The Research On Blood Micrometastasis And CTAL-4Gene Polymorphism For Patients With Non Small Cell Lung Cancer

Part One:The Correlation Study on Expression of CD44V6and CK19Gene to Micrometastasis in Non small Cell Lung CancerOBJECTIVE:To investigate the expression of CD44v6and CK.19gene in tumors and peripheral bood in patients with non-small cell lung cancer (NSCLC) and its clinical significance for micrometastasis.METHODS:Fifty-six patients with NSCLC (20cases as control) were randomly enrolled into the study. The expression of CD44v6and CK19was investigated by immunohistochemistry in cancer tissues, and Reverse Transcriptase-Polymcrase Chain Reaction (RT-PCR) was used to detect the expression of the two genes in peripheral bood.RESULTS:1. The expression of CD44V6in cancer tissues was significantly higher than that in normal lung tisssucs(66.07%vs.0%, P<0.001). Furthermore, its expression was correlated with clinical stage(P=0.005) and lymph node metastasis(P=0.024) for patients with NSCLC. High expression of CK19was also observed in cancer tissues for the56patients, but no statistical significance was founded when compared with the normal lung tissues in the control group (83.92%vs.90%, P=NS). There was no no correlations were observed between the expression of CK19and the clinicopathological factors in patients with NSCLC.2. In NSCLC group, the expression of CD44V6mRNA and CK19mRNA was detected in the peripheral blood of36(64.29%) patients, and32(57.14%) patients respectively. The expression of CD44V6mRNA and CK19mRNA in the peripheral blood of NSCLC was significantly higher as compared to the control group (P<0.05).The positive rate of CD44V6mRNA expression in peripheral blood of NSCLC patients was closely correlated with the pTNM stages and the status of lymph node metastasis (P=0.016,0.02, respectively). The positive rate of mRNA expression was35.71%,66.67%and86.7%in pTNM staging I, II and Ⅲ patients, respectively. Similarly, the expression of CK19mRNA was also significantly correlated with the pathological TNM stages (P=0.019). The positive rate of CK19mRNA expression was28.57%,59.30%and80.0%in pTNM staging Ⅰ, Ⅱ and Ⅲ patients, respectively.3. The expression of CD44V6was closely correlated with CK19in both cancer tissues and peripheral blood of NSCLC patients (P=0.014,P=0.003, respectively). Combined detection of CD44V6and CK19mRNA in peripheral blood can lead to a higher sensitivity of75.0%in patients with NSCLC.CONCLUSION:Detection of CD44V6and CK19in both cancer tissues and peripheral bood might be proper molecular markers for peripheral circular cancer cells in patients with NSCLC. Combined measurement of the two genes can further improve the diagnostic sensitivity of NSCLC.Part two:Impacts of Surgical Procedures for Pulmonary Lobectomy on Blood Micrometastasis of Non Small Cell Lung CancerObjectives:The aim of this study was to investigate the impacts of different sequences in ligating pulmonary vessels during pulmonary lobectomy on blood micrometastasis of NSCLCMaterials/Methods:Cytokeratin19(CK19) and adhesion molecule CD44V6mRNA was used as molecular markers, A total of30patients with NSCLC undergoing pulmonary lobectomy were randomly divided into two groups (15cased in each group) according to the ligating sequence of the pulmonary vessels during the procedure of lobectomy. In PA-first group, pulmonary artery was ligated first; as in PV-first group, pulmonary vein was ligated in the first hand. Fluorescent quantitative RT-PCR technique was used to detect the mRNA expressions of CK19and CD44V6in pulmonary venous blood during surgery at different time-points accordingly. ACt values were calculated. Meanwhile, the peripheral blood samples from15healthy volunteers were served as control. Results: ACt values of CD44V6mRNA and CK19mRNA in NSCLC groups at early period during surgery were7.93±1.70and10.76±2.74, compared to9.47±1.59and13.10±3.04in the control group. The expression of CD44V6mRNA and CK19mRNA in venous blood of NSCLC patients were significantly higher as compared to the control (P<0.05). In addition, the ACt values of CD44V6mRNA and CK19mRNA at early period during surgery in PA-first group were7.92±1.97and11.21±3.14, versus5.67±2.11and8.60±4.02respectively at the late period. In PA-first group.the expressions of CD44V6mRNA and CK19mRNA at late period were both significantly higher than those at the early period(P<0.05),while neither the ACt value of CD44V6mRNA nor that of CKl9mRNA at early and late periods in PV-first group displayed statistically significant differences (7.95±1.91vs7.74±2.10and10.60±3.15vs10.30±2.98, P>0.05).Conclusions: The surgical manipulation itself might stimulate the occurrence of blood micrometastasis, thus the ligation of pulmonary vein first during pulmonary surgery may help prevent blood micrometastasis.Part Three: Research of CTLA-4+49A>G Polymorphism in Non Small Cell Lung Cancer and its Relationship with Blood Micrometastasis.Objectives: It was reported that Cytotoxic T-lymphocyte antigen-4(CTLA-4). as a potential immunoregulatory molecule, can suppress anti-tumor response by down-regulating T-cell activation. And the+49A>G polymorphism of CTLA-4gene was associated with several autoimmune diseases. However, little is known about functional polymorphism of CTLA-4in NSCLC and the association with blood micrometastasis.The current section was aimed to investigate functional polymorphism of CTLA-4in NSLCL and the association with blood micrometastasis.Methods: The CTLA-4+49A>G polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in158patients with NSCLC and72healthy controls.RESULTS: The frequencies of CTLA-4+49GG, GA and AA were detected in44.30%,41.77%and13.92%patients with NSCLC, respectively. The frequency of the CTLA-4+49AA genotype was significantly higher in patients with NSCLC compared with the corresponding controls (P=0.046). The odds of carrying the CTLA-4+49AA genotype in NSCLC group were1.489(95%CI,0.977-2.060) as compared with the CTLA-4+49GG genotype. No significant association was observed between CTLA-4+49A>G polymorphism and clinicopathologic features of NSCLC patients including gender, histopathological type, clinical stage, tumor markers (CEA and CYFRA21-1), expressions of CK19mRNA and CD44V6mRNA (P>0.05).CONCLUSION:The polymorphism of CTLA-4+49A>G may be the susceptible factors o f NSCLC, and the AA genotype might be one of the susceptibility genes to NSCLC.