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33 Increased Sensitivity in the Detection of Circulating Prostatic Tumor Cells in Peripheral Blood after Immunobead enrichment and RT-PCR for Prostate Specific Membrane Antigen (PSMA) mRNA.

Our hypothesis is that immunobead enrichment followed RT-PCR increases the detection rate of PSMA mRNA in the blood of prostatic cancer patients. PSMA is a prostatic tissue specific marker highly expressed in metastatic tumors. We therefore compared the detection of a novel three step immunobead RT-PCR assay for PSMA mRNA with non-immunobead RT-PCR. The three step immunobead PSMA RT-PCR was able to detect one PC cell per one milliliter of Peripheral Blood (PB). Twenty-six duplicate samples of PB from twenty-four Prostatic Carcinoma (PC) patients with metastatic progressive disease were analyzed for the comparative study. One set of PB samples were incubated with magnetic beads coated with Ber-EP4 antibody directed against the Human Epithelial Antigen, a membrane antigen widely expressed in epithelial cells. The enriched magnetic fraction of epithelial cells was then used for the isolation of mRNA using oligo (dT) magnetic beads. Total RNA from the other set of PB samples was isolated using RNAzol. Nested RT-PCR for PSMA was performed on the mRNA-oligo (dT) complex and the total RNA isolated using RNAzol. The identify of the RT-PCR products was confirmed by Southern Blotting. The three-step immunobead PSMA RT-PCR was able to detect a higher number of cases (11/24; 46%) than in non-enriched samples (6/24; 25%). All the samples were positive for the presence of actin mRNA by RT-PCR confirming the presence of amplifiable RNA. It is our conclusion that the three-step immunobead PSMA RT-PCR can detect PC cells with higher sensitivity than non-immunobead RT-PCR. This novel PSMA RT-PCR assay may help better stratify patients with PC.

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