Enzyme Tests in Diseases of the Prostate

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 3 Copyright 1977, Institute for Clinical Science Enzyme Tests in Diseases of the Prostate RONALD M. TOWNSEND, M.D. Department of Laboratory Medicine, University of California Medical Center, San Francisco, CA ABSTRACT Enzyme tests in diseases of the prostate focus primarily on the use of serum acid phosphatase assays in patients with suspected or histologically proved adenocarcinoma of the prostate. The purpose of this review is to consider various potential clinical uses of the assay, to examine the data available concerning the performance of the test in given clinical situations and to define those situations in which the test is actually useful. Included in this discussion will be sources of false positive and false negative values, predictive values of the test in clinical settings and efforts to minimize shortcomings. Introduction For more than 40 years the clinical use of enzyme assays in prostate disease has focused on determination of serum acid phosphatase.7,22 Numerous reports7,13,14,24 have shown an increased incidence of elevated serum acid phosphatase levels in patients with adenocarcinoma of the prostate. Considerable controversy exists, however, concerning the clinical utility of such determinations.21 The purpose of this review is to attempt to define those clinical situations in which acid phosphatase determinations are useful. Such an analysis requires discussion from several points of view. First, the various clinical settings will be examined to determine what questions are being asked by the physician concerning prostatic disease. Next, data from the literature will be presented from the points of view of sensitivity, specificity and predictive value5 in an attempt to provide some measure of utility between differing approaches. This will include a discussion of the sources which limit the clinical utility of serum acid phosphatase determinations, as well as some of the methods employed to overcome the various shortcomings. Finally, there will be a brief review of some of the alternative enzymatic approaches for the diagnosis and management of prostate carcinoma. Uses of Cancer Tests Because neoplastic disease is, in general, more easily managed in its early stages, considerable effort has been expended seeking assays which will distinguish an individual with malignant disease from a healthy population. This ideal, the perfect screening test, remains

2 EN ZYM E TESTS IN DISEASES O F THE PROSTATE 255 elusive. Thus, the concept of screening a healthy population for cancer generally evolves to the goal of identifying patients with cancer among high risk individuals. By increasing the prior probability that a tested patient has disease, the predictive value of a positive test improves. Fewer false positives from healthy individuals are observed if other variables, such as age or sex, are used to select a population prone to disease. Thus, a logical question in a clinical setting might be: Does this elderly man with symptoms or physical signs suggestive of prostatic disease have carcinoma of the prostate? How useful is a serum acid phosphatase assay in this situation, to predict the presence or absence of prostate cancer? A second, potentially important application of a chemical assay is not simply to distinguish presence or absence of cancer, but to define the extent of tumor spread in a cancer patient at a given time. The development of a highly predictive noninvasive means of detecting metastatic disease would be of great benefit in management. Again, both high sensitivity and high specificity are desirable. A false negative test in this situation results in the likelihood of inadequate therapy, while a false positive may result in bypassing local, relatively safe, curative measures for riskier more aggressive approaches to management. In order to judge clinical utility in this situation, two questions must be answered. First, how useful is an elevated serum acid phosphatase in predicting the presence of metastatic disease? Finally, to what extent does a normal value imply limited disease? In addition to the potential use of determining disease stage, a further application is in monitoring the effects of antitumor therapy. Changes in the serum acid phosphatase levels during progression or regression of disease, as well as any changes in response to therapy, provide potential clinically useful information. The clinical value of such use, however, depends on the predictive value of positive and negative tests in the particular clinical situation. In this context, a fall in serum acid phosphatase levels during therapy might be predictive of tumor mass decrease and absence of residue. This in turn might be related to prognosis. Vital to management in such patients would be the predictive value of persistent elevations after therapy in implying persistent tumor. Similarly, the extent to which an increasing value implies tumor growth within an individual needs to be known before significant management decisions are based on such data. Serum Acid Phosphatase Levels in Prostate Carcinoma Compounding the difficulties inherent in comparing results of various studies evaluating serum acid phosphatase levels in prostatic disease is the fact that a variety of substrates and assay conditions are used. Nevertheless, several studies of large patient groups show relatively good agreement in terms of percent elevation of serum acid phosphatase within patient groups (table I). This is even more remarkable because not only are methodologies different, but means of specifying an elevated from nonelevated value vary considerably. It should be clear then, that even with advanced malignancy, a considerable number of false negatives, i.e., normal values in patients with bone metastasis, are observed. Similarly, if one were to preselect a population of patients with either benign prostatic disease or confined, presumably early, carcinoma, one would not be able to distinguish the two groups on the basis of serum acid phosphatase levels. The percent of false positives seen in the benign group approaches the percent true positives in the group with early carcinoma.

