Benign prostate-specific antigen (BPSA) in serum is increased in benign prostate disease.
The biological mechanism for the increased concentrations of free PSA in benign disease is not fully understood, but it is generally accepted that free PSA is composed of enzymatically inactive PSA. This hypothesis is supported by studies on BPH tissue nodules that showed that nodular PSA was more enzymatically inactive than seminal plasma PSA (8). PSA has been most widely characterized in seminal plasma, for which several internally clipped and inactive forms have been described (9). Although each of these inactive PSA forms may also be present in the serum at some concentration, the major percentage of free (noncomplexed) PSA in serum is now known to comprise at least three distinct forms of inactive PSA, which have been recently reviewed (10). This includes one form of PSA that has been identified as the proenzyme, or precursor form of PSA (pPSA), which is associated with cancer (11-14). A second PSA form appears to be composed largely of intact PSA that is similar to native, active PSA except that it is enzymatically inactive (15-17). A third form of free PSA, called "benign" PSA (BPSA), is an internally cleaved or degraded form of PSA that is more highly associated with BPH tissues (18). BPSA is simultaneously clipped at [Lys.sup.145] and [Lys.sup.182]. Additional studies have shown that BPSA is also present in seminal plasma (19). The BPSA in these studies was purified and analyzed by analytical HPLC methods because PSA is present at gram per liter concentrations in tissues and seminal plasma. The critical questions remained: was BPSA also found in the serum of men with BPH and, if so, at what concentrations? The measurement of BPSA in serum is far more difficult than in tissue or seminal plasma because PSA is present at only microgram per liter concentrations. This report describes the development and characterization of a research immunoassay to measure BPSA in serum and the first quantification of BPSA concentrations in men with BPH and cancer.
Materials and Methods
Murine monoclonal antibodies (mAbs) to BPSA were obtained by standard techniques of mouse immunization and hybridoma development (20). Hybridomas were screened by ELISA. Antibodies were selected based on their reactivity to BPSA and minimal PSA reactivity. Antibodies for assay development were purified from ascites with protein G-Sepharose (Amersham Biosciences Corp.). The BPSA assay is for research use only and is not for diagnostic use.
The BPSA assay protocol is as follows. Biotinylated capture anti-PSA mAb PSM773 (100 [micro]L; 0.25 mg/L), diluted in 10 mmol/L Tris-0.15 mol/L NaCl, pH 8.5, containing 10 g/L bovine serum albumin (mAb buffer), was incubated for 1 h with shaking in a streptavidincoated microtiter plate. The plate was washed with 10 mmol/L Tris-0.15 mol/L NaCl, pH 8.5, containing 0.5 mL/L Tween 20 and 50 [micro]L of blocking buffer (10 mmol/L Tris-0.68 mol/L NaCl, pH 8.5, containing 10 mL/L Tween 20,10 mL/L Triton X-100, 200 mL/L horse serum, and 47 g/L bovine serum), followed by 50 [micro]L of either the calibrator in mAb buffer or sample and allowed to incubate with shaking for 2 h. The plate was washed, and the alkaline phosphatase-labeled anti-BPSA mAb, PS2E290, was added (100 [micro]L; 2 mg/L) and allowed to incubate with shaking for 1 h. The plate was washed, and 100 [micro]L of 4-methylumbelliferyl phosphate was added and allowed to incubate with shaking for 5 min. The plates were then read at 5, 60, and 120 min. Relative fluorescence was measured in a Victor 1420 multilabel counter (Wallac, EG&G).
The serum cohorts tested included the following: BPH [biopsy-negative men with increased PSA (PSA, 1.5-17 [micro]g/L)]; cancer [biopsy-positive (PSA, 2.6-9.7 [micro]g/L)]; urologic outpatients [men who received outpatient examination but who were not suspected of having cancer or benign disease (PSA, 0.17-2.3 [micro]g/L)]; young men [men <30 years of age in apparent good health (PSA, 0.21-0.9 [micro]g/L)]; females [young women <30 years of age (PSA nondetectable)]; and prostatectomy [post-radical prostatectomy serum with minimal PSA (PSA, 0-0.34 [micro]g/L)]. PSA and free PSA were measured with Hybritech brand Tandem-MP PSA and FPSA assays (Beckman Coulter, Inc.). The analytical minimum detectable concentration (MDC) of these assays is 0.05 [micro]g/L. All patients providing serum signed an informed consent approved in advance by Western Institutional Review Board (Seattle, WA).
