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A comparative study on the diagnostic value of prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) in patients with carcinoma of the prostate gland
131
Clinica Chimica Acta, 174 (1988) 131-140 Elsevier
CCA 04149
A comparative study on the diagnostic value of prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) in patients with carcinoma of the prostate gland M.P. van Dieijen-Visser
a, K.P.J. Delaere b, A.H.J. and P.J. Brombacher a
Departments
Gijzena
of a Clinical Chemistry and b Urology, De Wever-Hospital, Heerlen (The Nether-lam&)
(Received 22 August 1987; revision received 25 January 1988; accepted after revision 1 February 1988) Key won&: Prostatic acid phosphatase; Prostatic specific antigen; Prostatic carcinoma; Receiver operating characteristic curve
Serum prostatic-specific antigen (PSA) and prostatic acid phosphatase (PAP) were determined simultaneously in 241 patients presented to the Urology Department. The patients consisted of 140 prostatic carcinoma patients (34 newly diagnosed and 106 previously treated) and 101 patients with benign prostatic hypertrophy (BPH). Prostatic acid phosphatase was measured by two different methods, an enzymatic method (PAP-EA, Boehringer) with tartrate inhibition and an immunoenzymetric assay (PAP-IEMA, Hybritech). The concentration of prostatic specific antigen in serum was measured using a recently introduced immunoradiometric assay (PSA-IRMA, Hybritech). Receiver operating characteristic curves were constructed to compare the diagnostic value of the different tests at different cutoff values. The diagnostic efficiencies of the PAP-EA and the PAP-IEMA appeared to be similar. A better diagnostic efficiency for PSA compared to PAP was found independent of the cutoff value. The upper-normal limit of 2.7 pg/l for PSA, as suggested by the manufacturer and mentioned in the literature introduces too many false-positive results. We therefore selected 10 pg/l as the upper-normal limit for PSA (sensitivity 57%, specificity 88%). Combined sensitivity found for PAP + PSA was 37% with a
Correspondence to: Dr. M.P. van Dieijen-Visser, Department of Clinical Chemistry, De Wever-Hospital, P.O. Box 4446, 6401 CX Heerlen, The Netherlands.
0009-8981/88/$03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
132
specificity of 97%. A literature survey is included to allow better comparison with data published elsewhere.
Introduction
Levels of the acid phosphatase isoenzyme, prostatic acid phosphatase (EC 3.1.3.2) in serum are frequently elevated in the presence of prostatic carcinoma and can be valuable in the follow-up of treatment. Methods for quantitation of serum PAP are either based on assay of enzyme activity, using specific substrates or inhibitors of the prostatic isoenzyme, or on immunochemical procedures. Various results with regard to detection of elevated serum PAP in prostatic cancer have been reported [l-20]. Due to improvement of reproducibility of the immunological assay, values close to the upper-normal limit can be interpreted with increased confidence [21,22]. However, convincing evidence that the immunological approach introduces a significant clinical benefit is still not available [21]. During the past few years prostatic specific antigen determination has been suggested as a specific test for monitoring carcinoma of the prostate [7,15,23-281. Commercial assays for PSA have recently been introduced. The Hybritech double monoclonal antibody assay was used in the present study. The purpose of this study is to evaluate and compare the diagnostic value of prostatic acid phosphatase (PAP) and/or prostatic specific antigen (PSA) for the detection of prostatic carcinoma. In common practice the accuracy of tumor markers to discriminate between benign and malignant samples is often evaluated using a single cutoff value. Different cutoff values have been used in different studies throughout the literature, making comparison on sensitivity and specificity of PAP and PSA difficult. Therefore in the present study receiver operating characteristic curves have been constructed to examine the influence of changes in cutoff values on the sensitivity and specificity and to select optimal cutoff values for diagnostic purposes. Patients and methods Patients
Blood samples, taken before rectal examination, were obtained from males attending the Urology Department of the De Wever Hospital in Heerlen. The patients consisted of 140 prostatic cancer patients (34 untreated and 106 previously treated) and 101 patients with benign prostatic hypertrophy (BPH). The diagnosis of prostatic carcinoma was established histologically, whereas the diagnosis BPH has been made on a clinical basis (history, rectal palpation, flowmetry, endoscopy). Bone metastases have been established by bone scan. The cancer group was classified according to the TNM system of the Union Intemationale Contre le Cancer: TO-4 describes the stage of the tumor; MO-3 describes the stage of the lymph nodes [l]; MO, no distant metastases; Ml, distant metastases present [29].
