Papers in Press. First published October 7, 2004 as doi: /clinchem

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1 Papers in Press. First published October 7, 2004 as doi: /clinchem Clinical Chemistry 50: (2004) Proteomics and Protein Markers Analysis of Subforms of Free Prostate-Specific Antigen in Serum by Two-Dimensional Gel Electrophoresis: Potential to Improve Diagnosis of Prostate Cancer Klaus Jung, 1* Janett Reiche, 1 Axel Boehme, 1 Carsten Stephan, 1 Stephan A. Loening, 1 Dietmar Schnorr, 1 Wolfgang Hoesel, 2 and Pranav Sinha 3 Background: The aim of this study was to develop a method to separate and quantify subforms of free prostate-specific antigen (fpsa) in serum by two-dimensional electrophoresis and to assess the diagnostic accuracy of these subforms for prostate cancer (PCa) diagnosis in comparison with total PSA (tpsa) and the ratio of fpsa to tpsa (%fpsa). Methods: Sera from 50 patients with and without PCa, respectively, were studied. PSA was isolated by immunoadsorption on streptavidin-coated magnetic beads with biotinylated anti-psa antibodies and separated by two-dimensional electrophoresis. After semidry blotting, the intensities of the fpsa spots were quantified by chemiluminescence using an imager analyzer. Results: The method detected subforms to a concentration of 0.1 g/l fpsa with an imprecision (CV) <16%. We detected 15 immunoreactive fpsa spots of different intensities. Spots F2 and F3 were present in all samples. F2 was lower in samples from non-pca patients (median, 23%) than in samples from PCa patients (49%), whereas F3 behaved inversely (non-pca, 73%; PCa, 45%). Ratios of F2 to F3 and F2/F3 to %fpsa, respectively, showed improved diagnostic accuracy compared with tpsa and %fpsa. Better differentiation by F2/F3 or 1 Department of Urology and 3 Institut of Laboratory Medicine and Pathobiochemistry, University Hospital Charité, Humboldt University, Berlin, Germany. 2 Roche Diagnostics, Penzberg, Germany. *Address correspondence to this author at: Department of Urology, Research Division, University Hospital Charité, Humboldt University Berlin, Schumannstrasse 20/21, D Berlin, Germany. Fax ; e- mail klaus.jung@charite.de. Current address: Institute of Medical and Chemical Laboratory Diagnostics, State Hospital, Klagenfurt, Austria. Received July 21, 2004; accepted September 9, Previously published online at DOI: /clinchem by F2/F3 to %fpsa was particularly evident in patients with %fpsa values >15%. There were no associations between the PCa grading scale and fpsa subforms. Conclusions: fpsa subforms separated by two-dimensional electrophoresis may improve both sensitivity and specificity in prostate cancer diagnostics compared with tpsa and %fpsa. The development of a practicable assay based on the immunologic properties of these different fpsa subforms seems to be promising American Association for Clinical Chemistry Prostate-specific antigen (PSA) 4 in serum is the most useful indicator for prostate cancer (PCa) (1). The concentration cutoff of 4 g/l is conventionally used to distinguish benign from malignant prostate diseases (2), but only 25% of men with PSA values 4 g/l have prostate cancer because various benign conditions also lead to increased serum PSA concentrations (3, 4). Histologic evidence is necessary for the final diagnosis of prostate cancer; therefore, better preselection of patients with possible PCa is needed to reduce the number of biopsies without evidence of malignancy. Several concepts, such as PSA density, PSA velocity, and age- or race-specific reference limits, have been recommended for improving the limited diagnostic specificity of PSA as a cancer marker (5, 6), and measurements of the molecular forms of PSA have been shown to be most promising in this regard (7). The latter procedure is based on the quantitative differences in molecular forms of PSA in serum of 4 Nonstandard abbreviations: PSA, prostate-specific antigen; PCa, prostate cancer; BPH, benign prostatic hyperplasia; tpsa, total PSA; fpsa, free PSA; %fpsa, percentage ratio of fpsa to tpsa; 2DE, two-dimensional polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; and AUC, area(s) under the curve(s). 1 Copyright 2004 by The American Association for Clinical Chemistry

