A CASE CONTROL STUDY ON THE EFFECTS OF MDM2 rs2279744

POLYMORPHISM ON HEAD AND NECK CANCER RISK IN NORTHEAST INDIAN

POPULATION

Thesis submitted in Partial Fulfilment of the requirement for the degree of Master of Science
in
BIOTECHNOLOGY
2022

UNDER THE SUPERVISION OF : SUBMITTED BY :

Dr. Debasish Borbora Dwijottam Sarma Bordoloi
Assistant Professor M.Sc Biotechnology
Dept. of Biotechnology Registration No. :- 20070710
Gauhati University Roll no. :- PS-201-805-0009
Guwahati-14, Assam

DEPARTMENT OF BIOTECHNOLOGY
GAUHATI UNIVERSITY
GUWAHATI-14
ASSAM
Prof. M.C. Kalita Phone: 0361-2700231
Professor & Head E-mail: mckalita@gauhati.ac.in

Date :

To
The Controller of Examination
Gauhati University
Guwahati -14

Sir,

I have the pleasure in forwarding the dissertation entitled “A Case control study on
the effects of MDM2 rs2279744 polymorphism on head and neck cancer risk in
Northeast Indian population” submitted to the Department of Biotechnology by
Dwijottam Sarma Bordoloi in partial fulfilment of the requirements for the degree of
“Master of Science in Biotechnology”. The work was carried out in the Department of
Biotechnology.

Yours faithfully

Date : (Prof. Mohan Chandra Kalita)

Place : Professor & Head
Department of Biotechnology
Gauhati University
 ACKNOWLEDGEMENT

I take this opportunity to thank the following people, whose immense help cannot go unnoticed.

First and foremost, I would like to thank Prof. Mohan Chandra Kalita, head of the department
of Biotechnology, for providing me this opportunity and guiding me to a successful completion
of my dissertation program. My heartfelt gratitude and thanks to my mentor Dr. Debasish
Borbora for giving me this wonderful opportunity to do a project under his guidance. I am
grateful to my lab-partners Ms. Adrija D Purkayastha and Ms. Geetashree Deka, Ms. Gauri
Priya Bora and Mr. Vishal Kumar for their amiable cooperation and help.

I express my special thanks to Dr. P.J. Handique as he shared his knowledge and enlightened
me with his experience. I am also grateful to Mr. Rantu Moni Sharma, Ms. Himashree Kalita
and Ms. Bandita Pathak for their priceless help and guidance throughout the journey. I would
like to offer my thanks to all the bearers, staff of Department of Biotechnology. I also
acknowledge the cooperation of Northeast Cancer Hospital & Research Institute, Guwahati.

Last but not the least; I would like to thank all members of the Biotech Hub, my parents and
friends I would also like to thank all members of the Biotech Hub, my parents and friends
whose moral support and guidance is unforgettable.

Thank you one and all.

Dwijottam Sarma Bordoloi

 LIST OF TABLES

Table no. Title Page no.

1 Various risk factors leading to development of HNC, From 12
(Marur and Forastiere 2008)
2 Estimated incidence and mortality in 2020 and 5-year 21
prevalence of HNC among both sexes of all ages (Source –
Head and Neck Cancer landscape in Asia-Pacific 2021, by
Novotech, The Asia Pacific CRO)
3 Estimated number of Head and Neck Cancers by Sex – 23
India – (2010-2020) (Source - Takiar, Ramnath et al. 2010)

4 Average Age Adjusted Incidence Rates for Head and Neck 23

Cancers for Various Registries by sex (Source - Yeole,
Balkrishna B. 2007)
5 Recipe for polyacrylamide gel 38
6 Genotyping, primer sequences, length of PCR products, 38
and restriction enzymes
7 Composition of a PCR 39
8 PCR conditions for the amplification of MDM2 promoter 39
309
9 Association of MDM2 rs2279744 polymorphism with the 44
risk of head and neck cancer
 LIST OF FIGURES

Figure no. Title Page no.

1 Tumours of varying sizes. (Source - AYDIN, Hülya AYIK, et 4
al. 2018)
2 Phenotypical progression in carcinogenesis of HNC (Source - 8
Argiris, Athanassios, et al. 2008)
3 Anatomy of the head and neck illustrating the locations of 8
various regions of HNC
4 Regions of salivary glands cancer occurence 10
5 Malignancies in the mouth (Source - Vokes, Everett E., et al 11
1993)
6 Regulation of MDM2 at the transcriptional and translational 25
levels (Source - Zhao, Yuhan et al. 2014)
7 Regulation of p53 by MDM2 (Source - Moll, Ute M., and 26
Oleksi Petrenko. 2003)
8 Gel image of gDNA isolated using Tris buffers 41
9 Gel image of gDNA isolated by PCI method 42
10 Gel image of PCR amplicons of MDM2 promoter 309 43
(rs2279744)
11 PCR-RFLP analysis of MDM2 rs2279744 polymorphism 43
 ABBREVIATIONS

μl microlitre
μM micrometre
μmol micromolar
AJCC American Joint Committee on Cancer
APC Adenomatous polyposis coli
APS Ammonium Persulfate
ASR Age standardized rate
bp base pair
BRCA Breast Cancer gene
BRCA1 Breast Cancer gene 1
BRCA2 Breast Cancer gene 2
cm centimetre
CRO Contract Research Organization
DNA Deoxyribonucleic acid
EC Esophageal cancer
EDTA Ethylenediaminetetraacetic acid
EtBr Ethidium Bromide
EtOH Ethanol
Fig Figure
FISH Fluorescence in situ hybridization
gDNA Genomic DNA
GDP Gross domestic product
GLOBOCAN Global Cancer Observatory
HNC Head and neck cancer
HNSCC Head and neck squamous cell carcinoma
i.e. Id est
IARC International Agency for Research on Cancer
INK4A INhibitor or CDK4
kDa kilo Dalton
MDM2 Murine double minute 2
min minutes
ml millilitre
MLH1 MutL protein homolog 1
mm millimetre
mmol millimolar
mRNA Messenger RNA
MSH2 MutS homolog 2
MSH6 MutS homolog 6
NE North East
NF1 Neurofibromatosis type 1
NF2 Neurofibromatosis type 1
ng nanogram
NIH NIH
PAGE PAGE
PBCR PBCR
PBS PBS
PCI PCI
NIH National Institutes of Health
PAGE Polyacrylamide gel electrophoresis
PBCR Population-based cancer registry
PBS Phosphate buffered saline
PCI Phenol Chloroform Isoamyl alcohol
PCR Polymerase Chain Reaction
RB1 Retinoblastoma protein
RFLP Restriction Fragment Length Polymorphism
rpm Revolutions per minute
SCC Squamous Cell Carcinoma
SDS Sodium dodecyl-sulfate
SNP Single Nucleotide Polymorphism
STE Sodium Chloride-Tris-EDTA
TAE Tris acetate EDTA
TB1 Tris buffer 1
TB2 Tris buffer 2
TBE Tris-borate-EDTA
TE Tris-EDTA
TEMED Tetramethylethylenediamine
TNM Tumour, Nodes, Metastasis
TP53 Tumor protein p53
UK United Kingdom
USA United States of America
UTR Untranslated region
UV Ultraviolet
VHL Von Hippel–Lindau syndrome
WHO World Health Organization
WT1 Wilms' tumor suppressor gene 1
WT2 Wilms' tumor suppressor gene 2
 CONTENTS
Contents Page no.
1. INTRODUCTION 1
1.1 Cancer 1-3
1.1.1 Tumour 3-4
1.1.1.1 Types of tumours 4-5
1.1.1.2 Causes of tumour 5
1.1.2 Types of cancer 6-7
1.1.3 Stages of cancer 7
1.2 Head and neck cancer 7-8
1.2.1 Types of head and neck cancer 8 - 11
1.2.2 Causes and risk factors of head and neck cancer 12 - 14
1.3 Inherited mutation in cancer development 14 - 16
1.4 Cell cycle, tumour suppressors and oncogenes 16 - 18
2. REVIEW OF LITERATURE 19
2.1 Incidence and prevalence of head and neck cancer 19
2.1.1 Global overview 19 - 22
2.1.2 Indian perspective 22 - 24
2.1.3 Scenario in Northeast India 24 - 25
2.2 The MDM2-p53 interaction 25 - 27
2.3 Polymorphisms in MDM2 gene 27
3. OBJECTIVES 28
4. MATERIALS AND METHOD 29
4.1 Approval of ethics committee 29
4.2 Study design 29
4.3 Study population 29
4.4 Materials 29
4.4.1 Preparation of reagents 29 - 30
4.5 Methods 30
4.5.1 Sampling and storage 30
4.5.2 Isolation of genomic DNA 30
4.5.2.1 Isolation using Tris buffers 30 - 32
4.5.2.2 Isolation by PCI method 32 - 34
 4.5.3 Ethanol reprecipitation of DNA 34 - 35
4.5.4 Determination of quality and quantity of genomic DNA 35
4.5.4.1 Agarose gel electrophoresis 35 - 36
4.5.4.2 DNA polyacrylamide gel electrophoresis 36 - 38
4.5.5 SNP Genotyping using PCR-RFLP 38
4.5.5.1 PCR reaction set up 38 - 39
4.5.5.2 MDM2 promoter 309T>G polymorphism (rs2279744) 40
4.5.6 Statistical analysis 40

