Abstract
The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples.
Highlights
In this report we describe the detection and quantitative analysis of very low amounts of EGP2 mRNA transcripts in bone marrow and peripheral blood by reverse transcriptase polymerase chain reaction (PCR) (RT-PCR)
After reverse transcriptase polymerase chain reaction (RT-PCR) all subsequent reaction products of the titration series were analysed by gel electrophoresis
A 316-bp product derived from the competitor RNA was readily distinguishable from the 514-bp product generated from the normal EGP2 mRNA transcript
Summary
The human small-cell lung cancer (SCLC) cell lines GLC-3, GLC4 and GLC-14, the human breast carcinoma cell line MCF-7 and the human colorectal cancer cell line Colo 320 were selected as representatives of different types of carcinomas. SCLC cell lines GLC-3, GLC-4 and GLC-14 were chosen because of the different expression levels of EGP2 as determined by immunocytochemistry. All SCLC cell lines have been established in our laboratory and have been described previously
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