Summary of Phd thesis: Clinical, subclinical features and resistance to tyrosine kinase inhibitor of lung cancer patients with EGFR mutations

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.

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

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

2 Objectives

1 To describe the clinical and subclinical features of lung cancer patients with EGFR mutations before TKIs therapy and in the relapsed period

2 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

3 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,

4 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

5 Thesis structure

The thesis includes 118 pages (without References and Appendix), divided into 7 parts:

- Introduction: 3 pages

- Chapter 1: Literature review, 35 pages

- Chapter 2: Methodology, 15 pages

- Chapter 3: Results, 29 pages

- Chapter 4: Discussions, 31 pages

- Conclusions: 4 pages

- Recommendations: 1 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

1 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 smokers, 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

non-2 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 6 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

3 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 2 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 hoặc gefitinib), having good response or stable in at least 6 months and relapsed after that

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 6 months

+ Relapsed or developed new lesions despite of treatment with erlotinib

- 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 time PCR

real Identification of MET amplication using FISH

 Analyzing clinical factors that could affect the development of T790M mutation and MET amplication leading to EGFR-TKIs resistance: age, gender, smoking status, histopathological feature

EGFR-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 <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

non-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)

- 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

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)

- 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)

- 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 14.9 months) and females (15 months; 95%CI 13.5-16,5 months) (p=0.180)

12.6-(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)