- Email: [email protected]
Combined Serum and Bone Marrow Radioimmunoassays for Prostatic Acid Phosphatase
0022-534 7/79/1223-0498$02.00/0 Vol. 122, October
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1979 by The Williams & Wilkins Co.
COMBINED SERUM AND BONE MARROW RADIOIMMUNOASSAYS FOR PROSTATIC ACID PHOSPHATASE J. FENIMORE COOPER, ANDRAS FOTI
AND
HARVEY HERSCHMAN
From the Division of Clinical Urology and Urological Research Laboratory, Kaiser Permanente Medical Center and the Department of Biological Chemistry and Nuclear Medicine, UCLA School of Medicine, Los Angeles, California
ABSTRACT
Combined serum and bone marrow radioimmunoassays for prostatic acid phosphatase provide a unique means for the early diagnosis and more accurate clinical staging of prostatic cancer. The combined screening technique appears to be helpful, particularly in providing a clinical assessment of the presence or absence of early, subclinical lymphatic and bone marrow metastases. Low titer elevations of bone marrow prostatic acid phosphatase by radioimmunoassay have been observed commonly in clinically understaged C prostatic cancer with normal technetium bone scans, indicating the presence of unrecognized stage D disease with bone metastases. The combined screening method also is of distinct clinical value in the early diagnosis of prostatic cancer and in monitoring the effects of specific therapy. In therapeutically responsive patients marked suppression of serum and bone marrow prostatic acid phosphatase is observed regularly with the radioimmunochemical method under study. The original observations of Flocks and associates, 1 Macalalag and Prout,2 Peel,3 Moncure and associates,4 and Moncure and Prout5 on the antigenic nature ofprostatic protein provided a firm basis for the subsequent development of potential immunochemical methods for the detection and quantitation of serum prostatic acid phosphatase. Our recent studies, based on the aforementioned data, have introduced a promising solid phase radioimmunoassay for serum prostatic acid phosphatase that has been used clinically as an aid in the early diagnosis, staging and post-treatment evaluation of prostatic cancer.6- 10 In addition, in the last year the original radioimmunoassay technique for serum prostatic acid phosphatase has been adapted to the measurement of bone marrow prostatic acid phosphatase. 11 This was necessitated by the clinical observation that standard enzymatic methods for bone marrow acid phosphatase are associated commonly with an unusually high incidence of falsely positive results, as reported by Dias and Barnett, 12 and Kahn and associates. 13 It should be emphasized that the inherent error with enzymatic methodology is owing to the cross-reactivity of non-prostatic acid phosphatases in erythrocytes, leukocytes, platelets and other bone marrow cells in maturation. As Dias and Barnett, 12 and Khan and associates13 have indicated transsacral and sternal bone marrow biopsies are traumatic in terms of cellular destruction. As a result, nonprostatic acid phosphatases biochemically contaminate the essential prostatic acid phosphatase on aspiration of the bone marrow sample. In contrast to radioimmunoassay methodology, current standard enzymatic techniques for the estimation of serum or bone marrow acid phosphatase are not able to discriminate between phosphatases of prostatic epithelial origin and those present in erythrocytes and normal bone marrow cells. Our study is devoted to preliminary observations on the clinical advantages of combining serum and bone marrow radioimmunoassays for prostatic acid phosphatase in the clinical diagnosis, staging and evaluation of therapeutic measures for prostatic cancer.
piration in the standard fashion. Five to 10 cc venous blood and 3 cc bone marrow aspirate were centrifuged at 3 to 5,000 revolutions per minute and the serum was separated. A 0.1 Inl. sample was used for radioimmunoassay by the solid phase method. 7 We originally reported the normal ranges for serum and bone marrow prostatic acid phosphatase as 0.5 to 6.4 ng./ 0.1 Inl. serum and 4.7 to 15.0 ng./0.1 Inl. bone marrow supernate.11 In the current study of bone marrow samples the controls were augmented to 54 samples and the total combined serum and bone marrow samples to 179. All bone marrow radioimmunoassay prostatic acid phosphatase values >15 ng./0.1 Inl. were considered abnormally elevated. All serum samples >6.5 ng./0.1 Inl. were considered pathologic. Mean values for serum and bone marrow were 4.8 and 9.2 ng./0.1 Inl., respectively. The patient is placed in a lateral decubitus position with the sacrum presenting to the physician. The lateral sacral prominence is identified and the overlying skin and periosteum are infiltrated with 2 cc 1 per cent lidocaine solution. A suitable bone marrow biopsy needle, preferably the J amshidi needle,* is introduced in a rotating maneuver through the cortex of the posterior iliac tubercle. As the marrow cavity is entered there is an abrupt loss of resistance to the penetration of the needle and aspiration of marrow substance can be secured with a tightly fitting glass or plastic 10 cc syringe. Then 3 cc bone marrow substance should be aspirated slowly, placed in a glass tube without preservative and transported to the laboratory for centrifugation as soon as possible. Only 0.1 cc serum is required for assay. The remainder of the sample can be placed in a serum bank at -25 to 70C for future referral. A routine bone marrow biopsy also can be secured with the J amshidi needle after bone marrow aspiration for radioimmunoassay purposes. However, the biopsy results have not been consistently reliable or productive in our experience even in the presence of stage D disease.
