An Analysis of the Expression of Bcl-2, Podoplanin and Lymph

& Experim al e Kaur and Gupta, J Clin Exp Pathol 2013, 3:3 ic n n t li a l DOI: 10.4172/2161-0681.1000145

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Research Article Open Access An Analysis of the Expression of Bcl-2, Podoplanin and Lymph Angiogenesis in Benign and Malignant Salivary Gland Tumours Harshaminder Kaur* and Sonal Gupta Department of oral pathology, M.M.College of Dental Sciences and Research, Mullana (Ambala), Haryana, India

Abstract Background: Salivary gland tumours comprise a significant proportion of oral tumours and are the next common neoplasm of the mouth after squamous cell carcinoma. These neoplasm’s have widely variable histopathologic and biologic characteristics, which makes it difficult to determine the pathogenesis and selection of therapeutic modalities. Alteration in some proto-oncogenes and tumour suppressor genes may lead to the development and progression of these tumours. Aim: The purpose of this study was to analyze the immune histochemical expression of Bcl-2 and epithelial podoplanin and lymph angiogenesis in benign and malignant salivary gland tumours. Material and method: The sample consisted of 20 formalin-fixed paraffin embedded blocks of benign and malignant salivary glands tumours. Immunohistochemical staining procedure was performed using monoclonal anti Bcl-2 antibody and D2-40 antibody. The sections were evaluated for Bcl-2 and epithelial podoplanin expression and D2-40 positive lymphatic vessels. Results and conclusion: Bcl-2 showed positive immune expression in all benign and malignant salivary gland tumours with mucoepidermoid carcinomas showing most intense expression. Podoplanin immunostaining was also assessed, highest recorded score was in peripheral mucoepidermoid carcinoma (10.4? 3.05). Lymphatic micro vessel density expressed by podoplanin showed intense score in central mucoepidermoid carcinoma (5.05? 5.0). Present investigation demonstrated positive Bcl-2, podoplanin response and Lymphatic micro vessel density in both benign and malignant salivary gland tumours suggesting that Bcl-2 and podoplanin alteration may be associated with the progression of these neoplasms.

Keywords: Bcl-2; Podoplanin; Apoptosis; Lymph angiogenesis; protein, mediates a pathway leading to a collective cell migration in Lymphatic micro vessel density vivo and vitro. Human podoplanin is a 38KDa type-I trans membrane glycoprotein consisting of 162 amino acids, nine of which form the Introduction intercellular domain. In normal human tissue, podoplanin is expressed Salivary gland tumours comprise a significant proportion of oral in kidney podocytes, in skeletal muscle, placenta, lung and heart, in my fibroblasts of the breast and salivary glands, in osteoblasts tumours and are the next common neoplasm of the mouth after squamous cell carcinoma. These neoplasm’s have widely variable and mesothelial cells [7]. D2-40 a commercially available antibody specifically recognizes human podoplanin and can be used to assess histopathologic and biologic characteristics, which makes it difficult to determine the pathogenesis and selection of therapeutic modalities [1]. podoplanin expression in tumour cells [8]. Podoplanin expression using D2-40 has been reported to appear in many types of human Apoptosis (programmed cell death) is a specific form of cell death cancers such as mesothelioma [9], skin cancer, carcinoma of the that constitutes an important mechanism of maintaining homeostasis. uterine cervix [10], ovarian cancer [11], thyroid cancer and head and Apoptosis occurs physiologically as well as in the course of many neck squamous cell carcinoma [12]. diseases. Alterations of apoptosis are always coupled with pathological conditions and/or oncogenesis [2]. Anti-apoptotic marker like Bcl-2 Podoplanin is highly and superficially expressed in lymphatic may potentially be able to predict the tumour behaviour. endothelial cells, but not blood vessel endothelium. The immunostaining of D2-40 is now widely used for the detection of tumour lymph Bcl-2 encoded by the proto-oncogene Bcl-2 and expressed in many angiogenesis in many human cancers. types of malignant tumours, protect cells from apoptosis induced DNA damaging agents. This anti-apoptotic effect is assumed to be caused by Thus, this study was attempted to investigate the retardation of cell proliferation due to cell’s accumulation in the G0 and

