title: Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy.
abstract: OBJECTIVE
  To establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC).
  
  
  METHODS
  HCC cells were bound by biotinylated asialofetuin, a ligand of asialoglycoprotein receptor, and subsequently magnetically labeled by antibiotin antibody-coated magnetic beads, followed by magnetic separation. Isolated HCC cells were identified by immunofluorescence staining using Hep Par 1 antibody. The system was used to detect CTCs in 5 mL blood. Blood samples spiked with Hep3B cells (ranging from 10 to 810 cells) were used to determine recovery and sensitivity. Prevalence of CTCs was examined in samples from HCC patients, healthy volunteers, and patients with benign liver diseases or non-HCC cancers. CTC samples were also analyzed by FISH.
  
  
  RESULTS
  The average recovery was 61% or more at each spiking level. No healthy, benign liver disease or non-HCC cancer subjects had CTCs detected. CTCs were identified in 69 of 85 (81%) HCC patients, with an average of 19 ± 24 CTCs per 5 mL. Both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, differentiation status, and the disease extent as classified by the TNM (tumor-node-metastasis) classification and the Milan criteria. HER-2 gene amplification and TP53 gene deletion were detected in CTCs.
  
  
  CONCLUSIONS
  Our system provides a new tool allowing for highly sensitive and specific detection and genetic analysis of CTCs in HCC patients. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision making.
authors.affiliation: Molecular Oncology Laboratory, and Departments of Comprehensive Treatment and Radio-intervention Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, PR China.
authors.forename: Wen
authors.initials: W
authors.lastname: Xu
authors.forename: Lu
authors.initials: L
authors.lastname: Cao
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Zhang
authors.forename: Hai-Hua
authors.initials: HH
authors.lastname: Qian
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Kang
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Liao
authors.forename: Le-Hua
authors.initials: LH
authors.lastname: Shi
authors.forename: Ye-Fa
authors.initials: YF
authors.lastname: Yang
authors.forename: Meng-Chao
authors.initials: MC
authors.lastname: Wu
authors.forename: Zheng-Feng
authors.initials: ZF
authors.lastname: Yin
mesh_terms: D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins / Q000097:blood; D019921:Biotinylation; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D005260:Female; D060748:Fetuins; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D009360:Neoplastic Cells, Circulating; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins; D060748:Fetuins; D018719:Erb-b2 Receptor Tyrosine Kinases; D016159:Tumor Suppressor Protein p53; C495901:TP53 protein, human; C017029:asialofetuin