3 256 TOW NSEND TABLE I Elevation of Serum Acid Phosphatase Patient Group Benign prostatic hyperplasia 7 Prostate carcinoma confined to prostate 5-10 Prostate carcinoma - locally invasive Prostate carcinoma with bone metastasis False Positives and False Negatives False positives acid phosphatase values, i.e. elevated serum levels in patients without prostate carcinoma, are generally explained in one of two ways. First, serum elevations may result from acid phosphatase derived and released from tissue of nonprostatic origin. Second, elevations may arise from certain nonneoplastic conditions of the prostate. Nonprostatic sources of serum acid phosphatase activity are normally present,11since sera from adults and children of both sexes display measurable activity.7 The activity in erythrocytes and platelets is particularly problematical.1,20 Hemolysis, both in vivo and in vitro, can elevate serum acid phosphatase activity. Similarly, increased in vivo platelet destruction, as in idiopathic thrombocytopenic purpura, or increased release of enzyme in clotted blood with abnormally high platelet counts, can serve as sources for false positive values. Other organs which serve as sources of clinically significant serum elevations are bone, liver, Nonprostatic Diseases with Serum Acid Phosphatase Elevation1 Bone Paget's disease Osteogenesis imperfecta Osteogenic sarcoma Osteopetrosis Osteoporosis Hyperparathyroidism Multiple myeloma Metastatic carcinoma Reticuloendothelial Gaucher's disease Liver Viral hepatitis Drug induced hepatitis Extrahepatic obstruction Cirrhosis Metastatic carcinoma Kidney Acute renal failure Chronic renal failure kidney and the reticulo-entotheleal system. In table II is shown the wide variety of nonprostatic diseases in which elevations of serum acid phosphatase are observed. The variety of sources for nonprostatic elevations of serum acid phosphatase has led to a quest for an assay which is specific for acid phosphatase of prostatic origin. Approaches to this problem have been directed toward substrate substitution or chemical inhibition. In table III are displayed the more prominent of the substrates used in acid phosphatase assays. In table IV are shown relative activities of various acid phosphatase isoenzymes as a function of substrate. Of particular note is the reduced hydrolysis rate observed with red cell derived acid phosphatase when a naphthol phosphate or sodium thymolphthalein monophosphate is used as substrate. With either of these two substrates, however, caution should be exercised with the term prostatic specific since such specificity is relative to other substrates, and interference from platelets, for instance, is on a similar order of magnitude for the two most specific substrates.4it would appear that sodium thymolphthalein monophosphate is, relative to other substrates, most specific for prostatic acid phosphatase. The observation that L-tartrate inhibited 95 percent of the activity of prostatic acid phosphatase, while activity from erythrocytes was unchanged, led to investigation of the utility of such a prostate specific method. Further studies revealed, however, that chemical nonspecificity remained a problem, although certainly of lesser magnitude.1 Acid phosphatase from liver and kidney are also inhibited by L-tartrate and may still act as sources of false positives. The clinical utility of any increased organ specificity will be discussed.

