Limitations of CD44v6 amplification for the detection of tumour cells in the blood of colorectal cancer patients.

Based on the important role of CD44 splice variants in colorectal cancer progression and metastasis, we evaluated the use of CD44v6 expression to detect and assess the metastatic potential of colorectal tumour cells circulating in peripheral blood. A nested amplification was designed that allowed to detect 10-100 colon cancer cells. This assay was applied to blood samples from healthy donors. Strong signals were detected in all cases, indicating that it cannot be used to detect colorectal carcinoma cells in whole blood. We then included an enrichment step based on the use of an anti-epithelial cells monoclonal antibody (BerEP4) coupled to magnetic beads. The CD44v6 reverse transcription polymerase chain reaction (RT PCR) assay was performed on cDNA synthesized from blood samples treated with these beads. We analysed 18 samples from 12 patients with a gastrointestinal disease, and 36 samples from ten patients with a colorectal cancer. None of the patients used as negative controls were found to contain epithelial cells in their blood as determined by cytokeratin 19 RT-PCR. By contrast, CD44 transcripts containing exon v6 were detected in nine out of the 18 samples tested (50%). For the colorectal cancer patients, six out of the seven samples (85.7%) that were cytokeratin 19-positive were CD44v6-negative, whereas ten samples out of the 29 not containing epithelial cells were CD44v6-positive (34.5%). This is probably due to the persistence of CD8+ leucocytes in the enriched preparations, as determined by PCR analysis of the CD8 alpha-chain. We conclude that detection of CD44v6 transcripts using a sensitive nested RT-PCR assay has no potential value to detect and characterize colorectal cancer micrometastases from blood, even following an initial enrichment step.