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Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2 ). In multiple rodent models, Actinium-225-labeled hu11B6-IgG 1 ([ 225 Ac]hu11B6-IgG 1 ) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [ 225 Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG 3 , the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [ 225 Ac]hu11B6-IgG 3 was a functionally enhanced alternative to [ 225 Ac]hu11B6-IgG 1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7 , ETV1 , NTS , and SCHLAP1 , we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2 , demonstrating efficacy of sequential [ 225 Ac]hu11B6 in a mouse model., Competing Interests: Competing interest statement: S.-E.S., A.-C.M.H., H.L., and D.U. are consultant/advisory board members for and hold ownership interest in Diaprost AB. S.-E.S. and D.U. are listed as coinventors on a several patents regarding the humanized form of 11B6, which is owned by Diaprost. C.G.R. is cofounder and holds equity in Sofie Biosciences and Trethera Therapeutics. Intellectual property has been patented by the University of California and has been licensed to Sofie Biosciences and Trethera Therapeutics. This intellectual property was not used in the present study. H.L. holds ownership interest (including patents) in OPKO Health, and reports other remuneration from OPKO Health. S.M.L. reports receiving commercial research grants from Regeneron and Telix, holds ownership interest (including patents) in Voreyda, Imaginab, and Elucida, and is a consultant/advisory board member for Johnson and Johnson. M.J.M. is a consultant/advisory board member for Bayer, Endocyte, Advanced Accelerator Applications, Blue Earth Diagnostics, Tolmar, and ORIC. Memorial Sloan Kettering Cancer Center has filed for IP protection for inventions related to α-particle technology of which M.R.M. is an inventor. M.R.M. was a consultant for Actinium Pharmaceuticals, Regeneron, Progenics, Bridge Medicines, and General Electric. M.E.P. is a cofounder and board member of Sofie Biosciences but does not hold ownership interest nor receive any funding from the company. M.E.P. also holds some ownership interest in InDi Molecular.

Purpose: The 4 kallikrein panel, commercially available as the 4Kscore®, is a statistical model that has been shown to accurately predict Gleason Grade Group 2 or greater (high grade) cancer on biopsy and the long-term risk of distant prostate cancer metastases. The panel includes 2 novel markers, namely intact prostate specific antigen and hK2. It has been questioned whether these 2 additional markers add discrimination to the clinical predictors of patient age, digital rectal examination and prior biopsy, and the established molecular markers total and free prostate specific antigen., Materials and Methods: We performed an individual patient data meta-analysis of published studies in which the 4 kallikrein panel was measured in men undergoing prostate biopsy. We assess the improvement in discrimination associated with including intact prostate specific antigen and hK2 along with total and free prostate specific antigen in the statistical model., Results: Included in analysis were 14,510 men from a total of 10 studies. The fixed effects meta-analytical estimate of the discrimination of the model without intact prostate specific antigen and hK2 was 0.742 (95% CI 0.727-0.756) compared to 0.813 (95% CI 0.801-0.825) for the full kallikrein model. The 95% CIs did not overlap and the difference in discrimination was highly statistically significant (0.069, 95% CI 0.057-0.080, p <0.0001). Intact prostate specific antigen (increase in discrimination 0.059, 95% CI 0.050-0.069) and hK2 (increase in discrimination 0.024, 95% CI 0.020-0.029, each p <0.0001) added independently to the model., Conclusions: The clinical value of the panel could not be replicated using data readily available to urologists without measuring intact prostate specific antigen and hK2., (Copyright © 2018 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers., (Copyright © 2016, American Association for the Advancement of Science.)

Background: The four-kallikrein panel and the Prostate Health Index (PHI) have been shown to improve prediction of prostate cancer (PCa) compared with prostate-specific antigen (PSA). No comparison of the four-kallikrein panel and PHI has been presented., Objective: To compare the four-kallikrein panel and PHI for predicting PCa in an independent cohort., Design, Setting, and Participants: Participants were from a population-based cohort of PSA-tested men in Stockholm County. We included 531 men with PSA levels between 3 and 15 ng/ml undergoing first-time prostate biopsy during 2010-2012., Outcome Measurements and Statistical Analysis: Models were fitted to case status. We computed calibration curves, the area under the receiver-operating characteristics curve (AUC), decision curves, and percentage of saved biopsies., Results and Limitations: The four-kallikrein panel showed AUCs of 69.0 when predicting any-grade PCa and 71.8 when predicting high-grade cancer (Gleason score ≥7). Similar values were found for PHI: 70.4 and 71.1, respectively. Both models had higher AUCs than a base model with PSA value and age (p<0.0001 for both); differences between models were not significant. Sensitivity analyses including men with any PSA level or a previous biopsy did not materially affect our findings. Using 10% predicted risk of high-grade PCa by the four-kallikrein panel or PHI of 39 as cut-off for biopsy saved 29% of performed biopsies at a cost of delayed diagnosis for 10% of the men with high-grade cancers. Both models showed limited net benefit in decision analysis. The main study limitation was lack of digital rectal examination data and biopsy decision being based on PSA information., Conclusions: The four-kallikrein panel and PHI similarly improved discrimination when predicting PCa and high-grade PCa. Both are simple blood tests that can reduce the number of unnecessary biopsies compared with screening with total PSA, representing an important new option to reduce harm., Patient Summary: Prostate-specific antigen screening is controversial due to limitations of the test. We found that two blood tests, the Prostate Health Index and the four-kallikrein panel, performed similarly and could both aid in decision making among Swedish men undergoing a prostate biopsy., (Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

Background: Treatment decisions can be difficult in men with low-risk prostate cancer (PCa)., Objective: To evaluate the ability of a panel of four kallikrein markers in blood-total prostate-specific antigen (PSA), free PSA, intact PSA, and kallikrein-related peptidase 2-to distinguish between pathologically insignificant and aggressive disease on pathologic examination of radical prostatectomy (RP) specimens as well as to calculate the number of avoidable surgeries., Design, Setting, and Participants: The cohort comprised 392 screened men participating in rounds 1 and 2 of the Rotterdam arm of the European Randomized Study of Screening for Prostate Cancer. Patients were diagnosed with PCa because of an elevated PSA ≥3.0 ng/ml and were treated with RP between 1994 and 2004., Outcome Measurements and Statistical Analysis: We calculated the accuracy (area under the curve [AUC]) of statistical models to predict pathologically aggressive PCa (pT3-T4, extracapsular extension, tumor volume >0.5cm(3), or any Gleason grade ≥4) based on clinical predictors (age, stage, PSA, biopsy findings) with and without levels of four kallikrein markers in blood., Results and Limitations: A total of 261 patients (67%) had significant disease on pathologic evaluation of the RP specimen. While the clinical model had good accuracy in predicting aggressive disease, reflected in a corrected AUC of 0.81, the four kallikrein markers enhanced the base model, with an AUC of 0.84 (p < 0.0005). The model retained its ability in patients with low-risk and very-low-risk disease and in comparison with the Steyerberg nomogram, a published prediction model. Clinical application of the model incorporating the kallikrein markers would reduce rates of surgery by 135 of 1000 patients overall and 110 of 334 patients with pathologically insignificant disease. A limitation of the present study is that clinicians may be hesitant to make recommendations against active treatment on the basis of a statistical model., Conclusions: Our study provided proof of principle that predictions based on levels of four kallikrein markers in blood distinguish between pathologically insignificant and aggressive disease after RP with good accuracy. In the future, clinical use of the model could potentially reduce rates of immediate unnecessary active treatment., (Copyright © 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Kallikreins are a family of serine proteases with a range of tissue-specific and essential proteolytic functions. Among the best studied are the prostate tissue-specific KLK2 and KLK3 genes and their secreted protease products, human kallikrein 2, hk2, and prostate-specific antigen (PSA). Members of the so-called classic kallikreins, these highly active trypsin-like serine proteases play established roles in human reproduction. Both hK2 and PSA expression is regulated by the androgen receptor which has a fundamental role in prostate tissue development and progression of disease. This feature, combined with the ability to sensitively detect different forms of these proteins in blood and biopsies, result in a crucially important biomarker for the presence and recurrence of cancer. Emerging evidence has begun to suggest a role for these kallikreins in critical vascular events. This review discusses the established and developing biological roles of hK2 and PSA, as well as the historical and advanced use of their detection to accurately and non-invasively detect and guide treatment of prostatic disease.

