MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY LE HOAN CLINICAL, SUBCLINICAL FEATURES AND RESISTANCE TO TYROSINE KINASE INHIBITOR OF LUNG CANCER PATIENTS WITH EGFR MUTATIONS SPECIALTY: PULMONARY MEDICINE CODE: 62.72.01.44 SUMMARY OF PHD THESIS HA NOI - 2020 THE RESEARCH WAS COMPLETED IN HANOI MEDICAL UNIVERSITY Supervisor: Prof NGO-QUY Chau, MD PhD Reviewer 1: A Prof NGUYEN Dinh Tien, MD PhD Reviewer 2: A Prof NGUYEN Tuyet Mai, MD PhD Reviewer 3: A Prof CHU Thi Hanh, MD PhD The thesis is defended in front of the Thesis Committee of the University at Hanoi Medical University The thesis can be accessed at: - Vietnam National Library - Hanoi Medical University Library INTRODUCTION Rationale Lung cancer is recently the leading cause of cancer related mortality in the world Until now, a number of gene mutations have been identified to be related to the pathogenesis of lung cancer with the most common ones are mutations of EGFR gene The targeted therapy with tyrosine kinase inhibitors (TKIs) is proved to be an effective therapy for non small cell lung cancer with EGFR mutation However, researches showed that after 12 to 24 months with EGFR-TKIs therapy, most of patients developed the drug-resistance Several mechanisms underlying drug-resistance to EGFR-TKIs have been identified such as the development of new mutations of EGFR gene, the amplification or increasing the expression of other genes; as well as the transformation of cancer cells Among those, the most important mechanisms are induced T790M mutation and MET amplification, which account for over 70% of EGFR-TKIs resistant cases In Viet Nam, EGFR mutations targeted therapies have been used recently and drug-resistance has been found in clinical practice Therefore, it is necessary to study the EGFR-TKIs resistance in lung cancer patients in order to identify the mechanism of the resistance and to point out the strategy for the next period of those patients Objectives To describe the clinical and subclinical features of lung cancer patients with EGFR mutations before TKIs therapy and in the relapsed period To identify the T790M mutation and MET amplification in patients with lung cancer and EGFR-TKIs resistance and the relationship with clinical and subclinical features Scientific and practical meanings Identification of the clinical and subclinical features of lung cancer patients with EGFR mutations help us to generalize specific features of this patients group, for individualizing in treatment Knowing the features of patients at the time of they develop EGFR-TKIs resistance can support for the recommendations of clinical practice to find out the resistance early Studying the mechanism underlying EGFR-TKIs resistance in lung cancer patients with EGFR mutations is the scientific evidence to choose the following therapy for those patients Patients with EGFR-T790M can be treated with the later generations of EGFR-TKIs such as afatinib, dacomitinib, osimertinib,…While patients with MET amplification can be treated with MET inhibitors like crizotinib, tivantinib, Novelty It is the first research in Viet Nam to study of clinical and subclinical features of lung cancer patients with EGFR mutations and the mechanism underlying EGFR-TKIs resistance The results of this research can provide clinical doctors with the view of targeted therapy including treatment outcome, drug resistance, therefore they can have a appropriate approach for patients developing drug resistance Thesis structure The thesis includes 118 pages (without References and Appendix), divided into parts: - Introduction: pages Chapter 1: Literature review, 35 pages Chapter 2: Methodology, 15 pages Chapter 3: Results, 29 pages Chapter 4: Discussions, 31 pages Conclusions: pages Recommendations: page The thesis includes 26 tables, 23 charts, 09 figures It has 148 references in Vietnamese and English Appendixes include Research Forms and the list of 66 lung cancer patients Chapter 1: LITERATURE REVIEW Overview of lung cancer Lung cancer is the most common and has the highest rate of mortality in all kinds of cancer According to the GLOBOCAN 2018 database, it was estimated 2.09 millions new lung cancer cases and 1.76 millions lung cancer deaths worldwide In the United States, lung cancer is the leading cause of cancer death and the second-ranked cancer incidence in both genders There was an estimated 228150 new lung cancer cases and 142670 lung cancer deaths in the US in 2019, account for 23% of all cancer deaths According to researches, lung cancer is more common in males In 2018, it was estimated that there were 1377500 lung cancer cases in males, accounting for 66% all lung cancer cases, with the gender rate of 1.94/1 In developing countries, the gender rate may be higher, while in developed countries, the prevalence of lung cancer in female is rising In the US in 2019, those numbers was 116440 cases and 111710 cases respectively In Viet Nam, according to GLOBOCAN 2018 database, there was an estimated 23667 new lung cancer cases, with the 2nd ranked in all cancers, after liver cancer Smoking is considered the major risk factor of lung cancer, that approximately 80- 85% of lung cancer patients in the world smoked Other risk factors of lung cancer include: air pollution, ionized radiation, occupational exposure, virus, diet, history of respiratory diseases Researches of molecular level revealed that the development of lung cancer had several periods with the interactions of some factors, the sensitization of genes, cumulative process of gene mutations of oncogenes and tumor suppressor