Kwok K. Yeung

MEASUREMENT OF COMPLEXED PSA IMPROVES SPECIFICITY FOR EARLY DETECTION OF PROSTATE CANCER

OBJECTIVES Prostate-specific antigen (PSA) is the most useful of all tumor markers. Although the sensitivity is impressive, low specificity results in a lack of cancer detection in a significant proportion of patients undergoing prostate biopsy. Several recent studies have addressed the need for improved specificity. Of all these approaches, the free/total PSA ratio appears to be the most promising. Given that most circulating PSA is complexed to alpha1-antichymotrypsin, and that this moiety represents a greater proportion of the total PSA in those men with carcinoma, we set out to determine whether complexed PSA would improve specificity in the detection of men with prostate cancer. METHODS Archival sera were obtained from 300 men, 75 of whom had biopsy-proved prostate cancer. All sera had been previously stored at -70 degrees C for variable periods. An investigative assay for complexed PSA (Bayer) was used. The Tandem-R free and total PSA assays (Hybritech) were used according to the manufacturers recommendations. RESULTS Among all patients, specificities for the total PSA, free/total PSA, and complexed PSA alone were 21.8%, 15.6%, and 26.7%, respectively, at cutoffs yielding 95% sensitivity. Similar equivalence or superior performance, in terms of specificity relative to the free/total PSA ratio, was seen at other sensitivity thresholds and other total PSA ranges. CONCLUSIONS Complexed PSA alone performs better than total PSA or the free/total PSA ratio and obviates the need for a second analyte determination. We believe this marker may offer significant enhancement in PSA testing with significant economic advantages.

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Measurement of four tumor marker antigens in the sera of pregnant women

We sought to determine the maternal serum levels of four tumor‐associated antigens during the three trimesters of pregnancy in healthy women. CEA, CA 228, CA 15‐3, and Her2/neu oncogene product p105 assay values were determined for 90 healthy pregnant women during the three trimesters of pregnancy at five participating evaluation sites. Results were compared to means and cut‐off values determined for healthy nonpregnant women. Differences in assay values in the 1st and 3rd trimester were analyzed for statistical significance (Students t‐test). CEA, CA 228 and CA 15‐3 assay values in general were found to be within the normal range. CA 15‐3 and Her2/neu p105 serum assay values were above the cut‐off (3.3% and 8.2%, respectively) and were significantly elevated in the 3rd trimester as compared to the 1st trimester of pregnancy (P < 0.05 and P < 0.001, respectively). CEA and CA 228 may be of potential value in monitoring pregnant women with malignant disease. Normal elevations in 3rd trimester serum Her2/neu p105 and CA 15‐3 assay values should be considered when monitoring a pregnant patient with malignant disease. J. Clin. Lab. Anal. 13:35–39, 1999.

Effect of Desialylation on Binding, Affinity, and Specificity of 56 Monoclonal Antibodies against MUC1 Mucin

We evaluated 56 monoclonal antibodies (MAbs), submitted to the ISOBM TD-4 Workshop, for changes in binding following desialylation of the MUC1 molecule and for epitope specificity. Antibody binding of MAbs was assayed by an ELISA method using microtiter plates coated with the MUC1 mucin obtained from supernatants of the ZR75-1 cell line. The MUC1 mucin was desialylated directly on the plate by treatment with neuraminidase. For each MAb, binding to untreated mucin was compared over a range of antibody concentrations. The concentration at which binding was half-maximal (K50) was determined for all antibodies whose binding reached saturation in the assay. Results showed that K50 values for MAb binding to untreated MUC1 mucin varied from 10–10 to 10–6 M. These data suggest that MAbs to MUC1 mucin bind with a broad range of intrinsic affinities. Desialylation was found to have variable effects on antibody binding, in that binding was either increased, decreased, or unchanged. No relationship was found between the apparent affinities for untreated mucin and changes in binding following desialylation. Among the 56 Workshop MAbs, 33 were found reactive with synthetic peptides which mimic the MUC1 tandem repeat. We determined the epitope specificity of the 33 MAbs by competitive binding using 10 amino acid peptides corresponding to various regions of the 20-amino acid tandem repeat domain of MUC1. All antibodies which recognized epitopes in the 1-10 amino acid region of the tandem repeat showed increased binding to desialylated mucin. Antibodies to other peptide epitopes showed no consistent pattern of change in binding following desialylation. Our results suggest that sialic acid residues on the MUC1 mucin may contribute either positively or negatively to antibody binding. In addition, our results suggest that improved antibody selection methods could provide MAbs with improved selectivity for cancer-derived mucin compared with mucin from normal tissues. This could form the basis of improved biomarker assays for breast cancer.

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1™ complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1™ PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10–day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02μg/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71–86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1–100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.

