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Clinical Chemistry 47: 1415-1423, 2001;
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(Clinical Chemistry. 2001;47:1415-1423.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Discrimination of Prostate Cancer from Benign Disease by Plasma Measurement of Intact, Free Prostate-specific Antigen Lacking an Internal Cleavage Site at Lys145-Lys146

Pauliina Nurmikko1a, Kim Pettersson1, Timo Piironen2, Jonas Hugosson3 and Hans Lilja4

1 Department of Biotechnology, University of Turku, Tykistökatu 6A 6th floor, FIN-20520 Turku, Finland.
2 The Finsen Laboratory, Rigshospitalet, 2100 Copenhagen, Denmark.
3 Department of Urology, Sahlgrenska University Hospital, 5-41345 Göteborg, Sweden.
4 Department of Clinical Chemistry, Lund University, University Hospital, S-20502 Malmö, Sweden

aAuthor for correspondence. Fax 358-2-3338050; e-mail pauliina.nurmikko{at}utu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The proportion of free prostate-specific antigen (PSA) is higher in the sera of patients with benign prostatic hyperplasia compared with patients with prostate cancer (PCa). We developed an immunoassay that measures intact, free PSA forms (fPSA-I), but does not detect free PSA that has been internally cleaved at Lys145-Lys146 (fPSA-N), and investigated whether this form could discriminate patients with PCa from those without PCa.

Methods: The assay for fPSA-I uses a novel monoclonal antibody (MAb) that does not detect PSA that has been internally cleaved at Lys145-Lys146. A MAb specific for free PSA was used as a capture antibody, and purified recombinant proPSA was used as a calibrator. The concentrations of fPSA-I, free PSA (PSA-F), and total PSA (PSA-T) were analyzed in EDTA-plasma samples (n = 276) from patients who participated in a screening program for PCa (PSA-T, 0.83–76.3 µg/L).

Results: The detection limit of the fPSA-I assay was 0.035 µg/L. Both the measured concentrations of fPSA-I and the concentrations of fPSA-N (calculated as PSA-F - fPSA-I) provided statistically significant discrimination of the two clinical groups. By contrast, PSA-F did not discriminate between these groups. Each of the ratios fPSA-I/PSA-F, fPSA-N/PSA-T, and PSA-F/PSA-T separated cancer samples from noncancer samples in a statistically significant manner (P <0.0001). The ratio fPSA-I/PSA-F was significantly higher in cancer (median, 59%) compared with noncancer samples (47%).

Conclusions: The ratio fPSA-I/PSA-F is significantly higher in cancer compared with noncancer. The percentages of both fPSA-N/PSA-T and fPSA-I/PSA-F may provide interesting diagnostic enhancements alone or in combination with other markers and require further studies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Measurements of serum prostate-specific antigen (PSA), 1 also known as human kallikrein 3, are widely used in prostate cancer (PCa) detection and screening. PSA is produced by the prostate gland epithelium and secreted into seminal fluid, where it can be found at concentrations of 0.2–5 g/L (1). The main biological function of PSA, a chymotrypsin-like serine protease, is thought to be the cleavage of the gel-forming proteins semenogelin I and II (2)(3). In young healthy males, the PSA concentrations in the blood are <2 µg/L. Increased PSA concentrations in the circulation suggest a disorder of the prostate, which can be prostatitis, benign prostatic hyperplasia (BPH), or PCa. Thus, PSA is not a very specific marker for PCa.

In the circulation, a major portion of PSA is complexed to {alpha}1-antichymotrypsin (ACT). PSA also occurs complexed to {alpha}2-macroglobulin (A2M); this form is not recognized or measured by conventional immunoassays because of steric shielding of the PSA epitopes by A2M under physiologic, nondenaturing conditions (4). In addition, a minor proportion of other serpin complexes has been detected in the blood, such as the PSA–{alpha}1-protease inhibitor complex (5). The ratio of free PSA to PSA-ACT complex has been shown to be higher in patients with BPH than in patients with PCa (6)(7).

