Nghiên cứu đột biến, mức độ biểu hiện gen EGFR và tình trạng METHYL hoá một số gen liên quan trên bệnh nhân ung thu biểu mô tuyến ở phổi tt tieng anh

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY --- NGUYEN NGOC QUANG STUDY ON MUTATION, PROTEIN EXPRESSION

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

NGUYEN NGOC QUANG

STUDY ON MUTATION, PROTEIN EXPRESSION

OF EGFR AND METHYLATION OF RELATED

GENES IN LUNG ADENOCARCINOMA PATIENTS

Major: Biotechnology Code: 942 02 01

SUMMARY OF BIOLOGY DOCTORAL THESIS

Hanoi - 2020

The research was completed at: Graduate University Science and Technology, Vietnam Academy of Science and Technology

Supervisor 1: Assoc Prof Chu Hoang Ha

Supervisor 2: PhD MD Nguyen Phi Hung

The thesis can be found at:

- Graduate university science and technology library

- Vietnam National library

suppressor genes such as BRCA1, MGMT, MLH1, RASSF1A are

associated with the initiation and development of non-small cell lung cancer The EGFR mutation has been reported in lung cancer partients

in many recent studies However, the molecular alterations of EGFR and the effect of methylation of tumor suppressor genes on these changes are not comprehensively investigated Therefore, the goal of this PhD research was aimed to investigate: "Study on mutations, expression of EGFR and methylation of related genes in lung adenocarcinoma patients"

Objectives

1 Analysis of mutation and protein expression of EGFR in patients

with lung adenocarcinoma

2 Investigation the hypermethylation frequency of EGFR, BRCA1,

MGMT, MLH1, RASSF1A and correlation of the methylation between

these genes with mutations and protein expression of EGFR

Dominant results

1 To collect medical records including lung adenocarcimoa and benign specimens using lung screen

2 To detect genetic and epigenetic alterations as well as protein expression of EGFR and to investigate the association of molecular abnormalities and clinicopathologic factors

3 To determine the methylated promoter of tumor suppressor genes

such as BRCA1, MGMT, MLH1, and RASSF1A and evaluate the

relationship of their methylation with clinicopathologic parameters

4 To analyze the interaction between molecular abnormalities of

EGFR and hypermethylation of BRCA1, MGMT, MLH1, or RASSF1A

CHAPTER 1 BACKGROUND 1.1 Lung cancer

1.1.1 Lung cancer facts

Lung cancer (LC) is the most common cancer in the world Vietnam is among the second highest rate of lung cancer in the world, with the incidence in men is 25.5 - 41.5 /100 000 people and

in women is 7.3 - 13.6 /100 000 people

1.1.2 Classification of lung cancer

Lung cancer is classified based on histopathological results into two groups: on-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) In which, adenocarcinoma (AD) is the most common form of lung cancer

1.1.3 Stages of lung cancer

Currently, the most commonly used lung cancer classification system is the TNM system (Tumor - Node - Metastasis), Depending upon tumor size, the number of lymph nodes metastasis and the degree of distant metastasis According to this classification system, the progression of lung cancer can be divided into four stages from 0 to IV

1.2 Adenocarcinoma lung cancer

1.2.1 Characteristics of adenocarcinoma lung cancer

Lung adenocarcinoma accounts for about 40% of lung cancer It usually starts in normal secretory cells such as mucus secretions and more common in non-smokers, females and young people

1.2.2 The histopathological subtypes in lung adenocarcinoma

Tumors in adenocarcinoma lung cancer differentiate with one

or more developmental forms including several types: lepidic predominant, acinar, papillary, micropapillary, solid …

1.3 EGFR alterations in lung cancer

1.3.1 EGFR gene structure and function

EGFR belongs to Epidermal Growth Factor Receptor (EGFR) with 3 parts: Extracellular ligand binding, transmembrane and tyrosine kinase activation domain EGFR plays an important role

in the process of multiplication, apoptosis, cellular, invasion, repair and interaction between cells

1.3.2 EGFR mutation in lung cancer

Up to date, more than 40 EGFR mutations have been found

scattered on 4 exons from 18 - 21 in the kinase domain, which are important in assessing the ability to treat lung cancer with TKIs

1.3.3 EGFR expression in lung cancer

Promoter methylation is closely related to protein expression EGFR expression study facilitates the development of useful biomarkers in clinical trials

1.4 Methylation of DNA in lung cancer

1.4.1 Methylation of DNA

Methylation of DNA is the phenomenon of attaching -CH3 to the 5'C position of nucleotides Methylation of DNA plays roles in normal cells such as gene expression, maintaining heterochromatin, maintaining an inactive state of an X chromosome in female mammals DNA methylation is divided into two categories: hypo-methylation and hyper-methylation

