High Percentage of False-Positive Results of Cytokeratin 19 RT-PCR in Blood: A Model for the Analysis of Illegitimate Gene Expression

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Abstract
Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amounts of information support the fact that it is also associated with a high percentage of false-positive results. In our study, we not only managed to demonstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely novel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the CK19 mRNA to exclude DNA contamination. Tumor dilution experiments were performed in order to analyze the specificity and sensitivity of the method. Control experiments using the blood of healthy donors were performed. Tumor cell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detection limit was 1 tumor cell in 105 mononuclear cells. RT-PCR of mononuclear cells from healthy blood donors gave false-positive results in 29% of the 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. These limitations have to be taken into account if RT-PCR is to be used for the detection of tumor cells either in blood or in bone marrow in clinical practice.