5. RESULTS 41 - 44
6. DISCUSSION 45
7. BIBLIOGRAPHY 46 - 49
8. APPENDIX 50 - 52
1. INTRODUCTION

1.1. CANCER

Our body is made up of trillions of cells of different types. An average person roughly contains
37.2 trillion human cells, with 200 different types of cells of varying weight and size, each with
their own function and size. These cells are constantly working, dying and being replaced by
new cells through a process called cell division, where the cells make copies of themselves.
This process usually happens in a very controlled way, so that we maintain the right numbers
of each type of cells. Our DNA (Deoxyribonucleic acid) is responsible for bearing the code of
instructions which carefully controls what each cell does, telling them when to die or when to
divide. However, errors can occur in the DNA instructions called mutations. A mutation can
be defined as a change that occurs in the DNA sequence as a result of error while copying of
DNA, or by external factors like UV light that can damage the DNA. These random errors
build up over time, which is why cancer risk increase with age. This damage can then build up
as we age and can lead to cancer. The incidence rates for cancer overall climb steadily as age
increases, and therefore, age is a big risk factor for the disease. DNA mutations are also caused
by environmental factors that damage the DNA, such as tobacco smoke, alcohol, obesity,
pollution, certain infections and sun exposure. The term “gene variant” is commonly used to
describe an altered DNA nucleotide sequence. Gene variations can be acquired during a
person's lifespan or passed down from parents. Inheritable variations are present in almost all
of a person's cells throughout their lifetime and are transferred from parent to child. Because
the parent's egg or sperm cells, which are also known as germ cells, contain these variants, they
are also referred to as germline variants. The fertilised egg cell that results from the union of
an egg and a sperm cell includes DNA from both parents. Any variants that are present in that
DNA will be present in the cells of the child that grows from the fertilized egg. Non-inherited
variations, on the other hand, appear at some point in a person's life and exist in only a subset
of the body's cells. Non-inherited variants are frequently referred to as somatic variants because
they typically develop in somatic cells (cells other than sperm and egg cells). The following
generation cannot inherit these mutations. Environmental influences like the sun's UV radiation
or mistakes made when DNA copies itself during cell division might result in non-inherited
variations. However, only a small proportion of the gene variants found in human body are
known to be responsible for genetic illnesses. In reality, a certain small portion of all variants
actually have a beneficial effect because they result in new protein variants that enable a person
1
to more effectively adapt to changes in their environment. An advantageous variety, for
instance, might produce a protein that defends against a novel bacterial strain for the current
generation as well as future generations. The majority of gene variations have no effect on
development or health. This is due to some variations that change the DNA sequence of a gene
without changing how the gene produces proteins. And those that could cause a genetic
disorder are repaired by certain enzymes before the gene is expressed and an altered protein is
produced. Enzymes can detect and correct DNA mistakes through specific routes that are
unique to each cell. Since our DNA is prone to damage by various factors, DNA repair thus
proves itself to be an important process by which the body protects itself from disease. But in
the case of a harmful gene variant, the mutation can cause a cell to copy and grow
uncontrollably, which is how a cancer starts. These abnormal cells keep creating copies of
themselves, growing into a clump or mass of cells called a tumour. Some tumours stay in one
place do not spread, known as benign or non-cancerous tumours, and these are less likely to
cause problems. Tumours can also be malignant or cancerous, which means they can spread to
other parts of the body. Malignant tumours are more likely to cause problems, as they invade
other body structures, affect the way the body works, or use up the body’s nutrients. Tumours
that have spread away from their primary site of the cancer are called “metastases”. Therefore,
it is really important to detect cancer at an early stage, as it is easier to treat before it has had a
chance to spread. Routine screening tests are used to pick up some common cancers. It is also
important to see a doctor if one experiences unexplained symptoms that could be a warning
sign of cancer. Cancer has become one of the leading causes of deaths worldwide as well as in
India. Annually, more than 7 lakhs new cases and 3 lakh deaths occur due to cancer.
Approximately 2-2.5 million cases of cancer prevail at a given point of time (Mathur, Garima
et al. 2015). When cancers occur in mucosal surfaces of the head and neck, they fall into the
category of Head and neck cancer. Areas of occurrence of head and neck cancers include the
mouth, throat and, the voice box, salivary glands and the sinuses. Cancers of the head and neck
occur commonly in individuals aged above 50. Approximately 9,00,000 cases and 4,00,000
deaths are accounted by head and neck cancer worldwide (Stenson, Brockstein et al. 2014) and
so, this class of cancer brings forth the need for diagnosis of victims all over the world. Various
investigations are used to diagnose cancer. These might include blood tests, imaging tests, and
a biopsy – where cells are taken for examination under a microscope. If a tumour is found,
these investigations help determine the type, size and spread of cancer, which is called its stage.
This is used to work out the most appropriate treatment. The aim of cancer treatment is to
remove or destroy the tumour cells in the body, while minimising damage to normal healthy
2
body cells. The main types of cancer treatment include : Surgery – Where the tumour is
removed from the body; Chemotherapy – Where anti-cancer medicine is taken to kill off cancer
cells; Radiotherapy – where radiation is targeted to destroy cancer cells. Other forms of cancer
therapy include : Immunotherapy, hormone therapy, and gene therapy. Some cancer treatments
also have an effect on normal body cells, particularly fast growing like the skin, hair, gut and
bone marrow. This can lead to a range of side effects, such as rashes, hair loss, nausea, and
tiredness, though these are generally temporary and usually resolve after treatment finishes.
Cancer treatment might significantly reduce the tumour size and ease symptoms, known as
remission. But it is often difficult to know whether all the cancer cells have been removed or
destroyed, and if some remain in the body, the cancer might return months or years later, which
is called recurrence. The overall prognosis i.e., chances of recovery vary a lot from person to
person, depending on the site, type and stage of cancer and the treatments available. Due to the
remarkable progress of medical science, advances in cancer therapy are happening all the time,
and these continue to lead to improvements in cancer survival worldwide.

1.1.1. TUMOUR

A tumour is a solid mass of tissue that forms when abnormal cells group together. Tumour is
also spelled as “tumor”. The NIH National Cancer Institute defines tumour as “an abnormal
mass of tissue that forms when cells grow and divide more than they should or do not die when
they should”. Tumours can affect bones, skin, tissue, organs and glands. In modern English,
the word “tumour” is used as a synonym for another word – “Neoplasm”. A Neoplasm is a type
of abnormal and excessive growth of tissue. Even if the primary reason is treated, a neoplasm
continues to grow abnormally and is not coordinated with the normal surrounding tissue. The
term used to describe the formation or production of a neoplasm is neoplasia.

The size of a tumour may range from 1 cm to more than 30 cm. It can be solid or fluid-filled.
As a tumor grows it can result in pain because of the compression of adjacent nerves and
organs. If a tumour spreads to the spine, it can cause pain by pressing on the nerves of the spinal
cord. If the tumour metastasizes, it can cause pain in other parts of our body. The common
symptoms of tumour include formation of a lump at the affected site such as a breast lump
accompanied by pain in the area, fatigue, fever, lack of appetite, unexplained weight loss and
night sweats.

3
Fig 1 : Tumours of varying sizes. Left – A 6cm small tumour, Right – A 30cm tumour arising
from the left ovary (Source - AYDIN, Hülya AYIK, et al. 2018)
To determine the progress of a prevalent growing tumour, doctors use a system called TNM
staging system for most types of cancer. It was developed and is now maintained and updated
by The American Joint Committee on Cancer (AJCC). The TNM system uses letters and
numbers to describe the tumour, lymph nodes and its metastatic activity by using the letters T,
N and M respectively. The letter T describes the size and location of the tumour. TX means the
tumour cannot be measured, T0 means there is no evidence of the tumour, Tis means the tumour
has not spread and can only be found in the cell of origin, and T1-4 describes the size and
location of the tumour on a scale of 1 to 4 (a higher number means a larger tumour or a deep
growing tumour). The letter N describes if the tumour has spread to the lymph nodes. It also
answers which lymph nodes and how many lymph nodes has the tumour spread onto. The
number after the letter N can be anywhere between 0 to 3. While N0 means there are no lymph
nodes involved, a higher number indicates more lymph nodes with cancer. The letter M and
the number after it describe if the cancer has spread to other parts of the body and if so, to
where and how much. M0 means it has not spread and M1 means has spread to other parts of
the body.

1.1.1.1 Types of tumours

There are mainly 3 types of tumours –

1. Benign - A benign tumour is a mass of cells that lacks the ability either to metastasize
or invade neighbouring tissue and as such, stay in one place. Benign tumours are non-
cancerous. They don’t usually grow back once removed. They tend to have a regular
and smooth shape and have a covering called a capsule. Although they cannot spread,
some types can be harmful such as brain benign tumour, which can be life-threatening.
The growth of benign tumours causes the compression of tissue and may cause nerve
damage, ischaemia (reduction of blood flow to an area of the body), necrosis (tissue

4
 death) and organ damage. If the tumour grows within enclosed spaces like the cranium,
sinus or the respiratory tract, the health effects of the tumour can be prominent.
Some examples of benign tumour are adenomas, fibroids, hemangiomas and lipomas.
2. Malignant – Malignant tumours are cancerous tumours. They grow in nearby tissues
and have cells that have ability to break away and travel through the lymphatic system
and spread to lymph nodes and distant parts of the body, and that is when the disease
becomes life threatening. When cancer cells spread and develop into new tumours, the
new tumours are called metastases.
Some examples of malignant tumours are carcinoma, sarcoma, germ cell tumor,
blastoma, and meningiomas.
3. Premalignant - Premalignant tumours are tumours that have cancer-free cells but have
the potential to develop into malignant ones if untreated. Some precancerous cells
transfer genetic changes and gradually develop abnormalities over a lengthy period of
time when they proliferate, eventually developing into cancer. Depending on how slight
or significant the alterations are, precancerous changes can be described in many ways.
Some examples of premalignant tumours are actinic keratosis, cervical dysplasia,
metaplasia of the lung and leukoplakia.

1.1.1.2. Causes of tumour

The common risk factors that cause tumours are –

a. Genetic mutations e.g., mutated BRCA genes cause breast cancer.

b. Inherited conditions e.g., Lynch syndrome is the cause of hereditary colon cancer.
c. Family history of certain cancer types.
d. Smoking, and exposure to secondhand smoke.
e. Exposure to toxins like asbestos and benzene can cause lung cancer and leukemia.
f. Exposure to radiation e.g., X-rays and gamma rays can cause thyroid cancer.
g. Viruses e.g., Epstein-Barr virus, human papilloma virus, hepatitis B virus.
h. Having obesity increases the risk of cancer because of the inflammation caused by the
fat that surrounds the vital organ.

5
 1.1.2. TYPES OF CANCER

On the basis of the type of cell that the tumor originates from, cancer is classified into the
following 6 types –

1. Carcinoma – In this type of cancer, malignancy develops in epithelial cells. Carcinoma

begins in tissue that lines the outer and inner surfaces of the body, and linings of internal
organs like liver and kidney. It is the most common type of cancer. Carcinomas can
metastasize, or be confined to the primary location. The several histological types of
carcinomas are adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma,
anaplastic carcinoma, large cell carcinoma and small cell carcinoma.
2. Sarcoma – It is a malignant tumour that arises from connective tissue like cartilage, fat,
muscle, blood vessels, fibrous tissue etc and all in bone. It can occur in both adults and
children. Some subtypes of bone sarcoma are osteosarcoma, chondrosarcoma,
hemangioendothelioma, angiosarcoma, fibrosarcoma, chordoma, adamantinoma,
leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma. Some subtypes of soft tissue
sarcoma include liposarcoma, dermatofibrosarcoma protuberans, giant cell
fibroblastoma, fibrosarcoma and many more.
3. Lymphoma – Lymphoma is a cancer of the lymphatic system. It develops from
lymphocytes, which is a type of white blood cell. These cells are in the lymph nodes,
spleen, thymus, bone marrow, and other parts of the body. There are 2 subtypes of
lymphoma namely Hodgkin and Non-Hodgkin Lymphoma and both of these types
grows at different rates and respond differently to treatment.
4. Leukemia – It begins in the bone marrow and is also a cancer of the lymphatic system.
In leukemia, the bone marrow produces an excessive amount of abnormal white cells
called blasts or leukemia cells, which are not fully developed. Leukemia is classified
into acute and chronic leukemia. In acute leukemia, the amount of immature blood cells
rapidly increases, preventing the bone marrow from producing healthy blood cells. In
chronic leukemia, there is excessive buildup abnormal cells which are relatively mature
and typically after months or years, it results in many abnormal white cells.
5. Germ cell tumour – Germ cell tumors can be cancerous or non-cancerous and they
begin from the germ cells. They mostly occur in the testicles and ovaries. The types of
germ cell tumours are teratomas, germinomas, yolk sac tumour, embryonal carcinoma
and choriocarcinoma.

6
 6. Blastoma – It is a type of cancer that arises from precursor cells called blasts. It is more
common in children. The types of blastoma are hepatoblastoma, medulloblastoma,
nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulomary blastoma,
retinoblastoma and glioblastoma multiforme.

1.1.3. STAGES OF CANCER

Staging is the assessment of the extent of spread of cancer inside and beyond its site of origin.
Before treating a patient’s cancer, it is important to find out what “stage” it has reached. The
information helps in developing a treatment plan, and providing some insight on how
aggressive and treatable the cancer can be.

There are 5 stages of cancer –

1. Stage 0 :- There is no cancer but only abnormal cells that can become cancerous.
2. Stage I :- The cancer is small. It is contained with the organ of origin. This is also called
early stage cancer.
3. Stage II :- The tumour is larger than Stage I but it hasn’t spread into surrounding tissues,
or has spread into lymph nodes close to the tumour.
4. Stage III :- The tumour has grown in size and entered neighbouring lymph nodes and
surrounding tissues.
5. Stage IV :- The tumour has spread to other areas of the body from its original location.
It is also known as metastatic or advanced cancer

1.2. HEAD AND NECK CANCER

“Head and neck cancer” is a broad term used to describe a number of different malignant
tumours that develop in or around the mouth or the throat. Cancer of the head and neck may
develop in areas such as the sinuses which are hollow spaces in the bones of our head around
our nose, the nasal cavity, the mouth (also known as the oral cavity), the voice box (also known
as the larynx) and the salivary glands which are organs that help us digest food. Most of these
structures are lined with squamous cells and hence the most common type of head and neck
cancer that begins in these cells is squamous cell carcinoma. Over time, the cancer cells grow
out of control and form a tumour which can then grow and spread, destroying healthy tissue.
Essentially, the part of the body that is responsible for speech, swallowing, communication is
affected. Often patients are seen with cancer originating in the thyroid glands or the salivary

7
glands. Head and neck cancer is the sixth leading cancer by incidence worldwide, and only 40-
50% of patients will survive for 5 years (Leemans, Braakhuis et al. 2011).

Fig 2 : Phenotypical progression in carcinogenesis of HNC (Source - Argiris, Athanassios, et

al. 2008)

The symptoms that come with this disease are – sore throat that doesn’t heal, a lump that
doesn’t go away, a sore throat that doesn’t go away, pain or difficulty in swallowing, ear pain,
hoarseness or change in voice, and trouble breathing.

1.2.1. TYPES OF HEAD AND NECK CANCER

Fig 3 :- Anatomy of the head and neck illustrating the locations of various regions of HNC

There are 5 main types of head and neck cancer, each named according to the part of the body
where they develop. They are -

8
1. Laryngeal and hypopharyngeal cancer – Both laryngeal and hypopharyngeal cancers
are throat cancers. In laryngeal cancer, the malignant cells form in the tissues of the
larynx. The larynx is a part of the throat located at the top of the windpipe, or trachea,
and is commonly called the voice box. It helps us speak, swallow and breathe. The
larynx is made up of a flexible tissue that builds a supportive framework called
cartilage. The upper part of the larynx is called supraglottis, the middle part is called
glottis, and the lower part is called subglottis.

Hypopharyngeal cancer is a rare form of throat cancer that takes place in the
hypopharynx. The hypopharynx lies within the lower neck and throat behind the larynx
just above the inlet to the esophagus i.e., the food pipe. The hypopharynx makes sure
that food goes around the larynx and into the esophagus and not into the larynx.

Symptoms of laryngeal and hypopharyngeal cancer include a persistent sore throat or

cough, difficulty or pain in swallowing, ear pain, lump in the neck or throat, and change
or hoarseness in voice.

2. Nasal cavity and paranasal sinus cancer - The nasal cavity is the area directly behind
the nose through which air travels as it moves toward the throat. It lies above the bone
that forms the roof of the mouth and curves down at the back to join the throat. The
paranasal sinuses are a group of four air-filled areas that surround the nasal cavity,
named after the bones in which they are located. They are the maxillary sinuses (located
under the eyes), the frontal sinuses (located above the eyes), the ethmoidal sinuses
(located between the eyes) and the sphenoidal sinuses (located behind the eyes).
Paranasal sinus cancer usually begins in the maxillary sinus

The nasal cavity and paranasal sinuses contain several different types of tissue, and
each contains several types of cells including squamous epithelial cells, minor salivary
glands, nerve cells, lymphocytes and blood vessel cells, and therefore, the cancer that
may develop from these cells include squamous cell carcinoma, adenocarcinoma,
melanoma, inverted papilloma, esthesioneuroblastoma, midline granuloma, lymphoma
and sarcoma. Symptoms of nasal cavity and paranasal sinuses cancer include nasal
congestion or stuffiness that worsens over time, pain above or below the eyes, blockage
of one side of the nose, post nasal drip, nosebleeds, pus draining from the nose,
problems with sense of smell, numbness or pain in part of the face, loosening or

9
 numbness of teeth, a lump on the face or the top of the mouth or inside the nose, constant
watery eyes, bulging of one eye, loss or change of vision, pain or pressure in one of the
eyes, hearing loss, headache, trouble open the mouth and enlarging lymph nodes in the
neck.

3. Nasopharyngeal cancer – Nasopharyngeal cancer starts in the nasopharynx, the upper

part of the throat behind the nose and near the base of skull. It is a box-like chamber
about 1½ inches on each edge which lies just above the soft part of the roof of the mouth
and just behind the nasal passages. It serves as a passageway for air traveling from the
nose to the throat. Most nasopharyngeal cancers are nasopharyngeal carcinoma.
Symptoms include formation of lump or mass on both sides of the neck towards the
back, hearing impairment, recurrent ear infections, nasal blockage or stuffiness,
nosebleeds, headaches, facial pain or numbness, trouble open the mouth, blurred or
double vision and difficulty in breathing or talking.
4. Oral and oropharyngeal cancer – Oral cancer takes place in the oral cavity with
includes the lips, gums, tongue, cheeks, roof and floor of the mouth. It is also called
mouth cancer. In oropharyngeal cancer, cancer cells form in the tissue of the
oropharynx, the part of the throat at the back of the mouth behind the oral cavity which
includes the back one-third of the tongue, the soft palate, the side and back walls of the
throat, and the tonsils. Symptoms of oral and oropharyngeal cancer include lumps in
the neck, chronic sore throat, difficulty in swallowing, opening the mouth, or moving
the tongue, swelling or thickening or erosion of the lips, gums or cheeks, white or red
patches in the mouth, unexplained bleeding in mouth, numbness or pain in areas around
the mouth, ear pain, unexplained weight loss and loosening of teeth.
5. Salivary gland cancer – The salivary
gland produces saliva, a fluid that is
released into the mouth which keeps
the mouth moist and helps in digestion
of food. In salivary gland cancer,
tumour growth begins in salivary
glands and these cells can be cancerous
Fig 4 : Regions of salivary glands cancer
as well as non-cancerous. There are 3 occurence
pairs of major salivary glands namely
parotid glands, the largest of all the salivary glands which are found in front of and just

10
below each ear and are associated with the occurrence of most salivary gland tumours;
sublingual glands, which are found under the tongue in the floor of the mouth; and
submandibular glands, which are found below the jawbone. There are also microscopic
salivary glands present in the lining of the mouth, nose and the larynx. The most
common type of salivary gland cancers is mucoepidermoid carcinomas, followed by
adenoic cystic carcinoma on the second most common place. Other types include acinic
cell carcinoma, polymorphous adenocarcinoma, secretory carcinoma and some rare
adenocarcinoma. Symptoms include usually a painless lump in the area of the ear,
cheek, jaw, lip or inside of the mouth, fluid draining from the ear, trouble swallowing
or opening the mouth fully, and numbness or pain in the face.

Fig 5 : Malignancies in the mouth (A) Leukoplakia in the hard palate of the mouth (B)
Leukoplakia in the floor of the mouth (C) SCC of the lateral border of the tongue (D)
SCC of the left vocal cord (Source - Vokes, Everett E., et al 1993)

11
1.2.2. CAUSES AND RISK FACTORS OF HEAD AND NECK CANCER

Table 1 :- Various risk factors leading to development of HNC,

From (Marur and Forastiere 2008)

The causes for the incidences of head and neck cancer around the world are -

1. Alcohol and tobacco use – Alcohol and tobacco are the two most important risk factors
for head and neck cancers, especially cancers of the oral cavity, hypopharynx, and voice
box. Upon consumption of alcohol, the enzyme alcohol dehydrogenase presently
mostly in the liver converts it into acetaldehyde, which is carcinogenic i.e., it can
potentially cause cancer. Acetaldehyde is known to interfere with DNA replication and
induce DNA damage and thus it promotes cancer development. On the other hand, upon
burning of tobacco, thousands of dangerous chemicals are released, and over 40 of them

