Quantitative Real-Time RT-PCR for Detection of Circulating Prostate-Specific Antigen mRNA Using Sequence-Specific Oligonucleotide Hybridization Probes in Prostate Cancer Patients

Abstract

Objective: A great number of studies have failed thus far to demonstrate that the presence of PSA-expressing tumor cells in the blood of prostate cancer (PC) patients is a highly sensitive prognostic marker, particularly after radical prostatectomy (RPX). These studies have only relied on qualitative or semiquantitative detection techniques, however. We report our initial experience in testing real-time RT-PCR for the detection of PSA mRNA using a quantitative online PCR system, the LightCycler^TM, and sequence-specific oligonucleotide hybridization probes. Methods: Blood samples were obtained before and after surgery from 129 patients undergoing RPX for localized PC and from 19 patients undergoing transurethral resection of the prostate for benign prostatic hyperplasia (BPH). Quantitative RT-PCR for the detection PSA mRNA was performed using the LightCycler system with RNA Amplification Kit Hybridization Probes and sequence-specific oligonucleotide hybridization probes. Results: PSA mRNA was detected by the LightCycler in 28 patients (39%) with pT2 tumors, in 22 patients (38%) with >pT2 tumors, but in only 3 patients (16%) with BPH. The mean values of the LNCaP cell equivalents were higher in >pT2 patients than in pT2 patients (37 × 10³ and 104 × 10³) or BPH patients (7.1 × 10³ and 4.8 × 10³) both preoperatively (333 × 10³/ml blood) and postoperatively (545 × 10³). Conclusion: Real-time RT-PCR with the LightCycler appears to be a promising method for the preoperative detection of circulating LNCaP tumor cells in PC as reflected by PSA mRNA. Considering the low detection rates in BPH patients, the method may also be suitable for patient monitoring after RPX and could thus play an important role in deciding on early radiotherapy or even hormone ablation therapy. Additional long-term follow-up will have to show whether patients with high expression of PSA mRNA actually have an increased risk or recurrence and whether the method is suitable as well to detect progression.