical analysis of CK 18 in dental follicles of impacted third molars: A pilot study. Indian J Dent Res [serial online] 2013 [cited 2020 Feb 25];24:168-71. Available from: http://www.ijdr.in/text.asp?2013/24/2/168/116671
Odontogenic tumors are lesions derived from epithelial, ectomesenchymal, and/or mesenchymal elements that still are, or have been, part of the tooth-forming apparatus. Approximately 80% of odontogenic tumors occur in the mandible, with a marked predilection for the posterior region, and are often associated with an unerupted tooth. [1] Controversy still exists regarding the removal of asymptomatic impacted teeth. The follicular sac surrounding the impacted tooth is interpreted in radiographs as pericoronal radiolucency. The differentiation between a normal and abnormal dental follicle is based on the width of this radiolucency. Both reduced enamel epithelium and odontogenic epithelium can be found in the tissue removed from the dental follicle at the time of extraction. These epithelia and/or their remnants are the origin of different odontogenic lesions, with distinct clinical behavior. Several studies have reported differences in the proliferative potential of odontogenic epithelial cells, with significant impact on the formation of odontogenic cysts and tumors. [2]

A number of two-dimensional gel electrophoresis experiments on keratin subunits extracted from various epithelial tissues have shown a total of 20 different subunits in any mammalian species, with molecular weights varying within the range 40-70 kDa, and categorized a total of 19 human epithelial keratins; an additional keratin 20 was subsequently identified. The keratins can be divided into low- and high-molecular forms based on molecular weight, and into acidic and basic forms based on isoelectric point. Not all keratins are synthesized simultaneously by any one cell; rather, different subsets of keratin are expressed during the course of terminal differentiation, in different stages of development, as well as in different epithelia. Thus, all epithelia (simple and complex) can be classified based upon cytokeratin (CK) protein expression. When an epithelium undergoes malignant transformation, its keratin profile usually remains constant. Therefore, since keratin expression varies so greatly among different epithelia, they have been widely used in the fingerprinting of various carcinomas. [3]

Numerous immunohistochemical studies have been undertaken in the past to compare the CK profile of dentigerous cyst epithelium with that of odontogenic keratocyst (OKC) and radicular cyst. Their results indicate that the dentigerous but not other cyst types may share with ameloblastoma the expression of CK 18, with the speculation that CK 18 positive cells could have a specific histogenetic origin and consequently distinct functional characteristics. Positive CK 18 expression was considered suggestive of oncofetal transformation. [4]

This study was planned to evaluate the potential oncofetal transformation in the odontogenic epithelium of the dental follicle in the pericoronal tissues of impacted third molars by CK 18 immunostaining.


   Materials and Methods Top


Impacted third molars were removed for a variety of reasons at the Department of Oral and Maxillofacial Surgery, A. B. Shetty Institute of Dental Sciences, Mangalore, South India. Ethical clearance was obtained, and informed consent from the patients was taken. The patients underwent preliminary intraoralperiapical (IOPA) radiography to determine if they met the study criteria. Fifty-four patients with age ranging from 16 to 30 years were selected. The inclusion criteria were: (1) presence of at least one impacted third molar and (2) pericoronal radiolucency of less than 2.5 mm in any dimension. Follicular tissue specimens were collected and fixed in 10% formalin, stained routinely with Hematoxylin and Eosin, and examined independently by two oral pathologists. Thirty cases showed fibrous connective tissue only, and thus 24 cases were taken for subsequent immunostaining. These cases showed either (a) reduced enamel epithelium (REE) with fibrous connective tissue with/without inflammatory cells or (b) stratified squamous epithelium with fibrous connective tissue with/without inflammatory cells.

Immunohistochemistry procedure

The 4-μm-thick sections were mounted on 3-aminopropyl triethoxysaline (APES) coated slides and incubated at 58°C overnight. The sections were then dewaxed in two changes of xylene for 15 min each, and rehydrated through a series of graded alcohols and distilled water. Antigen retrieval was performed by the conventional pressure cooker method in citrate buffer (pH 6.0) for 10-12 min. The slides were then rinsed in distilled water for 5 min and transferred to Tris-buffered saline (TBS) buffer (pH 7.5) for 10 min.

