MUTATIONAL ANALYSIS OF EPIDERMAL GROWTH

FACTOR RECEPTOR GENE IN LUNG CANCER PATIENTS

OF QUETTA, PAKISTAN
MS Synopsis submitted to

BALOCHISTAN UNIVERSITY OF INFORMATION TECHNOLOGY

ENGINEERING & MANAGEMENT SCIENCES
for the partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE (MS)

in

BIOTECHNOLOGY

by

FARWA ZAHRA

Supervisor

Dr. HALEEMA SADIA

Department of Biotechnology, Faculty of Life Sciences and Informatics BUITEMS

Co-supervisor

2019-2021

TABLE OF CONTENTS
i
Introduction 1-3
Review of literature 4-8
Problem statement 9
Objectives 9
Methodology 9-10
Justification and significance 10
References 11-19

ii
 INTRODUCTION

All living organisms are made up of cells that contains the genetic information in
form of DNA. These cells have a predetermined lifecycle but changes or any error in their
normal performance can lead to disease or death (Johnson et al., 2012). Cancer is a condition
where the cells surpass their distinctive lifespan and continue to divide in an uncontrolled
fashion (Philpott et al., 2017). Changes in the genome are one of the most important reasons
that lead to cells becoming cancerous (Rehman et al., 2020). DNA can acquire changes over
time or inherit them, this starts from a single cell that keeps on growing even when it should
not, after undergoing the change brought about by internal or external factors, such as
mutations or environmental factors (Zhang et al., 2018).

Environmental or external factors that make normal cells cancerous are termed as
non-genetic factors. They may include both a person’s diet and their lifestyle, such as:
consumption of alcohol and tobacco (Webb, 2015), exposure to chemicals, radiations or
ionizing substances (Jin et al., 2016) hormones, obesity, certain infectious agents; and even
the quality of air, water and soil are considered a risk factor of cancer (Falette et al., 2019).
Drinking of alcohol and use of tobacco are two of the mostly strongly evident substances that
are associated with cancers of the lung, mouth, oral cavity, the wind pipe and the upper
digestive tract (Friedman, et al., 2019). Liver and colon are also effected from alcohol
consumption (Copur and Manapuram, 2019). Research has also found that the patients who
have survived surgery and are in remission from lung cancer have a very high risk of
developing cancers of the head, neck and secondary lung cancers in a phenomenon called
Field Cancerization, where the cancerous cells can transform again into another type of
cancer (Lewandowska et al., 2018).

Lung cancer (LC) is a multifactorial set of diseases which can occur both due to the
genomic changes and the environmental factors such as smoking (Park et al., 2020). It is one
of the most common types of cancers that occur worldwide and has a higher mortality rate
compared to other cancers like colon and breast (Botling et al., 2013). Lung cancers have an
extremely low survival rate. In Pakistan, LCs are the third most commonly diagnosed cancers
which cause more than 6% deaths annually (Sarwar and Saqib, 2017; Majeed et al., 2019).
According to a report published by WHO (retrieved from Globocan France), the overall
mortality rate of LCs in Pakistan has risen to more than 5.6% (Idrees et al., 2018). The main
reason behind this high death rate is that LC does not show outwardly symptoms until the

1
cancer has reached its advanced stage, it usually happens when the trachea, bronchi or lung
cells undergo mutations (Najafi et al., 2020) and the cells continue to divide and metastasize
to other tissues and neighbouring organs (Chen et al., 2014). When the cancer is in its initial
stage and is in the lungs, it is termed as primary cancer but after it has spread out in the lungs
or to other parts of the body, it becomes secondary LC. LC is mainly of two types (Shea et
al., 2016): (i) NSCLC (non-small cell lung cancer) and (ii) SCLC (small-cell lung cancer).
The NSCLCs are further classified as: Squamous cell, which develop on the lung epithelium,
adenocarcinoma which occur in the mucous glands, and large cell carcinomas (Bowman et
al., 2020).

