ECTOPIC EXPRESSION OF TARGET GENES MAY REPRESENT AN INHERENT LIMITATION OF RT-PCR ASSAYS USED FOR MICROMETASTASIS DETECTION - STUDIES ON THE EPITHELIAL GLYCOPROTEIN GENE EGP-2

Our objective was to develop and study the feasibility of a quantitati ve, nested reverse-transcription polymerase chain reaction (RT-PCR) as say for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP-2 gene as a target, Several carcinoma cel l lines and peripheral blood samples of 10 healthy volunteers were scr eened for levels of EGP-2 mRNA, The assay included EGP-2, competitor m olecules, carrying an internal deletion, that had been titrated by lim iting dilution. Seven carcinoma cell lines showed a wide spectrum of E GP-2 mRNA expression levels, with the highest values (20-100 molecules /cell) seen in 3 breast-cancer cell lines. Unexpectedly, a consistent low level of EGP-2 mRNA expression (0.0004 molecules/cell) was observe d in peripheral blood mononuclear cells, probably representing ectopic non-functional expression, Because of this background level, spiking experiments with T47D breast-carcinoma cells added to blood mononuclea r cells exhibited a detection limit that was not better than approxima tely one tumor cell in 2 x 10(4) normal cells, Together with the consi derable variation of EGP-2 transcript levels that is observed in diffe rent carcinoma cell lines, the extent of expression in normal blood ce lls would prevent a reliable estimation of low numbers of carcinoma ce lls in clinical samples. A similar situation might well apply for othe r target genes, This emphasizes the need for a critical evaluation of the different steps involved in the methods used for RT-PCR detection of micrometastatic tumor cells. (C) 1997 Wiley-Liss, Inc.