Judith A. Finlay

Human kallikrein 2 (hK2) and prostate-specific antigen (PSA): Two closely related, but distinct, kallikreins in the prostate

Recent studies on human kallikrein 2 (hK2) have revealed striking similarities and significant differences with the closely related kallikrein PSA. Both PSA and hK2 are primarily localized to the prostate and share close structural similarities. Although both kallikreins are produced by the same secretory epithelial cells in the prostate, hK2 is associated more with prostate tumors than PSA and is highly expressed in poorly differentiated cancer cells. The potent trypsin-like activity of hK2 contrasts with the weak chymotrypsin-like activity of PSA. The inactive precursor form of PSA, proPSA, is converted rapidly to active PSA by hK2, suggesting an important in vivo regulatory function by hK2 on PSA activity. The high homology between hK2 and PSA results in significant cross-reactivity to hK2 by polyclonal and some monoclonal antibodies to PSA. Future studies on both PSA and hK2 need to take into account this potential for cross-reactivity. Specific monoclonal antibodies to hK2 have now demonstrated that serum levels of hK2, like PSA, are correlated with prostate cancer. The production of hK2 protein in active protease form and specific monoclonal antibodies to the hK2 antigen will allow extensive future studies delineating the physiological and clinical utility of this new prostate antigen.

Use of human glandular kallikrein 2 for the detection of prostate cancer: Preliminary analysis

OBJECTIVES Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha2-macroglobulin and alpha1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. METHODS Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritechs Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). RESULTS The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. CONCLUSIONS In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.

Use of percentage of free prostate-specific antigen to identify men at high risk of prostate cancer when psa levels are 2.51 to 4 ng/mL and digital rectal examination is not suspicious for prostate cancer: an alternative model

OBJECTIVES Currently, many clinicians do not recommend prostate biopsy for men with digital rectal examination (DRE) results that are not suspicious for cancer and prostate-specific antigen (PSA) values between 2.51 and 4 ng/mL. We propose a new model for the detection of prostate cancer using the percentage of free PSA (%FPSA) in the limited range of PSA values between 2.51 and 4 ng/mL that maximizes clinical specificity (ie, minimizes false-positive results). This model identifies higher risk patients in this relatively low-risk population. METHODS Three hundred sixty-eight archived serum samples from men evaluated and treated at two academic institutions were reviewed. All men had a histologic diagnosis, findings not suspicious for cancer on DRE, and PSA levels between 2.51 and 4 ng/mL. Samples were tested in Hybritechs Tandem-R PSA and Tandem-R free PSA (FPSA) assays in the same laboratory at each institution. RESULTS Various models for cancer detection using %FPSA when PSA is 2.51 to 4 ng/mL and DRE is not suspicious for cancer are proposed. These models recommend biopsy for only 10% to 36% of the men in this population and would identify as many as 30% to 54% of the detectable cancers. There is evidence that the cancers that would be detected are the most aggressive cancers in this population. CONCLUSIONS Our models identified men with a higher risk of prostate cancer in a relatively low-risk population that currently does not routinely undergo biopsy. This may allow for a more cost-effective way to increase cancer detection when PSA values are between 2.51 and 4 ng/mL and DRE is not suspicious for cancer. This model has the potential to detect a greater number of clinically important and potentially curable cancers than would be detected with current practice.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

Molecular forms of prostate-specific antigen and human kallikrein 2 (hK2) in urine are not clinically useful for early detection and staging of prostate cancer

