|Year : 2017 | Volume
| Issue : 3 | Page : 570-575
A study of the distribution of B-cell lymphoma/leukemia-2 in odontogenic cyst and tumors: Histochemical study
Khushboo Phull, Rashmi Metgud, Shrikant Patel
Department of Oral Pathology and Microbiology, Pacific Dental College and Hospital, Debari, Udaipur, Rajasthan, India
|Date of Web Publication||31-Aug-2017|
Department of Oral Pathology and Microbiology, Pacific Dental College and Hospital, Debari, Udaipur, Rajasthan
Source of Support: None, Conflict of Interest: None
Introduction: The odontogenic keratocyst is known for its aggressiveness, high recurrence rate, and transformation of keratinized epithelia to nonkeratinized squamous epithelium for which inflammation has been suggested to be responsible. B-cell lymphoma/leukemia-2 (Bcl-2) an anti-apoptotic protein, prolongs the life span of epithelial cells and allows proliferation, differentiation, and morphogenesis.
Materials and Methods: Study was carried out comprising of 90 cases; (30 ameloblastoma, 30 keratocystic odontogenic tumor (KCOT), and 30 radicular cyst). Bcl-2 expression was determined with respect to localization, area (percentage) and intensity of stained cells in epithelium, and connective tissue stroma by counting the endothelial, round, and fusiform cells.
Results: In epithelium bcl-2 expression in KCOTs was higher followed by ameloblastoma and lowest in the radicular cyst. Whereas, in connective tissue stroma bcl-2 expression was higher in KCOT and radicular cyst than ameloblastoma cases. Solid variants showed statistically higher expression as compared to the unicystic variants of ameloblastoma (P = 0.009, 0.033, 0.011, and 0.041).
Conclusion: High expression of bcl-2 in KCOT supports the general agreement that some features of KCOT are those of a neoplasia. The bcl-2 expression in connective tissue cells suggests that these cells may also be important as epithelial cells in the biological behavior odontogenic keratocyst.
Keywords: Ameloblastoma, apoptosis, B-cell lymphoma/leukemia-2, keratocystic odontogenic tumor, radicular cyst
|How to cite this article:|
Phull K, Metgud R, Patel S. A study of the distribution of B-cell lymphoma/leukemia-2 in odontogenic cyst and tumors: Histochemical study. J Can Res Ther 2017;13:570-5
|How to cite this URL:|
Phull K, Metgud R, Patel S. A study of the distribution of B-cell lymphoma/leukemia-2 in odontogenic cyst and tumors: Histochemical study. J Can Res Ther [serial online] 2017 [cited 2020 Sep 22];13:570-5. Available from: http://www.cancerjournal.net/text.asp?2017/13/3/570/183197
| > Introduction|| |
A dysfunctional apoptotic system can lead to either excessive removal or prolonged survival of cells. Therefore, dysregulation of apoptosis is involved in the pathogenesis of a variety of diseases such as cancers, viral infections, and immunological disorders.
The centerpiece of the apoptotic program and the major effector arm of the cell death program is the B-cell lymphoma/leukemia-2 (bcl-2) family of related proteins. The prototype member of this family is bcl-2, an acronym for B-cell lymphoma/leukemia-2 gene, that was first discovered at the breakpoint of the t(14;18) in a follicular non-Hodgkin's B-cell lymphoma. The proteins encoded by the bcl-2 family localize to the outer mitochondrial membrane, the nuclear membrane, and the endoplasmic reticulum.
Immunoreactivity for bcl-2 protein has been detected in enamel organs and dental lamina of tooth germs as well as in odontogenic lesions such as ameloblastoma, keratocystic odontogenic tumor (KCOT), and radicular cyst to elucidate any relationship between histological features and biological potential.
Ameloblastoma is the most significant odontogenic neoplasm of concern. Several recent studies have detected genetic and cytogenetic alterations in these epithelial odontogenic tumors; however, the detailed mechanisms of oncogenesis, cytodifferentiation, and tumor progression remain unknown.
KCOT, another common cyst arising from dental lamina or remnants of the dental lamina, has been deeply studied due to its aggressive clinical behavior with high recurrence rates, distinct histopathologic features, singular growth mechanism, and genetic alterations. The potential for aggressive clinical behavior and local recurrence resulted in its recent classification as a benign odontogenic tumor with a new nomenclature: KCOT. Several studies have demonstrated the higher proliferation activity of the epithelial lining in KCOTs in relation to odontogenic cysts. However, despite the fact that cell turnover is controlled by both cell proliferation and apoptosis, few studies have evaluated apoptosis-related proteins and apoptotic index in the epithelial lining of the odontogenic cysts.
