Cancer (Prostate-Specific Antigen) William J. Castellani Section of Biochemistry, The Cleveland Clinic Foundation, Cleveland, Ohio 44195-5130
Prostatespecific antigen (PSA) has become an important analyte in clinical medicine over the last decade. Serum PSA measurements have become routine as a marker for prostate disease, specifically for prostatic adenocarcinoma, since the first kit methods became available for in vitro testing in the mid-1980s. Over the past few years, our understanding of the biochemistry, physiology, and pathophysiology of this protein has grown immensely: it is no longer simply an “antigen”-defined by a specifk antibody reaction-but rather a protein with a biological role, evolutionary history, and clinical signiicance. Unfortunately, its clinical utility is limited by the complexity of its raison d’etre, the evaluation of prostatic carcinoma. No discussion of this marker is complete without also discussing prostatic carcinoma and the issues being debated currently about its significance, clinical history, and treatment. These considerations,more than any technical aspect, will determine how the use of PSA as a serum marker will evolve. The clinical significance of PSA detection and monitoring would not be clear without a discussion of prostate biology. Some terminology needs to be clarified at this point. The prostate contains secretory tissue composed of a folded sheet of cells called the glandular epithelium, which form lobules called glands. These glands are surrounded by supportive tissue. The entire structure, glands and supportive tissue, is an organ. In common usage, this organ is often called the “prostate gland. The term “gland can, therefore, refer to either the epithelial cells within the organ that actually secrete prostatic fluid or to the entire organ, and the correct interpretation is usually evident by context. In this paper, however, I will reserve the term “gland” for the glandular epithelium and refer to the entire prostate as an “organ” to differentiate between the prostate itself and its secretory component. BIOLOGY OF THE PROSTATE The prostate is located at the base of the bladder, surrounding the urinary outnow passage, the urethra. The male reproductive tract enters the urethra after passing through the prostate. Secretions from the glandular epithelium mix with seminal fluid to form semen. The prostatic secretions are a complex mixture of proteins, fructose, and other constituents produced by the glandular tissues of the organ. Development and function is supported by the male sex hormones (androgens),predominantly testosterone. Embryologically,the glandular tissue of the prostate develops from the cells lining the urethra as outpouchings. The external aspect of these glands is lined by a connective tissue layer called the basement membrane. This membrane separates the glands from the remaining tissue of the prostate, the stroma, which is composed of smooth muscle with some fibrous tissue. The basement membrane may serve to prevent gland secretions from escaping into the stroma as well as providing an anchoring point for gland cell attachment. The embryologic structures are present in all fetuses, but full differentiation into the male prostate is guided by in utero exposure to androgens. (In the female, the tissues remain undeveloped and are called “Skene’sglands”.) The prostate will not develop fully until puberty, when it increases in
size rapidly as part of the sexual maturation process. It usually continues to grow slowly throughout adult life. In later maturity, it may reach a sufficiently large size that it obstructs the urinary stream, a condition called benign prostatic hypertrophy. This may be due to proliferation of the glands, the stroma, or both. This continued growth is stimulated by circulating androgens. Prostate cancer is a malignant alteration in the glandular epithelium and is more appropriately termed prostatic adenocarcinoma. Although this is both the most common cancer and the second most common cause of cancer-related death in men it frequently causes no problems whatsoever (02, 03). The natural history of prostatic adenocarcinoma is extremely varied in its aggressiveness (04).Prostates that are removed for urinary obstruction or during routine autopsy examination may show microscopic areas of cancer, even though the rest of the tissue is completely benign. Untreated prostate cancer (when identified early in its course) is reported to have a high 10-year survival rate (03,indicating a long latent period in which it remains localized and slowly growing before it becomes aggressive. During this latent period, the abnormal tissue shows all the microscopic features of cancer, invading the stroma of the prostate by disrupting the basement membrane that surrounds the normal glandular epithelium and occupying an increasingly greater fraction of the prostate. At some point, the cancer will invade outside the prostate and spread metastatically to lymph nodes and bones. Survival with advanced and metastatic disease is poor, and current treatment methods in general have not been shown to improve survival (06). The alterations that are necessary to cause these aggressive changes are not currently known, but are presumably genetic in nature-similar to the genetic changes that occur in the development of adenocarcinoma of the colon (Dn-and appear to be random occurrences in any given patient. There is no k e d sequence or timetable: one man may have an extremely short course with rapid spread and death within a few