The detailed molecular nature of free PSA in the circulation has not yet been fully clarified, but free PSA has been concluded to be inactive because it is essentially nonreactive with the very large excess of inhibitors in the blood, mainly ACT and A2M. The two commonly proposed explanations for the presence of free PSA in the circulation are the proforms of the protein, which have been shown to possess little or no enzymatic activity (8)(9), and internal cleavage at Lys145-Lys146, which has been shown to inactivate PSA (10). It is not known whether internal cleavage at Ser85-Phe86 inactivates PSA (11)(12). Cleavage at Lys182-Ser183 was recently shown to be responsible for the inactive form of PSA, called BPSA, that was isolated from BPH nodules and was also found in seminal plasma (13)(14). In addition, there have been reports describing a mature, intact, inactive form of PSA detected in the spent medium of LNCaP cells (15)(16), in seminal plasma (12), and in serum (17)(18)(19). The reason for the lack of activity is unknown.

We recently reported the development and characterization of a novel PSA-specific antibody that loses recognition of its epitope when PSA is internally cleaved at Lys145-Lys146 (20). We have now developed an immunoassay based on time-resolved fluorescence that selectively measures free PSA that is not cleaved at Lys145-Lys146, now called intact fPSA (fPSA-I). Thus, our assay measures single-chain forms of free PSA, such as proPSA and inactive mature PSA, which has been shown to be nonreactive with inhibitors in blood (Fig. 1 ). This assay makes use of a free-PSA-specific antibody as a capture antibody and the novel fPSA-I-specific antibody as detection antibody. The assay was standardized with purified recombinant proPSA. With this assay we determined fPSA-I concentrations in the sera of patients with and without PCa. Samples were obtained from a population-based PCa screening study, and as a preliminary attempt we wanted to explore the extent to which cleaved and noncleaved PSA forms occur in these two conditions.



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  		Figure 1. Simplified representation of various molecular forms of free PSA and their relation to each other.

hK2, human kallikrein 2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
purified proteins, reagents, and instrumentation
The production of proPSA using the baculovirus expression system has been described previously (9). proPSA was affinity-purified according to the same protocol that we used in the purification of LNCaP PSA, which has been reported previously (16)(20). proPSA was used as a calibrator, and dilutions (0.07–57 µg/L proPSA) were made in Tris-saline-azide buffer (50 mmol/L Tris, 150 mmol/L NaCl, 0.5 g/L NaN3, pH 7.7) containing 75 g/L bovine serum albumin. Concentrations were determined using a Prostatus® PSA free/total reagent set (Perkin-Elmer Life Sciences). Microtiter plate wells were coated with streptavidin (1 µg/well) in citrate-phosphate buffer, pH 5.0, by overnight incubation at 35 °C. Delfia Assay Buffer (containing added mouse IgG to neutralize heterophilic interaction), Enhancement solution, Wash concentrate, and the 1234 Delfia Plate Fluorometer were from Perkin-Elmer Life Sciences.

monoclonal antibodies
We previously reported the characterization of the antibody 5C3 (20), which was raised against purified PSA produced by LNCaP cells. The antibody 5C3 was shown to bind to PSA at an area overlapping the internal cleavage site Lys145-Lys146 and was shown not to recognize PSA that had been cleaved at this site. Monoclonal antibody (MAb) 5C3 binds to proPSA and intact mature PSA with the same affinity. The characteristics of anti-PSA MAbs 5A10 [free-PSA (PSA-F) specific] and H117 [total PSA (PSA-T) specific] have been reported previously (21)(22).

antibody labeling and biotinylation
MAbs 5C3 and H117 were used as detection antibodies and were labeled with europium according to the instructions in the Delfia labeling reagent set (Perkin-Elmer Life Sciences). The labeling degree of the antibodies was 5–11 Eu3+/IgG. The capture antibody 5A10 was biotinylated using 50 mmol/L carbonate buffer, pH 9.8, containing a 40-fold molar excess of biotin-isothiocyanate diluted in dry N,N-dimethylformamide (10 mmol/L). Incubation was at room temperature for 4 h. The biotinylated MAb was purified using NAP5 and NAP10 columns and Tris-saline-azide buffer for equilibration and elution. Diethylenetriamine pentaacetic acid-purified bovine serum albumin was added to a final concentration of 1 g/L to purified biotinylated 5A10 and europium-labeled 5C3 and H117 (5C3-Eu3+ and H117-Eu3+).