2 2 Jung et al.: Subforms of fpsa patients with PCa and benign prostatic hyperplasia (BPH) (8 10). Total PSA (tpsa) circulates in serum in complexed forms (bound to protease inhibitors) and in an uncomplexed, free form (8 10). Approximately 70 90% of tpsa is bound to 1 -antichymotrypsin, 1 -protease inhibitor, or inter- -trypsin inhibitor (8 10); the remaining 10 30% of tpsa is not bound to serum proteins and is called free PSA (fpsa). The proportion of fpsa to tpsa is lower in PCa patients than in BPH patients. Numerous reports have demonstrated the high diagnostic validity of the percentage ratio of fpsa to tpsa (%fpsa) for differentiating between malignant and benign prostate diseases (7). There are several hypotheses for the decreased ratio of fpsa to tpsa in cancer patients. The exact reason for this phenomenon has not been elucidated, but it has been shown that the fpsa fraction is heterogeneous (11, 12). The so-called fpsa subforms propsa, BPH-associated PSA, and intact PSA have been characterized in detail and recommended for diagnostic purposes (13 20). However, an even larger number of PSA subforms could be separated as specific spots by two-dimensional electrophoresis (2DE) and immunoblotting (11, 21). Charrier and coworkers (11, 22) used the 2DE pattern of fpsa subforms obtained by this technique as a tool to differentiate between patients with PCa and BPH. However, because of the limited analytical sensitivity of their electrophoretic procedure, only combined groups of spots and not single spots could be evaluated with regard to their diagnostic relevance. The aims of our study, therefore, were (a) to develop a sensitive, reliable procedure for separating and quantifying fpsa subforms based on immunosorption and 2DE in men with tpsa concentrations of 2 20 g/l; (b) to compare the diagnostic validity of the electrophoretic fpsa subforms with conventional data (tpsa and %fpsa) for differentiating between patients with or without PCa; and (c) to assess the association of fpsa subforms with tumor stage and grade. Materials and Methods study groups and samples Groups investigated. We sued archived sera from 50 consecutive patients with PCa (median age, 65 years; range, years) and 50 consecutive patients with BPH (median age, 65 years; range, years) available as surplus unthawed serum with sufficient volume for additional measurements as shown in the method details. Specimens were collected between December 2002 and January 2004 and investigated between January 2003 and January PCa was diagnosed histologically by transrectal ultrasound-guided octant prostate biopsy as described previously (23) and stratified according to the TNM classification (24) and grading system according to Gleason et al. (25). Patients had tumor stages pt1 or T1 (n 6), pt2 or T3 (n 27), pt3 or T3 without metastasis (n 14), T3pN1M0 (n 2), and T3NxM1, respectively, with Gleason scores 7 (n 32) or 7 (n 16). Gleason scores were not available for two patients. In the group of the 50 BPH patients, the clinical diagnosis of BPH was histologically confirmed by use of tissue material obtained by octant biopsy, transurethral resection, or after open adenomectomy. Samples. Blood samples were collected in evacuated tubes (Monovette ; Sarstedt GmbH) before diagnostic procedures were started or 4 weeks (at the earliest) after digital examination, prostatic biopsy, or transrectal ultrasound and, in the case of PCa patients, before different treatment regimens (radical prostatectomy, radiotherapy, hormonal therapy). Samples were centrifuged at 1600g for 15 min at 4 C. The sera were frozen within 2 h after venipuncture and stored at 80 C until analysis. The use of blood samples for research purposes has been approved by the Ethical Committee of the Charité Hospital, Berlin. preparation of samples for 2de PSA from serum was isolated according to a direct immunoadsorption method (26). Briefly, we washed ml of a suspension of streptavidin-coated magnetic beads (10.7 g/l) three times with 1 ml of washing buffer (50 mmol/l Tris-buffered saline solution containing 10 g/l reduced Triton X-100, ph 7.4) and added to it 0.5 ml of biotinylated monoclonal anti-tpsa-m36-igg (Roche; 33.3 mg/l in buffered saline solution containing 10 g/l bovine serum albumin and 1 g/l Tween 20). We incubated the bead mixture for 30 min with slight rotation. The beads were separated, washed three times as mentioned above, and incubated with 1 ml of serum. After 1 h, the beads were collected and washed three times as described. To release the immunoadsorbed PSA, we incubated the beads in ml of rehydration solution {8.3 mol/l urea; 2 mol/l thiourea; 20 g/l Pharmalyte, ph 3 