|Year : 2012 | Volume
| Issue : 2 | Page : 232-237
Overexpression of MDM2 protein in ameloblastomas as compared to adenomatoid odontogenic tumor
A Krishna1, H Kaveri2, RK Naveen Kumar3, KL Kumaraswamy4, S Shylaja1, Sarvani Murthy3
1 Department of Oral and Maxillofacial Pathology, S.V.S Institute of Dental Sciences, Mahabubnagar, Andhra Pradesh, India
2 Department of Oral and Maxillofacial Pathology, S.D.M College of Dental Sciences and Hospital, Dharwad, India
3 Department of Oral and Maxillofacial Pathology, Sri Hasanamba Dental College and Hospital, Hassan, India
4 Department of Oral and Maxillofacial Pathology, Farooqia Dental College and Hospital, Mysore, Karnataka, India
|Date of Web Publication||26-Jul-2012|
Department of Oral and Maxillofacial Pathology, S.V.S Institute of Dental Sciences, Appanapally, Yenugonda, Mahabubnagar, Andhra Pradesh
Background: Recent studies on odontogenic tumors have identified various molecular alterations responsible for their development, and determination of epithelial proliferation is a useful means of investigating the differences in biologic behavior of these tumors. One such specific marker to identify proliferative activity and tumor aggressiveness by immunohistochemistry (IHC) is MDM2, 90-95kDa protein.
Objective: This immunohistochemical study using MDM2 expression was undertaken to understand better the diverse biological activity of two groups of odontogenic tumors namely ameloblastoma and adenomatoid odontogenic tumor (AOT) based on their cell proliferation activity.
Materials and Methods: A total of 50 cases, comprising of 36 ameloblastoma samples and 14 AOT samples, were subjected to heat-induced antigen retrieval method using citrate buffer in a pressure cooker. Consequently, the sections were stained with MDM2 monoclonal antibody and visualized using an LSAB+ kit.
Results: In ameloblastomas, statistically significant association was seen between plexiform ameloblastomas, follicular ameloblastomas with granular cell changes, desmoplastic and unicystic variants. The predominant nuclear staining by MDM2 revealed overexpression in ameloblastomas as compared to AOT.
Conclusion: The MDM2 overexpression noticed in plexiform ameloblastoma, follicular ameloblastoma with granular cell changes and acanthomatous ameloblastoma when compared to simple unicystic and desmoplastic ameloblastoma suggest a relatively enhanced proliferative phenotype of these solid multicystic variants of ameloblastomas. On overall comparison, higher expression was noted in ameloblastomas when compared to AOT. This indicates differences in the aggressive nature between these two groups of odontogenic tumors favoring the perception of a greater aggressive nature of ameloblastomas.
Keywords: Adenomatoid odontogenic tumor, ameloblastoma, immunohistochemistry, MDM2, odontogenic tumors
|How to cite this article:|
Krishna A, Kaveri H, Naveen Kumar R K, Kumaraswamy K L, Shylaja S, Murthy S. Overexpression of MDM2 protein in ameloblastomas as compared to adenomatoid odontogenic tumor. J Can Res Ther 2012;8:232-7
|How to cite this URL:|
Krishna A, Kaveri H, Naveen Kumar R K, Kumaraswamy K L, Shylaja S, Murthy S. Overexpression of MDM2 protein in ameloblastomas as compared to adenomatoid odontogenic tumor. J Can Res Ther [serial online] 2012 [cited 2013 May 24];8:232-7. Available from: http://www.cancerjournal.net/text.asp?2012/8/2/232/98976
| > Introduction|| |
Normal cell growth is regulated by a finely tuned balance between its growth promoting proto-oncogene and its growth constraining gene. Disturbance in this equilibrium either from a struck accelerator (an activated oncogene) or a defective breaking system (an inactivated growth suppressing gene) may propel the cells toward malignancy.  Determination of this epithelial proliferation, using immunohistochemistry (IHC), helps in investigating the differences between biological behavior of various tumors.
One such specific marker to identify proliferative activity and tumor aggressiveness by IHC is MDM2, a 90-95kDa protein coded by mdm2 gene which is mapped on chromosome 12q13-14.  MDM2 protein overexpression generally results from gene amplification and concomitant appearance of double minutes (hence the name Murine Double Minute).  Using IHC, MDM2 overexpression associated with poor prognosis has been reported in various human cancers including breast carcinomas, malignant melanomas, esophageal carcinomas, soft tissue sarcomas, osteosarcomas, lung and bronchogenic carcinomas, leukemias, lymphomas and head and neck cancers. 
