MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENSE VIETNAM MILITARY MEDICAL UNIVERSITY HO VAN SON STUDYING THE mRNA EXPRESSION OF CIZ1b, VEGF GENES AND EGFR MUTATIONS WITH MERKEL CELL VIRUS INFECTION IN PATIENTS WITH NON- SMALL CELL LUNG CANCER Major: Biomedical Science Code: 72 01 01 SUMMARY OF DOCTORAL THESIS IN MEDICINE HA NOI – 2020 THE THESIS IS COMPLETED AT THE VIETNAM MILITARY MEDICAL UNIVESITY Supervisors: Prof Dr NGUYỄN LĨNH TỒN Dr NGƠ TẤT TRUNG Reviewer 1: Prof Dr VAN DINH HOA Reviewer 2: Prof Dr PHAN THU PHUONG Reviewer 3: Prof Dr TA BA THANG The thesis is defended in front of the scientific committee at the Vietnam Military Medical University at on 2020 The thesis can be found at: - National Library - Library of the Vietnam Military Medical University INTRODUCTION Primary lung cancer (PLC) is one of the most common malignancies today and is the leading cause of death in cancer In Vietnam, lung cancer (LC) has increased rapidly According to the Global Cancer Research Organization (GLOBOCAN) in 2018, there are 23,667 new people in the country with LC About 90% of patients with PLC die in the first year Recently, CIZ1, a gene coding for intracellular protein involved in the initiation of DNA replication, has been studied CIZ1b, a variant of CIZ1, manifests itself in nonsmall cell lung cancer (NSCLC) Besides, Vascular endothelial growth factor (VEGF) plays a very important role in tumour growth EGFR mutation is a valuable molecular target in the use of Tyrosine Kinase inhibitors - TKIs in treatment of NSCLC Recently, the role of Virus Merkel cell (Merkel cell polyomavirus, MCV) in LC has been mentioned and researched We conducted the project: "Studying the mRNA expression of CIZ1b, VEGF and EGFR mutations with Merkel cell virus infection in patients with nonsmall cell lung cancer" with these objectives: Assessed the level of mRNA expression of CIZ1b, VEGF, EGFR mutations and the rate of Merkel cell infection in patients with nonsmall cell lung cancer Analysed the relationship between the level of mRNA expression of CIZ1b, VEGF, EGFR mutations with Merkel cell infection and some clinical and subclinical symptoms in patients with non-small cell lung cancer Rationale of the Study Study on mRNA expression of CIZ1b, VEGF, EGFR mutation and Merkel cell virus (MCV) infection contribute to early diagnosis, identify the cause of disease and its application in monitoring and treatment of NSCLC, contribute to improving the quality of life, reduce mortality for patients with NSCLC Significance of the Study This thesis is the first study to evaluate the mRNA expression of CIZ1b, VEGF and MCV infection rates in patients with NSCLC in Vietnam The thesis also raised the diagnostic value of two mRNA markers of CIZ1b, VEGF in NSCLC and the relationship of MCV with EGFR mutation as well as the risk of LC when being infected with MCV Layout The thesis has 120 pages, including: Introduction (2 pages), Chapter 1: Literature Review (31 pages), Chapter 2: Research subjects and Methodology (28 pages), Chapter 3: Results (33 pages), Chapter 4: Discussion (22 pages), Conclusion (2 pages), Recommendations (1 page) The thesis has 150 references (English: 146) CHAPTER 1: LITERATURE REVIEW 1.1 Overview of lung cancer Lung cancer is currently the most common type of cancer in both incidence and mortality In 2018, there were about 2.1 million new cases of lung cancer detected and an estimated 1.8 million deaths, accounting for more than 18% of all cancer deaths In Vietnam, LC accounted for 15.48% of all new cancers About 90% died in the first years Prevalence of male is higher than female (The ratio of male to female is approximately 2.5:1) The main risk factors associated with lung cancer include tobacco use, environmental pollution, Amian infection, Randon gas, infectious agents, interactions between the sensitive genome and the environment and The oncogene in NSCLC often involves four main functional pathways: cell proliferation, programmed resistance to death, invasion or metastasis and angiogenesis 1.2 Expression of CIZ1b in non-small cell lung cancer CIZ1b is a variant of the CIZ1 