1.4.2 Methylation of DNA in lung cancer

DNA methylation is a molecular marker in the early stage of cancer, supporting the diagnosis, prognosis and treatment of lung

cancer In particular, EGFR methylation is directly related to the

effectiveness of treatment with target drugs In addition, the

methylation of tumor suppressor genes such as BRCA1, MGMG, MLH1, RASSF1A i.e as been shown the role in predicting the

progression of lung cancer patients

1.5 Targeted therapy in lung cancer

1.5.1 Targeted therapy in cancer

The targeted treatment in cancer is divided into 3 main categories: Monoclonal antibodies, small molecule inhibitors and immunotoxicity

1.5.2 Targeted therapy in lung cancer

Currently, the FDA has approved EGFR monoclonal antibodies including Cetuximab and Panitumumab; TKIs inhibitors such as Erlotinib, Gefitinib, Osimertinib; TKK inhibitors of ALK such as Crizotinib, Ceritinib for lung cancer treatment

1.6 Methodological analysis of molecular alterations in lung cancer

1.6.1 Methodological analysis of mutation in lung cancer

Gene mutations in lung cancer are detected through many methods such as: DNA sequencing, Realtime PCR, DNA hybridization…

1.6.2 Methodological analysis of DNA methylation in lung cancer

Three common methods are used to analyze DNA methylation including: Immune precipitation; using methyl sensitive enzyme and based on the bisulfite treated DNA

1.7 Study on bio markers for lung cancer in Vietnam

In order to support the lung cancer targeted treatment in Vietnam, serveral studies have been performed to determine the

EGFR mutation in NSCLC For example, Vu A.H et al., Nguyen M

H et al., Mai T K et al studied on 332, 120 and 511 patients with

NSCLC and found that the EGFR mutation rate was 40.7, 35.7 and

40.1%, respectively Furthermore, there were serveral thesises

focusing on EGFR mutation lung cancer However, there is no simultaneous evaluation of EGFR molecular changes including

genetic mutations, DNA methylation, and protein overexpression along with methylation status of tumor suppressor genes such as

MGMT, MLH1, BRCA1, RASSF1A has been published This can be

considered a new field of research for lung cancer in Vietnam

CHAPTER 2 MATERIALS AND METHODS

2.1 Materials

139 samples of lung tumor, 5 adjacent lung cancer samples, healthy human blood samples were provided by Hospital K The use of patient samples was approved by the ethicial committee of Hospital K

The chemicals and kits used in the study were qualified for molecular biology analysis materials

CHAPTER 3 RESULTS

3.1 Patient characteristics

In total of 139 patients in this study, the medium age of the patient group was 57.4 Of which 94 patients are male and 45 patients are female There are 79 smoking patients, of which 76/79 cases were male Patients in the study was belong to three main histopathological subtypes: Acinar adenocarcinoma (56.8%), Papillary adenocarcinoma (15.8%) and Solid adenocarcinoma (24.5%) 104 samples were collected from primary tumors and 35 samples from metastasis tumors Most patients were in stage II&IV (127/139) and there were only 12 cases in stage I&II

3.2 EGFR molecular characteristics in lung adenocarcinomas

3.2.1 EGFR mutation and correlation with patient characteristics

EGFR mutation was detected in 35.3% (49/139) patients, with

12 different types The deletion mutation at exon 19 and the substitution mutation at exon 21 (L858R) accounted for 85.5% In addition, there were six cases carried two mutations simultaneously

The substitution mutation G719X usually occured concurrently with other mutations (4/5 cases) such as S768I, L861Q, L858R and 19

deletions EGFR mutation was higher in younger patients, women and

non - smokers At the same time, patients with solid lung adenocarcinoma had a lower rate of mutation compared to the other subtypes (Table 3.3)

Table 3.3 EGFR mutation and the correlation with clinicopathologic

3.2.3 EGFR expression and correlation with patient characteristics

immunohistochemistry The results showed that 57 (41.0%) samples were negative (IHC: 0, 1+) and 82 (59.0%) samples were positive (IHC: 2+, 3+) with EGFR expression There was no correlation between EGFR expression and patient characteristics including age, gender, smoking status, histologic subtype, metastasis status, or

pathologic stage (p>0.05) (Table 3.6)

Table 3.6 EGFR expression and correlation with

3.3 Methylation of some genes involved in lung adenocarcinoma

3.3.1 Methylation of EGFR and correlation with patient characteristics

Aberrant promoter methylation of EGFR was detected in 33

(23.7%) of a total of 139 lung adenocarcinomas No significant

association between EGFR methylation and clinicopathologic variables

was observed

3.3.2 Methylation of BRCA1 and correlation with patient characteristics

Aberrant promoter methylation of BRCA1 was determined in

41 (29.5%) of a total of 139 lung adenocarcinomas No significant

association between BRCA1 methylation and patient characteristics

was indicated

3.3.3 Methylation of MGMT and correlation with patient characteristics

Aberrant promoter methylation of MGMT was detected in 46

(33.1%) tumors of a total of 139 lung adenocarcinomas Furthermore,

aberrant MGMT methylations was associated with metastasis status (p<0.05), but not with other clinicopathologic features