The ameloblastoma is the second most common odontogenic tumor. According to 2005 WHO classification, it has been divided into four groups namely solid / multicystic, desmoplastic, unicystic and extra-osseous ameloblastoma.  Though ameloblastoma is benign, it deserves special attention because of its particular biological behavior exhibiting greater infiltrative potential, high recurrence rate (55-90%)  and capacity to metastasize or undergo malignant transformation when compared to its other epithelial counterpart-the adenomatoid odontogenic tumor (AOT). Molecular studies have offered interesting findings regarding ameloblastoma pathogenesis.  In a couple of studies, the IHC expression of MDM2 was examined only in ameloblastomas, , and only one study has been done so far to assess the IHC expression of MDM2 in odontogenic cysts and tumors, of which tumors comprised only a minor fraction consisting of only fifteen ameloblastomas with as little as only two histological subtypes and eight AOT cases.  As very minimal data exists on MDM2 expression in odontogenic tumors consisting of different subtypes of ameloblastomas and AOT, the current study was undertaken to assess the contribution of MDM2 in three histological variants of ameloblastomas namely solid / multicystic, desmoplastic and unicystic each having proliferative diversity. In addition, the present investigation by comparing the expressions of MDM2 between ameloblastoma and AOT aims to elucidate whether MDM2 could be used as a solitary marker to predict the different aggressive potentialities of these two groups of odontogenic tumor.
| > Materials and Methods|| |
A total of 50 cases was evaluated immunohistochemically for MDM2 protein expression consisting of buffered formalin fixed, paraffin embedded tissues of previously diagnosed 36 cases of ameloblastomas and 14 AOT. The samples used in our study were diagnosed based on their clinical features which included the presenting signs and symptoms, site, size, extent and duration of the lesion along with radiological and histopathological parameters. Amongst the 36 ameloblastoma cases, according to 2005 WHO classification, three histopathological variants were included comprising of 27 cases of solid / multicystic ameloblastomas, three desmoplastic and nine unicystic ameloblastomas out of which four showed mural proliferation with plexiform changes. Within the solid / multicystic ameloblastomas, the following subtypes were included consisting of seven plexiform, five acanthomatous, 12 follicular and three follicular with focal granular cell changes.
Monoclonal antibodies and immunohistochemical technique
Sections of 4μm thickness were taken on silanized slides and de-paraffinized. They were subjected to heat-induced antigen retrieval method using citrate buffer (10mM, pH 6.0) in a pressure cooker for 2 min. To block endogenous peroxidase activity, the samples were treated with 3% hydrogen peroxide solution for 15 min. Consequently, the sections were incubated for 4 h with primary monoclonal antibody MDM2 (clone SMP14) diluted 1:50 in TRIS-Hcl (pH 7.6) followed by 45 min in secondary antibody (rabbit antibody against mouse immunoglobulins) (DAKO, Denmark). It was then immersed in streptavidin-biotin peroxidase conjugate (DAKO) and incubated for 10 min with 3, 3-diaminobenzidine chromogen (DAKO). Harris hematoxylin was used as counter-stain. All the steps were carried out at room temperature and after each step the sections were washed with TRIS-Hcl buffer (pH 7.6). Omission of primary antibody was used as a negative control. A sample of fibrosarcoma with previously known reactivity was used as positive control.
A semi-quantitative analysis of MDM2 expression was done based on the presence of brown colored nuclear staining. The representative fields were selected in each case randomly and up to 500 tumor cells were counted under high-power magnification (x40) with binocular microscope fitted with an eyepiece (x10) hauling a grid containing 100 blocks (10x10). Two individual examiners (A and B) performed the counts to determine inter-observer reliability so that cell counting could be proficient and without bias. The cases were scored by counting the positive cells per minimum of 500 tumor cells per specimen. The percentage labelling index (L.I) (number of positive tumor cells / total number of tumor cells, expressed as percentage) was calculated per case.
Deducing the required values:
- Total number of tumor cells in each slide should reach a minimum of 500 which would be the sum of the denominators.
- Total number of positive tumor cells was obtained by adding the values in the numerator (A + B) that is cell count obtained by examiner A plus cell count obtained by examiner B.
- The percentage of positive tumor cells was obtained by the simple formula shown below
The average of the percentage thus obtained by observers for each slide was used for scoring and for further analysis.
The normal mucosa, subjected to the similar calculation, revealed less than 10% of positive cells. This decided the percentage that could be used for negative expression of MDM2. The tumors with more than 10% of positive cells were considered positive and those, which express less than 10% of cells, were considered negative.