4 EN ZYM E TESTS IN DISEASES O F THE PROSTATE 257 TABLE I I I Common Methods fo r Serum Acid Phosphatase1' 20 Eponym Substrate Product Measured King-Armstrong Phenyl phosphate Phenol Huggins-Talalay Phenolphthalein monophosphate Phenolphthalein Babson-Read Alpha naphthol phosphate Alpha naphthol Bessey-Lowry Paranitrophenyl phosphate Paranitrophenol Bodansky Beta glycerophosphate Phosphate Shinowara Beta glycerophosphate Phosphate Seligman Beta naphthol phosphate Beta naphthol Roy Sodium thymolphthalein monophosphate Sodium thymolphthalein An additional source of false positives in the diagnosis of prostatic carcinoma is non-neoplastic conditions of the prostate. Benign hypertrophy, infarcts and various manipulations, including rectal examination or prostatic massage, have all been reported to cause at least transient elevations of serum acid phosphatase. Two recent studies,6,23 however, provide evidence that manipulative efforts short of transurethral prostatectomy cause no significant elevations. Furthermore, transurethral prostatectomy caused elevations of less than one day duration. These studies utilized the relatively prostate specific substrate of a-naphthol phosphate and indicate any elevations which should not be discounted as the result of physical manipulation. Adding to the confusion in interpretation of serum acid phosphatase in the diagnosis of prostate carcinoma is the considerable percentage of false negatives, i.e., patients with prostate cancer but non-elevated levels of acid phosphatase. Contributing to this problem are considerations of analytical limitations as well as certain characteristics of the tumor being sought. Assay insensitivity is a potential source of false negatives. It is possible that even minute tumors, with vascular alterations making enzyme clearance from tumor possible, secrete acid phosphatase in quantities below the limits of detection. Of importance in this regard is the nonprostate sources of acid phosphatase detected in normal sera by all methods, thus rendering difficult the detection of minute differences of activity. Interest in immunologically based assay procedures may eventually provide more specific and sensitive analytic methods.12 An additional source of false negatives is improper handling of specimens. Acid phosphatase is unstable at warm (37 ) temperatures or even slightly alkaline conditions. Samples should be processed and analyzed promptly, or better, acidified to ph 6 soon after collection to insure stability of at least four hours.1 Further false negative values are observed in some tumors which are widely TABLE IV Relative Activities of Various Human Acid Phosphatase Isoenzymes as Measured with Substrates20 Source PP PMP NP PNPP GP Prostate Erythrocyte I Erythrocyte II Erythrocyte III Liver I Liver II Liver III Bone Kidney I Kidney II Kidney III Urine Platelets Values represent activities relative to sodium thymolphthalein monophosphate taken as unity. PP = phenyl phosphate PMP = phenolphthalein monophosphate NP = alpha-naphthol phosphate PNPP = paranitrophenyl phosphate GP = beta glycerophosphate

5 258 TOW NSEND TABLE V Total or Prostatic Specific Acid Phosphatase Values Total Acid Phosphatase Tartrate >2.5 KA Units Values (Mean + 3 SD) >0.60 Units Diagnosis Clinical Stage No. No. % No. % Benign hyperplasia Carcinoma Nodule or occult Confined to prostate Local extension Metastatic Total carcinoma disseminated, bulky and invasive. Although serum acid phosphatase and prostatic specific acid phosphatase are increased more frequentiy as the stage of tumor increases, some tumors apparently do not produce the enzyme at currently detectable levels. Another consideration governing detection of acid phosphatase as a tumor marker is the degree of differentiation of the tumor. Murphy et al13observed a tendency for well differentiated prostate adenocarcinoma to produce and to excrete the enzyme, while patients with poorly differentiated tumors less frequently displayed evidence of acid phosphatase elevations. Acid Phosphatase as a Diagnostic Test The study by Murphy et al13illustrates the value of either total or prostatic T A B L E VI Sensitivity, Specificity and Predictive Values of Total or Prostatic Specific Acid Phosphatase Values Total Tartrate Labile Acid Phosphatase Acid Phosphatase No. Pos No. Neg No. Pos No. Neg Metastatic carcinoma Benign hyperplasia Sensitivity 88% 73% Specificity 93% 97% p.v. (+)* 88% 94% P.V. (-)t 93% 86% Predictive value of a positive test. fpredictive value of a negative test. specific acid phosphatase in selected populations. In this study, 141 cases of benign prostatic hyperplasia and 185 cases of prostate carcinoma in various stages were selected for evaluation after the initial histologic diagnosis was confirmed by autopsy or three year follow up. Total serum acid phosphatase was determined using the non-specific phenylphosphate