133
Serum was separated from the clot by centrifugation at 1500 X g for 15 min at 4O C and for the PAP-EA serum was immediately transferred to tubes containing sodium citrate buffer (0.2 mol/l, pH 5.2). Samples were then stored at - 20 o C until examination. Methods Enzymatic assay for prostatic acid phosphatase (PAP-EA) Prostatic acid phosphatase can be distinguished from other acid phosphatases by inhibition of its enzymatic activity in the presence of 1-tartrate, using p-nitrophenyl phosphate as a substrate. The assay was performed according to the manufacturer’s instructions (Boehringer Mannheim, FRG (cat. no. 125.008). Immunoenzymetric assay for prostatic acid phosphatase (PAP-IEMA) A test kit from Hybritech (Instruchemie, Hilversum, The Netherlands, Tandem-E-PAP, cat. no. 4073) was used and the assay was performed according to the manufacturer’s instructions. The assay appeared linear up to concentrations of 30 pg/l. In our laboratory the within-assay variation was ll-5% in the range of 3-29 pg/l with a between-assay variation of 14-10%. All samples were analysed in duplicate. Measurement of the zero standard (0.1 f 0.06, mean k SD, n = 10) resulted in a detection limit of 0.3 I*g/l. Immunoradiometric assay for prostatic specific antigen PSA-IRMA A testkit from Hybritech (Instruchemie, Tandem-R-PSA, cat. no. 3813) was used and the tests were performed according to the manufacturer’s instructions. The assay was linear up to concentrations of 100 pg/l. In our laboratory the within-assay variation was 7-6% in the range of 2.8-40 pg/l with a between assay variation of 15-78. All samples were assayed in duplicate. Measurement of the zero standard (0.2 k 0.04, mean f SD, n = 10) resulted in a detection limit of 0.4 pg/l. Alkaline phosphatase (EC 3.1.3.1) from Instruchemie (cat. no. 3141) was determined on a Cobas-Bio centrifugal analyser, using p-nitrophenylphosphate as a substrate at 37 o C. The upper limit of the reference interval is 130 U/l. Statistics
Receiver operating characteristic curves are graphical presentations of pairs of sensitivity and specificity, when taking different cutoff values to determine positivity of tumor marker test results. The ROC-curve farthest to the upper left comer belongs to the best discriminating test for the problem under investigation [30]. Results
Repeated freezing and thawing (n = 5) did not significantly alter the PAP and PSA concentrations. A good correlation has been observed between PAP-EA and
134
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(a) and PSA-IRh4A values (b) in the different groups in untreated (0); prostatic carcinoma under treatment (k).
PAP-IEMA for all samples (r = 0.99). Between samples with maximum concentration of 30 pg/l a correlation coefficient of 0.61 has been found. Figures la and b represent the distribution of PAP-IEMA and PSA-IRMA levels in the different groups (BPH, treated and newly diagnosed prostate cancer patients) in relation to tumor stage. Figure 2 presents ROC-curves for PAP-EA, PAP-IEMA and PSA-IRMA. At equal specificity, a higher sensitivity has been observed for PSA compared to PAP. Table I presents the sensitivities, specificities and predictive values of the different tests using cutoff values as selected from the ROC-curves. Although less specific for prostatic carcinoma alkaline phosphatase concentrations were measured routinely as a possible marker for metastasis. Eight of the patients with BPH had increased alkaline phosphatase concentrations. For the treated patients 36, of the 106 (sensitivity 36%) and for the newly diagnosed patients 9 of the 34 (sensitivity 35%, specificity 92%) had increased alkaline phosphatase concentrations. For the 9 newly diagnosed patients with metastases 8 had increased
I
specificity
’ b ’ *
4.0 4.0 5.0 5.0 2.1 2.1 10.0 10.0 5.0 5.0
u/I U/l
7/100 7/100 lO/lOl lO/lOl 40/96 40/96 12/96 12/96 3/96 3/96
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*/9
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3/9 8/16
l/9 12/16
l/9 7/16
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l/l 22/39
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Stage of carcinoma
values of PAP and PSA to detect
positive test/patients with BPH. positive test/patients with prostatic cancer. positive test/patients with metastases. patients and for treated patients.