METHODS AND MATERIALS
RESULTS
Serum and bone marrow samples were secured simultaneously by antecubital venipuncture and posterior ilial crest as-
During an interval of 18 months we have had the opportunity to survey 179 prostatic cancer patients with combined serum and bone marrow radioimmunoassays for prostatic acid phosphatase (table 1). Of these 179 cases 67 had pristine stages A,
TECHNIQUE OF BONE MARROW BIOPSY
Accepted for publication September 8, 1978. Read at annual meeting of American Urological Association, Washington, D. C., May 21-25, 1978. * Kormed, Inc., Minneapolis, Minnesota. 498
499
RADIOIMMUNOASSAYS FOR PROSTATIC ACID PHOSPHATASE
B, C and D cancer before any form of therapy and are the subject of this report. Of the remainder 16 had stages Bl and B2 cancer after radical prostatectomy, 49 had stages Cl and C2 cancer after supervoltage radiation therapy and 47 had stage D cancer after endocrine manipulation with orchiectomy and diethylstilbestrol administration. The post-treatment cases served for the evaluation of serum and bone marrow radioimmunoassays in terms of therapeutic response. Four types of radioimmunoassay prostatic acid phosphatase screens have been observed for the 67 untreated cases of prostatic cancer reported. Type I screen: normal serum and normal bone marrow radioimmunoassays. Serum and bone marrow radioimmunoassay prostatic acid phosphatase values are observed to be within normal limits (serum radioimmunoassay 0.5 to 6.5 ng., bone marrow radioimmunoassay 4.0 to 15 ng.). A type I screen may indicate that a well differentiated prostatic cancer is not present, although a single microfocal Al cancer or a small, poorly differentiated, transcriptionally altered cancer of the prostate cannot be eliminated. Type II screen: positive serum and negative bone marrow radioimmunoassays. A positive type H screen may indicate the presence of a well differentiated prostatic cancer. A low titer serum radioimmunoassay of 7.0 to 15 ng. with a negative bone marrow suggests localized intraprostatic cancer, well differentiated, stages A2, Bl, B2 and ? Cl. A high titer serum radioimmunoassay of 15 to 50 ng. with a negative bone marrow may indicate extraprostatic cancer of the prostate in regional, pelvic or retroperitoneal lymph nodes. In a typical type II screen no elevation of bone marrow radioimmunoassay prostatic acid phosphatase should be encountered, indicating the absence of bone marrow metastases. In the presence of evidence ofregional lymph node metastases such findings should require restaging of the cancer to stage D. Type III screen: positive serum and bone marrow radioimmunoassays. A type III screen indicates an advanced, extraprostatic, well differentiated prostatic cancer in regional lymphatics and bone marrow. Low titers of <30 ng. for each radioimmunoassay denote a lesser tumor burden. High titers of 50 to 100 ng. for both radioimmunoassays indicate a significant extraprostatic tumor burden in lymphatics and bone marrow. Very high simultaneous titers for each radioimmunoassay, ranging from 300 to 400 ng., usually are evidence of far-advanced disease invading soft tissue, lymphatics and bone marrow. Type IV screen: negative serum and positive bone marrow radioimmunoassays. A type IV screen in our experience is a rare entity. It possibly may be seen in primary or secondary bone neoplasia, Gaucher's disease, and metastatic pancreatic 1. Clinical experience (1977-1978) with combined serum and bone marrow radio immunoassay prostatic acid phosphatase
TABLE
No. Cases Pristine Ca of prostate, stages A, B, C and D After supervoltage radiation, stages B and C After supervoltage radiation, recurrence After endocrine manipulation, stage D After radical prostatectomy, stages A2 and B After radical prostatectomy with recurrence 'rota!
TABLE
67 39 10
47 13 3 179
and pulmonary cancer, which may in rare in~tan~e~ ~roduce cross-reactive acid phosphatases ofnon-prostatic ongm m bone marrow. STAGE VERSUS SCREEN
Table 2 presents the relation of clinical stage to the radioimmunoassay screening method in 67 cases of prostatic cancer before treatment. A type I screen was found in 11 of 15 cases (73 per cent) of stage A cancer, 10 of 20 cases (50 per cent) of stage B cancer, 3 of 16 cases (18 per cent) of stage C cancer and 0 per cent of stage D cancer. A type II screen presented in 4 of 15 cases (27 per cent) of stage A cancer, 10 of 20 cases (50 per cent) of stage B cancer, 13 of 16 cases (82 per cent) of stage C cancer and 3 of 16 cases (19 per cent) of stage D cancer. The elevated radioimmunoassay values were in low titer in all of the aforementioned cases presenting at <30 ng. A type HI radioimmunoassay screen was not observed in 15 cases of stage A cancer, However, it was observed in 3 of 20 cases (15 per cent) of stage B cancer and 6 of 16 cases (38 per cent) of stage C cancer. These observations indicated that the 3 stage B and 6 stage C cancers obviously were understaged clinically ~nd we~e, in fact, probably early, unrecognized stage D prostatic mahgnancies. In 16 cases of stage D cancer 13 (81 per cent) demonstrated high titer serum and bone marrow radioimmunoassay values, each >50 ng. In many instances serum and bone marrow radioimmunoassay values were recorded in the hundreds of ng./0.1 ml. in advanced stage D disease. CLINICAL MISSTAGING
Figure 1 presents a review of the incidence of clinical misstaging in 104 cases of stage C prostatic cancer submitted for supervoltage radiation therapy in our institution. All cases had fulfilled the standard criteria for the clinical diagnosis of stage C cancer on rectal examination and, in addition, had manifested normal enzymatic acid and alkaline phosphatases, normal bone surveys and bone scans. No routine staging pelvic lymphadenectomies were done in this series. Within 5 years 33 patients (31 per cent) had stage D disease clinically with evidence of hyperphosphatasemia and positive bone scans, while 21 (20 per cent) had clinical evidence of metastatic bone disease within 3 years and 12 additional patients (11 per cent) within 5 years. There was an over-all clinical staging error in 104 stage C patients of 31 per cent. In our early clinical experience with the specific use of combined serum and bone marrow radioimmunoassay prostatic acid phosphatase screening methods for stages C and D cancer 6 exemplary cases presented with characteristic type III screens (table 3). Four were typical stage D radioimmunoassay screens with relatively high titer serum and bone marrow values for prostatic acid phosphatase, abnormal enzymatic acid and alkaline phosphatase values, and positive bone scans. The 2 patients clinically staged to have Cl and C2 cancer were observed to have low titer elevations of radioimmunoassay prostatic acid phosphatase in serum and bone marrow associated with normal values for enzymatic acid and alkaline phosphatase, and normal bone scans. The radioimmunoassay screen, thus, indicated evidence of early bone marrow metastases and each stage C cancer was, therefore, restaged to stage D and
2. Relation of clinical stage to serum and bone marrow radioimmunoassay screening technique in 67 cases of prostatic cancer
Screen Type I Type II Type III Type IV Total No. cases
Al-A2 No. Cases(%)
Bl-B2 No. Cases(%)
Cl-C2 No. Cases(%)
D No. Cases(%)
11 (73) 4 (27) 0
10 (50) 10 (50) 3 (15) (understaged) 0 20
3 (18) 13 (82) 6 (38)
3 (19)
0 15
* Serum <30 ng., bone marrow <50 ng.
t Serum >50 ng., bone marrow >50 ng.
