Assays for PSA-Α1-Antichymotrypsin Complex May Soon Stand Alone In

VOLUME 8 NUMBER 1 SUMMER 1994 A publication of SCRIPPS LABORATORIES, INC.

Assays for

PSA-a 1-Antichymotrypsin Complex May Soon Stand Alone in the Diagnosis of Prostate Cancer

PSA-ACT Complex This new marker for prostate cancer exhibits what serum assays for PSA lack: the PSA assays fail to differentiate prostate cancer from benign prostatic hyperplasia ability to differentiate Since 1981, the number of reported frequently, only extreme elevations of PSA prostate cancer from benign prostate cancer cases in the US has increased (>50µg/L) are specific for prostate cancer.6 prostatic hyperplasia. by 50%, and the number of related deaths by As a result, difficulty arises when distin- 40%. Approximately 130,000 cases are guishing prostate cancer from BPH in reported each year, resulting in over 30,000 patients with serum PSA levels in the 4- annual deaths.1 As the population ages and 20µg/L range. PSA’s lack of specificity for average life-span lengthens, these numbers prostate cancer can cause the BPH patient are expected to increase. As such, early diag- unnecessary anxiety and even unnecessary nosis and effective treatment of prostate can- surgery. cer are increasingly important. One of the solutions to PSA’s low speci- To date, Prostate Specific Antigen (PSA) ficity for prostate cancer, as presented by Dr. is the most effective marker for prostate can- W u3, is a relatively new discovery. Recent cer. Serum assays for PSA are used for early studies suggest the existence of a potentially detection of prostate cancer, monitoring dis- better serum marker for prostate cancer, ease progress, evaluation of therapy, and P S A -a 1-Antichymotrypsin Complex (PSA- detecting early recurrence of disease after ACT). In a report published in November radical prostatectomy.2 , 3 1993, measuring serum levels of PSA-ACT The goal of PSA serum assays is to detect improved the ability of PSA measurements to prostate cancer prior to metastasis, that is, differentiate prostate cancer from BPH. while still in the organ-confined state. Assay specificity increased from 55% to Detecting prostate cancer localized to the 73%, at a sensitivity of 90%. More impor- prostate gland is paramount for reliable surgi- tantly, specificity in the critical 4-20µg/L cal excision. PSA’s ability to do this has range was also significantly increased, with recently come into question. An excellent no loss of sensitivity.7 review of serum assays for PSA is presented PSA is a 33-34 kD glycoprotein with chy- by JT Wu, PhD3; in it, PSA’s short-comings motrypsin-like protease activity.8 N o r m a l l y , are discussed and solutions proposed. very low concentrations of PSA are released What has distinguished PSA from other into the bloodstream, approximately 0- cancer markers is its tissue-specificity; PSA 4ng/mL. This enzymatically active form of is produced only in the prostate epithelium, PSA complexes with the serum protease making it arguably the best serum marker inhibitor a 1-antichymotrypsin (ACT) and 3 available today for any type of cancer. represents the predominant form of PSA in Although PSA is tissue-specific, it is not, s e r u m .6 , 9 PSA also forms a complex with however, cancer-specific. Serum levels of the enzyme inhibitor a - m a c r o g l o b u l i n PSA are elevated in both prostate cancer and 2 (A M) in serum, but to a much lesser degree. benign prostatic hyperplasia (BPH).4 , 5 A n d 2 8-04 PSA-ACT Complex

