High percentage of false-positive results of cytokeratin 19 RT-PCR in blood: A model for the analysis of illegitimate gene expression

Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amount s of information support the fact that it is also associated with a high pe rcentage of false-positive results. in our study, we not only managed to de monstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely no vel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the C K19 mRNA to exclude DNA contamination. Tumor dilution experiments were perf ormed in order to analyze the specificity and sensitivity of the method. Co ntrol experiments using the blood of healthy donors were performed. Tumor c ell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detec tion limit was 1 tumor cell in 10(5) mononuclear cells. RT-PCR of mononucle ar cells from healthy blood donors gave false-positive results in 29% of th e cases. We conclude that a significant decrease in the sensitivity of CK19 RT-PCR occurs if it is performed in blood cells and that the illegitimate CK19 gene expression in normal cells can lead to false-positive results. Th ese limitations have to be taken into account if RT-PCR is to be used for t he detection of tumor cells either in blood or in bone marrow in clinical p ractice. Copyright (C) 2000 S. Karger AG, Basel.