10; 40 g/l 3-[3-cholamidopropyl)dimethylammonio]-1- propanesulfonate, 100 mmol/l dithiothreitol} for 10 min. After separation of the beads, the solution containing the released PSA was used for 2DE. 2de 2DE was performed with the Immobiline polyacrylamide system as described previously (27). For the first dimension, Immobiline polyacrylamide gels (3 meq L 1 ph 1, 3.75% T; 4% C; 12 cm) were prepared in the ph range by a procedure with computer-driven burettes (27). Before isoelectric focusing, the dry gel strips were rehydrated with the above-mentioned PSA-containing rehydration solution for 10 min at room temperature, for 4.5 h at 30 V, and for 4.5 h at 60 V. Isoelectric focusing was performed under the following conditions: 100 V for 1 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 30 min, an increase to 8000 V within 30 min, and then 8000 V for 5 h. For the second dimension, the strips were equilibrated for 10 min in a solution of 6 mol/l urea, 300 g/l glycerol, 0.5 mol/l Tris-HCl, and 20 g/l sodium dodecyl sulfate (SDS) con-

3 Clinical Chemistry 50, No. 12, taining 65 mmol/l dithiothreitol and subsequently for 10 min in the same solution containing mol/l iodoacetamide instead of dithiothreitol. The strips were then sealed on top of polyacrylamide gels (15% T; 0.5% C) with a solution of 5 g/l agarose, 1 g/l SDS, 24 mol/l Tris base, and 20 mol/l glycine. The electrophoresis was performed in a Novex XCell electrophoresis unit (Invitrogen) with a buffer solution containing 25 mmol/l Tris base, mol/l glycine, and 1 g/l SDS. western blot and image analysis After electrophoresis, the protein spots from the gels were transferred by semi-dry blotting (Hoefer) to polyvinylidene difluoride membranes (Millipore) for 1 h at 0.8 ma/cm 2. The cathodic buffer consisted of 40 mmol/l 6-aminohexanoic acid, 0.1 g/l SDS, 0.1 g/l sodium azide, 200 ml/l methanol; anodic buffers I and II consisted of 300 and 25 mmol/l Tris base, respectively, containing 0.1 g/l sodium azide and 200 ml/l methanol. The membrane was blocked with 10 g/l casein (Sigma) in buffered saline solution for 1 h at room temperature, washed, and then incubated for 1 h with a 2000-fold dilution of a rabbit polyclonal anti-psa antibody (no. A056201; Dako) in 5 g/l casein-buffered saline solution. The membrane was then washed twice in buffered saline solution containing 1 g/l Tween 20 and twice in casein-buffered saline solution, and incubated for 1 h with a 2000-fold dilution of a goat horseradish peroxidase-conjugated anti-rabbit IgG (Dako; no. P0448) in casein-buffered saline solution. After the membrane was washed four times in buffered saline solution containing 1 g/l Tween 20, the spots were visualized by a chemiluminescence reaction according to the instructions of the manufacturer (ECL-Plus system; Amersham) on the Fluor-S MultiImager (Bio-Rad) for 5 min. Image analysis was performed with the PDQuest software for Windows, Ver. 6.2 (Bio-Rad). Matched sets for BPH and PCa were built up from all respective scans to identify the spots and measure their intensities. A high-level matched set was made to compare the spots for the BPH and PCa patients. software. Differences were considered statistically significant at P Results analytical characteristics Immunoadsorption of serum PSA. The completeness of immunoadsorption of PSA was tested with five serum samples with tpsa concentrations of g/l and fpsa concentrations of g/l. tpsa and fpsa measured by Immulite and Roche assays before and after immunoadsorption revealed that at most 2.7% of the original concentrations remained in the supernatants after immunoadsorption. Characteristics of the 2DE assay method. Shown in Fig. 1 is an electronic composite of all fpsa spots found in the serum samples of BPH and PCa patients by our 2DE method. Details on the spots are discussed below. To characterize this 2DE method, however, we evaluated the limit of quantification, the linearity, and the between-run imprecision. The limit of detection, defined as the lowest fpsa concentration that produced fpsa spots after 2DE under the conditions described below, was analyzed by dilution of a pooled serum. As shown in Fig. 2, the two major fpsa spots (F2 and F3) could be detected to a concentration of 0.1 g/l fpsa. These dilution experiments also demonstrated the linearity of the fpsa separation method. The mean (SD) recovery for each dilution step in relation to the theoretical value, defined as the sum of the intensities