+ 10.0 + 10.0
@S/l)
Cutoff value
and predictive
Number of patients with a Number of patients with a Number of patients with a Values for newly diagnosed
PAP-EA PAP-EA * PAP-IEMA PAP-IEMA *** PSA-IRMA PSA-IRMA * .* PSA-IRMA PSA-IRMA *** PAP-IEMA + PSA PAP-IEMA + PSA *
Tumormarker
Sensitivity,
TABLE
6/9 36/11
7/9 45/71
5/9 33/13
7/9 23/13
Metastases ’
29 25 38 38 80 60 51 43 31 32
Sensitivity
93 93 90 90 58 58 88 88 91 97
Spccificity
59 79 51 80 38 60 59 79 19 92
PVpos
19 54 81 58 90 58 87 59 83 57
PVneg
E
Reference
1 2 3 3 4 5 6 7 8 5 1 1 2 3 9 10 10 10
Tumor marker
PAP-EA PAP-EA PAP-EA PAP-EA PAP-EA PAP-EIA PAP-EIA PAP-IEMA PAP-IEMA PAP-MA PAP-MA PAP-MA PAP-RIA PAP-WA PAP-RIA PAP-RIA PAP-RIA PAP-MA
4/68 l/48 2/29 17/84 6/68 5/36 2/36 S/129 19/213 4/36 3/14 2/16 3/16
O/36 8/129 9/213 II/213
BPH a
l/6 lo/23 7/12 15/36 11/25 11/23 26/33 26/33 6/21 13/41 l/11 9/24 7/20 5/18
2/8 7/31 l/26 4/12 6/25 8/31 12/24 8/24 4/18 7/58
O/9
O/7 o/12 l/10
5/33 2/21 6/41 S/41
B
3/24 l/18 3/58 3/58
A
Stage of carcinoma b
5/7 5/10 5/10 5/10
3/8 26/58 9/20 18/28 13/25 27/58 25/31 22/31 15/29 17/36
9/31 11/29 11/36 13/36
C
d
d
24/25 23/25 26/30 62/72 14/14 12/14 9/11 lO/ll
13/15 d 85/116 21/25
15/25 24/30 53/72 58/72 15/18
D
Sensitivity and specificity of different tumor markers to detect prostatic carcinoma
TABLE II
28 39 35 40 53 38 41 64 51 41 71 70 52 48 51 43 43 42
Sensitivity
100 94 96 95 97 94 98 93 80 91 86 94 94 91 89 79 88 81
Specificity
100 83 89 88 88 91 97 98 15 88 95 98 86 84 83 90 92 87
PVpos
31 67 60 62 81 48 52 28 58 48 54 50 72 64 63 24 33 32
PVneg
0.2 u/ml 1.5 u;1 4.0 u/1 0.8 U/1 0.85 U/l 0.65 U/l 2.4 15.5 2.0 3.0 66.0 80.0 3.2 1.2 2.5 3.3 2.0 2.0
(h%/l) c
Cutoff value
a b ’ d
10 11 8 12 4 13 14 4 6 15 14 23 7 7 7 15 15 28 27
70/352
l/16 13/19 12/29 3/29 O/29
l/48
5/8 8/12 7/12 o/12 16/26 8/26 44/70 12/45
4/9 O/26 4/26 6/28
3/28
64/90 11/16
O/9 27/34 25/36 17/36 4/36 37/45 16/45
O/9 4/14 7/12 9/45
8/28 5/10
O/l 219
17/84
5/9 15/19 5/10 10/20 27,‘61 17/19 43/56 22/28 lo/28 9/28 55/61 48/61 104/128 29/30
11/25
l/7 3/14
l/6 4/25
25/762 O/16
4/10
5/18 4/8 lo/25
l/11
2/15
Number of patients with positive test/patients with BPH. Number of patients with positive test/patients with prostatic cancer. Cutoff value dete rmining pathology, upper-normal limit. TNM classification: (15): TO-l; R; T3; T4 (11): TO-2; T3-4 (14,27): Tl-ZNOMO; Tl-3Nl-3MO; Tl-4NXMx; T2-4N x Ml.
