Experimental Study Of The Immunomagnetic Capture To Circulating Tumor Cells Of Lung Cancer And Its Clinical Significance

[Objective]:In order to discuss the correlation between levels of CTCs in lung cancer patients and its clinical stage, we use combined immunomagnetic enrichment technology and fluorescent cell staining to test and count circulating tumor cells (CTCs) in lung cancer patients peripheral blood.[Methods]:1.Select 5 different tumor cell families (ie:NCI-H460, NCI-H446.7901, Hela, MCF-7) to make climbing film, choose GRP to be antibodies. Testing the GRP expression between different cell families by immunohistochemical staining. Sorting different concentrations of tumor cell families by MACS method, testing the result and draw a Curve picture.2.Incorporat different volume of NCI-H446 cells into five healthy volunteers peripheral blood, MACS method separat them out and calculat the tumor cell to understand the recovery rate of this experimental method.3.To understand the specificity and sensitivity of this method, Collect 100 patients with lung cancer,40 cases of benign lung tumor, and blood samples from 30 healthy volunteers as a negative control, the application of MACS enrichment of circulating tumor cells in blood (CTCs), then cell fluorescence technique morphological analysis of circulating tumor cells lines and the cells were stained with HE method for identifying tumor cells.To test the high sensitivity and specificity of this method, circulating tumor cells in blood (CTCs)will be collected from the blood samples which were collected from 100 patients with lung cancer,40 cases with benign lung,and 30 healthy volunteers by immunomagnetic separation. The tumor cells were identified by cell fluorescence technique and HE staining of tumor cells.4.SPSS17.0 software package for statistical analysis. Correlation and regression method for detection of tumor cells in relation to the concentration of tumor cells,the variables normality test, the experimental results indicate ± s; non-normal distribution or variance not Qi data using non-parametric Wilcoxon test, correlation analysis using Spearman test, with P<0.05 was considered statistically significant, trends produced by EXCEL.[Results]:1.Different numbers of tumor cells detected by immunohistochemistry beads, with the concentrations of tumor cells increasing, the number of CTCs are increasing too. the best correlation of tumor cells is NCI-H446, and it is a significant correlation, 0.8667 (P＜0.001). Regression analysis of number of recovered NCI-H446 cells yielded a regression function:y=6.681X+26.795 and a coefficient of determination of R2=0.8667. The expression of cell gastrin-releasing peptide (GRP) was positive in the five kinds of cells seeded by immunohistochemistry.and the positive expression rate of the level has the same result with the recovery of circulating tumor cells in blood (CTCs) by immunomagnetic separation.2.The recovery rate was ranging from 72%-89% by spiking varying numbers of NCI-H446 lung cancer cells into 2ml blood samples of healthy volunteers. Regression analysis of number of recovered vs. Spiked NCI-H446 cells yielded a regression function:y=0.781X+11.307, and coefficient determination:R2=0.998.3.After analysis:(1)The recovery of circulating tumor cells in blood (CTCs) that gathered from 100 cases of lung cancer patients,40 cases of benign lung and and 30 cases of peripheral blood of healthy volunteers are caculated by immunohistochemistry beads, the experimental method sensitivity and specificity are as follows:85% and 71.4%.(2)Patients with lung cancer, lung benign tumor patients and healthy volunteers to compare two groups (P<0.05), a significant difference.(3) 100 cases of lung cancer compared with the clinical parameters of CTCs detected between:① age (<50 years and ≥50 years old group) between the two groups, P value of 0.769 (P> 0.05); male group and female group, P = 0.536 (P> 0.05), the difference was not statistically significant;② tumor diameter <3cm tumor diameter≥3cm group (P<0.05), there are significant differences;③ The detection of circulating tumor cells in patients of III stage, IV stage was significantly higher than TNM stage I and II, (P<0.05),with a significant difference;④ In this case,33 patients had distant metastases,40 cases of bone metastasis,80 cases of lung cancer patients with lymph node metastasis and 20 cases without metastasis, detection of CTCs compared to the situation and found that circulating tumor cell in metastasis group and the group of patients with bone metastasis were significantly higher than those without metastasis and lymph node metastasis, and the difference was statistically significant. And there is no difference with pathological type of lung cancer (P> 0.05).(4)CTCs positive rate has no interrelated with the patient’s age, gender,it has no significant relationship (P> 0.05); and it has a high interrelated with tumor size and clinical stage, the CTCs detection rate was significantly associated (P<0.05) when the cancer diameter greater than 3cm, there lymph, bone metastasis or distant metastasis, and has no4hing to do with the pathological type of lung cancer (P> 0.05). CTCs has a high interrelated with in several serum tumor markers:CEA, CA125, CA153,CA199 increased significantly correlated (P<0.05);and lymphocyte factor:CD3+CD4+, CD3 +CD8, NK, T cells in absolute value, Th cell absolute, Ts cells absolute, natural killer fine, CIKN correlated; and cytokines: IFN-y, IL-2, TNF-a, IL-4, IL-6, IL-10 has a significant correlation (P<0.05).[Conclusion]:1. It is proved that using MACS method can be successfully enriched the circulating tumor cells (CTCs) from the blood samples of patients with lung cancers;2. Circulating tumor cells (CTCs) in lung cancer patients has a high interrelated with tumor size and clinical stage, TNM stage, distant metastasis and serum tumor markers.This method can be used as an auxiliary indicators for clinical detection of patients with lung cancer.