12
 are known to cause cancer. Some of these compounds include polycyclic aromatic
hydrocarbons, N-nitrosamines, benzene, aldehydes and ethylene oxide and these
compounds react with the cells to degrade the DNA and hence promote cancer. Both
smoking and alcohol are known to influence mortality in general, in combination, they
are associated with about 75% of all cases of head and neck cancer. People who use
both tobacco and alcohol are at greater risk of developing these cancers than people
who use either tobacco or alcohol alone. Most head and neck squamous cell carcinomas
of the mouth and voice box are caused by tobacco and alcohol use. Findings from a
study shows that smoking status at the time of diagnosis of a head and neck cancer is
associated with worst survival (Beynon, Lang et al. 2018). The use of paan (betel quid)
in the mouth is a common custom in Southeast Asia and is actively consumed in China,
Taiwan, India, Myanmar, Thailand, Cambodia, the Philippines, Laos and Vietnam.
Betel nut, a component of paan is positively associated with oral cancer. Betel nut
contains alkaloids and other compounds that are known to contribute to carcinogenesis
by generation of reactive oxygen species, formation of DNA adducts and breaking of
DNA strands. Another study shows that long term use of smokeless tobacco is
associated with high risk of head and neck squamous cell carcinoma (Zhou, Michaud
et al. 2013).
2. Infection with cancer-causing viruses – For oropharyngeal malignancies that affect
the tonsils or base of the tongue, HPV type 16 poses a risk. Approximately 75 percent
of oropharyngeal malignancies are brought on by persistent HPV infection. Although
HPV can be found in other head and neck cancers, it alone seems to be the cause of
oropharyngeal cancer. The Epstein-Barr virus infection is a risk factor for
nasopharyngeal carcinoma and salivary cancer.
3. Occupational exposure - Occupational exposure to wood dust is a risk factor for
nasopharyngeal cancer. The increased risk of voice box cancer has been linked to
specific industrial exposures, such as exposure to asbestos and synthetic fibres, but this
association is still debatable. Voice box cancer may be more common in those who
work in specific jobs in the construction, metal, textile, ceramic, logging, and food
industries. Cancers of the paranasal sinuses and nasal cavity are at increased risk as a
result of occupational exposure to formaldehyde, nickel dust, or wood dust.
4. Radiation exposure – Radiation can disassemble atoms and damage DNA in cells,
thus promoting the growth of tumour. Ionizing radiation has enough energy to dislodge
electrons from atoms. X-rays can cause breakage in the DNA double-helix and lead to
13
 translocations, inversions and other types of chromosome damage. Radiation to the
head and neck, for noncancerous conditions or cancer, is a risk factor for cancer of the
salivary glands.
5. Ancestry – Ancestry has effect on mutation rates. Asian ancestry, particularly Chinese
ancestry, is a risk factor for nasopharyngeal cancer. Oral cancer occurs significantly
higher among Asians, while nasopharyngeal cancer among the Chinese population
(Warnakulasuriya, Johnson et al. 1999).
6. Underlying genetic disorders - Some genetic disorders, such as Fanconi anaemia, can
increase the risk of developing precancerous lesions and cancers early in life. Fanconi
anaemia is a rare inherited disease that affects the bone marrow.

1.3. INHERITED MUTATION IN CANCER DEVELOPMENT

Cancer is a genetic disease. The accumulation of genetic changes over the course of many years
results in the conversion of normal cells into cancerous cells. In the late nineteenth and early
twentieth century, certain studies implied that our genome plays a central role in cancer
development (Stratton, Michael R. et al. 2009). Presence of chromosomal aberrations were
observed while examining dividing cancer cells under the microscope which led to the proposal
that cancers are abnormal clones of cells caused by abnormalities of hereditary material.
Genetic alterations can be categorised into point mutations and translocation. In point mutation,
one single nucleotide base is added, removed or changed. Substitution mutation is a point
mutation in which one base pair is substituted for another, interrupting the formation of the
correct codon. Nonsense mutation occurs when the substituted nucleotide leads to the
formation of stop codon that stops the production of amino acid; missense mutation is the one
where the substituted nucleotide leads to the formation of an amino acid that is different from
the correct one; and silent mutation is the one where the nucleotide substitutes for another, but
the same amino acid is produced anyway. Other types of point mutation include insertions and
deletions, where an extra base pair is added to or deleted from a sequence respectively. These
two mutations change the three-base codon by affecting the sequence of amino acids, causing
frameshift mutation. In frameshift mutation, the addition or removal of a single nucleotide
shifts the entire sequence, thus affecting the triplet codon frame. In translocation, alterations of
large amounts of DNA takes place at a chromosomal level. It involves the breakage of two
different chromosomes, movement of chromosome fragments and formation of two new
chromosomes with new combination of genes. This leads to increase or decrease in the level

14
of transcription of the genes in their new location. Apart from point mutations and
translocations, DNA damage may occur due to gene amplifications, where extra copies of a
gene are produced resulting in expression of more of the product. Overexpression of a certain
gene that leads production of cell growth proteins may cause cancer.

The genetic changes that cause cancer in the hereditary material can occur because of the
following reasons:

1. Occurrence of random mistakes in our DNA as cells multiply

2. Alteration of DNA by external factors like tobacco smoke, UV rays, cancer-causing
viruses etc.
3. Inherited genetically mutated genes from parents

It is worth noting that genetic changes in cancerous cells cannot be passed down from parents
to children but a genetic change associated with an increased risk of cancer can be inherited by
a child from his/her parents, if present in either of them. This is why some cancers seem to run
in families. Among all cancers, around 10% may be caused by inherited genetic changes.
Inherited cancer related genetic change is not an absolute determiner of whether a person will
get cancer, but increases the risk of getting cancer on exposure to risk factors. Inherited genetic
variants in cancer related genes cause a disorder known as “family cancer syndrome” or
“hereditary cancer syndrome” in which the risk of developing a certain type of cancer among
the family members is higher than average.

In our body, except the egg and the sperm cells, all cells are somatic cells. Somatic cells are
diploid i.e., they contain two sets of chromosomes, which are inherited one each from a parent.
An average adult human body comprises of over 3 trillion somatic cells. On the other hand,
germ cells are cells that are capable of giving rise to gametes through sexual reproduction.
They are found in the testes of males and ovaries of females and are fewer in number compared
to somatic cells. The sequence of cells produced from germ cells to gametes is called germline.
Mutations that occur in the somatic cells are called somatic mutations, and those that occur in
the germ cells are called germline mutations. Somatic mutations occur after birth and are not
passed down from one generation to the other. Somatic mutations are not present in all cells
but occur in a particular type of cell. Germline mutations are the one that pass through
generations. They are present at birth and exist in all of the cells of the body. The genetic

15
change in the egg or the sperm gets incorporated into the DNA of every cell in the body of the
offspring.

1.4. CELL CYCLE, TUMOUR SUPPRESSORS AND ONCOGENES

Cell cycle refers to a series of events taking place in a cell as it grows and divides. It is the
process by which cells replicate and produce two new cells. Cell cycle has four phases namely
G1 (first gap phase), S (synthesis phase), G2 (second gap phase) and M (mitotic phase). At G1
phase, the cell prepares to divide, growing physically larger with copies of organelles.
Following this, the cell enters S phase where it copies all the DNA, synthesizing a complete
copy of it. At the G2 phase, the cell starts to organize and condense the genetic material and
prepares itself for division. And lastly, at the M phase, the cell separates the two copies of the
genetic material into two daughter cells. Each of the resulting cells enter their own interphase
and begin a new round of the cell cycle.

The development and progression of tumours are linked to a series of changes in the activity
of cell cycle regulators. Negative regulators of the cell cycle prevent cells from dividing when
conditions are not right, and so, less activity of these inhibitors can promote cancer. Similarly,
a high activity of positive regulators of cell division can lead to cancer. In most cases of cancer,
mutations in the genes that encode cell cycle regulator proteins are noticed. The development
of cancer is a multi-step process wherein multiple mechanisms fail before a critical mass is
reached and cells become cancerous. Tumour suppressors are negative regulators of cell cycle.
They block the progression of cell cycle and thus prevent the uncontrolled growth of cells.
Tumour suppressors are responsible for the prevention of the cancerous tumour formation
when they are operating properly, and therefore, any mutation in the genes that code for tumour
suppressor proteins may quite often lead to cancer. Examples of tumour suppress genes that
get mutated in cases of various types of cancers are RB1, TP53, INK4A, APC, MLH1, MSH2,
MSH6, BRCA1, BRCA2, WT1, WT2, NF1, NF2 and VHL. On the other hand, oncogenes are
mutated genes that contribute to cancer development. Before mutation, they exist as proto-
oncogenes which are normal genes present in everyone’s DNA in the body. They function as
a blueprint that codes for proteins that trigger cell growth. When proto-oncogenes are mutated
or undergo gene amplification due to DNA damage, the proteins produced as a result can cause
cancer as they affect the growth, proliferation and survival of the cell. The products of
oncogene can cause a mass of cells to grow in the absence of growth factors and thus outnumber
the surrounding cells, resulting in the formation of a tumour.
16
Human beings are diploid organisms, which means they contain two copies of each
chromosome, one inherited from each parent, and the genotype of a specific gene is represented
by each of the alleles. This implies that in a normal individual, a region of an allele that
represents a certain gene is also present in the other allele. In case of oncogenes, the mutation
of only one copy of the gene can elevate the risk of cancer. Therefore, oncogenes are most
often autosomal dominant. Whereas, in the case of tumour suppressor genes, a mutation must
occur in both the copies of the gene to elevate the risk of cancer and as such, tumour suppressor
genes are most often autosomal recessive.

One of the most important tumour suppressors is tumour protein p53. P53 plays a prime role
in the cellular response to DNA damage. It acts primarily at the G1 checkpoint where it halts
the cell cycle progression after being activated by a sensor protein. This buys time for repairing
the damaged DNA. Upon successful repair, p53 releases the cell back to continue its cell cycle.
However, if the damage is not repairable, p53 triggers apoptosis which results in cell death,
ensuring that the damaged DNA is not passed on. A mutant form of TP53 is therefore a marker
for potential cancerous cells in the case of most forms of cancer. It is the most commonly
mutated gene in many cancers and is related with all types of cancer in some way or another.

Another protein called MDM2 is a prime participant in the regulation of cell cycle. MDM2 is
encoded by the MDM2 gene and is also known as E3 ubiquitin-protein ligase Mdm2. MDM2
is a negative regulator of p53. It binds to p53, marking it ready for ubiquitin mediated
proteosome degradation, which is a process that involves a small 8.6 kDa protein called
Ubiquitin binding to the lysine residue of p53 and promote its degradation. The MDM2 protein
itself is not degraded and is utilized to degrade other p53 proteins. In normal cells where there
is no DNA damage, p53 induces the expression of MDM2 protein, forming a complex called
p53-MDM2 complex. This complex formation leads to the degradation of p53 by the action of
ubiquitin and thus the concentration of p53 is maintained low in normal cells. But when there
is a DNA damage, p53 is activated by phosphorylation and the concentration of p53 increases
inside the cell. The MDM2 protein fails to bind to phosphorylated p53 and therefore, the
formation of p53-MDM2 complex does not occur. As a result, p53 degradation is prevented
until the DNA damage is repaired after which the p53 gets dephosphorylated again, making
way for MDM2 to bind to it and degrade it by the action of ubiquitin.

