APPLICATION OF IMMUNOMAGNETIC BEADS IN COMBINATION WITH RT-PCR FOR THE DETECTION OF CIRCULATING PROSTATE-CANCER CELLS

Recently published protocols using Reverse Transcriptase Polymerase Ch ain reaction (RT-PCR) for prostate specific antigen (PSA) provide a se nsitive means for detecting circulating prostate cancer cells. Attempt s to use these assays for staging of prostate cancer have produced con flicting results. As a first step towards rectifying these discrepanci es, a modified immunobead-RT-PCR assay capable of detecting as few as 10 prostate cancer cells in 8cc of blood was developed. This 10 fold i ncrease in sensitivity was achieved in part by introducing two target cell enrichment steps. As a model system to assess sensitivity of the modified assay, template RNA was extracted from PSA positive human car cinoma cells suspended in human blood and isolated with immunomagnetic beads following incubation with an epithelium specific antibody. Afte r 45 cycles of PCR, product from as few as IO target cells could be re adily detected when displayed on a 2% agarose gel stained with SYBR Gr een fluorescent dye. The identity of amplified DNA fragments was confi rmed by Southern blot hybridization. When applied to blood samples fro m patients with proven metastatic disease, the immune-bead RT-PCR assa y was successful in detecting circulating PSA positive epithelial cell s, suggesting this assay may be useful for assessment of disease progr ession or recurrence. (C) 1997 Wiley-Liss, Inc.