Detection of disseminated prostate cells by reverse transcription-polymerase chain reaction (RT-PCR): technical and clinical aspects.

Abstract

Metastasis of prostate cancer (CaP) to bone is responsible for much of the morbidity and mortality associated with this disease. While the primary goal of current methods of treatment is to prevent, slow down or reverse metastatic spread, the sensitivity of today’s conventional staging techniques is insufficient to detect micrometastatic disease. Radical prostatectomy is intended to be a complete cure, but 15–30% of these cases recur, because CaP cells that have escaped the prostate prior to surgery give rise to metastases. Although macroscopic techniques such as the bone scan cannot detect micrometastases, new molecular approaches that are under development may prove to be capable of filling this need. There is now hope that detection of prostate cells in circulation may provide a new and highly sensitive staging tool. Because prostate-specific antigen (PSA) is produced almost exclusively by prostate epithelial cells (Clements and Mukhtar, 1994; Levesque et al., 1995a,b; Oesterling, 1991; Zarghami and Diamandis, 1996; Zarghami et al., 1997), the presence of PSA-positive cells in circulation clearly indicates an abnormal condition, and a strong correlation between the appearance of prostate cells in circulation and the advent of metastasis might be expected. Current models of metastasis hold that cells ‘‘shed’’ by primary tumors are borne by the circulation (blood and lymphatics) to other organs, where they are deposited and form secondary tumors. It is not known whether these circulating cells have metastatic potential, or whether they must undergo further changes to cause metastases. Protocols based on the reverse transcription-polymerase chain reaction (RT-PCR) have been developed in response to the need for identification and widespread adoption of superior methods for detecting these extra-prostatic cells, so that more systematic studies with long-term follow-up may be undertaken. Currently, the mRNAs encoding PSA, prostate-specific membrane antigen (PSMA), and human glandular kallikrein (hK2) are the 3 messages most frequently used for detection of prostate cells, but these messages, while prostate-specific, are not cancer-specific, and therefore yield little or no information about the metastatic potential of the cells detected. Markers related to cancer progression are desired from the viewpoints of both prognostic methods and basic understanding. The limit of detection (LoD) of RT-PCR is better than that of other methods in current use, such as flow cytometry and immunohistochemistry (Pelkey et al., 1996). There is therefore considerable interest in the use of RT-PCR for detection and possible quantification of prostate cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN) samples from CaP patients. However, despite the promise of this method, it is not yet clear whether RT-PCR will prove to be of clinical value in prostatecancer staging. In this review we will summarize published reports on RT-PCR detection of circulating prostate cells. Because the field is developing rapidly, there have been several significant advances since the last major reviews were published (Gomella et al., 1997; Olsson et al., 1997), including the use of longer primers with 2-temperature PCR for specificity, development of internal controls and targeting of the hK2 message. There have also been a number of additional reports describing RT-PCR protocols and clinical results in CaP. With a very few exceptions, we have restricted the scope of this review to published articles. The review is divided into 2 parts: a technical evaluation of RT-PCR assays used for detection of prostate cells and a discussion of the results of clinical studies using these procedures.