genes Genes regulations is originally smooth and tight, when it is impaired it can lead to the abnormal enhancement or inhibition of functional genes With whole genome sequencing of a clone of lung cancer cells, it is partly understood about intracellular mediators signaling pathway related to the activation of oncogenes and deactivation of suppressor genes The activation of oncogenes through the signaling pathway of EGFR and other tyrosine kinase receptors such as MET, Her-2, c-KIT, IGF-1R in addition with the following activation of RAS/RAF/MEK/MAPK, PI3K/AKT and JAK/STAT can lead to the nonstop proliferation, differentiation, invasion, metastasis and resistance to apoptosis In normal cells, the activation of EGFR is necessary for several crucial functions of cells such as the proliferation and differentiation But the extreme activation due to gene mutations can lead to the abnormal proliferation as well as the transformation of cells In addition, the impaired activity of EGFR due to gene mutation can lead to maglinant disorders The mutation in exon 18- 21 makes EGFR to be in activating status independent from mediators Features of EGFR mutations in NSCLC patients include: high rate in nonsmokers, more common in adenocarcinoma type compared to other types of NSCLC, females are more common than males, higher rate in East Asian patients compared to other races EGFR-TKIs therapy for lung cancer EGFR mutations had a quite high prevalence in NSCLC patients, especially the adenocarcinoma and in non-smokers Researches in Europe and North America showed the prevalence of mutations is approximately 17%, while it is up to 78.8% in East Asia With whole genome sequencing, researchers can identify a number of mutations of EGFR influencing the response to TKIs The common mutation of EGFR being sensitive to TKIs is LREA (exon 19) and L858R (exon 21) Some EGFR-TKIs being used recently are erlotinib, gefitinib, afatinib Clinical trials showed that EGFR-TKIs help prolong non-disease survival time better than standard chemotherapy phase III RCTs compared EGFR-TKIs with standard chemotherapy as the first step treatment for lung cancer patients with sensitive EGFR mutations, and all of those RCTs revealed the rate of response and disease-free survival in the group treated with EGFR-TKIs, but no difference of survival An important question is that which therapy should be chose if patients relapse when using TKIs It is still lack of strong evidence Various approach can be done, such as examining if the tumor transforms into small cell type (by another biopsy) or considering chemotherapy with platin if patients can tolerate it Thereafter, TKIs can be used again if the chemotherapy fails Other choices include continuing using TKIs if the tumor grows gradually, switching to another TKI, optical therapy or cell toxic therapy To understand the resistant mechanism, another biopsy should be done and clinical trial should be considered EGFR-TKIs resistance in lung cancer patients Clinical trials showed good results of EGFR-TKIs therapy for lung cancer patients with EGFR mutations But, after 12 to 24 months of treatment, the cancer relapsed in most of the patients who had good results before The targeted therapy was no longer effective becaused of EGFR-TKIs resistance Until now, several mechanisms underlying EGFR-TKIs resistance had been studied (Figure 1.1) The most common cause of EGFR-TKIs resistance is the development of mutations in EGFR itself It is the mutation of EGFR-T790M (the Threonin at acid amin position 790 was substituted by Methionine) at the exon 20 This mutation accounts for 40-55% of the cases Recently, there are mechanism suggested by researchers to explain the relationship between T790M mutation and the resistance of tumor cells At the aspect of structure, the substitution from Threonine to Methionin changed the reaction position of kinase, inhibited the binding of erlotinib and gefitinib At the aspect of interactions between biological particles, the T790M mutation restored the affinity of kinase with ATP while reduced the affinity of erlotinib and gefitinib One less common mechanism of EGFR-TKIs resistance is MET amplication MET encode for MET protein which plays a role of surface receptor to receive signal from HGF By the activity of tyrosine kinase, MET can lead to the phosphorylization of ERBB3, maintaining the activation of PI3K/Akt signaling pathway, which can activate the process of invasion, metastasis and vascular proliferation of the tumor In this case, in stead of relying on the EGFR, the signal for proliferation of tumor cells relies on MET and MET downstream signaling pathway As a result, EGFR-TKIs becomes deactivated Alongsides, some other mechanisms can be identified includes ERBB2 amplication, transformation from NSCLC to small cell lung cancer or KRAS, BRAF mutations Figure 1.1: Mechanism underlying EGFR-TKIs resistance Chapter 2: METHODOLOGY 2.1 Subjectives We recruited 66 patients with a diagnose of NSCLC with TKIs sensitive EGFR mutations, treated with TKIs (erlotinib gefitinib), having good response or stable in at least months and relapsed after that 2.1.1 Inclusion criteria - Patients diagnosed with NSCLC by histopathological evidence according to WHO - Patients were treated in Hospital K, Center of Nuclear Medicine and Oncology in Bach Mai Hospital and Respiratory Department in Hospital 108 from June 2014 to June 2019 - Patients met the criteria of American Society of Clinical Oncology to access the TKIs resistance of NSCLC: + Diagnosed with NSCLC with TKIs sensitive EGFR