immunofluorometric assay formats
The fPSA-I assay is a three-step immunofluorometric sandwich assay. Briefly, biotinylated 5A10 (200 ng/well in 200 µL of Delfia Assay buffer) is bound to streptavidin plates in a 1-h incubation, and the plates are washed four times. Delfia Assay buffer supplemented with mouse serum (0.5 µL mouse serum/100 µL of Delfia Assay buffer per well) is added, after which calibrators or samples are added (50 µL/well). Plates are incubated 2 h at room temperature with slow shaking. After a wash step, europium-labeled 5C3 is added (200 ng/well in 200 µL of Delfia Assay buffer), and plates are incubated overnight at 4 °C. After a wash step, Enhancement solution is added, and after a 5-min incubation, the signal is measured.

The assay for PSA-F (capture antibody 5A10 and detection antibody H117) was performed essentially in the same way as the fPSA-I assay except that a 25-µL sample volume and 100 ng of europium-labeled H117 (H117-Eu3+)/well were used.

sample material
The study population consisted of 276 men, ages 50–66 years, who had participated in a population-based PCa screening study in the area of Göteborg, Sweden and were invited for further examination because of serum PSA concentrations >3 µg/L (23). Before digital rectal examination, transrectal ultrasound (TRUS), and TRUS-guided sextant biopsy were performed, additional EDTA-anticoagulated plasma samples were obtained. Samples were centrifuged within 3 h after venipuncture and stored at -70 °C until analysis. Samples were analyzed without knowledge of the biopsy results. Histopathologic examination of the sextant biopsies showed that 79 of the 276 tested men had PCa. Prostate volumes (PVs) of the patients were determined by TRUS.

We determined the fPSA-I and PSA-F concentrations of the samples with in-house assays (described above) using duplicate samples. The PSA-T concentrations had been determined earlier using the Prostatus PSA free/total reagent set.

Female serum and EDTA- and heparin-plasma samples were collected by venipuncture from five healthy volunteers. Blood samples were centrifuged within 1 h after venipuncture, and serum, EDTA-plasma, and heparin-plasma samples were pooled and immediately frozen and stored at -20 °C.

analytical recovery and in vitro stability of fPSA-I
Ten male serum samples with low fPSA-I concentrations (0.086–0.484 µg/L) were used to study the recovery of fPSA-I. A 1/10 volume of a serum sample with high fPSA-I (44.2 µg/L) was added to a 9/10 volume of each sample with low fPSA-I. The analytical recovery was calculated by dividing the measured fPSA-I concentration by the calculated fPSA-I concentration.

Four EDTA-plasma and serum samples taken from four patients were used to study the stability of fPSA-I. The fPSA-I concentrations of these samples were 0.58–262 µg/L (median, 37.5 µg/L), PSA-F concentrations were 0.77–275 µg/L (median, 41.7 µg/L), and PSA-T concentrations were 8.5–506 µg/L (median, 154 µg/L). The samples had been frozen immediately after venipuncture, processed rapidly, and stored at -20 °C. The stability of fPSA-I was studied by storing aliquots of the samples at room temperature (22 ± 2 °C) for various times (0, 3, 5, 24, 72, 96, 108, 120, 144, 168, 192, and 240 h). After storage, the samples were frozen immediately at -20 °C, and the fPSA-I, PSA-F, and PSA-T concentrations were determined simultaneously for all samples in duplicate. The immunoreactivity at different time points was calculated by dividing the measured PSA concentration after storage with the initial PSA concentration.

In another stability study, proPSA was added at concentrations of 5 and 25 µg/L to pooled female serum, heparin-plasma, and EDTA-plasma, and samples were stored at 4 °C, room temperature (22 ± 2 °C), and 37 °C. Samples were taken at time points 0, 24, 72, and 168 h and frozen immediately at -70 °C until the concentrations of fPSA-I and PSA-F were determined simultaneously for all samples.