Background: Percentage of free-to-total prostate-specific antigen (%fPSA) is an independent predictor of risk for prostate cancer among men with modestly elevated level of total PSA (tPSA) in blood. Physiological and pathological factors have been shown to influence the %fPSA value and diagnostic accuracy., Materials/methods: To evaluate genetic determinants of %fPSA, we conducted a genome-wide association study of serum %fPSA by genotyping 642,584 single nucleotide polymorphisms (SNPs) in 3192 men of European ancestry, each with a tPSA level of 2.5 to 10 ng/ml, that were recruited in the REduction by DUtasteride of Prostate Cancer Events study. Single nucleotide polymorphisms (SNPs) with P < 10(-5) were further evaluated among the controls of a population-based case-control study in Sweden (2899 prostate cancer cases and 1722 male controls), including 464 controls having tPSA levels of 2.5 to 10 ng/ml., Results: We identified two loci that were associated with %fPSA at a genome-wide significance level (P <5 x 10(-8)). The first associated SNP was rs3213764 (P = 6.45 x 10(-10)), a nonsynonymous variant (K530R) in the ATF7IP gene at 12p13. This variant was also nominally associated with tPSA (P = .015). The second locus was rs1354774 (P = 1.25 x 10(-12)), near KLK2 at 19q13, which was not associated with tPSA levels, and is separate from the rs17632542 locus at KLK3 that was previously associated with tPSA levels and prostate cancer risk. Neither rs3213764 nor rs1354774 was associated with prostate cancer risk or aggressiveness., Conclusions: These findings demonstrate that genetic variants at ATF7IP and KLK2 contribute to the variance of %fPSA.

Most pretreatment risk-assessment models to predict biochemical recurrence (BCR) after radical prostatectomy (RP) for prostate cancer rely on total prostate-specific antigen (PSA), clinical stage, and biopsy Gleason grade. We investigated whether free PSA (fPSA) and human glandular kallikrein-2 (hK2) would enhance the predictive accuracy of this standard model. Preoperative serum samples and complete clinical data were available for 1,356 patients who underwent RP for localized prostate cancer from 1993 to 2005. A case-control design was used, and conditional logistic regression models were used to evaluate the association between preoperative predictors and BCR after RP. We constructed multivariable models with fPSA and hK2 as additional preoperative predictors to the base model. Predictive accuracy was assessed with the area under the ROC curve (AUC). There were 146 BCR cases; the median follow up for patients without BCR was 3.2 years. Overall, 436 controls were matched to 146 BCR cases. The AUC of the base model was 0.786 in the entire cohort; adding fPSA and hK2 to this model enhanced the AUC to 0.798 (p=0.053), an effect largely driven by fPSA. In the subgroup of men with total PSA

Both benign and malignant prostate diseases elevate total prostate-specific antigen (tPSA), and the incidence of benign disease increases markedly with age. There is evidence, however, that free-to-total PSA ratio (%fPSA) and human kallikrein 2 (hK2) more closely reflect the malignant process. We tested the hypothesis that tPSA levels are more strongly predictive of cancer in younger when compared to older men, whereas %fPSA and hK2 are more strongly predictive in men tested closer to diagnosis. The study included 13,676 men age >/= 44 in Sweden, where PSA screening was uncommon during the study period. fPSA, tPSA and hK2 were measured in archived plasma collected during 1974-1986 in 501 men subsequently diagnosed with prostate cancer up to 1999 and in 1,292 matched controls. The predictive value of tPSA was lower in older men (p = 0.003) but was not strongly affected by time to diagnosis (p = 0.3); the predictive value of hK2 was higher closer to diagnosis (p < 0.0005) but was not modified by age (p = 0.7). A model including tPSA, fPSA and hK2 was superior (p = 0.02) to tPSA alone in older (AUC 0.819 vs. 0.794), but not in younger men (0.758 vs. 0.759). Total PSA can be used as a single marker at early middle age to predict long-term risk of prostate cancer and thus to determine intensity of subsequent screening. In contrast, %fPSA and hK2 add important predictive value in older men and much closer to diagnosis. Strategies for prostate cancer screening should be based on thorough understanding of the interaction of kallikrein-related biomarkers with prostate pathobiology., ((c) 2007 Wiley-Liss, Inc.)

Background: We evaluated the association of total and free forms of serum human kallikrein 2 (hK2) and prostate-specific antigen (PSA) with prostate cancers of unfavorable prognosis., Methods: We retrospectively measured total PSA (tPSA), free PSA (fPSA), and total hK2 (thK2) in preoperative serum samples from 867 men [and assessed free hK2 (fhK2) measured in 577 of these men] treated with radical prostatectomy for clinically localized prostate cancer. Associations between biomarker concentrations and extracapsular extension, seminal vesicle invasion, and biochemical recurrence (BCR) were evaluated. A subset of patients with PSA < or =10 microg/L, the group most commonly seen in clinical practice in the US, was analyzed., Results: thK2 was the strongest predictor of extracapsular extension and seminal vesicle invasion (areas under the ROC curve [AUC], 0.662 and 0.719, respectively), followed by tPSA (AUC, 0.654 and 0.663). All biomarkers were significant predictors of BCR. hK2 forms, but not PSA forms, remained highly significant for predicting BCR in the low-PSA group. Combining tPSA, fPSA, and thK2 in a multivariable model improved prediction compared with any biomarker used individually (AUC, 0.711, 0.755, and 0.752 for this combination predicting extracapsular extension, seminal vesicle invasion, and BCR, respectively; P <0.001 for all)., Conclusions: Increased concentrations of hK2 in the blood are significantly associated with unfavorable features of prostate cancer, and thK2 is predictive of locally advanced and recurrent cancer in patients with PSA < or =10 microg/L. Independent of tPSA and fPSA, hK2 predicts unfavorable prognosis.

