Studying the mrna expression of ciz1b, vegf genes and egfr mutations with merkel cell virus infection in patients with non - Small cell lung cancer

ancer 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 receptor-dependent 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 do 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 TKI-sensitive 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 5 kb in size, divided into three main regions: a non-coding 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 Characteristic NSCLC group (n = 100) Control group (n = 51) P X ± SD Median X ± SD Median Age 60,0 ± 10,5 61,0 53,9 ± 2,9 53,0 0,158 Gender (Male/Femal) 73/27 35/16 0,47 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 (%) 1 Adeno carcinoma 91 91 2 Squamus cell carcinoma 9 9 Gland carcinoma makes up the majority (91%), barbed carcinoma accounts for only 9%. No large and mixed cell carcinoma. 3.1.3. Results of differentiation evaluation in the treatment group Table 3.3. Differentiation of tumour cells of NSCLC patients No. Differentiation (n=100) Quantity Percentage % 1 No rating 37 37 2 To be evaluated (n=63) Degree 1 6/63 9,52 Degree 2 35/63 55,56 Degree 3 22/63 34,92 Average differentiation is the most common, followed by high differentiation, low differentiation only accounts for 9%. Figure 3.1. Distribution of desease stage if NSCLC patients 3.1.4. Results of the phase evaluation of NSCLC group Stage IV had the highest rate (76%), followed by stage IIIB, early stages encountered less. 3.2. mRNA expression of CIZ1b and VEGF, EGFR mutation and prevalence of MCV infection in patients with KTBN 3.2.1. mRNA expression of CIZ1b and VEGF in two study groups Table 3.4. mRNA expression of CIZ1b and VEGF in two study groups VEGF expression (2-∆Ct) NSCLC group (n = 100) Control group (n = 51) p X ± SD Median X ± SD Median CIZ1b expression (2-∆Ct) 10,5 ± 6,3 8,9 7,8 ± 2,6 7,0 0,008 VEGF expression (2-∆Ct) 2,3 ± 2,3 1,7 1,5 ± 1,0 1,3 0,007 The level of mRNA expression of CIZ1b and VEGF in the treatment group was significantly higher that that of healthy people (p<0,05). 3.2.2. EGFR mutation in NSCLC group Table 3.5. EGFR mutation of the NSCLC patients group No. Mutant position Number of patients (n=51) Percentage % 1 Exon 19 11 21,57 2 Exon 20 2 3,92 3 Exon 21 13 25,49 4 No mutation 25 49,02 Mutations in exon 21 accounted for the highest percentage, followed by mutations in exon 19, mutations in exon 20 was only 4%. 3.2.3. Prevalence of MCV infection in NSCLC group Table 3.6. The prevalence of MCV in patients compared to healthy people group Result Treatment group(%) Control group (%) (Blood) OR (95% CI) P Blood MCV (%) Tissue MCV (%) Positive 25 (25) 25 (25) 3 (5,9) 5,33 (1,49-28,83) 0,004 Negative 75 (75) 75 (75) 48 (94,1) Total 100 (100) 100 (100) 51 (100) The rate of MCV infection in patients with NSCLC (25%) was higher than that of normal people (5.9%), people infected with MCV were 5.3 times more likely to be infected with UV than those without MCV infection. 3.3 Relationship between the level of mRNA expression of CIZ1b, VEGF, EGFR mutations and Merkel cell infection and some clinical and subclinical symptoms in patients with NSCLC 3.3.1. Relationship between mRNA expression of CIZ1b and VEGF and stage of disease Table 3.7. mRNA expression of CIZ1b and VEGF, stages of disease Stages of disease Gene Stage I+II+III (n = 22) Stage IV (n = 69) p X ± SD Median X ± SD Median CIZ1b 9,6 ± 5,1 8,1 10,7 ± 6,7 9,4 > 0,05 VEGF 2,2 ± 1,3 2,0 1,5 ± 1,3 1,6 > 0,05 No association between mRNA expression of CIZ1b and VEGF was observed. 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 Gene Adeno carcinoma (n=91) Squamus cell Carcinoma (n=9) p X ± SD Median X ± 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 Differentiation Gene Grad 1 (n = 6) Grad 2 (n = 35) Grad 3 (n = 22) P X ± SD Median X ± SD Median X ± 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 1,9±1,3 1,7 1,9±0,9 1,7 > 0,05 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 Table 3.10. mRNA espression of CIZ1b and VEGF and EGFR gene mutation EGFR gene mutation Gene Mutations (n = 26) No mutations (n = 25) p X ± SD Median X ± 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 Stage MCV p Positive n(%) Negative n(%) IA 0 2 (3) >0,05 IIA 0 4 (6) IIIA 0 2 (3) IIIB 3 (12) 11 (16,7) IV 22 (88) 47 (71,3) 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 Table 3.12. Relationship between tumour cell differentiation and MCV infection Differentiation MCV p Positive n(%) Negative n(%) Grad 1 1 (4) 5 (6,7) >0,05 Grad 2 8 (32) 27 (36) Grad 3 7 (28) 15 (20) Undefined 9 (36) 28 (37,3) Total 25 (100) 75 (100) 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 (n = 25) Negative MCV (n = 75) p X ± SD Median X ± SD 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 mutation EGFR mutation Positive MCV Negative MCV OR (95% CI) p Mutation 17 (85) 9 (29) 13,8 (2,8-86,5) Chi2(1): 15,24 0,0001 No mutation 3 (15) 22 (71) Total number of patients analysed 20 (100) 31 (100) 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 (n = 25) Negative MCV (n = 75) p ± SD Median ± SD 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 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. Studies showed that the CIZ1b variant was sensitive enough to allow accurate identification of patients with stage 1 cancer, in high-risk 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 real-time 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. 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 <0.05) but, there was no difference with other clinical and subclinical factors. In our study, analysis of diagnostic efficacy for identifying lung cancer patients showed that VEGF mRNA expression was able to identify lung cancer patients with AUC value = 0.615. Therefore, this study, together with previous studies, indicates that assessing the mRNA expression of VEGF in plasma is a non-intervention measure that can be used in combination with other diagnostic methods to diagnose and screen lung cancer in high-risk groups such as those with lung disease, a family history of lung cancer, or in groups that are frequently exposed to toxic agents such as those who smoke or group of workers in the mines. However, this factor can be used as an effective marker to monitor and evaluate the effectiveness of treatment in non-small cell lung cancer. 4.2.3. EGFR mutation in non-small cell lung cancer patients in the study EGFR mutation plays a crucial role in the pathogenesis of non-small cell lung cancer. Moreover, this is a molecular target for the use of small molecule inhibitors (TKIs) for treatment. Previous studies have shown that over 50% of non-small cell lung cancers with adenocarcinoma have EGFR mutations. In our study, there were 51 cases of adenocarcinoma identified with EGFR mutation and 26 cases with mutation, accounting for 51%. Although the number of patients who have an EGFR mutation was small, the percentage of patients with EGFR mutations in our study s also consistent with previous studies. 4.2.4. The prevalence of MCV in patients with non-small cell lung cancer in the study We proceeded to determine the presence of MCV virus in lung cancer tissues and in the peripheral blood of lung cancer patients. Results showed that the rate of MCV infection in patients with lung cancer (25%) was significantly higher than that in the control group (5.9%). The results of the correlation analysis showed that MCV infection was related to lung cancer. People who were infected with MCV had 5.33 times higher risk of lung cancer than those without MCV (OR = 5.33). Our research results are similar to some previous studies in the world. A previous study showed that 30 of 163 patients with adenocarcinoma cancer and 2 of 8 patients with squamous cell carcinoma were positive with MCV. NSCLC epidemiological studies conducted in the US and Europe had 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 tumor. These cases have LT antigen manifested in cancer cells and integrated with the cancer cell's chromosome genome. In our study, the MCV infection rate in healthy people was 7.8% and in the lung cancer patients group was 25%, much higher than the studies of other authors in the world. 4.3. Relationship between the level of mRNA expression of CIZ1b, VEGF, EGFR mutations with Markel cell virus infection and some clinical and subclinical symptoms in patients with non-small cell lung cancer. 4.3.1. Relationship between mRNA expression of CIZ1b and VEGF genes with clinical and subclinical in non-small cell lung cancer In this study, we did not find a correlation between mRNA expression of CIZ1b and VEGF with hematological index, ionic graph, abnormal indicators of liver function, renal function and glucose concentration. This suggests that mRNA expression of CIZ1b and VEGF has little to do with subclinical manifestations in patients with lung cancer. The association between mRNA expression of CIZ1b and VEGF with other subclinical characteristics such as lung cancer classification, differentiation of lung cancer tumors and stage of disease development was also not noted. The analysis showed no statistically significant correlation between the expression of CIZ1b and VEGF mRNA with the different forms of lung cancer, cell differentiation, and disease progression. The results of CIZ1b and VEGF mRNA expression between mutated lung cancer patients and the group of lung cancer patients without mutations on the EGFR gene were compared. However, the comparison results showed that the levels of mRNA expression of CIZ1b and VEGF in plasma of these two patient groups were not significantly different. This suggests that mRNA expression of CIZ1b and VEGF is not related to the mutation in EGFR gene. We also compared the mRNA expression of CIZ1b and VEGF among patients with NSCLC and MCV-free. However, the comparison results showed that the levels of mRNA expression of CIZ1b and VEGF in peripheral blood of these two patient groups were not different. This suggests that mRNA expression of CIZ1b and VEGF is not related to MCV factor. 4.3.2. Relationship between MCV infection and EGFR mutation in non-small cell lung cancer In this study, MCV infection was linked to the EGFR mutation rate. The percentage of EGGFR mutants (85%) was higher among NSCLC patients who had MCV infection than those without MCV (29%), and the difference was statistically significant (P = 0.0001). There are two hypotheses that explain this correlation. First of all, the virus' genetic material mutations often occur against the host's protection system or anti-virus agents, so viral infections create genome instability, leading to the occurrence of somatic mutations that cause cancer. It can be said that MCV infection increases the risk of EGFR mutation, which in turn leads to lung cancer. Xu et al. support this by showing that the risk of EGFR mutations was 5.71 times higher in the anatomical form (95% CI: 2.03–16.04, P = 0.001) and 7.84 times in the adenocarcinoma form ( 95% CI: 2.54–24.25, P = 0.0004). Our study showed that patients with MCV infection were 13.8 times more likely to have an EGFR mutation than patients wi