Simultaneous detection of circulating prostate specific antigen (PSA)-expressing and prostate specific membrane antigen (PSM)-expressing cells by a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay

It has yet to be determined whether the detection of prostate specific antigen (PSA)-expressing or prostate specific membrane antigen (PSM)-expressing cells in the circulation of prostate cancer patients is a more accurate predictor of clinical outcome. A method of evaluating both markers simultaneously would aid in the determination of the clinical relevance of reverse transcriptase polymerase chain reaction (RT-PCR) as a staging tool for prostate cancer. We describe the development of a multiplex RT-PCR assay that simultaneously detects the presence of both PSA-expressing cells and PSM-expressing cells, as well as a ubiquitously expressed internal control all within a single reaction. Both PSA cDNA and PSM cDNA were concurrently amplified by multiplex PCR using LNCaP mRNA as the starting template. When used as part of a nested PCR system, the multiplex RT-PCR assay identified one prostate cancer cell when placed in a background of one million cultured B lymphocytes. The multiplex assay was then applied to mRNA isolated from metastatic prostate cancer patients and from healthy male and female volunteers. While all were positive for the internal control G3PDH, three of seven prostate cancer patients were positive for both PSA and PSM expression and two more were positive for either PSA or PSM. None of the male or female volunteers were positive for either PSA or PSM. Multiplex RT-PCR allows for the amplification of both PSA and PSM cDNA within a single RT-PCR reaction, and this approach should allow a consistent comparison of the clinical utility of both PSA and PSM markers as staging tools and predictors of response to therapy.