Download annexure ii

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