Detection of prostatic specific membrane antigen messenger RNA using immunobead reverse transcriptase polymerase chain reaction

The present study was performed to detect circulating prostatic carcinoma ( PC) cells using a novel three-step immunobead reverse transcriptase (RT) po lymerase chain reaction (PCR) assay for prostatic specific membrane antigen (PSMA) messenger RNA (mRNA). The sensitivity and specificity of this techn ique was assessed and the incidence of immunobead RT-PCR positivity correla ted with progressive metastatic disease and serum prostatic specific antige n (PSA) levels. Fifty peripheral blood (PB) samples from 46 patients with P C were incubated with magnetic beads coated with Ber-EP4, antibody directed against the human epithelial antigen, a membrane antigen widely expressed by epithelial cells. The epithelial cell-enriched magnetic fraction was the n subjected to mRNA isolation using oligo-deoxythymidine (dT) magnetic bead s. Nested RT-PCR for PSMA was performed on the mRNA oligo-dT complex and th e identity of the RT-PCR products was confirmed by Southern blotting. Twent y-one PB samples from 8 control subjects without PC were also evaluated. Th ree-step immunobead PSMA RT-PCR was able to detect one PC cell per 1 mt of PB. The positivity rate of the RT-PCR assay was significantly higher (II of 25; 44%) in patients with metastatic tumor than in patients with non-metas tatic disease (1 of 21; 5%) (P = 0.003). In patients with metastatic PC, RT -PCR positivity was much higher in patients with progressive disease (10 of 13; 77%) than in patients with responding or stable disease (1 of 12; 8%) (P = 0.001). There was a statistically significant correlation between immu nobead PSMA PCR positivity and high levels of serum PSA (P = 0.005). All co ntrol subjects without PC tested negative for PSMA PCR. The three-step immu nobead RT-PCR for PSMA can detect circulating PC cells with high specificit y and sensitivity. Preliminary data show a strong correlation between immun obead PCR positivity, the presence of progressive metastatic disease, and h igh levels of serum PSA.