Technicon Immuno 1 PSA assay measures both free and alpha-1-antichymotrypsin-complexed prostate-specific antigen on an equimolar basis.

In this study, we investigated the immunoreactivity of the Technicon Immuno 1® PSA assay, a monoclonal‐polyclonal sandwich immunoassay, with free and ACT‐complexed PSA. Assay calibrators prepared with free PSA (standard immuno 1 calibrators) and calibrators consisting of 90% ACT‐complexed and 10% free PSA (90:10 calibrators) yielded virtually identical PSA recoveries at all concentrations tested. Concentrations of total PSA at ∼4 and 10 ng/mL, prepared in varying ratios of free PSA to PSA‐ACT complex, recovered from 92–104% at 4 ng/mL and from 98–102% at 10 ng/mL. Additionally, an excellent correlation of serum total PSA values from a panel of 40 prostatic cancer patient samples was obtained using calibration curves generated with the standard Immuno 1 calibrators or the 90:10 calibrators. These results demonstrate that the Technicon Immuno 1® PSA assay measures free and ACT‐complexed PSA on an equal molar basis.

Equivalent recognition of free and act-complexed PSA in a monoclonal-polyclonal sandwich assay is conferred by binding specificity of the monoclonal antibody

The Bayer Immuno 1TM PSA Assay measures free and ACT‐complexed PSA on an equimolar basis, although it uses a monoclonal antibody (MM1) for capture and polyclonal antibodies for detection. Competitive inhibition studies using antibodies directed at various epitopes on PSA and PSA‐ACT demonstrated that the capture antibody, MM1, does not bind to free PSA simultaneously with antibodies against Epitope E which is exposed only in free PSA. Affinity studies showed that the affinity constants of MM1 for both free PSA and PSA‐ACT are similar. One explanation for the properties of MM1 is that it precludes the binding of antibodies to Epitope E due to steric hindrance. Alternatively, the binding of MM1 causes a conformation change within the free PSA molecule, so that Epitope E is altered in a way that causes a loss of binding affinity. The unusual properties of MM1 are responsible for the equimolar response of this monoclonal‐polyclonal sandwich assay for free and ACT‐complexed PSA. J. Clin. Lab. Anal. 12:242–249, 1998.

BAYER IMMUNO 1 PSA ASSAY : AN AUTOMATED, ULTRASENSITIVE METHOD TO QUANTITATE TOTAL PSA IN SERUM

The Bayer Immuno 1™ PSA Assay measures total PSA in human serum and demonstrates excellent performance with an interassay CV ≤ 3.4% and a biological detection limit of 0.03 μg/L. No significant interference from common hormonal and chemotherapeutic drugs, kallikrein, prostatic acid phosphatase, and trypsin, or elevated levels of total bilirubin, hemoglobin, triglycerides, and IgG was observed. The 95th percentile values for healthy individuals increased with age from 3.0 μg/L for males 50–59 years and 3.3 μg/L for males 60–69 years, to 4.6 μg/L for males ≥ 70 years. Clinical studies with retrospective samples demonstrated correspondence between serial measurements of PSA and clinical outcome for 98% of 159 prostate cancer patients. Clinical sensitivity for patients with clinical evidence of disease, untreated at the time of specimen draw, increased with increasing stage from 77.5–100%. Specificity of 60–70% for BPH and other benign urogenital diseases was consistent with previous findings. Bayer Immuno 1 PSA Assay values for 2131 specimens from healthy subjects and patients with prostate cancer, BPH, and other malignant and nonmalignant diseases correlated well with the Abbott IMx® PSA Assay over the range 0.0–6,238 μg/L (Y = 1.10 × + 0.02). The Bayer Immuno 1 PSA Assay provides automated ultrasensitive, precise, and equimolar measurement of total PSA in human serum. J. Clin. Lab. Anal. 12:65–74, 1998.

Analysis of Monoclonal Antibodies to Prostate-Specific Antigen: Reactivity with Native and Recombinant Prostate-Specific Antigen

Epitope mapping analysis was performed on 53 antibodies submitted to the ISOBM TD-3 PSA Workshop. Western blotting and N-terminal amino acid sequencing, using both native and recombinant human prostate-specific antigen (rPSA), identified four different epitope groups for native PSA. Under reducing conditions native PSA was not recognized by 18/53 antibodies suggesting they reacted with conformation-dependent epitopes. Nine other antibodies reacted with a rPSA polypeptide doublet of 34–35 kD corresponding to different rPSA glycoforms. From sequence mapping studies 22/53 antibodies bound epitopes within amino acid residues 25–85, 3/53 antibodies bound to epitopes within residues 86–220, while 10/53 antibodies bound epitopes within residues 221–261. These results indicate that there are multiple immunogenic epitopes localized on the PSA molecule.