As a critical negative regulator of p53, mutations in MDM2 can seriously affect the growth and
proliferation of cells in our body. Higher than normal expression levels of MDM2 can
17
significantly decrease p53 protein levels and function, which will eventually lead to an
increased cancer risk and accelerate tumour formation and progression. This study is a
dedicated approach to learn and observe the possible association of MDM2 mutation with the
development of head and neck cancer.

18
2. REVIEW OF LITERATURE

2.1. INCIDENCE AND PREVALENCE OF HEAD AND NECK CANCER

2.1.1. GLOBAL OVERVIEW

A statistical report approved by Cancer.net editorial board states that an estimated 562,328
people were diagnosed with head and neck cancer in 2020. In USA, HNC accounts for 4% of
all prevailing cancers as of 2022. HNC is the eighth most common cancer in the United
Kingdom, which accounts for 3% of all new cancer cases, as of 2016-2018. The same statistical
data states that in the UK, every year around 12,400 HNC cases emerge, which means 34 cases
every day. It is the sixth most common cancer globally and the highest incidence is observed
in Asia. HNC are quite prevalent in developing nations, particularly in Southeast Asia, where
high rates of cigarette, areca nut, alcohol, etc. usage are the norm. In terms of age, site of the
disease, and molecular biology, HNSCC (Head and Neck Squamous Cell Carcinoma) in
underdeveloped nations differs from that in industrialised countries. The management of these
tumours is significantly hampered by poverty, illiteracy, lack of access to and infrastructure for
treatment for health care, and poor infrastructure. When compared to industrialised countries,
the annual GDP (gross domestic product) spent on healthcare in developing countries is quite
low. (Joshi, Poonam, et al. 2014).

According to a study done by researchers of ‘Surveillance and Health Services Research

Program’ of American Cancer Society, Atlanta, USA, records of HNC cases from 1983 to 1987
and 1998 to 2002 obtained from population-based registry data across five continents, the
incidence rates of HNC were assessed. HNCs were categories into oral cavity, oropharynx and
a third group that includes larynx and poorly specific tumours of the lip, oral cavity and
pharynx. It was found that during those time periods, there was a significant global variation
in HNC incidence trends based on site of cancer, country and sex. The rates of oral cavity
cancer seemingly increased in both men and women in some European countries like Czech
Republic, Estonia, Finland, Slovak republic, the United Kingdom, Denmark and Asian
countries like Japan. In countries like France and Italy, rates among men decreased but
increased among women. Incidence rates of oral cavity cancer declined among men and women
in several Asian registries as well as in Canada and USA. In countries with tobacco epidemics
that were peaking at the time, oral cavity cancer incidence rates showed increase in number
cases while in areas where tobacco usage peaked some time ago, the number of cases seemed
to decrease. Increase of oropharyngeal cancer was observed among both men and women in a

19
number of European countries like Czech Republic, Norway, Denmark, Belarus, Iceland,
Finland, Latvia and the United Kingdom while some Asian countries exhibited a steady
decline. The largest increase in oropharyngeal rates was observed among Brazilian men. Rates
of other HNCs varied significantly by country and sex (Simard, Edgar P. et al. 2014).

The National Central Cancer Registry of China, cancer statistics of China 2015 conducted a
study where it was found that the net incidence of oral cavity and pharyngolaryngeal cancer in
China was estimated as 48 cases per 1,00,000 persons, with a mortality of 22, while the
incidence of nasopharyngeal cancer was estimated as 61 cases per 1,00,000 with a mortality of
34 after assessing data from 72 local population-based cancer registries (Anwei, Zhao, et al.
2020). While it is an evident fact that immigrants of Chinese and South Asian ethnicity are at
an increased risk of developing HNC, individuals of Chinese ethnicity with HNC have a higher
survival advantage compared to South Asians and the general population (Noel, Christopher
W., et al. 2021). In Hong Kong, nasopharyngeal cancer was observed as the most common one
especially in young men aged between 20 to 44 years (Thomson, Peter, et al. 2021). In Taiwan,
the most common HNC is oral cancer, followed by nasopharyngeal cancer in the second place
and laryngeal cancer in the third place (Hsu, Wan-Lun, et al. 2017). In Thailand, 14 out every
1,00,000 males and 10 out of every 1,00,000 females are diagnosed with HNC (Vatanasapt,
Patravoot, et al. 2012). They are the top five leading cancers in Thailand. Compared to the
worldwide rates of HNC incidences, the people of Thailand have lower incidence of laryngeal
and thyroid cancers, but higher incidence of nasopharyngeal cancer (Tangjaturonrasme,
Napadon, et al. 2018). In South Korea, laryngeal cancer was observed as the most frequent one
among HNCs. India’s global contribution of head and neck cancer patients is 58%. HNC
accounts for 30% of all cancers in India (Kulkarni, Manik Rao. 2013). Due to increased use of
tobacco, India now contributes to 60% of HNC patients worldwide, and the percentage is
expected to double in the next 8 years.

20
 Table 2 :- Estimated incidence and mortality in 2020 and 5-year prevalence of
HNC among both sexes of all ages (Source – Head and Neck Cancer landscape in
Asia-Pacific 2021, by Novotech, The Asia Pacific CRO)

Head and neck cancer ranked 2nd in Africa as stated by a study where secondary data archived
by WHO and IARC in GLOBOCAN 2008 and 2012 global cancer statistics were reviewed.
Several factors seem to play important roles in the diagnosis and management of cancer in
Africa, including religion, cultural beliefs, educational levels, poverty and lack of reliable
public transport over great distances (Adeola, H. A. et al. 2018). In Sub-Saharan Africa, the
oral cavity was reported as the most commonly reported site of HNSCC (Head and neck
squamous cell carcinoma) by far (Faggons, C. E., et al. 2015). Reports on the pattern of HNC
incidences from different regions of the country stated that nasopharyngeal cancer is the most
common HNC, followed by sino-nasal cancer at the second place, while laryngeal cancer is the
third most common HNC. The majority of HNC was epithelial in origin and was mostly
squamous cell carcinoma (da Lilly-Tariah, et al. 2009).

21
An article published in 2016 states that in South America, Brazil took the first position in
highest incidence rates for oral and pharyngeal cancer for both sexes, followed by Cuba on the
second place, Uruguay at the third and Argentina on the fourth. Cuba had the highest incidence
and mortality rates of laryngeal cancer for both males and females. Overall, males had rates
about four times higher than those in females. HNSCC incidence and mortality rates in Central
and South America vary significantly across countries, with Brazil, Cuba, French Guyana,
Uruguay and Argentina having the highest incidence rates (Perdomo, Sandra, et al. 2016).

In Europe, the incidence rates of oral cancer in men range from over 29 per 100,000 in Hungary
to 4 per 100,000 in Greece and, from 5 per 100,000 in Hungary to 1 per 100,000 in Greece in
women, indicating the geographical variability in disease frequency. Similarly, differences in
rates of pharyngeal cancer were also observed in areas of Europe as the risk in men was highest
in parts of Eastern Europe, particularly in Hungary and Slovakia. 98% of the patients are aged
above 40 years. However, during the past decades, an alarming rise in the incidence of oral
cancer has been observed among younger men (Döbróssy, Lajos. 2005).

In Australia, over 4,000 people are diagnosed with a HNC every year, including about 1,400
people diagnosed with cancer in the mouth and tongue, 1,000 with pharyngeal cancer, 935 with
lip cancer, 600 with laryngeal cancer, 300 with salivary gland cancer, and 200 with nasal or
paranasal sinus cancer. Men are three times more likely to develop a head and neck cancer than
women. In 2017, the number of HNC cases was double the number of HNC cases in 1982 and
is expected to continue to rise (Koh, Joo, et al. 2019). In New Zealand, over 500 new cases are
diagnosed with HNC each year. It is more common in people aged over 50 and thrice as
common in men than in women (Yakin, Muhammed, et al. 2017).

2.1.2. INDIAN PERSPECTIVE

An attempt to project cancer cases for India by sex, years and cancer groups was made and
published in paper where the source that were utilized were incidence data generated by
population-based cancer registries (PBCRs) at Bangalore, Barshi, Bhopal, Chennai, Delhi and
Mumbai for the years 2001-2005 along with the latest incidence data of North Eastern
Registries for the year 2005-06. The study concluded that he estimates for HNC for the year
2010 for males and females are 122,643 and 53,148, respectively and by the year 2020, the
numbers will rise to 153,636 and 64,785 cases, as observed in Table 3. For the year 2020,
mouth (64,525), tongue (38,052) and larynx (33,855) are the sites in decreasing order of
contribution to HNC (Takiar, Ramnath, et al. 2010).

22
 Table 3 :- Estimated number of Head and Neck Cancers by Sex – India – (2010-
2020) (Source - Takiar, Ramnath et al. 2010)

To determine the change in HNC in urban and rural population of India, a study reviewed the
cancer registry data of an urban and a rural population over a period of 13 and 11 years,
respectively where age specific incidence rates were used for analysis of data. It was found that
oral cancers formed the majority of the HNCs with a high bias for tongue in urban males,
whereas in rural males the pharynx was the found to be the predominant site for tumour
development. Overall, a decrease in the incidence of HNSCC was observed, especially in urban
females and rural males. Among the other sites, oral cavity cancers exhibited a decreasing trend
in urban females and rural males. However, the incidence of tongue cancers showed an
increased trend. Pharyngeal cancer showed decrease in urban females, but an increase in rural
females (Elango, J. Kalavathy, et al. 2006).

In another study, an attempt was made to study the trends in the age adjusted incidence rates
for the sites of HNC in Mumbai, Bangalore, Chennai, Delhi, Bhopal, and Barshi registry’s
populations. The following table presents the average age adjusted incidence rates per 100,000
population for various sites taken for analysis of trend for each registry.
Table 4 :- Average Age Adjusted Incidence Rates for Head and Neck Cancers for Various Registries
by sex (Source - Yeole, Balkrishna B. 2007)

23
In males, the highest incidence rate was noted by Bhopal registry and lowest in Barshi for the
sites of tongue and mouth. In females the highest incidence was observed in Bangalore and the
lowest in Barshi for the site of mouth. For the site hypopharynx, no significant difference was
seen in the age adjusted incidence rate among the registries in males, except for Delhi registry
which had the lowest number of cases. For laryngeal cancer in males the highest incidence is
noted by Delhi registry and the lowest by Barshi registry (Yeole, Balkrishna B. 2007).

