Klee, George G.

Abstract Objective.—To determine if the levels of imprecision of the commonly used analytic methods for drug measurements are suitable for long-term therapeutic drug monitoring. Design.—In 1996, 4 identical lyophilized samples (2 in the first mailing and 2 in the second mailing 4 months later) were sent to laboratories participating in a nationwide proficiency testing program. Similarly, in 1999, replicates from a liquid pool of spiked sera were mailed 3 times, 4 months apart, to participating laboratories. For each of 11 drugs regulated under the Clinical Laboratory Improvement Amendments of 1988 and 1 metabolite, the total variance for each method was partitioned into within- and between-laboratory components. The total within-laboratory and the total survey coefficients of variation (CVs) for each method were then compared with the “acceptable” precision criteria of Glick, Burnett, and Fraser for each drug. Setting.—The first 2 mailings of the College of American Pathologists Therapeutic Drug Monitoring surveys for 1996, sets Z and ZM, and the 3 mailings of 1999, sets ZM, Z, and Z2. Main Outcome Measures.—For each drug studied, the CV of each method was compared with the various imprecision criteria, and if greater than any of the criteria, the method was then tabulated as not meeting that specific criterion. Participants.—The approximately 5000 participants of the survey. Results.—The number of methods deemed as not having acceptable total long-term within-laboratory precision by the various criteria ranged from 35% to 88% in 1996 and from 22% to 77% in 1999. Conclusion.—The number of failures possibly indicates that many of the reagent assays being utilized are not precise enough for long-term therapeutic drug monitoring of chronically administered drugs or that the published criteria used to evaluate the data in this study are too stringent.

Abstract Objective. —To determine the magnitudes and sources of analytic variation in testing for therapeutic drugs. Specifically, among laboratories using the same analytic method, to compare the within-laboratory variation (including both short- and long-term variation) with the between-laboratory variation. Design. —Four identical challenges were prepared from a lyophilized pool of spiked sera and were sent in pairs 4 months apart to laboratories participating in a nationwide proficiency-testing program. For each of 25 drugs, the variability in reported results from laboratories using the same method was investigated using nested analysis of variance. Setting. —The first 2 mailings of the College of American Pathologists Therapeutic Drug Monitoring Survey, 1996, sets Z and ZM. Main Outcome Measures. —For each drug, total variance was partitioned into within- and between-laboratory components for common methods. The within-laboratory component was further partitioned into short- and long-term components. Participants. —The approximately 5000 laboratories enrolled in the survey. Results. —For the 25 drugs, the average percentages of the total variance due to short-term, within-laboratory variance; long-term, within-laboratory variance; between-laboratory variance; and total laboratory variance were 25.0% (range, 8.8–50.6%), 57.8% (35.3–73.7%), 17.3% (5.0–35.4%), and 82.7% (64.6–95.0%), respectively. Conclusion. —For all drugs tested, the within-laboratory component of variance was greater than the between-laboratory component of variance. Within laboratories, the magnitude of the long-term component was generally greater than the magnitude of the short-term component. This information will be helpful in determining the clinical utility of various drug assays and in evaluating the appropriateness of regulations involving therapeutic drug testing.

Abstract Context. —Large disparities in prostate-specific antigen (PSA) results from different assays have been observed in the College of American Pathologists (CAP) Ligand Assay Survey, with interassay results varying severalfold. Survey specimens are predominately composed of free PSA and do not reflect the composition of typical patient specimens. Objectives. —To characterize a pilot material developed for CAP in which pooled sera samples were spiked with purified PSA and α 1 -antichymotrypsin–bound PSA at targeted concentrations and to compare it to CAP survey and reference materials. Design. —CAP survey, reference, and pilot materials were analyzed using 10 total PSA and 7 free PSA assays. These assays included Food and Drug Administration–approved assays and assays for research use only. Results. —Variability among the 10 total PSA methods was greatest for the 1997 ligand survey material (CV range, 56%–65%) followed by the pilot material (CV range, 10%–29%) and the reference material (CV range, 6%–13%). In contrast, interassay variability for the 7 free PSA methods was similar for the 3 preparations, with the exception of one specimen close to the limit of detection of the assays. As determined with the Hybritech Tandem-R method, the ligand survey specimens were essentially composed of all free PSA, whereas the reference and pilot materials were composed of approximately 10% and 35% free PSA, respectively. Conclusions. —The newly formulated pilot material prepared using a human base that contained defined concentrations of free PSA and α 1 -antichymotrypsin–bound PSA more closely resembled patient specimens and minimized differences among methods compared with the semen-supplemented original survey material.

Objectives To prospectively evaluate the performance of a pre‐specified statistical model based on four kallikrein markers in blood (total prostate‐specific antigen [PSA], free PSA, intact PSA, and human kallikrein‐related peptidase 2), commercially available as the 4Kscore, in predicting Gleason Grade Group (GG) ≥2 prostate cancer at biopsy in an international multicentre study at three academic medical centres, and whether microseminoprotein‐β (MSP) adds predictive value. Patients and Methods A total of 984 men were prospectively enrolled at three academic centres. The primary outcome was GG ≥2 on prostate biopsy. Three pre‐specified statistical models were used: a base model including PSA, age, digital rectal examination and prior negative biopsy; a model that added free PSA to the base model; and the 4Kscore. Results A total of 947 men were included in the final analysis and 273 (29%) had GG ≥2 on prostate biopsy. The base model area under the receiver operating characteristic curve of 0.775 increased to 0.802 with the addition of free PSA, and to 0.824 for the 4Kscore. Adding MSP to the 4Kscore model yielded an increase (0.014–0.019) in discrimination. In decision‐curve analysis of clinical utility, the 4Kscore showed a benefit starting at a 7.5% threshold. Conclusion A prospective multicentre evaluation of a pre‐specified model based on four kallikrein markers (4Kscore) with the addition of MSP improves the predictive discrimination for GG ≥2 prostate cancer on biopsy and could be used to inform biopsy decision‐making.

