| Part1 The Application of Immunomagnetic Bead Negtive Enrichment and Targeted Fluorescent Quantitative Polymerase Chain Reaction Technique in Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Non-small Cell Lung CancerObjective:To study the correlation between peripheral blood circulation tumor cells(CTCs)and clinicopathological data and operative staging in patients with non-small cell lung cancer(NSCLC),and to analyze the feasibility of its application in early clinical diagnosis.Methods:CTCs were captured from peripheral blood of 136 patients with non-small cell lung cancer,10 patients with benign lung diseases and 54 healthy volunteers by immunomagnetic bead negative enrichment targeting folic acid.The tumor specific folate ligand oligonucleotide conjugates were used to mark CTCs,which would be amplified by quantitative PCR.We analyzed the correlation between the CTCs value and the age,sex,tumor size,histological classification,and clinical TNM stage.We evaluated the sensitivity and specificity of CTCs detection in diagnosis of NSCLC.We compared the CTCs with serum tumor markers,CEA,CA125,CA724,CYFRA21-1 and NSE,in diagnostic efficacy.Results:(1)The median CTC was 4.95 FU/3ml,in healthy volunteers,7.55 FU/3ml in benign lung disease group and 11.21 FU/3ml in lung cancer group.The level of CTC in lung cancer group was significantly different from that in healthy group(Kruskal-Wallis test,P=0.000)and benign lung disease group(Kruskal-Wallis test,P=0.018).The level of CTC in healthy group was significantly different from that in benign lung disease group(Mann-Whitney U test,P=0.000).(2)The median CTC of lung cancer patients was stage ?11FU/3ml,stage ? 13.25FU/3ml,stage ? 14.77FU/3ml,stage ?17.89FU/3ml.Differences between the four groups were obvious statistical significance(Kruskal-Wallis test,P=0.001).There was no significant difference in CTC level among different T-staging,differentiated degree,age,sex,tumor diameter,and pathological subtype(Kruskal-Wallis test:T1 vs T2 vs T3 vs T4,P=0.254;G1 vs G2 vs G3,P=0.424;Mann-Whitney U test:<60 years old vs?60 years old,P=0.853;male vs female,P=0.739;?3cm vs>3cm,P=0.322;Squamous cell carcinoma vs Adenocarcinoma,P=0.497).(3)When CTC=8.70 FU/3mL was used as cutoff value,the sensitivity was 79.4%,the specificity was 98.1%,and the area under the curve was 0.953(95%CI:0.926,0.979).The diagnostic efficiency was better than other serum tumor markers such as CEA,CA125,CA724,CYFRA21-1,NSE and so on.There was no significant difference between CTC positive rate and age,sex,tumor maximum diameter,differentiation grade,pathological subtype and TNM stage(P=0,319,0.972,0.341,0.268,0.125,0.208).Conclusion:By analysing CTCs in peripheral blood of NSCLC patients with immunomagnetic bead negtive enrichment and targeted fluorescent quantitative PCR technique,this study puts forward the diagnostic advantage of CTCs detection,which can be used for lung cancer screening.Part2 The Application of Circulating Tumor Cells Detection Combined with pulmonary biopsy and 18F-FDG Positron Emission Tomography/Computed Tomography in Early Diagnosis of peripheral Pulmonary NodulesObjective:To compare the advantages and disadvantages between 18F-FDG PET/CT imaging,pulmonary biopsy and peripheral blood circulation tumor cells(CTCs)performing on patients with peripheral pulmonary nodules before surgery,and to analyze the feasibility of their applications in early clinical diagnosis.Methods:18F-FDG PET/CT examination,pulmonary biopsy and peripheral blood circulation tumor cells(CTCs)were performed before operation for all 44 patients with peripheral pulmonary nodules.The sensitivity,specificity and accuracy of the three methods were compared to infer the early diagnostic efficacy of peripheral pulmonary nodular disease.Results:(1)The value of 18F-FDG PET/CT SUV max was 2.35<2.5(positive diagnostic standard value).(Wilcoxon test,P=0.469>0.05).The sensitivity of 18F-FDG PET/CT was 36%,the specificity was 57.89%,the accuracy was 45.45%,kappa=0.058,and P=0.680>0.05.The consistency between PET/CT and the pathological results was poor,and there was no statistical significance.(2)The median CTC of this group was 9.35FU/3ml,the sensitivity was 88%,the specificity was 84.21%,the accuracy was 86.36%,kappa=0.722,and P=0.00.The consistency between CTC and the pathological results was average,and there was statistical significance.(3)The sensitivity of percutaneous lung biopsy was 84%,the specificity was 100%,the accuracy was 90.91%,kappa=0.819,and P=0.000.The consistency between percutaneous lung biopsy and the final pathological results was good,and there was statistical significance.(4)The ROC curve was drawn by CTC and SUVmax values.The AUC value was 0.965(95%CI:0.916,1.000)and 0.656(95%CI:0.487).The diagnostic efficacy of CTC was significantly better than that of PET/CT.Conclusion:For early malignant peripheral pulmonary nodular disease,especially ground-glass lesion,the intake of 18F-FDG PET/CT is low,which is likely to cause false negative results of PET/CT,resulting in low diagnostic efficacy.Pulmonary biopsy,although sensitive,specific,and accurate,is an invasive procedure and prone to complications.Peripheral blood CTC detection is fast,convenient,non-invasive,reproducible,and highly diagnostic.It could offset the shortcomings of the former two methods and make great contribution to the diagnosis of early peripheral lung cancer. |
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