(understaged) 0 16
0 13 (81) 0 16
Serum Radioimmunoassay
B_o1;1e Marrow Rad101mmunoassay
Neg. Low titer* High titert
Neg. Neg. High titert
Neg.
Pos.
500
COOPER, FOTI AND HERSCHMAN
treated by endocrine manipulation. Without the positive bone marrow radioimmunoassay prostatic acid phosphatase and in view, particularly, of the negative bone scan, the 2 misstaged C cancers would otherwise have been subjected to supervoltage radiation in the presence of unrecognized bone metastases. Identical findings were encountered subsequently in 7 additional cases of stage C cancer requiring restaging. On this basis it is recommended that all cases of stage C prostatic cancer under consideration for possible supervoltage or 1251 implanta-
I 04 STAGE C CANCERS NORMAL ENZYMATIC PTASE, BONE SURVEY, BONE SCAN
I
7000-7400 RADS SV-RT
I
/l~
STAGE D within 3yr 21/20%
STAGE D within 5 yr 12/11%
lDTAL STAGE D within 5 yr 33/31%
SV- RT = Supervoltage Radiation Therapy
Fm. 1. Clinical misstaging in stage C prostatic cancer submitted for supervoltage radiation. TABLE
3. Type III radioimmunoassay prostatic acid phosphatase screen-stages C to D
Serum Radioimmunoassay
Bone Marrow Radioimmunoassay
Cl C2 D
21.6 23.0 34.2
29.8 33.4 40.0
D D
162 240 345 0.5-6.5 ng.
186 265 200 2.5-15 ng.
Stage
tion therapy have specific radioimmunoassays for prostatic acid phosphatase in bone marrow aspirates to eliminate the possibility of early bone marrow metastases. Such a measure would avoid considerable physical distress incident to the proposed radiation and provide reduced cost to the patient. In addition, the more rarely misstaged A2, Bl and B2 cancers that are, in fact, clinically unrecognized stage D lesions can be detected commonly by the type III form of the combined serum and bone marrow radioimmunoassay screening technique. The effect of transurethral resection, radical prostatectomy and hormonal therapy on the serum radioimmunoassay prostatic acid phosphatase was noted (fig. 2). Serial serum samples drawn during transurethral resection of benign and malignant prostates demonstrate dramatically different curves during an interval of 72 hours. The radioimmunoassay prostatic acid phosphatase curve in benign tissue is of short duration, usually <24 to 30 hours, and increases to a peak of 200 to 250 ng. at the close of operation. Resolution to normal levels occurs usually within 24 hours. In contrast, in malignant prostates the curve increases to higher levels and there is a slower resolution to normal levels during an interval of 48 to 72 hours. The curve after radical prostatectomy for stage B cancer decreases rapidly to low levels in the range of 2.5 to 5 ng. and usually will remain at that level unless recurrent disease becomes apparent. A similar resolution to low levels of serum and bone marrow radioimmunoassay prostatic acid phosphatase is noted after a therapeutic clinical response to orchiectomy and diethylstilbestrol administration. Supervoltage radiation also will suppress the serum radioimmunoassay prostatic acid phosphatase level to low levels in radiosensitive stage C cancers.
Enzyme Phosphatase
Alkaline Phosphatase
Bone Scan
Therapy
38 35 72
Neg. Neg. Pos.
Restaged D Restaged D End manipulation
120 50 134 25-40 JL.
Pos. Pos. Pos.
End manipulation End manipulation End manipulation
Low titer values 0.7 0.2 1.4
High titer values
D Controls
4.5 12.8 21.5 0.1-1.1 JL.
