| Abstract|| |
Aim: To evaluate the use of proliferating cell nuclear antigen index in the different histopathological variants of ameloblastoma, such as the follicular, plexiform, and unicystic types, and in ameloblastic carcinoma by immunohistochemical staining. The proliferating cell nuclear antigen index values of the variants of ameloblastomas and ameloblastic carcinomas are compared in order to determine the biological behavior of these tumors.
Materials and Methods: For the present study, archival tissues that had been diagnosed as ameloblastoma and ameloblastic carcinoma were collected from the department of oral pathology. Specimens were embedded in paraffin wax and were sectioned at a thickness of 5 μm and stained with hematoxylin-eosin for reconfirming the histologic pattern. It was also stained immunohistochemically for anti-proliferating cell nuclear antigen antibody.
Results: Positive proliferating cell nuclear antigen expression is seen as a light brown, granular stain. The proliferating cell nuclear antigen values of ameloblastic carcinoma were almost five times the value of ameloblastoma. Analysis of variance test, Fischer's exact test/variance ratio test, and Student's t-test were performed and the probability values were determined.
Summary and Conclusion: This study showed that ameloblastic carcinoma had the maximum proliferative capacity. Among the variants of ameloblastoma, the plexiform variety had the maximum proliferative capacity, followed by the follicular and unicystic varieties. Altogether, these data indicate that proliferating cell nuclear antigen is related to the biological behavior and proliferation of tumor cells in the variants of ameloblastoma and ameloblastic carcinoma.
Keywords: Ameloblastoma, ameloblastic carcinoma, proliferating cell nuclear antigen
|How to cite this article:|
Maya R, Sekar B, Murali S. Comparative evaluation of expression of proliferating cell nuclear antigen in variants of ameloblastoma and ameloblastic carcinoma. Indian J Dent Res 2012;23:15-9
The odontogenic tumors  are lesions derived from epithelial, ectomesenchymal, and/or mesenchymal elements that are or have been a part of the tooth-forming apparatus. These tumors are found exclusively within the jaw bones or in the soft mucosal tissues overlying the tooth-bearing areas.
|How to cite this URL:|
Maya R, Sekar B, Murali S. Comparative evaluation of expression of proliferating cell nuclear antigen in variants of ameloblastoma and ameloblastic carcinoma. Indian J Dent Res [serial online] 2012 [cited 2014 Apr 24];23:15-9. Available from: http://www.ijdr.in/text.asp?2012/23/1/15/99031
Proliferating cell nuclear antigen (PCNA)  is a 36-kD nuclear protein and a polymerase expressed during the late G1 and S phases. PCNA is a good marker of the aggressiveness, malignant potential and chances of recurrence of ameloblastomas. In the present pioneering study, we counted the PCNA-positive cells in the variants of ameloblastoma and five ameloblastic carcinomas.
The aim of this study was to find the expression of PCNA, which can be detected by immunohistochemistry and be used as a proliferation marker in histological variants of ameloblastoma and ameloblastic carcinoma.
To evaluate the use of the PCNA index in the different histopathological variants of ameloblastoma (such as follicular, plexiform, and unicystic types) and in ameloblastic carcinoma. The PCNA index values of variants of ameloblastoma and ameloblastic carcinoma are compared in order to determine the biologic behavior of these tumors.
| Materials and Methods|| |
For the present study, archival tissues that had been diagnosed earlier as ameloblastoma and ameloblastic carcinoma were collected from the department of oral pathology. We were able to collect five follicular ameloblastomas, three plexiform ameloblastomas, seven unicystic ameloblastomas, and five ameloblastic carcinomas.
The specimens were embedded in paraffin wax and were sectioned at a thickness of 5 μm and stained with hematoxylin-eosin for reconfirming the histopathologic pattern.
