Download RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES ANNEXURE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
A
BRIEF RESUME OF THE INTENDED WORK
Incidence rates of OSCC has been increasing globally. It is
estimated that 35,310 new cases of OSCC were diagnosed in
Need for the study :
US during 2008. Head and neck malignancies constitute the
sixth most common malignancy, and more than 90 %
malignancies are squamous cell carcinoma. Oral and
oropharyngeal malignancies constitute 30-40 % of all types of
cancer in Indian population making it a leading cause of cancer
mortality . In one of the survey done in Karnataka cancer
therapy research institute, Navnagar Hubli carcinoma of the
oral cavity was the 3rd most prevalent after carcinoma of cervix
& oesophagus 2
Neoplasia is a result of imbalance between cell proliferation and
apoptosis. This further fecilitates results in tumor growth and is
influenced by angiogenesis, cell - cell and cell - extracellular
matrix(ECM)
interactions.
ECM
consists
of
proteins
and
polysaccharides distributed in many different tissues of the body.
ECM environment provides appropriate conditions for cell
growth, cell differentiation and survival of tissues. It constitutes
fibrous proteins such as collagen and elastin, elongated
glycoproteins such as fibronectin and laminin ,which provide
cell matrix adhesion. The role of ECM in the tumour
microenvironment is not limited to acting as a physical barrier
to neoplasia, but it also works as a reservoir for ligand proteins
and growth factor 1.
Matrix metalloproteinases are a family of zinc dependent
endo peptidases that are capable of degrading most components
of the extra cellular matrix (ECM). Degeneration of matrix
is a key invent in invasion and metastasis of malignant lesions
of the head and neck. A polymorphism is a genetic variant
observed in atleast 1% of population. It represents natural
sequence variants which may occur in more than one form.90%
of DNA polymorphisms are single nucleotide polymorphisms
(SNPs) due to a single base exchange 1. Common bi-allelic
SNPs have been found in the promoter region of several
MMPs in patients with HNSCC. Evaluation of such genetic
polymorphisms are vital because they can be used as biomarkers
for malignant lesions and thus may be involved in early
intervention and diagnosis of malignancies.
MMP-3 gene has been mapped to the long arm of chromosome
11q22.3 and the level of expression of this gene can be
influenced by single nucleotide polymorphisms(SNPs) in the
promoter region of their respective gene3.The promoter
region of MMP3 is characterized by a 5A/6A promoter
polymorphism at position-1171 in which one allele has six
adenosine(6A) and the second has five adenosine(5A).A single
adenosine insertion/ deletion polymorphism(5A/6A) at position
-1171 of the MMP-3 promoter region causes different
transcription of MMP-3.Invitro assays of promoter activity
showed that the 5A allele had a two fold higher promoter
activity than the 6A allele 3. The prevalence of 5A allele in
the European population 40-50%.The present study aims to
asses the association of 5A and 6A polymorphisms in the
MMP-3gene in patients suffering from oral squamous cell
carcinoma and compare with that of healthy controls.
B.
MATERIALS AND METHODS
Patients with OSCC and the age and sex matched control from
Source of data:
department of oral medicine and radiology, SDMCDS, Dharwad
and OPD, KCTRI, Navnagar would be taken up for the study.
30 patients with clinically and histopathologically diagnosed
Method of collection of
data(Including
procedure ):
OSCC and 20 healthy controls who are willing to participate in
the study will be enrolled. Detailed clinical and personal
sampling 7
information of each patient will be noted in standardized proforma.
7Information regarding the patients name, age, sex, occupation,
personal habits and present complain will be gathered. A detailed
7
information regarding addictions to arecanut, tobacco, gutkha
(mixture of arecanut, tobacco and lime) and alcohol consumptions
will be noted.
Inclusion criteria –
1. Patients with clinically and histopathologically diagnosed
OSCC.
2. Age and sex matched healthy individuals without the habits like
tobacco, gutkha, arecanut chewing.
3. patients who are willing to participate in the study.
