Forms Of Prostate-specific Antigen In Serum Research Articles

Proximity Ligation Measurement of the Complex between Prostate Specific Antigen and α1-Protease Inhibitor

Prostate specific antigen (PSA)-alpha1-protease inhibitor complex (PSA-API) is a minor form of PSA in serum. It may be useful for prostate cancer (PCa) diagnosis, but its specific detection is hampered by nonspecific background. To avoid this, we developed an immunoassay for PSA-API based on proximity ligation. We used a monoclonal antibody (mAb) to total PSA (tPSA) to capture PSA, while using another anti-tPSA mAb together with an anti-API mAb as probes. We measured PSA-API by quantification of amplified DNA strands conjugated to the probes. We measured serum PSA-API in 84 controls and 55 men with PCa who had PSA concentrations of 4.0-10 microg/L. The detection limit of the assay was 6.6 ng/L. The proportion of PSA-API to tPSA (%PSA-API) tended to be lower in men with PCa (2.8%) than without cancer (3.3%) but was not statistically significant (P = 0.363). When used alone, %PSA-API [area under the curve (AUC) 0.546] did not improve detection of PCa, whereas %fPSA (AUC 0.710) and the sum of %fPSA and %PSA-API (AUC 0.723) did. At 90% diagnostic sensitivity, the diagnostic specificity for cancer was not significantly better for %f PSA + %PSA-API than for %fPSA alone (36% vs 30%). Proximity ligation eliminated nonspecific background, enabling accurate measurement of PSA-API in serum specimens with moderately increased tPSA. The combined use of %PSA-API and %fPSA provided a modest improvement for PCa detection, but based on the current study cohort, it is uncertain whether the improvement has clinical utility. .

Les formes moléculaires du PSA

Molecular forms of serum PSA (prostate specific antigen) have been developped to improve total PSA sensitivity and specificity in prostate cancer diagnosis and staging. Total PSA is measured in bound (complexed PSA) and unbound (free PSA) molecular forms. Their levels in absolute values and in relation to total PSA (f/t PSA and c/t PSA) have been evaluated. The percentage of free PSA is more specific but less sensitive than tPSA and it is not recommended as a first line diagnostic test. It may be useful as a second-line test, prescribed by the urologist after a first series of negative biopsies. There is general agreement that at high sensitivity, cPSA provides higher specificity compared with tPSA in the gray zone (2-10 ng/ml). Nevertheless the widespread use of tPSA an the small benefit in terms of specificity explains why cPSA is not generally recommanded. Molecular derivates of free PSA have been identified: proPSA (precursor inactive form of PSA), intact PSA (an additionnal form of proPSA that is found intact and inactive), human Kallikrein 2 and BPSA (for benign PSA wich is associated to BPH) have been evaluated. Preliminary studies did not have demonstrate their ability to discriminate between cancer and BPH, and did not define cutoff values.

Inhibition of prostate-specific antigen (PSA) by alpha(1)-antichymotrypsin: salt-dependent activation mediated by a conformational change.

Prostate-specific antigen (PSA) and its SDS-stable complex with the serine proteinase inhibitor (serpin) alpha(1)-antichymotrypsin (ACT), which is the dominant form of PSA in serum, are in widespread use as markers for the diagnosis of prostate cancer, and there is increasing evidence for the involvement of PSA proteinase activity itself in the development of prostate and other cancers. However, both the formation and degradation of the PSA-ACT complex, denoted PSA*ACT* to indicate substantial changes in the structure of both proteins on complex formation, have been incompletely studied. Here we determine rate and equilibrium constants for the steps involved in PSA*ACT* formation and demonstrate that (a) the effects of added NaCl, polyamines, and Zn(2+) on this process parallel their effects on PSA catalytic activity [Hsieh, M.-C., and Cooperman, B. S. (2000) Biochim. Biophys. Acta 1481, 75-87], (b) the effect of added NaCl in dramatically increasing the rate of ACT inhibition of PSA correlates with salt-induced changes in PSA conformation, and (c) the PSA*ACT* complex is subject to proteolysis by human neutrophil elastase. Possible clinical implications of these findings are considered.

Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays

Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum. We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule. MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with alpha1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by alphaACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA. The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.