G1 phases of the cell cycle [3]. *Corresponding author: Harshaminder Kaur, Professor and head, Department Bcl-2 contributes to malignant cell expansion primarily by of oral pathology, M.M. College of Dental Sciences and Research, Mullana prolonging cell survival rather than by increasing the rate of cellular (Ambala), Haryana 133207, India, Tel: 091-9896219197; 091-9915333478; E-mail: proliferation, and accumulation of cells with an aberrant Bcl-2 [email protected] expression could be an important step in carcinogenesis [4,5]. Its over Received June 12, 2013; Accepted September 04, 2013; Published September expression has been reported in most human low grade tumours and 06, 2013 this inhibition of apoptosis has been regarded as being one of the most Citation: Kaur H, Gupta S (2013) An Analysis of the Expression of Bcl-2, common pathways of tumourigenesis [6]. Podoplanin and Lymph Angiogenesis in Benign and Malignant Salivary Gland Tumours. J Clin Exp Pathol 3: 145. doi:10.4172/2161-0681.1000145 Invasion of cells into the surrounding tissue and destruction of normal tissue architecture are two hallmarks of malignant tumours. Copyright: © 2013 Kaur H, et al.. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted Morphologically, two patterns of tumour invasion can be distinguished: use, distribution, and reproduction in any medium, provided the original author and single cell and collective cell invasion. Podoplanin, a small mucin like source are credited.

J Clin Exp Pathol Volume 3 • Issue 3 • 1000145 ISSN: 2161-0681 JCEP, an open access journal Citation: Kaur H, Gupta S (2013) An Analysis of the Expression of Bcl-2, Podoplanin and Lymph Angiogenesis in Benign and Malignant Salivary Gland Tumours. J Clin Exp Pathol 3: 145. doi:10.4172/2161-0681.1000145

