A QUANTITATIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION-BASED ASSAY TO DETECT CARCINOMA-CELLS IN PERIPHERAL-BLOOD

The presence of tumour cells in the circulation may predict disease re currence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and qua ntitative technique for the detection of carcinoma cells in blood, usi ng the reverse transcriptase polymerase chain reaction (RT-PCR) identi fying 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 c ells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochem istry with the anti-EGP2 MAb MOC31 could not identify any positively s tained cell. Analyses using this RT-PCR assay may prove to have applic ations to the assessment of circulating tumour cells in clinical sampl es.