The Clinical Application Of Liquid Biopsy In Non-small Cell Lung Cancer

BACKGROUND AND PURPOSELung cancer is currently the malignant tumor with the highest incidence and mortality,and non-small cell lung cancer(NSCLC)accounted for 85%.In addition,there are no methods with both high efficacy and little trauma for the early diagnosis of NSCLC,the prediction of metastasis and the differential diagnosis of pulmonary nodules.However,the emerging liquid biopsies,including ctDNA,CTC and exosomes,have been increasingly applied in the clinical application of various tumors due to their noninvasive,simple and repeatable advantages.Due to the lack of standardization of the methods and techniques,there is still a lack of a good biomarker for liquid biopsy in NSCLC,which could meet the clinical needs mentioned above.The first purpose of the study is to explore the ability of CTC as a biomarker for liquid biopsy to make differential diagnosis between the benignancy and malignancy of pulmonary nodules,and to be compared with PET/CT in the differential diagnosis.The second purpose of the study is to explore the potential of exosomes in the diagnosis of lung adenocarcinoma and in the prediction of lung cancer metastasis,and also to explore the relationship between the exosomes and the primary tumor protein expression and tumor burden.Through all these three parts of the study,I hope to comprehensively explore and analyze the potential of liquid biopsy in the clinical application in NSCLC patients.METHODSIn the first part,CTCs of enrolled patients were detected and counted by EpCAM-specific peptide-based nanomagnetic CTC isolation system.The CTC count was used as biomarker for the differential diagnosis of benignancy and malignancy of pulmonary nodules.ROC curve analysis was used to calculated the sensitivity and specificity of CTC counts in the differential diagnosis and also compared with PET/CT.In addition,subgroup analyses of were further performed according to the radiological features of the nodules in chest CT,to determine the optimal indications of the CTC detection technique.In the second part of the study,the exosomes of enrolled lung adenocarcinoma patients and healthy candidates were isolated by the aldehyde/sulphate latex beads based exosome isolation technique,and the exosomal EGFR and CXCR4 expression levels were quantitatively determined by the flow cytometry.The difference of expression level of exosomal EGFR between two groups were analyzed,and ROC curve analysis were conducted to determine the potential and sensitivity and specificity in the diagnosis of lung adenocarcinoma.In addition,the expression level of exosomal CXCR4 was used as an indicator of lung adenocarcinoma metastasis,and the same analyses were made.Also,the correlations between the expression of exosomal CXCR4 protein and the invasiveness of lung adenocarcinoma were investigated.Finally,by the comparison of the expression of proteins in the exosomes and primary tumor tissue,and also the expression of exosomal proteins of the patient before and after the surgery,the relationships between exosomal proteins and primary tumors and also tumor burden were explored.RESULTSIn the first part of the study,104 patients with pulmonary nodules(97 cases of lung adenocarcinoma,25 cases of benign lesions)were enrolled.The difference of CTC count between the benign group and malignant group was statistically significant(P<0.05),with a median of 0.5/mL and 2.0/mL respectively.The AUC of the ROC curve of CTC count to differentiate between malignancy and benignancy of pulmonary nodules was 0.651(P<0.05),and the sensitivity and specificity was 45.6%and 80.0%respectively when the threshold was set to be>0.5/mL.By subgroup analysis,the CTC count was found to be a good biomarker for differential diagnosis in the nodules which presented as pure GGO in the chest CT,with a median of 0/mL and 1.0/mL(P<0.01).The AUC of the ROC curve in the subgroup pGGO was 0.8631(P<0.01),and the sensitivity and specificity was 45.6%and 80.0%respectively when the threshold was set to be>0/mL.By comparison with PET/CT,the potential of CTC count in the differential diagnosis was found to be even better than PET/CT,of which the sensitivity and specificity were only 54.8%and 66.7%,respectively,which were significantly lower than those of CTC(P=0.039).In the second part of the study,68 subjects(38 cases of lung adenocarcinoma,30 healthy candidates)were enrolled.The expression of exosomal EGFR was not significantly different between the two groups.The expression of exosomal CXCR4 were found to be significantly correlated with invasiveness of lung adenocarcinoma.The correlation coefficients of exosomal CXCR4 fluorescence intensity in flow cytometry and the pathologic invasion score was 0.413(P<0.01).In addition,the difference of the expression of exosomal CXCR4 between groups of patients with and without metastasis were statistically significant(P<0.04).The AUC of ROC curves of exosomal CXCR4 positive rate and fluorescence intensity to predict the metastasis of lung adenocarcinoma were 0.697(P=0.04)and 0.770(P<0.01)respectively.The sensitivity of exosomal CXCR4 positive rate and fluorescence intensity were both 100%and the specificity were 40.9%and 59.1%,respectively,with>7.4%and>1429.39 as thresholds.The concordance with the expression level of primary tumor tissue determined by immunohistochemical staining and the exosomal protein expression were not observed.Still,the exosomal proteins expression level were not altered after the surgery and the difference of expression level of exosomal proteins before and after the surgery was not statistically significant.CONCLUSIONSBy comparing the CTC count with PET/CT in the differential diagnosis of benignancy and malignancy of pulmonary nodules,the CTC count by this technique was found to be a good biomarker for differential diagnosis.For nodules which presented as pure GGO in the chest CT,it was even better than PET/CT.In addition,the expression of exosomal EGFR in peripheral blood determined by flow cytometry,was found not capable of being a biomarker for the diagnosis of lung adenocarcinoma.Nevertheless,the expression of exosomal CXCR4 was significantly correlated with the invasiveness of lung adenocarcinoma.It was also found to have great potential to predict the metastasis of lung adenocarcinoma.The quantitative detection of exosomal proteins by this technique could not reflect the expression level in the primary tumor,and was incapable of monitoring the tumor burden in patients with lung adenocarcinoma.