Summary of Biology doctoral thesis: Study on mutations, expression of EGFR and methylation of related genes in lung adenocarcinoma patients

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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.

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Nội dung Text: Summary of Biology doctoral thesis: Study on mutations, expression of EGFR and methylation of related genes in lung adenocarcinoma patients

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 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. 1
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. 2
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 3
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 4
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. 5
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 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. 6
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 p Mutation Wildtype N 139 49 90 Age (57.4 ±10.8) 0.023 ≤57.4 67 30 37 >57.4 72 19 53 Gender
3.2.3. EGFR expression and correlation with patient characteristics EGFR protein expression was assessed by 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 clinicopathologic parameters EGFR overexpression p Negative Negative N 139 57 82 Age (57.4 ±10.8) 0.599 ≤57.4 67 29 38 >57.4 72 28 44 Gender 0.191 Male 94 35 59 Female 45 22 23 Smoking status 0.627 Smoker 79 31 48 Non-smoker 60 26 34 Histological subtypes Acinar 79 27 52 0.060 Papillary 22 9 13 0.992 Micropapillary 3 2 1 0.361 Solid 34 18 16 0.104 Mixed 1 1 0 0.229 Tumors 0.203 Primary 104 39 64 Metastasis 35 18 18 Stages 0.572 I & II 12 4 8 III & IV 127 32 95 8
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
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
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
that RASSF1A methylation was correlated with BRCA1 and MLH1 (Table 17). Table 3.15. Correlation of EGFR mutation and BRCA1, MGMT, MLH1, and RASSF1A methylation EGFR Mutation N Mutation Wildtype p Methylation 139 49 90 BRCA1 M 41 9 32 0.034 U 98 40 58 MGMT M 46 11 35 0.049 U 93 38 55 MLH1 M 28 8 20 0.408 U 111 41 70 RASSF1A M 41 7 34 0.004 U 98 42 56 1 in 4 M 90 26 64 0.041 U 49 23 26 2 in 4 M 41 6 35 0.001 U 98 43 55 3 in 4 M 17 1 16 0.004 U 122 48 74 4 in 4 M 5 0 5 0.106 U 134 49 85 CHAPTER 4. DISCUSSION 4.1. EGFR molecular characteristics in patients with lung adenocarcinoma 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 12
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 EGFR MLH1 RASSF1A MGMT M U p M U p M U p M U p BRCA1 M 11 30 0.580 10 31 0.419 21 20 0.001 15 26 0.571 U 22 76 18 80 20 78 31 67 MGMT M 13 33 0.378 13 33 0.093 17 29 0.175 U 20 73 15 78 24 69 RASSF1 M 10 31 0.907 17 24 0.001 U 23 75 11 87 MLH1 M 10 18 0.096 U 23 88 4.1.2. Overexpression of EGFR protein Evaluation of EGFR expression shows that 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 13
(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 14
mutation with methylation and EGFR protein expression. However, there was a significant association between EGFR promoter methylation and EGFR protein expression (Table 3.12). Cappuzzo F. et al. (2005) did not find the correlation between EGFR expression and EGFR mutation; In contrast, Liang Z. (2010) found a high level of EGFR expression in patients with EGFR mutations. The correlation of mutation, methylation and expression EGFR may be different between the research groups due to differences in ethicity, age, gender and stage of disease. Protein expression levels can be used in responding evaluation as well as prognosis in cancer treatment. Li et al. (2013) showed an increase in EGFR expression and programmed death in methylated EGFR cell lines cultured in media added 5-aza-CdR and TKIs. Therefore, regulation EGFR expression levels by methylated DNA which combines with TKIs targeted therapy could be a new direction not only in research but also in the treatment of lung cancer. 4.2. Methylation of tumor suppressor genes BRCA1, MGMT, MLH1 and RASSF1A in patients with lung adenocarcinoma 4.2.1. Aberrant promoter methylations of BRCA1 Methylated BRCA1 was commonly studied in breast cancer but had little attention in lung cancer. The hypermethylation of BRCA1 can be considered a tumorigenesis pathway in addition to the EGFR or KRAS mutation. The rate of BRCA1 methylation in various studies ranges from 4 - 54%. The frequency of methylated BRCA1 in this study was 29.5% which was higher than in American patients (4%) and lower than in Chinese (30 - 54%). This results could be explained by the differences in ethicity and geography of patient populations. In Asian populations, BRCA1 methylation tends to occur more commonly than Western patients. 15
