title: Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation.
abstract: OBJECTIVE
  We evaluated whether detection of human telomerase reverse transcriptase (hTERT) mRNA after immunomagnetic separation is useful to detect circulating cancer (CC) cells.
  
  
  METHODS
  Two ml of peripheral blood were collected from 55 cases with hepatocellular carcinoma (HCC), 20 cases with chronic liver diseases devoid of cancer, and 20 healthy volunteers. Then 1500 and 500 micro l were subjected to immunomagnetic separations using Ber-EP4 and anti-CD45 antibodies, harvested and supernatant cells were collected as epithelial and nonleukocyte fractions, respectively. Samples of each fraction were subjected to reverse transcription-PCR detecting beta-actin, interleukin-2 receptor (IL-2r), alpha-fetoprotein, and hTERT mRNAs. The cases were judged to be positive, equivocal, or negative for CC cells when hTERT positivity with IL-2r negativity, hTERT positivity with IL-2r positivity, or hTERT negativity was seen in epithelial and/or nonleukocyte fractions, respectively.
  
  
  RESULTS
  The dilution experiments revealed that our system could detect 10(0-1) HeLa cells involved in 2 ml of blood. The Ber-EP4-harvested cells from cases with distant metastasis were positive for immunostaining using Hep Par 1 monoclonal antibody. CC cells were judged to be positive in 29 of 55 (53%) HCC cases. On the contrary, no cases without HCC were determined to be positive. The frequency of positivity was significantly correlated with disease extent of HCC.
  
  
  CONCLUSIONS
  These results strongly suggest that detection of hTERT mRNA after immunomagnetic separation is a specific and sensitive tool to detect CC cells and that it would provide useful source for further investigation of cancer metastasis.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Cellular Function, Graduate School of Medical and Dental Science, Niigata University, Niigata 951-8122, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Waguri
authors.forename: Takeshi
authors.initials: T
authors.lastname: Suda
authors.forename: Minoru
authors.initials: M
authors.lastname: Nomoto
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kawai
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Mita
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuroiwa
authors.forename: Masato
authors.initials: M
authors.lastname: Igarashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Fukuhara
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Aoyagi
mesh_terms: D000368:Aged; D001772:Blood Cell Count; D006528:Carcinoma, Hepatocellular / Q000097:blood* / Q000175:diagnosis*; D002469:Cell Separation / Q000379:methods*; D004268:DNA-Binding Proteins; D005260:Female; D006367:HeLa Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008113:Liver Neoplasms / Q000097:blood* / Q000175:diagnosis*; D008297:Male; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D011379:Prognosis; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D019098:Telomerase; D017493:Leukocyte Common Antigens