Detection of Circulating Prostate Cells during Radical Prostatectomy by Standardized PSMA RT-PCR: Association with Positive Lymph Nodes and High Malignant Grade

Background: The purpose of this study was to establish a quantitative standardized RT-PCR for the detection of Prostate Specific Membrane Antigen (PSMA)-expressing circulating cells and to evaluate clinical relevance in patients undergoing radical retropubic prostatectomy (RRP) for clinically localized prostate cancer (PCa). Materials and Methods: An external standard molecule (PSMA MIMIC) was constructed for standardization of PSMA RT-PCR reactions. It has the same sequence as endogenous PSMA, except for a central 85 bp deletion, allowing the amplification of both targets simultaneously with nearly the same amplification characteristics as in a nested PCR. PSMA RT-PCR was performed from peripheral blood samples of 73 patients with clinically localized PCa, 4 with metastatic PCa, 27 with benign prostatic hyperplasia (BPH) and 27 controls. Pre, intra- and postoperative blood samples were tested for circulating PSMA expressing cells in 54 out of 73 patients with clinically localized PCa. We also tested intraoperative blood samples of 19 BPH patients treated by transurethral or open surgery. Results: Endogenous PSMA signals from PCa patients varied between 850 and 9900 transcript molecules, corresponding to 2-20 PSMA-expressing cells/ml blood. Standardized RT-PCR using the PSMA MIMIC molecule revealed a significant decrease of false-positives in cancer-free controls (p=0.004). Controls could clearly be distinguished from prostate cancer patients based on PSMA PCR positivity (p=0.003). Thirty-two % of patients with localized prostate cancer, 11% of BPH patients and 7% of healthy controls were positive in standardized assays compared to 48%, 30% and 27% without PSMA MIMIC, respectively. Preoperatively, a correlation with tumor stage (p=0.030), grade (p=0.035) and Gleason Score (p=0.03) could be demonstrated in clinically localized PCa patients. Dissemination of prostate cells during surgery occurred in 32% of the RRPs and 21% of BPH patients. Positive PCR signals from intraoperative blood samples correlated with positive lymph node status (p=0.007) and tumor grade (p=0.005). Postoperative positive results correlated with grade (p=0.012) and Gleason Score (p=0.035). Conclusion: Counseling the patient with clinically localized prostate cancer can be challenging. Surgery may be, in retrospect, inappropriate in a number of patients due to preoperative understating. This newly constructed external standard allows quantitative detection of circulating prostate cells, and therefore may open new perspectives for PSMA RT-PCR techniques as diagnostic assays and tools for post-therapeutic follow-up.