statistical analysis
The detection limit was calculated as 2 SD of the calibration diluent divided by the slope of the calibration curve. Differences in the concentrations of PSA-T, PSA-F, fPSA-I, and nicked, free PSA (fPSA-N) and in the ratios PSA-F/PSA-T (F/T), fPSA-I/PSA-F (I/F), and fPSA-N/PSA-T (N/T) between PCa and noncancer patients were determined by a Mann-Whitney nonparametric test. The Kruskal-Wallis nonparametric test was used to study whether the fPSA-I, fPSA-N, PSA-F, and PSA-T concentrations in plasma were significantly different among patients with different PVs. The amount of fPSA-N was calculated by subtracting fPSA-I from PSA-F. ROC plots, areas under the curves (AUCs), and specificity and sensitivity were determined using a Windows-based GraphROC program (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
precision, detection limit, and analytical recovery
Serial 1:5 dilutions of purified proPSA were used as calibrators in the fPSA-I assay (Fig. 2 ). The detection limit of the fPSA-I assay (based on 10 aliquots) was 0.035 µg/L, as calculated using 2 SD of the calibration diluent divided by the slope of the calibration curve. In the analysis of clinical samples, the mean of the CVs of the two duplicates was 8.8% (SD, 8.9%). The median recovery of fPSA-I was 84% in 10 male samples (range, 75%–96%; SD, 6.7%; P <0.0001).



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  		Figure 2. fPSA-I calibration curve ({diamondsuit}) and CVs of calibrators ({circ}).

stability of fPSA-I in vitro
Four EDTA-plasma and serum samples from male patients were used to study the stability of fPSA-I. The fPSA-I concentrations in the plasma samples were 86–101% (median, 96%) of the starting concentrations after 10 days at room temperature. The stabilities of PSA-F and PSA-T were similar: after 10 days, the concentrations were 87–131% (median, 94%) of the starting concentrations for PSA-F and 89–106% (median, 96%) for PSA-T. The changes in the concentrations of fPSA-I, PSA-F, and PSA-T were not statistically significant.

In another stability study, proPSA was added to female serum, EDTA-plasma, and heparin-plasma. Samples were stored for different times at different temperatures, after which separate samples were assayed simultaneously for fPSA-I and PSA-F. During the first 3 days at 4 °C, the concentrations of fPSA-I and PSA-F remained at 89–103% of the starting concentrations (median, 96%; Fig. 3 ). The percentages of decrease of fPSA-I after storage for 7 days at 4 °C were 41% in serum, 4% in EDTA-plasma, and 6% in heparin-plasma. The corresponding decrease in concentration for PSA-F was 39% in serum, 3% in EDTA-plasma, and 4% in heparin-plasma. At room temperature, the results were similar: after 3 days, the concentrations of fPSA-I and PSA-F remained at 93–107% of the starting concentration (median, 97%). After 7 days, fPSA-I had decreased by 26% in serum, 3% in EDTA-plasma, and 15% in heparin-plasma, and PSA-F had decreased by 23% in serum, 10% in EDTA-plasma, and 16% in heparin-plasma. At 37 °C, the concentrations of fPSA-I and PSA-F were 78%–104% (median 88%) of the starting concentrations for the first 3 days, after which there was a dramatic decrease in concentrations (data not shown). There was no dose-dependent effect (proPSA, 5 or 25 µg/L) on the stability of any of the samples.



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  		Figure 3. Stability study using recombinant proPSA added to female serum (A), EDTA-plasma (B), and heparin-plasma (C) and stored at 4 °C for 7 days.

{diamond}, immunoreactivity of fPSA-I; {diamondsuit}, immunoreactivity of PSA-F.

analysis of clinical samples
The concentrations of PSA-T, which had been determined earlier with Prostatus PSA free/total reagent set, were 0.83–56.9 µg/L (median, 4.0 µg/L) in EDTA-plasma samples from patients with BPH and 1.92–76.3 µg/L in samples from patients with PCa (median, 5.3 µg/L; P <0.0001). The median concentration of fPSA-I was 0.43 µg/L in BPH and 0.48 µg/L in PCa (P = 0.0063), and the median concentration of fPSA-N was 0.41 µg/L in BPH and 0.34 µg/L in PCa (P = 0.0271). Thus, in addition to PSA-T, fPSA-I and fPSA-N could separate the two patient groups in a statistically significant way.

