The Establishment Of ISET-ICC Technical System To Detect Circulating Tumor Cells And The Validation Of Its Clinical Application

BackgroundCirculating tumor cells (CTCs) exist in a variety of malignant tumors. CTCs status provides a real-time and minimal invasive approach for the detection of early disease and assessing prognosis, assessing theraputic effect and applying personalized therapy.At present, the main methods for the detection of circulating tumor cells including CellSearch Systerm and Isolating by size of epithelial tumor cells (ISET). The enrichment of CTCs by CellSearch Systerm based on the expression of different cell surface antigen markers such as EpCAM and CK. The advantage of this technique is that the higher specificity, and has been the commercialization of semi-automatic testing equipment, but lack of tumor-specific antigen expression may cause false negative results. While ISET applies the principle of significant size differences between the tumor cells and blood cells to identify the CTC.The equipment and technology of ISET do not require much of technic or equipment,and the process is easy to control, the separated tumor cell can keep a complete morphology, however, due to the lack of morphological diagnostic gold standard, relatively poor specificity is the Inadequate of the enrichment technology.Our research group have previously established modified ISET technical system to detect circulating tumor cells based on the principle of ISET. Mainly workings include: establish the modified ISET manual experiment device, to improve the dyeing method, improve the retention rate of the tumor cells. The results show that the detection effect of the system is ideal, tumor cell retention rate can reach above 80%, after staining the cells morphology discernible, distinguish typical of CTCS and normal white blood cells. But the system still has shortcomings, first of all is the morphological criteria for lack of identification of CTCS is easily influenced by subjective factors, followed by filtration process when plugging hole phenomenon, causing blood filtering slowly or not smooth.ObjectiveThe objective of his topic is to optimize the traditional ISET technology system, and joint immunofluorescence (ICC) techniques, to establish a new detection of circulating tumor cells in a reliable way; By comparison with CTC-Biopsy and CellSearch, verify the clinical application of ISET-ICC techniche system.Methods1. Optimize the traditional ISET technology system by increase erythrocyte lysis operation and increase the pore size of the filter membrane.2. By jointing immunofluorescence (ICC) techniques, to establish a new detection of circulating tumor cells in a reliable way, and Pre-Clinical study of detecting CTCs in 23 cases of III/IV stage tumor by ISET-ICC techniche system was coducted.3. Establish ISET combined with wax block immunohistochemical technology system to make sure whether there is a phenomenon of EMT in 23 cases of clinical samples through index of twist, vimentin and E-cadherin.4. Through detecting 74 cases of patients of lung cancer, esophageal cancer, gastric cancer, colon cancer, renal cell and liver cancer by ISET-ICC techniche system and verifying its clinical application by comparison with CTC-Biopsy and CellSearch.Results1. The traditional ISET technology system was optimized by increase erythrocyte lysis operation and increase the pore size of the filter membrane, the filtering and dyeing effect are more ideal.2. A new detection of circulating tumor cells was established by jointing ISET with ICC techniques.23 cases of Ⅲ-Ⅳ stage tumor patients were detected by peripheral blood circulating tumor cells, CTCs positive in 9 cases (9/23,39.1%), EMT-CTCs positive in 3 cases (3/23,13%), CTM3 cases (3/23,13%).3. Technology system of ISET combined with wax block immune was established, and the above 23 cases of clinical samples were detected, twist antigen expression was positive in 1 case and vimentin expression was positive in 2 cases.4. The overall positive rate of ISET-ICC technique systerm (13/74,17.6%) was between the cellsearch (9/74,12.2%) and CTC-Biopsy (14/74,18.9%) by detection of 74 cases of lung cancer, esophageal cancer, gastric cancer, colorectal cancer, renal cell and liver cancer. Specifically, CTCs overall positive rate was significant difference (χ2=10.21, p=0.007) between ISET-ICC techniche system and CellSearch Systerm, former is higher; CTCs overall positive rate was non-significant difference (χ2=3.926, p=0.062)) between ISET-ICC techniche system and CTC-Biopsy Systerm, but the consistency is poor(Kappa 0.230).Conclusions1. The CTCs detection system of ISET-ICC was established by optimizing the traditional ISET technology system.2. EMT was verified by the system of ISET combined with wax block immunohlstoc-hemical technology.3. ISET-ICC techniche system has feasible clinical applications, it can be used for the detection of lung cancer, esophageal cancer, stomach cancer, colorectal cancer, kidney cancer, liver cancer and other cancers.4. ISET-ICC techniche system also can detect circulating tumor cells with epithelial-mesenchymal transformation(EMT-CTCs) and circulating tumor microemboli (CTM), which are the main cause of false negative of CellSearch.5. ISET-ICC techniche system can assist in identification of CTCs enriched by CTC-Biopsy method.