Study clinical, paralcinical features and expression of vegf gene in serum of patients with non-small cell lung cancer – Ho Van Son

Journal of military pharmaco-medicine n 0 9-2018 101 STUDY CLINICAL, PARALCINICAL FEATURES AND EXPRESSION OF VEGF GENE IN SERUM OF PATIENTS WITH NON-SMALL CELL LUNG CANCER Ho Van Son1,2; Ngo Tat Trung3; Nguyen Linh Toan1 SUMMARY Objectives: To investigate clinical, subclinical features and expression of VEGF gene in non- small cell lung cancer in order to accurately diagnose the disease at an earlier stage, before the disease has developed, which can lead to a worse prognosis. Subjects and methods: One hundred patients with non-small cell lung cancer and 51 healthy subjects participated in an analytical cross-sectional study with comparison group. Results: The relative expression mRNA of the VEGF gene in the study group was significantly higher compared with the control group (p < 0.01). The level of mRNA expression of VEGF was the lowest at grade 1, that increased at grade 2, and it reached the highest level at grade 3 (p < 0.05). The mRNA expression of VEGF in serum was capable to distinguish the patients with lung cancer from healthy people by the AUC value reached 0.615 (p = 0.05). Conclusions: The significantly expression of mRNA of VEGF gene in non-small cell lung cancer patients contributes to diagnosis and prognosis of the disease. * Keywords: Lung cancer; Non-small cell lung cancer; Expression of VEGF gen; Clinical, paraclinical features. INTRODUCTON Primary lung cancer (PLC) is one of the most common malignant diseases today and is the leading cause of death in cancer diseases. According to the International Agency for Research on Cancer (IARC,) lung cancer accounts for about 12% of all types of cancers and death rates account for 18% of cancers [1, 4]. Among them, non-small cell lung cancer (NSCLC) accounts for more than 80% of PLC cases. In Vietnam, lung cancer has increased rapidly, related to smoking, air pollution, dust emissions and industrial waste. About 90% of patients with primary lung cancer die within the first five years [1]. The prominent feature of NSCLC is the increased manifestation of the epithelial growth factor receptor (EGFR) and the vascular endothelium growth factor receptor (VEGFR). The receptors of cells often carry tyrosine kinase activity which depends more on the interaction between the extracellular region and the growth factors or free- radicals in the cytoplasm. When VEGF is activated and transmitted to the cell nucleus, it promotes cell proliferation and also increases the probability of cancer cells to spread to neighboring tissues. Several studies have demonstrated that expressive VEGF gene 1. Vietnam Military Medical University 2. 175 Military Hospital 3. 108 Military Central Hospital Corresponding author: Nguyen Linh Toan (toannl@vmmu.edu.vn) Date received: 12/10/2018 Date accepted: 23/11/2018 Journal of military pharmaco-medicine n 0 9-2018 102 has the potential to be used in NSCLC diagnosis with sensitivity and specificity of 80 - 90% [3, 4]. This is one of the biomarkers being studied for use in clinical practice guidelines for lung cancer in developed countries around the world. Although studies had been shown the VEGF expresion in cancer tissue of patients with NSCLC. However, there are no published studies on the expression of the VEGF in serum of NSCLC in Vietnam. In this study, we conducted a study to: Evaluating the association of mRNA expression of VEGF in serum patients with NSCLC. SUBJECTS AND METHODS 1. Subjects. 100 patients with NSCLC were diagnosed with histopathology. Patients were grouped according to stage of disease following TNM classification criteria [5]. Excluding from the study group the patients who did not agree to participate in the study, had small cell lung cancer or metastatic lung cancer. The controls consisted of 51 healthy subjects who had a periodontal health examination with the same age group as those with lung cancer. The healthy people were examined for health and confirmed to be healthy and agreed to participate in the study. 2. Methods. * Preclinical and clinical examination: A cross-sectional descriptive study, comparative control, laboratory combination. * Clinical research methods: All patients and the control group were examined clinically and laboratory tests such as hematology, biochemistry, imaging (chest X-ray, chest CT) were performed. The overall condition of the participants was assessed using KPI (KPI: Karnofsky Performance Index from 0 - 100) [7]. Patients were classified according to the classification criteria [6]. The procedure for sampling and storing samples was conducted at 175 Military Hospital. 