In general, most of the immunohistochemical studies on bcl-2 expression included epithelial cell examination. There are a few studies in the literature examining bcl-2 expressions in odontogenic epithelium and connective tissue cells of oral lesions. Hence, the present study was aimed to evaluate bcl-2 expression and its distribution in the epithelial lining as well as connective tissue cells of ameloblastoma, KCOT, and radicular cyst.
| > Materials and Methods|| |
The present study was carried out on 120 formalin-fixed paraffin-embedded tissues forty each of ameloblastoma, KCOT, and radicular cyst samples, which were retrieved from the archives of Department of Oral and Maxillofacial Pathology. Relevant clinical details were obtained as per the case history Performa. All the other relevant patient clinical data were also tabulated. None of the KCOTs were associated with nevoid basal cell carcinoma syndrome.
Four-micrometer-thick sections were cut and mounted on poly-L-lysine-coated slides, air dried, and heated at 45°C overnight. After deparaffinization and rehydration, the sections were incubated in 0.01 M citrate buffer in a microwave oven for 15 min for antigen retrieval. The slides were then washed in phosphate-buffered saline (PBS) for 30 min at room temperature and incubated in 0.5% H2O2 in methanol for 10 min to block endogenous peroxidase activity. Nonspecific antibody binding was blocked with 3% normal horse serum in PBS. A mouse anti-human bcl-2 monoclonal antibody (Clone: Bcl-2-100, Dako, South San Francisco, CA, USA) with a 1/100 dilution was applied as primary antibody and placed in a humidified chamber at 4°C overnight. The sections were subsequently washed in PBS and processed for detection of the positive immunohistochemical reaction using the streptavidin-biotin complex immunoperoxidase technique. Rabbit-mouse antibody link solution (Dako ABL008, South San Francisco, CA, USA) was used as a secondary antibody to enhance the sensitivity of the procedure and diaminobenzidine was applied as the chromogen. Sections were finally counterstained with hematoxylin, cleared, and mounted. Negative controls consisted of PBS instead of primary antibody and a sample of lymphoma and inflammatory cells within the cyst wall was used as the external and internal positive controls.
Stained sections were examined in Olympus BX60 microscope attached with a color video camera (Olympus Analysis Five). All stained areas demonstrating positivity for bcl-2 were determined with respect to localization, area (percentage) and intensity of stained cells at a magnification of ×40, and the number of positively stained cells was counted in ten representative areas of the epithelium using a ×40 objective in a minimum of one hundred cells in the full length of the epithelium. The expression of bcl-2 was also determined in the connective tissue stroma, by counting the endothelial, round and fusiform cells at a magnification of ×40, and the number of positively stained cells was counted on ten representative areas as specified by Kichi et al. 2005.
The area of staining (immunoreactivity) in the epithelium was defined by Vered et al., 2009: (0) No staining, (1) low staining 1–10% positive, (2) intermediate staining 11–50% positive, and (3) high staining more than 50% positive. Whereas, the intensity of bcl-2 positivity was estimated as follows (Jahanshahi et al., 2006): (−) <5% stained cells, (±) 5–9% cells stained positively, (+) between 10% and 24% cells stained positively, (++) between 25% and 50% cells stained positively, and (+++) >50% of tumor cells stained positively. The degree of inflammation was assessed in the connective tissue stroma adjacent to the epithelial lining in which the cell count had been performed (magnification, ×100) and also approximal to bcl-2 negative cases (Jahanshahi et al., 2006). A lack of inflammatory cells was scored as (−), <30 inflammatory cells as (+), 30–59 inflammatory cells as (++), and 60 or more inflammatory cells as (+++).
The number of positive cells was divided into the total number of cells counted in the whole area. The result was multiplied by 100 to find the percentage of positive cells. Collected data were analyzed using the SPSS software for Windows (version 17, IBM Software, Company Head Quarters in New York, US). One-way ANOVA (F-test) was carried out for comparing the parameter for multiple groups. Comparison between groups was carried out using the Student's t-test. Correlations between bcl-2 positivity in different lesions and degree of inflammation were derived using Karl Pearson's, with the level of significance set at P ≤ 0.05.
| > Results|| |
Positive bcl-2 expression was considered in the epithelium of the odontogenic lesions when at least 10% of the cells were stained. All KCOTs [Figure 1]a and ameloblastoma [Figure 1]b and ten out of forty radicular cysts [Figure 1]c were positive for bcl-2.
|Figure 1: (a) Bcl-2 expression in the epithelium of ameloblastoma. (b) Bcl-2 expression in the epithelium of keratocystic odontogenic tumor. (c) Bcl-2 expression in the epithelium of radicular cyst. (d) Bcl-2 expression in the connective tissue of ameloblastoma. (e) Bcl-2 expression in the connective tissue of keratocystic odontogenic tumor. (f) Bcl-2 expression in the connective tissue of radicular cyst. Bcl-2: B-cell lymphoma/leukemia-2|
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In 26 of the 38 stained KCOTs, positively stained cells were observed in the basal layer while in the other bcl-2 positive KCOTs (14 of 40), the stained cells were in the basal/suprabasal region. In radicular cyst, positive cells were located in the basal/suprabasal layers of only four cases. In ameloblastomas, bcl-2 was detected mainly in the peripheral layer, whereas only a few cells were positively stained in the central layer of epithelial tumor islands (eight cases).