of spots F1, F2, F3, and F4, was 93.7 (11.3)%. The precision of the method was verified by 10 repeats of the total separation procedure using a pooled serum (9.1 g/l tpsa; 0.72 g/l fpsa), including immunosorption, electrophoresis, immunodetection, and imaging of the major spots F2 and F3. The imprecision (CV) for measurement of the F2 and F3 spots was 16% and 12%, respectively. Specificity of the immunodetection. The specificity of the fpsa spots was confirmed by addition of anti-psa antibody 13C9E9D6G8, which was used by Charrier and coworkers (11, 22), to the polyclonal anti-psa antibody psa assays tpsa and fpsa were measured using the IMMULITE analyzer (Diagnostic Products Corp.) as described previously (28). In recovery experiments, PSA measurements were also performed on the Elecsys 2010 analyzer (Roche). statistical analyses Data were analyzed with the statistical software SPSS for Windows (SPSS Inc.), MedCalc (MedCalc Software), and GraphPad Prism 4.02 for Windows (GraphPad Software). Nonparametric ANOVA analyses were performed with the Kruskal Wallis test, Mann Whitney U-test, and calculation of Spearman correlation coefficients. ROC analyses were performed with MedCalc Fig. 1. Overview of all fpsa subforms found in the study groups. Shown is an electronic composite of all fpsa spots found in the serum samples from the BPH and PCa patients studied. The frequencies of the spots are given in Table 1. The sector containing complexed PSA was not shown to ensure a clearly arranged display of all fpsa spots.

4 4 Jung et al.: Subforms of fpsa g/l, 29 patients had concentrations of 4 10 g/l, 9 had concentrations of g/l, and 1 patient had a tpsa concentration 20 g/l. The scatter plots of total and free PSA concentrations and the ratio of fpsa to tpsa are shown in Fig. 3. Fig. 2. Sensitivity and linearity of the combined immunoadsorption and two-gel electrophoresis method to detect fpsa subforms. Pooled serum with a fpsa concentration of 1.0 g/l was diluted to concentrations of 0.5, 0.25, 0.1, and 0.05 g/l, separated, and visualized as described in the Materials and Methods. A (Dako). As demonstrated in Fig. 1S in the Data Supplement that accompanies the online version of this article at spots similar to those detected with the Dako antibody were obtained. fpsa subforms in bph and pca patients Characteristics of the groups studied. Serum samples from 50 BPH and 50 PCa patients were investigated by three experienced laboratory scientists and technicians who were blinded to the clinical origin of the samples. In the group of PCa patients, 32 patients had tpsa values in the range 2 10 g/l, 25 patients had concentrations of 4 10 g/l, 11 patients had tpsa concentrations of g/l, and 7 patients had concentrations 20 g/l. In the group of BPH patients, 38 patients had tpsa values of 2 10 Qualitative data for fpsa subforms. At least 15 immunoreactive spots of different intensities were detected. Fig. 1 gives an overview of all spots. The spots were arbitrarily named by letters or a letter-number combination. Not all spots were found in all samples. The frequencies of the spots found in BPH and PCa patients are summarized in Table 1. Whereas spots F2 and F3, characterized by a mass of 33 kda and pi between 6.6 and 6.8, were present in all samples, spots F1, E, and G occurred more frequently in PCa patients. Spots A, B, C, D, showing a higher molecular mass, were seen only in PCa patients. Quantitative data for fpsa subforms. Scatter plots for the subforms F1, F2, F3, F4, and J, the most frequently observed spots, are shown in Fig. 4. The data are plotted as percentage values of the summarized intensities. The subforms F2 and F3 accounted for 95% of the total spot intensities. The subforms F1 and F4, with a molecular mass ( 30 kda) similar to those for F2 and F3, and the subform J, with a lower molecular mass ( 25 kda) did not exceed 5% of total fpsa in 75% of all cases. It is also striking that the percentages of F2 and F3 were directly opposite in BPH and PCa patients. F2 was lower in BPH (median, 23%; range, 3 68%) than in PCa (median, 49%; range, 15 86%), whereas F3 was higher in BPH (median, 73%; range, 32 99%) than in PCa (median, 45%; range, 14 77%). Thus, the ratio of F2 to F3 was lower in BPH (median, 0.32; range, ) than in PCa (median, 1.01; range, ). Fig. 3. Scatterplots of tpsa (A), fpsa (B), and %fpsa (C) in the study groups. Statistical differences were calculated using the Mann Whitney U-test.