Page 2 of 6 immunohistochemical expression of Bcl-2 and podoplanin in the was calculated as score: 8 or higher (Highly reactive), score: 4 to 7 tumour cells of benign and malignant salivary gland tumours and to (Moderately reactive) and score: 0 to 3 (Weakly reactive). assess lymph angiogenesis in these lesions. Quantification of lymphatic micro vessel density (LMVD) Materials and Methods For evaluating the lymphatic micro vessel density (LMVD) A total of 20 formalin fixed, paraffin embedded blocks of immunohistochemical D2-40 reactions were evaluated considering previously diagnosed cases of benign and malignant salivary gland the cytoplasmic staining in lymphatic endothelial cells. Evaluation tumours were selected from the archives of Department of Oral of positive reactions was performed by counting positive D2-40 and Maxillofacial Pathology, M.M College of Dental Sciences and lymphatic vessels, sitting around a visible lumen clearly separated from Research, Mullana, Ambala, to analyze immunohistochemical adjacent micro vessels and from other connective tissue components. expression of Bcl-2 and podoplanin (D2-40). The sample comprised Packed vessels were assumed as one lymphatic unit. The images were of 8 benign tumours (pleomorphic adenoma=8) and 12 malignant captured on to computer under 20X objectives, to evaluate the number tumours (mucopeidermoid carcinoma=9, adenocarcinoma NOS=2, of lymphatics. All the results were then statistically analyzed using ‘T Polymorphous low grade adenocarcinoma=1). test’, ‘Mann Whitney test’ and ‘Chi - square test’. Serial sections of 4µm thickness were taken on silanised slides Results meant for immunohistochemistry. All slides were then subjected to Bcl-2 showed positive immune expression in both benign and microwave heat- induced epitope retrieval (cycle 1: 98°C for 5 minutes, malignant salivary gland tumours. In benign neoplasm’s, all cases cycle 2: 95°C for 8 minutes). Endogenous peroxidise activity was showed weak immune expression. (Mean=1.8 ± 0.64) (Table 1). In blocked by treating the sections for 10-15 minutes in 0.3% peroxide malignant neoplasms, expression varied from weak to intense with block solution. Power block was applied for 10-15 minutes and 1 of 5 cases of peripheral MEC showing strong positive expression slides were then incubated with primary antibody, anti Bcl-2 (for (Mean=3.4 ± 1.95). All 4 cases of central type showed weak expression Bcl-2) antibody and D2-40 antibody (for podoplanin) for 1 hour in (Mean=2 ± 1.414). Other cases of malignant neoplasm’s studied a humidifying chamber and incubated with secondary monoclonal (adenocarcinoma NOS, polymorphous low grade adenocarcinoma) antibody for 30 minutes. DAB was applied to all slides till brown colour also showed weak Bcl-2 immune expression. Overall, malignant appeared and counterstained with haematoxylin. tumours showed a more intense Bcl-2 immune expression as compared to benign tumours. (Mean=2.58 ± 1.72) (Table 1). Evaluation of Bcl-2 immunohistochemical staining All cases of salivary gland neoplasm have showed positive All slides were assessed at a magnification of 40X of the light podoplanin immune expression. In benign neoplasm’s, expression microscope (Eclipse 80i) in 5 representative areas called hot spots of the varied from weak to intense with 3 out of 8 cases showing strong epithelium. Yellow to dark brown particles in the cytoplasm, indicated podoplanin immune expression (total mean=5.88 ± 3.271) (Table positive staining evaluation of positive reactions was performed 2). Malignant neoplasms also expression of podoplanin varied from using the criteria defined by Eslami B. et al. [12]. According to him, weak to high. 1 case of polymorphous low grade adenocarcinoma cumulative points were calculated as a product of Staining Intensity studied showed weak expression. (Mean=2) All 5 cases of peripheral Scores (A) and Proportion of Positively Stained Cell Score (B). mucoepidermoid carcinoma studied showed intense expression The staining intensity (A) was rated on a scale of 0 to 3 as score (mean=10.44 ± 3.05). 1 out of 4 cases of central mucoepidermoid 0 (colourless), score 1 (light yellow), score 2 (brown yellow), score 3 carcinoma showed high immune expression and 3 cases showing (brown). An average of 5 hotspots was then as the staining intensity (A) moderate expression. (Total mean=6.50 ± 3.786). 1 of 2 cases of of the slide. Proportion of stained tumour cells (B) was scored as score adenocarcinoma NOS showed intense expression (mean=6 ± 2.828). 1 (when less than 25% positive cells), score 2 (25 – 75% positive cells) Malignant neoplasm’s showed higher mean of podoplanin expression and score 3 (when more than 75% positive cells). The scores were based (7.66 ± 3.91) as compared to benign neoplasm (5.88 ± 3.271) (Table 2). on examination of the whole section in each biopsy. Cumulative score Lymphatic vessel density (LMVD) expressed by podoplanin in was then calculated (A x B) and scored as score: 0 negative (-), score: benign neoplasm was significant (5.1 ± 3.13). Lymph angiogenesis 1-4 weakly positive (+) and score : >4 strongly positive (++). was also pronounced in MEC and present in both types. Peripheral Evaluation of D2-40 positive tumour cells MEC showed a mean LMVD count of (4.2 ± 2.37) whereas central MEC showed higher count of mean (5.05 ± 5.021) (Table 3). The Cytoplasm and/or membrane immune reactivity were considered number of podoplanin positive lymphatic vessels was not higher in to indicate D2-40 expression. Analysis of positivity of epithelium adenocarcinoma NOS than other salivary gland tumours (Mean=2.60 was performed under 20X objective and 10X ocular lens (X200 ± 1.697). Both, Bcl-2 and podoplanin were weakly expressed in magnification). The scores were based on examination of the whole polymorphous low grade adenocarcinoma but it showed a significant section in each biopsy and immunostaining was scored under the LMVD count (Mean=3.40) (Table 3). Results were compared to following criteria. Quantitative scores of 0 to 5 were given as Score 0 haematoxylin, eosin stained sections of same tissues, and only those (0% of tumour cells are positive), Score 1 (1% to 10% of tumour cells vessels, which were recognized as endothelial cell, lined vessels in H & are positive), Score 2 (11% to 30% of tumour cells are positive), Score E stained sections were counted as positive lymphatic vessels. 3 (31% to 50% of tumour cells are positive), Score 4 (51% to 80% of tumour cells are positive) and Score 5 (81% to 100% of tumour cells Discussion are positive). The staining intensity is rated on a scale of 0 to 3 as score Tumour initiation, progression and invasion involve cellular 0 (negative), score 1 (weak), score 2 (moderate) and score 3 (strong). proliferation, apoptosis as well as cell adhesion and communication The data was then converted to a German Immunoreactive Score (IRS) that ensure cell survival, renewal and co-ordination. Salivary gland as a product of Quality Score and Staining Intensity Scores. IRS score neoplasms show a varied behaviour [7]. Benign salivary gland