Purpose: We examined whether prostate-specific antigen (PSA) forms and human kallikrein 2 (hK2) measured at age 44 to 50 years predict long-term risk of incident prostate cancer., Methods: From 1974 to 1986, 21,277 men age 50 years in Malmö, Sweden, enrolled onto a cardiovascular study (74% participation). The rate of PSA screening in this population is low. According to the Swedish Cancer Registry, 498 were later diagnosed with prostate cancer. We measured hK2, free PSA, and total PSA (tPSA) in archived blood plasma from 462 participants later diagnosed with prostate cancer and from 1,222 matched controls. Conditional logistic regression was used to test for association of prostate cancer with hK2 and PSA forms measured at baseline., Results: Median delay between venipuncture and prostate cancer diagnosis was 18 years. hK2 and all PSA forms were strongly associated with prostate cancer (all P < .0005). None of the 90 anthropometric, lifestyle, biochemical, and medical history variables measured at baseline was importantly predictive. A tPSA increase of 1 ng/mL was associated with an increase in odds of cancer of 3.69 (95% CI, 2.99 to 4.56); addition of other PSA forms or hK2 did not add to the predictive value of tPSA. tPSA remained predictive for men diagnosed > or = 20 years after venipuncture, and the predictive value remained unchanged in an analysis restricted to palpable disease., Conclusion: A single PSA test at age 44 to 50 years predicts subsequent clinically diagnosed prostate cancer. This raises the possibility of risk stratification for prostate cancer screening programs.

Objectives: The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard., Design and Methods: The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates., Results: Reproducibility was best when large copy numbers (>5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold., Conclusions: We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.

Various blood constituents can interfere with immunoassays, usually by binding the Fc portion of antibodies. Our previously developed assays for intact free prostate-specific antigen (PSA), free human kallikrein 2 (hK2), and total hK2 frequently yielded falsely high results despite including an excess of scavenger antibodies. We investigated whether this interference could be eliminated by replacing monoclonal capture or tracer antibodies with F(ab')2 or recombinant Fab fragments. Female heparin plasma samples (n = 1092), which should have negligible PSA and hK2, and male samples (n = 957) were analyzed to identify samples manifesting interference, which then were used to optimize protocols for the immunoassays. We compared original assays (monoclonal antibodies) versus optimized assays (F(ab')2 fragments: denatured mouse IgG added as scavenger) using another set of EDTA plasma (n = 113), heparin plasma (n = 160), and serum samples (n = 171). With the original assays, the frequency of falsely elevated hK2 and intact free PSA was 15 and 13%, respectively. The optimized assays eliminated 70-85% of these falsely elevated results and importantly reduced the magnitude in the remainder. F(ab')2 fragmentation was the most important factor in reducing interference. The optimized intact free PSA, free hK2, and total hK2 assays manifested high accuracy close to the lower limit of detection.

The human kallikrein locus on chromosome 19q13.3-13.4 contains kallikrein 1--the tissue kallikrein--and 14 related serine proteases. Recent investigations into their function and evolution have indicated that the present nomenclature for these proteins is inadequate or insufficient. Here we present a new nomenclature in which proteins without proven kininogenase activity are denoted kallikrein-related peptidase. Names are also given to the unique rodent proteins that are closely related to kallikrein 1.

Most models to predict biochemical recurrence (BCR) of prostate cancer use pretreatment serum prostate-specific antigen (PSA), clinical stage and prostate biopsy Gleason grade. We investigated whether human glandular kallikrein 2 (hK2) and free prostate-specific antigen (fPSA) measured in pretreatment serum enhance prediction. We retrospectively measured total PSA (tPSA), fPSA and hK2 in preoperative serum samples from 461 men with localized prostate cancer treated with radical prostatectomy between 1999 and 2001. We developed a regression model to predict BCR using preoperative tPSA, clinical stage and biopsy Gleason grade. We then compared the predictive accuracy of this "base" model with a model with fPSA and hK2 as additional predictors. BCR was observed in 90 patients (20%), including 48 patients with a pretreatment tPSA < or = 14 ng/ml (13%), and 28 patients (10%) with a pretreatment tPSA < or = 10 ng/ml. Overall, the predictive accuracy of the base model (bootstrap-corrected concordance index of 0.813) was not improved after the addition of fPSA or hK2 (0.818). However, for men with moderate tPSA-elevation (tPSA < or = 10 ng/ml), addition of fPSA and hK2 data increased predictive accuracy (from a base model concordance index of 0.756-0.815, p = 0.005). The improvement in accuracy was not sensitive to the threshold for "moderately elevated" PSA. For patients with a moderate tPSA-elevation (tPSA < or = 10 ng/ml), which closely corresponds to concurrent disease demographics, BCR-prediction was enhanced when fPSA and hK2 were added to the conventional model. Measurements of fPSA and hK2 improve on our ability to counsel patients prior to treatment as to their risk of BCR., ((c) 2005 Wiley-Liss, Inc.)

Background: Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for hK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an hK2-activated prodrug that was efficiently hydrolyzed by purified hK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to hK2 producing prostate cancer cells in vitro., Methods: In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the hK2-activated prodrug in vivo., Results: The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 micromole/kg) which produced peak serum concentration of approximately 36 microM and had a half-life of approximately 40 min. In addition, over a 24 hr period <0.5% of free L12ADT analog was observed in plasma. The prodrug demonstrated significant antitumor effect in vivo while it was being administered, but prolonged intravenous administration was not possible due to local toxicity to tail veins. Subcutaneous administration of equimolar doses produced lower plasma AUC compared to intravenous dosing but equivalent intratumoral levels of prodrug following multiple doses., Conclusions: The hK2-activated prodrug was stable in vivo. The prodrug, however, was rapidly cleared and difficult to administer over prolonged dosing interval. Additional studies are underway to assess antitumor efficacy with prolonged administration of higher subcutaneous doses of prodrug. Second-generation hK2-activated thapsigargin prodrugs with increased half-lives and improved formulations are also under development., ((c) 2005 Wiley-Liss, Inc)

An overview is given of serum and urine prostate cancer markers that are currently under investigation and subsequently the P-Mark project is introduced. There are many markers showing promise to overcome the limitations of prostate specific antigen (PSA). Eventually, these markers should be able to increase the specificity in diagnosis, differentiate between harmless and aggressive disease and identify progression towards androgen independence at an early stage. In the P-Mark project, several recently developed, promising markers will be evaluated using clinically well-defined biorepositories. Following successful evaluation, these markers will be validated on a sample set derived from two large, European, prostate cancer studies and used for the identification of special risk groups in the general population. In addition, novel markers will be identified in the same biorepositories by different mass spectrometry techniques.

Background: We investigated the association of different subfractions of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), such as total PSA (tPSA), complexed PSA (cPSA), free PSA (fPSA), "single-chain Intact fPSA" (fPSA-I), "multi-chain nicked fPSA" (fPSA-N), and total hK2 with volumes of total prostate gland, transition zone (tz), and prostate cancer (PCa) tissue in patients with benign and malignant prostatic disease., Methods: Serum samples were collected from men with negative biopsy (n=164) and PCa (n=252). Total and fPSA were measured using a commercially immunoassay. We measured hK2 and fPSA-I by previously reported in-house research assays specific for hK2 and single-chain, non-cleaved fPSA, respectively. Levels of fPSA-N (=fPSA-fPSA-I) and cPSA (=tPSA-fPSA) were calculated. Total prostate and tz volume were measured using transrectal ultrasound (TRUS); PCa volume was calculated using a computer assisted volumetric program. Association with tz and cancer volumes (CaVols) was performed by linear regression analysis., Results: All PSA subfractions and hK2 were associated with tz volume in multivariable linear regression analysis. Only hK2, fPSA, and fPSA-N were significantly associated with CaVol in multivariable analysis, fPSA-I seemed to be cancer related., Conclusions: The multi-chain fPSA-N subfractions of fPSA may be a valuable predictor of both benign prostate hyperplasia (BPH) and CaVol that is likely to be more useful in predicting tz volumes than CaVols. fPSA-I may provide information on cancer without being influenced by the presence of BPH., (Copyright (c) 2004 Wiley-Liss, Inc.)

