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

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An Analysis of the Expression of Bcl-2, Podoplanin and Lymph xp & E erim 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 C P f a o t h l o a Journal of l n o r g u y o J ISSN: 2161-0681 Clinical & Experimental Pathology 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
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