Exclusion criteria –
1. patients who have completed or under the course of treatment for
OSCC
2. patients suffering from any other malignancies other than OSCC
or having suffered from malignancies previously.
3.
Individuals with hematological diseases, skin diseases and
autoimmune disorders.
The blood samples (5ml) will be taken after obtaining patients
informed consent. Blood will be drawn into vacutainer tubes
containing EDTA and the collected sample will be transported to
the laboratory in -200C cryobox and stored at -200C deep freezer.
Isolation of Genomic DNA from collected samples: Genomic
DNA would be isolated from the samples using standard DNA
isolation kit, after standardizing the protocol to the laboratory
conditions.
DNA quantification: DNA will be quantified using nano drop
technique.
Primer design: Primers needed to detect MMP-3 would be
designed.
(FP 5’-GGTTCTCCATTCCTTTGATGGGGGGAAAGA-3’
RP 5’-CTTCCTGGAATTCACATCACTGCCACCACT-3’)
PCR amplification based MMP-3 detection and genotyping:
MMP-3 gene promoter was genotyped using polymerase chain
reaction-restriction fragment length polymorphism(PCR-RFLP).
PCR amplification of a 129 bp fragment was performed in the
promoter region of MMP-3 for the detection of -1171 5A/6A
polymorphism using genomic DNA, forward and reverse primers,
IX of Taq buffer, Taq polymerase and PCR conditions would be
standardized.
PCR based Restriction Analysis:
Tth 111 l digestion of MMP-3
The aliquot of PCR-RFLP product was amplified on agarose gel
electrophoresis stained
with Ethidium Bromide and 5A alleles
represented by DNA bands at 97 and 32bp and 6A alleles at 129bp.
Gel electrophoresis:10 µl of the product would be loaded into
agarose gel containing ethidium bromide for MMP-3 gene
amplification.
Statistical ananlysis and interpretation: Statistical analysis will
be done using chi-square test.
Does the study need any
investigation
or
Yes. (5 ml peripheral venous blood)
interactions
to
be
conducted on patients or
animals?
Has the ethical clearance
Yes.
been obtained from your
institution (in case of 7.3)?
C.
List of references:
1.Chaudhary A ,Singh M, Bharti A, Shukla S, Singh A,
Mehrotra R. Genetic polymorphisms of matrix metalloproteinases
and their inhibitors in potentially malignant and malignant lesions
of the head and neck. Journal of biomedical science2010;17:10
2.Kulkarni B, Hiremath S, Hallikeri U, Patil B, Gai P. Decades
of breast cancer-trends in patients profiles attending tertiary cancer
care centre in south india.Asian journal of epidemiology
2012.5(4):103-113
3.Chaudhary A ,Singh M, Bharti A, Shukla S, Singh A,
Mehrotra R.
synergistic effect of stromelysin-1(matrix
metalloproteinase-3)promoter(-1171 5A->6A)polymorphism in oral
submucous fibrosis and head and neck lesions.BMC Cancer
2010;10:369
Vairaktaris E,Yapijakis C, Vasiliou D, Nkenke E,
Skerefoglou Z, Vorris E, vylliotis A, Ragos V, Neukam
F,Patsouris E.Association of –1171 Promoter Polymorphism of
Matrix Metalloproteinase-3 with Increased Risk for Oral Cancer.
Anticancer research.2007.27:4095-4100.
4.
5.Tadbir azadeh A, Purshahidi S, Ebrahimi H, Khademi B,
Malekzadeh M, Mardani M, Taghva M, Sardari Y. Serum Level
of MMP-3 in Patients with Oral Skquamous Cell Carcinoma - Lack
of Association with Clinico-pathological Features. Asian pacific
journal cancer .2012.13(9):4545-4548.
6.Zhang C, Li C, Zhu M, Zhang Q, Xie Z, et al. Meta-Analysis
of MMP2, MMP3, and MMP9 Promoter Polymorphisms and Head
and Neck Cancer risk.2013. PLoS ONE 8(4).