Higher bcl-2 staining area and intensity of the connective tissue were found in KCOT as compared to radicular cysts and least in ameloblastomas. All KCOTs [Figure 1]d, 38 out of 40 ameloblastoma [Figure 1]e and 38 out of 40 radicular cysts [Figure 1]f were positive for bcl-2.
Mean and standard deviation values of bcl-2 staining area and intensity of the stained cells in the epithelium and connective tissue of studied odontogenic lesions are shown in [Table 1]. A significant difference was observed between ameloblastoma, KCOT, and radicular cyst (ANOVA, P = 0.00) [Table 1]. In multiple comparisons, bcl-2 staining area and intensity revealed a statistically significant difference between KCOT and ameloblastoma (Student's t-test, P = 0.008, 0.000, 0.012, and 0.000), [Table 2] and between ameloblastoma and radicular cyst (Student's t-test, P = 0.001, 0.001, 0.057, and 0.000) [Table 3]. There was no significant difference of bcl-2 staining area and intensity between radicular cyst and KCOT in the connective tissue (Student's t-test, P = 0.46, 0.497) [Table 4], whereas epithelial bcl-2 staining area and intensity were significantly higher in KCOTs (Student's t-test, P = 0.000) [Table 4].
|Table 2: Intercomparison between keratocystic odontogenic tumor and ameloblastoma|
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|Table 4: Intercomparison between keratocystic odontogenic tumor and radicular cyst|
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In both variants of ameloblastomas, solid ameloblastomas lesions had a statistically significant high score of bcl-2 staining area and intensity both in the epithelium and connective tissue (Student's t-test, P = 0.009, 0.33, 0.011, and 0.041) [Table 5] and [Figure 2]a and [Figure 2]b.
|Figure 2: (a) Bcl-2 expression in the variants of ameloblastoma solid or multicystic. (b) Bcl-2 expression in the variants of ameloblastoma unicystic. Bcl-2: B-cell lymphoma/leukemia-2|
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Radicular cyst displayed the highest degree of inflammation, KCOT also displayed a considerable inflammatory component, whereas ameloblastoma demonstrated a mild to intermediate degree of chronic inflammation (49.74 ± 17.93, 48.71 ± 23.51, and 26.35 ± 9.60, respectively) [Figure 3].
|Figure 3: (a) Degree of inflammation in multicystic ameloblastoma. (b) Degree of inflammation in unicystic ameloblastoma. (c) Degree of inflammation in keratocystic odontogenic tumor. (d) Degree of inflammation in radicular cyst|
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KCOT revealed an increased epithelial and connective tissue bcl-2 area and intensity in the presence of inflammation (Karl Pearson's coefficient, P = 0.006, 0.000, 0.005, and 0.000). In ameloblastoma and radicular cyst, comparison of degree of inflammation with bcl-2 expression showed an increased connective tissue expression and intensity of bcl-2 in the presence of inflammation, (P = 0.000) whereas epithelial bcl-2 area and intensity were not related to inflammation (P = 0.520, 0.110, 0.828, and 0.657).
| > Discussion|| |
In the present study, odontogenic epithelium of KCOT revealed strong positive staining for bcl-2 in the whole thickness of epithelium in basal and suprabasal layers and low expression of bcl-2 in the basal and suprabasal layers in radicular cysts cases. These findings are in accordance with earlier studies done by Piattelli et al. 1998 and Jahanshahi et al. in 2006.
Ameloblastoma in the present study revealed medium expression of bcl-2 in the peripheral cell layer of the tumor islands and low expression in the central stellate reticulum like layer. These findings are in accordance with earlier studies done by Mitsuyasu et al. 1997 and Sandra et al. 2001. In a study by Florescu et al. in 2012, bcl-2 expression was present in 88.23% of the investigated ameloblastomas, predominantly in columnar cells from the peripheral zone. Similar studies in the literature communicate that around 90% of ameloblastomas are positive for bcl-2 similar to our study, which indicates that bcl-2 expression may be related to differentiation and proliferation of odontogenic epithelium, and bcl-2 overexpression may be associated with the ameloblastoma development. These results indicate that in ameloblastomas bcl-2 protein could function primarily as an anti-apoptotic factor, which reflects the proliferative activity of neoplasms. Moreover, the expression of bcl-2 suggests the aggressive nature of odontogenic tumor and these results will be beneficial in the differential diagnosis of odontogenic tumors and other tumors that occur in the mouth. In addition, it is estimated that the bcl-2 protein may play a role in maintaining stem cell population in peripheral layers of tumor islands of which are recruited proliferating cells.
In the connective tissue stroma, bcl-2 area and intensity in KCOT and radicular cyst were higher than ameloblastoma. Low positivity in ameloblastoma may be attributed to the mature fibrous connective tissue stroma as compared to the highly inflamed stroma of the odontogenic cysts. These results are in contrast to study done by Kichi et al. in 2012 in which there was no significant difference in expression of bcl-2 between KCOT and ameloblastoma, as well as between radicular cyst and ameloblastoma. So far in the literature, no studies have been cited that have compared the staining intensity of bcl-2 in these three lesions only the area of bcl-2 staining has been compared.
In the present study, overall comparison for the expression of bcl-2 in all three odontogenic lesions revealed statistically significant higher expression in the whole thickness of epithelium of KCOT and predominantly in the peripheral cell layer of the tumor islands of eight ameloblastomas. On the other hand, radicular cyst samples revealed very low bcl-2 expression. These results are in accordance with that found by Mitsuyasu et al. in 1997 and Sandra et al. in 2001. This could lead to the aggressive growth pattern of KCOT and ameloblastoma.
In the present study, intercomparison between different variants of ameloblastoma revealed a significantly higher expression of bcl-2 area and intensity both in the epithelium and the connective tissue of solid or multicystic variants as compared to the unicystic variants of ameloblastoma. These results are in accordance with the study done by Vered et al. in 2009 where solid ameloblastoma (SAM) demonstrated intermediate and low scores in basal and stellate reticulum layers and only a few SAM samples had a high score similar to our study, on the other hand, unicystic ameloblastoma (UAM) samples demonstrated less positivity for bcl-2 expression and one case of UAM was negative for bcl-2.
A study by Vered et al. 2009, provide further support to the assumption that a KCOT can be of a neoplastic rather than a cystic nature. This is based not only on the expression of the Protein patched homolog 1 (PTCH) and its downstream factors, Smoothened, Frizzled Class Receptor (SMO), and GLI-1 but also on the analysis of the immunohistochemical profile of KCOT, which is comprised of the sonic hedgehog (SHH)- related proteins and the SHH-induced bcl-2 oncoprotein. The quality and quantity of the interactions between the SHH and other cell cycle regulatory pathways most probably work synergistically to define the individual phenotype and the corresponding biological behavior of KCOTs. The activation of at least two cell cycle regulatory systems is “switched on” in KCOTs as this lesion evolves. Investigations on the immunoreactivity of bcl-2 protein have been demonstrated in KCOTs, and recent studies report that bcl-2 positive cells are predominantly located basally, thus supporting the concept that apoptosis does not occur in the basal cells of the lining epithelium. However, TUNEL-positive cells have been detected exclusively in the surface layer of KCOTs, indicating marked levels of apoptosis. Thus, bcl-2 inhibits apoptosis to facilitate cellular proliferation in the basal and suprabasal layers, whereas apoptosis maintains the homeostasis of the thickness of the lining epithelium and allows the synthesis of large amounts of keratin in the surface layer of KCOTs. Considering that there is a regulated balance between cell proliferation, cell differentiation, and cell death in this type of lesion, this may explain why KCOTs, though portraying a neoplastic behavior, with an increase potential to proliferate, do not tend to form tumor masses.
| > Conclusion|| |
The results of the present study suggest that KCOT has a high proliferative and survival activity and that might be one of the reasons why KCOT has a high recurrence rate. The proliferation potential of the epithelium and the overexpression of various anti-apoptotic proteins in odontogenic epithelial tumors are quite significant for their clinical behavior providing further support to the assumption that a KCOT can be of a neoplastic rather than a cystic nature. We have also demonstrated that connective tissue cells may also be important as epithelial cells in the biological behavior of these odontogenic lesions. Although these results support that KCOT is a neoplasm, with an expression of bcl-2 similar to ameloblastoma rather than radicular cyst, there are not enough genetic studies and only two studies so far have directly compared the expression of bcl-2 in these three lesions altogether. Further studies on genomic changes may help better understanding of the pathogenesis of KCOTs.
I affirm that I/we have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements, or honoraria) or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript nor have any such arrangements existed in the past 3 years. Any other potential conflict of interest is disclosed.
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Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]