Study On Micrometastasas In Peripheral Blood Of Non-small Cell Lung Cancer

Background&Objective:Lung cancer is one of the most frequent malignancies.The incidence in China is gradually increasing yearly;it is especially higher in Xuanwei and Gej iu area of Yunnan province than the rest of the nation.Although the effect of surgical,radiotherapy and chemotherapy of lung cancer has increased in recent years, while,gene and biological technology to carry out,all result in the treatment of lung cancer in a new level.Unfortunately,long-term treatment outcome of lung cancer is still unsatisfactory.Study shows that local micro-metastasis not easily detected by routine clinical examinations is an important cause for tumor recurrence and metastasis.So in order to increase the effect of treatment with tumor,we have to diagnosis and treatment as earlier as we can,on the other hand we should also emphasize judge the rank of recurrence when at the first treatment and consider how to prevent and treatment of tumor recurrence on timely.On this study,we make use of CK19,LUNX,MUC1 three genes as molecular markers to monitor micro-metastasis in peripheral blood of NSCLC patients to be able to set up a simple non-invasive and effective method of screening,diagnosis,and prediction postoperative status of micro-metastasis.Methods:(1) To detect the presence of CTCs and evaluate their relationship to patients' clinicopathologic features,we used Trizol extraction total RNA in peripheral blood.In order to explore the possibility of three genes as micrometastases molecular markers in peripheral blood and relationship with clinicopathologic features,the expression of CK19mRNA,MUC-1 mRNA and LUNX mRNA were analyzed by nested RT-PCR in 60 NSCLC in peripheral blood,compared with 15 patients with benign lung disease and 20 healthy volunteers,we usedβ-actin as internal control. (2)On the base of the part one the expression of levels of molecular markers in peripheral blood of 60 NSCLC patients and 60 healthy volunteers were evaluated by real-time Q-PCR.The quantity of expression ratio of CK19mRNA was normalized toβ-actin.Receiver operating characteristic(ROC) curve analyses were carried out to determine cut-off value.Results:(1)This study used nested RT-PCR technology with sensitivity up to 10~6.The results of study show that:The Expressions of CK19mRNA,MUC-1 mRNA and LUNX mRNA were 51.6%(31/60 ),80.0%(48/60 ) and 55%(33/60) in peripheral blood of 60 NSCLC patients.In the control group,There was no expression of LUNX mRNA in 15 patients with benign lung disease and 20 healthy volunteers.One healthy volunteer expression CK 19mRNA,while the expressions of MUC-1mRNA in 15 patients with benign lung disease and 20 healthy volunteers were 60.0%(9/15) and 65.0%(13/20).(2)Using ROC curve to analysis the real-time Q-PCR results of CK19mRNA,when the cut-off value is 0.3031,its sensitivity and specificity was 91%and 80.1%respectively.Conclusions:(1)The use of nested RT-PCR technology to detect CTCs in peripheral blood of NSCLC patients is feasible and this method has high sensitivity and specificity.(2) CK19mRNA and LUNX mRNA might be specific and sensitive markers to detect CTCs in peripheral blood of NSCLC patients,while the value of MUC-1 as the marker of micrometastases in peripheral blood is not sure.(3)We have developed a real-time Q-PCR method to detect the CTCs in peripheral blood of NSCLC patients.(4)During the clinical diagnosis for NSCLC,the cut-off value of CK19 should be set by ROC curve analysis.However,a further study with long-term follow up in a larger number of patients is required to confirm the clinical application of this molecular marker.