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Cumulative score Negative Weakly positive Strongly positive S.No Lesion No. of cases No. of positive cases 4-Jan >4 Mean ± SD 0 Group I Benign Pleomorphic adenoma 8 8 - 8 - 1.8 ± 0.64 Peripheral mucoepidermoid carcinoma 5 5 - 4 1 3.4 ± 1.95 Central mucoepidermoid carcinoma 4 4 - 4 2 ± 1.414 Malignant Adenocarcinoma nos 2 2 - 2 - 2.5 ± 2.121 PLGA 1 1 - 1 - 1 Total 12 12 - 11 1 2.58 ± 1.72 A total of 20 cases formed the study group out of which Group I (Benign) comprising of 8 cases and group II (Malignant) comprising of 12 cases were each immunohistochemically stained for Bcl-2. All 8 cases of group I was positive and showed weak positivity. In group II, all 12 cases were positive and the expression varied from weak to intense with 1 case showing strong positive Bcl-2 expression. Overall, malignant tumours showed a more intense Bcl-2 expression compared to benign tumours.

Table 1: Malignant tumours showing more intense Bcl-2 immune expression as compared to benign tumours.

Cumulative score S no. Lesion No. Of total cases No. Of positive cases Weakly reactive Moderately reactive Highly reactive Mean ± sd 0-3 7-apr >8 Group I Plemorphic adenoma 8 8 3 2 3 5.88 ± 3.271 Benign Peripheral mucoepidermoid 5 5 - - 5 10.4 ± 3.05 carcinoma Central mucoepidermoid 4 4 - 3 1 6.50 ± 3.786 Malignant carcinoma Adenocarcinoma nos 2 2 - 1 1 6 ± 2.828 PLGA 1 1 1 - - 2 Total 12 12 1 4 7 7.66 ± 3.91 A total of 20 cases formed the study group out of which Group I (Benign) comprising of 8 cases and group II (Malignant) comprising of 12 cases were each immunohistochemically stained for podoplanin. All 8 cases of group I were positive and expression varied from weak to intense with 3 cases showing strong expression. In group II, all 12 cases were positive and the expression varied from weak to high with 7 cases showing strong positive podoplanin expression. Overall, malignant tumours showed a more intense podoplanin expression compared to benign tumours.

Table 2: Cases showing strong podoplanin immune expression.