The biological MDC for BPSA was calculated as the mean concentration of BPSA in 20 female sera plus 2 SD of the mean. The cross-reactivity of the BPSA assay was calculated as the slope of the BPSA calibration curve divided into the slope of the calibration curve for PSA that did not contain the internal clip at [Lys.sup.182] [[PSA.sub.182(-)]]. The concentrations of all free PSA forms were determined with the Hybritech brand Tandem FPSA (Beckman Coulter).
Transurethral resection of the prostate (TURP) tissue was frozen in liquid nitrogen and homogenized in phosphate-buffered saline (PBS) as described previously (18). PSA was purified from the filtered supernatant solution by passage over an immunoaffinity column containing the bound anti-PSA mAb, PSM773 (Beckman Coulter), at 5 g/L of resin. PSM773 has been shown previously to be a PSA-specific mAb and to have specificity for mature, cleaved, and precursor forms of PSA (21-23). The column was washed with 40 volumes of PBS containing 1 mL/L reduced Triton X-100 (Sigma), and the PSA was eluted with 100 mmol/L glycine, pH 2.5, containing 200 mmol/L NaCl. The eluate was immediately neutralized with 1 mol/L Tris, pH 8.0 (100 mL/L). For the purification of PSA from seminal plasma, seminal plasma was diluted 1:20 in PBS (1 mL of seminal plasma plus 20 mL of PBS), and the PSA was purified as described above.
High-performance hydrophobic interaction chromatography (HIC-HPLC) was performed with a polypropylaspartamide column [PolyLC; 250 X 4.6 mm (i.d.); pore size, 1000 A; distributed by Western Analytical]. Samples were applied in 1.5 mol/L ammonium sulfate and eluted with a gradient. Buffers were as follows: buffer A, 1.2 mol/L sodium sulfate-25 mmol/L sodium phosphate, pH 6.3; buffer B, 50 mmol/L sodium phosphate-50 mL/L 2-propanol. The gradient was 0-30% B over 1 min and 30-60% A over 10 min, followed by equilibration in buffer A. High-sensitivity peak detection was obtained with a Varian Model 9070 (Varian Medical Systems) scanning fluorescence detector with excitation at 232 nm and emission at 334 nm to detect the tryptophan residues in protein.
N-Terminal sequence analysis of the samples was performed on an Applied Biosystems Model 492 protein sequencer. Purified PSA and peaks collected by HICHPLC were applied directly to Prosorb cartridges (Applied Biosystems), which were then washed with three 0.1-mL volumes of 0.1 mL/L trifluoroacetic acid, and the membrane was applied to the sequencer.
PSA samples were incubated with 1 g/L lysyl endopeptidase C (LysC; Wako Chemicals USA) in PBS, pH 7, for 30 min to obtain comparable proportions of BPSA and PSA entirely cleaved at [Lys.sup.182] only [[PSA.sub.182(+)]]. The LysC reaction was quenched by the addition of a 10-fold molar excess of aprotinin before analysis by HIC-HPLC. The internal cleavage sites of the different HIC-HPLC peaks were determined by N-terminal sequencing.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4-20% gradient minigels (Invitrogen) under reducing or nonreducing conditions, as indicated. Samples for sequencing were resolved by SDS-PAGE and electroblotted onto polyvinyl difluoride membranes (Applied Biosystems). The bands were visualized with Coomassie Brilliant Blue G250, excised, and applied directly to the Applied Biosystems Model 492 protein sequencer.
PSA was affinity-purified from seminal plasma and further purified by HIC-HPLC to yield the single peak containing no BPSA [[PSA.sub.BPSA(-)]; Fig. 1, peak A], as has been described previously (19). The PSA in peak A contained ~35% PSA cleaved at [Lys.sup.145] and 4% cleaved at [Lys.sup.182], which are typical values for seminal plasma [PSA.sub.BPSA(-)]. Peak A contained all forms of seminal plasma PSA except BPSA. After digestion with LysC, the PSA of peak A was converted into two new peaks (Fig. 1, peaks A and C). N-Terminal sequencing confirmed that peak A represented PSA entirely cleaved at [Lys.sup.l82] only [[PSA.sub.182(+)]] and that peak C represented PSA cleaved at both [Lys.sup.145] and [Lys.sup.182] (the BPSA form of PSA).