5 mL of blood from each participant, both the patients and healthy people was stored at -80°C until use. * Expression of serum VEGF gene level: The expression level of VEGF in the serum of patients with lung cancer and control group was determined by realtime PCR. Total RNA was extracted from plasma samples of patients and healthy individuals using an RNA extraction kit following the manufacturer's instructions. cDNA were synthesized from RNA using the Thermo Scientific cDNA kit. Each realtime PCR reactions include Master Mix SYBR Maxima 2X, Primers 5 pm (VEGF and ABL), Water and cDNA template. The total volume of each reaction is 12 µL. The thermal cycle for the realtime PCR reaction for ABL and/or VEGF was 50°C for 2 mins, 95°C for 10 mins, 95°C for 15s, and repeat for 45 cycles. * Statistical analysis: Data were analyzed using SPSS v.19. The difference between groups was analyzed using Mann-Whitney U test for non-parameter variables. Spearman correlation coefficient (rs) was used to investigate the relationship between continuous variables. p < 0.05 was considered statistical significance. Journal of military pharmaco-medicine n 0 9-2018 103 RESULTS 1. Baseline characteristics of the study subjects. * Age and gender: Lung cancer patients had an average age of 60 years, while the control group had an average age of 53.88 years. No significant difference in age between patients with lung cancer and the healthy controls was observed (p = 0.158). In terms of gender distribution among the patients, 73 were male and 27 were female. There were 35 men, and 16 women in the control group. No significant difference in gender distribution between the two groups was found (p = 0.225). * Clinical symptoms: Cough was the most common symptoms accounted for 83%, followed by chest pain, made up 56%, dyspnea held 20%, weight loss accounted for 17% and lymph nodes accounted for 8%. * Location of cancer: Cancer happened more often in the left lung: 61/100 patients (61%) with tumors in the right lung (34 in the upper lobes, 9 in the middle lobe, and 18 in the lower lobe), 39/100 patients (39%) had left lung tumors (26 with tumors in the upper lobe, 13 patients with tumors in the lower lobes). * Histopathology: Histopathologic results showed 91/100 patients (91%) with adenocarcinoma, and 9/100 patients (9%) with squamous cell carcinoma. * Classification according to TNM: In 91 patients with NSCLC, the patients were classified into stage IA, IIA, IIIA, IIIB and IV according to the TNM classification system [5], the majority of patients were in the late stage IV (76%), 15% of patients were in stage IIIB, 5% of patients were in phase IIA, and only 2% of patients were in stages IA and IIIA. 2. Expression of VEGF in patients with lung cancer and healthy individuals. Figure 1: VEGF mRNA expression level in the study group The relative mRNA expression of the VEGF gene in NSCLC and healthy subjects was calculated by ∆Ct adjusted with the expression of the ALB gene. The analysis also showed that the relative expression of VEGF gene in the group of patients with lung cancer was significantly higher than that of the healthy group (p < 0.01). Journal of military pharmaco-medicine n 0 9-2018 104 3. Correlation between VEGF expression and clinical parameters. Table 1: Relationship between VEGF expression and tumor differentiation mRNA lung cancer. Differentiation mRNA Lung cancer (n = 63) Grade 1 (n = 6) Grade 2 (n = 35) Grade 3 (n = 22) p VEGF 2.68 ± 3.28 1.85 ± 0.65 1.96 ± 1.39 3.06 ± 2.88 < 0.05 Tumor differentiation reflects the degree of malignancy of cancer cells. To evaluate the association of VEGF gene expression with tumor differentiation, we compared VEGF expression levels between different degrees of differentiation including grade 1, grade 2, and grade 3. The relative level of VEGF expression was compared between the three groups. The relative levels of mRNA VEGF expression were the lowest in grade 1, increased in grade 2 and the highest in grade 3 (p < 0.05). The level of expression of VEGF increased with the degree of differentiation of the tumor and was closely related to the development and differentiation of lung cancer cells. Analysis of the association of VEGF expression with other subclinical indices showed no correlation between VEGF mRNA expression with hematological index, glucose concentration, electrolyte index such as Na+, K+ and Cl-, liver and kidney function and disease stage (p > 0.05). 