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Richard Douard, Stéphane Moutereau, Valérie Serru, Jean Patrick Sales, Philippe Wind, Paul-Henri Cugnenc, Michel Vaubourdolle, Sylvain Loric
Immunobead multiplex RT-PCR detection of carcinoembryonic genes expressing cells in the blood of colorectal cancer patients
Keywords: carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7), circulating cells, colorectal cancer, immunobead, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR)
Circulating cell detection using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques has been studied as a new prognostic factor in colorectal cancer patients. With the view of enhancing detection sensitivity, we developed a new multiplex RT-PCR assay for circulating cell detection based on the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5; formerly CEA) and CEACAM7 (formerly CGM2).
Between November 2002 and December 2003, 45 stage III-IV, 39 stage I-II colorectal cancer patients, 32 non-colorectal cancer patients and 41 healthy individuals were included. Positive selection using HEA-125 immunobeads was applied to blood samples before mRNA extraction, cDNA synthesis and a multiplex CEACAM5/CEACAM7 RT-PCR assay. For both CEACAM5 and CEACAM7, the limit of detection was found to be as low as 1 expressing cell in 10 6 nucleated blood cells.
The multiplex RT-PCR assay was negative for the 41 healthy individuals and the 32 non-colorectal cancer patients. The test was positive in 53/84 (63%) of the colorectal cancer patients for CEACAM5 and/or CEACAM7, whereas 32/84 (38%) were positive for both markers. Colorectal cancer patients were positive for one of the two markers in 80% of cases (36/45) for stage III-IV patients (CEACAM5 73%, CEACAM7 51%) and in 44% of cases (17/39) for stage I-II patients.
This multiplex RT-PCR assay with two markers proved to be more sensitive than use of a single marker in detecting circulating tumour cells. The discrepant expression of CEACAM5 and CEACAM7 may label circulating tumour cells that have different levels of differentiation and subsequent aggressive behaviour.
Clinical Chemical Laboratory Medicine, Walter de Gruyter
Print ISSN: 1434-6621
Volume: 43, 04/2005
Pages: 127 - 132