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Technical Briefs |
1
Department of Clinical Chemistry, The Johns Hopkins University, 600 N. Wolfe St., Baltimore, MD 21287-7065;
2
Department of Urology Research, Washington University, 10130 Wohl Clinic, St. Louis, MO 63110;
3
Scott Department of Urology and Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030;
4
Department of Immunology, University of Washington Medical Center, Box 357110, Seattle, WA 98195;
5
Nuclear Medicine In Vitro Laboratory, Loyola Medical Center, 2160 S. First Ave., Maywood, IL 60153;
6
Department of Clinical Chemistry, UCLA Medical Center, 10833 LeConte Ave., Los Angeles, CA 90095;
7
Department of Clinical Chemistry, Brigham & Women's Hospital, 75 Francis St., Boston, MA 02115;
8
Department of Research and Development, Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., P.O. Box 269006, San Diego, CA 92196-9006;
a author for
correspondence: fax 619-536-8058, e-mail KGLoveland{at}Beckman.com
Prostate-specific antigen (PSA) is present in serum in several
forms, most importantly free PSA (FPSA) and PSA complexed to
1-antichymotrypsin
(1)(2). These PSA forms are useful in assessing
prostate disease (3)(4)(5). When PSA is >10 µg/L (ng/mL),
the probability of prostate cancer is 50% (6); when PSA is
between 4 and 10 µg/L, the probability of prostate cancer is 25%.
Patients in the latter range are usually recommended for biopsy, but
here the low specificity leads to many unnecessary biopsies. The
percentage of FPSA (%FPSA) is used to enhance specificity. The
relative proportion of FPSA in serum may range from 5% to 50%
(7), but a lower %FPSA is associated with higher
probability of prostate cancer (8).
Published guidelines for the clinical use of %FPSA have been contradictory because of differences in assay systems and standardization, study designs, patient populations, and the number of subjects enrolled (9). A large well-controlled multicenter clinical trial was conducted to define a medically significant %FPSA cutpoint that would indicate the need for prostate biopsy. This report summarizes the assay performance from the largest clinical trial to date evaluating %FPSA in a patient population representative of those men in whom the test would be used in clinical practice. The data formed the basis for the Food and Drug Administration's approval of Hybritech's free PSA assays. Detailed clinical performance characteristics have been presented previously (10).
The objective of this clinical trial was twofold: to evaluate the performance of Hybritech's Tandem®-R free PSA assay as it is routinely used in laboratories, and to identify a %FPSA cutpoint with a high rate of cancer detection (clinical sensitivity) while avoiding unnecessary biopsies in men without cancer (clinical specificity) when total PSA is between 4 and 10 µg/L.
Subjects were enrolled prospectively at seven university medical centers in accordance with practices and ethical standards of each site's Institutional Review Board and the Declaration of Helsinki. The subjects were men 50–75 years of age with a nonsuspicious digital rectal examination, a Tandem PSA result between 4 and 10 µg/L, and a histologically confirmed diagnosis from a six-sector ultrasound-guided needle biopsy.
Blood was drawn before prostate treatment or biopsy. Serum was separated from the clot within 3 h, stored at 4 °C, and tested within 24 h. PSA was assayed and reported in duplicate using Hybritech's Tandem-E PSA for photon ERA® instrumentation (immunoenzymetric monoclonal antibody assay) or Tandem-R PSA (immunoradiometric monoclonal antibody assay) reagent sets. Three laboratories used Tandem-E PSA and four laboratories used Tandem-R PSA. Serum was then stored at -70 °C. After meeting all enrollment criteria, the same serum was thawed and assayed at each site in duplicate using the Tandem-R free PSA (immunoradiometric monoclonal antibody) assay (11). FPSA assays included kit controls (~1.0 and 15 µg/L) and three serum pools (~0.3, 2.0, and 6.5 µg/L FPSA). In this blinded study, FPSA was not reported to the physician, and laboratory personnel did not know the diagnosis of the patient. The percentage of FPSA was calculated as (FPSA/PSA) x 100% = %FPSA.