mutations + Treated with EGFR-TKIs (erlotinib or gefitinib) + Responsed or stable in at least months + Relapsed or developed new lesions despite of treatment with erlotinib or gefitinib + No systemic chemotherapy after stopping erlotinib or gefitinib and before a new episode of treatment 2.1.2 Exclusion criteria - Lung cancer patients treated with EGFR-TKIs without confirmed TKIs sensitive EGFR mutations - Patients without response or stable state after at least months of EGFRTKIs treatment - Patients refused to participate in the research 2.2 Methods 2.2.1 Study designs - Prospective descriptive study with case series 2.2.2 Study process 2.2.2.1 Describing clinical, subclinical features of NSCLC with EGFR mutations before TKIs therapy and after relapse Clinical, subclinical features were documented in a research medical report, including:  Basic information: - Age, Gender, Occupation, Address  Clinical features : - Respiratory symptoms: cough, sputum, hemoptysis, chest pain, dyspnea Systemic symptoms : fatigue, weight loss, lymph nodes Access ECOG according to WHO Invasive or local invasive symptoms: hoarse, choking, superior vena cava compression syndrome, Pancoast Tobias syndrome Metastatic symptoms: headache, abdominal pain, muscoskeletal pain, pleural effusion, pericardial effusion, etc Paraneoplastic syndromes Family history of lung cancer Patients„ history: smoking, comorbidity diseases  Subclinical features: - Location, size, number, morphological and invasive characteristics of tumors on chest Xrays and contrast enhanced chest CT Histopathology according to WHO criteria in 2015 for lung and pleural tumor TNM staging: According to TNM classification 8th edition 2018 - EGFR mutations analysis which EGFR mutations were TKIs sensitive 2.2.2.2 Identification of TKIs resistance of lung cancer patients with EGFR mutations  Accessing the duration to develop EGFR_TKIs resistance  Analyzing clinical factors that could affect the response to EGFR-TKIs: age, gender, smoking status, histopathological feature, TKIs sensitive gene mutations  Obtaining sample at the time developing drug resistance of patients who were suspected to have EGFR-TKIs resistance according to ASCO 2009, including: another biopsy under CT guided or bronchoscopy; lymph nodes biopsy; biopsy of metastatic lesion such as liver, bone, vetebra; cell-block of metastatic fluid  Gene analyzing to identify abnormalities related to EGFR-TKIs resistance was done at Gene and Protein Research Center at Hanoi Medical University, including: - Identification resistant mutations of EGFR using Scorpions ARMS realtime PCR - Identification of MET amplication using FISH  Analyzing clinical factors that could affect the development of EGFRT790M mutation and MET amplication leading to EGFR-TKIs resistance: age, gender, smoking status, histopathological feature 2.3 Data analysis - Data was managed, analyzed with the statistic software SPSS 22.0 - Using statistic algorithm including descriptive test to calculate the rates, average, comparison, analyzing - Difference was statistical significant with p 60 Yes No Yes No Yes n 22 16 17 21 21 17 20 18 38 % 33,3 24,2 25,7 31,9 31,9 25,7 30,3 27,3 57,6 No 0 No EGFR-T790M n % 12 18,3 16 24,2 11 16,7 17 25,7 13,6 19 28,8 18 27,3 10 15,1 27 40,9 1,5 p 0,169 0,425 0,053 0,244 0,424 Comments: The research did not find out any relationship between factors such as age, gender, smoking status, comorbidities, pathological feature and the development of EGFR-T790M with p > 0.05 Table 3.8: MET amplication and the relationship with clinical and subclinical features (n=66) MET amplication Features Gender Age Smoking Cormorbidities Adenocarcinoma Male Female ≤ 60 > 60 Yes No Yes No Yes No n 3 % 4,5 1,5 3,0 4,5 3,0 1,5 4,5 No MET amplication n % 34 51,6 29 43,9 27 40,9 36 54,6 30 45,5 33 50,0 36 54,6 27 40,9 62 94,0 1,5 p 0,108 0,615 0,156 0,615 0,955 Comments: The research did not find out any relationship between factors such as age, gender, smoking status, comorbidities, pathological feature and the development of EGFR-T790M with p > 0.05 Chart 3.3: Relationship between EGFR-T790M mutation and MET amplication with PFS (n=66) Comments: - Average PFS was not different between EGFR-T790M group (14.5 months; 95%CI 13.3-15.8 months) and non-EGFR-T790M group (14.1 months; 95%CI 12.7-15.6 months) (p=0,642) - Average PFS was not significantly different between MET amplication group (18.3 months; 95%CI 9.8-26.8 months) and non-MET amplication group (14.3 months; 95%CI 13.4-15.2 months) (p=00.80) Chapter 4: DISCUSSION Through a study of 66 lung cancer patients with EGFR mutations treated with EGFR-TKIs then having drug resistance, we have some discussions as follows: 4.1 Clinical and subclinical features of patients with lung cancer with EGFR mutations before TKIs therapy and in the relapsed period 4.1.1 General characteristics The average age of the study group was 60.6 ± 10.7 years, the youngest was 26 years old, the oldest was 80 years old (Table 3.1) This result is similar to some recent studies in Vietnam and around the world A study of 100 lung cancer patients with EGFR mutations at Vietnam National Cancer Hospital in 2019 showed that the average age of patients was 56.27 ± 7.9 years, ranging from 39 to 80 years old A study by Xu Q et al (2019) in China on 206 lung cancer patients with EGFR mutations recorded an average age of 58 years, ranging from 28 to 83 years Regarding the gender of the research group, there were 34 male patients (51.5 higher than that of female However, studies in the world with a larger sample size