Pro PSA: a more cancer specific form of prostate specific antigen for the early detection of prostate cancer

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1 REVIEW Pro PSA: a more cancer specific form of prostate specific antigen for the early detection of prostate cancer Stephen D. Mikolajczyk and Harry G. Rittenhouse Beckman Coulter, Inc., San Diego, CA, USA (Received for publication on December 16, 2002) Abstract. BPSA and propsa are distinct molecular forms of free PSA in serum. BPSA is a form of free PSA that is associated with BPH. The inactive precursor of PSA, propsa, is associated with prostate tumors. ProPSA is comprised of native propsa as well as truncated propsa forms, [C2]pPSA and [C4]pPSA, which have been shown to be more cancer-associated than native propsa. We have developed highly specific and sensitive research immunoassays for BPSA, and the different forms of propsa. Free PSA in prostate cancer serum contains a median of 28% BPSA and 32% propsa, though each form of PSA can range from 0 to more than 50% in individual samples. Early studies revealed that propsa significantly increases the specificity for prostate cancer, especially in the 2 4 ng/ml PSA range. It is estimated that 20 30% of men with PSA values from 2 4 ng/ml have prostate cancer. In the ng/ml PSA range propsa gave a receiver operating characteristic-area under the curve (ROC-AUC) of compared with free PSA (0.506) and complex PSA (0.509). At 90% sensitivity the specificity for propsa was 25% compared to 10% for %FPSA and cpsa (PFH0:001). ProPSA was superior to %FPSA and complexed PSA in the 4 10 ng/ml PSA range (AUC F 0:689, and 0.538, respectively). ProPSA represents a more cancer-specific form of PSA that better discriminates prostate cancer from BPH. (Keio J Med 52 (2): 86 91, June 2003) Key words: propsa, prostate cancer, prostate specific antigen, benign prostatic hyperplasia Introduction The detection and monitoring of prostate-specific proteins in the serum has revolutionized the management of prostate disease. The most widely used and successful serum marker is prostate-specific antigen (PSA). One reason for the success of this protein as a serum marker is that PSA is present in the prostate tissues mg per cc of tissue, but is almost undetectable in the serum of men with healthy prostates. The presence of elevated PSA in the serum is indicative of prostate trauma or prostate disease.1 PSA isolated from seminal plasma is a single chain glycoprotein with a polypeptide backbone of approximately 26 kda consisting of 237 amino acid residues.2 4 The true mass of PSAasdeterminedbymassspectrometry is approximately 28.4 kda, though it runs kda on SDS- PAGE.5 PSA is a serine protease with chymotrypsinlike hydrolytic activity towards the carboxy-terminal side of certain tyrosine and leucine residues.6 The biological function of PSA is believed to be liquifaction of the seminal clot formed by semenogelin I and II, and fibronectin in freshly ejaculated semen7 which aids in sperm motility and may have a role in fertility. In current medical practice men with serum PSA values above 10 ng/ml have greater than 50% chance of cancer.8 Men with PSA values between 4 10 ng/ml are considered candidates for prostate biopsy to confirm the presence of cancer. However, only 20 40% of the men within this range will be found to have prostate cancer.9 Traditionally men with PSA values below 4 ng/ml were not biopsied but in recent years it has been determined that 20 30% of men with 2 4 ng/ml serum PSA will also have prostate cancer.10,11 Therefore the current PSA range of interest for the early detection of cancer is from 2 10 ng/ml, with a growing emphasis on better cancer detection in the lower ranges from 2 6 ng/ml. Presented at the 1274th Meeting of the Keio Medical Society in Tokyo, September 17, Reprint requests to: Dr. Harry G. Rittenhouse, Beckman Coulter, Inc., 7730 Carroll Rd. San Diego, CA , USA, hgrittenhouse@beckman.com 86

