Calculations for Adjusting Endogenous Biomarker Levels During Analytical Recovery Assessments for Ligand-Binding Assay Bioanalytical Method Validation.

It is often necessary to adjust for detectable endogenous biomarker levels in spiked validation samples (VS) and in selectivity determinations during bioanalytical method validation for ligand-binding assays (LBA) with a matrix like normal human serum (NHS). Described herein are case studies of biomarker analyses using multiplex LBA which highlight the challenges associated with such adjustments when calculating percent analytical recovery (%AR). The LBA test methods were the Meso Scale Discovery V-PLEX® proinflammatory and cytokine panels with NHS as test matrix. The NHS matrix blank exhibited varied endogenous content of the 20 individual cytokines before spiking, ranging from undetectable to readily quantifiable. Addition and subtraction methods for adjusting endogenous cytokine levels in %AR calculations are both used in the bioanalytical field. The two methods were compared in %AR calculations following spiking and analysis of VS for cytokines having detectable endogenous levels in NHS. Calculations for %AR obtained by subtracting quantifiable endogenous biomarker concentrations from the respective total analytical VS values yielded reproducible and credible conclusions. The addition method, in contrast, yielded %AR conclusions that were frequently unreliable and discordant with values obtained with the subtraction adjustment method. It is shown that subtraction of assay signal attributable to matrix is a feasible alternative when endogenous biomarkers levels are below the limit of quantitation, but above the limit of detection. These analyses confirm that the subtraction method is preferable over that using addition to adjust for detectable endogenous biomarker levels when calculating %AR for biomarker LBA.

Serum pro prostate specific antigen improves cancer detection compared to free and complexed prostate specific antigen in men with prostate specific antigen 2 to 4 ng/ml.

PURPOSE: Pro prostate specific antigen (pPSA) is a precursor form of PSA enriched in tumor compared to benign prostate tissues that may be a more specific serum marker for prostate cancer. Serum pPSA was measured in the clinically relevant early detection PSA range of 2 to 10 ng/ml. MATERIALS AND METHODS: Research use immunoassays were used to measure native and truncated forms of pPSA. The subject cohort contained 1,091 serum specimens from men enrolled in prostate cancer screening studies at 2 sites who had undergone prostate biopsy and were divided into PSA ranges of 2 to 4 ng/ml (benign 320, cancer 235) and 4 to 10 ng/ml (benign 315, cancer 221). RESULTS: In PSA ranges 2 to 4, 2 to 6, 4 to 10 and 2 to 10 ng/ml, pPSA in a ratio with free PSA (%pPSA) gave the highest cancer specificity. At 2 to 4 ng/ml and 90% sensitivity, %pPSA spared 19% of unnecessary biopsies compared to 10% for free PSA and 11% for complexed PSA(p <0.001). Similar results were obtained at PSA 2 to 6 ng/ml. At 90% sensitivity in the PSA 4 to 10 ng/ml range, %pPSA spared 31% of unnecessary biopsies compared to 20% for % free PSA and 19% for complexed PSA (p <0.0001). In the combined 2 to 10 ng/ml range, %pPSA spared 21% of unnecessary biopsies compared to 13% for % free PSA and 9% for complexed PSA (p <0.0001). CONCLUSIONS: The %pPSA significantly improved specificity for cancer detection and decreased the number of unnecessary biopsies in the PSA 2 to 4 ng/ml range. This relative improvement of %pPSA compared to % free PSA and complexed PSA was maintained throughout the PSA range of 2 to 10 ng/ml.

Proenzyme forms of prostate-specific antigen in serum improve the detection of prostate cancer.

INTRODUCTION: Pro or precursor forms of prostate-specific antigen (PSA) have emerged as potentially important diagnostic serum markers for prostate cancer detection. Immunoassays were developed to measure specific proPSA forms containing propeptides of 2, 4, and 7 amino acids [(-2)proPSA, (-4)proPSA, and (-7)proPSA, respectively]. METHODS: Research-use dual monoclonal antibody immunoassays using europium-labeled detection monoclonal antibodies were developed for each form of proPSA. Sera from patients with prostate cancer or benign prostate disease containing 4-10 microg/L PSA were assayed and analyzed by area under the ROC curve (AUC) for specificity and sensitivity. RESULTS: The proPSA forms had quantification limits of 0.015-0.025 microg/L in serum, with cross-reactivities <1% with PSA. The sum of the proPSA forms divided by free PSA (percentage proPSA) had a higher AUC than did percentage of (-2)proPSA, free PSA, and complexed PSA with AUC (95% confidence intervals) of 0.69 (0.64-0.74), 0.64 (0.58-0.68), 0.63 (0.58-0.68), and 0.57 (0.51-0.62), respectively. The proPSA comprised a median of 33% of the free PSA in cancer and 25% in noncancer sera (P <0.0001). One-third (33%) of cancer samples had >40% proPSA, whereas only 8% of noncancer samples did (P <0.0001). In men with cancer and >25% free PSA, the (-2)proPSA had an AUC of 0.77 (0.66-0.86), with 90% sensitivity and 36% specificity at 0.04 microg/L. CONCLUSIONS: The percentage of proPSA gave better cancer detection in the 4-10 microg/L range than did percentage of free PSA and complexed PSA. (-2)proPSA significantly discriminated cancer in men whose serum had >25% free PSA, for whom there is currently no good marker for cancer detection.