showed that women were more common in the lung cancer group with EGFR mutations The study of Kim HR et al on 417 lung cancer patients in Korea noted that women accounted for 61.9% Wang JF et al conducted a meta-analysis of 478 lung cancer patients with EGFR mutations from 10 previous studies, the results also showed that there were 310 female patients, accounting for 64.9% Regarding smoking status in lung cancer patients with EGFR mutation, up to 54.5% did not smoke, only 30.3% were smokers and 15.2% had smoked but had quit (Table 3.1) This result is also consistent with records from studies of lung cancer with EGFR mutation, the disease was more common in the nonsmoking group A study by Xu Q et al (2019) in China on 206 lung cancer patients with EGFR mutation recorded a non-smoking rate of 60.7% 4.1.2 Clinical characteristics Respiratory symptoms are quite diverse but not specific in lung cancer patients Our study noted that chest pain (60.6%) and dry cough (48.5%) were the two most common respiratory symptoms at the time prior to EGFR-TKIs treatment Less common symptoms were dyspnea (30.3) and hemoptysis (12.1%) (Table 3.2) This result is also consistent with the records of other authors in Vietnam and around the world Nguyen Thanh Hoa et al studied over 100 lung cancer patients with EGFR mutations at Vietnam National Cancer Hospital in 2019: chest pain (73%) and dry cough (64%) were the two most common symptoms Regarding systemic symptoms, we noted that weight loss was the most common symptom (36.3%), followed by fatigue (27.3%) and peripheral lymph nodes (24.2%) (Table 3.2) This result is also consistent with previous studies on lung cancer in Vietnam as well as in the world The study of Kim HC et al on 489 lung cancer patients with EGFR mutations in Korea recorded 6.7% of patients showing weight loss The group of symptoms related to local diffusion, metastatic cancer, and paraneoplastic syndromes in lung cancer is also quite various Our study noted that at the time prior to EGFR-TKIs, headaches (24.2%) and spinal pain (21.2%) associated with cancer metastases were common symptoms Less common local diffusion symptoms include hoarseness (6.1%); choking, swallowing problems (4.5%) and Pancoast Tobias syndrome (3.0%) (Table 3.2) The patients in our study were all diagnosed with late-stage lung cancer, therefore it is appropriate to have symptoms of cancer metastases such as headache, spinal and bone pain By the time the disease progressed, dry cough (57.6%) and chest pain (48.5%) were the two most common respiratory symptoms However, dyspnea also appeared more frequently in these stages with 45.5% of patients (compared to the time of initial diagnosis of 30.3%) Kim HR et al studied over 360 patients with gefitinib-resistant lung cancer in 2014, which noted that at the time of recurrence, there were 34.4% of patients with dry cough, 27.2% of patients with dyspnea and 14.2% of patients with chest pain Signs of cancer metastases also often appear at the time of disease progression, which may be symptoms of previously recurrent cancer metastases or symptoms of newly emerging cancer metastases In a study of 66 lung cancer patients with EGFR mutations treated with EGFR-TKIs, at the time of disease progression we recorded 15.2% of patients with headache and 21.3% of patients with bone or spinal pain (Table 3.2) This result is also consistent with the records of some studies in the world Kim HR et al in South Korea studied 360 patients with gefitinib-resistant lung cancer in 2014, it was noted that at the time of recurrence, there were 10.3% of patients with headache and 15% of patients with bone pain Thus, in clinical practice, we also need to pay attention to the changing symptoms of patients during treatment Early recognition of these changes will suggest an EGFR-TKIs non-response, thereby identifying the cause of drug resistance and finding appropriate treatment options 4.1.3 Subclinical characteristics  Imaging characteristics Research on 66 lung cancer patients with EGFR mutation, at the time before EGFR-TKIs treatment, up to 87.9% of patients with lung lesions size > cm, of which up to 51.5% of patients have tumor size > cm; No cases of tumor size < cm were recorded This is also appropriate because our study subjects are late-stage lung cancer patients Suh YJ et al studied 524 lung cancer patients with EGFR mutations in Seoul, South Korea in 2018 showed up to 49.4% of patients with tumor size > cm Regarding lesion location, our study noted that lesions were most common in the right upper lobe (27.3%) and the left upper lobe (21.2%) This result was also consistent with the record of Rizzo S et al when studying 60 patients with lung cancer with EGFR mutations in Milan, Italy in 2016: the most common lesion was in the right upper lobe (37%) In general, lung cancer lesions may be encountered in any lung lobe, with no statistically significant difference in location About evaluating cancer metastatic lesions, at the time before EGFRTKIs treatment, we recorded the most metastatic lesions were brain metastases (24.2%), followed by pleural metastases (21.2%) and bone metastases (21.2%) This result is also consistent with studies on lung cancer in general as well as lung cancer with EGFR mutations Research by Nguyen Thanh Hoa et al on 100 lung cancer patients with EGFR mutations at Hospital K in 2019 recorded 55% of bone metastases, 20% of pleural metastases and 19% of brain metastases In term of imaging at the time of disease relapse, 63.6% of patients increased primary tumor size; 31.8% of patients