years, whereas others may live more than a decade with known cancer and die of other causes without ever having a symptom. Classifying a patient’s tumor into prognostically useful categories is currently the greatest challenge in the management of prostate cancer (02, 08, 09). For those unfortunate individuals with aggressive, widespread disease, though, a different challenge exists: there is no therapy that does more than ameliorate the relentless course of prostate cancer that has entered this phase of the disease (06). The prostate can be examined directly by two methods: The digital rectal examination @RE) allows the posterior portion of the organ to be felt by the physician’s finger, and the transrectal ultrasound (TRUS) produces an image of the prostate showing density changes detected by ultrasound echo. Both methods permit some estimate of the prostate size as an indication of urinary obstruction and detection of suspicious nodules that warrant further evaluation for cancer. DRE is limited to the posterior aspect of the organ; however, this is where most cancers arise (04).It is also limited, though, by the skill of the physician performing the examination. TRUS will image the organ and identify abnormalities based on localized changes in density-which is common, but not always present, in areas involved by cancer. Analytical Chemistry, Vol. 67,No. 12, June 15, 1995
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Unfortunately, both have limitations and low positive predictive value as screening tests for prostate cancer (09). The DRE has the advantage of low cost, limited to an examining glove and a few seconds of time. The TRUS, on the other hand, requires ultrasonography equipment, introduction of the transducer into the rectum, and a trained interpreter; it is most often used to locate suspected lesions. Both methods are unpleasant for the patient and the physician and tend to find too many “abnormalities”that are not cancer. PSA is a normal constituent of prostatic secretions; very few other tissues or organs have been demonstrated to produce this protein. Its history and biochemistry have been reviewed many times (010-012). PSA was originally d e h e d by antibodies raised to human semen; in 1980, this protein was demonstrated in the serum of men with advanced prostate cancer (013). Over the intervening 10 years, the protein was identified and defined (014).It is a 237-amino acid (-34kDa) glycoproteinand member of the kallikrein family of serine proteases-enzymes that cleave other proteins through the activity of a serine molecule at the active site. Although considerable homology exists between true kallikrein and PSA (also called KLK3 in this context) (018,the enzyme specificitiesare quite different, and PSA has no kallikrein activity. Rather, it cleaves at the carboxylic sides of leucine, phenylalanine, and tyrosine in any protein, nonspecifically. Its natural substrate, though, is semenogelin, a high molecular weight protein that is present in seminal fluid and rapidly forms a coagulum from semen (DIG. PSA is secreted into the lumen of the prostatic glands and joins then the seminal fluid as it passes through the prostate; it hydrolyses semenogelin, liquefying the coagulum and releasing spermatozoa (014). A trace amount seeps into the bloodstream, though the serum level may have no relationship to the amount of benign glandular tissue present in the organ (017). Since adenocarcinoma of the prostate originates from the cells of the glandular epithelium, it also produces PSA. However, cancer tissue has repeatedly demonstrated lower levels of PSA synthesis when compared to normal glands, based on comparisons of immunohistochemical staining for PSA (018) and messenger RNA levels for the PSAgene product (019). The increased serum PSA levels seen with prostate cancer have been attributed to the absence of the basement membrane investment that normally isolates the prostate glands from surrounding stroma, which is the hallmark of invasive cancer. The PSA that is synthesized by the malignant cells leaks directly into the surrounding tissues and readily enters the bloodstream. In general, the serum PSA concentration is proportional to the amount of cancer tissue present. However, extremely abnormal (“high-grade”)cancers may show very little evidence of prostatic function, producing little if any PSA and resulting in no or slight elevations of this marker in an individual with aggressive disease (D17).This situation is rare, but does occur. PSA activity has been described from other tissue sources. The periurethral glands in both men and women produce trace amounts of the enzyme (DZO), which is detectable in urine (021, 022) (though not currently a problem with serum measurements). Histologically, both benign and malignant salivary gland tumors (D23),as well as 30%of breast cancers (D24,have on occasion shown intracellular PSA by immunologicmethods-though serum elevations have never been demonstrated to be due to these lesions. Finally, a few women with cancer of the kidney have been 400R