5 Clinical Chemistry 50, No. 12, Spot Table 1. Frequencies of the various molecular fpsa subforms in sera from BPH and PCa patients. a BPH group with positive samples, n (%) PCa group with positive samples, n (%) A 0 (0) 3 (6) B 0 (0) 4 (8) C 0 (0) 4 (8) D 1 (2) 3 (6) E 0 (0) 6 (10) F1 20 (40) 32 (64) F2 50 (100) 50 (100) F3 50 (100) 50 (100) F4 23 (46) 29 (58) G 0 (0) 7 (14) H 0 (0) 1 (2) I 0 (0) 3 (6) J 36 (72) 38 (76) K 0 (0) 3 (6) L 0 (0) 1 (2) a The number of fpsa subforms (spots) in serum samples from 50 BPH and 50 PCa patients, as shown in Fig. 1, were counted. Values indicate the total number and percentage (in parentheses) of the respective spots in the particular group. b Frequency differences between the two groups were tested by the Fisher exact test. Associations with clinicopathologic data.there were no associations between the grading scale (Gleason sum) and percentages of F2, F3, and the ratio F2 to F3 (r s ; P ). A correlation existed between tumor classification (T1-T3) and the ratio of F2/F3 (r s 0.346; P 0.016). Because of the opposite behavior of F2 and F3 in BPH and PCa, the percentage of F2 was negatively and that of F3 positively (r s 0.415; P ) correlated with the ratio of fpsa to tpsa; the F2/F3 ratio was also negatively (r s 0.372; P ) correlated with the ratio of fpsa to tpsa. diagnostic validity ROC analyses were performed for all patients with tpsa values between 2 and 20 g/l and whose spots included the major subforms F2 and F3. The areas under the ROC curves (AUC) are given in Table 2. Absolute and percentage values for F2 and F3 as well as the ratios of F2 to F3, F2 to fpsa or %fpsa, and F2/F3 to %fpsa gave higher AUC values than did tpsa (Table 2 and Fig. 5A). A clear tendency to higher values for the ratios of F2 to %fpsa and F2/F3 to %fpsa was also observed compared with %fpsa (Table 2 and Fig. 5A). The additional improvement by the ratios of F2 to F3 or F2/F3 to %fpsa compared with %fpsa was especially evident in patients with %fpsa values 15% but not in patients with %fpsa 15% (Fig. 5, B and C). In the %fpsa range 15%, both variables gave significantly better differentiation between BPH and PCa, whereas tpsa and %fpsa could not distinguish between these groups (Fig. 5C). The sensitivities and specificities of fpsa subforms F2 and F3 and their derivatives compared with those for P b tpsa and %fpsa at the decision limits for 90% sensitivity and specificity in the tpsa range of 2 20 g/l are listed in Table 3. A complete list, including the values at the 95% confidence limits, is given as Table 1 of the online Data Supplement. Both the specificity and the sensitivity of the ratio F2/F3 and the ratios of absolute or percentage F2 and F2/F3, respectively, compared with %fpsa showed a tendency of better discriminatory power than %fpsa. The results were confirmed by logistic regression analyses showing that the overall correct classification of BPH and PCa increased from 60.2% based on tpsa to 66.7% with %fpsa, to 76.3% with F2/F3, and to 80.6% with the ratio of F2/F3 to %fpsa. When we used all four variables together (forward and backward procedure), only the ratio of F2/F3 to %fpsa remained as an independent variable (P ; odds ratio 3.04, 95% confidence interval, ) for differentiation, giving an overall correct classification of 80.6%. Discussion During the biosynthesis and processing of PSA, various PSA isoforms are generated (17, 29). PSA is synthesized as prepropsa with a 17-amino acid leader sequence that is cleaved, forming the enzymatically inactive propsa. ProPSA is secreted into the lumen of the prostate ducts, and the propeptide is removed by activated human kallikrein 2. The mature PSA of 237 amino acids is enzymatically active, and after diffusing into the blood stream, it is rapidly bound primarily by 1 -antichymotrypsin, generating PSA complexes (8, 9). Several inactive truncated