PAP-RIA PAP-RIA PAP-WA PAP-RIA PAP-RIA PAP-RlA PAP-RlA PAP-IRMA PAP-IRMA PAP-IRMA PAP-IRMA PSA-IEMA PSA-IEMA PSA/PAP PSA + PAP PSA-IRMA PSA-IRMA PSA-IRMA ySM-IEMA 27/34 12/15 d 16/19 d
5/9 27/34
19/25
10/14
38 36 44 31 55 56 32 60 40 37 41 84 79 45 34 84 60 80 57 86
80
72
31 8 29 20 19
75 99 30 83 52
54
28
(5): TO-ZNXMO; T3-4NXMo;
96 97 93 97 100
94 32 59 90 100
72
91
73 17 100 85 97
80
87
90 97 100 95 98
TO-2MO; T3-4MO; T2-4Ml
d 296/344 96/116 53/116 53/116 19/19 d 19/19 d 233/265 d
d
d
Tl-4NXMl
4.0 3.1 2.2 3.1 3.0 2.2 4.7 2.0 3.0 2.0 1.8 1.8 2.5 7.5/15.5 7.5 + 15.5 2.0 10.0 4.0 10.0
(6):
138
20
40
false
d0 positives
40
I%l
Fig. 2. Specificity-sensitivity diagram (ROC-curve) for PAP-EA (0), PAP-IEMA (*) and PSA-IRMA (0) for untreated newly diagnosed patients. Cutoff values for the different methods (gg/l for PAP-IEMA and PSA-IRMA and U/l for PAP-EA) are: a = 0.5; b = 1.0; c = 1.5; d = 2.0; e = 2.7; f = 3.0; g = 4.0; h = 5.0; i = 7.5; j = 10.0 and k =15.0.
alkaline phosphatase concentrations. For the 71 treated patients with metastases 35 had increased alkaline phosphatase concentrations. Discussion Table II presents a review of the literature available on sensitivities and specificities of PSA and different PAP procedures for detection of prostatic carcinoma and in different staging of the carcinoma. Differences in sample populations, methods and cutoff values used, makes comparison of the different studies difficult. Foti et al [l] described a very low sensitivity for the PAP-EA and a high sensitivity for the PAP-RIA. However, in their study a very low cutoff value has been chosen for PAP-EA (0.2 U/ml) compared to other studies [2-41, whereas an
139
extremely high cutoff value has been taken for the PAP-RIA (80 pg/l) compared to other investigations [2-5,8-141. The use of a high cutoff value (80 pg/l) compared to all other investigations should result in a lower sensitivity, instead of in a higher sensitivity. The extremely encouraging results the authors presented on the sensitivity of the PAP-RIA method compared to the PAP-EA have not been confirmed by other authors. This illustrates that the conclusions in the different studies are very much dependent on the populations under investigation and the cutoff values chosen. Therefore in our study ROC-curves have been constructed to compare paired sensitivities and specificities of the different tumor markers at different cutoff values in a group of newly diagnosed patients. Comparable results have been observed for the patients receiving treatment. Table II shows that sensitivities to detect prostatic carcinoma using PAP-EA or PAP-IEMA were very much comparable. In the present study higher sensitivities have been observed for PSA-IRMA compared to PAP-EA and PAP-IEMA, independent of the cutoff value chosen. However, the upper-normal limit of 2.7 pg/l as suggested by the manufacturer introduces too many false positives and also explains the low specificity observed by others [15,23]. Therefore, a cutoff value of 10 pg/l has been choosen in our investigation, as has also been suggested by Siddall et al [27]. PAP and PSA are two biochemically and immunologically distinct proteins of prostate tissue. The value of the combined use of the different test has also been suggested by Kuriyama et al [7] and our results confirm this conclusion. Stamey et al [31] recently suggested PSA to be a more useful parameter in monitoring responses and recurrence after therapy. For the detection of metastasis alkaline phosphatase appears a more reliable parameter, especially in the untreated group. Although over the years significant technical improvements of the different procedures have been made we conclude that, neither PAP nor PSA as such appear to be effective in screening