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described with measurable serum levels of PSA, using a polyclonal immunoassay-these same women were negative when a monoclonal method was used (D25). Currently, these alternate sources of PSA do not appear to affect serum levels in men. However, after removal or ablation of the organ, ultrasensitive methods may measure serum PSA that originates in nonprostatic tissue (OK?), an issue that will be considered later in this paper. PROSTATESPECIFICANTIQEN IN HEALTH AND DISEASE Four major issues are currently under investigation or evaluation in optimizing the diagnostic utility of serum PSAlevels. Since the normal prostate grows slowly throughout postpubertal life, the diagnostic threshold for the reference range must take this into account. In addition, though PSA in prostatic fluid is a single polypeptide chain, PSA in the bloodstream is partially complexed to specifc serum proteins, and the presence of these PSA complexes may have diagnostic significance. Another issue is the utility of “ultrasensitive”methods in evaluating patients after treatment for prostate cancer. Finally, perhaps the most important issue is the optimum use for PSA in screening for and diagnosing prostate cancer itself. In reality, none of these are truly “technical” issues-rather, they are all due to the complex and variable natural history of the disease. Prostate Screening and Diagnosis of Prostate Cancer. Screening for a disease implies the use of a simple test on people who have no symptoms to detect and treat that subset with the disease before problems arise. It is not as simple as that. Screening is not diagnosis (026). Screening tests typically identify those people at risk for having the disease - these individuals must then undergo additional testing to corhrm that they do, indeed, have the disease and need treatment. Much work has been published evaluating the efficacy of serum PSA in screening for prostate cancer, most often in comparison to digital rectal examination and transrectal ultrasound (08,027). PSA is much more sensitive than other screening modalities, but is prone to false positives. Disease confirmation involves biopsy of the prostate, usually by inserting a needle through the rectal wall into the organ and removing a core of tissue, usually guided by TRUS (028). If an elevated serum PSA level is the only indication of possible cancer, the physician has no idea where the disease might be in the organ. In this case, the standard procedure is to do several biopsies of the organ in a specifc pattern (such as a “sextant biopsy”) (029). Even with negative biopsy results, the patient may still have a small malignancy that was missed by the procedure, so these patients need to be followed. The biopsy and the followup for negative results are expensive and painful consequences of the false positive rate seen in PSA screening. Therefore, serum PSA measurements are controversial as a screening method; the literature includes cost/benefit decision analyses (030, 031) and a discussion of the legal aspects of screening for prostate disease (032-034). Until September 1994, the Food and Drug Administration approved serum PSAmethods only for monitoring patients with known prostate cancer. At that time, the FDA extended approval for one PSA method to include its use as an aid in the detection of prostate cancer in conjunction with the DRE based, in part, on a multicenter trial involving over 6000 men (035). There is no method currently approved for screening-presuming that there is a distinction between “aid in detection” and “screening”in practice. The American Urological
Society and the American Cancer Society have recommended measurement of serum PSA levels, together with the DRE,in all men over 50 years of age (08, 036). An alternate approach to the detection of prostate cancer using serum PSA is based on the rate at which the level increases in a person over time. Studies have been reported that both support (037-039) and refute (040,041) this approach. However, this is not a screening method: it is preventive medicine, requiring routine periodic evaluations. A method that discriminates between disease and health with a single measurement is always preferable, since it does not require long-term patient compliance and recordkeeping. As we better understand the behavior of serum PSA levels in prostate cancer, PSA screening may become effective enough to be routine. Two such approaches, adjusting the reference range for organ size and measuring PSA complexes, are discussed below. Age-Specific Reference Ranges. The most commonly cited reference range is 0-4.0 ng/mL, based on the range supplied by the manufacturer of the first method approved by the U.S. Food and Drug Administration for in vitro diagnostic testing. A “borderline” range between 4.1 and 10.0 ng/mL is currently accepted, based on a high frequency of false positives seen in routine practice in older men without demonstrable prostate cancer or symptomatology. This is necessary due to the benign enlargement common in the prostate glands of older men. However, in younger men, a “borderline” serum level is highly suspicious, since prostatic enlargement is rare in men in their 40s, when the most significantprostate cancers may develop.Two groups have published agestratified ranges by decades (042, 043); although the ranges are similar, they are not identical. Both studies, though, indicate that the traditional upper limit of 4.0 ng/ mL is too high for men in their 40s and 50s. Since prostate cancer takes around a decade to go from latent to virulent stage, this is the patient group that would most benefit from early diagnosis and treatment; they are the ones that are most likely to die of prostate cancer in their 50s, 60s, and 70s (044). In contrast, men in their 70s would be expected to have elevated PSA levels without any pathology-and both groups suggest an upper limit greater than 6.0 ng/mL for the reference range in this age group. However, two studies indicate that