SDS-PAGE of the peaks in Fig. 1 was performed under standard reducing conditions with 2-mercaptoethanol, and the identities of the bands were further confirmed by acid sequencing (Fig. 2). Lane 1 in Fig. 2 shows the purified PSA starting material, of which the majority is intact PSA but which also contains internal peptide bond cleavages. The bands at 22 and 10 kDa are the fragments containing residues 1-145 and 146-237, respectively, resulting from cleavage at [Lys.sup.145]. Lane 2 in Fig. 2 shows that peak B contains no intact PSA and is almost entirely cleaved at [Lys.sup.182] [[PSA.sub.182(+)]]. The fragments at 29 and 6 kDa represent fragments containing residues 1-182 and 183-237, respectively. Lane 3 in Fig. 2 shows the fragment pattern of peak C (BPSA). The main bands are found at 22, 6, and 4 kDa and represent fragments comprising residues 1-145, 146-182, and 183-237, respectively. These results demonstrate that very pure and homogeneous BPSA and [PSA.sub.182(+)] can be isolated after treatment of PSA with LysC.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
In addition to the natural BPSA present in seminal plasma, PSA also naturally contains minor amounts of [PSA.sub.182(+)] as demonstrated by the SDS-PAGE blot and N-terminal sequencing and Western blot analysis of peak A in Fig. 1. BPSA contains clips at [Lys.sup.145] and [Lys.sup.182] whereas [PSA.sub.182(+)] contains only a single clip at [Lys.sup.182] Seminal plasma PSA typically contains 7-10% BPSA compared with 3-4% [PSA.sub.182(+)], or approximately two to three times more BPSA than [[PSA.sub.182(+)]. Prostate transition zone tissues obtained from TURP samples were analyzed to determine whether this reflected similar proportions of BPSA and [PSA.sub.182(+)] in BPH tissue.
The HIC-HPLC profile of the PSA forms purified from TURP tissue is shown in Fig. 3A. The clearly resolved PSA and BPSA are equivalent to peaks A and C, respectively, in Fig. 1. To determine the concentration of any unresolved [PSA.sub.182(+)] that might be present in the PSA eluting at 10 min in Fig. 3A, an immunoaffinity column was constructed to purify all PSA forms containing the clip at [Lys.sub.182]. The mAb PS52E290 was used for the immunoaffinity column because this mAb was demonstrated to have high selectivity for PSA containing the [Lys.sup.182] clip (Fig. 4). This includes PSA with only the [Lys.sup.182] clip and PSA containing clips at [Lys.sup.182] and [Lys.sup.145].
The PSA from Fig. 3A was passed over the PS2E290 immunoaffinity column, and the elution profile of the PSA that bound to the column is shown in Fig. 3B. Because only BPSA containing the clip at [Lys.sup.182] is retained on the PS2E290 immunoaffinity column, Fig. 3B shows the relative proportions of BPSA and [PSA.sub.182(+)], both of which were confirmed by N-terminal sequencing. In the sample shown in Fig. 3, the [PSA.sub.182(+)] represented 21% of the eluted [Lys.sup.182]-clipped PSA forms, whereas BPSA represented the remaining 79%. The same analysis of six TURP tissues from different patients is shown in Table 1, which lists the relative percentages of BPSA and [PSA.sub.182(+)] in each of the individual TURP tissues. Despite the wide range of naturally occurring BPSA present in these tissues (3-24% of the total PSA), the [PSA.sub.182(+)] remained a relatively constant minority percentage of the BPSA, suggesting that both resulted from the same mechanism. Therefore, the [PSA.sub.182(+)] represented a mean (SE) of 31% [+ or -] 7% of the [Lys.sup.181]-clipped PSA in TURP tissue, whereas BPSA represented 69% [+ or -] 7%.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Mice were immunized with various NSA forms, including native BPSA purified from seminal plasma, in vitro prepared BPSA purified after treatment with LysC or trypsin, and [PSA.sub.182(+)] obtained after LysC treatment. The pure and precisely characterized forms of BPSA and [PSA.sub.182(+)] shown in Figs. 1 and 2 were used to evaluate the binding and specificities of the resulting mAbs. The negative-control PSA was [PSA.sub.BPSA(-)] (Fig. 1, peak A).
The antibodies with the highest specificity for BPSA and low cross-reactivity with [PSA.sub.BPSA(-)] were determined to have specificity for the presence of the [Lys.sup.182] clip in PSA. Some mAbs had a preference for BPSA compared with [PSA.sub.182(+)] or vice versa, but none was sufficiently specific between these two forms to allow for the development of an immunoassay specific for the individual BPSA or [PSA.sub.182(+)] forms. The mAb PS2E290 was selected as suitable for assay development because it is strongly and equally reactive to both BPSA and [PSA.sub.182(+)] but has negligible reactivity to PSA that does not contain the clip at [Lys.sup.182] (Fig. 4). In Fig. 4, the assay shows a minor reactivity to the [PSA.sub.BPSA(-)] (peak A in Fig. 1) because it contains a minor amount of [PSA.sub.182(+)]. When the [PSA.sub.BPSA(-)] was subsequently passed over the PS2E290 immunoaffinity column to remove the minor [PSA.sub.182(+)] contamination, the assay reactivity dropped to nearly zero (Fig. 4). The reactivity of this PSA containing no clips at [Lys.sup.182], termed [PSA.sub.182(-)], is the true negative PSA control for the BPSA assay. [PSA.sub.182(-)] is devoid of the ~10% of PSA containing the clip at [Lys.sup.182] but otherwise contains all of the PSA forms typically found in purified seminal plasma PSA.
[FIGURE 5 OMITTED]
The BPSA assay is therefore defined as specific for PSA that contains the clip at [Lys.sup.182], which includes both BPSA and [PSA.sub.182(+)]. The lower limit of detection of the assay in buffer was 6 ng/L. The biological MDC of the assay as determined with female serum was 0.023 [micro]g/L. Serum BPSA was linear on dilution (slope, 1.02; r = 0.998). BPSA was found to be relatively stable in serum. The analysis of BPSA assay control calibrators added to female serum and stored at -70 [degrees]C showed no significant variation from the mean after more than 6 months, with an intraassay CV of 5.1% and an interassay CV of 8.7% (n = 159). The BPSA concentrations in freshly thawed serum specimens showed no differences after 48 h of storage at 4 [degrees]C (data not shown).
The assay of BPSA forms in the serum of men with BPH is shown in Fig. 5. The term BPH in this case indicates men with increased PSA and negative biopsy but does not necessarily imply men with symptomatic BPH. These men had a mean (SE) International Prostate symptom score of 12.0 [+ or -] 8.5 and a [Q.sub.max] (maximum urine flow) of 11.1 [+ or -] 4.9 mL/s. The median percentage of free PSA in these men was 19% compared with 16% in the cancer cohort (Wilcoxon P = 0.007; Table 2). The BPSA ranged from 0% to 60% of the free PSA in both cohorts, and both had median BPSA /free PSA values of 25% (Wilcoxon P = 0.59). There was, however, no significant correlation between the percentage of free PSA and the percentage of the free PSA present as BPSA in either the benign (r = -0.14) or cancer (r = -0.20) groups. BPSA represented 4.9% of the total PSA in the benign group and 4.0% in the cancer group. The median BPSA/total PSA values were significantly different between the cancer and benign cohorts (Wilcoxon P = 0.014). The most representative control set for interfering substances for the cancer and benign cohorts was the urologic outpatient controls because these men comprised a random patient profile of men without significantly increased PSA or other evidence of prostate disease. Measurable PSA was found only in those outpatients with mildly increased PSA, but not in men without measurable PSA. The other control groups of young men, women, and post-radical prostatectomy patients served to demonstrate that the assay did not recognize significant artifacts in divergent sera. None of the points in these latter control groups were above the MDC. Table 2 provides the general values for these serum cohorts.
This report describes the development of a research immunoassay for BPSA and the first demonstration that this BPH-associated form of free PSA is found at increased concentrations in the blood. On average, BPSA comprised approximately one-fourth of the free PSA in serum. However, BPSA was low or undetectable in some specimens, whereas it represented >50% of the free PSA in other patients. These results were independent of whether cancer was present. Such wide variations in BPSA, together with a poor correlation with the percentage of free PSA and cancer diagnosis, suggests that BPSA may represent a more specific subpopulation of free PSA for studying the development or progression of benign conditions, BPH in particular. It should be noted that there is no evidence that BPSA itself has a functional role; rather, it is more likely a consequence of posttranslational proteolytic processes within the prostate.
In this study we have shown that BPSA formation in the tissues is closely paralleled by the cleavage at [Lys.sup.182] only, which suggests a common proteolytic pathway. As seen in Table 1, the proportion of [PSA.sub.182(+)] correlates with BPSA. For example, TURF samples containing either 3% or 24% of their PSA as BPSA both contained the same relative proportion (21%) of [PSA.sub.182(+)], suggesting that [PSA.sub.182(+)] formation is not independent of BPSA. It is possible that [PSA.sub.182(+)] is initially formed in BPH tissues as a precursor to BPSA and that the subsequent clip at [Lys.sup.145] occurs by the same proteolytic mechanism that usually produces the large percentage of PSA clipped only at [Lys.sup.l45] in the seminal plasma.
The understanding of these tissue forms of PSA is important because any PSA in the blood is thought to derive from the tissues. It is interesting that Western blot studies of free PSA forms in BPH serum suggest that BPH serum can contain significant quantities of a 29-kDa form of PSA consistent with [PSA.sub.182(+)] (24). The BPSA assay described in the present study measures both BPSA and [PSA.sub.182(+)] equally. It is possible that the development of individual assays that measure each form individually could offer some additional insight, but currently, such mAbs have not been identified. It is not anticipated that such mAbs will be pursued because the prevailing evidence suggests that that the clip at [Lys.sup.182] is the important feature of these BPH-associated forms of PSA and that both are formed in parallel. Thus, the BPSA assay measures the BPH-associated forms of PSA, which is functionally defined as PSA containing the clip at [Lys.sup.182].
There are some common features in the current work with a reported assay that measures PSA-I, defined as the free PSA that is not clipped at [Lys.sup.145] (17), but there are also some important differences. The PSA-I assay is reported to measure inactive forms of free PSA not clipped at [Lys.sup.l45], which would most likely include most proPSA forms. With the PSA-I assay, one can calculate, by difference, the remainder of the free PSA not detected by the PSA-I assay, i.e., free PSA that is clipped at [Lys.sup.145]. It is assumed that this latter calculation would substantially include BPSA but could also include some percentage of PSA clipped only at [Lys.sup.145]. However, from the present study it can be seen that approximately one-third of the PSA that is measured in the BPSA assay may be [PSA.sub.182(+)], which does not contain a clip at [Lys.sup.145]. The PSA-I assay for cancer detection may be confounded to some extent by the presumed measurement of the BPH-associated [PSA.sub.182(+)]. Thus, although there may be substantial overlap in the forms of free PSA measured in the BPSA assay compared with (the inverse of) the PSA-I assay, these assays clearly measure different populations of free PSA and these differences may be important. In the future, careful analysis of well-defined patient populations will be necessary to determine which forms of free PSA offer the greatest diagnostic value for different disease states.
BPSA represents a subpopulation of free PSA that is present in the serum of men with increased PSA but with no diagnosis of prostate cancer (i.e., biopsy-negative). BPSA is increased in the prostate tissues of men with enlarged prostates and pathologic BPH, as indicated by the presence of BPH nodules (18). However, because men with prostate cancer may also have enlarged prostates and coexisting BPH, this marker alone would not be expected to significantly distinguish prostate cancer from BPH. Stamey et al. (25) have proposed that most of the increased serum PSA in men with <9 [micro]g/L PSA is attributable to BPH. This limited comparison of biopsypositive and -negative sera indicates that BPSA is not increased because of cancer and is, in fact, significantly higher in benign serum as a percentage of total PSA (Table 2). However, these limited cohorts are not sufficiently powered to determine a clear role for BPSA discrimination of cancer or a potentially more specialized role in other aspects of cancer detection, such as men with very high or low free PSA concentrations. For cancer detection, we have previously shown that proPSA forms, and particularly the truncated proPSA forms of free PSA, are highly associated with prostate cancer (26).