Groups Lmvd Benign tumours Pleomorphic adenoma 5.1 ± 3.13 Malignant tumours Peripheral mucoepidermoid carcinoma 4.2 ± 2.37 Central mucoepidermoid carcinoma 5.05 ± 5.021 Adenocarcinoma nos 2.60 ± 1.697 PLGA 3.4 Total 4.15 ± 3.15 Lymphatic micro vessel density (LMVD) between group I (Benign tumours) and group II (Malignant tumours) mean value of lymphatic micro vessel density in group I was 5.1 ± 3.13 and mean value of lymphatic microvessel density in group II was 4.15 ± 3.15. Overall, lymphatic micro vessel density was found to be almost similar in both the groups.

Table 3: Peripheral MEC showed a mean LMVD count whereas central MEC showed higher count of mean. neoplasm’s like pleomorphic adenomas may show an indolent In the present study, all the 20 cases (100%) of salivary gland behaviour or grow to very large sizes with/without any signs of neoplasms showed positivity for Bcl-2 immune expression. Benign malignant changes. Malignant salivary gland tumours may also show salivary gland tumour which included 8 cases of pleomorphic adenomas a varied behaviour ranging from borderline malignancies like PLGA showed weakly positive Bcl-2 expression. This immune expression was to high grade MECs. Thus, in the present study, these neoplasms seen mainly in ductal cells (Figure 1). Studies have also shown positive were assessed for the expression of Bcl-2, an anti-apoptotic marker; staining for Bcl-2 in pleomorphic adenomas, indicating the possible and podoplanin, a small mucin like protein, which has been shown role of Bcl-2 as an anti-apoptotic agent in these neoplasm’s [13]. In a to mediate cell migration and invasion in various malignancies. An study performed by Yanez et al. [14] the immunohistochemical gene increase in number of lymphatic vessels in the tumours trauma has also Bcl-2 protein expression was determined in 27 salivary gland tumours. been shown to correlate with lymph node metastasis and is a predictor They found that Bcl-2 protein could be expressed in virtually all benign and malignant salivary gland tumours. This suggests Bcl-2 protein has of clinically meaningful outcomes in a number of malignancies. Thus, important role in the development of this tumour [14]. the role of lymph angiogenesis in salivary gland neoplasm was also assessed. In the present study, the immunohistochemical localization

