Detection And Quantitation Of Circulation Tumor Cells In Patients With Non-small Cell Lung Cancer Using Immunomagnetic Enrichment And Multiparameter Flow Cytometry

Background: Some patients even in early-stage disease underwent surgical resection develop recurrent disease and distant metastases. It is hypothesized that those must have developed form occult metastases which could not be detected by the routine methods at the time of diagnosis or before, leading an underestimation of the true tumor stage. The occult metastasis itself is a micro-metastasis characteristic and an ominous sign for prognosis, the tumor cells detached form their original tumor and give rise to minimal metastatic tumor in distant tissue and organ all over the body by circulating blood and lymphatic route. Lymph node metastasis finally disseminate tumor cell into blood circulation, form circulating tumor cells (CTCs) and lead to disease relapse tendency all over the body. The metastatic process is comprised of a series of sequential steps, the tumor cells enter into the blood circulation is one of the important step for distant metastases. Several methods are used to detect the CTCs of non-small cell lung cancer(NSCLC) with different accuracies. CTCs can be detected sensitively and specifically by immunomagnetic enrichment combined with flow cytometry. This study was to investigate the sensitivity, specificity and clinical value of this method in detecting CTCs in NSCLC.Methods: Twenty-five patients who underwent surgical resection for non-small cell lung cancer form Jan 2007 to Apr 2007 were selected. All the patients were staged and diagnosed by chest X-ray, endobrochial bronchoscopy, computerized tomography (CT), bone scan, operative lymph node dissection and size of tumor-free margin, they were not treated by chemotherapy and radiotherapy before detecting. Including 16 male patients and 9 female, mean ages 61.68 years, range form 43 to 78 years. For 25 patients histological evaluation was available and revealed 12 cases of Squamous cell carcinoma and 11cases of adenocarcinoma. The pTNM staging of patients was as follow: 12 cases of stageⅠ, 4 cases of stageⅡ, 4 cases of stageⅢA, 3 cases of stageⅢB and 2 cases of stageⅣ. 10 cases of benign pulmonary diseases (2 case of hamartoma and 8 case of pulmonary tuberculosis) and 10 healthy volunteers without evidence or history of malignant disease consist of control group. The pulmonary veins blood and peripheral vein blood were respectively collected, choosing the CD326 immunomagnetic beads and CK-fluorescein isothiocyanate (CK-FITC) as the marker antibodies of CTCs, firstly mononuclear cell marked by minibeads conjugated with CD326, the mononuclear cells were enriched and separated though Magnetic-activated cell separation(MACS), secondly the positive separation cells were marked by anti-CK-FITC and anti-leukocyte antibody CD45-phycoerythrine (anti-CD45-PE), finally those cells detected and analyzed by flow cytometry.Results: There were 16 cases of stageⅠand stageⅡpatients, CTCs were detected in peripheral vein blood of 5 (5/16, 31.25%)and were detected in pulmonary veins blood of 9 (9/16, 56.25%) after MACS enrichment by flow; 9 cases in stageⅢand stageⅣ, CTCs were detected in peripheral vein blood of 5 (5/9, 55.56%) and were detected in pulmonary veins blood of 9 (7/9,77.78%) after MACS enrichment by flow; among 13 stageⅠcases, CTCs were detected in peripheral vein blood of 3 (3/13, 23%) and were detected in pulmonary veins blood of 8 (8/13, 61.54%) after MACS enrichment; the whole CTCs positive detection rate of 25 cases in pulmonary veins blood was 64%(16/25)higher than the positive detection rate of peripheral vein blood(40%, 10/25)(p<0.05)。After following-up all the CTCs positive patients for 3-6 months, there were no recurrent diseases in all patients. For the follow-up term is short, the long-term prognosis should be observed.Conclusions: Detection and quantification of circulating tumor cells in non-small cell lung cells is a potential and useful indicator for the tumor cells develop, treatment reaction and prognosis. In this study, the method was set up by MACS combined with FCM to detect the CTCs of pulmonary veins blood and peripheral vein blood of patients with NSCLC, MACS combined with FCM may improve detection rate of CTCs. The sensitive and specificity are higher than other methods. With the discover of new specificity moleculer tumor marker for NSCLC, NSCLC cell line separating and setting up and understanding new biology characteristic about of non-small cell lung cancer, this technique appear to be an efficient technique to detect circulating tumor cells and may be important for clinical practice in the future.