forms of propsa in which any of the seven amino acids have been removed can be formed (20, 30).In the tpsa range of g/l, propsa was measured as the sum of the ( 2), ( 4), and ( 7) forms and averaged 40% of fpsa. The percentage of propsa tended to be higher in the PCa group (50.1%) compared with the non-pca group (35.5%) (31). ( 2)proPSA may represent an important new diagnostic marker for the early detection of PCa (30, 32). Inactive fpsa forms are also generated by internal cleavage. The most common internal cleavage site of the amino acid chain is at Lys 145 -Lys 146 ; another at Lys 182 -Ser 183 produces a fpsa form predominantly associated with BPH (15). The latter accounts for a significant percentage of the fpsa in BPH serum (33). In cancer, decreased processing of propsa to active PSA and of active to inactive PSA is probably one molecular reason for the reduced %fpsa in PCa patients. Additional inactive fpsa subforms refer to PSA that is similar to the intact and mature PSA but shows conformational changes that have made it enzymatically inactive (34, 35). Changes in the internally cleaved PSA forms or the structural changes of intact PSA do not seem to affect the epitopes of PSA that react with anti-psa antibodies. 2DE separates proteins according to two independent properties: their isoelectric points and to their molecular masses. Because we used this technique with isolated PSA from serum and detected PSA-specific spots by Western

6 6 Jung et al.: Subforms of fpsa Fig. 4. Scatterplots of the fpsa subforms F1 (A), F2 (B), F3 (C), F4 (D),J(E), and the ratio of F2 to F3 (F). Statistical differences were calculated using the Mann Whitney U-test. blotting, our study clearly demonstrates the molecular diversity of fpsa in serum. The tpsa and fpsa measurements before and after immunoadsorption of the sera indicated that 97% of PSA in the samples was available for 2DE. The completeness of PSA removal by the immunoadsorption procedure was confirmed by use of differ-

7 Clinical Chemistry 50, No. 12, Table 2. Areas under the ROC curves for major fpsa subforms F2 and F3 compared with those for tpsa and %fpsa in tpsa range 2 20 g/l. a Variable AUC (95% confidence interval) P, pairwise comparison of AUC for various variables with the AUC for tpsa %fpsa tpsa ( ) fpsa ( ) %fpsa ( ) fpsa subforms b F ( ) F2% ( ) F ( ) F3% ( ) F2/F ( ) %fpsa and subforms F2/fPSA ( ) F3/fPSA ( ) F2/%fPSA ( ) F3/%fPSA ( ) (F2/F3)/%fPSA ( ) (F2 F3)/%fPSA ( ) a This subgroup analysis included 43 PCa and 49 BPH patients. b The variables fpsa subforms as % refer to the percentages of the respective fractions in relation to the sum of the subforms J, F1, F2, F3, and F4 taken as 100%. Fig. 5. ROC curves for tpsa, %fpsa, and derivatives of fpsa subforms. (A), ROC analysis for 49 BPH and 43 PCa patients within the tpsa range 2 20 g/l with AUC given in Table 2. (B), ROC analysis for 19 BPH and 31 PCa patients within the tpsa range 2 20 g/l and %fpsa 15% with mean (SD) AUC for F2/F3 of (0.060), for the ratio of F2/F3 to %fpsa of (0.054), and for %fpsa of (0.064), showing significant differences from the AUC for tpsa [0.493 (0.085); P 0.002, , and , respectively], but no differences between F2/F3 or the ratio of F2/F3 to %fpsa and %fpsa (P and 0.471, respectively). (C), ROC analysis for 25 BPH and 11 PCa patients within the tpsa range 2 20 g/l and %fpsa 15% with AUC for F2/F3 of (0.072) and for the ratio of F2/F3 to %fpsa of (0.051), showing significant differences to the AUC for tpsa [0.602 (0.106); P and 0.004, respectively] and that for %fpsa [0.522 (0.105); P and 0.003, respectively]. ent PSA assays; it therefore can be concluded that the negligible loss of PSA did not affect the results of the subsequent experiments. We obtained at least 15 different immunoreactive fpsa spots of different molecular masses or different charges (Fig. 1 and Table 1). The