|Year : 2022 | Volume
| Issue : 2 | Page : 126-130
Immunohistochemical Analysis of Unicystic Ameloblastoma and Dentigerous Cyst: A Comparative Study
Gargi S Sarode1, Meena Kulkarni2, Namrata Sengupta1, Sachin Sarode1, Musarrat Khatri2
1 Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
2 Private Practitioner, Pune, Maharashtra, India
|Date of Submission||26-Apr-2022|
|Date of Acceptance||07-May-2022|
|Date of Web Publication||22-Aug-2022|
Gargi S Sarode
Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Sant-Tukaram Nagar, Pimpri, Pune - 411 018, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Diagnosis of odontogenic cystic lesions is challenging because their lining epithelia, which are basically stratified squamous epithelia, resemble each other, especially when they become hyperplastic from an inflammatory reaction. The histological distinction between unicystic ameloblastomas (UAs) and certain non-neoplastic odontogenic cysts can be problematic. Thus, Notch 2 expression has been observed in this immunohistochemical study to discriminate reliably and objectively UA from dentigerous cysts (DCs) in routine practice. Literature search has shown that Notch 2 has not been studied in any of the odontogenic lesions to date, and this study is the first of its kind both nationally and internationally. Thus, the aim of this study is to determine whether Notch 2 can be used to discriminate reliably and objectively cystic jaw lesions in routine practice. Materials and Methods: A total of 26 paraffin-embedded tissues of 13 UA and 13 DC were included, and serial 3 or 4 μm sections were produced from paraffin blocks. One section from each tissue was stained with a routine stain, hematoxylin and eosin to re-evaluate the final histopathologic diagnosis and the other section was expended for immunohistochemistry. Results: The Notch 2 immunoexpression in UA and DC tissue sections demonstrated a mixed result. Four cases of DC showed strong expression, whereas two cases of UA strongly expressed Notch 2. Conclusion: Notch 2 can form a reliable marker in differentiating DC from unicystic ameloblastoma. Thus, the study proved the utility of Notch 2 in differentiating unicystic ameloblastoma from DC.
Keywords: Dentigerous cyst, Notch 2, odontogenic cyst, odontogenic tumor, unicystic ameloblastoma
|How to cite this article:|
Sarode GS, Kulkarni M, Sengupta N, Sarode S, Khatri M. Immunohistochemical Analysis of Unicystic Ameloblastoma and Dentigerous Cyst: A Comparative Study. J Dent Res Rev 2022;9:126-30
|How to cite this URL:|
Sarode GS, Kulkarni M, Sengupta N, Sarode S, Khatri M. Immunohistochemical Analysis of Unicystic Ameloblastoma and Dentigerous Cyst: A Comparative Study. J Dent Res Rev [serial online] 2022 [cited 2022 Sep 28];9:126-30. Available from: https://www.jdrr.org/text.asp?2022/9/2/126/354208
| Introduction|| |
Histopathological diagnosis of odontogenic cystic lesions is perplexing since the lining epithelia, which are essentially stratified squamous epithelia, become difficult to distinguish and lose their characteristic features, specifically when they undergo inflammatory hyperplasia. The histological discrepancy between unicystic ameloblastomas (UAs) and certain nonneoplastic odontogenic cysts can be challenging. UA, Type I, i.e., luminal variant, is difficult to diagnose as sometimes it mimics other odontogenic cysts clinically, radiologically, and histopathologically. The origin of the lesion remains controversial with some authors favoring the origin from a pre-existing odontogenic cyst, usually dentigerous cyst (DC), but including keratinizing cystic odontogenic tumor and radicular cyst (RC), while others regard this lesion as a cystic neoplasm arising de novo. It is necessary to differentiate these lesions from each other as this may drastically change the treatment strategy. Thus, Notch 2 expression has been observed in this immunohistochemical study to discriminate reliably and objectively UA from DCs in routine practice.
Notch 2 belongs to the Notch family. The Notch family plays a major role in different processes during development by regulating cell fate. The Notch signaling plexus of the pathways is an evolutionary pathway and regulates connections and interactions between neighboring cells. On ligand stimulation with the Notch intracellular domain, there is the formation of a complex, transcriptional activator-RBPJ/RBPSUH, which initiates genes of the enhancer of split locus. It shows some effects on various programs such as differentiation, proliferation, and apoptotic programs, along with modulation of bone remodeling and maintaining homeostasis.,,
The Notch family includes Notch 1, 2, 3, and 4 and has diverse special effects on growth and tumor development. Notch signaling stimulates malignancy by upholding the proliferation of progenitor cells. Notch 1 demonstrates another function of tumor suppressor in various other tumors.