The human epidermal growth factor receptor (EGFR) gene is present on the p-arm of
the 7th chromosome at position 11.2, its NCBI gene ID is 1956 and has a total of 31 exons
(Greenhalgh et al., 2016). This gene codes for a 170kDA protein called the Epidermal
Growth Factor Receptor (EGFR) which is present on the cell membrane. It is a receptor
protein and its main function is to receive signals called ligands which are also protein in
nature (Lee, 2017). The receptor and ligands work like lock and key. When the EGFR is
bound to the ligand, the receptor forms a dimer with another EGFR receptor present nearby
(Gazzeri, 2018). When both the receptors dimerize, the receptor complex is activated
resulting in a cascade of signals which make the cells grow and proliferate (Lee, 2017;
Passaro et al., 2020).

EGFR belongs to the protein kinase family and has 3 domains, i.e., the tyrosine kinase
domain, the transmembrane domain (hydrophobic), and the ligand binding domain that is
present outside the cell surface (Chen et al., 2016). This protein is also known as Human
Epidermal Growth Factor Receptor 1 (HER-1) and its family includes HER-2, HER-3 and
HER-4, all having similar structure (Landre et al., 2020). EGFR binds a total of six ligands,
including its own EGF and Transforming Growth factor -α (TGF-α) (Agarwal et al., 2018).
Even when lung cancers are in initial stages, surgery and chemotherapy are the only option
available for treatment but the chances of recurrence still remain and it has become critical
that mutations and rearrangements be identified timely to increase lifespan of patients and
provide palliative care (Schmit and Michiels, 2018).

The identification of mutations can help in designing targeted and personalized

therapy strategies which is the biggest need for cancer patients worldwide (Tan et al., 2015)
in general and the population of Pakistan in particular. Since the EGFR is responsible for cell

2
survival and growth, it has a vital role in cell cycle and any mutation or dysregulation in the
EGFR gene or its resultant protein can directly lead the cells toward cancer is reported to be
mutated in a number of cancers (Vanderlaan et al., 2017). Evidence from the literature
suggests that the epidermal growth factor gene’s exons 18 to 21 and protein are frequently
mutated in LCs (Harari, 2004; Scaltriti and Baselga, 2006; Sasaki et al., 2013; Tan et al.,
2015; Luo et al., 2018; Roh et al., 2020; Yutaka et al., 2020), so this study will aim to detect
the mutations of EGFR exons in LCs in context of Quetta, Pakistan.

3
 REVIEW OF LITERATURE

The lungs are very vital organ of our body that play role in providing oxygen via
blood to every other organ, they are involved in the process of gaseous exchange and make
up our respiratory system (Leeman et al., 2014; Jameson, 2018). Cancers of the lung are
reported to be one of the most common and deadly group of diseases in the world with
approximately 18.4% of the total deaths due to cancer worldwide (Jamal-Hanjani et al.,
2017). NSCLC or the Non Small-Cell Lung Cancer account for more than 80% of the total
diagnosed lung cancers (Bray et al., 2018). It has now become evident that cancers do not act
alone, instead they are a group of diseases that can occur either from genetic mutations, or
environmental factors; or both (Crosbie et al., 2013). These changes in the genome cause the
cancer to initiate, progress and metastasize to other parts of the body but the genomic and/or
proteomic analyses have resulted in improved responses in lung cancer therapeutics
(Jameson, 2018).

Almost all cancers, but especially lung cancers have a very poor prognosis and are
usually diagnosed when the disease has advanced to later stages (Tlemsani et al., 2019). It
has now become critical to identify alternative ways, which can help in early detection of
LCs. It is reported in a large number of clinical trials that early detection of mutations can
grant a positive therapeutic response and increase the quality of life of patients in contrast to
those being treated by chemotherapeutics, since they not only degrade the cancers but also
disturb the normal surrounding cells (Pan et al., 2019).

Approximately 90% lung cancers originate from the lung epithelium as a result of
multifactorial changes that arise in the epithelial cells, like smoking and irregular expression
of various genes and their products, e.g. the mutations in EGFR and the Ras oncogene family
(Liu and Liu, 2020). According to a study conducted by Crosbie et al, in 2013, it was found
that lung cancers, especially the non-small cell ones cause more deaths worldwide than
cancers of the colon and breast (Crosbie et al., 2013). Pan et al, reported that the Lung
cancers have no outward symptoms and when they are detected, the process of metastasis has
already occurred in them and almost 60% of them have become inoperable at the time of
discovery (Pan et al., 2019).