500 BPH-TURP*
200
CAP-TURP* Stage C
200
z
~
ii:
100
100
10
10 0
0 OP
24
500
~
ii:
72 Hours
OP
24
48
72 Hours
Orchiectom}'. + DES* Stage D
Radical Prostatectom}'. * Stage 82
z
48
200
200
100
100
10
10
o-~-----,rt---i OP
24
48
72 Hours
6 Months
2
3
4 Months
Fm. 2. Effect of various forms of therapy on serum radioimmunoassay levels of prostatic acid phosphatase
RADIOIMMUNOASSAYS FOR PROSTATIC ACID PHOSPHATASE
DISCUSSION
Preliminary clinical experience with the immunochemical approach for human serum and bone marrow acid phosphatase has provided reliable clinical data with a minimum of falsely positive results. 9•11 Serum and bone marrow samples are relatively simply acquired from suspect patients and, when performed simultaneously as a screening technique, may provide a unique clinical method for more precise staging of the disease process. When combined with staging pelvic lymphadenectomy and technetium bone scan they may provide the ultimate in clinical staging technique. However, the radioimmunoassay methodology in its present form is patently developmental and may well require subsequent modification as clinical experience demands. In the present state of the art radioimmunoassay prostatic acid phosphatase results may, at times, appear discordant in application. The inconsistencies are due to variations in the specificity of prostatic acid phosphatase antibody. As Amon has indicated all enzymes are characterized by the presence of multiple specificity determinants on the antigenic protein molecule, which may induce measurable heterogeneity in antibody production. 14 Subsequent individual prostatic acid phosphatase antisera may, therefore, vary in their capacity to react with the antigenic sites on the prostatic acid phosphatase molecule. Macalalag and Prout,2 Peel,3 and Flocks and associates 1 were among the first to describe inconsistent specificities with canine antisera to prostatic proteins. Such technical problems may be resolved in great part with improved methods for isolation and purification of prostatic acid phosphatase antigen and rigid standardization of prostatic acid phosphatase antibody. Federal Drug Administration regulations will require such measures before commercial radioimmunoassay kits for prostatic acid phosphatase can be released for general clinical and laboratory use. Inconsistent clinical results will relate invariably to these basic principles. Application of the serum and bone marrow radioimmunoassays as a screening technique in prostatic cancer has demonstrated considerable utility in the initial staging of the primary disease process. It is appapent that the differentiation of intraprostatic from extraprostatic cancer is feasible by means of the relative characteristics of the types II and III radioimmunoassay screens. Certainly, it is apparent that a type II screen is mandatory when considering stages A2, B or C patients for radical prostatectomy, 125I implants or supervoltage radiation therapy, since with a normal bone marrow radioimmunoassay for prostatic acid phosphatase one can assume confidently that no bone marrow metastases are present. It is possible that lymph node metastases may be differentiated clinically from bone metastases by a type II radioimmunoassay screen presenting with a relatively high titer serum radioimmunoassay and normal bone marrow radioimmunoassay. Regional lymph node and early bone marrow metastases also may be substantiated by a type III screen when the serum radioimmunoassay prostatic acid phosphatase is elevated moderately and the bone marrow radioimmunoassay prostatic acid phosphatase is of low titer, <25 to 50 ng. Such information might well be invaluable in a clinically understaged stage C cancer under consideration for radiation since the data would indicate that early bone marrow metastases are probably present. Very high titers for serum and bone marrow radioimmunoassays in type III screens usually are an absolute indication of heavy tumor burden in soft tissues, lymph nodes and bone. In contrast, the intraprostatic cancers A2, Bl and B2 confined within the prostatic capsule are typically type II radioimmunoassay screen with low titer serum elevations and normal bone marrow values. A relatively high titer prostatic acid phosphatase serum concentration in a type II screen may indicate regional lymph node metastases. Such an observation would indicate the necessity for restaging and should be confirmed by
501
pelvic lymphadenectomy and retroperitoneal lymphatic evaluation. The possibility of falsely positive bone marrow radioimmunoassay prostatic acid phosphatase results is certainly of concern to the authors. However, cross-reactivity with normal acid phosphohydrolases in normal bone marrow cells has not been apparent in our experience to date. No falsely positive bone marrow radioimmunoassays were encountered in the early intracapsular prostatic cancers reported herein. However, we have reported falsely positive radioimmunoassay prostatic acid phosphatase values in the serum of patients with advanced bronchogenic cancers but in no other forms of malignancy. A double blind approach to this problem would be advantageous but was not used in this study. Resolution of abnormal types II and III radioimmunoassay screens to normality has been observed commonly in stages B, C and D patients responding well to specific therapies, such as radical retropubic prostatectomy, supervoltage radiation therapy and endocrine therapy. Recurrence and progression of the disease to stage D usually are accompanied by the development of a typical type III radioimmunoassay screen. It is probable that continuing clinical experience with the immunochemical measurement of human serum and bone marrow prostatic acid phosphatase may provide the clinician with significant amplification of diagnostic and therapeutic options in the management of prostatic cancer. In the brief period of time and the modest number of cases studied with the combined serum and bone marrow radioimmunoassay prostatic acid phosphatase method described it is apparent clinically that a more precise and sensitive staging technique for prostatic cancer may now be feasible. Clinical confirmation and specific delineation of the concept by other prostatic oncologists are recommended. REFERENCES
1. Flocks, R. H., Urich, V. C., Patel, C. A. and Opitz, J.M.: Studies on the antigenic properties of prostatic tissue I. J. Urol., 84: 134, 1960. 2. Macalalag, E. V., Jr. and Prout, G. R., Jr.: Anti-canine prostatic fluid serum and prostatic degeneration. Invest. Urol., 4: 321, 1967. 3. Peel, S.: An immunologic study of dog prostate and the effects of injecting antidog prostate serum. Invest. Urol., 5: 427, 1968. 4. Moncure, C. W., Prout, G. R., Jr. and Blaylock, W. K.: Prostatic acid phosphatase antisera. Invest. Urol., 5: 331, 1968. 5. Moncure, C. W. and Prout, G. R., Jr.: Antigenicity of human prostatic acid phosphatase. Cancer, 25: 463, 1970. 6. Cooper, J. F. and Foti, A.: A radioimmunoassay for prostatic acid phosphatase. I. Methodology and range of normal male serum values. Invest. Urol., 12: 98, 1974. 7. Foti, A., Herschman, H. and Cooper, J. F.: A solid-phase radioimmunoassay for human prostatic acid phosphatase. Cancer Res., 35: 2446, 1975. 8. Cooper, J. F., Foti, A. and Imfeld, H.: Production of specific antibody to purified prostatic acid phosphatase. Urol. Res., 4: 111, 1976. 9. Foti, A. G., Cooper, J. F., Herschman, H. and Malvaez, R. R.: Detection of prostatic cancer by solid-phase radioimmunoassay of serum prostatic acid phosphatase. New Engl. J. Med., 297: 1357, 1977. 10. Cooper, J. F., Foti, A., Herschman, H. H. and Finkle, W.: A solid phase radioimmunoassay for prostatic acid phosphatase. J. Urol., 119: 388, 1978. 11. Cooper, J. F., Foti, A. G. and Shank, P. W.: Radioimmunochemical measurement of bone marrow prostatic acid phosphatase. J. Urol., 119: 392, 1978. 12. Dias, S. M. and Barnett, R. N.: Elevated bone marrow acid phosphatase: the problem of false positives. J. Urol., 117: 749, 1977. 13. Kahn, R. M., Turner, B., Edson, M. and Dolan, M.: Bone marrow acid phosphatase: another look. J. Urol., 117: 79, 1977. 14. Amon, R.: Immunochemistry oflysozyme. In: Immunochemistry of Enzymes and Their Antibodies. Edited by M. R. J. Salton. New York: John Wiley & Sons, Ltd., p. 1, 1977.