Variation in the Quantitation of Prostate-specific Antigen in Reference Material: Differences in Commercial Immunoassays,

Prostate-specific antigen (PSA) is a widely used biochemical marker for prostate cancer (1). PSA, a serine protease, forms complexes with serine protease inhibitors such as α2-macroglobulin and α1-antichymotrypsin (ACT) (2). The major immunoreactive forms of serum PSA are PSA-ACT complex and free PSA. PSA complexed to α2-macroglobulin is not measured by current immunoassays (3). Because the proportion of PSA complexed to ACT is higher in men with prostate cancer than in men with benign prostatic hyperplasia (BPH) (3)(4), the use of a ratio of free-to-total PSA may improve discrimination between men with BPH and prostate cancer (5)(6). Results obtained with different PSA immunoassays are not interchangeable (7)(8)(9)(10). The variation among immunoassays reflects, in large part, the specificity of different PSA antibodies (7). To a lesser extent, the composition of the calibrator, the PSA values assigned to the calibrator, the test diluent, and the physical design of the immunoassays contribute to intermethod differences (6)(7). Assays using a monoclonal-monoclonal sandwich format yield, in general, an equimolar response to free and ACT-complexed PSA, whereas monoclonal-polyclonal formats tend to yield higher values for samples containing greater proportions of free PSA (8). We have recently described the Bayer Immuno 1TM PSA Assay (Bayer Corporation), which uses a monoclonal-polyclonal antibody format that yields an equimolar response to varying proportions of free and complexed PSA (11). This has now been found to be a result of the unique binding properties of the monoclonal antibody used for the capture of PSA (manuscript in submission). Proficiency testing among clinical laboratories has indicated large intermethod variability in the quantitation of total PSA (12). The aim of this study was to determine the magnitude and cause of this intermethod variation. Here we report our multi-site analysis of …

Individual prostate-specific antigen (PSA) forms as prostate tumor markers

Prostate-specific antigen (PSA) is a kallikrein-like serine protease mainly expressed in the human prostate. It is responsible for the proteolysis of the gel-forming proteins in human semen. Two major extracellular protease inhibitors, alpha-1-antichymotrypsin (ACT) and alpha-2-macroglobulin (AMG) may inactivate PSA escaping from the prostate. The predominant immunodetected form of PSA in serum is complexed to ACT but PSA exists also in a free non-complexed form despite the large excess of inhibitors. The concentrations of PSA in serum are normally less than 4 μg/l, but elevated concentrations are found in a majority of patients with prostate cancer (CAP) and the analysis of PSA in serum has become invaluable in the detection and monitoring of patients with CAP. However, it is not an ideal tumor marker in the sense that there are CAP patients with normal PSA concentrations in serum and patients with benign hyperplasia of the prostate (BPH) with elevated PSA concentrations. Analysis of the various PSA forms in serum attracts much interest as there is a higher proportion of PSA in complex with ACT in patients with CAP than in those with BPH. Optimal combinations of monoclonal antibodies have been used to design sensitive noncross-reacting immunoassays for the detection of free PSA, PSA-ACT complexes and the detection of both free PSA and PSA complexes in an equimolar fashion (i.e. total PSA). Several studies have demonstrated that the analysis of the proportions of the free-to-total PSA in serum may increase the diagnostic specificity by 15–20% without significant loss in the sensitivity for detection of CAP.

Comparison of analysis of the different prostate-specific antigen forms in serum for detection of clinically localized prostate cancer

Objectives To compare different forms and ratios of serum prostate-specific antigen (PSA) to determine which form or ratio provides optimal diagnostic specificity and sensitivity in distinguishing between benign prostatic hyperplasia (BPH) and clinically localized prostate cancer. Methods Serum samples were obtained from 47 patients with BPH and 39 with clinically localized prostate cancer. Patients with BPH underwent either transurethral resection of the prostate or transurethral microwave thermotherapy. Patients with prostate cancer, all of whom had no metastases on radionucleotide bone scans and no pelvic lymph node involvement, underwent either radical external beam radiation therapy or radical retropubic prostatectomy. All patients had pretreatment serum PSA levels between 1 and 20 ng/mL. The different forms of serum PSA (free PSA [PSA-F], PSA complexed to alpha 1-antichymotrypsin [PSA-ACT], and total PSA [PSA-T]) were measured using different monoclonal antibodies against PSA and ACT and immunofluorometric assay techniques. Furthermore, three ratios (PSA-F/PSA-T, PSA-ACT/PSA-T, and PSAF/PSA-ACT) were calculated. Results By receiver operating characteristic curve analysis, the performance of the different forms and ratios were compared. The PSA-F/PSA-T ratio had the greatest area under the curve (AUC, 0.776), significantly larger than that for PSA-T (0.612; P=0.024). For PSA-ACT/PSA-T, the AUC was 0.695 ( P=0.283 versus PSA-T) and 0.773 for PSA-F/PSA-ACT ( P=0.051 versus PSA-T). At a cutoff level <0.17, PSA-F/PSA-T had a sensitivity of 79%, a specificity of 66%, and a positive predictive value of 66% compared with 74%, 38%, and 50%, respectively, for PSA-T at a cutoff level >4.0 ng/mL. Conclusions The PSA-F/PSA-T ratio gives the best diagnostic performance compared with that for other forms and ratios of PSA and will reduce the number of prostatic biopsies in patients with BPH.