had new lung lesion; 21.2% of patients had pleural effusion Distant metastases were most commonly reported as brain metastases (13.6%), followed by bone metastases (12.1%), liver metastases (7.5%) and adrenal metastases (7.5%) The study of Kim HR et al on 360 patients with lung cancer relapse after first-step treatment showed that 58.4% of patients with primary tumor size increased; 26.3% of patients had new lung lesion; 14.2% of patients had pleural effusion; 14.8% of patients had central nervous system metastases; 13.1% of patients had bone metastases; 4.2% of patients had liver metastases  Pathological characteristics Our study noted that up to 98.5% of patients were identified as adenocarcinoma, only case was identified as adenosquamous carcinoma This result is also consistent with the medical literature in the world when studying lung cancer with EGFR mutations, the most common histopathological lesion is still adenocarcinoma The study of Kim HR et al on 110 non-smoking lung cancer patients with EGFR mutations in Seoul, South Korea recorded 95% of patients with adenocarcinoma; squamous cell carcinoma was 1.8% and large cell carcinoma was 0.9% A study of Hata A et al On 78 lung cancer patients with EGFR mutations in Kobe, Japan recorded 92.2% of cases were adenocarcinoma  EGFR gene mutation analysis Our study noted that the most common mutations were LREA at exon 19 (54.5%) and L585R at exon 21 (43.9%) We encountered one case of EGFR mutation at exon 18 G719S, which is also a sensitive mutation for EGFR-TKIs All of these patients did not detect an EGFR-T790M mutation that caused EGFR-TKIs resistance at the time prior to treatment Our findings are also consistent with the findings of several recent studies: Nguyen Thanh Hoa et al recorded the two most common mutations: LREA at exon 19 (54%) and L585R at exon 21 ( 36%) The study of Hata A et al on 78 lung cancer patients with EGFR mutation in Kobe, Japan in 2013 recorded the LREA mutation at exon 19 in 53.8% of patients and mutation L585R at exon 21 in 42.3% of patients 4.2 Identification T790M mutation of EGFR gene, MET amplication in lung cancer patients with EGFR-TKIs resistance and the relationship with clinical and subclinical features 4.2.1 The effect of EGFR-TKIs on lung cancer patients with EGFR mutations The common EGFR-TKIs currently used clinically are erlotinib, gefitinib, afatinib, osimertinib and dacomitinib In Vietnam, two first-generation EGFRTKIs, including erlotinib and gefitinib, are indicated for the first-step treatment for most lung cancer patients with EGFR mutations Our study recorded 53% of patients treated with erlotinib and 47% of patients treated with gefitinib In a study of 66 lung cancer patients with EGFR mutations treated with EGFR-TKIs, we recorded a lot of adverse effects, of which the most common were skin rash (22.7%); nausea, vomiting (21.2%); fatigue, anorexia (18.2%); elevated liver enzymes (16.7%) and stool disorders (15.1%); other adverse effects such as nail inflammation, hair loss, and muscle aches are less common This result is similar to that recorded in recent studies in Vietnam as well as in the world Nguyen Thanh Hoa et al studied over 100 lung cancer patients with EGFR mutations at Vietnam National Cancer Hospital noted that 59% of patients had skin rash; 31% of patients had nail inflammation; 21% of patients had digestive disorders; 6% of patients had elevated liver enzymes Although many of the adverse effects of EGFR-TKIs are reported, compared with traditional chemotherapy, EGFR-TKIs are considered safer In respect of assessing the effectiveness of treatment with EGFR-TKIs after months, we noted that 87.9% of patients responded partially and 12.1% of patients achieved stable disease, no cases completely responded after months (Table 3.3) Hata A et al studied 78 lung cancer patients with EGFR mutations treated by EGFR-TKIs in Japan in 2013 recorded up to 70.5% of patients met the disease response and 29.5% of patients achieved stable disease When evaluating the performance status of patients treated with EGFRTKIs after months, we recorded a significant increase in the number of patients with PS 0-1 and a significant reduction in the number of patients with PS 2-4 (Table 3.4) The improvement of performance status after treatment EGFR-TKIs compared with before treatment was statistically significant with p< 0.05 Thus, through analysis of the above results, we can see the effect of EGFR-TKIs on lung cancer patients with EGFR mutations After months of treatment with EGFR-TKIs, all patients were assessed for partial or stable response according to RECIST criteria, improved clinical symptoms, improved performance status and did not have many adverse effects However, after 12-24 months of response to treatment, resistance to EGFR-TKIs appears in most patients Our study noted that the average time from the start of EGFR-TKIs treatment to recurrence was 14.48 ± 3.9 months; the earliest time to develop recurrence is months and latest is 26 months The majority of patients had a response or stabilization period between 13-18 months (42.4%) and 6-12 months (39.3%) The median progression-free survival (PFS) in our study was 14 months (Chart 3.1) This result is similar to the record of some authors in the world The study of Jaiswal R et al on 90 lung cancer patients with EGFR mutations treated with EGFR-TKIs had median PFS of 12.45 months While the study of Oxnard GR et al on 93 lung cancer patients with EGFR mutations in the US treated with EGFR-TKIs had median PFS of 13 months When analyzing the factors