2 Keio J Med 2003; 52 (2): Fig. 1 Representation of the Yin and Yang of PSA forms. BPSA is associated with benign prostatic hyperplasia (BPH), while ppsa is associated with prostate cancer. The primary shortcoming of PSA as a serum marker of prostate cancer is that PSA is found in the serum due to benign prostate diseases such as BPH and prostatitis. The improvements in PSA cancer detection have come from an understanding of the molecular forms of PSA in the serum. It was initially assumed that the PSA measured in the serum by the immunoassays for PSA was the kda form of the protein containing 237 amino acids. It was later discovered that PSA in the serum was largely covalently attached to the serum protease inhibitor alpha1-antichymotrypsin (ACT) and that only a relatively minor portion of the PSA was present as the non-complexed form.12,13 Thus, the terms free PSA (FPSA) and complexed PSA (cpsa) are used to distinguish non-complexed from complexed PSA. Immunoassays were developed to distinguish free and total PSA (free plus complexed PSA) and it was discovered that a lower ratio of FPSA correlates with a higher risk of prostate cancer.14,15 By contrast, a higher percentage of FPSA was correlated with benign disease such as benign prostatic hyperplasia (BPH). Many different PSA antibodies have been developed which can distinguish free PSA from cpsa and total PSA.16 Despite the success of FPSA in cancer detection, several limitations remain and better serum markers are needed. Because FPSA is the serum component that provides better cancer discrimination, investigations have focused on further discriminating FPSA into different molecular forms. This is because FPSA represents inactive PSA that cannot form a complex with ACT. Recent studies have confirmed the hypothesis that FPSA contains different molecular forms of inactivepsaandthattheseformsaremorehighlycorrelated with specific aspects of prostate disease than the total pool of FPSA. Different molecular forms of FPSA have been identified that are associated with either cancer or BPH.17,18 By contrast the cpsa is relatively homogeneous in serum,19 and is present in only trace amounts in benign or malignant prostate tissues.20 Therefore the study of cpsa appears to offer no inherent cancer-specificity beyond FPSA. In recent years the term molecular forms of PSA has expanded beyond free and complexed PSA to include the subforms of free PSA: BPSA and ppsa.17 BPSA is a form of free PSA that is identical to native mature PSA, contains 237 amino acids like PSA, but has 2 internal peptide bond cleavages at Lys182 and Lys BPH and prostate cancer tend to be localized within different regions or zones within the prostate: BPH typically is associated with nodular hyperplasia within the inner portion, or transition zone of the prostate, while cancers typically develop within the outer portion, or peripheral zone.25 BPSA is elevated in the prostate transition zone and is associated with pathologic BPH. A second form of PSA, the zymogen or propsa (ppsa), is associated with prostate tumor.26 Together these forms represent individual forms of free PSA that are more disease-specific than FPSA or cpsa. Immunoassays have been developed for BPSA and ppsa (Beckman Coulter, Inc., San Diego, CA, [For research use only, not for diagnostic procedures]). Because BPSA and ppsa are associated with opposing disease states in the prostate, cancer versus benign, they are sometime referred to as the yin and yang of PSA forms as shown in Fig. 1. ProPSA in Tissues and Serum The initial attempts to confirm the presence of ppsa in serum were unsuccessful and the investigators concluded that ppsa was not a significant component of serum PSA.27,28 Several subsequent studies have conclusively demonstrated the presence of ppsa forms in serum Like other serine proteases, PSA is translated as an inactive pre-pro-psa precursor. During passage through the secretory pathway, the signal peptide is released to yield the pro-psa. PSA is normally secreted from prostate luminal epithelialcellsasthepro formcontaininga7aminoacidn-terminalproleader peptide (APLILSR) in addition to the 237 aa of mature PSA.33,34 The ppsa in serum and tissues was found to be comprised of several truncated ppsa forms, containing from 1 to 5 amino acids in the pro leader peptide instead of the native 7 amino acids.26,29 31 The truncated ppsa forms containing pro leader peptides of 4 and 2 amino acids, [ 4]pPSA and [ 2]pPSA, respectively, are of particular interest (Table 1). One potential explanation for the enrichment of truncated ppsa forms is that the more truncated forms of ppsa are more resistant to activation than the intact ppsa.31 The truncated ppsa forms are therefore more stable