OBJECTIVES Prostate-specific antigen (PSA), a member of the human kallikrein (hK) family, is the most important tumor marker for early detection, staging, and monitoring of men with prostate cancer today. However, the sensitivity of serum PSA is not sufficient to be used alone for prostate cancer screening. Recently, it was reported that the serum-to-urinary total PSA ratio improves the detection of men with prostate cancer, especially in men with a serum total PSA level between 4.0 and 10.0 ng/mL. We tested this hypothesis by evaluating the clinical usefulness of this PSA ratio as well as the use of the different molecular forms of PSA and human kallikrein 2 (hK2) in urine for detection and staging of prostate cancer. METHODS One hundred ten fresh, midstream urine specimens (prostate cancer 62, benign prostatic hyperplasia [BPH] 38, healthy male control 5, women 5) were collected. Serum total PSA, urine total PSA, urinary free PSA, urinary alpha 1-antichymotrypsin-bound PSA, and urinary hK2 levels were determined by monoclonal antibody assays (Hybritech Inc.). The serum-to-urinary total PSA ratio was calculated. RESULTS The serum-to-urinary total PSA ratio did not accurately distinguish between men with BPH and men with prostate cancer. There was no significant difference between the urinary levels of any of the molecular forms of PSA or hK2 between men with prostate cancer and men with BPH. Among men with prostate cancer, neither urinary hK2 nor urinary levels of any of the molecular forms of PSA correlated with age, pathologic stage, or Gleason grade. CONCLUSIONS In our study, the serum-to-urinary total PSA ratio did not improve the detection of men with prostate cancer. Furthermore, measurement of the molecular forms of PSA and hK2 in urine did not improve the detection or staging of prostate cancer over serum PSA alone.

Standardization of Two Immunoassays for Human Glandular Kallikrein 2

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.

Polyclonal and monoclonal antibodies to prostate-specific antigen can cross-react with human kallikrein 2 and human kallikrein 1

OBJECTIVES The human tissue kallikrein family contains three closely related proteases: human kallikrein 1 (hK1), human kallikrein 2 (hK2), and prostate-specific antigen (PSA). The structural homology between these three proteins suggests potential cross-reactivity interference when different immunologic techniques are used. This study evaluated PSA and hK2 monoclonal antibody (mAb) and polyclonal antibody (pAb) reactivities to hK1, hK2, and PSA. METHODS mAbs and pAbs to hK2 and PSA were evaluated using Western blot analysis on hK1, hK2, PSA, and seminal plasma. RESULTS pAbs to PSA and hK2 recognized all three human kallikreins, as well as fragments of hK2 and PSA. An mAb with minimal (less than 0.4%) cross-reactivity between PSA and hK2 and a cross-reactive mAb were found. mAbs specific to PSA or hK2 did not cross-react with the less homologous hK1 protein. A PSA mAb raised specifically to PSA fragments recognized both PSA and hK2 but did not cross-react with hK1. pAbs to hK1 cross-reacted slightly with PSA and not at all with hK2. CONCLUSIONS Both pAbs and mAbs to hK2 and PSA may exhibit immunocross-reactivity. pAbs to PSA or hK2 react with all three human tissue kallikreins. The potential for cross-reactivity should be considered in any clinical or research procedures that use hK1, hK2, and PSA antibodies.

Comparison of Two Assays for Human Kallikrein 2

BACKGROUND We compared two recently developed research assays for the measurement of human kallikrein 2 (hK2) in serum: one fully automated assay (Beckman Coulter Access immunoanalyzer) and one manual assay based on the DELFIA technology. METHODS We used two subsets of clinical specimens consisting of 48 samples from prostate cancer patients and 210 samples from participants in an ongoing screening study (ERSPC). Both subsets were measured in the Rotterdam laboratory, and the prostate cancer samples were used for analytical comparison with the originating sites for the assays: Beckman Coulter Research Department (San Diego, CA) and Turku University (Turku, Finland). RESULTS Both the Beckman Coulter and the Turku assays performed very similarly between the Rotterdam laboratory and the originating sites: the R(2) value for both comparisons was 0.99, and the slope difference between sites was <20%. Deming regression analysis of the DELFIA (y) and Access (x) assays yielded the following: for the prostate cancer group, y = 1.17x - 0.01 (R(2) = 0.88; n = 48); and for the ERSPC group, y = 0.62x - 0.01 (R(2) = 0.77). Breakdown of the latter group into subgroups (nondiseased, benign prostatic hyperplasia, and prostate cancer samples) gave only minor differences. The Access calibrators were underrecovered by 13% in the DELFIA assay, whereas the DELFIA calibrators were overrecovered by 45% in the Access assay. CONCLUSION The DELFIA and Access assays for hK2, which have similar analytical features, show differences that cannot be explained by calibration.