Purpose: Previously human glandular kallikrein 2 (hK2) has been implicated to predict pathologically organ confined prostate cancer (PCa) in patients with stage T2 disease. Now we evaluated the usefulness of hK2, as measured by 2 entirely different immunoassay designs, to enhance the discrimination of pathologically organ from nonorgan confined clinical stage T1c PCa., Materials and Methods: A consecutive series of pretreatment serum from 148 men with clinical stage T1c PCa was used in 2 equally sensitive and specific methods to measure total hK2 with independent reagents and entirely different assay designs. Total prostate specific antigen (tPSA) and free PSA (fPSA) were measured and percent fPSA was calculated. We determined the algorithm, hK2*tPSA/fPSA, from data generated by each hK2 assay, calculated means, medians and ranges for each analyte and algorithm, and calculated the significance of differences on univariate analysis. Using pretreatment PSA, clinical stage and biopsy Gleason grade we then developed a multivariate logistic regression base model to predict organ confined cancer and we compared predictions of the base model supplemented by the different hK2 measurements., Results: hK2 and hK2 based algorithms obtained by each hK2 assay were significantly different for pT2a/b vs pT3a or greater PCa (p = 0.034 to 0.0001) compared to tPSA (p = 0.06), fPSA (p = 0.90) or percent fPSA (p = 0.059). However, AUC (0.67 to 0.70) calculated by ROC analysis of the 4 models containing hK2 derived information was not significantly larger than that of the base model (AUC = 0.64, p = 0.52)., Conclusions: The current data confirm that hK2 alone or hK2*tPSA/fPSA measured by 2 immunoassays is significantly lower in men with pT2a/b vs pT3a or greater PCa compared to tPSA, fPSA or percent fPSA on univariate analysis of a validation set of clinical stage T1c prostate cancer treated at an American center of excellence for prostate cancer surgery. However, the incorporation of preoperative hK2 into multiparameter predictive models for pT2 cancers did not increase predictive accuracy in this cohort of men.

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. In the literature, there are reports of nonprostatic expression of PSA that potentially can affect early diagnosis. However, the results are scattered and inconclusive, which motivated us to conduct a more comprehensive study of the tissue distribution of PSA and the closely related protein human glandular kallikrein 2 (hK2). RT-PCR, in situ hybridization and immunohistochemistry were used to detect expression of both PSA and hK2 in secretory epithelial cells of trachea, thyroid gland, mammary gland, salivary gland, jejunum, ileum, epididymis, seminal vesicle and urethra, as well as in Leydig cells, pancreatic exocrine glands and epidermis. Immunometric measurements revealed that the concentration of PSA in nonprostatic tissues represents less than 1% of the amount in normal prostate. Pronounced expression of PSA was detected in the Paneth cells in ileum, which prompted us to compare functional parameters of PSA in ileum and prostate. We found that in homogenates from these 2 tissues, PSA manifested equivalent amidolytic activity and capacity to form complexes with protease inhibitors in blood in vitro. Thus, PSA released from sources other than the prostate may add to the plasma pool of this protein, but given the lower levels detected from those sites, it is unlikely that nonprostatic PSA normally can interfere with the diagnosis of prostate cancer. Nevertheless, this risk should not be neglected as it may be of clinical significance under certain circumstances. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Comparisons of the glandular kallikreins loci in human, mouse and rat revealed remarkable differences. For example, the mouse and the rat lack the genes encoding prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). In contrast, the intergenic region between KLK1 and KLK15 is devoid of genes and spans only 1.5 kb in humans, but encompasses 23 KLK1-like genes spanning 290 kb in the mouse. To further elucidate the evolution of glandular kallikrein genes, we investigated the structure and organization of these genes in the cotton-top tamarin (Saguinus oedipus), a New World monkey. We conclude that this species has no PSA gene. Moreover, the ortholog of the hK2 gene is a pseudogene, as it contains several mutations that preclude formation of a functional serine protease. The expression of this gene was probably silenced by a 15-bp deletion observed in an androgen response element in the upstream promoter region. Replacing the deleted base pairs in vitro with nucleotides from the human counterpart dramatically restored the transcriptional activity to a level that even surpassed that of the human ortholog. We also determined the nucleotide sequence of KLK15 and the intergenic region between this gene and KLK1 in the cotton-top tamarin. The region between KLK1 and KLK15 is conserved between the cotton-top tamarin and humans, and there are no signs of the extension seen in the mouse. KLK15 appeared to be functional, thus, we predict that it generates a protease with specificity similar to that of the human ortholog.

Objective: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation-independent apoptosis through dysregulation of intracellular calcium levels., Methods: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity., Results: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 micromol/L, kcat of 1.09 s(-1), and a kcat/Km ratio of 41,132 s(-1) mol/L(-1). The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2., Conclusion: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity., Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer., Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 microg/L. The median total hK2 concentration was 0.022 microg/L (range, 0.0015-0.37 microg/L). hK2 concentrations were 0.1-58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41-50 and 51-60 years of age. The ratio of hK2 to PSA steadily decreased from 5-30% at PSA <1 microg/L to 1-2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 microg/L (range, 0.0015-16.2 microg/L). The median free hK2 concentration was 0.070 (range, 0.005-12.2) microg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%)., Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

Purpose: The low molecular mass and short half-life of free (f) prostate specific antigen (PSA) implies elimination from blood by glomerular filtration. In addition, patients with terminal renal failure have increased fPSA in serum but there have been sparse data reported on the rates and pathways of elimination of PSA complexes and human kallikrein 2 (hK2). We studied glomerular filtration dependent elimination of fPSA and hK2 in patients with renal insufficiency undergoing successful renal transplantation., Materials and Methods: We studied 14 patients with immediate onset of renal function after renal transplantation. Blood samples were obtained before and at regular intervals up to 160 hours after transplanted kidney reperfusion. Measurements of fPSA, total PSA and hK2 were performed with immunofluorometric assays and complexed PSA was determined by a chemiluminiscence assay. Glomerular filtration rates were monitored by analyzing serum creatinine and cystatin C. NONMEM, a multivariate pharmacokinetic approach, was used to determine the elimination rates of fPSA and hK2 after renal transplantation., Results: Serum fPSA and hK2 but not PSA complexes, decreased rapidly after renal transplantation. Significant reductions in fPSA and hK2 were observed after only 16 and 8 hours, respectively. fPSA and hK2 showed similar elimination patterns, decreasing to 42% and 44% of their original levels compared to cystatin C, which was at 44% after 160 hours. The median half-lives of fPSA and hK2 were 17.4 and 11.5 hours, respectively., Conclusions: These results verify the hypothesis that fPSA and hK2 are eliminated from the blood circulation by glomerular filtration and severe renal failure influences the levels of the 2 proteins in serum.

