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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. NAME OF THE CANDIDATE AND ADDRESS (IN BLOCK LETTERS) Dr. P. SRILAKSHMI POST GRADUATE STUDENT DEPARTMENT OF ORAL & MAXILLOFACIAL PATHOLOGY, THE OXFORD DENTALCOLLEGE, BOMMANAHALLI, HOSUR ROAD, BANGALORE-560068. 2. NAME OF THE INSTITUTION THE OXFORD DENTAL COLLEGE BOMMANAHALLI, HOSUR ROAD, BANGALORE-560068. 3. COURSE OF STUDY AND SUBJECT MASTER OF DENTAL SURGERY(MDS) ORAL & MAXILLOFACIAL PATHOLOGY 4. DATE OF ADMISSION TO COURSE 25th MAY 5. TITLE OF THE TOPIC EXPRESSION OF P63 IN LEUKOPLAKIA AND ORAL SQUAMOUS CELL CARCINOMA -AN IMMUNOHISTOCHEMICAL STUDY 6. BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED FOR THE STUDY: Over 90% of oral malignancies are known to arise from pre-existing potentially malignant lesions, such as Leukoplakia and Erythroplakia. Yet little is known about genetic events involved in the progression of these precancerous states to cancer. A large number of risk markers have so far been examined to assess their value in predicting which pre-cancers may progress to cancer with time. Leukoplakia is the most commonly diagnosed potentially malignant lesion in the oral cavity, with a rate of malignant transformation between 17% and 24% during periods of up to 30 years.1 Prevention of malignant transformation is particularly important in view of the poor prognosis associated with OSCC. Approximately 94% of all oral malignancies are SCC.2 P63 is a member of p53 gene family. Gene is located in 3q27-29 chromosome. It has six isoforms, α, β, γ sub divided into two groups: TAp63 and ∆Np63. It draws attention for depicting crucial impact on development, proliferation, differentiation, maintenance and maturation of stratified epithelium in addition to its implication on prevention of oral cancers. It is frequently amplified and over expressed in head and neck SCC suggesting that p63 may act as an oncogene, making p63 a useful marker for the OSCC.3 The risk of malignant transformation of potentially malignant lesion is difficult to assess. Thus the study is to assess p63 expression in Leukoplakia and its role as a marker of oral cancer risk and also to estimate the usefulness and diagnostic yield of the p63 marker in OSCC. 6.2 REVIEW OF LITERATURE: An Immunohistochemical study using isoforms of p63 in 23 cases of human primary well differentiated buccal carcinoma. They found that ∆NP63 expression was seen in all 23 cases, whereas TA isoform expression was seen in 5 out of 23 cases. But the pattern of expression remained same in the peripheral cells of tumor nests. This study concluded that ∆NP63 isoform may play a role in epithelial differentiation and proliferation in human oral carcinogenesis and specific isoform of p63 may be associated with oral squamous cell carcinoma.4 An immunohistochemical study conducted on expression of p63 protein and mRNA in oral epithelial dysplasia. P63 staining was compared for samples from 90 male patients with buccal epithelial dysplasia and 15 healthy individuals and results showed nuclear p63 staining in basal layers of epithelium of normal mucosa and staining was not restricted to basal layer, extending to middle spinous layer for mild category. Expression of p63 was observed across almost full thickness of dysplastic epithelium. They conclude p63 is associated with the severity of oral epithelial dysplasia and in human oral tumorigenesis.3 A study conducted on p63 over expression associates with poor prognosis in head and neck squamous cell carcinoma. Ninety four cases of oral squamous cell carcinoma and 10 cases of normal mucosa were analyzed for p63 expression. Normal oral mucosa showed a basal and parabasal expression of p63.There was also a statistically significant correlation between p63 expression and tumor differentiation .The expression was amplified in poorly differentiated tumors. The statistical analysis showed no significant correlation between p63 expression, sex, age, tumor size, staging, recurrence and metastasis. The data suggest p63 expression may be useful to identify cases of oral squamous cell carcinoma with invasive phenotype providing novel diagnostic and prognostic information on individual patient survival with oral cancers.5 A study conducted on p63 over expression. This study predicts the development of oral cancer in patients with Leukoplakia using immunohisto-chemistry in 152 patients with oral precancerous lesion. The associations between p63 expressions as well as with other potential risk factors for oral cancer development were analyzed. They concluded that ∆Np63 over expression in oral