Abstract
Objective: To evaluate the clinical utility of using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify prostate-specific antigen (PSA) mRNA in peripheral blood samples from patients with prostate cancer as a predictor of extraprostatic extension of the disease and to assess any correlations with known predictive markers of this condition. Methods: Immediately before radical prostatectomy, peripheral blood samples were taken from 42 men with clinically localized prostate cancer and analysed for PSA and 18S ribosomal (endogenous control) genes using real-time RT-PCR (with gene expression assays and the comparative C T -cycle threshold-method for quantifing). A total of 30 healthy male blood donors aged o50 y was taken as a control group.The relationships between PSA mRNA values, pathological and clinical features were analysed. PSA mRNA value, PSA level and biopsy Gleason score were then compared as predictors of extraprostatic extension. Results: PSA gene expresion was 3.73 times significantly higher in patients with clinically localized prostate cancer than in healthy men (Po0.05). There was no relationship between PSA real-time RT-PCR values and pathological stage pT2 or pT3 (P ¼ 0.5), and no association between PSA mRNA value and serum PSA level (P ¼ 0.9) or the Gleason score of the preoperative biopsy (P ¼ 0.9). Conclusion: There was no significant advantage in using the real-time RT-PCR assay of PSA mRNA before surgery to stage prostate cancer and to discriminate between organ-confined and extraprostatic extension.
Introduction
Despite advances in the clinical staging of prostate cancer, understaging is one of the commonest problems in the clinical management of patients with such disease. In surgical series, as many as 30-50% of patients with clinically localized disease are understaged before the time of surgery. 1 Attempts in order to improve clinical staging by means of molecular techniques (RT-PCR) have been made: prostate-specific antigen (PSA), 2, 3 prostate specific membrane antigen (PSM) 4 and human kallikrein type 2 (hK2) 5, 6 have been used as gene markers for detecting circulating prostate cells in peripheral blood circulation as a metastatic feature.
Introduction
RT-PCR is a powerful tool that can detect a small number of circulating cells by amplifying a targeted mRNA. The amplification of PSA mRNA or another prostate-specific marker gene (such as PSM or hK2) allowed the detection of benign or malignant prostatic epithelial cells. An association has been reported between PSA RT-PCR positivity and metastatic disease, 2 but the published results on the ability of PSA RT-PCR to distinguish between organ-confined and extraprostatic extension are contradictory. 3, 4 Although the use of such methods for clinical staging can be foreseen in the next future, further developments of quantitative RT-PCR methods are needed to make this technology reproducible and reliable in all laboratories.
Introduction
Thus, the present prospective and longitudinal study aimed to test the ability of the quantitative (real time) PSA RT-PCR assay to detect prostate cells in blood specimens of patients with prostate cancer, to predict extraprostatic extension, 7 and to assess any associations with known predictive markers of extraprostatic extension of the disease.
Materials and methods
This prospective and longitudinal study was conducted between July 2002 and December 2003. A total of fortytwo patients (mean age 60.0774.29 y, median 59, range 53-71) with clinically localized prostate cancer (T1-T2b, TNM classification, 2002) 8 and scheduled for radical retropubic prostatectomy were enrolled in the study. Peripheral blood samples were taken from 30 healthy male blood donors aged o50 y as a control group, assuming that the underlying prostate cancer incidence was null.
Materials and methods
According to the recommendations of the UICC, all patients were staged with a DRE, serum PSA assay (Tandem E, Hybritech Inc., San Diego, CA, USA), TRUS, bone scintigraphy and abdominal CT (for lymph nodes) when indicated. None of the patients received hormonal treatment before surgery.
Materials and methods
Immediately before radical prostatectomy, blood samples (10-12 ml) were collected in tubes containing EDTA, transported to the laboratory at room temperature and processed within 4 h. On receipt, mononuclear cells were obtained on a gradient using Lymphoprep (Nycomed, Pharma AS, Oslo, Norway). The total RNA content of the blood samples was isolated using the RNeasy minikit (Qiagen, CA, USA) following the manufacturer's instructions. The total RNA was dissolved in 30 ml of RNase-free water and the concentration and purity determined by ultraviolet spectrophotometry at 260 nm and 260/280 nm.
Materials and methods
To analyze the expression of PSA by real-time PCR, 1 mg of the total RNA from each sample was primed with random primers and first-strand cDNA was synthesized by MultiScribe reverse transcriptase (High-capacity, Applied Biosystems, Madrid, Spain). All PCR reactions were performed using a fluorescence temperature cycler (iCycler, Bio-Rad Laboratories, Hercules, CA, USA) with 25 ml reaction mixture containing 12.5 ml Universal PCR Master Mix (Applied Biosystems, Madrid, Spain), 1.25 ml of a mix of unlabelled PCR primers (18S) and Taqman MGB probe (Assays-on-Demand, Applied Biosystems, Madrid, Spain). Each data point was repeated three times. Relative values were obtained from the threshold PCR cycle number (C T ; cycle threshold), where the increase in signal associated with an exponential growth for PCR product becomes detectable. PCR was performed at 951C for 10 min, 40 cycles of denaturation at 951C for 15 s and annealing/elongation at 601C for 1 min. To determine relative mRNA levels of PSA in each sample the results were normalized to its 18S (used as an internal standard) content and the comparative C T method was used. 9 The relative expression level of the target gene is given by 2 ÀDDC T , where DDC T ¼ DC T target gene ÀDC T18S . For the comparative C T calculation to be valid, the efficiency of the target amplification must approximately equal the reference amplification, and hence the absolute value of the slope of log input amount cDNA vs C T must be o0.1. The slope for PSA and 18S passed this test with input amount cDNA for PSA (range 2-200 ng) and 18S (range 0.1-0.0001 ng). Once this was proven, we used the C T calculation for the relative quantitation of the target.