affecting PFS of patients treated with EGFRTKIs such as gender, smoking status, number of metastases, EGFR mutation type or EGFR-TKIs type used, we did not record any statistically significant difference (p> 0.05) However, our study noted a difference in mean PFS between the age group ≤ 60 years (15.7 months; 95% CI 14.3-17.1 months) and the age group> 60 years (13.3 months; 95% CI 12.1-14.5 months) was statistically significant (p = 0.028) (Chart 3.2); and between the group without co-morbidity (15.6 months; 95% CI 14.2-17.0 months) and the group with comorbidity (13.4 months; 95% CI 12.3-14.6 months), these differences are statistically significant (p = 0.039) (Chart 3.2) The study by Li W et al on 54 lung cancer patients with EGFR mutations treated with EGFR-TKIs in China reported a difference in mean PFS between men and women; and between the age group > 60 years and ≤ 60 years Meanwhile, a study of Xu Q et al on 206 lung cancer patients with EGFR mutations treated with EGFR-TKIs, recorded four factors including gender, smoking status, number of metastases and EGFR mutation type which affected PFS with statistical significance.In general, there are many factors that affect the PFS of lung cancer patients with EGFR mutations, hence we need to carefully analyze those factors to make a prognosis for each patient 4.2.2 Some causes of EGFR-TKIs resistance To determine the cause of resistance to EGFR-TKIs, we need to have cancer specimens at the time of disease progression One of the most effective measures to identify the cause of resistance is re-biopsy of cancer lesions Depending on the specific case, people may have a re-biopsy of the primary tumor or metastatic biopsy In our study, we collected histopathological specimen or cell block of cancer metastases to look for the cause of resistance Results showed that 42.4% of patients underwent pulmonary biopsy under CT scan instructions; 21.2% of patients had lung biopsy through bronchoscopy; 18.1% of patients were tested for pleural fluid cell block; 12.1% of patients had peripheral lymph node biopsy; 9.1% of patients were tested for pericardial fluid cell block; 3.0% of patients underwent vertebra biopsy and 1.5% of patients had liver biopsy (Table 3.5) Helena A Yu et al studied resistance to EGFR-TKIs in 155 lung cancer patients with EGFR mutations in New York, and recorded 50.6% of patients underwent lung re-biopsy; 8.6% of patients were tested for pleural fluid; 8.0% of patients had a liver biopsy; 5.5% of patients had bone biopsy and 5.5% of patients had lymph node biopsy After diagnostic procedures, we noted that 27.3% of patients had pain at the biopsy site; 12.1% of patients coughed up blood; 3% of patients had mild pneumothorax and 60.1% of patients had absolutely no complication after the procedure (Table 3.6) All cases of post-biopsy complications only need internal medicine management Thus, it can be seen that the biopsy of cancer lesions at the time of recurrence is very necessary and safe Pathological results at the time of EGFR-TKIs resistance recorded: 63.6% of patients were identified as adenocarcinoma; 33.3% of patients were identified as metastatic adenocarcinoma; and especially patients (3.1%) were identified as small cell carcinoma Of these patients, patient was diagnosed by pleural fluid cell block and patient was diagnosed by lung biopsy Both patients were immunohistochemically stained and confirmed a cell transformation from adenocarcinoma to small cell carcinoma Our study did not report any case of cell transformation from carcinoma to mesothelioma This result is also consistent with some records from other studies around the world Research by Chen HJ et al on 29 EGFR-TKIs-resistant lung cancer patients in China recorded 25 cases of re-biopsy with lesions of adenocarcinoma; cases of re-biopsy with lesions were squamous cell carcinoma and cases of re-biopsy with lesions were unspecified carcinoma Kuiper JL et al studied 66 lung cancer patients with EGFR-TKIs resistance in the Netherlands recorded 65 cases (98.5%) of re-biopsy results of remaining adenocarcinoma and case of cell transformation from adenocarcinoma to small cell carcinoma Helena A Yu et al studied 155 lung cancer patients with EGFR-TKIs resistance in New York, USA, reporting that the transformation from adenocarcinoma to small cell carcinoma accounted for 4% Conducting genetic analysis for the cause of EGFR-TKIs resistance, we recorded 54.5% of patients with EGFR-T790M mutations and 4.5% of patients with MET gene amplification This result is also consistent with the records of some other authors in the world Jaiswal R et al studied over 90 patients with EGFR-TKIs resistant lung cancer in India in 2019 recorded an EGFR-T790M mutation rate of 52.2% Helena A Yu et al studied 155 lung cancer patients with EGFR-TKIs resistance in New York, USA in 2013 recorded a high rate of EGFR-T790M mutation of 63% and 5.3% of patients had amplification status of MET gene Thus, it can be seen that EGFR-T790M mutation is the most common cause of EGFR-TKIs resistance secondary to NSCLC patients with EGFR mutations The detection of a patient with a secondary EGFR-T790M mutation that is resistant to first-generation EGFR-TKIs is the basis for further selection of next-generation EGFR-TKIs such as afatinib, dacomitinib, osimertinib, etc for patients to continue EGFR-targeted therapy In addition, amplification of MET gene is considered to be a less common cause of target drug resistance Detecting MET gene amplification in patients with EGFR-TKIs resistance will allow patients to