substrate, while L-tartrate inhibition was the method chosen to increase organ specificity. Elevations were defined as greater than a mean +3 S.D., as determined on a healthy population. In table V are depicted Murphy s data in tabular form. From this data if distinguishing benign prostatic hypertrophy from clinically manifested metastatic prostate carcinoma were the question being asked, then the assf.y would be of some utility. In table VI are tabulated sensitivity, specificity and predictive values for those two patient population and data. However, the important clinical question is whether or not the patient with symptoms or signs suggestive of early curable tumor can be designated as having benign or malignant disease using acid phosphatase levels. In table VII are shown results addressed to this question. Of importance is that although specificity, the percent of negative values in patients without cancer, is approximately 95 percent, the predictive value of a negative test is only around 75 percent. This emphasizes the importance of disease prevalence on predictive value of laboratory tests. Furthermore, given the limited sensitivity of the test in early carcinoma, use as a screening test cannot be advocated. Given a predictive value of either a positive and negative test as approximately 70 percent and the fact that the consequences of mistakes are clinically costly, the test appears inadequate in this context. The next question one may ask is the utility of acid phosphatase assays in dis

6 EN ZYM E TESTS IN DISEASES O F THE PROSTATE 259 tinguishing patients with metastatic prostate carcinoma from non-metastatic disease. These data are shown in table VIII. Use in this context demonstrates that even by optimizing predictive value by use of the more prostate specific assay, one is still confronted with approximately 20 percent false positives and 20 percent false negatives. Again, the cost of such errors in a clinical situation is heavy. Of note in tables VI, VII and VIII is the observation that the tartrate labile assay for acid phosphatase, not surprisingly, provides slightly more specific information at the cost of sensitivity. To approach the clinical utility of isoenzymes as seen in the diagnosis of acute myocardial infarction, considerably better predictive value is needed. Because of the importance, in management, of detecting metastatic disease, a further application of acid phosphatase assays has been suggested. By use of a relatively prostate specific substrate and by analyzing material from bone marrow aspirates, a more sensitive and specific means of staging is being sought.2'8,15,19 There are several inherent difficulties in demonstrating that a given procedure is more sensitive for metastatic disease. Primary among these difficulties is knowing with any certainty that patients with positive results, do, in fact, have metastatic disease in spite of no positive evidence from other clinical means. Follow-up or autopsy are the major means of verification. In a study comparing bone marrow acid phosphatase, bone biopsy and radiological techniques, it was claimed that bone marrow acid phosphatase was more sensitive2 in detecting metastatic disease in patients with prostate carcinoma. Several interesting observations are noted in this study. In table IX it is shown that bone marrow elevations are at least as sensitive as bone biopsy for detecting marrow involvement, since 41 out of 41 patients TABLE V I I Symptoms of Benign or Malignant Disease from Acid Phosphatase Levels Total Acid Phosphatase N o. Pos N o. Neg Tartrate Labile Acid Phosphatase N o. Pos N o. Neg Occult or confined carcinoma Benign hyperplasia Sensitivity 38% 12% Specificity 93% 97% P.V. (+)* 71% 67% P.V. (-)t 77% 71% Predictive value of a positive test. tpredictive value of a negative test. with positive marrow biopsies displayed elevated marrow acid phosphatase. In contrast, skeletal survey or serum acid phosphatase was elevated in 75 percent of these patients. This study also examined a group of patients with acid phosphatase measured in serum and bone marrow, compared with bone scans. Nine of 55 had positive bone scans, with one of these being a false positive from osteoarthritis. Acid phosphatase in bone marrow was elevated in all eight patients with true positive bone scans. Serum acid phosphatase was elevated in only five of eight. This is further evidence that bone marrow assays are at least as sensitive as biopsy or scanning methods to stage patients. T A B L E V I I I Use of Acid Phosphatase Assay to Distinguish Metastatic from Non-metastatic Carcinoma Total Acid Phosphatase No. Pos No. Neg Tartrate Labile Acid Phosphatase No. Pos No. Neg Metastatic carcinoma Non-metastatic carcinoma Sensitivity 88% 73% Specificity 55% 84% P.V. (+)* 60% 78% P.V. (-) + 85% 80% Predictive value of a positive test. tpredictive value of a negative test.