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produce podoplanin and glycosylate with polysaccharides in the Golgi apparatus, and transport it to the cell membrane. Podoplanin may be involved in maintaining the homeostasis of myoepithelial cells through its characteristic mucin type transmembrane protein. Various findings have suggested that podoplanin is an antigen for salivary gland myoepithelial cells and that the immunostaining of podoplanin in salivary glands directly reflects myoepithelial cell shapes [18]. The immunohistochemical expression of podoplanin in malignant tumours varied from weak to intense with mucopeidermoid carcinomas showing the most intense expression followed by adenocarcinoma NOS and polymorphous low grade adenocarcinoma. It was found that 5 peripheral mucopeidermoid carcinomas showed high expression for podoplanin and 3 out of the 4 central mucoepidermoid carcinomas showed moderate expression suggesting Figure 1: Photomicrograph Showing Weak Positive Bcl-2 Expression in a more intense expression of podoplanin in peripheral than central Pleomorphic Adenoma (4x). MECs. Positive podoplanin expression in MECs was restricted to the epidermoid cells while the mucous cells did not show any podoplanin of Bcl-2 in malignant tumours varied from weak to intense with expression. The intermediate cells showed a variable staining pattern mucopeidermoid carcinomas showing most intense expression (Figure 4). Podoplanin has been reported to enhance tumour invasion followed by adenocarcinoma NOS and polymorphous low grade adenocarcinoma. Soini et al. [15] compared Bcl-2 expression in pleomorphic adenomas and in malignant salivary gland tumours comprising mainly of Adenoid cystic carcinoma and Mucoepidermoid carcinoma and found 100% positivity in pleomorphic adenoma and 64% positivity in malignant tumours [15]. They attributed 100% positivity in pleomorphic adenoma to the large size and long duration of the tumour at the time of diagnosis, thus emphasizing the association of increased cell survival with increased Bcl-2 expression. In addition, cell survival is generally more in malignant neoplasms than benign neoplasms. The benign neoplasm (pleomorphic adenomas) in this study showed overall weaker expression of Bcl-2 when compared to malignant salivary gland tumours. PLGA showed generalized weak expression, which may be associated with the low grade malignant potential of the neoplasm. A recent report of Bcl-2 expression in MEC by Yin et al concluded that Bcl-2 is one of the potentially useful markers for survival in patients with MEC in minor salivary glands. The expression of Bcl-2 in cells in Figure 2: Photomicrograph Showing Strong Positive Bcl-2 Expression In MEC may be linked to the degree of differentiation. Terminal differ Mucoepidermoid Carcinoma (10x). entiated cells like the mucous cells, normal acinar cells, salivary gland duct cells also do not express Bcl-2, but basal cells of the normal oral epithelium express Bcl-2 [16]. In our study, Bcl-2 immunohistochemical localization in malignant tumours mostly showed weak expression with only 1 out of 7 mucoepidermoid carcinomas showing strongly positive expression. Immunoreactivity was mainly found in the peripheral tumour cells and cells surrounding tubulo-ductal structures (Figure 2). In the present study, all cases of salivary gland neoplasm’s showed positive podoplanin expression in the tumour cells. All pleomorphic adenomas studied in this case showed positivity for podoplanin expression. 3 out of 8 cases of pleomorphic adenoma showed high staining intensity and 2 cases showed moderate staining intensity. Podoplanin immunoreactivity was mainly seen in myopeithelial cells, also seen in cells surrounding the tubulo-ductal structures (Figure 3). Kanner [17] in their study showed that myopeithelial cells in breast and salivary gland and basal cells in prostrate consistently demonstrate podoplanin immune reactivity, but typically less intensely than lymphatic’s. Cutaneous and mucosal based basal cells also showed podoplanin expression but often in a patchy, focal manner [17]. Figure 3: Photomicrograph Showing Strong Positive Podoplanin Expression in Pleomorphic Adenoma (40x). Salivary gland myoepithelial cells have been reported to constantly

Page 5 of 6 by enhancing cell motility. Cancer cell migration and invasion involves NOS showed LMVD counts similar to MECs. Though, Bcl-2 and D2- active remodelling of actin cytoskeleton, however, podoplanin does not 40 were weakly expressed in polymorphous low grade adenocarcinoma interact directly with actin but via ERM proteins such as ezrin, radixin but it showed a significant LMVD count. and moesin. It has been shown that over expression of podoplanin Thus, the present investigation demonstrated positive Bcl-2, leads to increased phosphorylation of ezrin, which links to the observed podoplanin response and LMVD in both benign and malignant salivary rearrangement of actin cytoskeleton. Podoplanin also increases activity gland tumours suggesting that Bcl-2 and podoplanin alteration may be of Rho family GTPases, which have also been linked to tumour cell associated with the progression of these neoplasms. But no statistically motility [19]. It was also found both the cases of Adenocarcinoma NOS significant difference was observed with regard to intensity of Bcl- showed positive podoplanin expression with 1 showing pronounced 2 and podoplanin staining in benign and malignant salivary gland expression. tumours. Studies of a larger sample with a wide variety of benign as In addition, lymphatic vessel density (LMVD) expressed by well as malignant salivary gland tumours comprising of all subtypes or podoplanin in pleomorphic adenomas was significant suggesting variants of tumours are required to understand the significance of Bcl- lymphangiogensis to be pronounced in this neoplasm. Lymph 2 and podoplanin expression in the prognosis and clinical outcome for angiogenesis was also pronounced in MEC and present in both types the future generations. (Figure 5). Mean of lymphatic micro vessel density count was found to References be almost similar in both central and peripheral MEC. 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J Clin Exp Pathol Volume 3 • Issue 3 • 1000145 ISSN: 2161-0681 JCEP, an open access journal