In North India, the incidences of these HNC are thought to be closely linked to the use of
tobacco and alcohol. A study found that the larynx was the most common site observed in
patients, followed by the nose. 99% of these biopsies had squamous cell carcinoma (Mehrotra,
Ravi, et al. 2005).

2.1.3. SCENARIO IN NORTHEAST INDIA

The north-eastern region of India lacks proper infrastructure with respect to specialized
treatment facilities, human resources, as seen by the low 5-year survival of HNC relative to the
rest of India. A huge number of patients with HNC from the NE region travel outside the NE
for treatment and diagnosis. Local cultural factors and lifestyle choices are thought to
contribute to the difference in incidence rates compared to the rest of the nation. Long-term
exposure to carcinogens can cause the accumulation of somatic mutations and thus increases
the chance of developing cancer. However, research has also demonstrated the critical function
of germline influence in HNSCC, particularly given the surge in incidence of young patients
under the age of 45. For many centuries, the Mizo people have consumed tobacco in the form
of smoking, dipping (sahdah), chewing (gutkha), and smokeless-infused water (tuibur), along
with other customs including consuming smoked food, alcohol, and areca nut chewing. The
risk of developing cancer increases in a dose-dependent manner with the increase in years and
frequency of cigarette smoked. A study conducted to find the association of risk factors and
HNSCC in a small tribal population of Mizoram, NE India indicates a significant interaction
of risk factors between lifestyle habits and family history of cancer. The high incidence among
non-smoking and non-drinking patients having family history of cancer seemed to impose
family history of cancer as an attributable factor for development of HNC (Pachuau,
Lallianmawii, et al. 2022).

Another 5-years retrospective study of cancer patients from 2007 to 2011 registered in Civil
Hospital, Shillong shows that oropharynx and oral cancer were the most common HNC among
the patients. A total 1,438 HNC cases were registered out of which, 1,118 were male patients

24
and 320 were female patients. Oropharyngeal cancer constituted 24.2% of HNC with a male to
female ratio of 7.92:1 while oral cancer constituted 23.92% of HNC with male to female ratio
of 1.27:1. Prevalence of hypopharyngeal cancer was 18.42% of the total HNC patients with a
male to female ratio of 8.81:1, followed by laryngeal cancer constituting 15.61% of total HNC
cases with male to female ratio of 7.62:1. 50 and 26 cases of nasopharyngeal carcinoma were
recorded in male and female respectively forming 5.28% of HNC and 25 cases of paranasal
sinuses and nose cancer cases with male to female ratio of 1.27:1. In case of male HNC,
oropharynx was the most common site whereas in females, the oral cavity and lips were the
most common site of HNC. 40-49 years was the most common age group for the prevalence of
HNC followed by 50-59 years (Shunyu, Neizekhotuo Brian, et al. 2013).

2.2. THE MDM2-P53 INTERACTION

Fig 6 ; Regulation of MDM2 at the transcriptional and translational levels. (A) P53 binds to
the P2 promoter region of MDM2 gene to induce its expression. (B) Increased expression of
aberrantly spliced MDM2 variants give rise to MDM2 isoforms such as A, B and C. (C) A
group of microRNAs bind to 3’-UTR of the MDM2 mRNA inhibiting its translation. (Source
- Zhao, Yuhan et al. 2014)

The human MDM2 gene is located on the long arm 13–14 of chromosome 12 (12q13-14) and
was first discovered by Oliner et al. in 1992 using an MDM2 gene probe (Oliner, J. D., et al.

25
1992). It encodes a 483 amino acid protein that has a molecular weight of approximately
90 kDa. Originally, the MDM2 gene was identified on double-minute chromosomes of
spontaneously transformed mouse cell line 3T3 and was later found to be associated with p53
physically and having critical role in controlling p53. MDM2 protein has several substrates out
of which, p53 is a major one. The N-terminal of MDM2 contains region I which is the binding
site for p53. In response to stress signals that emerge due to DNA damage, p53 gets activated
which then transcriptionally induce MDM2 by binding to p53-responsive elements present in
the P2 promoter of the MDM2 gene as shown in Fig 6. Both p53 and MDM2 form an auto-
regulatory negative feedback loop as they regulate each other.

Fig 7 : Regulation of p53 by MDM2 (Source - Moll, Ute M., and Oleksi Petrenko. 2003)

In addition to the overexpression of the whole MDM2, numerous cancer forms, including
breast cancer, paediatric rhabdomyosarcoma, and soft tissue sarcoma, frequently exhibit
enhanced expression of alternatively and aberrantly spliced MDM2 variants. MDM2 isoforms
A, B, and C are the most frequently over-expressed of the more than 40 MDM2 spliced variants
that have been found in human malignancies thus far. Over-expression of MDM2 spliced
variants is frequently linked to tumour development that is already far along and a bad
prognosis.

26
Another mechanism that has been suggested to cause over-expression of MDM2 in cancerous
cell is the enhanced translation of the MDM2 transcripts in addition to MDM2 transcriptional
regulation. An important group of microRNAs which target for MDM2 are responsible for
regulation of MDM2 translation. They are a group of small non-coding RNAs that regulate the
translation of gene products by binding to partially complementary sites in the 3’-UTRs of
target mRNAs. MicroRNAs like miR-143/145, miR-605, miR-25, miR-32, miR-18b, and miR-
661 target MDM2 (Zhao, Yuhan, et al. 2014).

2.3. POLYMORPHISMS IN MDM2 GENE

Since the over-expression of MDM2 has been noticed in several human tumours, it is also
considered as an oncogene. As a critical negative regulator of p53, increased levels of MDM2
leads to decreased in p53 protein levels and function, resulting in elevated cancer risk and
tumour formation. Gene amplification mainly causes this increased expression of MDM2 in
human tumours.

An example of naturally occurring polymorphism in MDM2 gene is the change of T to G in

SNP309, a common SNP present in the P2 region of the MDM2 gene as shown in Fig 6. This
change enhances the binding affinity of transcription factor Sp1 which leads to a 2-4-fold
increase of transcription of MDM2 in humans. Thus, p53 function and activity is inhibited. The
female sex hormone estrogen enhances the binding of Sp1 towards the MDM2 SNP309 G
allele, leading to a significantly high expression levels of MDM2 expression (Zhao, Yuhan, et
al. 2014).

In a study conducted to evaluated the amplification of MDM2 gene in case of esophageal

cancer, it was found that 6.3% of the EC cases showed pronounced MDM2 gene amplification,
which were validated by a cluster-like signal pattern by FISH (Fluorescent in-situ
hybridisation). Moreover, a qPCR screening approach detected 15 borderline ECs with a
moderate MDM2 gene amplification (Michalk, Maike, et al. 2016).

Another study demonstrated an earlier onset of HNSCC when MDM2 promoter and p53 codon
72 are mutated. The finding further suggested in detail that both MDM2 promoter
polymorphism and p53 codon 72 polymorphisms may increase the risk of non-oropharyngeal
cancer and that MDM2 SNP309 G allele and p53 codon 72 SNP may elevate the development
of non-oropharyngeal cancer in women (Yu, Hongping, et al. 2011).

27
3. OBJECTIVES

I. To isolate DNA from whole blood samples of participants

II. To determine the quality of genomic DNA using agarose gel electrophoresis
III. Amplify select locus using PCR and genotyping the SNP using PCR-RFLP
IV. To evaluate the association between MDM2 mutation and the risk of head and neck
cancer.

28
4. MATERIALS AND METHOD

4.1. APPROVAL OF ETHICS COMMITTEE

The project is pending approval from the Northeast Cancer Hospital and Research
Institute.
4.2. STUDY DESIGN
The study was a hospital-based case-control based study.
4.3. STUDY POPULATION
The study population comprised of Male and Female participants of Northeast Indian
ethnicity. Cases were participants presenting malignancies in the head and neck
excluding those presenting malignancies affecting the brain and eyes. Controls were
participants of the same age range who visited the tertiary health care facility with minor
ailments.
4.4. MATERIALS
4.4.1. Preparation of reagents :-
1. For isolation of DNA using Tris Buffers –
i. Stock solutions
a) 1M Tris HCl solution (pH 8)
b) 1M KCl solution
c) 0.5M EDTA solution (pH 8)
d) 1M MgCl2 solution
e) 10% SDS solution
f) 5M NaCl solution
g) 0.1N HCl solution
h) 0.1N NaOH solution
ii. Tris buffer 1 (TB1)
iii. Tris buffer 2 (TB2)
iv. TE buffer (pH 8)
v. Ice cold 100% EtOH solution
vi. 70% EtOH solution
2. For ethanol reprecipitation of DNA –
i. 3M CH3COONa solution

29
 ii. TE buffer (pH 8)
iii. Ice cold 100% EtOH solution
iv. Ice cold 75% EtOH solution
3. For DNA isolation by PCI method –
i. STE buffer
ii. PBS buffer (pH 7.4)
iii. 20% SDS solution
iv. TE buffer (pH 8)
v. Chloroform: Isoamyl alcohol (24:1)
vi. 3M CH3COONa solution
vii. Ice cold 95% EtOH solution
viii. 70% EtOH solution
4. For Agarose gel electrophoresis –
i. 50X TAE stock solution
ii. 1X TAE running buffer
iii. 10X TBE stock solution
iv. 1X TBE running buffer
v. 5mg/ml EtBr solution

4.5. METHODS
4.5.1. Sampling and storage :-

All blood samples were collected before administration of chemotherapy. One millilitre of
peripheral whole blood was collected in K3 EDTA vials from the participants. EDTA stands
for Ethylenediaminetetraacetic acid and the K3 potassium salt of EDTA functions as an
anticoagulant by binding calcium in the blood. After proper mixing with the anticoagulant, the
blood sample was aliquoted into a 1.5 ml cryovials and kept at -80oC until further use.