7 260 TOW NSEND TABLE IX Acid Phosphatase Levels8 32 Patients 41 Patients Positive Random Positive Random Bone Biopsy Bone Biopsy Pos. Skeletal Survey Acid Phosphatase Acid Phosphatase Serum Bone Marrow Serum Bone Marrow Elevated Normal The problems involved in deciding whether or not the test is better are seen in data from a group of 39 patients with negative bone scan and skeletal survey. Sixteen of these patients had elevated marrow acid phosphatase. This could be interpreted as increased sensitivity for metastatic disease or decreased specificity. Since five of these 16 patients subsequently developed overt metastatic bone involvement, the present authors postulate that elevation of bone marrow acid phosphatase is the earliest available indication of bone metastases and may contraindicate radical therapy. Further comparative studies would be of obvious importance. Although acid phosphatase assays may have limitations as screening or diagnostic tools within large groups of patients, serial determinations in patients with prostate carcinoma do serve as a guide to response to therapy. Post treatment falls in patients with favorable responses are seen with such regularity that acid phosphatase determination, although subject to limitations, would seem to have a role.7,14 Of interest in this regard is the study by Ishebe et al10 who studied five year survival in relation to changes in total and prostate specific acid phosphatase in 58 patients with histologic diagnosis of prostatic carcinoma who were treated with orchiectomy and estrogens. His findings were that there was no difference in survival with high versus normal pretreatment acid phosphatase levels. What was observed is that post-therapy changes, either assayed as total or prostate specific acid phosphatase, were related to survival. Those patients whose values decreased post-therapy greater than 50 percent of initial values had better five year survival figures than those who showed no change. The group with poorest survival figures were those patients whose acid phosphatase levels increased. Whether or not one used total acid phosphatase or prostate specific values was unimportant. This indicates that for monitoring a given patient, the quest for organ sensitivity in assays may not be so crucial. Other Enzymatic Approaches Recent interest has been shown in immunological11,12 and electrophoretic18,24 approaches to develop a sensitive and specific test for the presence of acid phosphatase from prostatic carcinoma. Clinical experience with these techniques is limited, but such studies may prove alternative techniques more satisfactory than current chemical methods. Another potentially useful area of investigation is the examination of lactic dehydrogenase isoenzymes in prostatic fluid. Interest in this arises from the observation16that when surgically removed prostates were studied, 86 percent of benign prostates showed lactic dehydrogenase (LDH) 5 < 1, while 78 percent of malignant specimens showed LDH 5 > 1. As might be expected, clinical3,9 studies involving LDH isoenzyme analysis of prostate fluid showed a similar sensitivity and specificity in detecting occult malignancy. Whether or not this is a significant advance is debatable. Summary Experience with acid phosphatase assays over the past 40 years has repeatedly

