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Molecular Pathology and Genetics |
1
Laboratory of Clinical Molecular Biology, Department of Biochemistry, Clos-Chapelle-aux-Champs, UCL 30.46, and Department of Urology, Ave. Hippocrate, 30, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, 1200 Bruxelles, Belgique.
2
Queen Astrid Military Hospital, Rue Bruyn, 2, 1120
Bruxelles, Belgique.
3
Hôpital Necker, Laboratory of
Clinical Chemistry, 149, rue de Sèvres, 75015 Paris, France.
a Address correspondence to this author at: Laboratoire de Biologie Moléculaire Clinique, Clos Chapelle-aux-Champs, 30 - UCL/30.46, B-1200 Brussels, Belgium. Fax 00/32.2.764.39.59; e-mail gala{at}sang.ucl.ac.be.
Circulating prostate cells can be detected in cancer patients by using reverse transcriptase–PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.
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