Sensitivity or specificity of reverse transcriptase-polymerase chain reaction assays: The real challenge for molecular staging of prostatic carcinomas

Molecular staging by RT-PCR analysis for PSA and PSMA in peripheral blood and bone marrow samples is an independent predictor of time to biochemical failure following radical prostatectomy for clinically localized prostate cancer

“…This controversy was attributed to the highly variable experimental protocols used by different groups and certain key technical pitfalls, including (a) major differences in terms of primer selection, sensitivity, and specificity of the RT-PCR protocol; (b) different timing of molecular staging, often performed shortly after diagnostic TRUS-biopsy (which can confound the results due to biopsy-related transient presence of prostatic cells in the bloodstream [7,8]); (c) high rate of false positive results [31][32][33][34], resulting from illegitimate PSA or PSMA transcripts expressed by non-prostate tissues (e.g., hematopoietic cells) in PBL or BM samples; (d) the evaluation, in most studies, of only one prostaterelated transcript (only PSA or only PSMA); (e) the focus of most studies on analysis of only PBL or only BM samples; and (f) the sampling for RT-PCR analysis, not BM biopsies, but of BM aspirates, which are often significantly contaminated with PBL [5], and thus are not representative samples of the BM tissue. Each of these technical pitfalls has significant adverse effect on the prognostic accuracy of RT-PCR analyses (which explains the conflicting conclusions from different studies), because it results in misclassification of many patients' molecular staging status (e.g., patients without circulating or distant micrometastatic disease are inappropriately classified as 'positive for molecular staging', or the opposite), miscalculation of post-surgical outcome, and incorrect assessment of the prognostic significance of RT-PCR analyses.…”

Combined androgen blockade therapy can convert RT-PCR detection of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) transcripts from positive to negative in the peripheral blood of patients with clinically localized prostate cancer and increase biochemical failure-free survival after curative therapy

“…This controversy is mainly attributed to the highly variable experimental protocols used by the different groups and to certain technical pitfalls, including (a) major differences in terms of primer selection, sensitivity and specificity of the PCR protocols; (b) different timing of molecular staging, often performed shortly after diagnostic transrectal ultrasonographyguided biopsy (TRUS-biopsy) or during and immediately after radical surgery, which can confound the results due to transient dissemination of prostatic cells in the bloodstream (19,20); (c) high rate of false positive results of ultra-sensitive PCR techniques, resulting from the detection of PSA and prostatespecific membrane antigen (PSMA) transcripts which are expressed by non-prostate cells (illegitimate transcription) in the peripheral blood (17,18,21,22); (d) the use of more than 0.8 mg of RNA in the PCR test that results in high rate detection of the illegitimate transcripts for PSA and PSMA (23); and (e) the evaluation, in most studies, of only one prostate-related transcript (PSA or PSMA), separately.…”