Blocking of endogenous peroxide was done by flooding the slides with 3% hydrogen peroxide for 15 min, followed by washing in three changes of TBS buffer for 5 min each. Power block was used for 15 min to block non-specific reaction with other tissue antigens. The sections were then flooded with CK 18 prediluted primary antibody (BioGenex Laboratories Inc., Fremont CA, USA) for 1 h.

This was followed by three changes of TBS buffer for 5 min each to remove unbound antibodies. Super enhancer was used for duration of 30 min to enhance the reaction, followed by TBS buffer wash. The sections were then treated with polymer Horseradish Peroxidase (HRP) for 30 min, followed by a TBS buffer wash. Chromogen was then added, followed by washing in TBS buffer and tap water. Counterstaining was done with Mayer's hematoxylin and mounting with distrene polystyrene xylene (DPX).

Two oral squamous cell carcinoma sections and one breast carcinoma section were used as positive controls. In addition, four adenomatoid odontogenic tumors (AOTs) and two dentigerous cysts were included for comparison. Sections without primary antibody served as negative controls. All sections were screened by two pathologists. A diagnosis was registered only when the interpretation of both pathologists was in concordance.

The immunohistochemistry (IHC) staining was scored in the following manner:

  • Score I: No cells stained
  • Score II: Weak staining or few cells stained (11-25%), +
  • Score III: Moderate staining or many cells stained (26-75%), ++
  • Score IV: Intense staining or most cells stained (more than 76%), +++.

   Results Top


The REE showed negative (Score I) CK 18 expression in all 24 cases [Figure 1]. One breast carcinoma and both oral squamous cell carcinomas showed high expression (Score IV/+++) and served as positive controls [Figure 2]. Four AOTs and both dentigerous cysts were negative (Score I).
Figure 1: Photomicrographs of (a) reduced enamel epithelium and (b) AOT; both were negative (Score I) for CK 18

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Figure 2: Photomicrographs of (a) breast carcinoma and (b) oral squamous cell carcinomas showed high expression (Score IV/+++) for CK 18

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   Discussion Top


The removal of asymptomatic or pathology-free mandibular third molar teeth is controversial. In 2000, the National Institute for Clinical Excellence had published guidance to practitioners, recommending against the removal of pathology-free third molars in the United Kingdom. A counterclaim has been made that waiting for pathology to cause damage is a lower standard of care when early preventive measures may be possible. [5] This study was undertaken to test the veracity of the latter claim.

It has been stated that the potential transformation of unerupted teeth into cystic or neoplastic lesions is related to the constituent structures of the follicle, in particular, the REE and remnants of the dental lamina located in its connective tissue wall. Radiographically, the pericoronal follicles present as slight semicircular radiolucencies around unerupted teeth; however, enlargements or asymmetries can occur, which may be misinterpreted. Pericoronal radiolucency is one of the most important factors to be borne in mind in deciding whether to retain or remove an impacted tooth, and the presence of radiolucency may be the only indication for this procedure. A radiographic pericoronal radiolucency of 2.4 mm or less is considered as normal in the literature; a cut-off point of 2 mm for periapical radiographs and 2.5 mm for panoramic radiographs is accepted as pathologically widened coronal spaces. [6]

Despite the importance of the radiographic findings, disease conditions may be found in minute follicular spaces; also, in enlarged radiolucent areas there may be histologically normal tissues, so histopathologic examination is imperative. The dental follicles of asymptomatic impacted third molars might be dormant or actively proliferating. It has been noted that the inflammation observed in the mesenchymal components of dental follicles up-regulates the cell turnover of odontogenic epithelium, leading to proliferation. [7] It is well documented that some dentigerous cysts transform into ameloblastoma or, rarely, even undergo malignant transformation to central mucoepidermoid carcinoma. [8] It is, however, important not to misinterpret dental follicles with a lot of odontogenic epithelial remnants on histology for the mural variant of unicystic ameloblastoma. The latter would show peripheral palisading and nuclear polarization. Calcifying cystic odontogenic tumor (CCOT) may show proliferation of odontogenic epithelium in the adjacent connective tissue and one-third of CCOTs are associated with an unerupted tooth. The immunoprofile similarities of odontomas and dental germs suggest that the AOT originates from the REE. [7],[8],[9]