Materials and methods
Univariate relationships between pathological stage (organ-confined or extraprostatic extension) and several clinical tumour features were analyzed using Student's t-test, w 2 -test, linear regression and ANOVA when indicated. Analysed features were DC T PSA, PSA level, free PSA, free to total PSA ratio, biopsy Gleason score and clinical stage. Multivariate logistic regression analysis was also performed to identify which of the mentioned tumour features were independent predictors of extraprostatic extension. Statistical significance level was achieved when Po0.05.
Results
The clinical and pathological characteristics of the 42 patients are given in Table 1 . The PSA gene expression was 3.73 times significantly higher in patients with clinically organ-confined prostate cancers than in healthy males (Student's t-test, P ¼ 0.025). There was no significant association between quantitative real-time PSA RT-PCR (DC T PSA) and pathological stage T2 or T3 (Student's t-test, P ¼ 0.5) with a PSA expression rate 0.83 times greater in pT3 than pT2 (Table 2) .
Results
On univariate analysis of the real-time PSA RT-PCR results did not correlate with serum PSA level (linear regression, P ¼ 0.9), free PSA (t-test, P ¼ 0.97), free/total PSA ratio (w 2 , P ¼ 0.74), biopsy Gleason score (ANOVA,
Results
The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the ROC curve (AUC) of the PSA mRNA real-time PCR in relation to the presence of prostate cancer with different cutoff points of the DC T value are shown in Table 3 . The best cutoff point to discriminate prostate cancer from the healthy males is a DC T value of 8.52 (AUC ¼ 0.598). Table 4 shows the sensitivity, specificity, PPV, NPV and the AUC of the PSA mRNA real-time PCR, PSA, biopsy Gleason score and clinical stage in relation to the presence of extraprostatic extension. No independent predictors of extraprostatic disease were found after the multivariate analysis (logistic regression). Previous studies of RT-PCR strongly suggest that PSA positive cells detected in the peripheral circulation are associated with metastatic prostate cancer. 10, [11] [12] [13] [14] [15] [16] From the hypothesis that prostate cancer metastasizes early in its natural history, there should be an increased detection rate of PSA mRNA transcripts according to the pathological stage. 17 The effort to identify early metastatic disease provided the incentive to use RT-PCR in prostate cancer.
Results
PSA mRNA positivity was reported as 0-39% in patients with clinically localized prostate cancer. 18 Despite the correlation reported between PSA mRNA positivity and the pathological stage the published results are contradictory and there is a trend to consider that this technique is not useful in predicting the pathological stage of the tumor. 19 Also, the classical procedure for the identification of prostatic cells, the nested RT-PCR of PSA mRNA in peripheral blood of prostate cancer patients, suffers from some important troubles: an increased number of false positive because of the possible cross-contamination in the laboratory, the illegimate transcription of mRNA in leukocytes (especially with PSMA mRNA 20, 21 ) and the positivity in nonprostatic tissues. 22, 23 So, the next step must be to quantify the number of prostatic cells in the peripheral blood, lymph nodes or bone marrow with quantitative RT-PCR methods. Table 2 Relative quantitation using the comparative C T method for PSA as target gene and 18S as internal standard Table 3 The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the ROC curve (AUC) of the PSA mRNA real-time PCR in relation to the presence of prostate cancer 
PSA for detection of prostatic cells L Llanes et al
Different quantitative methods have been proposed, based on relative or absolute quantification of target gene mRNA. In general, there are some reports that showed a relationship between the quantity of circulating prostatic cells and the pathological stage, 24, 25 but other studies did not find an improvement in the preoperative prostate cancer staging. 26, 27 From few years ago, real-time PCR has emerged like the best method for the quantitation of a target gene mRNA. Initial reports with quantitative nonested RT-PCR for PSA mRNA (LightCycler system applying the SYBR Green protocol) showed significant quantitative differences among stage pT2 disease, greater than stage pT2 disease and benign prostatic hyperplasia in percentage of patients that expressed PSA mRNA and the amount of it. 28 Recently, a highest detection of PSA mRNA in stage pT3 than pT2 and BPH has been reported, but the percentage of patients detected is similar in both groups(38 vs 39%). 29 The present prospective study was designed to establish the possible benefit of using PSA RT-real-time PCR in the preoperative staging of clinically localized prostate cancers. According to our results, the real-time detection of PSA mRNA in blood samples was 3.73 times higher in prostate cancer patients when compared to healthy males, but it failed to distinguish between organconfined (or pT2 stage) and extraprostatic extension (or pT3 stage) because the detection of PSA mRNA is 0.83 times higher in cancers with extraprostatic extension (pT3). To explain this lack of reliability between our results and the previous reports, it could be hypothesized that a selection bias towards less differentiated tumors can be responsible for the lack of positive results, 30 but this explanation is not valid for the studied population, since up to 92.9% of them had a Gleason score lower than 8.
PSA for detection of prostatic cells L Llanes et al
Thus, in our experience, the usage of quantitative realtime RT-PCR to determine PSA mRNA in peripheral blood does not improve the discrimination between organ-confined and extraprostatic extension before surgery. Further developments of quantitative RT-PCR methods or another prostate-specific marker gene are needed to make this technology reproducible and reliable in all laboratories. Maybe, the next step will be to improve the characterization of the circulating prostatic epithelial cells in order to facilitate diagnostic tools and more effective therapies. PSA for detection of prostatic cells L Llanes et al
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