Evaluation of a two-site immunoradiometric assay for measuring noncomplexed (free) prostate-specific antigen

Serum prostate-specific antigen (PSA) in men is present as two different molecular forms separable by gel-filtration chromatography (GFC). We have evaluated a two-site IRMA that measures only the noncomplexed (free) form of PSA (F-PSA). Verification that the F-PSA assay measures solely F-PSA was obtained by assaying GFC-fractionated serum samples with both the F-PSA IRMA and a commercial PSA assay that measures total PSA (T-PSA: F-PSA plus alpha 1-antichymotrypsin-complexed PSA). The F-PSA assay detected only the 30-kDa peak corresponding to the free form of PSA, whereas the T-PSA assay detected two peaks: complexed PSA at approximately 90 kDa and F-PSA at approximately 30 kDa. The F-PSA assay had an analytical detection limit of 0.03 microgram/L and a measuring range up to 50 micrograms/L. The intraassay CV was 1.7-10% in the concentration range of 0.2-30 micrograms/L. The interassay CV was 3.4-12.5% in the same concentration range. Dilution and recovery studies showed no significant deviation from linearity across the assay range. The assay was insensitive to interference from hemoglobin, bilirubin, and total lipids up to concentrations of 5, 0.2, and 10 g/L, respectively. No significant loss of immunological activity (analyte stability) was seen day-to-day ( < or = 5) or after repeated freeze/thaw ( < or = 5) cycles. We conclude that the F-PSA IRMA is an accurate, precise, and reliable tool for measuring F-PSA in human serum.

Clinical utility of measurements of free and total prostate-specific antigen (PSA): a review.

Prostate-specific antigen (PSA) is a widely-used tumor marker to aid in the early detection of prostate cancer. PSA testing has appreciable false-positive and false-negative results, particularly in the 2.5-10.0 ng/ml range. Measurements of the percentage of nonprotein-bound (free) PSA in serum, which is lower in patients with prostate cancer, has been evaluated as a method for increasing the accuracy of PSA testing. The literature on forms of PSA in serum, as it relates to issues of clinical utility for prostate cancer screening, was reviewed and summarized through May 1996. Measurements of the percentage of free PSA in serum increase the accuracy of PSA testing for prostate cancer in men whose total PSA levels are 2.5-10.0 ng/ml. Cutoffs for screening are affected by prostate volume and total PSA levels. One study also demonstrated a correlation between percentage of free PSA and pathologic features of cancer aggressiveness. Measurement of free PSA in serum has potential clinical utility for increasing the sensitivity and specificity of PSA screening. Insufficient data are available to establish cutoffs to be used in clinical practice. Cutoffs are affected by total PSA level and prostate volume. The prevalence rate of cancer in the screened population (age, race, previous biopsy history, etc.) will also influence screening cutoffs. Percentage of free PSA may also correlate with the potential aggressiveness of early-stage prostate cancer.