age-specific reference ranges have no particular advantage over an absolute reference limit of 4.0 ng/mL (039, 041). Nonetheless, age-specific reference ranges, when refined, may be optimized to detect as many men with prostate cancer early enough to be beneficial to them without causing excessive pain and suffering-and increased medical costs-in those who test as false positives. An alternate approach to accommodating the benign prostatic growth that occurs with age is called the prostate-specific antigen density (PSAD). The volume of the prostate is approximated by assuming that the prostate is a geometric solid and taking the measurements necessary to calculate the volume from the transrectal ultrasound. Dividing this into the measured serum PSA “corrects” for the amount of gland tissue present. Since 1g of carcinoma usually yields a much higher serum PSA than 1 g of normal glandular epithelium, the PSAD is elevated in the presence of prostatic cancer. Many studies have shown that this method gives improved diagnostic information when compared to serum PSA by itself (045, 046); unfortunately, other studies suggest that this is not so (039, 041). Intraexaminer variation between the individuals performing the ultrasound may be a
confoundingfactor in these studies. A standardized,reproducible method for determining prostate volume may rectify the situation, though no such method has been proposed. Prostate-Specific Antigen Complexes. Serine proteases will attack a wide range of protein substrates and, in the wrong location, can cause considerable damage. The bloodstream often absorbs enzymes that escape from the site where they perform their normal activities. Several inhibitor proteins are present in circulation that bind to and block such enzymes. PSA, with its chymotrypsin-like activity, is bound by several serum inhibitors, predominantly al-antichymotrypsin ( A m and armacroglobulin (A2M) (047). (Inter-a-hypsin inhibitor will also bind to PSA, but this has not been investigated or discussed in any depth in the literature.) Exogenous PSA added directly to serum in vitro binds slowly to A2M and more slowly to ACT. PSA is buried within the A2M molecule and dficult to detect in PSA-MM complexes. PSA-ACT complexes, on the other hand, leave the PSA molecule partially exposed, and antibodies can bind to the complexed PSA protein (047). Unbound (free) serum PSA is not seen unless added in sufficient amount that it overwhelms the inhibitors present in the serum. Circulating PSA that enters the bloodstream directly from the prostate shows very a e r e n t binding characteristics. An unbound PSA fraction is typically present, even though there is enough serum A2M and ACT to complex with this free fraction. The PSA molecules in this free fraction may have been somehow altered so that it will not bind to serum inhibitors (011). More significantly, benign conditions on average show a higher proportion of free PSA in the serum than is seen in cancer (048). Recent studies have shown that malignant prostate cells will synthesize ACT (which is usually produced only by the liver); benign prostate tissue does not show any evidence of ACT synthesis (049). Therefore, some of the PSA released by prostate cancer into the bloodstream may be prebound to ACT, resulting in a lower ratio of free to complexed PSA in malignant disease. However, there is considerable overlap between the ratios of free to total (or complexed) PSA in the serum seen in individual patients with benign and malignant prostate conditions (047). Several groups have published clinical studies that indicate improved discrimination between benign and malignant prostate conditions by evaluating either the PSA-ACT complex or the ratio of free to bound (or total) PSA present in the serum (048,050, 051). In contrast, one preliminary retrospective report, published as a letter to the editor and based on small numbers of samples, suggests that measuring the complexed fraction does not increase diagnostic sensitivity over total PSA alone (052). Currently, there is insufficient evidence to support routine use of free PSA ratios in evaluating for prostate disease. Since the ACT-bound PSA is the only complex that can be readily quantified in serum, it is possible to design immunometric methods that detect only unbound PSA, PSA-ACT complexes, or both simultaneously, by selecting appropriate pairs of antibodies. However, in the case where the both free and complexed PSA are detected, the method is usually more sensitive to one of the fractions. The d ~ e r e n commercially t available assays vary in their specificity for the free and complexed fractions (011,053, 054). This appears to have only limited impact on patient results, given the variability seen in the ratios of the two fractions (055). There is a much greater significance, however, when calibrators and controls are compared; in the past, these have given Analytical Chemistry, Vol. 67,No. 12,June 15, 1995
