Tóm tắt Luận án Study on mutation, protein expression of egfr and methylation of related genes in lung adenocarcinoma patients

n lung cancer 1.6.1. Methodological analysis of mutation in lung cancer 5 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. 6 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. 7 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 <0.001 Male 94 23 71 Female 45 26 19 Smoking status 0.005 Smoker 79 20 59 Non-smoker 60 29 31 Histological subtypes Acinar 79 32 47 0.155 Papillary 22 7 15 0.811 Micropapillary 3 2 1 0.284 Solid 34 7 27 0.042 Mixed 1 1 0 0.353 Tumors 0.787 Primary 104 36 68 Metastasis 35 13 22 Stages 0.263 I & II 12 6 6 III & IV 127 43 84 8 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 9 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 10 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 characteristics Methyl RASSF1A p M U N 139 41 98 Age (57.4 ±10.8) ≤57.4 67 20 47 0.930 >57.4 72 21 51 Gender Male 94 29 65 0.613 Female 45 12 33 Smoking status Smoker 79 29 50 0.032 Non-smoker 60 12 48 Histological subtypes Acinar 79 22 57 0.625 Papillary 22 7 15 0.795 Micropapillary 3 1 2 0.883 Solid 34 11 23 0.674 Mixed 1 0 1 0.521 Tumors Primary 104 26 78 0.045 Metastasis 35 15 20 Stages I & II 12 4 8 0.760 III & IV 127 37 90 3.4. Correlation between mutation and expression of EGFR with methylation of related genes in lung adenocarcinoma 11 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 methylation of EGFR EGFR methylaion EGFR mutaion M U p + - p 33 106 49 90 EGFR expression - 21 36 0.002 20 37 0.973 + 12 70 29 53 EGFR mutation + 15 34 0.160 - 18 72 3.4.2. Correlation between mutation and expression of EGFR with methylation of BRCA1, MGMT, MLH1 and RASSF1A The distribution of EGFR mutations and BRCA1, MGMT, or RASSF1A methylation were significantly exclusive in lung adenocarcinomas. Furthermore, EGFR mutation inversely correlated 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 12 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 13 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 14 (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 15 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. 16 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 17 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 18 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. 19 Methylation of RASSF1A has been associated to smoking habit, and the correlation between RASSF1A methylation and smoking status of patients has also been published in several studies, such as Yanagawa N. et al. (2011) and Lee S.M. et al. (2011). Therefore, smoking can be the cause of methylated RASSF1A in patients with lung cancer. Besides the EGFR mutation, methylation of RASSF1A may be an important mechanism in the carcinogenesis and development of lung cancer. Previously, RASSF1A has also been showed to inhibit the invasion and metastasis of lung cancer cell lines by prohibiting YAP (Yes-associated protein) protein. In our study, the level of RASSF1A methylation in metastatic tumors was higher than in primary tumors. The results proved that methylation of RASSF1A is not only involved in tumorigenesis but also plays an important role in tumor metastasis in lung cancer. Therefore, the identification of RASSF1A methylation status is becoming increasingly necessary for patients, contributing to the diagnosis as well as prognosis. 4.3. Correlation of EGFR alterations with aberrant promoter methylations of BRCA1, MGMT, MLH1, and RASSF1A in lung adenocarcinoma 4.3.1. Correlation of EGFR mutation with methylation of tumor suppressor genes Gene mutations and DNA methylation are the two main pathways for cancer generation and development. The interaction of these two pathways has been found in many types of cancer. In this study, we analyzed the interaction between EGFR mutation and methylation of four tumor suppressor genes typical of lung adenocarcinoma. Results showed that EGFR mutation and methylation of tumor suppressor genes tended to occur in opposite directions and 20 mutually exclusive (Table 3.1). The inversed correlation between EGFR mutation and RASSF1A methylation has been mentioned in a number of previous studies. Yanagawa N. et al. (2011) and Hoque M.O. et al. (2010) showed an inversed association simultaneously between RASSF1A methylation with smoking status and EGFR mutation. This study has confirmed the mutual exclusion of these two phenomena. At the same time, with the above analysis, EGFR mutation occurs in non-smokers, whereas methylation of RASSF1A is common in smokers. Therefore, it can be hypothesized that in non-smokers, adenocarcinoma lung cancer may be established by EGFR mutation. In contrast, lung adenocarcinoma in smokers are formed via the methylation of RASSF1A. In addition, in the study, we also showed the inversed correlation between EGFR mutation and methylation BRCA1 and MGMT (Table 3.15). The study of methylation of BRCA1 and MGMT has received limited attention in lung cancer. However, our results showed that BRCA1 methylation and MGMT methylation were common in lung cancer, and occured primarily in patients without EGFR mutations. RASSF1A methylation not only is considered one of the pathways for the formation of lung adenocarcinoma, the methylated BRCA1 and MGMT can also be an important step in tumor formation and development in patients without EGFR mutations. This theory was further strengthened by the results of analyzing the correlation of methyl and the four genes BRCA1, MGMT, MLH1 and RASSF1A with EGFR mutations (Table 3.15). The inversed correlation of EGFR mutation with random methylation of an individual gene, simultaneous 2, 3 or 4 genes found a clearer observation of genetic and epigenetic changes in lung adenocarcinoma. Our results have also 21 showed that gene mutation and DNA methylation of tumor suppressor genes are two pathways leading to the formation and development of lung cancer. Therefore, it can be considered that DNA methylation needs to be researched and tested as a target for cancer treatment. 4.3.2. Correlation of EGFR expression with methylation of tumor suppressor genes In this study, we found that EGFR expression increased in patients without methylated BRCA1. Although the mechanism of correlation between BRCA1 methylation and EGFR protein expression has not been clarified, it can be seen that methylaed BRCA1 is involved in inhibiting EGFR expression. However, there were a few studies on lung cancer have been conducted to clarify the mechanism of