In addition, a significant percentage of PSA has a very low detection limit of 0.002µg/L; with other diagnostic procedures, like digital is present unbound to any inhibitor in serum. this should prove useful in the detection of rectal exam and transrectal ultrasonography, This form of PSA possesses no enzymatic residual disease after radical prostatectomy. will considerably reduce the morbidity and activity and, thus, does not interact with The second report also describes a mortality of prostate cancer. 1 5 serum protease inhibitors like ACT and A2M. mono:poly immunofluorometric assay. Recently, the different complexes of PSA What makes this assay unusual, however, is with various serum protease inhibitors have that it concurrently measures free PSA and References been identified and characterized. 9 , 1 0 , 1 1 PSA-ACT, providing separate serum levels Although the reported percentage of each for each analyte. This greatly simplifies the 1. Crawford ED, DeAntoni EP. PSA as a Screening determination of the percentage of PSA-ACT Test for Prostate Cancer. Urol Clin of N Amer, PSA-complex varies from study to study, a 1993, 20(4): 637-646. 12 to total PSA in the serum of prostate cancer table presented by EP Diamandis, MD PhD 2. Bjartel A, Björk T, Matikainen MT, et al. patients. In fact, the authors found this assay nicely summarizes the various forms of PSA Production of a 1-Antichymotrypsin by PSA- (see Table 1). to reduce the frequency of false-positive Containing Cells of Human Prostate Epithelium. Urology, 1993, 42(5): 502-510. These studies led to the discovery that the results due to BPH by 38%. 3. Wu JT. Assay for Prostate Specific Antigen (PSA): ratio of PSA-ACT to total PSA increases in An issue that remains to be resolved is the Problems and Possible Solutions. J Clin Lab Anal, the serum of prostate cancer patients and that calibration of the PSA-ACT assay. An assay 1994, 8: 51-62. the percentage of free, unbound PSA decreas- standard containing the appropriate ratio of 4. Drago JR, Badalament RA, Wientjes MG, et al. PSA-ACT to free PSA is inherently complex. Relative Value of Prostate-Specific Antigen and es in prostate cancer. That these ratios Prostatic Acid Phosphatase in Diagnosis and remain normal in the serum of BPH patients One must take into account the normal serum Management of Adenocarcinoma of Prostate. is of particular clinical importance. In a ratio of PSA-ACT to free PSA, as well as any Urology, 1989, 34: 187. study by Stenman, et al1 0, prostate cancer interference presented by other PSA- 5. Catalona WJ, Smith DS, Ratlief TL, et al. 1 3 Measurement of Prostate-Specific Antigen in patients had a significantly higher proportion inhibitors like A2M. Zhou, et al d e s c r i b e Serum as a Screening Test for Prostate Cancer. NE of PSA-ACT than those patients with BPH. yet another sensitive mono/poly immunoas- J Med, 1991, 324: 1156. In a related experiment, Christensson, et al6 say for PSA-ACT that employs calibrators 6. Christensson A, Björk T, Nilsson O, et al. Serum Prostate Specific Antigen Complexed to a 1- measured the proportion of free PSA to total prepared by adding purified PSA to female Antichymotrypsin as an Indicator of Prostate PSA and found the ratio to be 36% lower in serum that had been treated with immobilized Cancer. J Urol, 1993, 150: 100-105. prostate cancer than in BPH. Although the antibody to A2M. Using such a calibrator, 7. Lilja H. Significance of Different Molecular Forms mechanism is not yet understood, both stud- of Serum PSA. Urol Clin of N Amer, 1993, 20(4): they reported a recovery of >98% of PSA 681-686. ies indicate that prostate malignancies release values throughout the standard curve. In 8. Loor R, Watt KWK. Clinical Aspects of Prostate more enzymatically active PSA than other addition, the authors cite a December 1992 Cancer. Elsevier Science Publishing Co, New conditions. This leads to a higher percentage international standardization conference York, 1989: 147. of PSA-ACT in the serum of prostate cancer where it was recommended that PSA stan- 9. Lilja H, Christensson A, Dahlen U, et al. Prostate- patients. Specific Antigen in Serum Occurs Predominantly dards should include serum protease in Complex with a 1-Antichymotrypsin. Clin At present, commercially available PSA inhibitors so that patient sera is more accu- Chem, 1991, 37(9): 1618-1625. assays do not consistently recognize PSA- rately reproduced. 10. Stenman UH, Leinonen J, Alfthan H, et al. A ACT, nor do they give compatible PSA Given the evidence presented above, it Complex Between Prostate-Specific Antigen and a 1-Antichymo-trypsin is the Major Form of results from the same patient sample. Two seems undeniable that the determination of Prostate-Specific Antigen in Serum of Patients with excellent reviews summarize these issues, serum levels of PSA-ACT is invaluable in the Prostatic Cancer: Assay of the Complex Improves 3 , 13 Clinical Sensitivity for Cancer. Can Res, 1991, 51: which will not be discussed in this review. differential diagnosis of prostate cancer from 222-226. Two interesting reports were published in BPH. Most importantly, PSA-ACT determi- 11. Wood WG, van der Sloot E, Böhle A. The 1993 describing newly developed assays for nations have significantly greater specificity Establishment and Evaluation of Luminescent- PSA-ACT. One of the studies details a mon- Labelled Immunometric Assays for Prostate- for prostate cancer than serum PSA evalua- Specific Antigen-a 1-Antichymotrypsin oclonal-polyclonal antibody immunofluoro- tions alone. Optimistically, further study will Complexes in Serum. Eur J Clin Chem Clin Biochem, 1991, 29: 787. metric assay that equally recognizes both free show that PSA-ACT immunoassays, coupled PSA and PSA-ACT.14 This mono:poly assay 12. Diamandis EP. An Update on Prostate Specific Antigen. Clin Chem News, 1993, Oct: 18-19. 13. Zhou AM, Tewari PC, Bluestein BI, et al. Multiple Forms of Prostate-Specific Antigen in Serum: 12 Differences in Immunorecognition by Monoclonal Table 1. Molecular Forms of PSA in Human Serum and Polyclonal Assays. Clin Chem, 1993, 39(12): 2483-2491. Approximate 14. Yu H, Diamandis EP. Ultra-sensitive Time- Molecular Form Mol. Weight % of Total Comments Resolved Immuno-fluorometric Assay of Prostate- Specific Antigen in Serum and Preliminary Free PSA 30 kD 10-40 Percentage of free PSA Clinical Studies. Clin Chem, 1993, 39(10): 2109- decreases in the serum of 2114. 15. Leinonen J, Lövgren T, Vornanen T, et al. Double- cancer patients as total Label Time-Resolved Immunofluorometric Assay PSA increases of Prostate-Specific Antigen and of its Complex with a 1-Antichymotrypsin. Clin Chem, 1993, 39(10): 2099-2103 PSA-ACT 100 kD 60-90 Percentage of PSA-ACT complex increases as total PSA increases

PSA-a 1-proteinase 190 & 80 kD < 1 Present only when PSA inhibitor exceeds 40 mg/L

PSA-A2M 800 kD < 0.1 Not measured by commercial PSA kits

PSA-inter-a - 250 kD < 0.1 Not measured by trypsin inhibitor commercial PSA kits