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inconsistent results when used with assays other than the one used for standardization (056). Protocols have been proposed to standardize control and calibrator material so that they more closely mimic true patient samples, which should alleviate many of the discrepancies that have been seen (056,057). Whether this is currently being followed by the industry is unclear at present. Ultrasensitive PSA Methods. Standard methods for serum PSA have lower limits of detection (LLDs) in the 0.2-0.5 ng/mL range; after removal of the prostate, serum levels often drop below this limit (058). Unfortunately, this drop does not necessarily signify cure if prostate cancer was present in the organ; a fair number of men go on to have rising PSA levels and eventual relapse due to either incomplete removal of the cancer at the surgical site or distant metastatic spread. Several authors have shown that relapse can be detected earlier by ultrasensitive methods that have increased low-end sensitivity (LLD 5 0.1 ng/ mL) (058, 059). A fully automated, FDA-approved method for PSA with a working LLD of less than 0.1 ng/mL exists, as documented by independent studies (060-062),though there is considerable lot-tdot variation in the performance of the method in the ultrasensitive range. This demonstrates that there is no technical obstacle to the development of routine automated ultrasensitive methods. Although there is considerable discussion about the possible utility of ultrasensitive methods, current approaches to the management of prostate cancer does not use this information effectively. Prognostic and diagnostic schemes based on serum PSA levels prior to therapy all have decision points well above standard assay LLDs. Patients who relapse with prostate cancer does not benefit from early detection of relapse: treatment methods can only control cancer in relapse temporarily. If more effective treatment protocols can be devised, both for cancer in full relapse (palliative therapy) and postsurgical treatment of clinically nonevident residual disease (adjuvant therapy), then ultrasensitive methods will play an essential role in the care of men after definitive therapy. In addition, monitoring would be more costeffective if the postsurgical serum PSAlevel can be used to identify patients that are probable cures, requiring less aggressive monitoring. However, alternate sources of serum PSA, such as the periurethral glands and salivary glands, may then become signiiicant. Extremely small, but detectable, increases in serum PSA may be due to disease processes in these sites (or others that have not yet been discovered), rather than cancer relapse. This is speculative-as is, in fact, all of the possible uses of ultrasensitive PSA methods. Until these methods enter routine use, their advantages and possible disadvantages cannot be fully determined. Other Issues. The PSA molecule shows considerable variation. Isoforms can be identified that represent glycosylation microheterogeneity and a nicked molecule has been identified at the Lys14j-Lys146 bond ( D l l , 057); neither of which has been shown to be useful in evaluating for prostate disease. NGlycosylation variants have been observed, though one study failed to show any difference in concanavalin A binding between patients with benign and malignant prostate disease (063). Even though these appear to be dead ends, the molecule remains under study and may yield diagnostic surprises in the future. 402R
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PROSTATE-SPECIFICANTIGEN AND MICROMETASTASES Cells that contain PSA messenger RNA can be assumed to be synthesizing the protein and are probably prostatic in origin. If these cells are located outside the organ in a patient with known prostate cancer, then this is evidence of metastatic spread and of a dire prognosis. Three groups have used the reverse transcriptase-polymerase chain reaction method to demonstrate the presence of PSA mRNA and, presumably, micrometastases in lymph nodes (064) and circulating blood (065, D66). This approach may identify those patients with disseminated disease who will not benefit from localized therapy, preventing considerable expense and pain in men who have no chance of cure. The utility of this approach remains to be proven, though one of the investigationalgroups has acquired an international patent for this method (067). CONCLUSION This review is heavily biased toward clinical studies rather than analytical techniques, which typifies the situation between prostate cancer and serum PSA testing. The literature raises concerns about whether current methods will be suitable when we fully understand the significance of serum PSA, PSA complexes, and ultrasensitive PSAmonitoring. The limitation at present, however, is not in our ability to measure serum PSA; it lies in the fact that we often cannot determine from this measurement, or any other clinical finding, what is in the best interest of the patient. The major issues are separating men with benign conditions from those with malignant prostate disease, identifying those with indolent cancer from those with aggressive disease, and develop ing improved treatment methods for men with advanced or progressive prostate cancer. It remains to be seen if any PSAbased method, or any other serum marker, will improve this situation. Clinical diagnosis and management techniques for prostate cancer will have to evolve considerably before the current analytical limitations of serum PSA testing become evident. William J. CastelZuni is staffin the Section of Biochemisty, Division of Pathology and Laborato Medicine, at The Cleveland Clinic Foundation. He aduatedfiom x e University of Mfchigan Medical School in 1980. I f completed a combzned anatomzc and clznzcal pathology residency at the Cleveland Clinic Foundation in 1984 and a 2year @llowship in biochemisty in 1986 at [he same institution. His research znterests include trend a n a l p s zn patzent care and he has responszbzlzty for the metabolic laboratoy zn the Sectaon.
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