8 EN ZYM E TESTS IN DISEASES OF THE PROSTATE 261 demonstrated elevations in patients with prostatic carcinoma. However, the extent to which elevations are seen in patients without malignancy has limited the assay s clinical usefulness, as well as stimulated efforts for more specific assays. The fact that patients with even advanced prostatic carcinoma may not manifest abnormal levels has stimulated efforts for development of sensitive techniques. At the present time, the problem is not solved, and it would appear best to emphasize its use in monitoring therapy of patients with histologically demonstrated carcinoma, rather than use the test in the context of a screening or diagnostic test. References 1. Ba t s a k is, J. G., B r i e r e, R. O., and M a r k e l, S. F.: Diagnostic Enzymology. American Society of Clinical Pathologists Commission on Continuing Education, Chicago, C h u a, D. T., V e e n e m a, R. J., M u g g ia, F., and G r a f f, A.: Acid phosphatase levels in bone marrow: value in detecting early bone metastases from carcinoma of the prostate. J. Urol. J03: , C l a r k, S. S. and Sr in iv a s a n, V.: Correlation of lactic dehydrogenase isoenzymes in prostatic tissue with serum acid phosphatase, digital examination, and histological diagnosis. J. Urol, 209: , E w e n, L. M. and Sp it z e r, R. W.: Improved determination of prostatic acid phosphatase (sodium thymolphthalein monophosphate substrate). Clin. Chem. 22: , G a l e n, R. S. and G a m b i n o, S. R.: Beyond normality: The predictive value and efficiency of medical diagnosis. New York, J. Wiley and Sons, G r e e n e, F. T. and T h o m p s o n, I. M.: The effects of various manipulations on serum phosphatase levels in benign disease. J. Urol. 222: , G u t m a n, A. B.: The development of the acid phosphatase test for prostatic carcinoma. Bull. N.Y. Acad. Med. 444-,63-76, G u r s e l, E. O., R e s v a n, M., Sy, F. A., and VEENEMA, R. J.: Comparative evaluation of bone marrow acid phosphatase and bone scanning in staging of prostatic cancer. J. Urol. Ill: 53-57, H e i n, R. C., G r a y h a c k, J. T., and G o l d b e r g, E.: Prostatic fluid lactic dehydrogenase isoenzyme patterns of prostatic cancer and hyperplasia. J. Urol. 223: , 19??. 10. I s h ib e, T., U s u i, T., and N i h e r a, H.: Prognostic usefulness of serum acid phosphatase levels in carcinoma of the prostate. J. Urol. 112: , Li, C., C h u d a, R. A., and L a m, W. K. W.: Acid phosphatase in human plasma. J. Lab. Clin. Med. 82: , M o n c u r e, C. W J o h n s o n, C. L., Sm i t h, M. J. V., and K o o n t z, W. W.: Im m u n o lo g ic a l and histological evaluation o f m arrow aspirates in p atie n ts w ith p ro static c a rc in o m a. J. U rol. 208: , 19??. 13. M u r p h y, G. P., R e y n o s o, G., Ke n n y, G. M., and G a e t a, J. F.: Comparison of total and pro s tatic fraction serum acid phosphatase levels in patients with differentiated and undifferentiated prostatic carcinoma. Cancer 23: , M u r p h y, G. P.: The diagnosis of prostatic cancer. Cancer 37: , P o n t e s, J. E., A l c o r n, S. W., T h o m a s, A. J., and PIERCE, J. M.: Bone marrow acid phosphatase in staging prostatic carcinoma. J. Urol. 114: , O l i v e r, J. A., E l h i l a l e, M. M., B e l e t s k y, P., and M a c K in n o n, K. J.: L D H isoenzymes in benign and malignant prostate tissue: the LD H V/I ratio as an index of malignancy. Cancer 25: , P r o u t, G. R.: Chemical tests in the diagnosis of prostatic carcinoma. J. Amer. Med. Assoc. 209: , R e i f, A. E., Sc h l e s in g e r, R. M., F i s h, C. A., and ROBINSON, C. M.: Acid phosphatase isoenzymes in cancer of the prostate. Cancer 31: , R e y n o l d s, R. D., G r e e n b e r g, B. R., et al: Usefulness of bone marrow serum acid phosphatase in staging carcinoma of the prostate. Cancer 32: , R o y, A. V., B r o w e r, M. E., and'hayden, J. E.: S o d iu m th y m o lp h th a le in m o n o p h o sp h ate : a new acid phosphatase substrate w ith greater specificity for the prostatic enzym e in serum. C lin. C hem. 27: , Sc h w a r t z, M. K.: Enzymes in cancer. Clin. Chem. 29:10-22, S c h w a r t z, M. K.: Laboratory aids to diagnosis-enzymes. Cancer 37: , W i e d e r h o r n, A. R. and PICKENS, R. L.: Serum acid phosphatase levels following prostatic massage: a réévaluation. J. Urol. 209: , Y a m, L. T.: Clinical significance of the human acid phosphatase: a review. Amer. J. Med. 56: , 1974.