Literature search has shown that Notch 2 has not been studied in any of the odontogenic lesions to date, and this study is the first of its kind both nationally and internationally. Thus, the aim of the present research was to study the expression of Notch 2 in the lining epithelium of odontogenic cystic lesions of the jaws and to determine whether this can be used to reliably and objectively diagnose the odontogenic cysts in routine practice.
| Materials and Methods|| |
The present research was carried out in Oral Pathology and Microbiology Department, Dr. D. Y. Patil Dental College and Hospital, Pimpri, Pune. Total 26 paraffin-embedded tissues (13 blocks of UA and 13 blocks of DC) were included and serial three or four micrometer sections were produced from paraffin blocks. One section from each tissue was stained with a routine stain, hematoxylin and eosin to re-evaluate the final histopathologic diagnosis, and the other section was expended for immunohistochemistry (IHC).
Dewaxed sections were pretreated for recovery or retrieval of the epitope. The activity of endogenous peroxidase was impeded with 3% hydrogen peroxide for 10 min at around room temperature of around 20°C. Then, slides were given treatment with tris-buffered saline (TBS). Overnight incubation was done using a primary antibody, anti-Notch 2 at 48°C. Once incubated with the primary antibodies, TBS was used to rinse the sections, and then incubation was done with the appropriate secondary antibodies (anti-rabbit or anti-mouse immunoglobulins) conjugated with peroxidase-labeled dextran polymers for about a time of 30 min at again room temperature, i.e., around 20°C. Sections were rinsed with TBS and subject to treatment with a 0.5 μg/mL 3, 3'-diaminobenzidine solution incorporating 0.001% hydrogen peroxide to demonstrate the resulting output. Counterstaining was performed for a short time using Mayer's hematoxylin. Then, the sections were subjected to dehydration. The final step was completed by mounting the slide. The stained sections of UA and DC were compared for Notch 2 expression.
Evaluation of immunohistochemistry staining
The stained sections were observed under a microscope and the immunostaining pattern was scored. The scoring was done by a semi-quantitative method. Scoring applied was centered on the IHC staining intensity and was marked as 0 = absent, 1 = weak, 2 = moderate, 3 = strong, and 4 = very strong.
The section stained with hematoxylin and eosin was observed for determining the histologic diagnosis.
| Results|| |
The Notch 2 immunoexpression in UA and DC tissue sections demonstrated a mixed result [Table 1] and [Table 2]. Thirteen sections were of UA and 13 were of DC. Out of 13, 2 sections (15.38%) of UA showed strong immunostaining with Notch 2, 8 (61.54%) showed moderate staining intensity and 2 (15.38%) were weak in expressing Notch 2 immunostaining. The remaining 1 section showed a moderate immunostaining intensity in the epithelium and weak in the connective tissue.
DC tissue sections showed strong immunostaining expression in 3 sections (23.07%), moderate in 4 sections (30.77%), and weak in 5 sections (38.46%). One section expressed strong staining intensity in the epithelium and moderate in the connective tissue [Figure 1], [Figure 2], [Figure 3], [Figure 4]
|Figure 1: Comparison of Notch 2 expression in UA and DC. UA: Unicystic ameloblastoma, DC: Dentigerous cyst|
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| Discussion|| |
DC and UA are odontogenic cysts and tumors, respectively, both arising from the odontogenic epithelium. Some researchers believe that the odontogenic islands present within the connective tissue capsule of DCs undergo potential transformation to give rise to ameloblastoma. UAs are a kind of cystic ameloblastomas that have an aggressive and destructive nature with the ability to cause erosion of bone and invasion of adjacent structures. Although histologically, the lesion shows ameloblastomatous epithelium, still it resembles jaw cysts in many aspects.
DC and UA are often related with the coronal part of an unerupted/impacted tooth. Clinically and radiographically, they often mimic each other and present a unilocular radiolucency surrounding the coronal part of an impacted tooth. UA is a rare lesion that even on gross pathology resembles DC, thus making its diagnosis challenging. The diagnosis of DC also becomes difficult when it is secondarily infected. The inflammatory features make it resemble like other inflammatory cysts such as periapical and paradental cysts. Thus, it is very important to differentiate these odontogenic lesions from each other, and immunohistochemical studies help in their better diagnosis.
Many such IHC studies have been done and reported in the literature, which has compared the different odontogenic cysts and tumors and made their diagnosis a little less challenging. Koneru et al. have studied the manifestation and function of calretinin in the odontogenic epithelium and in the pathogenesis of odontogenic tumors respectively. Ameloblastoma showed a stronger expression of calretinin than odontogenic keratocyst, whereas a negative expression was exhibited by an adenomatoid odontogenic tumor. Cytokeratin expression was also studied by Stoll et al. to differentiate the different odontogenic cysts. Odontogenic keratocysts (OKC) showed cytokeratin expression and it helped to differentiate OKC from DC and radicular cysts.