Purpose: We measured concentrations of human glandular kallikrein 2 (hK2), total prostate specific antigen (tPSA), free PSA (fPSA) and percent fPSA to evaluate their relationship to total prostate gland volume, benign prostatic hyperplasia (BPH) volume, total prostate cancer (PCa) volume (CaVol) and the volume of Gleason grades 4/5 cancer (CaVolGl4) in the serum of 256 patients with PCa undergoing radical retropubic prostatectomy and 185 with negative systematic sextant biopsies., Materials and Methods: Free and total PSA was measured using the Delfia Prostatus (Perkin-Elmer, Turku, Finland) total/free PSA assay and hK2 was measured using a research immunofluorometric assay. Transrectal ultrasound was used to determine total prostate and BPH volume. Total CaVol and CaVolGl4/5 were calculated using a volumetric program in specimens from 158 men with pT2a/b and 98 with pT3a or greater PCa. The Pearson correlation was performed after logarithmic conversion of PSA and hK2 levels. Benign gland, and pT2a/b and pT3a or greater PCa cases were subdivided into small vs large prostate gland volumes (42 cc or less vs greater than 42 cc)., Results: Total prostate and BPH volumes correlated closely with free PSA (r = 0.64 to 0.65, p <0.0001) in 143 patients with negative biopsy and a prostate of greater than 42 cc. Correlations of hK2 and tPSA with total prostate and BPH volumes were weaker (r = 0.35 to 0.36 and 0.45 to 0.46, respectively). In pT2a/b and pT3a or greater PCa cases hK2 most closely correlated with CaVol (range 0.31 to 0.62, p = 0.0072 and <0.0001) and with CaVolGl4/5 (range 0.26 to 0.56, p = 0.021 and <0.0001, respectively). The tPSA level correlated significantly with CaVol and CaVolGl4/5 except in glands 42 cc or greater harboring pT2a/b PCa (p = 0.08). Free PSA correlated significantly with CaVolGl4/5 only in pT3a or greater PCa (p <0.05), and with CaVol in pT3a or greater PCa and in small prostates harboring pT2a/b PCa., Conclusions: Large benign prostate gland volume affects fPSA more than tPSA in serum. In PCa hK2 more closely correlates with total cancer volume and high grade PCa volume compared with tPSA or fPSA.

Purpose: We investigated the value of serum measurements for glandular kallikrein 2 (hK2), and free (f) and total (t) prostate specific antigen (PSA) in a second round of biannual screening for prostate cancer., Materials and Methods: In 1995 to 1996, 5,853 of 9,811 randomly selected men in Göteborg, Sweden 50 to 66 years old had PSA measurements. Of 660 men 611 with tPSA 3 ng/ml or greater underwent biopsy and 145 had cancer. All were re-invited 2 years later for PSA testing, and 506 of 596 men with tPSA 3 ng/ml or greater underwent biopsy and 113 cancers were detected. We analyzed hK2, tPSA and fPSA in 423 of 453 (93%) men who underwent biopsy in 1997 to 1998 who were also screened in 1995 to 1996., Results: The 99 of 423 (23%) men who underwent biopsy diagnosed with prostate cancer in 1997 to 1998 had significantly different tPSA, percent fPSA and hK2 x tPSA/fPSA compared to the men with negative biopsies from 2 years earlier. The largest area under curve was obtained for hK2 x tPSA/fPSA in serum from 1995 to 1996 and from 1997 to 1998, but the difference was not significant compared to tPSA and percent fPSA. In serum from 1997 to 1998 measurements of hK2 x tPSA/fPSA gave significantly higher specificity than tPSA at 85% sensitivity, and significantly higher specificity than tPSA and percent fPSA at 70% to 75% sensitivity. In addition, levels of hK2 and hK2 x tPSA/fPSA manifested a significantly greater 2-year increase in men with cancer compared to those with benign biopsies., Conclusions: In men with tPSA levels 3.0 ng/ml or greater who were not diagnosed with cancer during a first round of screening, hK2 measurements enhanced specificity compared to tPSA testing at moderately high sensitivity, and manifested a greater 2-year increase in men with cancer.

Background: Measurement of human kallikrein 2 (hK2) has improved early detection and staging of prostate cancer. However, reported concentrations of hK2 among currently used assays have not been standardized in any way. We compared two hK2 assays and five different recombinant hK2 variants (rhK2) and suggest a common calibrator as an important step and putative reference substance in hK2 assay standardization., Methods: We measured 146 sera by two hK2 assays, using assay-specific calibrators to assess the difference between the two assays. Serial dilutions of five rhK2 preparations were measured repeatedly, with one preparation assigned as calibrator and the others as unknowns to define which variant provided the closest match between the two assays. This rhK2 variant was used to recalibrate both assays. We measured hK2 concentrations in the same 146 patients to evaluate the change in the difference., Results: Use of assay-specific calibrators for comparison of the two assays yielded a Deming regression equation of: y = 0.789 (95% confidence interval, 0.674-0.922)x + 0.014 (0.004-0.025) micro g/L; R(2) = 0.667. Analysis of five rhK2 variants revealed that the enterokinase (ek)-rhK2 form provided the best match between both assays. Using the ek-rhK2 as a common calibrator, we observed a change in the slope of the regression curve to: y = 1.106 (0.872-1.340)x + 0.006 (-0.002 to 0.016) micro g/L; R(2) = 0.648, suggesting an increase in the mean estimate of agreement between the two assays., Conclusion: Calibration with a common calibrator substantially increased agreement between the assays. The ek-rhK2 variant provided the best performance of all tested rhK2 variants and should undergo mass spectrometry and amino acid analysis for exact mass determination and value assignment to evaluate its potential as a reference material for immunoassays for hK2.

Background: In recent studies serum levels of human glandular kallikrein 2 (hK2) demonstrated significant differences in pathologically organ-confined versus non-organ-confined prostate cancer (PCa). In this study we investigated whether hK2 adds independent information when considered together with traditionally used parameters to predict organ confined (pT2a/b) PCa., Methods: Serum levels of hK2, total and free prostate-specific antigens (PSA) were obtained one day before radical prostatectomy in 245 consecutive men. These were included with clinical stage and biopsy Gleason grade into univariate analysis and multivariate logistic regression models., Results: pT2a/b PCa was found in n = 148 patients. In univariate analysis all preoperative parameters demonstrated significant association with the presence of pT2a/b PCa. Using multivariate logistic regression model hK2 (P = 0.022), clinical stage (P < 0.0001), and Gleason grade (P < 0.0001) were independent predictors of pT2a/b PCa whereas PSA (P = 0.3) was not. In bootstrap corrected logistic regression based nomograms the addition of hK2 density marginally enhanced predictive accuracy when PSA, PSA density, clinical stage, and Gleason grade were considered (AUC = 0.879 without hK2 density and 0.883 with hK2 density)., Conclusions: hK2 and hK2 density could independently predict pT2a/b PCa. However, improvement in predictive accuracy was marginal when nomograms based on traditional variables were complemented with this serum marker., (Copyright 2002 Wiley-Liss, Inc.)

Anti-thrombin, a member of the serpin family and an inhibitor of thrombin and blood coagulation factor Xa, was recently shown to inhibit angiogenesis and tumor growth. In the present study, we examined the expression of anti-thrombin in benign and malignant prostate gland. Using immunohistochemistry, anti-thrombin was found in prostate epithelium and stroma cells. Tissue microarrays of tumors (n = 112) and three different prostate cancer cell lines (PC-3, LNCaP, and DU-145) were all positive for anti-thrombin. Abundant expression in a population of prostatic tumor cells was further evidenced by in situ hybridization experiments. The immunostaining for anti-thrombin was confined to the cytoplasm, was most intense in Gleason grade 3 tumors, and in part overlapped with that of prostate-specific antigen. Western blotting of benign and malignant tissue homogenates revealed a predominant 58-kd anti-thrombin immunoreactive component. In vitro, anti-thrombin formed complexes more readily with human kallikrein 2, particularly in the presence of heparin, and less efficiently with prostate-specific antigen. Both complexes could be recognized by polyclonal and monoclonal IgGs against anti-thrombin. We conclude that anti-thrombin is widely expressed in prostate cancer but is gradually lost in tumors of high Gleason grade. Anti-thrombin may act as a local anti-angiogenic factor, the effect of which is partially lost in poorly differentiated prostatic tumors.