gene, due to the deficiency of eight amino acids at the beginning of the C end CIZ1 gene encodes for an intracellular protein that plays a role in controlling cell proliferation: regulate cell cycle, regulate transcription CIZ1 is an intermediate molecule that connects cyclin E and A to cyclin dependent kinase proteins such as CDK2 and p21, to accelerate mammalian DNA replication This protein also plays an indirect role in DNA replication by regulating the expression of genes It can bind directly to DNA molecules or act as a co-activator of transcription factors In LC, CIZ1b manifested much in NSCLC samples when compared to adjacent tissue samples By quantifying the concentration of CIZ1b in plasma (using Western blot technique), Higgins et al were able to distinguish 98% of LC cases from normal people In addition, compared patients with stage NSCLC to the smoking control group of the same age or the group of benign lung tumours, this marker can also classify 95% of cases Thus, CIZ1b shows the potential of a molecular marker that can detect NSCLC early with high clinical accuracy CIZ1b affected the DNA replication function of lung cancer cells Using RNAi technique to reduce the expression of CIZ1b (without affecting other forms of CIZ1) in the SBC5 lung cancer cell line can inhibit the proliferation of this cell line The same result was obtained with in vivo experiments, when implanting this cancer cell line in mice, CIZ1b depletion could reduce tumour growth These experiments have shown that CIZ1b variant plays a role in controlling proliferation of cancer cells 1.3 Manifestations of VEGF in non-small cell lung cancer VEGF, which has been shown to play an important role in lung cancer, is strongly correlated with abnormal blood vessel formation and promotes tumour growth This factor has been applied to differential diagnosis in patients with chronic obstructive pulmonary disease and lung cancer, benign and malignant pleural effusion Notably, for patients whose tumours are detected by a chest X-ray or CT scan, the level of VEGF expression in bronchial lavage is a marker that can be used to diagnose primary lung cancer 1.4 EGFR mutation in non-small cell lung cancer Epidermal growth factor receptor (EGFR) has a molecular weight of 170 kiloDaltons (kDa) When the epithelial growth factor (EGF) binds to the receptor (EGFR), the two EGFR molecules combine with each other (dimerization) to activate phosphorylation of the tyrosine kinase region, which activates specific tyrosins and EGFR receptordependent intracellular signal proteins that induce transcription of target genes to promote proliferation, programmed resistance, invasion, metastasis and neovascularization EGFR is especially important in the pathogenesis of non-small cell lung cancer, especially adenocarcinoma In people who not smoke or rarely smoke, women and Asian people, EGFR manifestations occur in more than 50% of cases of lung adenocarcinoma However, they also make malignant cells susceptible to TKIs, and even predict response to broad-spectrum TKI drugs like erlotinib and gefitinib In addition to the TKIsensitive mutation, EGFR also has a mutation that helps cancer cells resist this drug, such as the T790M mutation that occurs in exon 20 About 50–60% of recurrent patients have a T790M mutation This mutation reduces the effectiveness of the first generation of TKI drug, but so far the third generation TKI can help treat drug-resistant patients 1.5 Merkel cell Carcinoma Virus (MCV) in non-small cell lung cancer 1.5.1 Merkel cell virus and carcinogenic mechanism MCV belongs to the Polyomaviridae family and is listed on the list of class 2A carcinogens by the world cancer research fund international The MCV genome is a double-stranded circular DNA molecule, about kb in size, divided into three main regions: a noncoding control region (NCRR) containing the replication centre of virus Transcription factors are located between two regions: the early coding region and the late coding region Early coding region, coding for antigens: LT (Large T), ST (Small T) The late coding region encodes for the capsid proteins, VP1 