Tandem (total) PSA and Tandem free PSA assays are manufactured in several formats at Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., San Diego, CA.
The lower limit of detection was calculated at each laboratory as the FPSA concentration corresponding to the signal 2 SD greater than the mean of 20 replicates of the zero calibrator. Results for the lower limit of detection at the seven sites were 0.03, 0.02, 0.04, 0.03, 0.02, 0.01, and 0.02 µg/L FPSA, and met the manufacturer's claim of 0.05 µg/L or less.
A proficiency panel prepared at Hybritech was used for interlaboratory method comparison. Each laboratory received identical sets of 43 test samples for analysis in duplicate using Tandem free PSA and total-PSA assays. Assessment of >720 results demonstrated that equivalent results, laboratory to laboratory, were obtained for both total and free PSA. The Levene test for homogeneity of variance showed no significant differences in dispersion among sites for FPSA (P = 0.20) or PSA (P = 0.98). A repeated measures ANOVA showed no significant differences across the sites for results of the proficiency samples (FPSA, P = 0.81; PSA, P = 0.99). Therefore, pooling of results across sites and the use of any Tandem PSA format was acceptable.
Three elements of precision and long-term reproducibility were determined:
).
|
is a graph of controls and pools and shows consistent and
reproducible recovery over time.
|
After successful completion of method validation procedures, each laboratory reported free- and total-PSA results on subjects meeting study enrollment criteria. ROC curves were generated using data from the 773 subjects enrolled in the trial (379 prostate cancer and 394 benign prostatic disease). A cutpoint of 25% FPSA was determined to yield a sensitivity of 95%. The use of this cutpoint (performing biopsies only in patients with values less than or equal to the cutpoint) would have avoided biopsies in 20% of men with benign disease.
The probability of cancer was determined from PSA and %FPSA results on these subjects with a biopsy-based diagnosis. For men with PSA between 4 and 10 µg/L, the risk of cancer is 25% (6)(12). %FPSA is useful to further stratify the probability of cancer in this group. For subjects with %FPSA values of 0–10%, 10–15%, 15–20%, 20–25%, and >25%, cancer was found on biopsy in 56%, 28%, 20%, 16%, and 8%, respectively.
Percentage of FPSA studies may be affected by numerous factors (9). One important factor is sample stability. The handling of samples in this study followed published data. PSA and FPSA are reported to be stable when the serum is separated from the clot within 3 h, stored at 4 °C, and tested within 24 h. Freezing and thawing serum does not affect recovery (13)(14). It is important to freeze serum promptly after pipetting to preserve FPSA integrity. Samples may be stored at -70 °C for up to 2 years (15).
PSA and %FPSA results from other manufacturers may provide values different from those obtained in this study. The use of assays from multiple manufactures or applying the cutpoint derived from one set of assays to that of another manufacturer can lead to erroneous clinical conclusions (5)(16)(17). Differences may be attributable to assay calibration, antibody specificity, or non-equimolarity of the total-PSA assay.
In summary, results from this large multicenter clinical trial show that %FPSA can be applied in two ways: (a) use of a single cutpoint, or (b) determination of individual risk for cancer. With the first approach, a single cutpoint of 25% is recommended for men 50–75 years of age with PSA results between 4 and 10 µg/L with benign findings in a digital rectal examination. Men with 25% FPSA or below would be recommended for biopsy. This approach would detect 95% of cancers (clinical sensitivity) and would spare 20% of men with benign disease from biopsy (clinical specificity). The second approach provides an individual patient's risk (probability of cancer). Risk would be stratified based on the %FPSA value when total PSA is between 4 and 10 µg/L. The risk of cancer is high (56%) when %FPSA is 0–10%, and the risk decreases as %FPSA increases. The risk of cancer is low (8%) when %FPSA is >25%. This approach allows the physician and patient to discuss individualized management options.
References
1-antichymotrypsin. Clin Chem 1991;37:1618-1625.
1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.
1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.
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