3 88 Mikolajczyk SD and Rittenhouse HG: Serum propsa improves cancer detection Table 1 Description of Native ppsa and the Cancer-associated Truncated Forms of ppsa. Native ppsa Contains a 7 Amino Acid Pro Leader Peptide. Immunoassays for the Truncated Forms of ppsa, [ 4]pPSA and [ 2]pPSA, Show Increased Cancer Specificity Over the Native ppsa 1) APLILSR fflfflfflfflfflfflffl{zfflfflfflfflfflfflffl} -PSA (native propsa, 244 amino acids33,34) ½ 7ŠpPSA 2) ILSR fflffl{zfflffl} -PSA (truncated propsa, 241 amino acids; the first ½ 4ŠpPSA form of ppsa identified in serum29) 3) SR-PSA (truncated propsa, 239 aa; cannot be activated; most associated with aggressive cancer31) {z} ½ 2ŠpPSA Fig. 2 Mean percentages of the free PSA forms in cancer serum containing 4 10 ng/ml PSA. In a sample set of 157 men with biopsyproven cancer, and PSA values from 4 10 ng/ml, the %FPSA represented 16% of the total PSA. BPSA represented 28% of the FPSA while ppsa comprised a mean of 33% of the FPSA. The remaining portion of FPSA is described as inpsa (inactive, intact PSA) since this form of FPSA is not ppsa and appears to be largely comprised of intact (not internally clipped) but inactive PSA that does not form a complex with serum proteins. The inpsa is calculated by subtracting BPSA and ppsa from FPSA. The proportions of the 3 forms of ppsa are shown. The assay for native ppsa, [ 7]pPSA also recognizes a slightly truncated form containing 5 amino acids, [ 5]pPSA, which is biochemically indistinguishable from [ 7]pPSA.31 The assay for these combined forms is indicated as [ 5/ 7]pPSA. as ppsa forms, and are not converted to mature PSA. Overall ppsa represents about a third of the FPSA in cancer serum, comprised of the three major ppsa sub-forms (Fig. 2). The [ 7]pPSA and a slightly truncated ppsa form, [ 5]pPSA are recognized equally by the assay for [ 7]pPSA and so ppsa forms recognized by this assay are designated as [ 5/ 7]pPSA. The [ 5]pPSA form cannot easily be distinguished from [ 7]pPSA by any of the analytical procedures employed. Both the [ 7]pPSA and [ 5]pPSA forms are rapidly activated by human kallikrein 2, hk2, and appear to have identical biochemical properties.31 In contrast, the [ 4]pPSA and [ 2]pPSA are resistant to hk2 activation and have distinct biochemical properties. BPSA in Rissues and Serum The PSA in BPH tissues has been shown to have higher level of internal peptide bond cleavages and is more enzymatically inactive.35 A study by Mikolajczyk et al. identified a distinct degraded form of PSA termed BPSA (Benign PSA) since it was present at much lower levels in cancer tissue from the same prostate.21 BPSA is highly correlated with the presence of BPH nodules in the prostate, the primary pathological feature of BPH. For this reason BPSA is thought to represent a more specific marker of the biochemical processes relatedtobph.bpsaischaracterized by 2 internal peptide bond cleavages at residues Lys145 and Lys182, and is enzymatically inactive. BPSA is not known to have any specific biological function, but rather is the result of post-translational proteolytic cleavage in the prostate tissues. BPSA is therefore a marker of BPH but is not known to play a biological role itself. BPSA comprises about 25% of the free PSA in biopsy-negative men with elevated PSA.24 Of more interest is that BPSA can range from non-detectable to greater than 50% of the free PSA. There is no specific correlation between the %FPSA and the percentage of BPSA (BPSA/FPSA), indicating that BPSA is not the same as FPSA, and is not clinically identical to FPSA. BPSA is distinct, and is more related to the biochemical properties of BPH and BPH nodules than FPSA. Serum BPSA alone would not be expected to provide specific discrimination of prostate cancer from BPH since prostate cancer and BPH frequently co-exist. However the relationship of BPSA (more BPH associated) and ppsa (more cancer associated) as shown in Fig. 1 indicates that the ratio or other mathematical relationship between BPSA and ppsa may provide additional insight into disease diagnosis. The more unique aspects of BPSA involve the possibility that BPSA could serve as a marker to monitor the progression or treatment of BPH. While the overall %FPSA is correlated with the presence of BPH instead of cancer, the %FPSA provides little specific insight into clinical BPH. Immunoassays for Different Forms of PSA Many different PSA assays can be used to measure serum PSA. However, there can be significant differences between different manufacturers, especially with regard to the measurement of free PSA.36,37 The Hybritech Tandem-E PSA assay is equimolar for free and complexed forms of PSA and is considered to be the gold standard for the measurement of serum PSA.37 The Access 2 Hybritech PSA assay (Beckman Coulter, Inc., Fullerton, CA) is the automated chemiluminescence version of the Tandem-E, and is equivalent and equimolar to the Tandem-E.37,38 The relationship between free, complexed and total PSA appears to be additive using different assays.39 That is, one can

4 Keio J Med 2003; 52 (2): calculate FPSA by subtracting cpsa from total PSA,40 and one can calculate cpsa by subtracting FPSA from total PSA.41 Complexed PSA can also be measured directly using the Immuno I cpsa assay (Bayer Corporation, Tarrytown, NY). There appears to be no clinical difference between the cancer detection using either calculated or directly measured FPSA or cpsa. One should, however, use the same assay to measure free and total PSA, or complexed and total PSA since different assays are calibrated differently and can also be skewed for different forms of PSA.36 The research immunoassays for the study of BPSA and the ppsa forms (Beckman Coulter, Inc. San Diego, CA) are microtiter plate immunoassays with sensitivity less than ng/ml and cross-reactivity with one another or mature PSA of less than 0.2%. Clinical Studies of ppsa and BPSA in Men with ng/ml PSA Serum measurements of ppsa forms in clinically documented and blinded serum samples containing from ng/ml PSA have revealed that ppsa is a more cancer specific marker than %FPSA and cpsa.infig.3athe%[ 2]pPSA ([ 2]pPSA/FPSA), %FPSA (FPSA/total PSA) and cpsa are shown. The %[ 2]pPSA gives a 25% specificity for cancer detection at 90% sensitivity. Both %FPSA and cpsa give no area above the 0.5 AUC line, indicating a 50/50 probability of cancer detection, or in other words, only random chance of cancer detection. In Fig. 3B the %[ 2]pPSA gives comparable AUC to %FPSA, though both have significantly higher AUC than cpsa. Fig. 3C shows the combined samples over the entire range of ng/ml. Again, %[ 2]pPSA gives significantly higher AUC than %FPSA or cpsa. These results demonstrate that %[ 2]pPSA has greater cancer specificity in the ng/ml PSA range and equivalent specificity to %FPSA in the 4 10 PSA range. Overall %[ 2]pPSA gives the best cancer detection across the PSA range from ng/ml. In these samples the %ppsa (the sum of each of the 3 ppsa forms) gave results comparable to [ 2]pPSA for the ng/ml range, though %ppsa had a slightly lower AUC than %[ 2]pPSA in the PSA range and slightly higher AUC in the 4 10 ng/ml PSA range. These studies showed that serum containing higher percentages of ppsa in the FPSA were more associated with prostate cancer. In these samples the BPSA was also measured. The %BPSA (BPSA/total PSA) gave a slightly higher AUC than %FPSA but this was not significant. This is consistent with the hypothesis that BPSA is a marker of BPH and BPH frequently co-exists in the older men regardless of whether cancer is present.24 Fig. 4 shows Fig. 3 Receiver operator characteristic (ROC) curves for %[ 2]pPSA, %FPSA and cpsa. The %[ 2]pPSA is defined as [ 2]pPSA/FPSA, while the %FPSA is the ratio FPSA/total PSA. Panel A: total PSA range ng/ml; Panel B: 4 10 ng/ml PSA; Panel C: ng/ml PSA. the ratio of BPSA to ppsa (the sum of all 3 ppsa forms). The AUC for BPSA/pPSA was equivalent to the %[ 2]PSA across all ranges as seen in Fig. 3, though only the range for ppsa/bpsa is shown in Fig. 4. This analysis is interesting because it does not use total or free PSA. The measurement of ppsa and

5 90 Mikolajczyk SD and Rittenhouse HG: Serum propsa improves cancer detection disease, but upon closer examination the presence of ppsa may help explain why free PSA is not more benign-specific. For instance, 8% of men with %FPSA greater than 25% are predicted to have prostate cancer, while 44% of the men with less than 10% free PSA will still not have cancer.14 ThepresenceofpPSAorBPSA in the FPSA may contribute to the misdiagnosis of benign disease or cancer in some patients with only a %FPSA measurement. Preliminary evidence indicates that ppsa is significantly elevated in men with greater than 25% FPSA who have been diagnosed with cancer. Therefore ppsa may help discriminate cancer in those men with high %FPSA who would normally not be biopsied. Another area of promise for ppsa is the discrimination of aggressive cancers from more indolent cancer. In summary, the current evidence indicates that ppsa is a more cancer specific form of PSA and extends the current utility of PSA to detect prostate cancer. Fig. 4 Receiver operator characteristic (ROC) curves for the ratio of ppsa/bpsa, %FPSA and cpsa for PSA ng/ml. The ppsa is the sum of [ 2]pPSA, [ 4]pPSA, [ 5/ 7]pPSA. The ppsa/bpsa does not use FPSA, cpsa or total PSA but is obtained by direct measurement of ppsa and BPSA. BPSA alone provide better cancer discrimination than any combination of PSA, free PSA or cpsa. Summary The ppsa forms are more highly enriched in prostate tumors and are a more cancer specific marker of prostate cancer. The ppsa forms are especially useful in the ng/ml PSA range, where the other PSA forms show little diagnostic utility. Serum ppsa, as a percentage of free PSA, has a higher specificity for cancer than the individual PSA assays or ratios of free, complexed or total PSA. The ratio of ppsa and FPSA mayalsoprovideamorestableparameterthanindividual measurements of FPSA or cpsa.42 In Fig. 3 the %[ 2]pPSA is shown, but %ppsa, (the sum of all three ppsa forms) gave similar results that were superior to %FPSA and cpsa. Individual assays for the different ppsa forms have been employed in initial studies in order to determine if any of the individual ppsa forms has greater ultility than other ppsa forms. In some cases it may be desirable to measure only the sum of all ppsa forms and in this case a single research assay has also been developed that can measure all forms of ppsa. ppsa is a subform of free PSA and the probability of cancer increases as the percentage of ppsa in the FPSA increases. This may at first seem paradoxical since the free PSA is normally associated with benign References 1. Tchetgen MB, Oesterling JE: The effect of prostatitis, urinary retention, ejaculation, and ambulation on the serum prostatespecific antigen concentration. Urol Clin North Am 1997; 24: Wang MC, Valenzuela LA, Murphy GP, Chu TM: Purification of a human prostate specific antigen. Invest Urol 1979; 17: Sensabaugh GF: Isolation and characterization of a semenspecific protein from human seminal plasma: a potential new marker for semen identification. J Forensic Sci 1978; 23: Watt KW, Lee PJ, M Timkulu T, Chan WP, Loor R: Human prostate-specific antigen: structural and functional similarity with serine proteases. Proc Natl Acad Sci USA 1986; 83: Rittenhouse HG, Finlay JA, Mikolajczyk SD, Partin AW: Human Kallikrein 2 (hk2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate. Crit Rev Clin Lab Sci 1998; 35: Robert M, Gibbs BF, Jacobson E, Gagnon C: Characterization of prostate-specific antigen proteolytic activity on its major physiological substrate, the sperm motility inhibitor precursor/ semenogelin I. Biochemistry 1997; 36: Lilja H: A kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein. J Clin Invest 1985; 76: Catalona WJ, Smith DS, Ratliff TL, Dodds KM, Coplen DE, Yuan JJ, Petros JA, Andriole GL: Measurement of prostatespecific antigen in serum as a screening test for prostate cancer. N Engl J Med 1991; 324: Emiliozzi P, Longhi S, Scarpone P, Pansadoro A, DePaula F, Pansadoro V: The value of a single biopsy with 12 transperineal cores for detecting prostate cancer in patients with elevated prostate specific antigen. J Urol 2001; 166: Catalona WJ, Partin AW, Finlay JA, Chan DW, Rittenhouse HG, Wolfert RL, Woodrum DL: Use of percentage of free prostate-specific antigen to identify men at high risk of prostate cancer when PSA levels are 2.51 to 4 ng/ml and digital rectal examination is not suspicious for prostate cancer: an alternative model. Urology 1999; 54: Roehl KA, Antenor JA, Catalona WJ: Robustness of free prostate specific antigen measurements to reduce unnecessary biopsies in the 2.6 to 4.0 ng/ml range. J Urol 2002; 168:

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