and VP2 The LT and ST antigens play an important role in tumour formation caused by MCV LT carries domain J (associated with thermal shock protein), retinoblastoma binding motif (RB) (inhibits RB family members), domain affixes replication centre at C end and domain helicase / ATPase (necessary for DNA replication of virus) Much of the carcinogenic function of LT antigen is due to its high affinity for RB, causing isolation and inactivation of this tumour suppressor gene The RB binding function of MCV LT is essential for the sustainable growth of MCC-positive MCV tumours both in vitro and in experimental models 1.5.2 Relationship between Merkel virus and lung cancer Based on the histological similarities between MCC and small cell lung cancer (SCLC), the relationship between MCV and SCLC has been studied Two studies in Germany showed the presence of MCV DNA by PCR with detection rates of 6.7% (2/30) and 38.9% (7/18) For NSCLC, epidemiological studies conducted in the US and Europe detected MCV DNA at 16.7% (5/30), 4.7% (4/86) and 9.1% (10/110) patients with NSCLC Recently, Hashida et al first published the incidence of MCV in Asian lung cancer and detected MCV DNA in 17.9% (20/112) of NSCLC patients in Japan In particular, the Japanese team found two cases of NSCLC infection with MCV bearing the characteristic mark of the tumour These cases have the LT antigen expressed in cancer cells and integrate the viral genome into the cell chromosome and cause cancer modification Thus, integrated or mutated forms of MCV have been shown in a specific cancer other than MCC, suggesting that MCV is associated with the onset of an NSCLC group However, no studies have been conducted in Vietnam to detect MCV in patients with NSCLC CHAPTER 2: RSEARCH SUBJECTS AND METHODOLOGY 2.1 Research subjects 2.1.1 Patient group: 100 patients with non-small cell lung cancer, identified by histopathology, aged 27- 83 2.1.2 Healthy people (control group): 51 healthy people (without cancer), ages 48-60, are similar in age and gender to the patient group 2.1.3 Exclusion: SCLC, treated NSCLC, metastatic LC, COPD, asthma, acute and chronic bronchitis, patients receiving corticosteroids, pregnant women, patients with autoimmune disease and non-cooperative participants in the study 2.2 Research Methods 2.2.1 Research design - Cross-sectional descriptive and controlled study, combined with clinical examination and laboratory analysis 2.2.2 Location and study time - Research location: Department of Pathophysiology - Military Medical Academy, Military Hospital 175 and Central Military Hospital 108 - Research period: From October 2014 to February 2018 2.2.3 Sampling method - Sample selection method: Convenience sampling 2.2.4 Data collection - Selected patients, established research medical records in the unified form, coded the patient symptom information, extracted information, collected registration data, and entered the records 2.2.5 The implementation techniques 2.2.5.1 Diagnosis of non-small cell lung cancer Based on the guidelines: the diagnosis and treatment of non-small cell lung cancer of the Ministry of Health of Vietnam applies to medical examination and treatment facilities nationwide 2.2.5.2 Diagnosis of stage of disease WHO TNM classification for lung cancer 2015 2.2.5.3 Histopathological diagnosis, differentiation Based on WHO classification of lung cancer 2015 2.2.5.4 Using real-time PCR technique, PCR technique, direct sequencing technique to determine: - The level of mRNA expression of CIZ1b and VEGF in peripheral blood - Identify EGFR mutations - Determine the presence of Merkel cell virus in peripheral blood of healthy people, in blood and cancer tissue of patients 2.2.6 Evaluation criteria - Level of expression of mRNA VEGF and mRNA CIZ1b in LC group and the control group - EGFR mutation rate - The prevalence of Merkel cell virus infection in the LC group and the control group - The relationship between the expression level of mRNA VEGF, mRNA CIZ1b and some subclinical factors: renal function, liver function, blood sugar, CEA, CYFRA21-1, EGFR mutation, disease stage and tissue type