In this study, there was no correlation between methyl BRCA1 and patient characteristics. In previous studies, they were not although fully studying the features, but most of them showed no correlation between clinicopathologic variables and methylation BRCA1. Gao W. et al. (2016) indicated that BRCA1 methylation tended to increase in stage II and III than stage I; in patients with lymph node metastasis compared with patients without lymph node metastasis. From these results can be assumed that methylated BRCA1 occurs in parallel with the progression of the disease, the more the disease progresses the greater the level of methylation. In this study, we also found an increase in the methylation status of BRCA1 with disease progression but it is not significant. Therefore, methylation of BRCA1 can be considered as one of the causes leading to the tumorigenesis and development of lung adenocarcinoma and needs to be studied in detail. 4.2.2. Aberrant promoter methylations of MGMT MGM methylation has been observed in many cancers including lung cancer. The percentage of MGMT methylation was in various studies from 8 to 50%. The frequency of methylated MGMT in this study was 33.1%; It was higher than the reports of Feng Q. et al. (2008) (8%), Kim et al. (2005) (17%); and similar to other researches which were performed on Chinese patients (30-50%). MGMT methylation inhibits protein expression and reduces the MGMT DNA repair function leading to increase the mutation establishment. The results of correlation analysis showed that MGMT methylation is not associated with the patient characteristics in the study excepted metastasis status. Reports on Korean and Chinese patients have showed similar results. In this study, we found an increase in the MGMT methylation in metastatic tumors. Although the mechanism for 16
promoting methylated MGMT in metastatic cells in lung cancer has not been elucidated, it can be seen that MGMT methylation may contribute to accelerating tumor metastases and disease progression. MGMT methylation could be considered as one of the standard markers supporting the prognosis and treatment of lung cancer. 4.2.3. Aberrant promoter methylations of MLH1 Study on MLH1 methylation is popular in lung cancer, but the rate of methylation varies widely among the studies. The previously published MLH1methylation frequency ranges from 0 to 58%. The ratio of methylataed MLH1 in our research was 20.1% which is higher than a report of Tang M. et al. (2006) (0%); and lower than publishes in Asian patients (30-50%). In addition, Seng T.J. et al. (2008) compared survival rates of methylated and unmethylated MLH1 patients, and suggested that patients with the MLH1 methylation had a poor prognosis and lower survival time than the unmethylated group. MLH1 methylation could be one of the standard markers in the prognostic assessment for lung cancer patients along with other markers. MLH1 methylation studies on different patient groups have been performed and showed no correlation between MLH1 methylation with patient features such as age, gender, smoking status. The reports often did not go into the different subtypes of adenocarcinoma, but only focus on patients with adenocarcinoma. However, the status of methylated MLH1 at different stages of the disease showed inconsistencies between the studies. In the publication of Seng T.J. et al. (2008), the author found that the rate of methylation MLH1 in patients with stage II was higher than in stage I (40/86 compared to 46/153). In contrast, Wang Y.C. et al. (2003) indicated that the 17
difference in MLH1 methylation level in the group of patients with advanced stage and early stage patients (stage III and IV compared with stages I and II) was not statistically significant. In this study, we also did not observe differences in the level of methylation MLH1 at different stages of the disease (patients in the study were predominantly in late stages) as well as at the tumor status (primary or metastatic tumors). With the function of DNA mismatches repair in replication process, it is possible to hypothesize that the MLH1 methylation occurs mainly during the invasive stage of the tumor when cancer progress from stage I to stage II. In this process, the DNA mismatch repair function of the cell is impaired and the secondary mutations are generated before entering the metastatic stage. 4.2.4. Aberrant promoter methylations of RASSF1A RASSF1A is involved in regulating many important processes in the cell including the cell cycle as well as the programmed death process. Methylation of RASSF1A is crucial to inhibiting expression of this protein. Therefore, RASSF1A methylation has become a standard marker in the diagnosis and treatment of lung cancer. The rate of methylated RASSF1A in the previous studies varies from 20 to 80%. The frequency of RASSF1A methylation in our study was 29.5% (Table 2.17). As reported by de Fraipont F. et al. (2012) on overall survival and progression-free survival of two groups of patients after chemotherapy. The results showed that the methylated RASSF1A group had shorter survival time than the unmethylated group (progression- free survival 16.7 versus 61.2 months and overall survival 32.9 compared to 84 months). It can be seen that determination of RASSF1A methylation status should be considered as one of the prognostic factors for lung cancer patients. 18