premalignant lesion is associated with increased oral cancer risk.1 An Immunohistochemical study conducted on diagnostic utility of p63 tumor markers in squamous cell carcinomas of head and neck. Thirty cases of squamous cell carcinoma of head and neck region diagnosed on the bases of H&E staining where examined along with sixty cases of head and neck region biopsies other than SCC, negative on H&E staining, taken as a control. They conclude p63 gene plays important role in normal cellular and carcinogenic proliferation. It can be used as confirmatory diagnosis of the squamous cell carcinoma.2 6.3 OBJECTIVES OF THE STUDY : 1. To study the immunohistochemical expression of p63 in normal oral mucosa. 2. To study the immunohistochemical expression of p63 in Leukoplakia. 3. To study the immunohistochemical expression of p63 in Oral Squamous cell carcinoma. 4. To compare the expression of p63 in normal oral mucosa , Leukoplakia and OSCC. 7. MATERIALS AND METHODS 7.1 SOURCE AND METHOD OF COLLECTION OF DATA: 20 paraffin embedded tissue blocks of each, clinically and histopathologically confirmed cases of Leukoplakia and oral squamous cell carcinoma, 10 paraffin embedded tissue blocks of oral normal mucosa as control will be retrieved from the Department of oral and Maxillofacial Pathology and Microbiology, The Oxford Dental College and Hospital, Bangalore. 7.2 EQUIPMENTS USED: • Immunohistochemistry kit (primary antibody, secondary antibody, blocking agents, DAB Chromogen etc.,) • Primary antibody- p63 • Soft tissue Microtome • Water bath • EDTA buffer solution • Microwave • 3% Hydrogen peroxide • Acid alcohol • H & E stains • Xylene • Slides and cover slips • DPX solution • Compound microscope 7.3 METHODOLOGY: 1. 10% neutral buffered formalin-fixed and paraffin-embedded tissue will be cut into 3µm thick sections using a microtome for immunohistochemical staining. 2. Prostate would be taken as positive external control for immunohistochemical staining of p63. 3. For antigen retrieval, sections on slides will be placed in covered jars containing EDTA buffer and heated until boiling point in a microwave oven. 4. Sections will be cooled and then washed with Tris buffer saline [TBS]. 5. After tapping excess buffer, the sections will be covered with 3% hydrogen peroxide [to block endogenous peroxide activity], followed by washing with TBS. 6. Sections will then be covered completely with pre-diluted monoclonal antibody p63. 7. Tissue sections will be washed with TBS and counterstained with haematoxylin for 10min, washed gently under running water, and differentiated by dipping in acid alcohol. 8. Sections will be dehydrated and immersed in a Xylene bath. 9. The slides will be mounted in DPX and sections covered with a clean cover slip. 10. All stained sections will be examined under microscope and 20X magnification five hotpots areas within the slide would be photographed. 11. The expression would be scored based on the following equation. X= Total Number p63 positive cells in the photomicrograph Total number of cells counted in the photomicrograph 12. The mean values of the score would be calculated for all the cases and Chisquare test would be administered. 7.4 Does the study require any investigation or interventions to be made on patients or other humans or animals? If yes please describe briefly. NO 7.5 Has ethical clearance been obtained from your institution? YES 8. LIST OF REFERENCES: 1) Saintigny p et al., ΔNp63 Overexpression, Alone and in Combination with Other Biomarkers, Predicts the Development of Oral Cancer in Patients with Leukoplakia. Clin Cancer Research 2009; 15:6284-91. 2) Khan N R et al. Diagnostic Utility of p63 (Ab-1) and (Ab-4) Tumor Markers in the Squamous Cell Carcinomas of Head and Neck, Asian Pacific Journal of Cancer Prevention, 2012; 13, 975-8. 3) Chen Y K, Shui-Sang Hsue, li-Min Lin. Expression of p63 protein and mRNA in Oral epithelial dysplasia. J Oral pathol med 2005; 34:232-9. 4) Chen Y K, Hsue S. S, M.Lin L. Expression of p63 (TA and ∆N isoforms) in human primary well differentiated buccal carcinomas. Int. J. Oral Maxillofac. Surg, 2004; 33: 493-7. 5) Lo Muzio Lorenzo et al. p63 overexpression associates with poor prognosis in head and neck squamous cell carcinoma. Human Pathology 2005; 36, 187-94. 9. Signature of the candidate: 10. Remarks of the guide: 11. Name and Designation of 11.1 Guide Dr. N. CHAITANYA BABU PROFESSOR 11.2 Signature 11.3 Co-Guide Dr. SHOBITH . R. SHETTY SENIOR LECTURER 11.4 Signature 11.5 Head of the Department 11.6 Signature 12. Dr .T .V. NARAYAN PROFESSOR & HOD 12.1 Remarks of the Principal 12.2 Signature DR. PRIYA SUBRAMANIAM