pathology, differentiation and associated factors with MCV infection 2.3 Data processing - Processed data using SPSS 20.0 software CHAPTER 3: RESEARCH RESULTS 3.1 General characteristics of the research team 3.1.1 Age and gender Table 3.1 Comparing age and gender of the two groups Character istic NSCLC group (n = 100) ± SD Median Control group (n = 51) ± SD P Median Age 60,0 ± 10,5 61,0 53,9 ± 2,9 53,0 0,158 Gender (Male/Fem 73/27 35/16 0,47 al) There is no difference in age and gender in the two groups (disease and control) 3.1.2 Histopathological results of the treatment group Table 3.2 Histopathology of the NSCLC group No Histopathology results (n=100) Quantity Percentage (%) Adeno carcinoma 91 91 Squamus cell carcinoma 9 12 3.3.2 Relationship between mRNA espression of CIZ1b and VEGF with tumour histopathology Table 3.8 mRNA expression of CIZ1b and plasma VEGF and tumour histopathology Histopathology Adeno carcinoma (n=91) Squamus cell Carcinoma (n=9) ± SD Median Gene p ± SD Median CIZ1b 10,1 ± 6,1 8,6 14,5 ± 9,5 13,8 > 0,05 VEGF 2,3 ± 2,4 1,7 1,9 ± 1,2 1,2 > 0,05 No association between mRNA expression of CIZ1b and VEGF with histopathology was observed 3.3.3 Relationship between mRNA expression of CIZ1b and VEGF with tumour cell differentiation Table 3.9 mRNA expression of CIZ1b, VEGF and tumour cell differentiation Differenti ation Gene Grad (n = 6) ± SD Grad (n = 35) Median Grad (n = 22) P ± SD Median ± SD Median CIZ1b 11,8±5,8 10,9 11,5±6,7 9,9 9,3±6,9 7,7 > 0,05 VEGF 6,1±6,5 3,8 1,7 1,9±0,9 1,7 > 0,05 1,9±1,3 No association between mRNA expression of CIZ1b and VEGF with tumour cell differentiation was observed 3.3.4 Relationship between mRNA expression of CIZ1b and VEGF with EGFR gene mutation 13 Table 3.10 mRNA espression of CIZ1b and VEGF and EGFR gene mutation EGFR gene mutation Gene Mutations (n = 26) No mutations (n = 25) ± SD Median p ± SD Median CIZ1b 10,3 ± 4,9 8,3 10,4 ± 7,7 9,0 > 0,05 VEGF 1,6 ± 0,8 1,4 2,3 ± 1,8 1,9 > 0,05 No association between mRNA expression of CIZ1b and VEGF with EGFR mutation was observed 3.3.5 Relationship between MCV infection and the stages of NSCLC Table 3.11 Relationship between stages of NSCLC and MCV infection MCV Stage Positive n(%) Negative n(%) IA (3) IIA (6) IIIA (3) IIIB (12) 11 (16,7) IV 22 (88) 47 (71,3) p >0,05 Total 25 (100) 66 (100) No association between MCV infection and stages of NSCLC was observed 3.3.6 Relationship between tumour cell differentiation and MCV infection 14 Table 3.12 Relationship between tumour cell differentiation and MCV infection MCV Differentiation Positive n(%) Negative n(%) Grad 1 (4) (6,7) Grad (32) 27 (36) Grad (28) 15 (20) Undefined (36) 28 (37,3) Total 25 (100) 75 (100) p >0,05 No association was found between MCV infection and tumour cell differentiation 3.3.7 Relationship between tumour cell differentiation and MCV infection Table 3.13 Relationship between MCV infection and cancer marker Characteristics Positive MCV Negative MCV (n = 25) (n = 75) ± SD p ± SD Median Median CEA (ng/mL) 121,7±250,2 6,9 95,2±273,8 6,02 >0,05 CYFRA 21.1 (ng/mL) 24,7±70,5 2,69 8,98±21,5 3,8 >0,05 No association was found between MCV infection and expression of cancer markers 3.3.8 Relationship between MCV infection with EGFR mutation Table 3.14 Relationship between MCV infection with EGFR 15 mutation EGFR mutation Positive MCV Negative MCV OR (95% CI) Mutation 17 (85) (29) 13,8 (2,8-86,5) No mutation (15) 22 (71) Chi2(1): 15,24 Total number of patients analysed 20 (100) 31 (100) p 0,0001 The percentage of EGFR mutations in the group of NSCLC infected with MCV was significantly higher than the group without the mutation, in patients with NSCLC infected with MCV, the risk of EGFR mutation increased by 13.8 times 3.3.9 Relationship between MCV infection with the level of gene expression in VEGF and CIZ1b Table 3.15 Relationship between MCV infection with gene expression level of VEGF and CIZ1b Characteristics Positive MCV Negative MCV (n = 25) (n = 75) ± SD Median ± SD p Median VEGF (2-∆Ct) 2,07±1,1 1,89 2,33±2,5 1,66 >0,05 CIZ1b (2-∆Ct) 12,15±7,0 9,45 9,77±5,9 8,34 >0,05 No association between MCV infection and mARN of CIZ1b and VEGF expression was found CHAPTER 4: DISCUSSION 16 4.1 Some common characteristics of the NSCLC group 4.1.1 Age of illness According to the majority of domestic and foreign authors, the disease is usually occur with people aged between 50 and 70 In this study, the average age of disease was 60 ± 10.5, of which there were 61 patients from 55 to 74 years old, accounted for 61% of the total number of patients studied We found that the age of the disease in this study is lower than that of Patricia M.de Goot, which may be because our statistics are not big enough It may also be because people with lung cancer in Vietnam are getting younger 4.1.2 Gender Lung cancer is more common in men than women, in this study the ratio of male to female was 3.7:1 With this ratio, the amount of women having lung cancer is increasing In Vietnam, the incidence of lung cancer is also tending to balance between men and women, according to Globocan 2018 the rate of men over women is 2.5:1 The difference in the incidence between men and women in this study and Globocan's overall rate was probably due to the fact that our sample was not large enough, moreover we only got data from one cancer treatment center, this also affected the proportional distribution between men and women 4.2 The level of mRNA expression of CIZ1b, VEGF, EGFR mutations and the rate of Merkel Cell virus infection in patients with non-small cell lung cancer 4.2.1 Levels of mRNA expression of CIZ1b gene in patients with non-small cell lung cancer In previous research conducted by Higgins et al in 2012, CIZ1b was proved to be a valuable biomarker in the diagnosis of lung cancer CIZ1b was only found in lung cancer patients but not in normal people Therefore, CIZ1b was proposed as a suitable marker for early stage lung cancer 17 Studies showed that the CIZ1b variant was sensitive enough to allow accurate identification of patients with stage cancer, in highrisk groups, including patients with benign lymphadenopathy, pneumonia, asthma, chronic obstructive pulmonary disease) and smokers It is also found that cancer patients have much higher levels of CIZ1b than normal people without cancer When analyzed by stage of cancer, CIZ1b concentration also increased by stage in patients with non-small cell lung cancer In this study, specific primers had been designed to amplify and quantify mRNA expression of the CIZ1b gene in the blood by realtime PCR The method was based on the PCR technique, which results in fast and accurate expression of CIZ1b mRNA in the blood Our research results showed that the level of mRNA expression of CIZ1b gene in plasma in patients with non-small cell lung cancer was significantly higher than that of control group These results confirm that CIZ1b gene expression is affected by lung cancer development and CIZ1b gene play an important role in lung cancer development From this result, the CIZ1b gene has great potential to be used as a Biomarker to diagnose, to monitor lung cancer development as well as to monitor the prognosis of lung cancer treatment 4.2.2 Levels of mRNA expression of VEGF gene in patients with non-small cell lung cancer One study assessed the mRNA expression of VEGF in three groups of patients including squamous cell carcinoma, adenocarcinoma, and undifferentiated cell carcinoma The results showed that in 65% of cases, VEGF mRNA expression was higher in cancer tissue than normal tissue mRNA expression of VEGF was higher in non-squamous cell carcinoma and higher in tumors with lymph node metastases Similarly, this study also showed that mRNA expression of VEGF in peripheral blood was also significantly higher in lung cancer patients compared to the control group 18 In this study, we did not observe the association of VEGF mRNA expression with clinical features such as liver dysfunction, Glucose disorder, tumor histopathology, stage of disease We also did not see the difference in mRNA expression of VEGF between differentiation stages of the tumor (p> 0.05) This shows that the level of VEGF expression increased significantly between lung cancer group and healthy people (p