Background: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease., Methods: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1-pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 10(2)-10(6) mRNA copies., Results: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann-Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (< or =5) were negative for both PSA and hK2 (P = 0.02, Mann-Whitney U-test)., Conclusions: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.

Prostate-specific antigen (PSA or hK3) is a glandular kallikrein with abundant expression in the prostate that is widely used to detect and monitor prostate cancer (PCa), although the serum level is frequently elevated also in benign and inflammatory prostatic diseases. PSA testing is useful for early detection of localized PCa and for the detection of disease recurrence after treatment. However, PSA has failed to accurately estimate cancer volume and preoperative staging. There is no PSA level in serum that definitively distinguishes men with benign conditions from those with prostate cancer, although PCa is rare in men with PSA levels in serum < 2.0 ng/ml. This prompted searches for enhancing parameters to combine with PSA testing, such as PSA density, PSA velocity, and age-specific reference ranges. Due to the protease structure, PSA occurs in different molecular forms in serum and their concentrations vary according to the type of prostatic disease. Human glandular kallikrein 2 (hK2) is very similar to PSA, but expressed at higher levels in prostate adenocarcinoma than in normal prostate epithelium. Blood testing for hK2 combined with different PSA forms improves discrimination of men with benign prostatic disease from those with prostate cancer. Many data have also been reported on the extra-prostatic expression of both PSA and hK2, and it is now believed that they may both have functions in tissues outside the prostate.

Background: We measured serum levels of human glandular kallikrein 2 (hK2) in patients treated with radical retropubic prostatectomy (rrP) for clinically localized prostate cancer (PCa) with a total PSA (tPSA)-level below 10 ng/ml to investigate whether hK2 can be applied to preoperatively distinguish organ-confined (pT2a/b) from nonorgan-confined (> or = pT3a)-PCa more accurately than total PSA. Further, we evaluated hK2, free- and tPSA-concentrations in all pathologic stages of PCa., Methods: 161 serum samples from men scheduled for rrP were collected 1 day before surgery prior to any prostatic manipulation. Pathologic work-up revealed > or = pT3a-PCa in 48 and pT2a/b-PCa in 113 patients. HK2-levels in serum were measured using an immunofluorometric assay with an analytical sensitivity of 0.5 pg/ml, a functional sensitivity of 5 pg/ml and insignificant cross-reactivity with PSA (< 0.005%). Total (tPSA) and free PSA (fPSA) levels were measured using a commercially available assay from which we calculated %fPSA and an algorithm that combined hK2 and PSA-levels [hK2] x [tPSA/fPSA]. Means, medians, and ranges were calculated for pT2a/b vs. >/= pT3a-PCa and for all pathologic stages. Statistical significance of differences was calculated using Mann-Whitney-U and Kruskal-Wallis tests. Calculation of receiver-operator-characteristic (ROC) curves were performed for hK2, [hK2] x [tPSA/fPSA] and tPSA to compare diagnostic performance., Results: A mean tPSA level in serum of 6.12 ng/ml in > or = pT3a-PCa was not significantly different (P = 0.366) from 5.78 ng/ml in pT2a/b-PCa. Also, there were no statistically significantly different levels of fPSA (P = 0.947) or %fPSA (0.292) for these two groups. By contrast, mean hK2-level in pT2a/b-PCa of 80 pg/ml was significantly different (P = 0.004) from a mean hK2 level of 120 pg/ml in > or = pT3a-PCa as shown by Mann-Whitney-analysis Moreover, the algorithm of [hK2] x [tPSA/fPSA] was significantly lower (P = 0.0004) in pT2a/b-PCa vs. > or = pT3a-PCa. Calculation of areas under curve (AUC) by receiver-operator-characteristics (ROC) demonstrated that the AUC for hK2 (0.64) was larger and the AUC for [hK2] x [tPSA/fPSA] (=0.68) significantly larger (P = 0.007) compared to the AUC of tPSA (0.55). Furthermore, Kruskal-Wallis Test revealed a highly significant correlation to pathologic stage using hK2 (P = 0.008) and [hK2] x [tPSA/fPSA] (P = 0.0015) compared to no significant differences in serum concentration of tPSA (P = 0.296). Also at tPSA-levels from 10-20 ng/ml, the hK2-levels in pT2a/b-PCa were close to significantly different (P = 0.051) from those in men with >/= pT3a-PCa, while the algorithm of [hK2] x [tPSA/fPSA] in that tPSA-range was significantly lower (P = 0.002) in pT2a/b-PCa compared to > or = pT3a0-PCa., Conclusions: Highly significant differences in serum concentration enable hK2 to be a powerful predictor of organ-confined disease and pathologic stage of clinically localized prostate cancer, especially in the PSA-range below 10 ng/ml. As such, there are important clinical consequences for the application of hK2 for the adequate treatment of prostate cancer patients, i.e., the option of nerve-sparing surgery. (c) 2001 Wiley-Liss, Inc.

We present a multiplexed and internally calibrated quantitative reverse transcription-PCR (QRT-PCR) assay to detect human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) transcripts in blood samples from healthy subjects and prostate cancer (PC) patients. The assay detected 50 copies of hK2 and PSA mRNA, and 1 PSA- and 10 hK2-expressing LNCaP cells in the presence of 2.5 x 10(6) PSA- and hK2-negative cells. In PC patients, 20 of 25 and 19 of 25 gave detectable PSA and hK2 mRNAs, respectively. Number of hK2 mRNA copies was significantly higher than that of PSA mRNA copies in patients with biochemically progressive (P = 0.02) PC, and with locally advanced and metastasized (P = 0.004) PC. Patients with rapidly progressive and hormone refractory PC gave detectable hK2 mRNA only in 2 of 8 and PSA mRNA in 3 of 8 patients. Neither PSA nor hK2 mRNAs were detected in 16 healthy subjects. PSA and hK2 discriminated PC patients with biochemically progressive and advanced disease from the controls and from the aggressive distant metastatic disease. The assay provides a reliable quantification of the number of hK2 and PSA mRNA copies, allows to discriminate PC cases from healthy subjects, and offers a tool for further studies on molecular staging of PC.