for prostatic carcinoma in a general population or to determine whether a patient has or does not have metastases. However, both parameters are suitable in monitoring patients receiving treatment, especially in evaluating new treatments where it is important to have early warning of progression of cancer. Although PSA seems to be a preferable parameter, combined use of PAP and PSA can also be of clinical significance, since PAP and PSA are supposed to be expressed independently by cancer cells. References 1 Foti AG, Cooper JF, Hers&man H, Malvaez PR. Detection of prostatic cancer by solid-phase radioimmunoassay of serum prostatic acid phosphatase. N Engl J Med 1977;297:1357-1361. 2 Lindholm GR, Stirton MS, Liedtke RJ, Batjer JD. Prostatic acid phosphatase by radioimmunoassay. Sensitivity compared with enzymatic assay. J Am Med Assoc 1980;244:2071-2073. 3 Griffiths JC. Prostate-specific acid phosphatase; re-evaluation of radioimmunoassay in diagnosing prostatic disease. Clin Chem 1980;26:433-436. 4 Davies SN, Go&man N. Evaluation of a monoclonal antibody-based immunoradiometric assay for prostatic acid phosphatase. Am Sot Clin Pathol 1983;79:114-119. 5 Davies SN, Griffiths JC. Prostate-specific acid phosphase: further studies with immunological techniques. Clin Chim Acta 1982;122:29-38. 6 Writh MP, Gomes de Ohveira J, Frohmuller HGW. Monoclonal antibody-based radioimmunoassay
140
compared with conventional enzyme immunoassay in the detection of prostatic acid phosphatase. Eur Urol 1984;10:326-330. 7 Kuriyama M, Wang MC, Lee CL, et al. Multiple marker evaluation in human prostate cancer with the use of tissue-specific antigens. JNCI 1982;68:99-105. 8 Griffiths J, Rippe DF, Panfile PR. Comparison of enzyme-linked immunosorbent assay and radioimmunoassay for prostate-specific acid phosphatase in prostatic disease. Clin Chem 1982;28:183-186. 9 Klein LA, Shapiro P. Role of acid phosphatase measurement in management of prostatic cancer Urology 1981;17:550-553. 10 Wright GL, ScheBhammer PF, Brasil DN, Sieg SM, Leffell MS. Comparison of countercurrent immunoelectrophoretic assay with commercial radioimmunoassay. Kits for measuring prostatic acid phosphatase. Clin Chem 1981;27:1747-1752. 11 Huber PR, Zaugg T, Lmder E, Hagmaier V, Rutishauser G. Creatine kinase isoenqme (CK-BB) in combination to prostatic acid phosphatase measured by RIA in the diagnosis of prostatic cancer. Urol Res 1982;10:75-80. 12 Huber PR, Rist M, Hering F, Biedermann C, Rutishauser G. Tissue polypeptide antigen (TPA) and prostatic acid phosphatase in serum of prostatic cancer patients. Urol Res 1983;11:223-226. 13 V&ho P, Kontturi M, Lukkarinen 0, Ervasti J, Vikho R. Screening for cancer of the prostate. Cancer 1985;56:173-177. 14 Sturgeon CM, Hussey AJ, Beynon L, et al. Comparison of radioimmunoassay and immunoradiornettic assay for serum prostatic acid phosphatase. Chn Chim Acta 1986;161:47-57. 15 Siddah JK, Cooper EH, Newling DWW, Robinson MRG, Whelan P. An evaluation of the immunochemical measurement of prostatic acid phosphatase and prostatic specific antigen in carcinoma of the prostate. Eur Urol 1986;12:123-130. 16 Choe BK, Pontes ET, Dong MK, Rose NR. Doubleantibody immuno-enzyme-assay for human prostatic-acid phosphatase. Chn Chem 1980;26:1854-1859. 17 Maatman TJ, Gupta MK, Montie JE. The role of serum prostatic acid phosphatase as a tumor marker in men with advances adenocarcinoma of the prostate. J Urol 1984;132:58-60. 18 V&ho P, Kontturi M, Lukkarinen 0, Ervasti J, Viio R. Immunoreactive prostatic acid phosphatase in prostatic cancer; diagnosis and follow-up of patients. J Urol 1985;133:979-982. 19 Kaplan L, Chen I, Sperhng M, Bracken B, Stein EA. Clinical utility of serum prostatic acid phosphatase measurements for detection, diagnosis and therapeutic monitoring of prostatic carcinoma assessment and radioimmunoassays Am J Clin Path01 1985;84:334-339. 