| Background:Solitary pulmonary nodule(SPN)is defined as single nodule which has a maximum diameter that no more than 3.0cm,and surrounded by gas-filled lung tissue,without atelectasis,hilar enlargement or thoracic cavity fluid.Imaging features are characterized by higher density or ground-glass shadows.With the wide use of low-dose spiral CT(LDCT),more and more people undergo lung screening examination,and the detection rate of patients with solitary pulmonary nodules increases.The question then arises that a substantial proportion of the pulmonary nodules were hardly to be diagnosed by experience.The evaluation methods for SPN mainly include clinical informations,imaging,tumor markers,biopsy pathology and clinical lung cancer probability.So far the biopsy pathology is still the golden standard for SPN diagnosis,with the shortcoming of a larger trauma to patient.Tumor markers are widely used in SPN diagnosis as non-invasive methods,while the relatively low sensitivity and the resulting false negative rate has restricted its application.Therefore it is urgent to establish new non-invasive methods to improve the sensitivity of diagnosis and for prognosis prediction.Circulating tumor cells(CTCs)are cells that shed from a primary tumor and are carried around the body in the blood circulation.The detection sensitivity of CTCs for monitoring cell surface folate receptor(FR)by using targeted PCR technology is higher than other CTCs detection methods and conventional tumor markers,and can reach overthan 80%.The detection rate of stage ? lung cancer can reach about 67.2%,so it has certain value for diagnosis of early lung cancer.Circulating tumor DNA(ctDNA)is a characteristic tumor biomarker can be characterized,quantified and traced,and will be used in early diagnosis,development process monitoring,prognosis judgment and personalized medication guidance.Play an important role.Method:The subject of this thesis is divided into two parts.The first part is 80 patients with solitary pulmonary nodules treated in the First Affiliated Hospital of Suzhou University from September 2016 to June 2017.Before treatment,3mL whole blood samples were taken for CTCs detection,and automatic chemical analyzer was used to detect Carbohydrate antigen 125,(CA125),neuron specific enolase,(NSE),carcinoembryonic antigen(CEA)and cytokeratin fragment 21-1,(CYFRA21-1).Analysed the level of peripheral blood CTCs and the relationship of lung cancer-specific tumor markers between benign and malignant pulmonary nodules.Due to the high incidence of lung adenocarcinoma,120 patients with lung adenocarcinoma were enrolled in the Department of Respiration,Thoracic Surgery and Oncology of the First Affiliated Hospital of Svuzhou University from September 2016 to June 2017.The relationship between CTCs in peripheral blood and tumor size,subtypes and staging of lung adenocarcinoma was analyzed.The second part is 30 patients with lung adenocarcinoma undergone operation diagnosed by the First Affiliated Hospital of Suzhou University from September 2016 to June 2017.Using tumor DNA(tDNA)and plasma ctDNA as samples,frequent mutations in plasma ctDNA and tDNA and fewer mutations in other genes were identified by sequencing,consistency analysis was performed,and ctDNA mutation frequency and ctDNA were analyzed before and after surgery.Compare with the sensitivity of lung cancer tumor markers.Results:1.In patients with solitary pulmonary nodules,50 cases(62.5%)had malignant SPN,30 cases(37.5%)had benign SPN,and the level of CTCs in patients with malignant SPN was 9.79(8.98-10.60)units,which was significantly higher than the level of CTCs in patients with benign SPN 6.66(5.81-7.51)units,the difference was statistically significant(P<0.001).2.The multivariate logistic regression analysis was performed with SPN benign and malignant as the dependent variable.The results showed that the maximum diameter of the nodules,CEA and CTCs were independent risk factors for malignant SPN(P<0.05).3.The level of CTCs in the lung adenocarcinoma group was 10.65(9.93-11.37)units.The level of CTCs in the benign SPN group was 6.66(5.81-7.51)units.The Mann-Whitney U test showed that the CTCs level in the lung adenocarcinoma group was higher than that in the benign SPN group(P<0.001).4.In patients with lung adenocarcinoma,the peripheral blood CTCs level of patients with primary tumors>2.0cm was higher than the maximum diameter ?2.0cm,the difference was statistically significant(P=0.001).The level of CTCs in peripheral blood of patients with stage ?