Background: Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of an extensive kallikrein family of proteases. Both proteases are secreted as zymogens or proenzymes containing a seven amino acid propeptide that must be proteolytically removed for enzymatic activation. The physiological proteases that activate pro-hK2 and pro-PSA are not known., Methods: The pro-hK2 peptide sequence is Val-Pro-Leu-Ile-Gln-Ser-Arg (VPLIQSR). For PSA, the amino acid sequence of the propeptide is Ala-Pro-Leu-Ile-Leu-Ser-Arg (APLILSR). Fluorescent substrates were made by coupling these peptide sequences to 7-amino-4-methylcoumarin (AMC). The hydrolysis of the VPLIQSR-AMC and APLILSR-AMC substrates by hK2, PSA, and a panel of purified proteases was determined., Results: HK2 readily cleaved the pro-hK2 peptide substrate VPLIQSR-AMC with a rate of hydrolysis that was approximately 8-fold higher than an equimolar amount of purified trypsin. HK2 also had the highest hydrolysis rate from among a group of other trypsin-like proteases. In contrast, neither hK2 nor PSA was able to appreciably cleave the pro-PSA substrate APLILSR-AMC. The pro-PSA substrate was most readily hydrolyzed by urokinase and trypsin., Conclusions: HK2 can hydrolyze the pro-hK2 substrate suggesting that maturation of pro-hK2 to enzymatically active hK2 involves autoprocessing. As expected, PSA, a chymotrypsin-like protease, was unable to hydrolyze either of the propeptide substrates. Therefore, it is unlikely that PSA can auto-process its own enzymatic function. HK2 has trypsin-like specificity but was unable to hydrolyze the pro-PSA substrate. These results raise the possibility that an additional processing protease may be required to fully process PSA to an enzymatically active form.

Background: Our recently reported finding of rapid bi-exponential elimination of free prostate-specific antigen (PSA) after radical retropubic prostatectomy in patients with moderately elevated PSA levels, which contrasted a very slow, linear elimination of PSA complexed to alpha-1-antichymotrypsin (ACT), prompted us to study whether these elimination rates were applicable for patients selected for castration treatment with very high pretreatment concentrations of PSA in serum. In addition, serum concentrations of hK2, the activator of proPSA, were measured., Methods: Pretreatment serum was obtained from 21 previously untreated prostate cancer patients due for hormonal treatment with a GnRH-analog. Samples were also collected during treatment up to a minimum of 24 weeks at 2-week intervals and analyzed with immunofluorometric assays for free PSA (PSA-F), PSA complexed to alpha-1-antichymotrypsin (PSA-ACT), total PSA (PSA-T), and human kallikrein 2 (hK2). For pharmaco-kinetic analysis the serum concentrations of hK2 and PSA forms for each patient were plotted against time both before and after logarithmic transformation and the half-lives were calculated as ln2/k., Results: Median pretreatment serum concentrations were 322 ng/ml (range, 1.9-2210) for PSA-T, 27.8 ng/ml (range, 1.14-259) for PSA-F, and 207 ng/ml (range, 0.8-2080) for PSA-ACT. All patients had castrate levels of serum testosterone (< 2.5 nmol/l) in less than 21 days after initiation of GnRH-analog treatment. It was possible to evaluate data from 19/21 patients which showed an exponential decrease of all PSA concentrations in serum, with mean half-lives of 12.9 days (range, 7.3-30) for PSA-T, 15.5 days (range, 7.7-37.5) for PSA-F, and 12.3 days (range, 6.6-30) for PSA-ACT. Median pretreatment percent free PSA (PSA-F/PSA-T) was 12% compared to 18% at nadir. The median pretreatment level of hK2 was 3.5 ng/ml (range, 0.29-30.3). There was an exponential decrease in hK2 concentrations in serum after initiation of hormonal treatment with a mean half-life of 18.7 days (range, 7.5-37.5)., Conclusions: For the majority of patients with hormonally treated prostate cancer the serum concentrations of PSA-T, PSA-F, PSA-ACT, and hK2 decreased slowly in parallel and mono-exponentially after initiation of treatment. Mean half-lives were between 12 and 19 days.

Objectives: To design protocols for specific and quantitative immunohistochemical detection of human kallikrein 2 (hK2) using lanthanide chelate-labeled monoclonal antibodies (Mabs) and time-resolved fluorescence imaging., Methods: Anti-prostate-specific antigen (PSA) Mabs were tested in microtiterplate assays for their ability to prevent PSA from cross-reacting with the anti-hK2 Mab 6H10. Europium-labeled 6H10 and terbium-labeled anti-PSA Mab 2E9, selected as the best blocker antibody, were used for dual-label immunodetection in routinely fixed benign (n = 7) and malignant (n = 5) prostate specimens. The amounts of IgG bound in tissue were calculated from drops containing known Mab concentrations., Results: The use of anti-PSA Mab 2E9 for blocking diminished the cross-reaction from 5% to 0.3%. In the analyzed tissues, there was considerable variation in staining intensity for both proteins; PSA signals varied from 0.1 to 36.6 times that of hK2, with on average 10-fold more bound anti-PSA Mab than anti-hK2 Mab. In malignant tissue, the amounts of bound IgGs were lower and more variable than in benign tissue using both the anti-PSA Mab and the anti-hK2 Mab. The variation in signal intensities for PSA and hK2 correlated significantly in benign tissue (P >0.05), but not in benign hyperplastic and malignant specimens (P <0.05)., Conclusions: Quantification of two lanthanide chelate-labeled antibodies bound in the same tissue section enabled comparison of PSA and hK2 content in individual cells. The average cellular content of hK2 relative to that of PSA was consistent with previous mRNA studies. The time-resolved fluorescence imaging-based quantification method has universal applicability in fixed tissue specimens.

Purpose: We measured serum levels of human glandular kallikrein 2 (hK2) in patients with prostate cancer treated with radical retropubic prostatectomy for clinically localized prostate cancer to determine whether preoperative hK2 levels discriminate stage pT2a/b from pathological stage T3a or greater cancer. This finding would help to predict preoperatively the organ confined versus non-organ confined growth of prostate cancer., Materials and Methods: A total of 68 consecutive men underwent radical retropubic prostatectomy for clinically localized prostate cancer. Serum was obtained 1 day preoperatively before prostatic manipulation. hK2, and total and free prostate specific antigen (PSA) were measured using immunofluorometric assays. Mean, median and range of hK2, total and free PSA, and the ratio of free-to-total PSA (percent free PSA) were calculated. Each analyte or combination of analytes was evaluated to determine whether it significantly contributed to enhance the discrimination of organ confined from non-organ confined cancer. We calculated the statistical significance of observed differences using the Mann-Whitney U and Kruskal-Wallis tests. Sensitivity and specificity calculations were performed for hK2, total PSA and the algorithm, (hK2) x (total PSA/free PSA) in addition to receiver operating characteristics curves and the respective areas under the curves. Multivariate logistic regression analysis was done for hK2, and total and free PSA RESULTS: Disease was organ and non-organ (extraprostatic extension) confined in 38 and 30 men, respectively. In organ confined cancer mean hK2 was significantly lower than in non-organ confined cancer (0.09 ng./ml., range less than 0.03 to 0.23 versus 0.30, range 0.04 to 0.94, p <0.0001). In addition, there was significantly higher free and total but not percent free PSA in non-organ than in organ confined cases. There were also statistically significant differences in hK2, free PSA and total PSA at each pathological disease stage (p <0.001, <0.01 and <0.05, respectively). Sensitivity for detecting organ confined disease was 37% at 100% specificity (correct identification of all non-organ confined cancer) using hK2 measurements compared with a sensitivity of 14% for total PSA. At a specificity of 95%, sensitivity was 40% for hK2 versus 23% for total PSA, which was a statistically significant gain in sensitivity (p <0.05). Receiver operating characteristics curves demonstrated that hK2 had the largest area under the curve, followed by the algorithm, (hK2) x (total PSA/free PSA), and total PSA (0.76, 0.75 and 0.72, respectively). However, none of area under the curve differences was statistically significant., Conclusions: Compared with total and free PSA hK2 testing improved the preoperative evaluation of patients who underwent radical retropubic prostatectomy due to the superior discrimination of organ from non-organ confined cancer.

