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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
Author(s) -
De Graaf Hiltje,
Mælandsmo Gunhild M.,
Ruud Paal,
Forus Anne,
Øyjord Tove,
Fodstad Øystein,
Hovig Eivind
Publication year - 1997
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19970703)72:1<191::aid-ijc27>3.0.co;2-l
Subject(s) - micrometastasis , peripheral blood mononuclear cell , biology , cell culture , microbiology and biotechnology , circulating tumor cell , gene expression , gene , ectopic expression , reverse transcription polymerase chain reaction , real time polymerase chain reaction , cancer research , messenger rna , carcinoma , cell , cancer cell , cancer , metastasis , in vitro , genetics
Our objective was to develop and study the feasibility of a quantitative, nested reverse‐transcription polymerase chain reaction (RT‐PCR) assay for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP ‐2 gene as a target. Several carcinoma cell lines and peripheral blood samples of 10 healthy volunteers were screened for levels of EGP‐2 mRNA. The assay included EGP‐2 competitor molecules, carrying an internal deletion, that had been titrated by limiting dilution. Seven carcinoma cell lines showed a wide spectrum of EGP‐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 observed 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 mononuclear cells exhibited a detection limit that was not better than approximately one tumor cell in 2 × 10 4 normal cells. Together with the considerable variation of EGP‐2 transcript levels that is observed in different carcinoma cell lines, the extent of expression in normal blood cells would prevent a reliable estimation of low numbers of carcinoma cells in clinical samples. A similar situation might well apply for other 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. Int. J. Cancer 72:191–196, 1997. © 1997 Wiley‐Liss Inc.