CK patterns differ in the different types of oral epithelium. Numerous studies have been done on CK expression and other tissue markers in dysplastic and neoplastic oral epithelium. [3] CK 18 is potentially both a quantitative and qualitative biomarker for cell death. CKs 8, 18, and 19 are expressed by most types of carcinomas, including those of the breast, prostate, lung, colon, and ovary. It is speculated that CK 18 positive cells could have a specific histogenetic origin and could consequently have distinct functional characteristics, and CK18 immunostaining is suggestive of oncofetal transformation. [4] Other studies have suggested that the expression of CK 18 could be indicative of initiation of abnormal cell differentiation and CKs are involved in cell signaling responses to stress and apoptosis. [10] Neoplasms and tumors related to the odonotogenic apparatus may be composed only of epithelial tissue or epithelial tissue associated with odontogenic ectomesenchyme. The immunohistochemical detection of different CK polypeptides and vimentin has made it easier to explain the histogenesis of many epithelial diseases. [11] CK 18 expression has been seen in oral submucous fibrosis (OSF) samples as well as in oral cancer (OC) tissues. CK 18, as a single marker, has been identified as an independent prognostic factor for disease free- and overall survival in patients with stage II/III OC tumors. Morphologically normal tissues can also show CK 18 expression. Thus, aberrant expression of CK 18 in these tissues could be indicative of initiation of abnormal cell differentiation. [12]

According to Yoon et al., apart from the reflection of organ type and degree of differentiation, CK 18 may have some regulatory role in malignant transformation. Up-regulation of CK 18 may merely reflect integrated transcriptional activation of such transcription factors. Taken together, a significant increase of CK 18 expression in ameloblastic carcinoma could be interpreted as a change in differentiation (dedifferentiation) of tumor cells or as evidence of malignant transformation, whether or not the up-regulation of CK 18 is directly related with the induction of transformed phenotype. [13] A dentigerous cyst is one that encloses the crown of an unerupted tooth by expansion of its follicle (accumulation of fluid) and, attached to its neck, some unerupted teeth have a slightly dilated follicle in the pre-eruptive phase. This does not signify a cyst or even necessarily a potential cyst unless the pericoronal width is at least 3-4 mm. There would therefore appear to be some evidence of an inflammatory etiology in the pathogenesis of some dentigerous cysts. Nevertheless, individual cases need to be assessed critically. Attachment of the cyst wall to the neck of the associated tooth is an essential feature, and microscopically, the cyst lining should demonstrate a readily identifiable component of REE before a diagnosis of dentigerous cyst is made. Histological examination usually shows a thin fibrous cyst wall which, being derived from dental follicle, consists of young fibroblasts widely separated by stroma and ground substance rich in acid mucopolysaccharide. The epithelial lining, which is in fact REE, consists of two to four cell layers of flat or cuboidal cells. Localized proliferation of the epithelial lining may occur in response to inflammation. Occasional bud-like thickenings of the epithelium may be seen in the absence of inflammation, and sometimes there may be budding of the basal cells into the fibrous capsule. Nests, islands, and strands of odontogenic epithelium are often seen in the capsule. [4]

Whether squamous epithelium in follicular tissue in an impacted tooth with a pericoronal radiolucency of less than 2.4 mm should be considered a dentigerous cyst is debatable.

Significant increase of CK 18 expression is considered suggestive of oncofetal transformation, malignant transformation, or initiation of abnormal cell differentiation. [4],[10],[11],[12],[13] Although the REE and odontogenic epithelia of dental follicles showed negative (Score I) expression in all cases, suggesting there was no oncofetal transformation in the present study, however histologic study is mandatory for all follicular tissue after extraction. Unfortunately, most surgeons discard follicular tissue after extraction rather than submitting them for histopathologic evaluation, thus losing a potentially valuable informative source. Larger sample studies are required to improve our knowledge of the role of CK18 as abnormal cell differentiation/oncofetal transformation with REE and/or odontogenic epithelium of dental follicle.