-? lung adenocarcinoma was higher than that of patients with stage ?-? lung adenocarcinoma(P=0.000).In different pathological subtypes,peripheral blood CTCs levels of patients with microinvasive adenocarcinoma and invasive adenocarcinoma were higher than those of patients with adenocarcinoma in situ,and the difference was statistically significant(P=0.03,P=0.002).5.Multivariate Logistic regression analysis was performed using benign pulmonary SPN and lung adenocarcinoma as dependent variables and CTCs and clinical biomarkers as independent variables.The results showed that CTCs and CEA were independent risk factors for lung adenocarcinoma(P=0.021,P=0.001).6.All patients with lung cancer were selected as the experimental group,and patients with pulmonary benign SPN were selected as the control group to draw ROC curve.The Youden index was used to find the best cutoff value of 8.35 and 8.35 units were taken as the cutoff value for diagnosis.And the diagnostic sensitivity and specificity of all lung cancer patients were 70.2%and 79.3%respectively.7.CtDNA mutations in plasma before operation were compared with those in tumor tissues.Twenty-two patients had the same results in 10 positive and 12 negative cases.The coincidence rate was 73.33%,the sensitivity was 76.92%(95%Cl 49.74%-91.82%)and the specificity was 70.59%(95%Cl 46.87%-86.72%).8.CtDNA mutations in plasma after operation were compared with those in tumor tissues.Twenty patients had the same results in 5 positive and 15 negative cases.The coincidence rate was 66.67%,the sensitivity was 38.46%(95%CI 17.71%-64.48%)and the specificity was 88.24%(95%Cl 65.66%-96.71%).9.A total of 18 mutations were found in 18 samples,of which EGFR(13/18,72.21%),KRAS(3/18,16.67%),MET(1/18,5.56%),BRAF(1/18,5.56%).Among all the mutated genes,the average frequency of plasma etDNA mutation before surgery was 4.53%(0.00-26.44%),and the average frequency of plasma etDNA mutations after surgery was significantly reduced to 0.61%(0.00-5.68%).The average frequency of ctDNA mutation after operation was significantly lower than that before operation(p=0.0408).10.Preoperative ctDNA mutation rate had no significant correlation with age,gender and smoking history(p>0.05).Compared with the other serum tumor biomarkers,ctDNA has a higher sensitivity to lung adenoearcinoma induced by lung nodules.Conclusion:CTCs combined with CEA and the maximum diameter of nodules can improve the diagnostic efficacy of patients with malignant isolated pulmonary nodules.The quantitative of CTCs ean reflect the tumor load in the body of patients with lung adenocarcinoma,and can be used as an indicator to evaluate the severity of pathological subtype and cancer stage of patients,as well as an evaluation indicator of therapeutic effect.The results of preoperative and postoperative ctDNA and tDNA tests were highly consistent,and provided patients who could not get tumor specimens with opportunities for diagnosis and targeted therapy.Targeted sequencing detection of ctDNA can detect the mutation gene and mutation frequency of lung adenocarcinoma patients,guide the selection of targeted therapeutic drugs,and reflect the effect of surgical treatment of patients,with a certain degree of reliability.CTCs and ctDNA can be used as a new type of tumor markers,which have the advantages of small trauma and repeatability,and play an important role in the diagnosis,treatment and prognosis of patients with isolated pulmonary nodules.There is a close relationship between CTCs and the diagnosis of benign and malignant pulmonary nodules,the size of lung nodules in malignant patients,and the subtypes of malignant patients.At the same time,targeted detection of ctDNA can detect mutations in lung adenocarcinoma patients,and indicate the effect of surgical treatment of patients,it has certain reliability.This method of monitoring ctDNA changes in peripheral blood has certain application value for clinical management of patients with lung adenocarcinoma. |
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