Procedure for immunohistochemical staining
The technique used in this study is based on the labeled streptavidin-biotin method. After routine processing of the specimen and sectioning at a thickness of 3-5 μm, the endogenous peroxidase activity of the tissue is blocked by placing the tissue in 3% hydrogen peroxide for 5 min. The tissue is then incubated with an appropriate primary antibody. In this case, the primary antibody used was monoclonal anti-PCNA (PC10, BioGenex, USA). After this step, the nonspecific binding sites were blocked with nonimmune goat serum. Biotinylated secondary antibody and streptavidin- peroxidase conjugate were added sequentially, with the specimen being incubated for 30 min in each of them. Finally, the chromogen, diluted in the substrate buffer, was added. In order to ensure that all kit reagents were working properly, a known positive control specimen and a negative control specimen were stained along with each batch. A previously diagnosed poorly-differentiated squamous cell carcinoma stained with PCNA was taken as positive control and a tissue from the present study, without staining with primary antibody, was taken as the negative control. Positive PCNA expression is seen as light brown granular stain.
| Results|| |
Representative fields were randomly selected in each case of follicular, plexiform, and unicystic ameloblastoma and ameloblastic carcinoma. These areas included only well-preserved and maximum PCNA-positive epithelial areas in follicular ameloblastoma, plexiform ameloblastoma, and ameloblastic carcinoma, and cyst linings in unicystic ameloblastoma. Areas of necrosis, inflammation and stromal cells were excluded.
Clear brown nuclei were regarded as indicating PCNA-positive cells. The positive cells were counted manually, using an eyepiece graticule and a ×40 objective (×400 magnification). For each case, three microscopic fields were examined and the cells positive for PCNA in at least 1000 tumor cells were counted.
The PCNA values of ameloblastic carcinoma were almost five times the value of ameloblastoma. The interesting feature noticed in this study was that each square in the eyepiece graticule showed 3-4 positive cells in ameloblastic carcinoma, whereas the variants of ameloblastoma showed only 1-2 positive cells.
ANOVA test, Fischer's exact test (F-test)/variance ratio test, and Student's t-test were performed and the probability value (P value) was determined.
Values of the variants of ameloblastoma were compared. Values obtained for group I (follicular) show an average of 251, with a variance of 9494 and SD of 97.44; values for group II (plexiform) show an average of 280.33, with a variance of 433.33 and SD of 20.82; and values for group III (unicystic) shows an average of 127.75, with a variance of 3416.5 and SD of 58.46 [Table 1], [Graph 1].
Separate comparisons were made between ameloblastic carcinoma and each of the variants of ameloblastoma using the Student's t-test. Comparison of ameloblastic carcinoma and plexiform ameloblastoma showed 4 degrees of freedom (df), t-stat value of -7.46911 and P value of .001717 [Table 2], [Graph 2]. Comparison between ameloblastic carcinoma and follicular ameloblastoma showed 8 degrees of freedom (df), t-stat value of -6.241791, and P value of .000248 [Table 3], [Graph 3]. Comparison of ameloblastic carcinoma and unicystic ameloblastoma showed 8 degrees of freedom (df), t-stat value of -6.74196 and a P value of .000146 [Table 4], [Graph 4].
|Table 2: Ameloblastic carcinoma vs plexiform ameloblastoma t-test: Two-sample assuming equal variances|
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|Table 3: Ameloblastic carcinoma vs follicular ameloblastoma t-test: Two-sample assuming equal variances|
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|Table 4: Ameloblastic carcinoma vs unicystic ameloblastoma t-test: Two-sample assuming equal variances|
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| Discussion|| |
The present study was conducted on archival tissues that had been diagnosed earlier as ameloblastoma and ameloblastic carcinoma.
PCNA in ameloblastic carcinoma has been studied earlier. Kim et al.,  in 1994 and Setsuko et al.,  in 1999, studied one ameloblastic carcinoma each and reported very high PCNA values. Deeb et al.,  in 2003, studied three ameloblastic carcinomas by PCNA and AgNOR and reported that either could be used to distinguish between ameloblastoma and ameloblastic carcinoma. However, it should be noted that they studied the three cases by comparing the intensity of staining. The present study can be considered as a pioneering study, where the PCNA-positive cells in different variants of ameloblastoma and five ameloblastic carcinomas were counted.
The present study showed the maximum PCNA-positive cells in ameloblastic carcinoma specimens. PCNA-positive cells were found in the basal and parabasal layers as well as in the stellate reticulum areas of ameloblastic carcinoma showing follicular and plexiform patterns [Figure 1].The interesting feature noticed in this study was that each square in the eyepiece graticule showed 3-4 positive cells in ameloblastic carcinoma, whereas the variants of ameloblastoma showed only 1-2 positive cells. The results were similar to the results of Kim et al.,  in 1994 and Setsuko et al.,  in 1999, who studied one ameloblastic carcinoma each. They reported high PCNA values in their studies and opined that PCNA values may be useful for determining the possibility of malignant transformation in recurrent cases of ameloblastoma.
