Expression Of Ciz1b In Patients With Non-Small Cell Lung Cancer – Ho Van Son

Journal of military pharmaco-medicine n 0 8-2018 130 EXPRESSION OF Ciz1b IN PATIENTS WITH NON-SMALL CELL LUNG CANCER Ho Van Son1,2; Ngo Tat Trung1,3; Nguyen Linh Toan1 SUMMARY Introduction: Primary lung cancer is one of the most common malignant diseases. The expression of Ciz1b appears to be limited to tumor cells, suggesting that this variant can be used as a marker for the cancer. This study is to evaluate the expression and diagnostic role of Ciz1b in lung cancer patients. Subjects and methods: 100 patients with non-small cell lung cancer and 51 healthy controls were recruited for this study. Relative expression of Ciz1b mRNA was determined by realtime PCR. Results: Increased Ci1zb mRNA expression was observed in patients with non-small cell lung cancer compared to healthy individuals (p = 0.008). The expression of Ciz1b mRNA was negatively correlated with the ALT enzyme levels (Spearman's r = -0.22, p = 0.045) and with the AST levels (Spearman' r = -0.2, p = 0.068). The level of mRNA expression of the Ci1zb gene was significantly associated with tumor cell differentiation in patients with non-small cell lung cancer. Ciz1b mRNA expression in serum was capable of distinguishing lung cancer patients from healthy control (AUC = 0.602, p = 0.05). Conclusion: The expression of Ciz1b is regulated by the occurrence of lung cancer and Ci1zb plays an important role in lung cancer development, as well as has the potential as a biomarker for diagnosis and prognosis of lung cancer. * Keywords: Lung cancer; Non-small-cell lung carcinoma; Ciz1b; mRNA expression. INTRODUCTION Primary lung cancer is one of the most common malignant diseases and also is one of the leading causes of death from cancers. Lung cancer accounts for about 12% of all types of cancers and 18% of cancer-related deaths [1, 2]. Of these, non-small-cell lung carcinoma (NSCLC) accounts for more than 80% of lung cancer. In Vietnam, lung cancer has increased rapidly due to smoking, air pollution, dust emissions and industrial waste. According to the Ministry of Health, there are about 20,000 to 30,000 new cases of lung cancer annually and about 90% of primary lung cancer patients die in the first five year [1]. Ciz1 (Cip1-interacting zinc finger protein) enhances the activation of DNA replication in mammals by interacting with cyclin- dependent E and A protein kinases. It interacts directly with E and A cyclins, CDK2, and p21 cyclin-dependent kinase inhibitors. It also plays an important role in DNA replication by regulating the expression of genes, including cyclin D, which influence cell proliferation [3]. Normally, Ciz1 is attached to the salt- and nuclease-resistant protein 1. 175 Military Hospital 2. Vietnam Military Medical University 3. 108 Military Central Hospital Corresponding author: Nguyen Linh Toan (toanntl@gmail.com) Date received: 02/08/2018 Date accepted: 20/09/2018 Journal of military pharmaco-medicine n 0 8-2018 131 component of the nucleus called a "nuclear matrix". Ciz1 is in the spatial structure of DNA replication. Some recent studies described a Ciz1 variant “missing the C-terminal region associated with the nuclear matrix attachment and the expression of this stable variant appears to be limited to tumor cells, suggesting that this variant can be used as a marker for cancer”. Recent studies showed that determining this variant of Cip1 can be used for early diagnosis of lung cancer [3]. A previous study measured the expression of Ciz1 using Western blot and found that Ciz1 was present in the lung tumor, but was absent in the nearby tissues. The study also found that the Ciz1 variant accurately identified patients with stage I lung cancer [4]. The data showed that the Ciz1 variant was an important candidate for a specific marker of cancer, which can detect early stage lung cancer in the risky groups [5]. However, there are no reports of the expression of this gene at diagnosis or prognosis for NSCLC in Vietnam. This study is to Evaluate the expression and diagnostic role of Ciz1 in lung cancer patients. SUBJECTS AND METHODS 1. Subjects. The study was conducted in a cross- sectional model, described in combination with clinical examination and laboratory analysis. 100 patients with NSCLC were diagnosed by histopathology or cytology. Patients were grouped according to the stages of the disease according to TNM classification criteria [6]. The control group consisted of 51 healthy individuals who had the corresponding age as those with lung cancer. The group of healthy people was examined for health and were confirmed to be healthy and agreed to participate in the study. 