stop EGFR-TKIs but switch to Tyrosine kinase inhibitors targeted to MET such as crizotinib and capmatinib 4.2.3 T790M mutation of EGFR gene, MET gene amplification and its relationship with some clinical and subclinical characteristics Although there is no strong enough evidence to show the relationship between clinical or subclinical characteristics of patients before EGFR-TKIs treatment and the occurrence of EGFR-T790M mutations when the disease relapsed, there are some EGFR-TKIs resistance research addresses this problem In our study, we also analyzed the relationship between gender, age, smoking status, co-morbidity and histopathological lesion before EGFR-TKIs treatment and the occurrence EGFR-T790M mutation (Table 3.7) However, the analysis has not recorded a statistically significant difference (p> 0.05) between these factors and the occurrence of EGFR-T790M mutations Joo WJ et al analyzed over 111 lung cancer patients with EGFR mutations resisting to EGFR-TKIs, the results also did not record statistically significant differences between groups with and without EGFR-T790M mutations regarding characteristics such as gender, age, smoking status, gene mutation type before treatment In this study, we also analyzed the difference in the average of progression-free survival between groups with and without EGFR-T790M mutations (Chart 3.3) The results showed that the average of PFS did not have statistically significant difference between the group with EGFR-T790M mutation (14.5 months; 95% CI 13.3-15.8) and the group without EGFRT790M mutation (14.1 months; 95% CI 12.7-15.6) (p = 0.642) This result is also consistent with the findings of Huang YH et al studying on 205 lung cancer patients with EGFR mutations in Taiwan, there was no difference in mean PFS between groups with and without EGFR-T790M mutations (p = 0.235) Meanwhile, the study Oya Y et al in Japan on 181 lung cancer patients with EGFR mutations treated with EGFR-TKIs recorded a difference in mean PFS between groups with and without EGFR-T790M mutations (p = 0.0041) Thus, the above analysis shows that the appearance of secondary EGFRT790M mutations in lung cancer patients on EGFR-TKIs treatment may be more or less related to some clinical, paraclinical characteristics, and at the same time, may affect treatment response of patients with EGFR-TKIs This can be further clarified in studies with large sample sizes or meta-analysis in the near future Similarly, we also analyzed the relationship between factors such as gender, age, smoking status, co-morbidity, histopathological lesion before EGFR-TKIs treatment and the occurrence of MET gene amplification Results showed that there was no relationship between some factors such as age, gender, smoking status, co-morbidity and histopathology with the occurrence of MET gene amplification causing EGFR-TKIs resistance with p > 0.05 (Table 3.8) This result is also consistent with the record of Baldacci S et al studying 42 lung cancer patients with EGFR-TKIs resistance, there was no statistically significant difference in characteristics such as gender, age, smoking status, histopathological lesion between the group with and without MET gene amplification (p> 0.05) Regarding mean progression-free survival between the group with and without MET gene amplification, we found that the average PFS had a difference between the group with the MET gene amplification (18.3 months; 95 % CI 9.8-26.8) and the group without MET gene amplification (14.3 months; 95% CI 13.4-15.2) but not statistically significant (p = 0.080) (Chart 3.4) Similarly, the study of Lai GGY et al on 200 EGFR-TKIs-resistant lung cancer patients in Singapore also recorded no difference in mean PFS between MET gene amplification group and non-MET gene amplification group with p = 0.076 Thus, the above analysis has not recorded the occurrence of MET gene amplification in lung cancer patients on EGFR-TKIs treatment related to some clinical and paraclinical characteristics, and may affect treatment response of patients with EGFR-TKIs However, we can expect this relevance to be more evident in studies with larger sample sizes or future meta-analysis CONCLUSION Through a study of 66 lung cancer patients with EGFR mutations treated with EGFR-TKIs then having drug resistance, we have some conclusions as follows: Clinical and subclinical features of patients with lung cancer with EGFR mutations before TKIs therapy and in the relapsed period 1.1 General characteristics - Men accounted for 51.5%; women accounted for 48.5%; the ratio of male to female is approximately 1:1 - The average age is 60.6 ± 10.7 years; the youngest is 26 years old, the oldest is 80 years old 1.2 Risk factors - 54.5% of non-smokers, 30.3% of current smokers and 15.2% of former smokers had quit - 24.2% of patients had cardiovascular disease; 18.2% of patients had respiratory disease and 15.2% of patients had metabolic disorders - 4.5% of patients whose biological parents had cancer and 6.1% of patients whose siblings had cancer 1.3 Clinical characteristics - At the time prior to EGFR-TKIs treatment, common clinical symptoms include: chest pain (60.6%), dry cough (48.5%), dyspnea (30.3%), weight loss ( 36.3%), fatigue (27.3%), peripheral lymph nodes (24.2%), headache (24.2%) and spinal pain (21.2%) - At the time of disease progression, common clinical symptoms include: dry cough (57.6%), chest pain (48.5%), dyspnea (45.5%), fatigue (18.2%), weight loss (15.2%), peripheral lymph nodes (12.1%), headache (15.2%) and bone or spinal pain (21.3%) 1.4 Subclinical characteristics 