   Conclusions Top


Many impacted mandibular third molars remain asymptomatic for years, but are often prophylactically extracted to prevent development of future complications and pathologic conditions. Thorough clinical and radiographic examination of all such impacted molars and histopathologic examination of the dental follicular tissue is essential. Although CK 18 immunostaining did not decorate the epithelial elements in the follicular tissue in this study, other proliferative immunohistochemical markers or histomorphometric methods might provide further assistance in the diagnosis of abnormalities associated with odontogenic epithelia of dental impactions.

 
   References Top

1.Philipsen HP, Reichart PA, Slootweg PJ, Slater LJ, Gardner DG. Neoplasms and tumour-like lesions arising from the odontogenic apparatus and maxillofacial skeleton: Introduction, Ameloblastomas. In: Barnes L, Evson JW, Reichart P, Sidransky D, editors. World Health Organization classification of tumors. Pathology and genetics head and neck tumors. London: IACR Press; 2005. p. 285-96.  Back to cited text no. 1
    
2.Oliveira MG, Silva I, Lauxen A, Chaves CM, Rados PV, Manoel SF. Odontogenic epithelium: Immunolabeling of Ki-67, EGFR and survivin in pericoronal follicles, dentigerous cysts and keratocystic odontogenic tumors. Head Neck Pathol 2011;5:1-7.  Back to cited text no. 2
    
3.Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms Histopathology. 2002;40:403-39.  Back to cited text no. 3
    
4.Shear M, Speight P. Cyst of the oral and Maxillofacial Regions in dentigerous cyst Fo ed Oxford, England, UK: Blackwell Munkgaard Publication; 2007. p. 58-74.  Back to cited text no. 4
    
5.Hughes AJ, Vudiniabola ST, McMillan BD, Smith AC. Rotational drift of mandibular third molar teeth: A series of four cases. Aust Dent J 2009;54:54-6.  Back to cited text no. 5
[PUBMED]    
6.De Oliveira DM, de Souza Andrade ES, da Silveira MM, Camargo IB. Correlation of the radiographic and morphological features of the dental follicle of third molars with incomplete root formation Int J Med Sci 2008;5:36-40.  Back to cited text no. 6
    
7.Cabbar F, Guler N, Comunoglu N, Sençift K, Cöloðlu S. Determination of potential cellular proliferation in the odontogenic epithelia of the dental follicle of the asymptomatic impacted third molars. J Oral Maxillofac Surg 2008;66:2004-11.  Back to cited text no. 7
    
8.Vered M, Peleg O, Taicher S, Buchner A. The immunoprofile of odontogenic keratocyst that includes expression of PTCH, SMO, GLI-I and bcl-2 is similar to ameloblastoma but different from odontogenic cysts J Oral Pathol Med 2009;38:597-604.  Back to cited text no. 8
    
9.Crivelini MM, Felipini RC, Coclete GA, Soubhia MP. Immunoexpression of keratins in the calcifying cystic odontogenic tumor epithelium. J Oral Pathol Med 2009;38:393-6.  Back to cited text no. 9
    
10.Altun SZ, Akhisarolu ST, Batu J, Ate H, Giray H, Koçtürk S. Discrimination effectiveness of CK18 on cell death modes in colon cancer cells. Turk J Biochem 2010;35:208.  Back to cited text no. 10
    
11.Crivelini MM, Araujo VC, Sousa SOM, de Arau NS. Cytokeratins in epithelia of odontogenic neoplasms. Oral Dis 2003;9:1-6.  Back to cited text no. 11
    
12.Ranganathan K, Kavitha RS, Sawant S, Vaidya MM. Cytokeratins in oral submucous fibrosis. J Oral Pathol Med 2006;35:25-32.  Back to cited text no. 12
    
13.Yoon HJ, Jo BC, Shin WJ, Cho YA, Lee JI, Hong SP, et al. Comparative immunohistochemical study of ameloblastoma and ameloblastic carcinoma Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:767-76.  Back to cited text no. 13
    

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Correspondence Address:
Anand S Tegginamani
Department of Oral Pathology, Coorg Institute of Dental Sciences, Virajpet
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.116671

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