Our in-house PSA-F assay detected ~12% higher PSA-F concentrations than those obtained with the Prostatus PSA free/total reagent set. When analyzing the results, we used the PSA-F concentrations obtained from our in-house PSA-F assay to make the PSA-F and fPSA-I assays comparable because the same capture antibody was used in both assays. PSA-F concentrations did not separate the two patient groups (median concentrations, 0.88 µg/L in BPH and 0.94 µg/L in PCa; P = 0.43; Fig. 4A ). When fPSA-I and PSA-F were combined as an I/F ratio, the value was significantly higher in PCa (median, 59%) than in BPH (median, 47%; P <0.0001). The same discriminatory potential could be obtained with F/T and N/T ratios, which were significantly lower in PCa than in BPH (P <0.0001 for both; Fig. 4B and Table 1 ).



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  		Figure 4. Box plots showing distribution of PSA-T, PSA-F, fPSA-I, and fPSA-N concentrations (µg/L; A) and F/T, I/F, and N/T ratios (as percentages; B) among noncancer patients (BPH; {square}) and patients with PCa () in the whole study material.

*, significant (P <0.05); **, highly significant (P <0.01). {circ}, outliers.


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  		Table 1. Median and 25th and 75th percentiles for serum PSA-T, PSA-F, fPSA-I, and fPSA-N1 concentrations and the F/T, I/F, and N/T ratios in sera from BPH and PCa patients with PSA concentrations of 0.83–76.3 and 0.83-10 µg/L.

When plasma samples with PSA-T concentrations <10 µg/L were analyzed (n = 187 for BPH; n = 54 for PCa), the concentration of PSA-T, PSA-F, or fPSA-I alone was unable to differentiate PCa from noncancer (P = 0.35, 0.0581, and 0.82, respectively), although a trend could be seen with the borderline P obtained for PSA-F. By contrast, when we used the concentrations of fPSA-I subtracted from PSA-F to calculate fPSA-N, the concentrations of fPSA-N discriminated cancers from noncancers in a statistically significant manner (median concentration, 0.39 µg/L in BPH compared with 0.27 µg/L in PCa; P = 0.0043; Table 1Up ).

To compare the discriminative power of three free PSA measurements in the whole study material, namely PSA-F, fPSA-I, and fPSA-N, ROC plots and AUCs were calculated. fPSA-I and fPSA-N had significantly larger AUCs (0.6052 and 0.5851, respectively) than PSA-F (AUC = 0.5301; P = 0.0001 and 0.0137, respectively). Thus, greater discriminative power was achieved by measuring subforms of free PSA rather than total free PSA.

To further evaluate the diagnostic performance, we analyzed the ROC plots in the whole study material and calculated the AUCs for PSA-T and the F/T, I/F, and N/T ratios. The I/F and N/T ratios offered discriminative diagnostic power similar to that of the F/T ratio. The AUCs (with confidence intervals) were 0.6605 (0.59–0.73), 0.7238 (0.66–0.79), 0.6761 (0.60–0.75), and 0.7336 (0.67–0.80) for PSA-T, N/T, I/F, and F/T, respectively. The AUCs for the ratios F/T, I/F, and N/T were larger, but not significantly larger, than the AUC for PSA-T when the whole study material was used in the analysis.

At PSA-T concentrations <10 µg/L (Fig. 5 and Table 2 ), the AUCs for the F/T ratio (0.6684) and N/T ratio (0.6674) were significantly larger than the AUC for PSA-T (AUC = 0.5417; P = 0.0305 and 0.0273, respectively). The AUC for I/F (0.6391) was larger but not significantly larger than the AUC for PSA-T. Interestingly, measurement of fPSA-N also gave a significantly larger AUC (0.6275) than the AUC of PSA-T, with a borderline P of 0.0568.



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  		Figure 5. ROC curves for differentiation of patients with PCa from those with BPH.

Plots represent (1 - specificity) at different sensitivities for PSA-T, F/T (%), N/T (%), and fPSA-N for patients with PSA concentrations of 0.83–10 µg/L. Calculated areas (AUC) are shown.


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  		Table 2. AUCs1 with confidence intervals for PSA-T, PSA-F, fPSA-N, and F/T, I/F, and N/T ratios at PSA-T <10 µg/L.

