title: Circulating Tumor Cells Enriched by the Depletion of Leukocytes with Bi-Antibodies in Non-Small Cell Lung Cancer: Potential Clinical Application.
abstract: BACKGROUND
  It has been considered that the detection methods for circulating tumor cells (CTCs) based on epithelial cell adhesion molecule (EpCAM) underestimate the number of CTCs and may miss a metastatic subpopulation with cancer stem cell (CSC) properties. Therefore, we investigated EpCAM-positive and -negative CTCs in non-small cell lung cancer (NSCLC) patients at different stages, assessed the clinical value of these CTCs and explored their capacity in the following CSC model.
  
  
  METHODS
  CTCs were enriched by the depletion of leukocytes with bi-antibodies using a magnetic bead separation technique and then identified by the expression of EpCAM and cytokeratin 7 and 8 using multi-parameter flow cytometry. We determined the distribution of CTCs classified by the expression of EpCAM in 46 NSCLC patients with stages I to IV, assessed the diagnostic value of these CTCs by longitudinal monitoring in 4 index patients during adjuvant therapy and characterized the stemness of these CTCs by the expression of CXCR4 and CD133 in 10 patients.
  
  
  RESULTS
  EpCAM-negative (E-) CTCs were detected to be significantly higher than EpCAM-positive (E+) CTCs in stage IV (p = 0.003). The patients with the percentage of E-CTCs more than 95% (r > 95%) were detected to be significantly increased from 13.3% in stage I-II to 61.1% in stage IV (p = 0.006). Kaplan-Meier analysis indicated that the patients with r > 95% had significantly shorter survival time than those with r ≤ 0.95 (p = 0.041). Longitudinal monitoring of CTCs indicated that the patients with a high percentage of E-CTCs in the blood were not responsive to either chemotherapy or targeted therapy. Further characterization of CTCs revealed that a stem-like subpopulation of CXCR4+CD133+ CTCs were detected to be significantly more prevalent in E-CTCs than that in E+CTCs (p = 0.005).
  
  
  CONCLUSIONS
  The enrichment of CTCs by the depletion of leukocytes with bi-antibodies is a valuable method for estimating the number of CTCs, which can be potentially applied in predicting the prognosis, monitoring the therapeutic effect of NSCLC patients and further analyzing the biology of CTCs.
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Jian
authors: J
authors: Yin
authors: Department of Respiratory Diseases, Nanjing Chest Hospital, Nanjing, China.
authors: Yi
authors: Y
authors: Wang
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Hanlu
authors: H
authors: Yin
authors: Department of Respiratory Diseases, Nanjing Chest Hospital, Nanjing, China.
authors: Wenping
authors: W
authors: Chen
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Guangfu
authors: G
authors: Jin
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Hongxia
authors: H
authors: Ma
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Juncheng
authors: J
authors: Dai
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Jiaping
authors: J
authors: Chen
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Yue
authors: Y
authors: Jiang
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Hui
authors: H
authors: Wang
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Zhian
authors: Z
authors: Liu
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Zhibin
authors: Z
authors: Hu
authors: Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
authors: Hongbing
authors: H
authors: Shen
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018033:Antibodies, Bispecific / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000175:diagnosis / Q000473:pathology* / Q000628:therapy; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D005500:Follow-Up Studies; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007962:Leukocytes / Q000166:cytology* / Q000276:immunology*; D008175:Lung Neoplasms / Q000097:blood / Q000175:diagnosis / Q000473:pathology* / Q000628:therapy; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000473:pathology
chemical_list: D018033:Antibodies, Bispecific; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule