| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Hybritech, Inc., San Diego, California 92121 [S. D. M., K. M. M., L. S. M., A. K., M. S. S., J. K. P., C. L. E., C. L. G., H. J. L., H. G. R.], and University of California, Irvine Medical Center, Department of Pathology, Orange, California 92868 [P. C.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The biological mechanism for the variable levels of the percentage of free PSA in serum is unknown. The serum PSA that has become complexed is likely to be relatively homogeneous, because this represents enzymatically active, intact PSA. This assumption was confirmed by analysis of the PSA released from the PSA-ACT complex in PCa and BPH serum, which was found to be indistinguishable from seminal plasma PSA (8) . It follows that free PSA may offer better biochemical insight and that a characterization of the molecular isoforms of free PSA could help elucidate their prostatic origin and mechanism of release into the serum. However, attempts to purify and characterize the low levels of PSA from serum in the diagnostically relevant range near 10 ng/ml have not generally been considered feasible with current technologies. The development of pPSA mAbs for use in immunoassays has been particularly difficult (9) .
One approach used by ourselves and others (10) to identify possible disease-specific isoforms of PSA that might be in serum has been to analyze diseased prostate tissue, either cancerous or benign, such as BPH. PSA is present at µg/mg levels in prostate tissues. In previous studies we identified different molecular isoforms of PSA in benign and cancerous prostate tissues. Nodular BPH tissue from the prostate transition zone contains a specific molecular form of PSA that is characterized by internal peptide bond cleavage at Lys145 and Lys182 (11) . This form of PSA has altered immunological properties compared with all of the other isoforms of PSA (12) , which has allowed the development of an immunoassay to detect this specific isoform of PSA in serum.3
In prostate tumor we also identified a truncated form of pPSA that is elevated compared with BPH tissue (13) . This truncated pPSA form contained a serine-arginine leader peptide instead of the normal 7 aa pro leader peptide. Minor levels of other truncated pPSA isoforms were also detected. PSA is normally secreted with a 7 aa pro leader peptide, APLILSR, that is necessary for secretion from mammalian cells and which is removed extracellularly to produce active, mature PSA (14, 15, 16) .
In previous studies to identify PSA isoforms in serum, we and others have focused primarily on serum from men with unusually high levels of PSA, with hundreds or thousands of ng/ml PSA. We reported the presence of a truncated form of pPSA in pooled PCa serum containing 63 ng/ml total PSA (17) . A recent study by Peter et al. (18) , used mass spectrometry to identify multiple isoforms of pPSA in PCa serum containing >6000 ng/ml total PSA. However, other groups have failed to detect pPSA in serum containing similar levels of PSA (19 , 20) .
Whereas the presence of these truncated pPSA isoforms in serum and tissue now appears well established, the question remains as to how relevant these isoforms might be in diagnostic assays. PSA isolated from such high serum samples is suggestive but may not reflect the kind or percentage of PSA that is typically present in the early stages of disease where PSA is 
10 ng/ml. High levels of PSA released from large primary tumor lesions or metastatic disease may have different biochemical properties than PSA released from early, possibly lower-grade disease. Alternately, pPSA forms may not appear in the serum until PSA levels are 20 or 30 ng/ml, making it of little diagnostic value as a serum PCa marker.
To be useful for clinical detection of early PCa, such pPSA isoforms must be present at significant levels in serum that contains diagnostically relevant levels of total PSA near 10 ng/ml. The current study was designed to determine the relative quantification of pPSA isoforms in biopsy-positive men and to compare these levels to biopsy-negative men. In addition to serum analysis, we also examined the tissue distribution of pPSA isoforms by immunostaining with different pPSA mAbs. Activation studies were also undertaken to understand the nature of the truncated pPSA stability compared with the full length pPSA. These results add important new insight into the diagnostic potential of pPSA in PCa detection and mechanism of truncated pPSA formation and stability.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
20% cross-reactivity to the mature PSA protein by Western blot. However, the cross-reactivity dropped to 5% under nonreducing conditions, and so these conditions were used for detection of [-2]pPSA in the "Results." mAbs to full-length [-7]pPSA were obtained by mouse immunization with purified recombinant chimeric protein consisting of the PSA pro leader peptide attached to hK2 (14 , 22) . The expression vector consisted of PSA prepro gene sequence attached to mature hK2. After immunization with the chimeric pPSA-hK2 protein, the antibody clones were screened on their recognition of native recombinant pPSA and no recognition of native mature PSA. These mAbs were found to have high specificity for [-7]pPSA by immunoassay but to recognize both [-7] and [-4]pPSA proteins equivalently on Western blots.
Isolation of Recombinant pPSA Isoforms from Mammalian Cells.
Recombinant PSA was expressed in mammalian AV12 cells as described earlier (14)
. The spent medium was passed over the PSA-specific mAb, PSM773. PSM773 has been shown previously to have specificity for mature, clipped, and precursor forms of PSA (14
, 23
, 24)
. The column was washed with 40 volumes of PBS containing 0.1% reduced Triton X-100 and bound protein eluted with 100 mM glycine (pH 2.5) containing 200 mM sodium chloride. The eluant was immediately neutralized with 10% v/v 1 M Tris (pH 8.0). The purified PSA contained no mature PSA but contained [-7], [-5], [-4], and [-2]pPSA molecular isoforms of pPSA that were purified by HIC-HPLC as described in the "Results."
PSA Characterization by Immunoassay and SDS-PAGE.
The concentration of PSA in serum and in purified preparations was determined by Tandem-MP PSA and Tandem-MP free PSA assays (Hybritech, Inc.). SDS-PAGE was performed using 4–20% gradient minigels (Invitrogen, Carlsbad, CA) under reducing or nonreducing conditions, as indicated. Samples were electroblotted onto nitrocellulose using standard procedures. Primary pPSA mAbs, PS2P446 ([-7/-4]pPSA), PS2V476 ([-4]pPSA), and PS2X373 ([-2]pPSA), were used at 5 mg/ml and incubated with the blots overnight at 4°C. Bands were detected with a secondary antibody cocktail consisting of goat antimouse heavy and light chain-HRP 1:50,000 (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). The immunoreactive signals were detected by SuperSignal West Dura Extended Duration Substrate (Pierce Chemical Co.) according to manufacturer’s instructions. Image capture and densitometry analysis of the bands on the developed film were performed using the ChemImager 4000 (Alpha Innotech Corp., San Leandro, CA).
Immunostaining of Prostate Tissues.
Formalin-fixed, paraffin-embedded sections were placed on capillary gap slides and deparaffinized with Histoclear and rehydrated through decreasing concentrations of isopropyl alcohol. Avidin-biotin complex immunoperoxidase reactions were performed using Immunotech 500 automated immunostainer (Ventana Systems Inc., Tucson, AZ) according to manufacturer’s instructions. Briefly, the automated steps included blockage of endogenous peroxidase with 3% hydrogen peroxide and reaction with PS2X373 (anti-[-2]pPSA) or PS2P446 (anti-[-4/-7]pPSA) at 120 and 9 µg/ml, respectively. The reaction was followed by a biotinylated goat-antimouse IgG secondary antibody and then an avidin-biotin peroxidase complex. The chromogen was 3,3'-diaminobenzidine.
Amino Acid Sequencing and HIC-HPLC.
NH2-terminal sequence of the samples was performed through nine cycles on a PE-Applied Biosystems Model 492 amino acid sequencer (PE-Applied Biosystems, Foster City, CA). HIC-HPLC was performed as described previously (13
, 25)
. Purified PSA and peaks collected by HIC-HPLC were applied directly to polyvinylidene difluoride membranes using the Prosorb cartridges (PE-Applied Biosystems), washed three times with 0.1 ml 0.1% trifluoroacetic acid, and applied to the Model 492 sequencer.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
shows the Western blot and immunoassay analysis of these samples. In panels A–C, all of the samples are shown in the same order. The first five samples were the PCa samples, and the last three samples were from biopsy-negative men. The panel A histogram shows the ng/ml of total PSA and the percentage of free PSA in the serum before immunoaffinity purification. The percentage of free PSA was generally lower in the cancer samples compared with biopsy-negative or benign serum, which is in agreement with a predicted trend that benign samples should contain a higher percentage of free PSA. Panel B shows the percentage of [-2]pPSA as the percentage of the free PSA ([-2]pPSA/free PSA) where [-2]pPSA was determined by densitometry after Western blot analysis, and the free PSA was determined by immunoassay. Panel C shows the Western blots of these samples for [-2]pPSA. It should be noted that the percentage of free PSA measured in these affinity purified samples was unchanged from the original serum values as shown in panel A, confirming that the purification procedure had no selectivity for either free or complexed forms of PSA (data not shown).
|
shows that the five cancer samples contained the highest overall levels of [-2]pPSA. Using densitometry to provide semiquantitative values for [-2]pPSA indicated that 95% of the free PSA in Lane 2 was [-2]pPSA. The [-2]pPSA was estimated to comprise 42, 49, 40, and 25% of the free PSA in Lanes 3–6, respectively. These are only approximate values estimated by densitometry, but they clearly indicate that significant levels of [-2]pPSA were present in the serum. Lane 7, the first biopsy-negative sample, contained 19% pPSA, whereas Lanes 8 and 9 contained only 6 and 8%, respectively. Because the PS2X373 mAb had a minor cross-reactivity of 5% with mature PSA under these Western blot conditions, it is possible that some or all of the apparent band in Lanes 8 and 9 were attributable to PSA cross-reactivity. A second blot of the same samples was probed with PS2P446, which had equal specificity for [-4]pPSA and [-7]pPSA on Western blots. Lanes 3 and 5 contained [-4/-7]pPSA at levels comparable with the [-2]pPSA, whereas other cancer lanes were negative. The biopsy-negative samples in Lanes 7–9 showed trace levels of [-4/-7]pPSA similar to the [-2]pPSA band intensity in Lanes 8 and 9 (data not shown). Thus, in Lanes 3 and 5 a high percentage of the free PSA was comprised of multiple isoforms of pPSA. The mAbs with specificity for [-4]pPSA only had relatively poor sensitivity on blots and did not generate quantifiable results. These experiments suggest that whereas other pPSA isoforms may be present at significant levels in selected serum samples, [-2]pPSA appears to correlate more consistently with PCa. As a control for the detection of pPSA isoforms, female serum was passed over the immunoaffinity column and worked up identically with the male serum. Female serum showed no detectable pPSA bands (data not shown).
Immunostaining of pPSA in Prostate Tissues.
We tested the pPSA mAbs for staining on prostate tissues. The PS2P446 ([-7]/[-4]pPSA specificity) and PS2X373 ([-2]pPSA specificity) had similar staining intensities, whereas PS2V476 ([-4]pPSA specificity) was not suitable for tissue staining. Both PS2X373 and PS2P446 showed a generally similar staining pattern, though only PS2X373 is shown in Fig. 2
. Cancer tissues showed consistent epithelial staining in the nine cancers tested. Prostatic intraepithelial neoplasia also showed consistent strong staining. Benign tissues stained less intensely than the cancer in general, though the surrounding benign tissues were highly variable and, in some slides, stained similarly to cancer. Fig. 2A
demonstrates that truncated pPSA isoforms can be detected in the epithelium of fixed prostate tissues, which provides additional support for our earlier work by showing that the truncated pPSA isoforms were not an artifactual result of tissue extraction but were naturally present in prostate tissues. In the slide shown in Fig. 2A
, the cancer tissue shows strong staining, whereas nearby benign glands show little stain. One interesting observation was that [-2]pPSA showed consistently stronger staining in cancer secretions (Fig. 2B)
, which could explain the prevalence of this form in the serum.
|
. PSA is normally expressed with a 7 aa pro leader peptide consisting of APLILSR, but NH2-terminal sequencing of the three peaks resolved by HIC-HPLC in Fig. 3A
revealed clipped or truncated isoforms of pPSA. Peak 3 contained approximately equal levels of the 7 aa pro leader peptide APLILSR ([-7]pPSA) and a clipped, truncated 5 aa leader peptide containing LILSR ([-5]pPSA) that were not resolved from one another. Peak 2 contained the 4 aa pro leader peptide ILSR ([-4]pPSA). Peak 1 contained the 2 aa leader peptide SR ([-2]pPSA) in addition to 30% of PSA missing the first 4 aa of the mature sequence. Pure [-2]pPSA could be obtained by collecting the back half of this peak. The decrease in retention time of these proteins resulting from the incremental truncation of the pro leader peptide is most likely because of conformational changes in the pPSA protein and not attributable to the minor surface changes induced by removal of the indicated NH2-terminal amino acids.
|
shows an overlay from several individual chromatograms that demonstrates that a single chromatographic gradient can resolve all of the pro and mature forms of PSA from one another and from hK2, the second major kallikrein found in the prostate. Therefore, it was possible to incubate the hK2 with each of the pPSA isoforms and to monitor the activation to mature PSA by HIC-HPLC. All of the peaks resulting from incubation with hK2 were confirmed by NH2-terminal sequencing.
Fig. 4
shows the percentage of conversion of the pPSA isoforms to mature PSA by hK2. The [-5/-7]pPSA was activated most rapidly, whereas the [-4]pPSA was activated more slowly. Most significantly, [-2]pPSA was not converted to PSA by hK2. Fig. 5
shows the chromatographic profile after extended incubation of 40% hK2 with the individual isoforms of pPSA. After 5 h of incubation at 37°C, [-2]pPSA still showed no evidence of conversion to mature PSA (Fig. 5A)
. After incubation for 2 h, the majority of the [-4]pPSA was converted (Fig. 4B)
. By contrast, the [-5] and [-7]pPSA isoforms were converted to mature PSA in < 1 h (Fig. 5C)
. It should be noted that the ratio of the [-5] and [-7]pPSA species contained within the 12 min peak in panel C was unchanged throughout the activation process, as determined by NH2-terminal sequencing, indicating that these two species were biochemically indistinguishable as substrates for hK2. Therefore, the first major alteration in both the HIC-HPLC retention time and in the activation kinetics of pPSA isoforms occurred on the removal of the single [-5]leucine residue in the change from [-5]pPSA to [-4]pPSA. These differences between the [-5] and the [-4]pPSA isoforms suggests that the [-5]leucine plays an important role in the properties of pPSA.
|
|
Thus, the pro leader dipeptide on [-2]pPSA appears to be resistant to cleavage by proteases that are otherwise capable of cleaving the [-4] and [-5/-7] pro leader peptides. None of the pPSA isoforms formed a complex with a 4 x excess of ACT after 5 h of incubation at 37°C (data not shown). This indicated that all of the truncated and full-length pPSA isoforms were enzymatically inactive and would, thus, be expected to remain as free PSA in serum.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A notable example of improved cancer discrimination can be seen by the comparison of Lanes 6 and 8 in Fig. 1
, which represent the kinds of serum samples that are particularly difficult to distinguish by current PSA assays. Each of these serum samples contained comparable levels of total PSA, 6 ng/ml, which is in the diagnostic "gray zone" of 4–10 ng/ml. In addition, both samples also contained a percentage of free PSA of 15–20%, in the diagnostic gray zone between 10 and 25% (6
, 7)
. Thus, with current assay protocols using only free and total PSA, there is no clear differential and no clear indication of PCa in either sample. However, the [-2]pPSA was at least 4 times higher in the cancer sample of Lane 6 and provides clear discrimination between the two samples.
In our previous study using pooled cancer serum containing 63 ng/ml total PSA, we identified the truncated [-4]pPSA using HIC-HPLC (17)
. In the current investigation we have shown that [-2]pPSA may be the more predominant form. However, there is no conflict with our earlier results, because in that work we used chromatographic methodologies to identify the different isoforms of PSA, as shown in Fig. 3
. The HIC-HPLC approach required that elution fractions be collected and the PSA measured by immunoassay. Because PSA and [-2]pPSA elute closely (see Fig. 3
), it was not possible to distinguish these two isoforms of PSA in the previous work because of the loss of peak resolution incurred by fraction collection. Therefore, only the [-4]pPSA form was clearly resolved from the PSA peak. It is not known but it is likely that the PSA peak contained some percentage of [-2]pPSA in that study.
Recent studies by Peter et al. (18) identified [-1], [-2], [-4], [-5], and [-7]pPSA forms in PCa sera. They reported that the [-7/-5/-4]pPSA forms were consistently present in all of the samples and could be either higher or lower than the [-2]pPSA form in individual samples. Such differences from our study may reflect mere sample variability in a small population, or it may reflect real differences between their samples containing >6000 ng/ml total PSA and our serum samples containing nearly 1000-fold lower PSA levels from 6–24 ng/ml. However, their reliance on serum samples containing extremely high levels of PSA precluded the critical comparative analysis with benign serum containing mildly elevated PSA, as in our study, because benign samples rarely rise above 20 ng/ml. The one unambiguous finding from both studies is that pPSA forms appear to be a significant and important feature of PCa serum.
The reason for the enrichment of a stable, truncated form of pPSA in tissues, and ultimately serum, was suggested by the activation studies. It has been demonstrated previously that pPSA can be cleaved to active PSA by hK2 and trypsin (14, 15, 16)
. Because hK2 and PSA are colocalized in the prostate columnar epithelial cells (27)
, it has been speculated that hK2 may be the endogenous protein responsible for the activation of PSA. The process of pPSA activation is normally an extremely efficient process, because pPSA isoforms are undetectable in seminal plasma (data not shown). Fig. 4
shows that hK2 has no ability to activate [-2]pPSA and has reduced activity on [-4]pPSA compared with [-5/-7]pPSA. Even trypsin, which has a much wider substrate specificity range and activates pPSA at least 10 times more rapidly than hK2, was unable to activate [-2]pPSA (data not shown). Extended incubation with trypsin resulted in cleavage at other internal sites in PSA without significant cleavage of the serine-arginine pro leader peptide. Whereas there is no direct evidence that hK2 is responsible for the activation of pPSA in vivo, the failure of both hK2 and trypsin to activate [-2]pPSA makes it less likely that [-2]pPSA remains a viable substrate for other activation proteases.
The immunostaining of prostate tissues (Fig. 2)
supports our earlier studies with tissue extracts by showing that [-2]pPSA is naturally present in the tissues and is not the artifactual result of tissue extraction methodologies. The observation of more consistently intense staining of the cancerous luminal secretions suggests a preferential enrichment of the [-2]pPSA form and may help to explain its more consistent presence in serum. We have found no pPSA forms in seminal plasma of men with PCa,4
which may suggest that the cancerous ductal system is impaired. Once the inert and stable [-2]pPSA is formed in cancerous lesions, it is possible that the primary path of release is into the serum as free PSA. A more comprehensive immunohistological study will be required to determine the relationship of the pPSA isoforms to each other, cancer grade, BPH, and other histological aspects of prostate tissues.
Because elevated levels of free PSA have been shown to better correlate with benign disease (6
, 7
, 28)
, it may seem counter intuitive that cancer serum contains elevated levels of pPSA isoforms, which are forms of free PSA. However, Fig. 1
shows that pPSA represents a minor percentage of the free PSA in benign disease and a major percentage in cancer. The cancer samples had a lower percentage of free PSA than the benign samples but a much higher relative percentage of the free PSA was [-2]pPSA. As the percentage of free PSA increased in the biopsy-negative samples it was apparently comprised of increasing amounts of inactive PSA forms other than [-2]pPSA. Thus, pPSA makes up a progressively smaller percentage of the free PSA that derives from benign disease. Because men with PCa can also develop BPH and vice versa, it will be important to determine the relative contribution of pPSA in each disease state.
The current study of pPSA isoforms in serum was necessarily limited because of the considerable effort required to process individual serum samples of >100 ml. However, complementary results were recently obtained using a specific immunoassay for [-2]pPSA developed in our laboratory. Preliminary results showed [-2]pPSA to have a mean and median 3-fold higher (P < 0.001) in the serum of 20 biopsy-positive compared with 20 biopsy-negative men with total PSA levels from 2 to 22 ng/ml (data not shown). Immunoassays specific for [-4] and [-7]pPSA are also under development and together with [-2]pPSA will allow a comprehensive study of pPSA forms in cancer serum. Whereas we have demonstrated several interesting properties for the [-2]pPSA isoform in the present study, it will be important to measure all three of the major forms of pPSA in much larger patient populations to determine what role, if any, the different pPSA forms may play in the detection of PCa. It is not known, for instance, whether [-2]pPSA alone or together with other pPSA isoforms would offer the greatest diagnostic value and whether this would be realized as the percentage of total PSA, as a percentage of the free PSA, or as an independent marker of PCa in the serum. The possible role of the different pPSA isoforms in the discrimination of cancer grade or stage is particularly intriguing. In addition to the pPSA assays, we have recently developed an immunoassay to detect serum BPSA, the BPH associated form of PSA (29) . The use of pPSA immunoassays for PCa in combination with a BPSA assay for BPH may add even greater discrimination of PCa from BPH.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 To whom requests for reprints should addressed, at Hybritech, Inc., P. O. Box 269006, San Diego, CA 92196-9006. Phone: (858) 621-4601; Fax: (858) 621-4610; E-mail: sdmikolajczyk{at}beckman.com 
2 The abbreviations used are: PSA, prostate-specific antigen; PCa, prostate cancer; BPH, benign prostatic hyperplasia; pPSA, precursor prostate-specific antigen; mAb, monoclonal antibody; hK2, human kallikrein 2; ACT, a1-antichymotrypsin; aa, amino acid; HIC-HPLC, high performance hydrophobic interaction chromatography.
Received 3/22/01. Accepted 7/11/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(1)-antichymotrypsin (PSA-ACT). Clin. Chem., 46: 474-482, 2000.This article has been cited by other articles:
|
|
 |
|
  D. Nedelkov, U. A. Kiernan, E. E. Niederkofler, K. A. Tubbs, and R. W. Nelson Population Proteomics: The Concept, Attributes, and Potential for Cancer Biomarker Research Mol. Cell. Proteomics, October 1, 2006; 5(10): 1811 - 1818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Jung, J. Reiche, A. Boehme, C. Stephan, S. A. Loening, D. Schnorr, W. Hoesel, and P. Sinha Analysis of Subforms of Free Prostate-Specific Antigen in Serum by Two-Dimensional Gel Electrophoresis: Potential to Improve Diagnosis of Prostate Cancer Clin. Chem., December 1, 2004; 50(12): 2292 - 2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Mikolajczyk, W. J. Catalona, C. L. Evans, H. J. Linton, L. S. Millar, K. M. Marker, D. Katir, A. Amirkhan, and H. G. Rittenhouse Proenzyme Forms of Prostate-Specific Antigen in Serum Improve the Detection of Prostate Cancer Clin. Chem., June 1, 2004; 50(6): 1017 - 1025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Wu, L. Zhu, U.-H. Stenman, and J. Leinonen Immunopeptidometric Assay for Enzymatically Active Prostate-Specific Antigen Clin. Chem., January 1, 2004; 50(1): 125 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Haese, V. Vaisanen, J. A. Finlay, K. Pettersson, H. G. Rittenhouse, A. W. Partin, D. J. Bruzek, L. J. Sokoll, H. Lilja, and D. W. Chan Standardization of Two Immunoassays for Human Glandular Kallikrein 2 Clin. Chem., April 1, 2003; 49(4): 601 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. G. Blijenberg, M. F. Wildhagen, C. H. Bangma, J. A. Finlay, V. Vaisanen, and F. H. Schroder Comparison of Two Assays for Human Kallikrein 2 Clin. Chem., February 1, 2003; 49(2): 243 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Linton, L. S. Marks, L. S. Millar, C. L. Knott, H. G. Rittenhouse, and S. D. Mikolajczyk Benign Prostate-specific Antigen (BPSA) in Serum Is Increased in Benign Prostate Disease Clin. Chem., February 1, 2003; 49(2): 253 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Balk, Y.-J. Ko, and G. J. Bubley Biology of Prostate-Specific Antigen J. Clin. Oncol., January 15, 2003; 21(2): 383 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Isono, T. Tanaka, S. Kageyama, and T. Yoshiki Structural Diversity of Cancer-related and Non-Cancer-related Prostate-specific Antigen Clin. Chem., December 1, 2002; 48(12): 2187 - 2194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Leinonen, P. Wu, and U.-H. Stenman Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries Clin. Chem., December 1, 2002; 48(12): 2208 - 2216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Niemela, J. Lovgren, M. Karp, H. Lilja, and K. Pettersson Sensitive and Specific Enzymatic Assay for the Determination of Precursor Forms of Prostate-specific Antigen after an Activation Step Clin. Chem., August 1, 2002; 48(8): 1257 - 1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stephan, H. Cammann, A. Semjonow, E. P. Diamandis, L. F.A. Wymenga, M. Lein, P. Sinha, S. A. Loening, and K. Jung Multicenter Evaluation of an Artificial Neural Network to Increase the Prostate Cancer Detection Rate and Reduce Unnecessary Biopsies Clin. Chem., August 1, 2002; 48(8): 1279 - 1287. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