Scoring of percentage positive tumor cells were carried out as following → <10% staining as negative, 10-30% as Grade I, 30-50% as Grade II and >50% as Grade III.
The resultant values were then submitted for statistical analysis comprising of rank sum two-sample test (Mann-Whitney U test) and P value of < 0.05 was considered to be statistically significant.
| > Results|| |
The statistical analysis data on comparison between the study groups are tabulated from [Table 1], [Table 2], [Table 3] and [Table 4].
|Table 3: Comparison of overall ameloblastomas and AOT by Mann– Whitney U test|
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|Table 4: Statistical comparison of MDM2 staining results between AOT and various types of ameloblastomas by Mann– Whitney 'U' test|
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The staining was almost confined to nuclei except on occasions where cytoplasmic staining was observed. The results of immunohistochemical analysis of MDM2 expression in different groups of ameloblastomas are summarized in [Table 1] and similarly for AOT the results are tabulated in [Table 2]. The mean and standard deviation results in different types of ameloblastoma and AOT are illustrated in Graph 1. The MDM2 positive nuclei were found predominantly in peripheral ameloblast-like cells and in few cases it was also seen in central stellate reticulum like cells [Figure 1] and [Figure 2]. Majority of ameloblastomas not only showed positivity but also a score of grade III as compared to a very minor fraction of AOT [Figure 3] which were positive and had a score of grade III. The same is depicted in graphical representations [Graph 2 and 3]. The central cells of acanthomatous and granular cell ameloblastomas which are the subtypes of solid / multicystic ameloblastomas along with cystic epithelial lining in unicystic ameloblastomas showed negative MDM2 expression whereas the proliferating luminal and intra-mural strands in unicystic ameloblastoma showed a positive expression [Figure 2]a. Statistically highly significant difference (P=0.0048) in MDM2 expression was noted between ameloblastomas and AOT at 1% level of significance (LOS) (P < 0.01) [Table 3]. Amongst the histopathological variants of ameloblastomas, significant association was observed between solid / multicystic ameloblastomas and desmoplastic ameloblastoma. Under solid / multicystic ameloblastomas, significant association was noticed between the plexiform and desmoplastic ameloblastomas (P=0.0167) and also between follicular ameloblastoma with granular cell changes and desmoplastic ameloblastoma (P= 0.0495). Statistically significant association was also seen between solid / multicystic ameloblastoma subtype namely plexiform and unicystic ameloblastomas (P=0.0262). All these cases showed association between the groups at 5% LOS (P < 0.05). The statistically significant association observed between AOT and the three subtypes of solid / multicystic ameloblastoma include AOT and follicular ameloblastomas with granular cell changes (P=0.0233), AOT and acanthomatous ameloblastomas (P=0.0519) at 5% LOS (< 0.05) and a highly significant association which was seen between AOT and plexiform ameloblastomas (P= 0.0017) at 1% LOS (P < 0.01) [Table 4].
|Figure 1: MDM2 immunoreactivity exhibiting positive nuclear staining in (a) Strands of plexiform ameloblastoma (×250; Inset – lower magnification of same field ×100); (b) Follicular ameloblastoma (×250)|
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|Figure 2: (a) Positive MDM2 nuclei in the intra-mural proliferating strands and negative staining of the cystic lining in unicystic plexiform ameloblastoma (×100); (b) Positive MDM2 expression showing nuclear staining of peripheral ameloblast like cells and few central stellate reticulum like cells in desmoplastic ameloblastoma. Mature bony trabeculae adjacent to odontogenic epithelial island showing negative expression (×100; Inset – higher magnification of same field ×400)|
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|Figure 3: Predominantly negative MDM2 expression in adenomatoid odontogenic tumor (×250)|
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| > Discussion|| |
Recent studies have identified various molecular alterations responsible for development and progression of odontogenic tumors. Amongst the potentially oncogenic elements of cell cycle, machinery expression of MDM2 is associated with cell cycle progression and apoptosis suggesting that MDM2 plays a central role in cancer development and progression. 
The p53-mdm2 paradigm represents the best studied relationship between a tumor suppressor gene and an oncogene. These two genes form an auto-regulatory feed-back loop in which p53 positively regulates MDM2 levels and MDM2 in-turn negatively regulates p53 and its activity by inhibiting p53 functions as transcription factor and also inducing intracellular p53 degradation.  It has been demonstrated that single nucleotide polymorphism 309 (SNP 309) resulting in a T to G alteration within the first intron of the intronic MDM2 gene promoter can enhance MDM2 expression and therefore lead to a reduced tumor suppressor function of p53.  Since MDM2 is a negative regulator of p53 activity, the overproduction of MDM2 protein gives rise to a greater risk in the development of malignancy.  MDM2 protein also interacts with other cellular proteins involved in cell cycle regulation including pRb, E2F1/DP1 and p19(ARF). Therefore MDM2 plays a crucial role in malignant transformation and tumor growth indicating that the mdm2 oncogene is a potential molecular target for cancer therapy. 
