Establishment Of Quantitative Detection Of Circulating Tumor Cells In Non - Small Cell Lung Cancer And Clinical Significance Of Dynamic Monitoring Before And After Operation

BackgroundNon-small cell lung cancer is one of the highest prevalence and mortality disease.Currently radical surgery combined adjuvant chemotherapy is the main treatment, but afterdecades of efforts, the disease is yet to see5-year survival was significantly improved.Non-small cell lung cancer is the leading cause of death in patients with postoperativemetastasis and recurrence. Clinical studies show that the non-small cell lung cancerpatients present in the peripheral blood of tumor cells is an important factor leading todisease progression and exacerbation, and is an important indicator of prognosis, so thedetection of non-small cell lung cancer patients, intraoperative pulmonary vein bloodcirculating tumor cells which have very important clinical implications. Most research iscurrently used to detect metastatic tumor cytology peripheral venous blood cells exist, butthese methods because of its sensitivity limits, so that the presence of the specimen can bemore accurate cancer diagnosis and analysis of large aggregates. With the rapid advancesin molecular biology and cell biology, general use of quantitative PCR, and continue tofind a variety of tumor markers, high sensitivity makes it possible to detect circulatingtumor cells. In recent years, various improved methods using FQ-PCR and detection ofcirculating tumor cells in breast cancer research methods, the use of both colon cancer,the sensitivity of these methods are generally higher than conventional cytology.Although there are varying degrees of difference between the results of different researchgroups, and some even diametrically opposite conclusions, but do not doubt that,FQ-PCR for the detection of circulating tumor cells has brought a new approach. For thisreason, it is necessary to further non-small cell lung cancer circulating tumor celldetection method developed FQ-PCR testing.Taqman probe FQ-PCR with high sensitivity and specificity in the detection of geneexpression. On the basis of this study, by detecting intraoperative pulmonary venous peripheral blood and intracellular levels of EpCAM expression established to detectnon-small cell lung cancer circulating tumor cells, and in vitro models in laboratorymethod validation, and ultimately the use of the technique to detect peripheral blood ofpatients with non-small cell lung cancer and intraoperative blood circulating tumor cells,and to explore its clinical significance in non-small cell lung cancer patients withcirculating characters of clinical and pathological factors.MethodsPart I: A549, NCI-H1975, NCI-H1299non-small cell lung cancer line was establishednon-small cell lung cancer circulating tumor cells in vitro model, and for the detection ofEpCAM target genes using Taqman probe FQ-PCR method for detecting in vitro modelcirculating tumor cells, and to verify the specificity and sensitivity of the method.Part II: The first part of the detection method to detect outside the establishednon-small cell lung cancer patients with peripheral venous and intraoperative pulmonaryvenous blood circulating tumor cells, and some non-small cell lung cancer patients withimmunohistochemical staining to detect non-small cell lung cancer tissues of CD34expression, and statistical analysis within the primary tumor cells of solid tumormicrovessel counts and microvascular diameter ratio, and analyze the relationship betweenlevels of circulating tumor cells in patients with clinical and pathological factors. Andcomparative research component benign lung disease and healthy volunteers.ResultsThis study successfully established Taqman probe FQPCR method to detectnon-small cell lung cancer circulating tumor cell technology, EpCAM in circulatingtumor cells expressed for the detection of targets, using Taqman probe FQPCR detectionin vitro model of non-small cell lung cancer circulating tumor cells, and its sensitive thelowest degree of cancer cells can be detected5cells from107cells.Taqman probe FQPCR assay in peripheral blood of patients with preoperative220cases of non-small cell lung cancer circulating tumor cells,215cases positive, thepositive rate was97.73%; postoperative circulating tumor cells in peripheral blood of188 patients were positive, positive rate to82.45%; intraoperative pulmonary venous bloodcirculating tumor cells,201cases positive, the positive rate was91.36%;When Non-small cell lung cancer patients before surgery, postoperative detection ofcirculating tumor cells in peripheral blood is related with patient’s age, TNM stage, locallymph node metastasis of tumor cells, pathological type situation significantly;Former non-small cell lung cancer patients after surgery there are obvious differencesin peripheral blood circulating tumor cells in peripheral blood circulation and the positiverate of non-small cell lung cancer tumor cells was significantly higher in patients withbenign lung disease and healthy volunteers specimens;The preoperative detection of circulating tumor cells in peripheral blood of the resultsof220cases of non-small cell lung cancer as a result of the detection of circulating tumorcells in peripheral blood of patients with40control patients with benign lung disease forcomparison analysis, when used as benign and malignant lung shadows sensitive to themethod of100.00%, a specificity of88.89%;Test results prior to surgery in patients with non-small cell lung cancer tumor cellscirculating in peripheral blood and compare the results of postoperative surgical pathologywithin the organization paraffin microvascular invasion of tumor cells showed tumor cellcounts microvascular and microvascular diameter(CV) compare with the presurgeryperipheral blood circulating tumor cells number of cells regression results are obvious,microvascular invasion of tumor cells circulating in peripheral blood of cancer patientsthere is a direct relationship between the number of cells within the tumor;Conclusions1. Do establish a method Taqman probe FQ-PCR technique quantitative detection ofnon-small cell lung cancer circulating tumor cells, the sensitivity and specificity of themethod were high;2. CTCs quantitative detection method can be used for benign and malignant lungshadows differential diagnosis, the sensitivity was97.73%, specificity was100%;3. Before non-small cell lung cancer patients undergoing dynamic monitoring ofcirculating tumor cells, confirming the number of CTCs compared with preoperative decreased significantly compared to patients, suggesting that the technology can be usedfor early diagnosis of non-small cell lung cancer patients with postoperative recurrence ormetastasis.