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1979 by The Williams & Wilkins Co.
COMBINED SERUM AND BONE MARROW RADIOIMMUNOASSAYS FOR PROSTATIC ACID PHOSPHATASE J. FENIMORE COOPER, ANDRAS FOTI
AND
HARVEY HERSCHMAN
From the Division of Clinical Urology and Urological Research Laboratory, Kaiser Permanente Medical Center and the Department of Biological Chemistry and Nuclear Medicine, UCLA School of Medicine, Los Angeles, California
ABSTRACT
Combined serum and bone marrow radioimmunoassays for prostatic acid phosphatase provide a unique means for the early diagnosis and more accurate clinical staging of prostatic cancer. The combined screening technique appears to be helpful, particularly in providing a clinical assessment of the presence or absence of early, subclinical lymphatic and bone marrow metastases. Low titer elevations of bone marrow prostatic acid phosphatase by radioimmunoassay have been observed commonly in clinically understaged C prostatic cancer with normal technetium bone scans, indicating the presence of unrecognized stage D disease with bone metastases. The combined screening method also is of distinct clinical value in the early diagnosis of prostatic cancer and in monitoring the effects of specific therapy. In therapeutically responsive patients marked suppression of serum and bone marrow prostatic acid phosphatase is observed regularly with the radioimmunochemical method under study. The original observations of Flocks and associates, 1 Macalalag and Prout,2 Peel,3 Moncure and associates,4 and Moncure and Prout5 on the antigenic nature ofprostatic protein provided a firm basis for the subsequent development of potential immunochemical methods for the detection and quantitation of serum prostatic acid phosphatase. Our recent studies, based on the aforementioned data, have introduced a promising solid phase radioimmunoassay for serum prostatic acid phosphatase that has been used clinically as an aid in the early diagnosis, staging and post-treatment evaluation of prostatic cancer.6- 10 In addition, in the last year the original radioimmunoassay technique for serum prostatic acid phosphatase has been adapted to the measurement of bone marrow prostatic acid phosphatase. 11 This was necessitated by the clinical observation that standard enzymatic methods for bone marrow acid phosphatase are associated commonly with an unusually high incidence of falsely positive results, as reported by Dias and Barnett, 12 and Kahn and associates. 13 It should be emphasized that the inherent error with enzymatic methodology is owing to the cross-reactivity of non-prostatic acid phosphatases in erythrocytes, leukocytes, platelets and other bone marrow cells in maturation. As Dias and Barnett, 12 and Khan and associates13 have indicated transsacral and sternal bone marrow biopsies are traumatic in terms of cellular destruction. As a result, nonprostatic acid phosphatases biochemically contaminate the essential prostatic acid phosphatase on aspiration of the bone marrow sample. In contrast to radioimmunoassay methodology, current standard enzymatic techniques for the estimation of serum or bone marrow acid phosphatase are not able to discriminate between phosphatases of prostatic epithelial origin and those present in erythrocytes and normal bone marrow cells. Our study is devoted to preliminary observations on the clinical advantages of combining serum and bone marrow radioimmunoassays for prostatic acid phosphatase in the clinical diagnosis, staging and evaluation of therapeutic measures for prostatic cancer.
piration in the standard fashion. Five to 10 cc venous blood and 3 cc bone marrow aspirate were centrifuged at 3 to 5,000 revolutions per minute and the serum was separated. A 0.1 Inl. sample was used for radioimmunoassay by the solid phase method. 7 We originally reported the normal ranges for serum and bone marrow prostatic acid phosphatase as 0.5 to 6.4 ng./ 0.1 Inl. serum and 4.7 to 15.0 ng./0.1 Inl. bone marrow supernate.11 In the current study of bone marrow samples the controls were augmented to 54 samples and the total combined serum and bone marrow samples to 179. All bone marrow radioimmunoassay prostatic acid phosphatase values >15 ng./0.1 Inl. were considered abnormally elevated. All serum samples >6.5 ng./0.1 Inl. were considered pathologic. Mean values for serum and bone marrow were 4.8 and 9.2 ng./0.1 Inl., respectively. The patient is placed in a lateral decubitus position with the sacrum presenting to the physician. The lateral sacral prominence is identified and the overlying skin and periosteum are infiltrated with 2 cc 1 per cent lidocaine solution. A suitable bone marrow biopsy needle, preferably the J amshidi needle,* is introduced in a rotating maneuver through the cortex of the posterior iliac tubercle. As the marrow cavity is entered there is an abrupt loss of resistance to the penetration of the needle and aspiration of marrow substance can be secured with a tightly fitting glass or plastic 10 cc syringe. Then 3 cc bone marrow substance should be aspirated slowly, placed in a glass tube without preservative and transported to the laboratory for centrifugation as soon as possible. Only 0.1 cc serum is required for assay. The remainder of the sample can be placed in a serum bank at -25 to 70C for future referral. A routine bone marrow biopsy also can be secured with the J amshidi needle after bone marrow aspiration for radioimmunoassay purposes. However, the biopsy results have not been consistently reliable or productive in our experience even in the presence of stage D disease.