LCs are one of the most recurrent type of cancers all over the world. Ferlay et al,
described them in their study to cause more than 18.4% of all the deaths occurring from
cancers worldwide and their incidence rate among all cancers is 11.6% (Ferlay et al., 2018).

4
Of all the lung cancers, adenocarcinoma is reported to be extremely communal throughout
the globe and has a very low 5-year survival rate, which is reported to be 15% only (Toschi
et al., 2017). It accounts for more than 20% of the deaths that occur due to cancers in the
United States (Ferlay, et al., 2016).

A large number of genes and their products are found to be mutated in lung cancers
(Grigoriu et al., 2015; Jung et al., 2017), among them, the most common type is a family of
proteins called the Epidermal Growth Factor Receptors (EGFR) (Birkman et al., 2016). This
receptor is coded as 1210 amino acids containing residue by the EGFR gene located on the
short p-arm of chromosome 7, when matured, its N-terminal is cleaved resulting in 1186
amino acids containing mature transmembrane protein (Cho et al., 2018). The gene has 31
exons, the first 16 coding for the domain that lies outside the cell and works as a ligand
binding site, the 17th exon encodes the transmembrane domain which is hydrophobic, and
exon 18 to 28 are responsible for the C-terminus and the most critical Tyrosine Kinase (TK)
domain coding (Chen et al., 2016; Gazzeri, 2018; Wee and Wang, 2017; Mehlman et al.,
2019).

Fig.1: EGFR structure, starting from the N-terminus, Ligand binding domain, extracellular domain, TM domain
and the intracellular TK domain ending in the C-terminus (Chen et al., 2016)

As soon as the ligand attaches to the receptor, the receptor forms homo or
heterodimers with the neighbouring EGF receptor and gets activated (Furago et al., 2017).
Ligand binding and dimerization of the receptor leads to conformational changes and

5
activation of the TK domain resulting in the activation of downstream proliferation signals
that instruct the cells to divide and grow (Sigismund et al., 2018). Under normal conditions,
the receptor which is a glycoprotein, regulates cell proliferation and differentiation by starting
a signalling pathway (Lee et al., 2020). There are three other members in the EGFR family,
HER2, 3 and 4. The EGFR works like a lock and has specific ligands that help in its
activation, after activating the signals, the normal receptor is degraded by endocytosis
(Yamada et al., 2020).

Fig. 2: Blue: Indels, Orange: Point mutations in the EGFR gene (Harrison et al., 2020)

The EGFR is reported as an oncogene in a number of solid tumors, especially the lung
cancers. It can attain oncogenic properties due to a variety of reasons, such as changes in the
TK domain (Lee et al., 2020), over-expressed ligand and mutations in the receptor, in which
it is either always turned on or is overexpressed (Henson et al., 2017). In LCs, it is reported
that more than 60% of the patients have one or more significantly mutated gene (SMG), and
of those, EGFR Kinase domain mutations are most reported worldwide (Lee et al., 2020).
The TK domain acts as a molecular hotspot of mutations in the lung cancers (Henson et al.,
2017).

Fig.3: Difference between mutant and wild type EGFR activities

6
 Zhang et al, reported in 2016 that in NSCLCs, 40% patients have mutations in exon
21 where arginine replaces leucine at 858th codon (L858R), while 45% patients are reported
to have mutated exon 19; exon 18 and 20 mutations are less common. They also reported that
around 1/3rd of all the known NSCLC cases have mutation in one or more exons of EGFR.
Another finding from their study was that non-smoker, Asian females and people having
adenocarcinoma are more prone to EGFR mutations (Zhang et al., 2016).
A study conducted by Wee and Wang in 2017 reported that around 80% non-small
cell lung cancers (NSCLCs) occur due to the overexpression and point mutations in the
EGFR, these mutations usually occur in the TK domain and are observed in exon 19 of the
gene (Wee and Wang, 2017).