In our study, solid / multicystic ameloblastomas showed MDM2 reactivity mainly in peripheral columnar or cuboidal cells and occasionally in central stellate reticulum like cells suggesting that the peripheral cells have an anti-apoptotic and proliferative phenotype unlike the central cells. These findings are compatible with studies on ameloblastomas using various proliferation markers like PCNA and Ki-67. 
The keratinizing cells of acanthomatous ameloblastoma in the present study showed less MDM2 reactivity. A similar observation is evident using MDM2 and p14(ARF)  suggesting terminal differentiation and non-proliferative phenotype of these central cells.
Our cases showed higher percentage of MDM2 positive cells in solid / multicystic ameloblastoma subtypes namely plexiform and follicular ameloblastomas with granular cell changes as compared to desmoplastic variant. Kumamoto et al, also describe increased apoptosis associated with high reactivity for Caspase 3 and Fas in desmoplastic as compared to low reactivity for these markers in other ameloblastoma variants. Interpretation of these results indicates lesser aggressive nature of desmoplastic ameloblastomas.
In the current study, statistical comparison between unicystic and the plexiform variant of solid / multicystic ameloblastoma showed a higher percentage of positivity in the latter group. In addition, our study not only revealed a lesser percentage of MDM2 positive cells in unicystic ameloblastomas but also showed different percentage of positive cells in different areas of unicystic ameloblastomas. A higher number of MDM2 positive cells were noted in proliferating luminal and mural strands as compared to the negative expression in cystic lining. This indicates that the simple unicystic ameloblastomas are low-grade tumors with a low proliferation index. It also supports the contention of lower recurrence rates in simple unicystic ameloblastomas as compared to solid multicystic variants and also unicystic plexiform ameloblastoma subtypes.
Statistical evaluation between various groups of ameloblastomas and AOT in our study revealed significant association between the solid / multicystic subtypes like follicular ameloblastoma with granular cell changes, acanthomatous ameloblastoma and AOT along with a highly significant association between plexiform ameloblastoma and AOT. Overexpression of MDM2 in ameloblastomas noted in the present study indicates that an increased production or decreased breakdown of protein leads to disturbance in growth regulation. An extensive search of literature showed only a solitary study so far comparing immunohistochemical expression of MDM2 in ameloblastomas and AOT conducted by Carvalhais et al.  Their study included only fifteen cases of ameloblastomas with two histological subtypes and eight AOT cases, of which all AOT cases demonstrated a negative expression. Apart from being only a second study to compare IHC expression of MDM2 in odontogenic tumors the exclusivity in the present study is that, it includes as many as 36 cases of ameloblastomas with three histological subtypes according to the 2005 WHO classification and also 14 cases of AOT. In addition to this, we would as well like to highlight that a minor fraction of AOT cases in our study showed positivity predominantly in whorls and also to a lesser extent in other cell patterns like ducts and sheets as compared to complete negative expression seen in previously mentioned single study by Carvalhais et al.  However, in absentia of any staining in flat and ovoid cells, the neoplastic nature of the tumor can be ruled out. Another possibility that might explain this negative result is that AOT may not represent a true neoplasm, but rather a developmental outgrowth. 
| > Conclusion|| |
In summary, our study with higher expression of MDM2 in solid / multicystic ameloblastoma as compared to desmoplastic and unicystic variants suggests a divergent epithelial proliferative activity within these types and helps to elucidate the differences in their biological behavior. Similarly higher percentage of MDM2 positivity noted in ameloblastomas when compared to AOT indicated the differences in aggressive nature between these two groups of odontogenic tumors, favoring the universal perception of a greater aggressive nature of ameloblastomas. Further studies, such as molecular and cytogenetic analysis using larger odontogenic tumor samples, have to be done to elucidate and confirm MDM2's potential as a proliferative and prognostic marker, which in turn will generate more effective alternative approaches in diagnosis and treatment of these lesions.
| > Acknowledgements|| |
The authors are grateful to Dr. C. Bhasker Rao, Dean, S.D.M College of Dental Sciences and Hospital, Sattur, Dharwad 580009, Karnataka, India for providing institutional support to this study.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]