Objectives: To investigate the clinical value of human glandular kallikrein 2 (hK2) compared with free (f) and total (t) prostate-specific antigen (PSA) in the early detection of prostate cancer (PCa)., Methods: In PCa screening conducted in 1995 to 1996 in Göteborg, Sweden, 5853 of 9811 randomly selected men (aged 50 to 66 years; median 61) accepted PSA testing; those with tPSA levels of 3. 0 ng/mL or greater were offered digital rectal examination, transrectal ultrasound, and sextant biopsies. Serum from 604 of 611 biopsied men (18% with positive digital rectal examinations, tPSA range 3.0 to 220 ng/mL, 144 men with PCa) was analyzed for hK2 (research assay) and tPSA and fPSA (Prostatus). Sera were stored at -20 degrees C for a maximum of 2 weeks for tPSA and fPSA and 3 years for hK2., Results: hK2 levels and hK2 x tPSA/fPSA values were significantly elevated in men with PCa. Receiver operating characteristic data revealed that the area under the curve for hK2 x tPSA/fPSA was significantly greater than that for tPSA and greater, but not significantly greater, than that for percent fPSA. Also, the cancer-detecting sensitivity was significantly improved (P <0.05) using hK2 x tPSA/fPSA compared with tPSA and percent fPSA at specificity levels of 75% to 90%. At 75% specificity, a sensitivity of 74% was obtained compared with 64% or 54% using percent fPSA or tPSA; at 90% specificity, the corresponding sensitivity level was 55%, 41%, and 36%, respectively., Conclusions: Discrimination of men with and without PCa in a randomly selected population was improved by measuring hK2 in addition to tPSA and fPSA.

Background: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2., Methods: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA., Results: The analytical detection limit was <10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4. 8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-alpha(1)-antichymotrypsin (ACT) were detected in vitro with -6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4-19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor., Conclusions: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.

Purpose: To investigate the clinical value of measuring human glandular kallikrein 2 (hK2) compared with free and total prostate specific antigen (PSA-F and PSA-T) in serum from patients with prostate disease., Materials and Methods: Serum from healthy controls, from men with benign prostate hyperplasia (BPH), clinically localized prostate cancer (PCa), and advanced PCa were analyzed for hK2 (using an in-house-research assay with detection limit of 0.05 ng./mL and <0.1% cross-reaction with PSA) and for PSA-F and PSA-T (using the Dual Prostatus assay from EG&G Wallac)., Results: HK2 concentrations were <0.05 ng./mL in 50/50 healthy volunteers but significantly higher (p <0.0001) and > or =0.05 ng./mL in 28/54 (52%) patients with BPH. In comparison to these men, the hK2 levels were significantly higher (p <0.0001, median 0.085 ng./mL) and > or =0.05 ng./mL in 100/136 (74%) men with clinically localized PCa. Compared with localized PCa, the hK2 levels were significantly higher (p <0.0001, median 0.57 ng./mL) and > or =0.05 ng./mL in 55/57 (96%) patients with advanced PCa. The median hK2 levels ranged from 1.3 to 1.6% of those of PSA-T in all three patient groups, whereas percent hK2/PSA-F and hK2 x PSA-T/PSA-F levels were significantly higher in cancer patients compared with those with BPH. In the discrimination of clinically localized PCa from BPH, hK2 x PSA-T/PSA-F gave the largest area under the receiver operating curve (AUC = 0.81) and significantly (p = 0.025) larger AUC than PSA-T alone (0.70). Further, at 95% sensitivity there was significant gain in specificity, and at specificity levels of 90 to 95% there was significant gain in sensitivity using the measurements of PSA-T+PSA-F+hK2 compared with analysis of PSA-T and/or percent free PSA., Conclusions: Discrimination of patients with benign prostate disease from those with prostate cancer was significantly enhanced using measurements of hK2 in addition to those of PSA-T and PSA-F. Percent hK2/PSA-F was higher in PCa than in BPH, a phenomena not yet understood.

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 microgram.mL-1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.

Purpose: We studied plasma concentrations and elimination rates of prostate specific antigen (PSA) complexed to alpha1-antichymotrypsin and alpha2-macroglobulin, free PSA, total PSA (free PSA plus PSA alpha1-antichymotrypsin) and human glandular kallikrein 2 before, during and after radical retropubic prostatectomy for clinically localized prostate cancer., Materials and Methods: Plasma was collected and frozen within 10 minutes after sampling from 18 patients undergoing radical retropubic prostatectomy for prostate cancer. One sample was drawn preoperatively. Subsequent sampling intervals were 5 to 20 minutes perioperatively, 2 to 4 hours during the first 12 postoperative hours and 24 to 48 hours until postoperative day 14. Free PSA, PSA alpha1-antichymotrypsin, total PSA, PSA alpha2-macroglobulin and human glandular kallikrein 2 were measured with time resolved immunofluorometric assays., Results: Preoperatively PSA alpha2-macroglobulin was undetectable (less than 2 ng./ml.) in 17 of 18 patients. Human glandular kallikrein 2, free PSA and total PSA but not PSA alpha1-antichymotrypsin were significantly higher in patients with extraprostatic cancer (pT3a-pT4a, pN1) compared to those with organ confined cancer (pT2a/b). Surgical manipulation of the prostate caused no detectable elevation of human glandular kallikrein 2, PSA alpha1-antichymotrypsin or PSA alpha2-macroglobulin. In contrast, a mean 9.6-fold increase (range 3.4 to 22) in free PSA was noted 5 minutes after prostatectomy. Free PSA was eliminated from plasma in a biphasic exponential pattern with an early plasma half-life of 55 minutes and a late plasma half-life of 18 hours. PSA alpha1-antichymotrypsin decreased slowly, whereas human glandular kallikrein 2 was detectable only 12 hours after prostatectomy. PSA alpha2-macroglobulin remained at insignificant, nondetectable concentrations during the entire perioperative and postoperative period., Conclusions: Release of free PSA contributes to the elevation of plasma total PSA after prostatectomy. Free PSA is enzymatically inactive as the release does not result in subsequent elevation of PSA alpha1-antichymotrypsin or PSA alpha2-macroglobulin. Biphasic exponential elimination of free PSA may be explained by rapid extracellular redistribution (early half-life) and glomerular filtration in the kidneys (late half-life). Our data suggest rapid metabolism of human glandular kallikrein 2 but do not support suggestions of the significance in vivo of complex formations with alpha2-macroglobulin as a major means to eliminate PSA from plasma in patients with clinically localized prostate cancer.

Seventy-nine monoclonal antibodies submitted to the ISOBM TD-3 PSA Workshop were tested for their reactivity with recombinant human kallikrein-2 (rhK2). A sandwich immunofluorometric assay using polyclonal anti-prostate-specific antigen (PSA) antiserum-coated plates was used to capture rhK2 and subsequently the test antibody. The response of each test antibody was compared with 3 reference antibodies (H50, H117 and 5E4) known to react with hK2. Nine antibodies from the workshop panel failed to react with purified PSA and rhK2 in this assay and were subsequently excluded. From the remaining panel of antibodies, 11/70 showed strong reactivity with rhK2, 9/70 showed weak reactivity with rhK2, while 50/70 antibodies did not react with rhK2 in this assay format. All antibodies binding to rhK2 recognized both free and complexed PSA.