Deeb et al.,  in 2003, studied PCNA in three ameloblastic carcinomas and the variants of ameloblastoma and reported that the highest mean value of PCNA-positive cells was demonstrated in ameloblastic carcinoma followed by follicular ameloblastoma. He could not find a significant difference in the number of PCNA-positive cells between ameloblastic carcinoma and the other variants of ameloblastoma. However, in the present study, a significant difference was noted when the PCNA values of variants of ameloblastoma were compared with that of ameloblastic carcinoma separately. This difference may be attributed to the method adopted by Deeb et al.,  since their comparison was based on the difference in the percentage of cells with positive staining (classified as absent, weak, moderate and strong staining) rather than on the counts of PCNA-positive cells (the counting procedure was adopted by them only in AgNOR staining).
In the present study, the PCNA values of plexiform ameloblastoma were second only to that of ameloblastic carcinoma. PCNA-positive cells were seen in the basal and suprabasal layers with the stellate reticulum area showing relatively less number of positive cells [Figure 2]. Our results were comparable with the results of Liu et al.,  who reported in 2002 that the PCNA labeling indices were significantly higher in the peripheral cells of tumor nests and strands than in the central cells. Funaoka et al.,  in 1996, reported that the PCNA index in the follicular type was higher than in the plexiform type, which is contradictory to our result. This could be due to the cystic degeneration present in our cases, which resulted in low PCNA values. Our results were also comparable with that from the studies of Hirayama et al.,  in 2005, who reported that the plexiform type exhibited greater expression of PCNA protein than the follicular type.
The PCNA values of follicular ameloblastoma were slightly lower than that of plexiform ameloblastoma. In this study, PCNA-positive cells were found in the basal and suprabasal layers. Scattered positive cells were seen in the stellate reticulum area of the island [Figure 3]. The results were similar to that of Li et al.,  in 1995, who reported that the labeling indices of solid ameloblastomas of the follicular type were significantly higher than those of the cystic type and of Liu et al.,  in 2002, who reported that peripheral cells showed higher PCNA values than central cells. Small and irregular islands showed more positive cells. This could be due to the better nutritional supply and the absence of the stellate reticulum in small islands.
The least number of PCNA-positive cells was seen in unicystic ameloblastoma. This study demonstrated PCNA-positive cells in the intramural tumor islands. Cystic tumor lining had relatively few PCNA-positive cells [Figure 4]. Our results were similar to results of Funaoka et al.,  in 1996, who reported that the cystic type showed a low positive PCNA index. It was also similar to results of Piatelli et al.,  in 1998, who reported that unicystic ameloblastoma showed lower values than acanthomatous, plexiform and follicular ameloblastomas. Our results were however contradictory to the results of Meer et al.,  who reported in 2003 that unicystic ameloblastomas showed significantly higher PCNA labeling indices than the solid and multicystic variants. The inconsistent result is probably due to the difference in methodology, especially the counting protocol used.
| Summary and Conclusion|| |
This study shows that ameloblastic carcinoma has the maximum proliferative capacity. Among the variants of ameloblastoma, the plexiform variety has the maximum proliferative capacity, followed by the follicular and unicystic varieties. Altogether, these data indicate that PCNA is related to the biologic behavior and proliferation of tumor cells in variants of ameloblastoma and ameloblastic carcinoma.
The present study was aimed at finding the expression of PCNA in biopsies of variants of ameloblastoma and ameloblastic carcinoma as it can be detected by immunohistochemistry and has the potential to be used as a proliferation marker.
The immunohistochemical analysis was carried out using the streptavidin-biotin method. There was an increase in the scores of positively stained cells in an incremental order, i.e., unicystic ameloblastoma < follicular ameloblastoma < plexiform ameloblastoma < ameloblastic carcinoma. This can be correlated with the biological behavior of these tumors. The PCNA expression, along with the clinical features, can predict the aggresiveness, chances of recurrence and malignant potential of these tumors and this should help in better management of the patient.
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Department of Oral Pathology, VMSDC, Salem, Tamil Nadu
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]