MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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 Reviewer 1: … Reviewer 2: … Reviewer 3: … The thesis will be defended in front of the Academy Thesis Evaluation Council at graduate university science and technology, vietnam academy of science and technology at …, date … month … year The thesis can be found at: - Graduate university science and technology library - Vietnam National library OBJECTIVES Lung cancer is the leading cause of cancer mortality in many countries The activating mutations in the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) are considered as a therapeutic target for lung cancer Targeted treatment regimen by tyrosine kinase inhibitors based on EGFR mutations have been widely used and contributed to improve clinical management On other hand, EGFR protein expression is controlled by methylation level of it promoter Along with EGFR, aberrant epigenetic alterations of tumor 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 Analysis of mutation and protein expression of EGFR in patients with lung adenocarcinoma 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 To collect medical records including lung adenocarcimoa and benign specimens using lung screen To detect genetic and epigenetic alterations as well as protein expression of EGFR and to investigate the association of molecular abnormalities and clinicopathologic factors 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 To analyze the interaction between molecular abnormalities of EGFR and hypermethylation of BRCA1, MGMT, MLH1, or RASSF1A CHAPTER 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 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 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 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: hypomethylation 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 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 MATERIALS AND METHODS 2.1 Materials 139 samples of lung tumor, 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 2.2 Equipments Specialized equipments for molecular biology analysis 2.3 Methods This thesis research was performed at the key gene technology laboratory, Institute of Biotechnology and Molecular Biology laboratory, Pathology – Molecular Biology Center, Vietnam National Cancer Hospital It has been carried out by cross-sectional description method, using techniques including: Total DNA extraction, determination of DNA concentration; total DNA bisulfite treatment; PCR; MS-PCR; electrophoresis; identify EGFR mutations; CHAPTER 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 parameters EGFR mutation Mutation N Age (57.4 ±10.8) ≤57.4 >57.4 Gender Male Female Smoking status Smoker Non-smoker Histological subtypes Acinar Papillary Micropapillary Solid Mixed Tumors Primary Metastasis Stages I & II III & IV p Wildtype 139 49 90 67 72 30 19 37 53 94 45 23 26 71 19 79 60 20 29 59 31 79 22 34 32 7 47 15 27 104 35 36 13 68 22 12 127 43 84 0.023 0.05) (Table 3.6) Table 3.6 EGFR expression and correlation with clinicopathologic parameters EGFR overexpression p Negative N Age (57.4 ±10.8) ≤57.4 >57.4 Gender Male Female Smoking status Smoker Non-smoker Histological subtypes Acinar Papillary Micropapillary Solid Mixed Tumors Primary Metastasis Stages I & II III & IV Negative 139 57 82 67 72 29 28 38 44 94 45 35 22 59 23 79 60 31 26 48 34 79 22 34 27 18 52 13 16 104 35 39 18 64 18 12 127 32 95 0.599 0.191 0.627 0.060 0.992 0.361 0.104 0.229 0.203 0.572 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