Clinical, subclinical features and resistance to tyrosine kinase inhibitor of lung cancer patients with egfr mutations

zing - Difference was statistical significant with p <0,05. 2.4. Research ethnic - Participants were voluntary and had the right to withdraw of the research - Patients‟ personal data was secured - Techniques, procedures performed on patients were warranted to be right according to Ministry of Health. - The research was conducted for the scientific purpose but not any other one. - The research was approved by the Ethnic Committee of Hanoi Medical University. Chapter 3: RESULTS By studying 66 lung cancer patients with EGFR mutations, treated with EGFR-TKIs, thereafter developed EGFR-TKIs resistance, the results were as below: 3.1. Clinical and subclinical features of patients with lung cancer with EGFR mutations before TKIs therapy and in the relapsed period 3.1.1. Basic characteristics Table 3.1: Basic characteristics of research patients (n=66) Characteristics n % Age: 60,6 ± 10,7 years (min: 26; max: 80 years) Gender Male 34 51.5 Female 32 48.5 Smoking Non-smoker 36 54.5 Ex-smoker 10 15.2 Current smoker 20 30.3 Cormobidities Cardiovascular diseases 16 18.2 Pulmonary diseases 12 24.2 Metabolic disorders 10 15.2 Comments: Gender ratio was approximately 1/1; 54.5% of patients were non- smokers; 57.6% of patients had cormobidities 3.1.2. Clinical features of patients with NSCLC with EGFR mutations Table 3.2: Clinical features of patients with NSCLC with EGFR mutations (n=66) Symptoms Before therapy Relapse n % n % Chest pain 40 60.6 32 48.5 Cough 32 48.5 38 57.6 Dyspnea 16 24.2 30 45.5 Hemoptysis 8 12.1 0 0 Weight loss 24 36.3 10 15.2 Fever 8 12,1 2 3.0 Peripheral lymph node 16 24.2 8 12.1 Headache 16 24.2 10 15.2 Spinal pain 14 21.2 8 12.2 Muscoskeletal pain 12 18.2 6 9.1 Abdominal pain 6 9.1 6 9.1 Hoarse 4 6.1 5 7.6 Hiccup 3 4.5 0 0 Choking 3 4.5 0 0 SVC compression syndrome 3 4.5 0 0 Pancoast Tobias syndrome 2 3,0 0 0 Comments: - The most common respiratory symptoms were chest pain, cough and dyspnea - The most common systemic symptoms were weight loss, peripheral lymph node - The most common metastatic symptoms were headache, spinal pain, muscoskeletal pain. - In general, symptoms when relapse were similar to those before therapy but with lower rate. 3.1.2. Subclinical features of patients with NSCLC with EGFR mutations  Imaging characteristics - Size of the primary tumor: 12.1% of patients had tumor diameter of >2 to ≤3cm; 36.4% of patients had tumor diameter of >3 to ≤5cm; 45.4% of patients had tumor diameter of >5 to ≤7cm; 6.1% BN had tumor > 7cm, no patient had tumor < 2cm. - Location of the primary tumor: 27.3% of patients had tumor of upper lobe of the right lung; 12.1% of patients had tumor of middle lobe of the right lung; 21.2% of patients had tumor of lower lobe of the right lung; 21.2% of patients had tumor of upper lobe of the left lung; 18.2% of patients had tumor of lower lobe of the left lung. - Secondary lesions: 21.2% of patients had pleural effusion; 9.1% of patients had pericardial effusion; 21.2% of patients had brain metastasis; 21.1% had bone or vertebra metastasis; 18.2% had adrenal metastasis and 9.1% of patients had liver metastasis. - Imaging at the time of disease relapse: 63.6% of patients increased primary tumor size; 31.8% of patients had new lung lesion; some of patients developed from previous metastatic lesions, others had new metastatic lesions.  Histopathologic characteristics: 98.5% were adenocarcinoma; 1.5% was adenosquamous carcinoma.  EGFR mutations: 54.5% had LREA; 44% had L858R and 1.5% had G719S. 3.2. Identification T790M mutation of EGFR gene, MET amplication in lung cancer patients with EGFR-TKIs resistance and the relationship with clinical and subclinical features 3.2.1. Effectiveness of EGFR-TKIs in treating lung cancer patients with EGFR mutations  2 first generation EGFR-TKIs including erlotinib (53%) and gefitinib (47%) were used as first step therapy for patients with EGFR mutations and sensitive to TKIs.  Adverse effects of EGFR-TKIs: Skin rash (22.7%); nausea, vomit (21.2%); fatigue, appetite loss (18.2%); elevated liver enzyme (16.7%); stool disorders (15.1%); paronychia (6.1%); hair loss (4.5%); muscle pain (1.5%).  Patients’ response after 6 months of treating with EGFR-TKIs: Table 3.3: Patients’ response after 6 months of treating with EGFR-TKIs (n=66) Patients’ response n % Totally response 0 0 Partly response 58 87.9 Stable 8 12.1 Relapse 0 0 Total 66 100 Comments: - Most of patients had partly response after 6 months treating with EGFR- TKIs, accounting for 87.9% - 12.1% of patients were stable after 6 months treating with EGFR-TKIs - No patient had totally response after 6 months treating with EGFR-TKIs Table 3.4: Performance status before and after treatment (n=66) Performance status Before After p n % n % PS 0-1 40 60.6 52 78.8 p=0.035 PS 2-4 26 39.4 14 21.2 Total 66 100 66 100 Comments: - After 6 months, number of patients having PS0-1 increased significantly - After treatment, patients‟ performance status improved significantly (p=0.035)  Time until relapse Chart 3.1: Progression-free survial (PFS) (n=66) Comments: - Average time from the beginning of EGFR-TKIs therapy until relapse was 14.48 ± 3.9 months. - Median PFS was 14 months; min: 8 months, max: 26 months. - 39.3% of patients relapsed after 6-12 months; 42.4% after 13-18 months; 15.3% after 19-24 months; 3% after over 24 months.  