3.3.4 Methylation of MLH1 and correlation with patient characteristics

Our results showed that aberrant promoter methylation of

MLH1 was detected in 28/139 (20.1%) of a total of 139 lung adenocarcinomas No significant association between MLH1

methylation and patient characteristics was observed

3.3.5 Methylation of RASSF1A and correlation with patient characteristics

Aberrant promoter methylation of RASSF1A was detected in

41 (29.5%) tumors of a total of 139 lung adenocarcinomas The

statistically significant association between RASSF1A methylation and

smoking status was observed Prevalence of methylation in smokers

was higher than that in non-smokers (p<0.05) Furthermore, aberrant RASSF1A methylation were also associated with metastasis status (p<0.05) (Table 3.11)

Table 3.11 Methylation of RASSF1A and correlation with patient

3.4.1 Correlation between mutation, protein expression and DNA methylation of EGFR

The mutation status exhibited no significant association with

promoter methylation and protein overexpression of EGFR (p>0.05) but statistically correlated between EGFR methylation status and its protein expression was observed (p<0.05) (Table 3.12)

Table 3.12 Correlation between mutation, protein expression and DNA

with the methylation of at least one of the four genes, at least two of four genes or three genes (Table 15)

On other hand, EGFR expression did not correlate with

MGMT, MLH1, or RASSF1A methylation However, BRCA1

methylation was indicated to correlate with EGFR expression

3.4.3 Correlation of methylation between genes related to lung adenocarcinoma

In more detail, we analyzed the relationship between methylation of each set of two out of five genes The results showed

that RASSF1A methylation was correlated with BRCA1 and MLH1

4.1.1 EGFR mutation at Tyrosine Kinase Domain region

Analysis of 139 patients, we found that 35.3% of samples

occurred EGFR mutations, It is similar to previous reports in some

Asian countries such as Japan 32%, South Korea 36.4%; lower than some other countries in the region such as Thailand, 57.4%, Taiwan is

55% and higher than the America and Europe (10 - 15%) EGFR

mutation occurs with high frequency in patients with lung adenocarcinoma at National Cancer Hospital and more often in young people, women and non-smokers The solid adenocarcinoma has a lower rate of mutation than other adenocarcinoma types (Table 3.3)

Midha A et al (2015) Summarized and analyzed 139 studies of EGFR

mutations around the world and indicated that although there are differences in mutation rates among groups of people, geographic

regions, the rate of EGFR mutations is always higher than in women

and non-smoker patients

Table 3.17 Correlation of tumor suppressor genes methylation

M U p M U p M U p M U p BRCA1

4.1.2 Overexpression of EGFR protein

Evaluation of EGFR expression showsthat 59.0% (82/139) of tumor samples were positive EGFR expression in this study is in the same level with those reported from previous studies by Cappuzzo F

et al (2005) and Hirsch F.R et al (2008) Furthermore, we found that EGFR expression did not correlate with clinicopathologic variables

(Table 3.6); which was also observed in the study of Cappuzzo F et al (2005) and Liang Z (2010) In addition, we found that overexpression

of EGFR (3+) in men was higher than in women However, the mechanism of this phenomenon has not been clarified Moreover, the level of EGFR expression in metastatic tumors tended to increase compared to those in primary tumors Thus, it can be assumed that the higher degree of malignancy in metastasis tumors is the result of EGFR overexpression

4.1.3 Methylation of EGFR promoter region

The frequency of methylated EGFR in this study was 23.7%,

that was lower than that of Li J et al (2015) (36.8%) and Pan Z.Y et

al (2015) (35.7%) We did not find a correlation between EGFR

methylation with patient characteristics such as age, gender, smoking status, histological subtypes, stage of disease and tumor status; The similar results were also indicated the previous publications However,

Li J et al (2015) found that patients in stage III have lower levels of

methylated EGFR than stages I and II

4.1.4 Correlation between EGFR molecular characteristics

EGFR mutations lead to changes in amino acid sequence as

well as protein activity thereby affecting cell growth and development However, the level of protein expression is controlled by many pathways in the cell including promoter methylation Therefore, studying the correlation of mutations, methylation and protein expression will contribute to elucidating the mechanism of cancer formation and development

Studied on the correlation between gene mutation and methylation as well as EGFR expression showed very different results Analysis of 139 patients, we found that there is no correlation between