To see how the concentrations of different forms of PSA in plasma correlate with PV, we divided the BPH patients according to PV into three groups: PV <30 mL (n = 29); PV = 30–60 mL (n = 124); and PV >60 mL (n = 38; Table 3 ). Six BPH patients were excluded because of missing PV data. All three free PSA forms as well as PSA-T increased with PV. The relative increases between the low- and high-volume categories were 234% for PSA-F, 121% for fPSA-I, and 326% for fPSA-N compared with 70% for PSA-T. Of the three ratios calculated, N/T and F/T showed positive volume dependencies (P <0.0001), whereas the I/F ratio decreased with increasing gland volume (P = 0.0123). Regression analysis showed highly significant positive correlations between PSA-T, PSA-F, fPSA-I, F/T, or N/T, and PV (P <0.0001). For the I/F ratio, a negative correlation was found (P = 0.0008).


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  		Table 3. Median concentrations of PSA-F, fPSA-I, fPSA-N, and PSA-T (µg/L) and I/F, N/T, and F/T ratios (%) in BPH patients divided into three groups based on PVs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report the development of a specific immunoassay for the measurement of fPSA-I and the preliminary evaluation of its capability of separating cancerous from noncancerous samples in a screening cohort. Our assay measures a free PSA subfraction that has not been internally cleaved at Lys145-Lys146. On the basis of our studies with a patient-screening population (n = 276) with PSA-T concentrations of 0.83–76.3 µg/L, the fPSA-I concentrations were significantly higher in PCa compared with noncancer samples (P = 0.0063). This fPSA-I accounts for ~47% of the free PSA in the plasma of BPH patients and 59% of the PSA in the plasma of PCa patients (Table 1Up ). Interestingly, both fPSA-I and fPSA-N (calculated by subtracting fPSA-I from PSA-F) discriminated cancer from noncancer in a statistically significant way, although PSA-F alone did not. This was also seen when the AUCs for three free PSA measurements were compared; the AUCs for fPSA-I and fPSA-N measurements were significantly larger than the AUC for the PSA-F measurement.

Free PSA containing an internal cleavage site at Lys145-Lys146 is undetectable in our fPSA-I assay. This cleavage renders PSA enzymatically inactive (10) and incapable of forming complexes with ACT. The fPSA-I subfraction of free PSA measured by our assay therefore remains free and uncomplexed in the circulation for some reason other than the inactivation that results from internal cleavage at Lys145-Lys146. Intact PSA detected by our assay could be the mature nonreactive form of PSA that has no enzymatic activity, which has been found in seminal plasma (12), in the spent medium of LNCaP cells (15)(16), and in serum (17)(18)(19). The reason for the inactivity of this mature form of PSA is unknown. Our assay also measures the proform of PSA that is devoid of enzymatic activity and has been characterized as an intact, single-chain form of PSA (9). Other intact, enzymatically inactive forms of PSA are the N-terminally truncated forms of PSA (25) and a PSA variant resulting from alternative splicing of PSA mRNA (26). These different forms of PSA, if present, would remain free in the circulation because of their lack of enzymatic activity.

Cleaved forms of PSA have previously been associated with the serum of noncancer patients compared with PCa patients. With the aid of two-dimensional electrophoresis, Charrier et al. (27) showed that sera from BPH patients contained more internally cleaved forms of PSA than sera from PCa patients. This result is in agreement with our findings that fPSA-N (and the proportion of fPSA-N to PSA-T) is significantly higher in BPH than in PCa patients.

We studied the relationship between PV and concentrations of different PSA forms in BPH patients. All free PSA forms and PSA-T showed highly significant positive volume dependencies, which however, were more pronounced for the free forms than for PSA-T. As a logical consequence of this, BPH patients with larger prostates have higher blood F/T ratios, which is in agreement with previous reports (28). Among the three free PSA forms of this study, fPSA-N concentrations increased on average 326%, or 2.7 times more than fPSA-I between the low- and high-PV groups. Thus, fPSA-N constitutes the fraction of PSA-F that accounts most for the volume dependency of PSA-F and the F/T ratio. It is therefore conceivable that the new information provided by fPSA-I determinations would be especially helpful in correctly identifying PCa lesions in large-volume prostate glands. This will be a target for our future investigations.