METHODS AND MATERIALS
RESULTS
Serum and bone marrow samples were secured simultaneously by antecubital venipuncture and posterior ilial crest as-
During an interval of 18 months we have had the opportunity to survey 179 prostatic cancer patients with combined serum and bone marrow radioimmunoassays for prostatic acid phosphatase (table 1). Of these 179 cases 67 had pristine stages A,
TECHNIQUE OF BONE MARROW BIOPSY
Accepted for publication September 8, 1978. Read at annual meeting of American Urological Association, Washington, D. C., May 21-25, 1978. * Kormed, Inc., Minneapolis, Minnesota. 498
499
RADIOIMMUNOASSAYS FOR PROSTATIC ACID PHOSPHATASE
B, C and D cancer before any form of therapy and are the subject of this report. Of the remainder 16 had stages Bl and B2 cancer after radical prostatectomy, 49 had stages Cl and C2 cancer after supervoltage radiation therapy and 47 had stage D cancer after endocrine manipulation with orchiectomy and diethylstilbestrol administration. The post-treatment cases served for the evaluation of serum and bone marrow radioimmunoassays in terms of therapeutic response. Four types of radioimmunoassay prostatic acid phosphatase screens have been observed for the 67 untreated cases of prostatic cancer reported. Type I screen: normal serum and normal bone marrow radioimmunoassays. Serum and bone marrow radioimmunoassay prostatic acid phosphatase values are observed to be within normal limits (serum radioimmunoassay 0.5 to 6.5 ng., bone marrow radioimmunoassay 4.0 to 15 ng.). A type I screen may indicate that a well differentiated prostatic cancer is not present, although a single microfocal Al cancer or a small, poorly differentiated, transcriptionally altered cancer of the prostate cannot be eliminated. Type II screen: positive serum and negative bone marrow radioimmunoassays. A positive type H screen may indicate the presence of a well differentiated prostatic cancer. A low titer serum radioimmunoassay of 7.0 to 15 ng. with a negative bone marrow suggests localized intraprostatic cancer, well differentiated, stages A2, Bl, B2 and ? Cl. A high titer serum radioimmunoassay of 15 to 50 ng. with a negative bone marrow may indicate extraprostatic cancer of the prostate in regional, pelvic or retroperitoneal lymph nodes. In a typical type II screen no elevation of bone marrow radioimmunoassay prostatic acid phosphatase should be encountered, indicating the absence of bone marrow metastases. In the presence of evidence ofregional lymph node metastases such findings should require restaging of the cancer to stage D. Type III screen: positive serum and bone marrow radioimmunoassays. A type III screen indicates an advanced, extraprostatic, well differentiated prostatic cancer in regional lymphatics and bone marrow. Low titers of <30 ng. for each radioimmunoassay denote a lesser tumor burden. High titers of 50 to 100 ng. for both radioimmunoassays indicate a significant extraprostatic tumor burden in lymphatics and bone marrow. Very high simultaneous titers for each radioimmunoassay, ranging from 300 to 400 ng., usually are evidence of far-advanced disease invading soft tissue, lymphatics and bone marrow. Type IV screen: negative serum and positive bone marrow radioimmunoassays. A type IV screen in our experience is a rare entity. It possibly may be seen in primary or secondary bone neoplasia, Gaucher's disease, and metastatic pancreatic 1. Clinical experience (1977-1978) with combined serum and bone marrow radio immunoassay prostatic acid phosphatase
TABLE
No. Cases Pristine Ca of prostate, stages A, B, C and D After supervoltage radiation, stages B and C After supervoltage radiation, recurrence After endocrine manipulation, stage D After radical prostatectomy, stages A2 and B After radical prostatectomy with recurrence 'rota!
TABLE
67 39 10
47 13 3 179
and pulmonary cancer, which may in rare in~tan~e~ ~roduce cross-reactive acid phosphatases ofnon-prostatic ongm m bone marrow. STAGE VERSUS SCREEN
Table 2 presents the relation of clinical stage to the radioimmunoassay screening method in 67 cases of prostatic cancer before treatment. A type I screen was found in 11 of 15 cases (73 per cent) of stage A cancer, 10 of 20 cases (50 per cent) of stage B cancer, 3 of 16 cases (18 per cent) of stage C cancer and 0 per cent of stage D cancer. A type II screen presented in 4 of 15 cases (27 per cent) of stage A cancer, 10 of 20 cases (50 per cent) of stage B cancer, 13 of 16 cases (82 per cent) of stage C cancer and 3 of 16 cases (19 per cent) of stage D cancer. The elevated radioimmunoassay values were in low titer in all of the aforementioned cases presenting at <30 ng. A type HI radioimmunoassay screen was not observed in 15 cases of stage A cancer, However, it was observed in 3 of 20 cases (15 per cent) of stage B cancer and 6 of 16 cases (38 per cent) of stage C cancer. These observations indicated that the 3 stage B and 6 stage C cancers obviously were understaged clinically ~nd we~e, in fact, probably early, unrecognized stage D prostatic mahgnancies. In 16 cases of stage D cancer 13 (81 per cent) demonstrated high titer serum and bone marrow radioimmunoassay values, each >50 ng. In many instances serum and bone marrow radioimmunoassay values were recorded in the hundreds of ng./0.1 ml. in advanced stage D disease. CLINICAL MISSTAGING
Figure 1 presents a review of the incidence of clinical misstaging in 104 cases of stage C prostatic cancer submitted for supervoltage radiation therapy in our institution. All cases had fulfilled the standard criteria for the clinical diagnosis of stage C cancer on rectal examination and, in addition, had manifested normal enzymatic acid and alkaline phosphatases, normal bone surveys and bone scans. No routine staging pelvic lymphadenectomies were done in this series. Within 5 years 33 patients (31 per cent) had stage D disease clinically with evidence of hyperphosphatasemia and positive bone scans, while 21 (20 per cent) had clinical evidence of metastatic bone disease within 3 years and 12 additional patients (11 per cent) within 5 years. There was an over-all clinical staging error in 104 stage C patients of 31 per cent. In our early clinical experience with the specific use of combined serum and bone marrow radioimmunoassay prostatic acid phosphatase screening methods for stages C and D cancer 6 exemplary cases presented with characteristic type III screens (table 3). Four were typical stage D radioimmunoassay screens with relatively high titer serum and bone marrow values for prostatic acid phosphatase, abnormal enzymatic acid and alkaline phosphatase values, and positive bone scans. The 2 patients clinically staged to have Cl and C2 cancer were observed to have low titer elevations of radioimmunoassay prostatic acid phosphatase in serum and bone marrow associated with normal values for enzymatic acid and alkaline phosphatase, and normal bone scans. The radioimmunoassay screen, thus, indicated evidence of early bone marrow metastases and each stage C cancer was, therefore, restaged to stage D and
2. Relation of clinical stage to serum and bone marrow radioimmunoassay screening technique in 67 cases of prostatic cancer
Screen Type I Type II Type III Type IV Total No. cases
Al-A2 No. Cases(%)
Bl-B2 No. Cases(%)
Cl-C2 No. Cases(%)
D No. Cases(%)
11 (73) 4 (27) 0
10 (50) 10 (50) 3 (15) (understaged) 0 20
3 (18) 13 (82) 6 (38)
3 (19)
0 15
* Serum <30 ng., bone marrow <50 ng.
t Serum >50 ng., bone marrow >50 ng.
