Abstract
To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of β2-mircoglobulin (β2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to β2M mRNA, to CEA mRNA/β2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1–104 IS174T cells) of cDNA and β2M mRNA external standards prepared with tenfold serial dilutions (102–107 leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes’ B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.
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Abbreviations
- CEA :
-
Carcinoembryonic antigen
- Cp :
-
Crossing point
- CTC :
-
Circulating tumor cell
- MNC :
-
Mononuclear cell
- PBL :
-
Peripheral blood leukocyte
- PCR :
-
Polymerase chain reaction
- RT :
-
Reverse transcription
- β2M :
-
β2-Mircoglobulin
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Acknowledgements
This work was supported by Key Doctoral Fund of Ningbo Municipality (02J20101-07), Medical Sciences Research Fund of Zhejiang Province (2002A073), and Key Research Project of Ningbo University (z0214015). We thank Dr. Dieter Soll, Yale University, USA, for his kind help in editing the manuscript.
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Guo, J., Xiao, B., Zhang, X. et al. Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers. J Mol Med 82, 768–774 (2004). https://doi.org/10.1007/s00109-004-0590-8
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DOI: https://doi.org/10.1007/s00109-004-0590-8