2. Methods. * Preclinical and clinical examination: 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 points) [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 -20°C until use. * Expression of Ciz1b mRNA: The expression level of Ciz1b 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 don’t need this were synthesized from RNA using the Thermo Scientific cDNA kit. You have already said this at the beginning of the paragraph. Each realtime PCR reactions include Master Mix SYBR Maxima 2X, Primers 5 pm (Ciz1b and ABL), Water and cDNA template. The total volume of each reaction is 12 μL. Journal of military pharmaco-medicine n 0 8-2018 132 The thermal cycle for the realtime PCR reaction for ABL and Ciz1b was 50°C for 2 mins, 95°C for 10 mins, 95°C for 15s, and repeat for 45 cycles. * Statistical analysis: Data were analyzer using SPSS V.19. The diference between groups was analyzed using Mann-Whitnay U test for non- parameter variables. Sperman correlation coefficient (RS) was used to investigate the relationship between continuous variables. p < 0.05 was considered statistical significance. RESULTS 1. Baseline characteristics of the study subjects 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 males and 27 females. 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). 2. Clinical characteristics of lung cancer patients. On admission, the common clinical symptoms were coughing, chest pain, difficulty breathing and weight loss. 49 patients (49%) had coughing, 43 patients (43%) had chest pain, 12 patients (12%) had shortness of breath, 7 patients (7%) had weight loss and 24 patients (24%) had other clinical symptoms. Of the 100 patients with lung cancer, 34 had tumors in the upper lobes of the right lung, 26 had lobular tumors in the left lung, 18 had lobular tumors in the right lung, 13 had tumors in the lower lobes of the left lung and 9 patients with tumors in the right lobe of the lung. According to the TNM classification system [6], patients were classified into stages IA, IIA, IIIA, IIIB and IV. The majority of patients were in stage IV (76%), 15% in stage IIIB, 5% of patients was in stage IIA, and only 2% of patients were in stages IA and IIIA. In terms of tumor cell differentiation, 56% of patients had grade 2, 35% of patients had grade 3, and 9% of patients had grade 1. These results indicated that the majority of patients were at the late stages. General ultrasound was performed for 82 patients and we found 38 patients (46%) had abnormal images. Bone morphogenesis was performed for 63 patients, 34 of whom accounted for 54% of abnormal images. Radiography revealed that 49/51 patients (96.1%) had abnormal images. CT images showed 82/93 patients (88%) with tumors, 18/93 patients (19%) with pleural effusion, and 8/93 patients (8.6%) with thickened lung membranes. PET-CT was performed for 26 patients and 100% of patients had abnormal images, of which 16/26 patients (61.5%) had tumors, 7/26 patients (26.92%) had pulmonary effusion, 13/26 patients (50%) had malignant lesions, and 17/26 patients (65.4%) had lymph nodes. Journal of military pharmaco-medicine n 0 8-2018 133 3. Expression of Ciz1b in patients with lung cancer and healthy individuals. Figure 1: Expression levels of Cizb1 mRNA in patients and controls. The relative expression of the Ciz1b gene in the plasma of patients with lung and healthy cancers is calculated by the ΔCt method with the expression of ALB gene as the reference. The analysis showed that the expression of the Ciz1b gene in patients with lung cancer (10.02 ± 4.83) was significantly higher than that in the controls (7.75 ± 2.63) (p = 0.008). 4. Correlation between Ciz1b expression and clinical parameters. Table 1: Association between Ciz1b expression and differentiation of tumor. Gene Lung cancer (n = 63) Differentiation p Grade 1 (n = 6) Grade 2 (n = 35) Grade 3 (n = 22) Ciz1b relative expression 10.02 ± 4.83 6.55 ± 2.49 9.98 ± 4.34 11.5 ± 1.19 < 0.05 Tumor differentiation reflects the degree of malignancy of cancer cells. To assess the association of Ciz1b expression with tumor development, we compared the expression level of Ciz1b with degrees of differentiation including grade 1, grade 2, and grade 3. The results showed that Ciz1b mRNA expression was the lowest in the group with grade 1, increased in the group with grade 2 and the highest in the group with grade 3 (p < 0.05). These results suggested that the expression levels of Ciz1b increased with the degree of differentiation of the tumor and was closely related to the development and differentiation of lung cancer cells. Journal of military pharmaco-medicine n 0 8-2018 134 Figure 2: Correlation between Ciz1b mRNA expression and liver enzyme levels. We also analyzed the association of Ciz1b expression with other clinical parameters. Results showed that Ciz1b mRNA expression was negatively correlated with ALT enzyme levels (Spearman's r = -0.22, p = 0.045) and with the AST levels (Spearman' r = -0.2, p = 0.068). However, there was no association between Ciz1b mRNA levels with analyzed clinical parameters (p > 0.05). In addition, Ciz1b mRNA expression was not correlated with the level of hemoglobin, hematocrit, red blood cells, lymphocytes, platelet counts and monocytes (p > 0.05). Ciz1b mRNA expression was also not correlated with the ion index tests such as Na+, K+ and Cl- concentrations, kidney function tests such as urea and creatinine, and with cancer markers such as CEA and CyFra 21.1 (p > 0.05). 