The application of BPSA toward the study of BPH is complex, and many questions remain. It is not clear why some men have almost no BPSA whereas in others the BPSA comprises more than one-half of the free PSA in serum. Because BPSA is associated with pathologic BPH at the tissue level, it is intriguing to speculate about a serum correlation with prostate volume, nodular development, symptomatic BPH properties such as bladder obstruction, prostate hormonal balance, or any number of other potential indicators or consequences of BPH. Another important area for investigation is the serum BPSA response to BPH drug treatment.
We wish to thank Carleton Gasior and Diksha Katir for excellent technical assistance. Dr. Marks is a paid consultant for, and all other authors are paid employees of Beckman Coulter, Inc.
Received August 8, 2002; accepted November 18, 2002.
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HARRY J. LINTON,  LEONARD S. MARKS,  LISA S. MILLAR,  CHRISTINE L. KNOTT,  HARRY G. RITTENHOUSE,  and STEPHEN D. MIKOLAJCZYK *
 Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., San Diego, CA 92121.
 Department of Urology/ Surgery, UCLA School of Medicine, Los Angeles, CA 90095.
 Nonstandard abbreviations: PSA, prostate-specific antigen; BPH, benign prostatic hyperplasia; BPSA, BPH-associated PSA containing internal clips at [Lys.sup.145] and [Lys.sup.182]; mAb, monoclonal antibody; MDC, minimum detectable concentration; [PSA.sub.182(-)], PSA purified to remove all forms of PSA containing the clip at [Lys.sup.182]; TURP, transurethral resection of the prostate; PBS, phosphate-buffered saline; HIC-HPLC, hydrophobic interaction-HPLC; LysC, lysyl endopeptidase C; [PSA.sub.182(+)], PSA totally clipped at [Lys.sup.182]; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and [PSA.sub.BPSA(-)], PSA purified by HIC-HPLC to remove BPSA.
* Address correspondence to this author at: Beckman Coulter, Inc, 7330 Carroll Rd., San Diego, CA 92121-2302. Fax 858-621-4610; e-mail email@example.com.
Table 1. Relative proportions of BPSA and [PSA.sub.182(+)] determined as single values in six different prostate BPH transition zone tissue specimens obtained from TURP. Proportion of PSA forms clipped at [Lys.sup.182], (b) % Sample (% BPSA) (a) BPSA [PSA.sub.182(+)] T1 (3%) 79 21 T2 (24%) 79 21 T3 (21%) 65 35 T4 (19%) 81 19 T5 (5%) 38 62 T6 (12%) 70 30 (a) Values in parentheses are the percentages of BPSA (BPSA/total PSA) for each TURP sample, T1-T6. determined by HIC-HPLC as shown in Fig. 3A. (b) Relative proportions of BPSA (clipped at [Lys.sup.145] and [Lys.sup.182]) and [PSA.sub.182(+)] (clipped only at [Lys.sup.182]). Each sample was affinity-purified, and proportions were determined by HIC-HPLC as shown in Fig. 3B. Table 2. Median values for total PSA, free PSA, and BPSA in men with BPH (biopsy-negative men with increased PSA) compared with prostate cancer (biopsy-positive) and control sera. Group n Total PSA, Free PSA, Free PSA/ [micro] g/L [micro] g/L Total PSA BPH 79 4.8 0.83 0.19 Cancer 91 4.2 0.72 0.16 Wilcoxon P (BPH vs cancer) 0.53 0.079 0.007 Urologic outpatients 57 0.59 0.18 0.27 Young men 37 0.63 0.064 0.14 Females 20 0 0 0 Post-radical prostatectomy 10 0 0 0 Group BPSA, BPSA/Free BPSA/Total [micro] g/L PSA PSA BPH 0.22 0.25 0.049 Cancer 0.17 0.25 0.040 Wilcoxon P (BPH vs cancer) 0.053 0.59 0.014 Urologic outpatients <0.02 Young men <0.02 Females <0.02 Post-radical prostatectomy <0.02
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|Title Annotation:||Cancer Diagnostics|
|Author:||Linton, Harry J.; Marks, Leonard S.; Millar, Lisa S.; Knott, Christine L.; Rittenhouse, Harry G.; Mi|
|Date:||Feb 1, 2003|
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