(understaged) 0 16
0 13 (81) 0 16
Serum Radioimmunoassay
B_o1;1e Marrow Rad101mmunoassay
Neg. Low titer* High titert
Neg. Neg. High titert
Neg.
Pos.
500
COOPER, FOTI AND HERSCHMAN
treated by endocrine manipulation. Without the positive bone marrow radioimmunoassay prostatic acid phosphatase and in view, particularly, of the negative bone scan, the 2 misstaged C cancers would otherwise have been subjected to supervoltage radiation in the presence of unrecognized bone metastases. Identical findings were encountered subsequently in 7 additional cases of stage C cancer requiring restaging. On this basis it is recommended that all cases of stage C prostatic cancer under consideration for possible supervoltage or 1251 implanta-
I 04 STAGE C CANCERS NORMAL ENZYMATIC PTASE, BONE SURVEY, BONE SCAN
I
7000-7400 RADS SV-RT
I
/l~
STAGE D within 3yr 21/20%
STAGE D within 5 yr 12/11%
lDTAL STAGE D within 5 yr 33/31%
SV- RT = Supervoltage Radiation Therapy
Fm. 1. Clinical misstaging in stage C prostatic cancer submitted for supervoltage radiation. TABLE
3. Type III radioimmunoassay prostatic acid phosphatase screen-stages C to D
Serum Radioimmunoassay
Bone Marrow Radioimmunoassay
Cl C2 D
21.6 23.0 34.2
29.8 33.4 40.0
D D
162 240 345 0.5-6.5 ng.
186 265 200 2.5-15 ng.
Stage
tion therapy have specific radioimmunoassays for prostatic acid phosphatase in bone marrow aspirates to eliminate the possibility of early bone marrow metastases. Such a measure would avoid considerable physical distress incident to the proposed radiation and provide reduced cost to the patient. In addition, the more rarely misstaged A2, Bl and B2 cancers that are, in fact, clinically unrecognized stage D lesions can be detected commonly by the type III form of the combined serum and bone marrow radioimmunoassay screening technique. The effect of transurethral resection, radical prostatectomy and hormonal therapy on the serum radioimmunoassay prostatic acid phosphatase was noted (fig. 2). Serial serum samples drawn during transurethral resection of benign and malignant prostates demonstrate dramatically different curves during an interval of 72 hours. The radioimmunoassay prostatic acid phosphatase curve in benign tissue is of short duration, usually <24 to 30 hours, and increases to a peak of 200 to 250 ng. at the close of operation. Resolution to normal levels occurs usually within 24 hours. In contrast, in malignant prostates the curve increases to higher levels and there is a slower resolution to normal levels during an interval of 48 to 72 hours. The curve after radical prostatectomy for stage B cancer decreases rapidly to low levels in the range of 2.5 to 5 ng. and usually will remain at that level unless recurrent disease becomes apparent. A similar resolution to low levels of serum and bone marrow radioimmunoassay prostatic acid phosphatase is noted after a therapeutic clinical response to orchiectomy and diethylstilbestrol administration. Supervoltage radiation also will suppress the serum radioimmunoassay prostatic acid phosphatase level to low levels in radiosensitive stage C cancers.
Enzyme Phosphatase
Alkaline Phosphatase
Bone Scan
Therapy
38 35 72
Neg. Neg. Pos.
Restaged D Restaged D End manipulation
120 50 134 25-40 JL.
Pos. Pos. Pos.
End manipulation End manipulation End manipulation
Low titer values 0.7 0.2 1.4
High titer values
D Controls
4.5 12.8 21.5 0.1-1.1 JL.
500 BPH-TURP*
200
CAP-TURP* Stage C
200
z
~
ii:
100
100
10
10 0
0 OP
24
500
~
ii:
72 Hours
OP
24
48
72 Hours
Orchiectom}'. + DES* Stage D
Radical Prostatectom}'. * Stage 82
z
48
200
200
100
100
10
10
o-~-----,rt---i OP
24
48
72 Hours
6 Months
2
3
4 Months
Fm. 2. Effect of various forms of therapy on serum radioimmunoassay levels of prostatic acid phosphatase
RADIOIMMUNOASSAYS FOR PROSTATIC ACID PHOSPHATASE
DISCUSSION
Preliminary clinical experience with the immunochemical approach for human serum and bone marrow acid phosphatase has provided reliable clinical data with a minimum of falsely positive results. 9•11 Serum and bone marrow samples are relatively simply acquired from suspect patients and, when performed simultaneously as a screening technique, may provide a unique clinical method for more precise staging of the disease process. When combined with staging pelvic lymphadenectomy and technetium bone scan they may provide the ultimate in clinical staging technique. However, the radioimmunoassay methodology in its present form is patently developmental and may well require subsequent modification as clinical experience demands. In the present state of the art radioimmunoassay prostatic acid phosphatase results may, at times, appear discordant in application. The inconsistencies are due to variations in the specificity of prostatic acid phosphatase antibody. As Amon has indicated all enzymes are characterized by the presence of multiple specificity determinants on the antigenic protein molecule, which may induce measurable heterogeneity in antibody production. 14 Subsequent individual prostatic acid phosphatase antisera may, therefore, vary in their capacity to react with the antigenic sites on the prostatic acid phosphatase molecule. Macalalag and Prout,2 Peel,3 and Flocks and associates 1 were among the first to describe inconsistent specificities with canine antisera to prostatic proteins. Such technical problems may be resolved in great part with improved methods for isolation and purification of prostatic acid phosphatase antigen and rigid standardization of prostatic acid phosphatase antibody. Federal Drug Administration regulations will require such measures before commercial radioimmunoassay kits for prostatic acid phosphatase can be released for general clinical and laboratory use. Inconsistent clinical results will relate invariably to these basic principles. Application of the serum and bone marrow radioimmunoassays