Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation.
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.
Waguri
Nobuo
N
Division of Gastroenterology and Hepatology, Department of Cellular Function, Graduate School of Medical and Dental Science, Niigata University, Niigata 951-8122, Japan.
Suda
Takeshi
T
Nomoto
Minoru
M
Kawai
Hirokazu
H
Mita
Yusaku
Y
Kuroiwa
Takashi
T
Igarashi
Masato
M
Kobayashi
Makoto
M
Fukuhara
Yasuo
Y
Aoyagi
Yutaka
Y
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*
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D019098:Telomerase; D017493:Leukocyte Common Antigens