4. The diagnostic value of lung cancer of serum mRNA VEGF expression. Analysis of the diagnostic value of serum mRNA VEGF expression for lung cancer, we analyzed the area under the mRNA VEGF expression curve between lung cancer patients and healthy people (figure 2). Figure 2: The area under the AUC curve expressing of serum mRNA VEGF. The mRNA VEGF expression level in serum was capable of distinguishing patients with lung cancer and healthy people by the area under the AUC curve reached 0.615 (sensitivity 0.7, specificity 0.56, p = 0.05). Journal of military pharmaco-medicine n 0 9-2018 105 DISCUSSION Primary lung cancer is one of the most common types of malignancy and is the leading cause of death in cancer. Our research showed that the mRNA expression of VEGF gene in the serum of lung cancer patients was significantly higher than that of healthy individuals. This suggested that determining the level of VEGF gene expression in plasma may be one of the potential non-invasive methods for the screening of lung cancer. Of all the vascular factors involved the process of vessel formation related to tumor, VEGF is known to be one of the most important factors in normal physiological and pathological conditions. Evaluating the expression of VEGF gene in cells or tissues is very important in studying the role of VEGF in vascularization [7]. Studies showed that VEGF expression is closely related to NSCLC [8]. A study evaluating the expression of mRNA VEGF in three groups of patients included squamous cell carcinoma, adenocarcinoma, and no cognitive cell carcinoma. Results showed that in 65% of cases; the expression of mRNA VEGF was higher in cancer tissue than in normal tissue. mRNA VEGF expression was higher in non-squamous cell carcinoma and higher in tumors with lymph node metastases [9]. Similarly, our study also showed that peripheral blood mRNA VEGF expression was significantly higher in patients with lung cancer than in the control group. In a collective analytical study of 5,386 lung cancer patients from 51 different studies worldwide, it has been shown that overexpression of VEGF has a negative impact on the survival time of both cancer patients with NSCLC and small cell lung cancer [10]. A study evaluated the potential for applicability in diagnosis of mRNA VEGF for lung cancer. Studies have shown that the expression of mRNAs VEGF is significantly higher in the cancer group than in the benign group and the signal of mRNAs VEGF gained more than 89% of sensitivity and 90% accuracy [11]. Another study evaluating the role of VEGF in the growth and maturation of lung cancer found that the expression of VEGF not only enhanced the proliferation of epidermal cells, but also stimulated differentiation of lung cancer cells [12]. In this study, there was a marked increase in mRNA VEGF expression in patients with lung cancer compared to healthy subjects (p < 0.01) and a correlation between mRNA VEGF expression with the differentiation of the tumor (p < 0.05). This suggested that the level of VEGF expression increased with the differentiation of the tumor (p < 0.05). Comparative analysis showed that VEGF gene played an important role in the development and differentiation of cancer cells. In our study, the diagnostic value analysis of lung cancer patients showed that serum mRNA VEGF expression was capable of identifying lung cancer patients with an AUC of 0.615. Therefore, this study, together with previous studies, has shown that evaluating mRNA VEGF expression in plasma is a non-intervention that allows diagnosis of lung cancer in high-risk groups, as in patients with lung disease, a family history of lung cancer, or in groups frequently exposed to toxic agents such as smokers, mining workers. Journal of military pharmaco-medicine n 0 9-2018 106 CONCLUSION There is a marked increase in mRNA VEGF expression levels in plasma in patients with NSCLC compared with healthy controls (p < 0.01). The expression level of VEGF gene was associated with tumor cell differentiation of NSCLC patients (p < 0.05). REFERENCES 1. Nguyễn Hải Anh, H.H.T. Tình hình ung thư phổi tại Khoa Hô hấp, Bệnh viện Bạch Mai trong 10 năm từ 1991 - 2000. Tạp chí Y học Thực hành. 2005, 513, tr.3. 2. Hưng Đ.K. Nghiên cứu về lâm sàng, X quang phổi chuẩn và một số kỹ thuật xâm nhập để chẩn đoán ung thư phổi nguyên phát. Luận án Tiến sỹ Y học. Học viện Quân y. Hà Nội. 1995. 3. Khoa T.Đ. Nghiên cứu giá trị của phương pháp xạ hình SPECT 99mTc-MIBI trong chẩn đoán và đánh giá kết quả điều trị ung thư phổi. Luận án Tiến sỹ Y học. Học viện Quân y. Hà Nội. 2011. 4. Dubey S, C.A. Powell. Update in lung cancer 2006. Am J Respir Crit Care Med. 2007, 175 (9), pp.868-874. 5. Sobin L.H G, Wittekind C.H. 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