Immunobead multiplex RT-PCR detection of carcinoembryonic genes expressing cells in the blood of colorectal cancer patients

Abstract

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.