Notch 2 gene is well-studied till now in the literature and plays a vital role in various developmental processes. It has a major part in the growth and invasion of cells., In vertebrates, four Notch receptor proteins (Notch 1, 2, 3, and 4) and five membrane-bound ligand proteins (Delta 1, 2, and 4, Jagged1 and 2). Information through stimulus is carried forward between adjacent cells by attaching the ligand with its related or associated receptor. It begins with a short array of proceedings comprising discrepancy, multiplication, and apoptotic proceedings at all developmental stages. It leads to regulating organ development and morphogenesis. Disturbance in Notch signaling regulation has also been linked to various disturbances or disorders evolving during development and various neoplasms.
Notch 2 expression has been studied in cell growth and invasion. A study reported that it had a role to play in the pathogenesis of hepatoblastomas., Significance of Notch 2 was also studied in gastric carcinomas, wherein strong expression of Notch 2 in early tumors showed a better prognosis.
A study of Notch 2 expression in odontogenic lesions has not yet been mentioned in the literature. Hence, an attempt was made in this IHC study to observe whether Notch 2 is capable in distinguishing two odontogenic lesions (UA and DC) which clinically, radiographically and gross pathologically mimic each other. It is quite evident from the results that Notch 2 is expressed in the odontogenic epithelium. About 19.23% of the cases of UA and DC showed strong expression of Notch 2, whereas 46.15% of the cases exhibited moderate expression of Notch 2.
Notch signaling plays a major role in the specification related to cell fate in calcifying cystic odontogenic tumor (CCOT)-associated ghost cells (GCs). Immunohistochemical staining for four Notch receptors (Notch 1–4) and three ligands (Jagged1, 2 and Delta 1) was done on CCOT archival tissues. Outcomes disclosed that GCs exhibited excessive Notch 1 expression and Jagged1. It indicates that Notch 1-Jagged1 signaling could be the chief transduction pathway in decisions related to cell fate in GCs seen in CCOT. GCs demonstrated positivity for Notch 2 suggesting that the calcification process is related to upregulation of the said molecule. The expression of Notch 2 was weaker to totally absent in GCs and the epithelium seen in CCOT.
The activation of Notch in the final functions of cell fate works through three different mechanisms of action or pathways: lateral inhibition, binary cell fate, and lateral induction.,,
Cells with a determined fate restrain their adjoining cells from embracing the same fate. This indicates that the signal produced by interaction concerning the Notch ligand and the receptor on an adjoining cell constrains ligand manufacturing in the receptive cell through a negative feedback loop.
Binary cell fate
Irregular arrangement of Notch pathway sections regulates distinctive fates of the cells.
In this, signaling of Notch ligands to their receptors produces positive feedback promoting Notch ligand manifestation and triggering on both the adjoining cells. A cell to cell dispatch is used to transfer Notch signals in this mechanism.,,
Notch signaling is intricated in the upholding of the form of the tissue stem cell and controlling of the differentiating property and is accountable for the cytological controlling of the fate of the cells and developmental growth. A study has proved the use of IHC to assess Notch expression in odontogenic tumors and salivary gland neoplasm, pleomorphic adenoma (PA). Notch signaling was intricated in the property of differentiating of the epithelial cell in the salivary ducts of salivary gland tumors and also in the odontogenic neoplasms in the case of ameloblast-like cells. Notch signaling was also complexed in squamous metaplasia as well. In odontogenic neoplasms, Notch signaling was involved in epithelial–mesenchymal interactions and thus linked to tumorigenesis. The pathway is also suggested to be correlated with the malignant change of ameloblastomas. Notch signaling also directs the development of the distinguishing cellular structure and histological characteristic features of various odontogenic and non-odontogenic tumors.
Notch signaling is intricated in the cellular differentiation of ameloblast-like columnar cells in odontogenic tumors and cysts. Notch signaling sometimes demonstrates a cross talk with Wnt signaling and thus is known to function in various events such as differentiation of the neoplastic cell. This cross talk concerning Wnt and Notch is suggested to be associated with progression of the tumor in case of odontogenic tumors such as ameloblastoma.
Thus, Notch signaling is known to function in various neoplasms of epithelial origin (ameloblastoma), mesenchymal origin (odontogenic myxoma), mixed origin (ameloblastic fibroma), odontogenic cysts like calcifying odontogenic cyst, odontogenic origin malignancies like ameloblastic carcinoma and salivary neoplasms like PA.
| Conclusion|| |
Notch 2 can form a reliable marker in differentiating DC from unicystic ameloblastoma Type 1. It is important to differentiate both the lesions as unicystic ameloblastoma does not just need to be excised, but a stringent follow-up is necessary. Thus, the study proved the utility of Notch 2 in differentiating unicystic ameloblastoma from dentigerous cyst.
The ethical clearance number is DPU/Research 159 (17)/2013.
We would like to thank Mrs. Hema Chougule, our laboratory technician, for preparing the IHC slides.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]