March 2007 Clinical Laboratory News: PSA Isoforms

  
March 2007: Volume 33, Number 3

PSA Isoforms
The Next Generation of Prostate Cancer Detection
By Stacy Loeb, MD, and William J. Catalona, MD

Prostate-specific antigen (PSA) is a serine protease and a member of the human kallikrein family. This now widely measured marker of prostate cancer was first described in the forensic medicine literature as a marker for human semen and was originally called gamma-seminoprotein. In the late 1970s, researchers purified PSA from prostate tissue extracts, and subsequent experiments demonstrated that PSA could be measured in human serum. Interest in PSA as a marker of prostate cancer grew after Chu (Buffalo, N.Y.) and Stamey (Palo Alto, Calif.) and their respective research teams reported that PSA levels were elevated in a high percentage of men with newly diagnosed, untreated prostate cancer; they also reported that PSA levels increased with advancing clinical stage and in proportion to the estimated volume of the tumor.

The first application of PSA testing, approved by the Food and Drug Administration (FDA) in 1986, was for monitoring prostate cancer recurrence after definitive therapy. In the late 1980s, however, Catalona (St. Louis, Mo.) initiated the first clinical studies to examine the use of PSA as a first-line screening test for prostate cancer (1). These and subsequent studies confirmed that screening with both PSA and digital rectal examination (DRE) was superior to screening solely with DRE. Consequently, in 1994 the FDA approved the PSA test—with a threshold of 4 ng/mL as the upper limit of normal—to be used in combination with DRE to aid in the early detection of prostate cancer. Since that time, PSA has become the most commonly used screening test for prostate cancer. However, a prostate biopsy is always required to verify a diagnosis of cancer in men whose PSA value is above the threshold value.

Over the years, many encouraging epidemiological trends have supported PSA’s utility as a screening test. For example, the proportion of men in the U.S. with metastatic disease at the time of diagnosis has decreased from 16% during the period 1985–1989 to 4% in 2003, and the age-adjusted prostate cancer-specific mortality rate has decreased by 32.5% between 1995 and 2003, according to the most recent data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results Program (2). The World Health Organization’s global statistics also reflect these trends, with decreasing prostate cancer mortality rates in countries where PSA screening and active treatment are widely practiced, and continued increases in mortality rates where PSA screening is not practiced. The most striking example of this trend is in Austria where the initiation of free PSA screening in the Tyrol region has led to a significant reduction in prostate cancer-specific mortality compared to the remainder of the country where PSA screening was not widely practiced (3).

Limitations of Total PSA Measurements

Although PSA-based screening has dramatically shifted the profile of newly diagnosed prostate cancer, its performance as a screening test has several limitations. First, approximately one-third of prostate cancers detected at a PSA level at or above 4 ng/mL have already spread to the prostate capsule or beyond, and approximately 15% of men with a PSA level <4 ng/mL have prostate cancer that is detectable by needle biopsy (4). Specifically, the Prostate Cancer Prevention Trial reported that in men who underwent an empiric biopsy at PSA levels of <=0.5 ng/mL, 0.6–1.0 ng/mL, 1.1–2.0 ng/mL, 2.1–3.0 ng/mL, and 3.1–4.0 ng/mL, the prostate cancer detection rates were 6.6%, 10.1%, 17.0%, 23.9%, and 26.9%, respectively.

These data highlight the difficulty of using PSA in clinical practice, since no absolute PSA threshold exists below which the risk of prostate cancer is eliminated. Although many clinicians now use a PSA measurement of 2.5 ng/mL as the absolute threshold for recommending a prostate biopsy, the issue of a threshold value remains controversial. In fact, accumulating evidence suggests that a measure of the change in the total PSA level over time, such as PSA velocity, may actually be a more important predictor of significant prostate cancer than the absolute PSA level (5).

Free and Complexed PSA

In order to improve the performance characteristics of PSA-based screening, researchers have focused on related biomarkers, especially PSA derivatives. PSA exists in serum in several different forms: the so-called “free” or unbound form, and forms in which PSA is complexed to other molecules, such as α-1-antichymotrypsin and α-2 macroglobulin. In the early 1990s, researchers discovered that the proportion of PSA in the free form—known as percentage free PSA—is significantly higher among men with benign prostatic hyperplasia (BPH) than men with prostate cancer (6). Subsequent multi-institutional trials demonstrated that fewer than 8% of men with a percentage free PSA greater than 25% had prostate cancer on biopsy (7). By contrast, 56% of men with a percentage free PSA less than 10% had prostate cancer. Based on these studies, in 1997 the FDA approved the free PSA test as an aid to prostate cancer detection.