as a screening technique in prostatic cancer has demonstrated considerable utility in the initial staging of the primary disease process. It is appapent that the differentiation of intraprostatic from extraprostatic cancer is feasible by means of the relative characteristics of the types II and III radioimmunoassay screens. Certainly, it is apparent that a type II screen is mandatory when considering stages A2, B or C patients for radical prostatectomy, 125I implants or supervoltage radiation therapy, since with a normal bone marrow radioimmunoassay for prostatic acid phosphatase one can assume confidently that no bone marrow metastases are present. It is possible that lymph node metastases may be differentiated clinically from bone metastases by a type II radioimmunoassay screen presenting with a relatively high titer serum radioimmunoassay and normal bone marrow radioimmunoassay. Regional lymph node and early bone marrow metastases also may be substantiated by a type III screen when the serum radioimmunoassay prostatic acid phosphatase is elevated moderately and the bone marrow radioimmunoassay prostatic acid phosphatase is of low titer, <25 to 50 ng. Such information might well be invaluable in a clinically understaged stage C cancer under consideration for radiation since the data would indicate that early bone marrow metastases are probably present. Very high titers for serum and bone marrow radioimmunoassays in type III screens usually are an absolute indication of heavy tumor burden in soft tissues, lymph nodes and bone. In contrast, the intraprostatic cancers A2, Bl and B2 confined within the prostatic capsule are typically type II radioimmunoassay screen with low titer serum elevations and normal bone marrow values. A relatively high titer prostatic acid phosphatase serum concentration in a type II screen may indicate regional lymph node metastases. Such an observation would indicate the necessity for restaging and should be confirmed by
501
pelvic lymphadenectomy and retroperitoneal lymphatic evaluation. The possibility of falsely positive bone marrow radioimmunoassay prostatic acid phosphatase results is certainly of concern to the authors. However, cross-reactivity with normal acid phosphohydrolases in normal bone marrow cells has not been apparent in our experience to date. No falsely positive bone marrow radioimmunoassays were encountered in the early intracapsular prostatic cancers reported herein. However, we have reported falsely positive radioimmunoassay prostatic acid phosphatase values in the serum of patients with advanced bronchogenic cancers but in no other forms of malignancy. A double blind approach to this problem would be advantageous but was not used in this study. Resolution of abnormal types II and III radioimmunoassay screens to normality has been observed commonly in stages B, C and D patients responding well to specific therapies, such as radical retropubic prostatectomy, supervoltage radiation therapy and endocrine therapy. Recurrence and progression of the disease to stage D usually are accompanied by the development of a typical type III radioimmunoassay screen. It is probable that continuing clinical experience with the immunochemical measurement of human serum and bone marrow prostatic acid phosphatase may provide the clinician with significant amplification of diagnostic and therapeutic options in the management of prostatic cancer. In the brief period of time and the modest number of cases studied with the combined serum and bone marrow radioimmunoassay prostatic acid phosphatase method described it is apparent clinically that a more precise and sensitive staging technique for prostatic cancer may now be feasible. Clinical confirmation and specific delineation of the concept by other prostatic oncologists are recommended. REFERENCES
1. Flocks, R. H., Urich, V. C., Patel, C. A. and Opitz, J.M.: Studies on the antigenic properties of prostatic tissue I. J. Urol., 84: 134, 1960. 2. Macalalag, E. V., Jr. and Prout, G. R., Jr.: Anti-canine prostatic fluid serum and prostatic degeneration. Invest. Urol., 4: 321, 1967. 3. Peel, S.: An immunologic study of dog prostate and the effects of injecting antidog prostate serum. Invest. Urol., 5: 427, 1968. 4. Moncure, C. W., Prout, G. R., Jr. and Blaylock, W. K.: Prostatic acid phosphatase antisera. Invest. Urol., 5: 331, 1968. 5. Moncure, C. W. and Prout, G. R., Jr.: Antigenicity of human prostatic acid phosphatase. Cancer, 25: 463, 1970. 6. Cooper, J. F. and Foti, A.: A radioimmunoassay for prostatic acid phosphatase. I. Methodology and range of normal male serum values. Invest. Urol., 12: 98, 1974. 7. Foti, A., Herschman, H. and Cooper, J. F.: A solid-phase radioimmunoassay for human prostatic acid phosphatase. Cancer Res., 35: 2446, 1975. 8. Cooper, J. F., Foti, A. and Imfeld, H.: Production of specific antibody to purified prostatic acid phosphatase. Urol. Res., 4: 111, 1976. 9. Foti, A. G., Cooper, J. F., Herschman, H. and Malvaez, R. R.: Detection of prostatic cancer by solid-phase radioimmunoassay of serum prostatic acid phosphatase. New Engl. J. Med., 297: 1357, 1977. 10. Cooper, J. F., Foti, A., Herschman, H. H. and Finkle, W.: A solid phase radioimmunoassay for prostatic acid phosphatase. J. Urol., 119: 388, 1978. 11. Cooper, J. F., Foti, A. G. and Shank, P. W.: Radioimmunochemical measurement of bone marrow prostatic acid phosphatase. J. Urol., 119: 392, 1978. 12. Dias, S. M. and Barnett, R. N.: Elevated bone marrow acid phosphatase: the problem of false positives. J. Urol., 117: 749, 1977. 13. Kahn, R. M., Turner, B., Edson, M. and Dolan, M.: Bone marrow acid phosphatase: another look. J. Urol., 117: 79, 1977. 14. Amon, R.: Immunochemistry oflysozyme. In: Immunochemistry of Enzymes and Their Antibodies. Edited by M. R. J. Salton. New York: John Wiley & Sons, Ltd., p. 1, 1977.