The detection limit of our assay was 0.035 µg/L with purified calibrators. However, we could detect some nonspecific background in the patient samples. During the initial development of the assay, we found a frequent overestimation of fPSA-I concentrations, but this problem was reduced and almost abolished by the use of mouse serum in the sample incubation. It could not, however, be eliminated totally: 5 of 276 samples (1.8%) gave higher fPSA-I than PSA-F concentrations. Both assays used the same anti-free-PSA capture antibody, so the result could not be explained by the different ways in which samples bound to the capture antibody. Nonspecific background was also detected when female EDTA-plasma samples were tested with our fPSA-I assay. Some female samples devoid of free PSA gave minor but measurable fPSA-I concentrations, but these were almost abolished after the addition of mouse serum to samples during incubation (data not shown). The slightly low analytical recovery of fPSA-I in male serum samples (median, 84%) also indicates that in some serum samples, some type of interference can be found. We are currently trying to solve this problem without compromising the optimal clinical performance of the assay.

To ensure that the results were not affected by low PSA concentrations, we analyzed the data only from samples with PSA-T concentrations >2 µg/L (n = 78 for PCa and n = 184 for BPH). However, there was no statistically significant difference in the results obtained when we used only the samples with PSA-T >2 µg/L compared with the results analyzed from the data including the samples with PSA-T <2 µg/L (data not shown).

To study the stability of fPSA-I, we diluted proPSA in female serum, EDTA-plasma, and heparin-plasma. After a short period of storage, no significant changes in fPSA-I or PSA-F were detected, especially when plasma was used as a diluent (Fig. 3Up ). The concentrations of fPSA-I (or PSA-F) did not decrease, and the stability of fPSA-I correlated well with the stability of PSA-F. In another stability study where male serum and EDTA-plasma samples were stored at room temperature for 10 days, we could not detect significant changes in the concentrations of fPSA-I. The patient samples that we used in this study were EDTA-plasma samples. Thus, we can conclude that the concentrations of fPSA-I did not change during assay procedures at room temperature and that the stability of fPSA-I is similar to previously reported results on the stability of free PSA (29).

Currently, to our knowledge, there are no immunoassays available to measure different subfractions of free PSA. All previous studies investigating the nature of free PSA in blood were performed using samples from patients with advanced cancer to obtain the high amounts of PSA needed in the purification steps. Our novel immunoassay, on the other hand, enables accurate measurements of the concentrations of fPSA-I and fPSA-N from independent patient samples with low PSA concentrations.

In conclusion, we have developed an immunoassay that measures the single-chain form of free PSA, fPSA-I, present in serum or plasma. On the basis of our preliminary results with a small screening population, concentrations of fPSA-I differ statistically in noncancer and in PCa samples. fPSA-I seems to be a more cancer-related form of free PSA and conversely, fPSA-N is associated more with benign disease. We have now shown that free PSA in blood consists of at least two forms. It remains to be seen whether fPSA-I or fPSA-N alone or in combination with other markers improves the diagnostic accuracy of PCa. A large screening population will be tested to better understand the nature of fPSA-I and its relevance in the diagnosis of PCa.


   Acknowledgments
 
This work was supported by a grant from the Academy of Finland (Project 45252). In addition, this work was supported in part by grants from the Swedish Medical Research Council (Project 7903); the Swedish Cancer Society (Project 3555 and Grant 3792-B96-01XAB); the Faculty of Medicine, Lund University; the Research Fund and the Cancer Research Fund of the University Hospital, Malmö; and Fundacion Federico.


   Footnotes
 
1 Nonstandard abbreviations: PSA, prostate-specific antigen; PCa, prostate cancer; BPH, benign prostatic hyperplasia; ACT, {alpha}1-antichymotrypsin; A2M, {alpha}2-macroglobulin; fPSA-I, intact, free PSA; MAb, monoclonal antibody; PSA-F, free PSA; PSA-T, total PSA; TRUS, transrectal ultrasound; PV, prostate volume; fPSA-N, nicked, free PSA; F/T, ratio of PSA-F to PSA-T; I/F, ratio of fPSA-I to PSA-F; N/T, ratio of fPSA-N to PSA-T; and AUC, area under the curve.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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