The influence of clinical and subclinical factors on PFS - Average PFS was not different between males (13.8 months; 95%CI 12.6- 14.9 months) and females (15 months; 95%CI 13.5-16,5 months) (p=0.180) (Chart 3.2A). - Average PFS was different significantly between ≤60 year-old group (15.7 months; 95%CI 14.3-17.1 months) and >60 year-old group (13.3 months; 95%CI 12.1-14.5 months) (p=0.028) (Chart 3.2B). - Average PFS was not different between smokers (13.8 months; 95%CI 12.6-15.1 months) and non-smokers (14.8 months; 95%CI 13.4-16.1 months) (p=0.324) (Chart 3.2C). - Average PFS was different significantly between non-cormobidities group (15.6 months; 95%CI 14.2-17.0 months) and cormobidities group (13.4 months; 95%CI 12.3-14.6 months) (p=0.039) (Chart 3.2D). - Average PFS was not different between LREA group (13.7 months; 95%CI 12.4-14.9 months) and L858R group (15.2 months; 95%CI 13.8-16.6 months) and G719S group (12.0 months) (p=0.280) (Chart 3.2E). - Average PFS was not different between erlotinib group (13.7 months; 95%CI 12.4-15.0 months) and gefitinib group (15.0 months; 95%CI 13.7- 16.3 months) (p=0.287) (Chart 3.2F). Chart 3.2: The influence of clinical and subclinical factors on PFS (n=66) 3.2.2. Identification of EGFR-TKIs resistance causes  Methods of collecting sample at the time of drug resistance Table 3.5: Methods of collecting sample at the time of EGFR-TKIs resistance (n=66) Methods n % CT guided lung biopsy 28 42.4 Bronchoscopy lung biopsy 14 21.2 Cell block of pleural fluid 12 18.2 Peripheral lymph node biopsy 8 12.1 Cell block of pericardial fluid 6 9.1 Vetebra biopsy 2 3.0 Liver biopsy 1 1.5 Comments: Lung biopsy under the guidance of CT or bronchoscopy were the most common methods to confirm the relapse of cancer at the time of drug resistance development (63.6%). Table 3.6: Complications of methods of collecting sample at the time of EGFR-TKIs resistance (n=66) Complications n % Pain at the biopsy site 18 27.3 Hemoptysis 8 12.1 Mild pneumothorax 2 3.0 None 40 60.1 Comments: Another lung biopsy or metastatic lesion biopsy were safe, with 60.1% of patients did not have any complications.  Histopathological characteristics at the time of EGFR-TKIs resistance development - 63,6% of patients had lung adenocarcinoma. - 33,3% of patients had metastatic adenocarcinoma from lung. - 2 cases had transformation from lung adenocarcinoma into small cell lung cancer after treatment  Gene analysis - 54,5% of patients had EGFR-T790M mutation - 4,5% of patients had MET amplication. - 2 cases having transformation from lung adenocarcinoma into small cell lung cancer did not have neither EGFR-T790M mutation nor MET amplication.  The relationship between GFR-T790M mutation, MET amplication and clinical and subclinical features Table 3.7: EGFR-T790M mutation and the relationship with clinical and subclinical features (n=66) Features EGFR-T790M No EGFR-T790M p n % n % Gender Male 22 33,3 12 18,3 0,169 Female 16 24,2 16 24,2 Age ≤ 60 17 25,7 11 16,7 0,425 > 60 21 31,9 17 25,7 Smoking Yes 21 31,9 9 13,6 0,053 No 17 25,7 19 28,8 Cormorbidities Yes 20 30,3 18 27,3 0,244 No 18 27,3 10 15,1 Adenocarcinoma Yes 38 57,6 27 40,9 0,424 No 0 0 1 1,5 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) Features MET amplication No MET amplication p n % n % Gender Male 0 0 34 51,6 0,108 Female 3 4,5 29 43,9 Age ≤ 60 1 1,5 27 40,9 0,615 > 60 2 3,0 36 54,6 Smoking Yes 0 0 30 45,5 0,156 No 3 4,5 33 50,0 Cormorbidities Yes 2 3,0 36 54,6 0,615 No 1 1,5 27 40,9 Adenocarcinoma Yes 3 4,5 62 94,0 0,955 No 0 0 1 1,5 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 non- smoking 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 2 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 > 3 cm, of which up to 51.5% of patients have tumor size > 5 cm; No cases of tumor size < 2 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 > 3 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 EGFR- TKIs 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 1 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 EGFR- TKIs, 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 6 months, we noted that 87.9% of patients responded partially and 12.1% of patients achieved stable disease, no cases completely responded after 6 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 EGFR- TKIs after 6 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 6 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 8 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 EGFR- TKIs 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 co- morbidity (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