In addition to free PSA tests, in vitro diagnostic manufacturers have developed assays for measuring complexed PSA, and some supporters of these tests claim that measurement of complexed PSA alone provides as much risk-assessment information as measurements of free and total PSA together. However, since total PSA is equal to the sum of the free and complexed PSA, knowledge of any two of these parameters should provide more risk assessment information than knowledge of any single marker.

Free PSA Isoforms

Taking the analysis of PSA derivatives further, recent research has shown that free PSA is actually comprised of several different isoforms. Two such isoforms are an intact but enzymatically inactive form, called i PSA, and BPH-related PSA, which is an internally cleaved or degraded form, known as B-PSA. In 2000, Mikolajczyk et al. reported that nodular hyperplastic tissue from the transition zone of patients with BPH has an elevated proportion of B-PSA relative to the concentration found in normal transition zone or peripheral zone tissue (8). Canto et al. later showed that the serum level of B-PSA has a stronger correlation with transition zone volume than the level of either total PSA or free PSA (9). Moreover, they reported a relationship between the serum B-PSA level and the International Prostate Symptom Score, suggesting that B-PSA measurement could have potential utility in the management of patients with BPH.

Figure 1
Distribution of PSA Forms in
Prostate Epithelial Cells and Serum

All the PSA forms shown here are measurable by immunoassay methods.

Another PSA isoform is pro-PSA, which is the proenzyme precursor of PSA. Immunoassays are available for the entire seven amino acid leader peptide, as well as truncated forms such as [-2] pPSA, [-4] pPSA, and [-5/-7] pPSA. In contrast with the other isoforms discussed earlier, pro-PSA appears to be preferentially elevated in the serum of men with prostate cancer. Measurement of pro-PSA is frequently expressed as the ratio of pro-PSA to free PSA, or percentage pro-PSA.

Considerable evidence now suggests that pro-PSA augments the specificity of PSA-based screening across a variety of total PSA ranges. In men with total PSA levels between 2.5 ng/mL and 4.0 ng/mL, Sokoll et al. reported that the mean percentage pro-PSA was 50.1% among 31 men with prostate cancer, compared to 35.5% in 88 men without prostate cancer on biopsy (10). In a separate study of men with total PSA levels of 4.0 ng/mL–10 ng/ml, the percentage pro-PSA was 32.7% in prostate cancer, versus 25.3% in men with a negative biopsy (11). Furthermore, the combination of total PSA, free PSA, and pro-PSA had an area under the curve of 0.766 (p<0.001) for prostate cancer detection.

In a later study, Khan et al. examined the use of PSA isoforms in men with a percentage free PSA less than 15% who underwent prostate biopsy (12). Even within this population of men who were already at high risk by virtue of having a low proportion of free PSA, the use of PSA isoforms helped to further distinguish which men had prostate cancer. Specifically, the ratio of pro-PSA to B-PSA was 0.9 in men with a negative biopsy, compared to 1.33 in men with prostate cancer.

In a recent study, we evalutated PSA isoforms in 1,091 men who underwent prostate biopsy as part of screening studies at two separate sites. In men with total PSA levels of 2 ng/mL–4 ng/mL, the [-2] pPSA to free PSA ratio was 0.0325 among 320 men with a negative biopsy, compared to 0.0422 in 235 men with prostate cancer (p<0.001). Furthermore, the overall pro-PSA to free PSA ratio was 0.30 versus 0.385 in the two groups, respectively (p<0.001). In men with total PSA levels of 4 ng/mL–10 ng/mL, the [-2] pro-PSA to free PSA ratio was also significantly higher among men with prostate cancer (0.044 versus 0.031, p<0.001), as was the ratio of overall pro-PSA to free PSA (0.35 versus 0.25, p<0.001).

In addition to increasing the specificity of PSA-based screening, PSA isoforms may be associated with prostate cancer aggressiveness. For example, among men treated with radical prostatectomy, an increased proportion of pro-PSA was significantly associated with a high Gleason grade and extracapsular tumor extension (13). Due to the recent discovery of these isoforms, long-term follow-up has not yet been reported. Nevertheless, a preliminary analysis from our surgical database suggests that the 7-year progression-free survival is significantly lower among men with elevated preoperative levels of [-2] and [-7] pro-PSA.

An Alternative Marker: hK2

As mentioned at the beginning of this article, PSA is a member of the human kallikrein family. Another member of the human kallikrein family, hK2, is also elevated in the serum of men with prostate cancer (14). As with the PSA isoforms, the ratio of hK2 to free PSA may be useful to increase specificity in prostate cancer screening. One limitation to the use of hK2 in clinical practice, however, is that the serum levels are approximately 2% of the total PSA concentration, making measurement of hK2 more difficult.