4.5.2. Isolation of genomic DNA :-

Two methods were used for the isolation of DNA –
4.5.2.1. Isolation using Tris Buffers
Brief overview -
It is a rapid one-day method for isolating DNA from whole blood. It is very economical
and safe as it does not require the involvement of any hazardous organic solvents to

30
deproteinize cell digests. This method involves the use of detergents like Triton X-100
and SDS for the lysis of cells.
Equipments and instruments –
i. Pipettes, sterile pipette tips, sterile 1.5ml microcentrifuge tubes
ii. Sterile micropestles
iii. Centrifuge
iv. Vortex mixer
v. Dry bath heat block
Procedure –
1. 500μl of blood was taken from stock cryovials and put onto sterile 1.5ml
microcentrifuge tubes. A suitable amount of 0.9% NaCl was added in cryovials
containing less than 500ml of blood to make up the final volume of sample to
500μl.
2. The blood samples were then grinded properly using sterile micropestles to break
down clotted blood and create a uniform viscosity of the blood in the tubes.
3. The microcentrifuge tubes containing the samples were then subjected to
centrifugation at 2200rpm for 10 mins.
4. The supernatant was discarded leaving the pellet of cells at the bottom of the
tubes. 1ml of TB1 was added each of the tubes followed by thorough mixing
using vortex mixer.
5. Steps 3-4 were repeated for 3-4 times until the colour of the pellet turned pinkish.
6. 400μl of TB2 was added to the pellet in each tube.
7. The tubes were incubated at 57°C for 10 minutes in dry bath heat block.
8. 300μl of 5M NaCl was added to each of the tubes, followed by centrifugation at
12000rpm for 5 minutes.
9. After centrifugation, the supernatant from each tube was poured onto two new
microcentrifuge tubes in equal volumes, i.e., for each sample, the supernatant
was divided into two parts.
10. 2 volumes of ice cold 100% ethanol were added to precipitate DNA, followed
by inversion of the tubes for around 4-5 times.
11. After a short spin of 5 seconds at 5000rpm, 500μl of 70% ethanol was added to
the tubes and mixed well.
12. The tubes were then centrifuged at 12000rpm for 5 minutes.

31
 13. The supernatant in each tube was discarded carefully, leaving the DNA pellet at
the bottom of the tube.
14. 50μl TE buffer was added and the tubes were stored in 4°C for overnight
precipitation.

4.5.2.2. Isolation by PCI method

Brief overview –
PCI stands for Phenol Chloroform Isoamyl alcohol. This method is a 3-day process and
involves the use of organic solvents like phenol, chloroform and isoamyl alcohol to
separate DNA from proteins and lipids. Prior to that, overnight digestion of proteins is
done by Proteinase K to release DNA from the cells. It is a slow but effective method
to isolate DNA as it ensures a proper degradation of cellular proteins.
Equipments and instruments :-
i. Pipettes , sterile pipette tips, sterile 1.5ml microcentrifuge tubes
ii. Sterile micropestles
iii. Centrifuge
iv. Vortex mixer
v. Dry bath heat block
Procedure :-
Day 1
1. 300μl of blood sample was taken from stock cryovials and poured into sterile
1.5ml microcentrifuge tubes. The sample was grinded using separate
micropestles for each of the tubes to break down clotted blood and create a
uniform viscosity of the blood in the tubes.
2. 600μl of PBS buffer was added to the tubes containing the samples, followed by
thorough mixing of the contents inside the tubes using the vortex mixer.
3. The tubes were then centrifuged at 14000rpm for 5 minutes.
4. The PBS was then discarded off, leaving the pellet of cells behind at the bottom.
5. Steps 2-4 were repeated for two more times.
6. 340μl of STE buffer was added to the tubes, followed by the addition of 40μl
20% SDS solution.
7. 20μl of Proteinase K was added to each of the tubes. Thorough mixing of the
contents was done to ensure proper distribution of the buffers inside the tube.

32
8. The tubes were incubated at 60°C for 4 hours in dry bath heat block. During the
4-hour incubation period, the tubes were inverted and vortexed every 15 mins
for good results.
9. After the completion of the 4-hour incubation period, another 20μl of Proteinase
K was added to all the tubes, followed by their overnight incubation at 37°C for
proper digestion.

Day 2

10. The tubes were centrifuged at maximum speed (~14000rpm) for 15 minutes.
11. The supernatant from a tube was poured onto a new clean sterile microcentrifuge
tube. This was done for all the tubes containing the samples.
12. 400μl of saturated phenol was added to all the tubes containing supernatant.
13. Proper mixing of the contents was done by inverting the tubes several times by
hand. The tubes were then left on ice for strictly 10 minutes and not more than
that, as over exposure of the supernatant to phenol will cause the degradation of
DNA. This step was done to precipitate SDS.
14. The tubes were then centrifuged at 14000rpm for 2 minutes. This step separated
the mixture inside each of the tubes into two phases.
15. From each tube, the top layer containing the DNA was pipetted out by using a
200μl pipette very carefully and expelled into a new clean tube. It was made sure
that the bottom phenol layer and the middle interface protein layer was left
untouched.
16. Steps 12-15 were repeated once again, but this time the tubes were kept at ice for
5 minutes only.
17. 400μl of chloroform:isoamyl alcohol solution (24:1) was added to the tubes
followed by mixing by hand.
18. The tubes were then centrifuged at 14000rpm for 2 minutes. The top layer from
each tube was pipetted out by using a 200μl pipette very carefully and expelled
into new clean tubes for each sample.
19. A volume of ice cold 95% ethanol equivalent to double the amount of sample
present in each tube was added and the tubes was inverted 4-5 times.
20. 3M CH3COONa solution was added at an amount that is equal to 4% of the total
volume of sample in each tube. The tubes were inverted 4-5 times.
21. The tubes were then kept for overnight at -20°C to ensure precipitation.

33
 Day 3

22. The tubes were centrifuged at 14000rpm for 30 minutes to pellet DNA.
23. The supernatant was discarded and 500μl of 70% ethanol was added to each tube
to wash the DNA pellet.
24. The tubes were inverted by hand, followed by centrifugation at 14000rpm for 2
minutes.
25. The supernatant was discarded and the DNA pellet was dried by keeping the tube
caps open, laid down inside a laminar air flow hood for about 45-60 minutes.
26. The DNA was resuspended using 50μl TE buffer, and the tubes were stored at
4°C.

4.5.3. Ethanol reprecipitation of DNA

Brief overview – Ethanol is a good concentrating and de-salting agent for DNA
isolation. Sometimes after gel electrophoresis, smearing is observed in gels. This can
be due to protein contamination of presence of excess salt in the sample. In cases of
poorly resolved bands of DNA where a lot of smearing can be observed, ethanol
reprecipitation is done to obtain a better resolution of DNA bands in agarose gel
electrophoresis.

Equipments and instruments –

i. Pipettes
ii. Centrifuge
iii. Vortex mixture

Procedure –

1. 0.1 volume of 3M CH3COONa was added to the desired sample.

2. 2.5-3 volumes of ice cold 100% EtOH were added. The sample was mix using
vortex mixture.
3. The sample was precipitated at -20°C for 1 hour or overnight, or -80°C for 1
hour.
4. It was then centrifuged at 13000rpm for 30 minutes at 4°C.

34
 5. The supernatant was discarded and the pellet was washed with 0.5ml of ice cold
75% EtOH. The pellet was again spined down at 4°C for 10 mins. This step was
repeated once more.
6. The ethanol was disposed and any trace amount of ethanol was removed after a
quick spin.
7. The DNA pellet was air dried by keeping the tube laid down under a laminar air
flow hood for 30 minutes.
8. The DNA was resuspended by adding 50μl of TE buffer.

4.5.4. Determination of quality and quantity of genomic DNA

The purity and yield of DNA was assessed using the following 2 methods –

4.5.4.1. Agarose gel electrophoresis

Brief overview - Agarose gel electrophoresis was routinely done after DNA
isolations and PCR amplification to assess DNA quality and yield, however the
percentage of agarose gel used depended on the nature of the DNA fragment. To
observe the quality and yield of DNA after DNA isolation, TBE buffer was used,
whereas to assess the yield and quality of PCR amplicons, TAE buffer was used to
cast the agarose gel.

Equipment and instruments –

i. Gel casting tray, comb

ii. Electrophoresis tank
iii. Microwave
iv. Pipette
v. UV transilluminator

Procedure –

1. A 1% agarose gel was prepared by mixing 1g of agarose in 100 ml of 1X TAE

or 1X TBE buffer, depending upon the need. The agarose was then dissolved
by heating using a microwave.

35
 2. The molten agarose was allowed to cool to about 600 C and 8-10μ EtBr was
added to it using a 10μl pipette and mixed well.

3. In the meantime, the comb was cleaned and inserted into the casting tray in
such a way that 1mm gap between the teeth and the surface of tray could be
made.

4. The molten agarose was gently poured into the casting frame. Air bubbles (if
any) were removed using a disposable micropipette tip.

5. After the gel had solidified, it was kept at 4o C for some time and then the comb
was removed gently. The gel was then placed in the electrophoresis tank.

6. The tank was filled with 1X TAE (or 1X TBE running buffer, as per the need)
such that gel gets submerged by 5mm.

7. For assessing quality of DNA in the case DNA isolation, 5μl of sample was
mixed with 1μl of loading dye (sample : dye = 5:1) and the mixture was loaded
onto the well. For assessing quality and yield of DNA in the case PCR
amplicons, 2μl of DNA samples was mixed with a loading dye and diluted with
nuclease-free water to achieve a final 1X concentration of the loading dye. The
first well was loaded with a 100 bp DNA ladder.

8. Apply loading all the samples, the power supply was connected and
electrophoresis was performed at 60-70 volts. The run was continued until the
tracking dye had migrated at least ¾th of the gel towards the positive electrode.

9. The gel was visualized using gel documentation system or a UV

transilluminator.

4.5.4.2. DNA polyacrylamide gel electrophoresis

Brief overview –
In PAGE (polyacrylamide gel electrophoresis), the resolution capacity of the gel is
higher than that of agarose gel. Molecules of DNA differing by as little as 0.1% in
length can be separated via PAGE.
Equipments and instruments –
i. Bio-Rad Mini-protean tetra cell casting system
ii. 50ml tubes

36
iii. Pipettes
iv. Electrophoresis tank
v. Gel documentation system
Procedure –
1. Polyacrylamide gels were cast and electrophoretic runs were made using the
Bio-Rad Mini-protean tetra cell casting system.

2. The glass plates and spacers were cleaned meticulously with deionized water
and ethanol and then dried.

3. The glass plates were then assembled in the gel caster.

4. Based on the requirement, gels were prepared with desired polyacrylamide

percentage according to the recipe mentioned in Table 5, which was sufficient
to cast two mini-gels of 1 mm thickness.

5. The mix was prepared in an airtight tube, as the polymerization of acrylamide

is a free radical-mediated reaction and is sensitive to oxygen. After the addition
of tetramethylethylenediamine (TEMED), the mix was quickly dispensed
between the two glass plates using a pipette.

6. The comb was carefully inserted into the gel, preventing any air bubbles from
getting trapped under the teeth.

7. The gel was allowed to polymerize for about an hour.

8. For the electrophoretic run, the gels were removed from the caster and placed
in the electrophoretic tank which was filled with 1X TBE. The combs were then
removed carefully without distorting the wells.