4565 Background: The efficient delivery of adjuvant therapy after radical prostatectomy (RP) in patients with prostate cancer is limited by the lack of biomarkers, beyond clinicopathologic factors, that are able to assess the risk of clinically significant disease progression. Previously, routine FFPE patient specimens from the Mayo Clinic Radical Prostatectomy Registry with long term follow-up were selected to develop a genomic classifier (GC) to predict clinical progression. Here, we present the validation of a GC in a cohort of patients at high risk of disease progression. Methods: A case-cohort study of high-risk RP patients from the Mayo Clinic (N=219) was used to validate the genomic classifier (GC) for predicting clinical progression (defined by positive bone or CT scan post-RP). Its performance was compared to a multivariable clinical classifier (CC) and a genomic-clinical classifier (GCC) which combines GC with established clinicopathologic variables. Concordance index, Cox modeling and decision curve analysis were used to compare the different models. Results: GC and GCC were predictive of clinical progression in the high-risk cohort with c-indices of 0.79 and 0.82, respectively, compared to the clinical classifier (0.70). Multivariable survival analysis showed that the majority of prognostic information of GCC came from the GC with a minor contribution from Gleason score. Decision curve analysis showed that GCC had a higher overall net benefit compared to CC over a wide range of ‘decision-to-treat’ thresholds for the risk of progression. Conclusions: In this high-risk cohort, GC and GCC classifiers showed improved performance over CC in prediction of clinical progression. GC is an independent prognostic factor in this cohort and captures the majority of prognostic information. GC and GCC’s prognostic performance and their usefulness in guiding decision-making in the adjuvant setting after RP need further testing in studies of additional prostate cancer risk groups.

5017 Background: The majority of men with prostate cancer are diagnosed with cancers with low mortality. Such men are treated with radical prostatectomy, external beam radiotherapy, or brachytherapy and followed by serum PSA evaluations. Some men with a rising PSA therapy will have local recurrence or metastasis, but many will have no other evidence of recurrent disease other than a rising PSA. The PSA doubling time has been used to determine which of these men deserve adjuvant hormonal ablation, radiation therapy, or observation. We hypothesize that additional biomarkers will predict which men with a rising PSA post-definitive therapy would benefit from additional therapy. Methods: We designed a custom array containing 526 RNA targets whose expression has been reported to be altered in association with prostate cancer progression. We included targets from Mayo Clinic prostate cancer research. Together with a second commercial array, 530 genes implicated in prostate cancer progression and 420 other cancer-related genes were evaluated. A case-control design was used to test the association of the expression results with outcome. Cases were men post-radical prostatectomy who developed systemic progression within 5 years after PSA recurrence (N=213). Controls were matched men post-radical prostatectomy with PSA recurrence but no evidence of clinical progression within 5 years (N=213). Results: Of 426 eligible patients, paraffin blocks were available on 418 (98.1%). RNA was prepared from all 418 blocks, and both arrays were both successful on 405 (96.9%) RNAs. Upon univariate analysis, 40 genes were highly significantly over- or under-expressed in the cases versus controls (1x10-22 < p < 1x10-7). Recursive partitioning (RP) selected 4 genes (TPX2, FAM13C1, TOPO2A and TSP2) that distinguished cases from controls. Random Forest analysis selected 24 genes (including 3 of the RP 4). A multivariable ROC analysis using these 24 genes generated an AUC of 0.80 (95% CI: 0.75–0.84). Conclusions: A specific gene expression pattern was significantly associated with systemic progression after PSA recurrence. The measurement of gene expression pattern may be useful for determining which men may benefit from additional therapy after PSA recurrence. No significant financial relationships to disclose.

AbstractBackground: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-α1-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease.Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions.Results: The minimum detectable concentration in the thK2 assay was 0.008 μg/L, and PSA cross-reactivity was &lt;0.001%. The assay detected prohK2 and three different hK2–serum protease complexes. The median serum concentration of thK2 in control samples (0.013 μg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 μg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at −70 °C.Conclusions: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

AbstractBackground: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to α1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results.Methods: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS® analyzer.Results: The detection limit of the assay was 1.5 ng/L, the “functional sensitivity” (day-to-day CV &lt;15%) was &lt;4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was &lt;7 ng/L and was &lt;15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations.Conclusion: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.

AbstractThe efficacy of endocrine tests depends on the choice of tests, the preparation of the patients, the integrity of the specimens, the quality of the measurements, and the validity of the reference data. Close dialogue among the clinicians, the laboratory, and the patients is a key factor for optimal patient care. The characteristics of urine and plasma samples and the advantages and limitations of paired test measurements are presented. The importance of test sequence strategies, provocative or inhibitory procedures, and elimination of drug interferences is illustrated with four cases involving Cushing syndrome, pheochromocytoma, primary aldosteronism, and hypercalcemia. For each of these scenarios, key clinical issues are highlighted, along with discussions of the best test strategies, including which medications are likely to interfere. The importance of targeting laboratory tests to answer well-focused clinical decisions is emphasized. The roles of some time-honored provocative procedures are questioned in light of more sensitive and specific analytic methods. The importance of decision-focused analytical tolerance limits is emphasized by demonstrating the impact of analytic bias on downstream medical resource utilization. User-friendly support systems to facilitate the implementation of test strategies and postanalytic tracking of patient outcomes are presented as essential requirements for quality medical practice.