Improving Prostate Cancer Detection

PSA-based screening has been temporally associated with many favorable epidemiological trends in recent years, including a pronounced stage migration and a decreased prostate cancer-specific mortality rate. Although the total PSA level reflects the spectrum of prostate cancer risk and aggressiveness, its usefulness may be confounded by conditions such as BPH and prostatitis. As such, PSA derivatives can be valuable adjuncts in increasing the specificity for detecting clinically significant prostate cancer.

One of the latest developments in prostate cancer detection is the measurement of free PSA isoforms. Specifically, the overall percentage of the proenzyme precursor of PSA (pro-PSA), or in particular the [-2] pro-PSA truncated form, increases the specificity of PSA-based screening, and can therefore be used to reduce the number of unnecessary prostate biopsies. An increased proportion of pro-PSA is also significantly associated with adverse pathological tumor features. Additional research is needed to further examine the performance characteristics of these new free PSA isoforms and their relationship to long-term treatment outcomes.

REFERENCES

  1. Catalona WJ, Smith DS, Ratliff TL, Dodds KM, CoplenDE, Yuan J J, et al. Measurement of prostate-specific antigen in serum as a screening test for prostate cancer. N Engl J Med 1991; 324: 1156–1161.
  2. Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov) SEER*Stat Database: Incidence - SEER 17 Regs Public-Use, Nov 2005 Sub (1973-2003 varying), National Cancer Institute, DCCPS, Surveillance Research Program, Cancer Statistics Branch, released April 2006, based on the November 2005 submission. Accessed August 28, 2006.
  3. Bartsch G, Horninger W, Klocker H, Reissigl A, Oberaigner W, Schonitzer D, et al. Prostate cancer mortality after introduction of prostate-specific antigen mass screening in the Federal State of Tyrol, Austria. Urology 2001; 58: 417–424.
  4. Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL, et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med 2004; 350: 2239–2246.
  5. D'Amico AV, Chen MH, Roehl KA, Catalona WJ. Preoperative PSA velocity and the risk of death from prostate cancer after radical prostatectomy. N Engl J Med 2004; 351: 125–135.
  6. Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson O, Pettersson K, et al. Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem 1991; 37: 1618 –1625.
  7. Catalona WJ, Partin AW, Slawin KM, Brawer MK, Flanigan RC, Patel A, et al. Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial. JAMA 1998; 279: 1542–1547
  8. Mikolajczyk SD, Millar LS, Wang TJ, Rittenhouse HG, Wolfert RL, Marks LS, et al. "BPSA," a specific molecular form of free prostate-specific antigen, is found predominantly in the transition zone of patients with nodular benign prostatic hyperplasia. Urology 2000; 55: 41–45.
  9. Canto EI, Singh H, Shariat SF, Lamb DJ, Mikolajczyk SD, Linton H J, et al. Serum BPSA outperforms both total PSA and free PSA as a predictor of prostatic enlargement in men without prostate cancer. Urology 2004; 63: 905–910.
  10. Sokoll LJ, Chan DW, Mikolajczyk SD, Rittenhouse HG, Evans CL, Linton HJ, et al. Proenzyme psa for the early detection of prostate cancer in the 2.5-4.0 ng/ml total psa range: preliminary analysis. Urology 2003; 61: 274–276.
  11. Khan MA, Partin AW, Rittenhouse HG, Mikolajczyk SD, Sokoll LJ, Chan DW, et al. Evaluation of proprostate specific antigen for early detection of prostate cancer in men with a total prostate specific antigen range of 4.0 to 10.0 ng/ml. Urology 2003; 170: 723–726.
  12. Khan MA, Sokoll LJ, Chan DW, Mangold LA, Mohr P, Mikolajczyk SD, et al. Clinical utility of proPSA and "benign" PSA when percent free PSA is less than 15%. Urology 2004; 64: 1160–1164.
  13. Catalona WJ, Bartsch G, Rittenhouse HG, Evans CL, Linton HJ, Horninger W, et al. Serum pro-prostate specific antigen preferentially detects aggressive prostate cancers in men with 2 to 4 ng/ml prostate specific antigen. Urology 2004; 171: 2239–2244.
  14. Finlay JA, Evans CL, Day JR, Payne JK, Mikolajczyk SD, Millar L S, et al. Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity. Urology 1998, 51: 804–809.


Stacy Loeb, MD, is a resident in the Department of Urology at the Georgetown University School of Medicine, in Washington, D.C. Email: stacyloeb@gmail.com

William J. Catalona, MD, is a professor in the Department of Urology at the Northwestern University Feinberg School of Medicine and is Director of the Clinical Prostate Cancer Program of Northwestern's Robert H. Lurie Comprehensive Cancer Center in Chicago, Ill. Recognized as one of the pioneers of the PSA test, Dr. Catalona has received numerous awards and honors for his work and is often quoted in the press. Email: wcatalona@nmff.org.

This study was supported in part by the Urological Research Foundation and Beckman Coulter, Inc. (Fullerton, Calif.).

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