The role of prostate-specific antigen in the chemoprevention of prostate cancer

An understanding of the natural history of changes in prostate-specific antigen (PSA) may be valuable as a surrogate view of prostate dynamics, as a method to differentiate between benign and malignant growth, and as a means to assess the use of PSA as a tool for monitoring activity of chemoprevention agents. Although PSA appears to be useful as a noninvasive marker of prostatic growth, PSA changes should not be confused with a direct measure of tumor growth. Serum PSA levels are a function of tumor volume but are also influenced by the volume of benign epithelium, grade of carcinoma (if any), inflammation, androgen levels, growth factors, and the extracellular matrix. The biological functions of PSA in the prostate and in its secretions need to be more completely elucidated in order that PSA measurements may more accurately describe prostate dynamics. The expression of PSA is androgen-regulated. It is one of the most abundant prostate-derived proteins in the seminal fluid. Seminogelin, a major protein in seminal fluid, is cleaved by PSA, and this cleavage is important in the liquefaction of semen. Less is known about other PSA substrates. Current PSA studies indicate that cancer cases exhibit an early slow linear PSA phase followed by a rapid exponential phase, and that PSA levels begin to increase exponentially approximately 7-9 years before diagnosis. The establishment of age-specific PSA reference ranges (ASRR) and of PSA velocity (PSAV) rates provide elements of a baseline from which prediction models could measure malignant potential of a prostatic carcinoma. Moreover, recent discoveries of different molecular forms of PSA in serum may allow a much more accurate differentiation of benign and malignant growth as well as a more potent measure of the impact of chemoprevention agents. If PSA doubling time is approximately 2.4-3.0 years and accurately reflects tumor doubling time, and if the average man has less than 0.5 ml of latent prostatic tumor tissue and the average stage T2 cancer is approximately 4 ml when detected, then the available PSA data suggest that the 3 doublings necessary to change from 0.5-4.0 ml, would take 7-12 years for a typical small volume tumor to reach the size of most stage T2 tumors. The findings that histologic cancers appear at much younger ages than previously known is disturbing. It indicates that disease initiation may begin sooner than ever thought likely. "Normal" PSA levels for younger men (< 40 years of age) may need to be studied, and an emphasis upon premalignant lesions in this age group may be necessary. Younger men may represent the most appropriate population and premalignant lesions the most relevant clinical factor for prostate cancer chemoprevention studies and trials. The molecular composition and molecular changes of PSA derived from premalignant lesions have yet to be elucidated, but such investigations may lead to a more complete understanding of the possible progression or transformation of normal prostate cells to premalignancy and subsequently to carcinoma. High grade prostatic intraepithelial neoplasia (PIN) in and of itself does not account for elevated serum PSA levels, but subtle changes in the molecular dynamics of PSA may reveal the influence of androgens and the impact of chemopreventive agents.

The risks outweigh the benefits of radical prostatectomy in localised prostate cancer: the argument for.

Prostate cancer represents a major menace to Western society. Since it is asymptomatic in its early stages and there is no curative therapy for advanced stages, our only hope to decrease the mortality rate from prostate cancer is through annual prostatic evaluations of men who agree to undergo such testing. It is important to emphasise that these patients should be carefully informed about the possible consequences. Since prostate cancer is a slow growing tumour and its clinical significance decreases with advancing in age, only young men with a life expectancy of 10-15 years or more should participate in early detection efforts. These men should be evaluated as diligently as possible through the combined use of digital rectal examination and the newly described prostate-specific antigen testing, i.e. analysis of the ratio between different forms of serum prostate-specific antigen. Radical prostatectomy, when performed on men with a life expectancy of 15 years or more who truly have organ-confined prostate cancer, is an acceptable and effective treatment for this group of patients who would benefit most from a radical approach to their disease.

Standardization of PSA determinations

Standardization of determinations for prostate specific antigen (PSA) has become an important issue due to the widespread use of these determinations for prostate cancer screening. Standardization of this assay is complicated due to the occurrence of two major forms of PSA in serum, the free antigen and a complex between PSA and alpha 1-antichymotrypsin (PSA-ACT). These two forms of PSA are recognized differently by different antibodies, but by careful selection of antibodies, it is possible to design methods that measure each form equally. It is suggested, that standards for PSA and PSA-ACT should be prepared and established as international standards. Furthermore, reference methods should be established on the basis that these standards and carefully selected reference antibodies.

Prostate specific antigen predominantly forms a complex with alpha 1-antichymotrypsin in blood. Implications for procedures to measure prostate specific antigen in serum.

Prostate specific antigen (PSA) is a zymogen of a 33-kilodalton (kD) serine proteinase with extensive similarity to glandular kallikreins. The mechanism responsible for converting the zymogen into active proteinase has not been defined, but active PSA may be irreversibly inactivated in vitro by two of the major proteinase inhibitors in blood: alpha 1-antichymotrypsin and alpha 2-macroglobulin. Procedures have been designed to characterize the different molecular forms of PSA in serum. One assay detects PSA epitopes available on both PSA binding to serine proteinase inhibitors and PSA not binding to a proteinase inhibitor. A second assay only detects PSA in complex with alpha 1-antichymotrypsin. A third assay mainly detects PSA not binding to a proteinase inhibitor. In serum samples, an 80-kD to 90-kD species of PSA in complex with alpha 1-antichymotrypsin is the predominant molecular form and a minor molecular form of serum PSA was an approximately 30-kD fraction not binding to a proteinase inhibitor. The benefits of detecting different molecular forms of serum PSA should be investigated regarding possibilities to facilitate differential diagnosis of carcinoma of the prostate (CAP) and benign prostatic hyperplasia (BPH).