Fig.4: EGF receptors on cell surface and the cascade they activate
Xia studied the EGFR mutations in the younger and older patients of NSCLCs. In
their study, they found that the younger generation and patients who are below 40 years of
age lied at 52.6%, while persons above this age limit had a positive mutation rate of 52%.
They concluded that age has no relation with mutations in receptor and stated that it would be
beneficial of both clinical practice and research to compare the age of patients of LCs with
mutated EGFR and find whether there is any difference in rate of mutations (Xia et al., 2019).
Evans et al, also conducted a mutational analysis of 18,920 patients tested positive with non-
small cell lung cancers. Their results came at 93.9%, which clearly describes the involvement
of EGFR mutations in LCs. They also reported 9 novel mutations from their data and
described the trend that mutation rates increased with age. The deletion mutation of exon 19
was more common in the younger patients, while exon 21 mutation L858R (arginine
replacing leucine) was more frequent in adults (Evans et al., 2019).

7
 Chan et al, tested 104 patients with NSCLC and found 44% positive for the exon 18
(T790M) mutation, while the rest of the sample did not show mutations at first, but after
subsequent blood and tissue testing, 12 more patients were found to have the same mutation,
which brought the overall positive rate to 56.7% (Chan et al., 2020). A similar study was
conducted by Ito et al, where 1105 patients were enrolled. Out of these, 697 had exon 19
mutations, exon 21 L858R was found in 537 patients and 114 remaining patients had either
some minor mutation or a combination of both exon 19 and 21 mutations, which suggests the
critical role of these mutations in the occurrence and progression of lung cancers (Ito et al.,
2020).
All the above mentioned studies have identified that mutations in the Epidermal
Growth Factor Receptor gene is one of the leading causes of Lung Cancers in patients
worldwide, so it has become critical to identify whether these mutations are present in Quetta
region. A number of studies, like those conducted by Kwapisz, Saiyaros et al, Yamada et al,
Miyake et al, and Yutaka et al, (Kwapisz, 2017; Miyake et al., 2019; Saiyaros et al., 2019;
Yamada et al., 2020) have all reported that PCR and Sequencing based techniques are a very
useful and precise tool for the identification of mutations, so this study will aim to combine
the knowledge that mutations occur in exon 18 to 21 and polymerase chain reaction and
sequencing can be used to identify them, and try to identify similar mutations in context of
Quetta Pakistan.

8
 PROBLEM STATEMENT

One of the most commonly reported cancers in the world and Pakistan is the cancer of

Lungs. It is not only deadly but also shows no signs until its already too late. It is reported to

be a leading cause of deaths due to cancer in Pakistan, and occurs mainly due to mutations in

the Epidermal Growth receptor factor gene and protein. So, this research will try to find

whether these mutations occur in the patients from Quetta and to what extent.

RESEARCH OBJECTIVES

Following are the proposed objectives of this research:

 To find the mutations in exon 18 to 21 of the EGFR gene in lung cancer patients of

Quetta, Pakistan using PCR and Sequencing based methods.

 To determine the demographic features of lung cancer patients of Quetta, Pakistan.

METHODOLOGY

1. Ethical approval:

Permission to conduct this study will be obtained from the Ethical Review Board of

Balochistan University of Information Technology, Engineering and Management Sciences

(BUITEMS) before starting.

2. Patient selection:

For the purpose of this study, patients with diagnosed LCs will be identified from

different hospitals of Quetta, Balochistan. Questionnaire will be designed to obtain relevant

information regarding demographics of patients, tumor stage, grade, age at the time of

diagnosis, area, caste, gender of patients and other life style and health conditions.

3. Sample collection:

9
 Blood from the diagnosed patients with familial LCs will serve as a sample for this

research. It will be collected from different hospitals of Quetta in sterile EDTA tubes. The

collected samples will be stored at -20°C until the DNA is extracted. Written consent from

the patients or dependents will be taken before sample collection.

4. DNA extraction and PCR:

Total DNA from the blood will be isolated and PCR will be performed after optimization

of the reaction conditions.

5. Primers:

Primers defined and used by Luo et al, for exon 18 to 21 will be synthesized and used for

this study (Luo et al., 2018).

6. Sequencing:

Samples will be send for Sanger sequencing to detect the presence of mutations.

7. Statistical Analysis:

Demographic data will be analysed using SPSS.

JUSTIFICATION AND SIGNIFICANCE

Lung Cancer is a rapidly spreading and an extremely lethal disease which is highly

prevalent in Pakistan. This research will be helpful in filling the research gap as mutational

analysis of exon 18 to 21 is not previously conducted in context of Quetta Pakistan. A large

scale genetic and molecular study after this preliminary study will help to design biomarkers

for early diagnosis.

10
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