major spots F2 and F3 were characterized by a molecular mass of 33 kda and pi ranging from 6.6 to 6.8. There were also more acidic spots (e.g., spot J) with lower molecular masses but also spots (A, B, C, D) with higher molecular masses of 35 kda detected only in PCa patients. These results generally confirm the previous 2DE data of Charrier and coworkers (11, 22), who did not describe the spots in more detail. However, similar to our study, the fpsa forms with molecular masses 30 kda also accounted for only 5 10% of the total fpsa (11, 22). Combining the fpsa subforms with a molecular mass of 32 kda as standard forms and those below that molecular mass as lower molecular mass forms, Charrier and coworkers (11, 22) showed a higher percentage of the latter fpsa forms in BPH sera than in PCa sera. Similar conclusions concerning the higher proportions of smaller PSA fragments in BPH sera were drawn by Hilz et al. (12), whereas other 2DE studies did not report that these forms were regularly found in seminal fluid, possibly because of the low analytical sensitivities of the detection methods used after 2DE (21, 36). However, our approach of performing 2DE after immunoadsorption of PSA allowed us to differentiate single subforms in more detail (Figs. 1 and 4; Table 1). That procedure produced pictures of fpsa patterns similar to those described by Charrier and coworkers (11, 22).

8 8 Jung et al.: Subforms of fpsa Table 3. Diagnostic sensitivity and specificity of tpsa, %fpsa, and of the ratios of fpsa subforms at the 90% limits to distinguish PCa and BPH patients. a Sensitivity (95% confidence interval), % Specificity (95% confidence interval), % tpsa, g/l (77 97) 11 (3.8 25) (8.6 34) 90 (77 99) %fpsa (77 99) 27 (15 43) (39 70) 90 (77 99) F2/F (77 99) 39 (24 55) (53 82) 90 (77 99) F2/%fPSA (77 99) 52 (37 68) (51 80) 90 (77 99) %F2/%fPSA (77 99) 59 (43 74) (58 86) 90 (77 99) (F2/F3)/%fPSA (77 99) 57 (41 72) (55 84) 90 (77 99) a Data from ROC analysis performed with samples from 49 BPH and 43 PCa patients (tpsa range, 2 20 g/l). Using the monoclonal antibody 13C9E9D6G8, which was used by those authors (11, 22) instead of the polyclonal antibody customarily used in our immunoblot analysis system, we confirmed the comparability of the PSA patterns (Fig. 1S in the online Data Supplement). In this study, we preferred a conventional polyclonal antibody to a monoclonal antibody because polyclonal antibodies generally recognize not only mature PSA but also a wide range of cleaved PSA forms (21). One limitation of the 2DE method used in this study should be considered, similar to that of the procedures described above (11, 12, 22), i.e., PSA not covalently bound to proteins could appear as fpsa forms. However, only a negligible error is likely because very little PSA is bound in such a way, e.g., to inter- -trypsin inhibitor (8). A recent study investigating PSA heterogeneity by 2DE, mass spectrometry, and amino acid sequencing suggested a catalog of molecular forms of PSA to analyze PSA heterogeneity (21). Such a catalog could be helpful for comparative purposes in the future to identify PSA spots. However, the catalog was established with PSA from seminal fluid, which does not contain propsa forms and could therefore not identify those forms (21). Thus, because antibodies against the different propsa forms were not available for this study, an exact relationship between the spots and the distinct propsa or other forms could not be established at this time. Spots F4 or G could represent propsa forms, as already assumed by Charrier et al. (22). In addition, spots E, F1, F2, and F3, which have similar molecular masses but different pi values, may be at least partially explained by differences in glycosylation (37 39). The charge heterogeneity of PSA from seminal fluid was found mainly to be a product of different degrees of sialylation (40), but recent studies showed that not only the glycan content but particularly the structures of the sugar chains affect the differences in glycosylation of PSA forms (38). Thus, to examine this possibility of different glycosylation patterns, e.g., for the major spots F2 and F3, a detailed structure analysis would be necessary, which was beyond the scope of this study. In contrast, spots J, K, and L represent lower molecular mass forms of PSA produced either by internal cleavage of PSA as mentioned above or by alternative splicing of the hklk3 gene, which codes for PSA (41, 42). However, to date, proteins corresponding to these splice variants were demonstrated only in tissue but not in serum (42). The use of %fpsa as a primary decision tool for first-time biopsies in men with nonsuspicious digital rectal examination has been proposed (43, 44). The use of %fpsa can significantly improve specificity by 15 25% compared with tpsa, with only a minimal loss in sensitivity in detecting PCa (45 47). This has been shown for the 4 10 g/l tpsa range as well as for the lower ranges of or g/l tpsa (48, 49). Thus, all new assays for the diagnosis of PCa are only a real diagnostic improvement if they indicate a further advantage to the well-established ratio of fpsa/tpsa. Our study showed that the 2DE subforms F2 and F3 are more suitable to use in differentiating between both groups of patients because their intensities are directly opposite in BPH and PCa patients. F2 was lower in BPH than in PCa, whereas F3 behaved inversely, a further sign of the contrasting association of various fpsa subforms with carcinoma as shown for ( 2)proPSA or BPSA (15, 30). The heterogeneity of fpsa subforms appears to be related not only to the molecular mass but also to that specific pattern and the occurrence of the cancer-specific spots A, B, C, and D, with higher molecular masses of 35 kda (Fig. 1 and Table 1). At the moment, the clinical significance of these spots detected only in PCa patients is still unknown. However, from the analytical point of view, the advantage of subforms F2 and F3 is that they could always be detected, in contrast to the lower molecular forms. The latter account for 0% of the fpsa in a high proportion of PCa patients (22). ROC analyses, the calculation of specificity and sensitivity at certain cutoff points, and logistic regression analyses confirmed that the ratios of F2 to F3 or of F2 as well as of F2/F3 to %fpsa improve the differentiation between both prostate-specific diseases (Tables 2 and 3). Although there was no relationship between the aggressiveness of the tumor (Gleason score) and the above-mentioned variables, the ratio of F2/F3 to %fpsa remained as the sole independent variable in the logistic regression model for predicting PCa. It was most important that in men with 15% fpsa, the ratio of F2/F3 to %fpsa differentiated between BPH and PCa, whereas tpsa and fpsa% did not (Fig. 5C). For ( 2)proPSA, a

9 Clinical Chemistry 50, No. 12, similar effect was noticed recently in men with %fpsa 25% (30). With regard to the clinical application, 2DE is not suitable for routine use because it is expensive in terms of human labor and consumables and needs substantial experience and a sophisticated technique. At present, we would consider it only as a special tool in controversial cases. However, the study showed the discriminating potential of fpsa subforms; thus, more detailed characterization of the fpsa forms in spots F2 and F3 by mass spectrometry could be useful for raising antibodies against defined components and for developing assays. We believe that, in addition to the already characterized propsa forms (20, 30) BPSA (33) and intact fpsa (35), still other forms could also be of diagnostic value. In conclusion, 2DE separated at least 15 different fpsa spots. 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