9. The wells were flushed with the running buffer and a blank run was made which
removes polymerized acrylamide. The DNA samples were mixed with the gel
loading dye and loaded into the wells using a micropipette.

10. Electrophoresis was performed at 120 volts until the tracking dye had migrated
the desired distance.

11. The gels were stained for 15 minutes by placing them in a tank containing
ethidium bromide (5μl of EtBr in 100 ml distilled water). The gels were then
destained using distilled water and viewed using a gel documentation system.

37
 Table 5 :- Recipe for polyacrylamide gel

Reagents Volume

Nuclease-free water 5 ml

Acrylamide monomer 5 ml

10X TBE 1.25 ml

80% Glycerol 1.25 ml

APS (10%) 60µl

TEMED 7 µl

4.5.5. SNP Genotyping using PCR-RFLP

The study genotyped a few polymorphisms affecting genes involved in telomere length
homeostasis, to evaluate their unique as well as combined effect on tumour risk. The
polymorphisms were analyzed using polymerase chain reaction-restriction fragment length
polymorphism technique (PCR RFLP). Genotyping methods, primer sets and annealing
temperatures used for RFLP are shown in Table 6. A PCR reaction mixture of 25 µl was set up
using GoTaq Hot start Green 2X master mix consisting of 1 µl a 10 µmol/L solution of each
primer and about 200 ng template DNA.

Table 6 :- Genotyping, primer sequences, length of PCR products, and restriction enzymes

Annealing Genotyping Size Restriction Reference

Gene Primer sequence
temperatu (bp) enzyme
re
MDM2
5’-CGC GGG AGT TCA GGG TAA AG-3’ 62°C RFLP 237 Ohmiya et al.
promoter
309 5’-AGC TGG AGA CAA GTC AGG ACT TAA C-3’ MspA1I - 37o C
(rs2279744)

4.5.5.1. PCR reaction set up

Procedure –

1. Primer rehydration : To make 100μM stock solution of primer, appropriate volume

of TE buffer was added. This volume was calculated by multiplying the number
of nanomoles of primer in the tube by 10.

38
2. Primer dilution : 90μl of TE buffer was mixed with 10μl of 100μM stock solution
of primer to achieve a 1/10 dilution, which is the standard dilution for PCR.
3. Reaction set up :
i. The inside of the laminar air flow hood was thoroughly cleaned by wiping
the surfaces and pipettes to be used with 70% ethanol. The UV light was
kept switched on for at least 30 minutes.
ii. The tubes containing samples, primers and mastermix was brought to the
site of work i.e., the laminar air flow hood, in clean trays and was tapped
properly before usage.
iii. The samples, mastermix and primers were added in correct amount as
shown in Table 7, and mixed in different PCR tubes for different samples.
iv. The tubes were then put in the Thermocycler and the appropriate conditions
for PCR cycle as shown in Table 8, were set accordingly.
v. After completion of the PCR process, the products were run in agarose gel
electrophoresis in a 1% TAE gel to examine the quality and yield of
products.

Table 7 :- Composition of a PCR

Mastermix 6.25μl

Forward primer 0.25μl

Reverse primer 0.25μl

Nuclease free water 4μl

Sample 2μl

Table 8 :- PCR conditions for the amplification of MDM2 promoter 309

Sl. No. 1 2 (1/3) 3 (2/3) 4 (3/3) 5 6

Step Initial Denaturation Annealing Extension Final Hold

denatur- extension
ation
Tempr 94°C 94°C 62°C 72°C 72°C 4°C
Time 4 min 30s 30s 30s 7 min ∞
No. of - 35 - -
cycles

39
 4.5.5.2. MDM2 promoter 309T>G polymorphism (rs2279744)

The MDM2 promoter 309T>G polymorphism was genotyped using the method

originally described by Ohmiya et al. (Ohmiya et al., 2006). A 237 bp fragment was generated

using specific primers (Table 6). The PCR cycles were 30 seconds at 94°C, 30 seconds at 62°C

and 30 seconds at 72°C for a total of 35 cycles. The amplicon was digested with MspA1I (New

England Biolabs) at 37o C overnight and subjected to electrophoresis on an ethidium bromide

stained 2.5% agarose gel. The wild-type allele produces a single 237 bp fragment and the

variant allele produces two fragments of 189 and 48 bp.

4.5.6. Statistical analysis

All statistical analyses were done using IBM SPSS Statistics 20 (SPSS Inc., Chicago,

USA). The association between MDM2 polymorphism and cancer risk was estimated using

univariate logistic regression to determine the crude odds ratios (OR) and their 95%

confidence interval (CI). All statistical tests were two-sided, and associations were considered

statistically significant at P < 0.05.

40
5. RESULTS

Fig 8 : Gel image of gDNA isolated using Tris buffers

Agarose gel electrophoresis of gDNA isolated using Tris Buffers showed very little to almost
no yield of DNA in majority of the samples indicated by poor band quality (443, 411, 458, 435,
418, 456); while in case of some samples, a lot of smearing was observed in the bands
indicating a possible contamination of proteins in the samples (436, 438).

41
 Fig 9 : Gel image of gDNA isolated by PCI method

Agarose gel electrophoresis of gDNA isolated by PCI method showed comparatively better
results as nearly clean bands were observed in some samples (329, 45, 283, 116, 382, 328, 41);
however, smearing still seemed to be prevalent in certain cases (381, 146, 242) along with poor
quality and yield in certain others (241, 112).

42
 Fig 10 : Gel image of PCR amplicons of MDM2 promoter 309 (rs2279744)

Clean bands observed after agarose gel electrophoresis of PCR products implied the presence
of amplicons indicating the successful amplification of MDM2 promoter 309 (rs2279744) via
PCR.

Fig 11 : PCR-RFLP analysis of MDM2 rs2279744 polymorphism.

The amplicon was digested with MspA1I. The wild-type allele produces a single 237 bp
fragment and the variant allele produces two fragments of 189 and 48 bp.

43
 Table 9 : Association of MDM2 rs2279744 polymorphism with the risk of head and neck
cancer

Genotyp Cases Controls Crude OR p

e N % N % (95% CI)
MDM2 promoter 309T>G polymorphism (rs2279744)
TT 16 32.0 12 24.0 Ref.
TG 21 42.0 26 52.0 0.61 (0.24-1.56) 0.298
GG 13 26.0 12 24.0 0.81 (0.27-2.40) 0.707
TG+GGd 34 68.0 38 76.0 0.67 (0.28-1.61) 0.374
TT+TG 37 74.0 38 76.0 Ref.
GGr 13 26.0 12 24.0 1.11 (0.45-2.75) 0.817
d, dominant model; r, recessive model

A statistical analysis for evaluation of association of MDM2 rs2279744 polymorphism with the
risk of head and neck cancer in this case study has been depicted in Table 9. In this statistical
analysis we have compared the frequency of the genotypes – wildtype, hetero, and homozygous
mutant among case and controls.

The OR (odd ratio) in all the cases except the recessive model GGr were found to be less than
1. The P value in all the cases were found to be more than 0.5.

The study found no significant association between MDM2 rs2279744 polymorphism and the
risk of head and neck cancer.

44
6. DISCUSSION

It was concluded that a slightly elevated risk has been found in case of recessive model,
although it was statistically insignificant. An OR value of higher than 1 indicates elevated risk
of the happening of the event, while a value lower than 1 means the factor is protective. The
study showed a value of 1.11 crude OR with 95% confidence interval in recessive model, but
with a P value of 0.817, the data was not supported by the statistical significance, which is why
no association of MDM2 rs2279744 polymorphism was found with the risk of head and neck
cancer.

In 2011, Yu, Hongping, et al. found that MDM2 SNP309 individually or synergistically with
p53 codon 72 may accelerate the development of non-oropharyngeal cancer in women. They
discussed that it is likely due to the acceleration of tumour formation by SNP309 G-allele
allowed by the female sex hormone estrogen (Yu, Hongping, et al. 2011).

In 2016, Michalk, Maike, et al. found out in their study that MDM2 gene amplification was
associated with esophageal carcinoma. Out of 127 cases of ECs, 8 of them showed highly
elevated quantity of MDM2 gene (Michalk, Maike, et al. 2016)

45
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8. APPENDIX
Reagents :

1. Tris buffer 1 (TB1)

For 1000ml
Ingredients Amount
1M Tris HCl solution (pH 8) 10ml
1M KCl solution 10ml
1M MgCl2 solution 10ml
0.5M EDTA solution (pH 8) 4ml
Triton X-100 25ml

The total volume was made up to 1000ml using distilled water.

2. Tris buffer 2 (TB2)

For 1000ml
Ingredients Amount
1M Tris HCl solution (pH 8) 10ml
1M KCl solution 10ml
1M MgCl2 solution 10ml
0.5M EDTA solution (pH 8) 4ml
5M NaCl 80ml
SDS 0.6g

The total volume was made up to 1000ml using distilled water.

3. TE buffer (pH 8)

For 100ml
Ingredients Amount
0.5M EDTA solution 0.2ml
1M Tris HCl solution (pH 8) 1ml

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 The pH was adjusted to 8 using 0.1N HCl and 0.1N NaOH as per requirement. The
total volume was made up to 100ml using distilled water

4. STE buffer

For 100ml
Ingredients Amount
1M Tris HCl solution 2ml
5M NaCl 1ml
0.5M EDTA solution 0.2ml

The total volume was made up to 100ml using distilled water. The solution was
mixed well, autoclaved and stored at 4°C.

5. PBS buffer (pH 7.4)

For 1000ml
Ingredients Amount
NaCl 8g
KCl 0.2g
Na2HPO4 1.44g
KH2PO4 0.24g

The pH was adjusted to 7.4 using 0.1N HCl and 0.1N NaOH as per requirement. The
total volume was made up to 1000ml using distilled water. The solution was mixed
well and autoclaved.

6. TAE buffer

50X stock solution, for 1000ml

Ingredients Amount
Tris base 242g
Acetic acid 57.1ml
0.5M EDTA solution (pH 8) 100ml

The total volume was made up to 1000ml using distilled water.

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 1X running buffer, for 100ml
Ingredients Amount
50X stock TAE solution 2ml
Distilled water 98ml

7. TBE buffer

10X stock solution, for 1000ml

Ingredients Amount
Tris base 108g
Boric acid 55g
0.5M EDTA solution (pH 8) 40ml

The total volume was made up to 1000ml using distilled water.

1X running buffer, for 100ml

Ingredients Amount
10X stock TBE solution 10ml
Distilled water 90ml

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