Serum pro-prostate specific antigen preferentially detects aggressive prostate cancers in men with 2 to 4 ng/ml prostate specific antigen.

PURPOSE: Pro forms of prostate specific antigen (PSA) have been reported to be more cancer specific markers of prostate cancer than total PSA and they also may preferentially detect the more aggressive forms of the disease. MATERIALS AND METHODS: Research immunoassays with high specificity for pro-PSA forms were used to study 1091 retrospective serum specimens, including 555 with 2 to 4 and 536 with 4 to 10 ng/ml PSA, from men enrolled in prostate cancer screening studies who underwent prostate biopsy. RESULTS: In the 2 to 4 ng/ml PSA range the ratio of pro- to free-PSA (percent pro-PSA) using a cutoff of 1.8% for recommending prostate biopsy detected 90% of cancers, including 16 of 16 extracapsular tumors and 28 of 29 tumors with a pathology Gleason score of 7 or greater, while avoiding 19% of unnecessary biopsies. Serum percent pro-PSA was significantly increased for Gleason score 7 or greater vs less than 7 (p = 0.0018). In the PSA range of 4 to 10 ng/ml percent pro-PSA had the highest cancer specificity, avoiding 31% of unnecessary biopsies, while detecting 34 of 35 cancers with a pathology Gleason score of 7 or greater and 29 of 31 extracapsular tumors. Neither percent free PSA nor complexed PSA enhanced the detection of aggressive cancers in the 4 to 10 ng/ml PSA range. CONCLUSIONS: Percent pro-PSA was superior to percent free and calculated complexed PSA for the detection of prostate cancer in the PSA range of 2 to 10 ng/ml and it had selectivity for detecting more aggressive cancers, as indicated by Gleason score 7 or greater and/or extracapsular tumor extension.

Clinical utility of proPSA and "benign" PSA when percent free PSA is less than 15%.

OBJECTIVES: To investigate the clinical utility of the subforms of free prostate-specific antigen (PSA), namely proPSA and "benign" PSA (BPSA), to improve cancer detection when the percent free PSA level is less than 15%. Percent free PSA, while maintaining sensitivity, has greatly improved the specificity of PSA for the early detection of prostate cancer. A low percent free PSA value indicates a greater risk of cancer, but only 30% to 50% of men with percent free PSA levels of less than 15% actually have cancer at biopsy. METHODS: Archived sera from 161 consecutive men who were prospectively enrolled in our Early Detection Research Network prostate cancer early detection biomarker program with a percent free PSA value of less than 15% were included in the study. Total PSA, free PSA, proPSA, and BPSA were measured for each sample. RESULTS: The mean total PSA was 6.1 ng/mL (range 1.8 to 24.0). The mean age of the study group was 62 +/- 7 years. Prostate cancer was detected in 66 (41%) of 161 men. The area under the curve-receiver operating characteristic for total and percent free PSA was 0.51 and 0.54, respectively. BPSA and proPSA/BPSA both improved cancer detection compared with percent free PSA alone; the improvement was statistically significant (P <0.001) . The area under the curve-receiver operating characteristic for proPSA/BPSA was 0.72, giving a sensitivity and specificity of 90% and 46%, respectively. CONCLUSIONS: The results of our preliminary studies have suggested that the ratio of proPSA and BPSA can distinguish cancer with greater accuracy when the percent free PSA value is very low (less than 15%), and may, therefore, provide better clinical utility in this lower range of percent free PSA.

Proenzyme psa for the early detection of prostate cancer in the 2.5-4.0 ng/ml total psa range: preliminary analysis.

OBJECTIVES: To determine the clinical utility of using proenzyme prostate-specific antigen (pPSA) for early detection of prostate cancer in the 2.5 to 4.0 ng/mL total PSA range. pPSA, the precursor form of PSA that contains a 7 amino acid leader peptide, and truncated forms such as [-2]pPSA and [-4]pPSA can be measured in serum by research immunoassay. METHODS: Archival serum from 119 men (noncancer, 88; cancer, 31), obtained before biopsy and in the total PSA range of 2.5 to 4.0 ng/mL, were assayed for total PSA, free PSA (fPSA), and pPSA. pPSA was defined as the sum of the [-2], [-4], and [-7] forms, and the percent pPSA (%pPSA) was defined as pPSA/fPSA. RESULTS: pPSA averaged 4.6% +/- 0.4% (SEM) of total PSA and 39.3% +/- 3.5% of fPSA. PSA and %fPSA values were similar between the noncancer and cancer groups, and %pPSA tended to be higher in the cancer group (50.1% +/- 4.4%) compared with the noncancer group (35.5% +/- 6.7%; P = 0.07). Using receiver operating characteristic analysis to assess clinical utility, the area under the curve for %pPSA was 0.688 compared with 0.567 for %fPSA. At a fixed sensitivity of 75%, the specificity was significantly greater for %pPSA at 59% compared with %fPSA at 33% (P <0.0001). CONCLUSIONS: In the 2.5 to 4.0 ng/mL total PSA range, 75% of cancers can potentially be detected with 59% of unnecessary biopsies being spared using %pPSA; use of %fPSA would result in sparing only 33% of unnecessary biopsies. A large prospective clinical trial is needed to confirm these preliminary findings.