1.4.1 At the time prior to EGFR-TKIs treatment: - Imaging characteristics: 87.9% of patients had lung lesions size > cm, of which 51.5% of patients had tumor size > cm; N2 lymph nodes (36.4%), N3 lymph nodes (30.3%); brain metastases (24.2%), pleural metastases (21.2%) and bone metastases (21.2%) - Pathologic characteristics: adenocarcinoma (98.5%), adenosquamous carcinoma (1.5%) - EGFR mutation type: LREA mutation at exon 19 (54.5%), L858R mutation at exon 21 (43.9%) and G719S mutation at exon 18 (1.6%) 1.4.2 At the time of relapse: - Imaging characteristics: increase in size of primary tumor (63.6%), new lung lesions (31.8%), pleural effusion (21.2%), brain metastases (13.6%), bone metastases (12.1%), liver metastases (7.5%) and adrenal metastases (7.5%) - Pathologic characteristics: 63.6% of patients were identified as adenocarcinoma; 33.3% of patients were identified as metastatic adenocarcinoma Especially, patients (3.1%) were identified as small cell carcinoma Identification frequency of EGFR-T790M mutation, MET amplication in lung cancer patients with EGFR-TKIs resistance and the relationship with clinical and subclinical features 2.1 The effect of EGFR-TKIs on lung cancer patients with EGFR mutations - 53% of patients treated with erlotinib and 47% of patients treated with gefitinib - Adverse effects when treating EGFR-TKIs: skin rash (22.7%); nausea, vomiting (21.2%); fatigue, anorexia (18.2%); elevated liver enzymes (16.7%) and stool disorders (15.1%); other adverse effects such as nail inflammation, hair loss, and muscle aches are less common - 87.9% of patients had partial response and 12.1% of patients achieved stable disease, none of them completely responded after months of treatment with EGFR-TKIs 2.2 EGFR-TKIs resistance status in lung cancer patients with EGFR mutations 2.2.1 Time until relapse - The average time from initial EGFR-TKIs treatment until relapse was 14.48 ± 3.9 months; median PFS is 14 months; the earliest time to develop recurrence is months and latest is 26 months - No statistically significant differences in the effects of factors such as age, gender, smoking status, EGFR gene mutation type, EGFR-TKIs type on average PFS were recorded 2.2.2 The method of collecting cancer specimens at the time of drug resistance - Lung biopsy: 42.4% of patients underwent a lung biopsy under the instructions of CT scans; 21.2% of patients had lung biopsy through bronchoscopy - Metastatic lesion biopsy: 12.1% of patients had peripheral lymph node biopsy; 3.0% of patients had a vertebra biopsy; 1.5% of patients had a liver biopsy - Cell block test: 18.1% of patients were tested for pleural fluid; 9.1% of patients were tested for pericardial fluid - Post-procedure complications: 27.3% of patients have pain at the biopsy site; 12.1% of patients coughed up blood; 3% of patients had small pneumothorax and 60.1% of patients had absolutely no complication after the procedure 2.2.3 Some causes of EGFR-TKIs resistance - 54.5% of patients had EGFR-T790M mutations - 4.5% of patients had MET gene amplification - 3.1% of patients had cell transformation from adenocarcinoma to small cell carcinoma 2.3 T790M mutation status of EGFR gene, MET amplification and its relationship with some clinical and paraclinical characteristics - There has been no relationship between a number of factors such as age, gender, smoking status, co-morbidity and histopathology with the presence of EGFR-T790M mutation causing EGFR-TKIs resistance (p > 0.05) - Mean PFS did not differ statistically between group with and without EGFRT790M mutations (p = 0.642) - There has been no relationship between several factors such as age, gender, smoking status, co-morbidity and histopathology with the occurrence of MET gene amplification causing EGFR-TKIs resistance (p > 0.05) - Mean PFS did not differ statistically between groups with and without MET amplification (p = 0.080) SCIENTIFIC WORKS PUBLISHED IN THE SCOPE OF THE THESIS Le Hoan, Nguyen Minh Ha, Tran Van Khanh, Ngo Quy Chau, Ta Thanh Van, Tran Huy Thinh (2015) “Acquired resistance of lung adenocarcinoma to gefitinib is associated with a second mutation in EGFR kinase domain” Journal of medical research, 96 (4): 137-143 Le Hoan, Nguyen Minh Ha, Tran Van Khanh, Vuong Ngoc Duong, Ngo Quy Chau, Mai Trong Khoa, Ta Thanh Van, Tran Huy Thinh (2015) “Acquired resistance to EGFR tyrosine kinase inhibitors by the EGFRT790M mutation in non-small cell lung cancer patients in Vietnam” Journal of medical research, 97 (5): 123-128 Tran Quoc Dat, Tran Huy Thinh, Le Hoan, Tran Van Khanh, Ta Thanh Van (2016) “Targeting TKI resistance in non - small cell lung cancer patients caused by secondary EGFR T790M mutation” Journal of medical research, 105 E (7): 11-19 Le Hoan, Ngo Quy Chau, Tran Khanh Chi, Tran Huy Thinh (2020) “EGFR-T790M mutation is associated with acquired resistance to tyrosine kinase inhibitors in non-small cell lung cancer patients” Journal of medical research, 126 (2): 7-15 ... Reviewer 2: A Prof NGUYEN Tuyet Mai, MD PhD Reviewer 3: A Prof CHU Thi Hanh, MD PhD The thesis is defended in front of the Thesis Committee of the University at Hanoi Medical University The thesis can... SCIENTIFIC WORKS PUBLISHED IN THE SCOPE OF THE THESIS Le Hoan, Nguyen Minh Ha, Tran Van Khanh, Ngo Quy Chau, Ta Thanh Van, Tran Huy Thinh (2015) “Acquired resistance of lung adenocarcinoma to gefitinib... medical research, 96 (4): 137-143 Le Hoan, Nguyen Minh Ha, Tran Van Khanh, Vuong Ngoc Duong, Ngo Quy Chau, Mai Trong Khoa, Ta Thanh Van, Tran Huy Thinh (2015) “Acquired resistance to EGFR tyrosine