5. Potential diagnostic function of Ciz1b expression for lung cancer. Figure 3: Diagnostic potential of Ciz1b mRNA expression in lung cancer. Journal of military pharmaco-medicine n 0 8-2018 135 Ciz1b mRNA expression was significantly different between lung cancer patients and controls, and was associated with the differentiation of lung cancer tumor. Therefore, Ciz1b mRNA expression may have a potential for screening and prognosis of lung cancer. We analyzed the effectiveness of Ciz1b expression for identification of lung cancer from the control group. The results showed that Ciz1b mRNA expression in serum was capable of distinguishing lung cancer patients from healthy control. However, the diagnostic efficacy was not high, and the AUC was only 0.602 (p = 0.05). DISCUSSION Primary lung cancer is one of the most common malignant diseases and also is one of the leading causes of death from cancers. Ciz1 plays an important role in triggering DNA replication by regulating the function of cyclin E and A-dependent protein kinases. Ciz1 also interacts directly with cyclins E, cyclins A, CDK2 (cyclin-dependent kinase 2) and with the cyclin-dependent p21 kinase inhibitor to regulate DNA replication and the expression of genes involved in the cell cycle including cyclin D [8]. However, only a few studies have addressed the role of the Ciz1b gene in lung cancer. The level of Ciz1 expression has been shown in early cell differentiation. Some variants of Ciz1 that are involved in cancer development are deficient in exon 4 and the expression of different Ciz1 variants is closely related to the expression of the cyclin A1 protein. This is a vital protein in the development and differentiation of cells [9]. A previous study showed that the Ciz1b had the potential as a biomarker that was usually found in patients with lung cancer but not in healthy individuals [3]. In that study, the authors used the Western blot method for protein detection, which was quite complex and was not optimized to be a method used for diagnostic purposes. Therefore, theELISA method of quantifying serum protein levels has been established [3]. In our study, specific primers were designed to amplify and quantify the expression of Ciz1b mRNA in the serum by realtime PCR, which provides a rapid and accurate result regarding the expression of Ciz1b mRNA in the serum. Another recent study has shown that Ciz1b was sensitive enough to identify patients with stage I cancer in high-risk groups, including patients with benign pulmonary lung disease, pneumonia, asthma, chronic obstructive pulmonary disease (COPD) and smokers [3]. This study indicated that cancer patients had significantly higher levels of Ciz1b compared to those in controls and the Ciz1b levels also increase with age in patients with NSCLC. The study also found that Ciz1b levels in patients with stage I lung cancer were significantly higher compared to smokers, and Ciz1b levels were the highest in patients with basal cell carcinoma followed by adenocarcinoma, pneumothorax and patients with benign neoplasia. In our study, the level of mRNA expression of the Ciz1b gene in the serum of patients with NSCLC was significantly higher than that of controls. This result confirms Ciz1b gene expression is affected by the Journal of military pharmaco-medicine n 0 8-2018 136 disease status, suggesting that Ciz1b plays an important role in the development of lung cancer. The results also suggest that Ciz1b gene may be used as a biomarker for diagnosis and prognosis of lung cancer [5]. The study showed that Ciz1b levels help diagnose cancer patients with an accuracy threshold of 98% for patients with NSCLC and controls (AUC = 0.96). When comparing patients with NSCLC in stage I with asymptomatic smokers, or those with benign pulmonary lymph nodes, the AUC reached 0.913, and 0.905, respectively. In this study, we also analyzed the diagnostic potential of Ciz1b for patients with NSCLC and the results showed that Ciz1b was capable of detecting patients with NSCLC (AUC=0.602). Therefore, determining the presence of Ciz1b in the serum of high-risk groups is an effective non-invasive method for diagnosing, screening and predicting lung cancer. CONCLUSION Increased Ci1zb mRNA expression in patients with NSCLC was observed compared to healthy individuals. Ci1zb mRNA expression levels were associated with tumor cell differentiation and correlated with liver enzyme levels. Ci1zb plays an important role in lung cancer development and has the potential as a biomarker for diagnosis and prognosis of lung cancer. REFERENCES 1. Nguyễn Hải Anh, H.H.T. 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