20 Stromme JH, Haffner F, Johannessen NB, Talseth T, Frederichsen P, Theodorsen L. Diagnostic efficiency of biological markers in blood serum on prostate cancer: a comparison of four different markers and 12 different methods. Stand J Chn Lab Invest 1986;46:443-450. 21 Griffiths JC. The laboratory diagnosis of prostatic adenocarcinoma. Crit Rev Clin Lab Sci 1983;19:187-204. 22 Pontes JE. Biological markers in prostate cancer. J Urol 1983;130:1037-1047. 23 Kuriyama M, Wang MC, Papsidero LD, et al. Quantitation of prostate-specific antigen in serum by a sensitive enzyme immunoassay. Cancer Res 1980;40:4658-4662. 24 Takeuchi T, Kuriyama M, Fujihiro S, Fujimoto Y, Okano M, Nishiura T. Evaluation of serum prostate-specific antigen in urologic cancers. J Surg Oncol 1983;24:157-160. 25 Liedte JR, Batjer JD. Measurement of prostate-specific antigen by radio-immuno-assay. Clin Chem 1984;30:649-652. 26 Killian C, Yang N, Emrich LJ, et al. Prognostic importance of prostate-specific antigen for monitoring patients with stages B2 to Dl prostate cancer. Cancer Res 1985;45:886-891. 27 Siddall JK, Shetty SD, Cooper EH. Measurements of serum seminoprotein and prostate specific antigen evaluation for monitoring carcinoma of the prostate. Chn Chem 1986;32:2040-2043. 28 Tumormarkers in prostate cancer. Symposium Proceedings, May 30, 1986, Hybritech. 29 UICC, Union International Contre le Cancer. TNM classification of malignant tumors, 3rd ed. Geneva: International Union Against Cancer, 1978. 30 Sackett DL, Haynes RB, Tugwell P. Clinical epidemiology (1985) Boston/Toronto: Little Brown Co., 1985. 31 Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine BA. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 1987;317:909-916.
Clinica Chimica Acta, 174 (1988) 131-140 Elsevier
CCA 04149
A comparative study on the diagnostic value of prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) in patients with carcinoma of the prostate gland M.P. van Dieijen-Visser
a, K.P.J. Delaere b, A.H.J. and P.J. Brombacher a
Departments
Gijzena
of a Clinical Chemistry and b Urology, De Wever-Hospital, Heerlen (The Nether-lam&)
(Received 22 August 1987; revision received 25 January 1988; accepted after revision 1 February 1988) Key won&: Prostatic acid phosphatase; Prostatic specific antigen; Prostatic carcinoma; Receiver operating characteristic curve
Serum prostatic-specific antigen (PSA) and prostatic acid phosphatase (PAP) were determined simultaneously in 241 patients presented to the Urology Department. The patients consisted of 140 prostatic carcinoma patients (34 newly diagnosed and 106 previously treated) and 101 patients with benign prostatic hypertrophy (BPH). Prostatic acid phosphatase was measured by two different methods, an enzymatic method (PAP-EA, Boehringer) with tartrate inhibition and an immunoenzymetric assay (PAP-IEMA, Hybritech). The concentration of prostatic specific antigen in serum was measured using a recently introduced immunoradiometric assay (PSA-IRMA, Hybritech). Receiver operating characteristic curves were constructed to compare the diagnostic value of the different tests at different cutoff values. The diagnostic efficiencies of the PAP-EA and the PAP-IEMA appeared to be similar. A better diagnostic efficiency for PSA compared to PAP was found independent of the cutoff value. The upper-normal limit of 2.7 pg/l for PSA, as suggested by the manufacturer and mentioned in the literature introduces too many false-positive results. We therefore selected 10 pg/l as the upper-normal limit for PSA (sensitivity 57%, specificity 88%). Combined sensitivity found for PAP + PSA was 37% with a
Correspondence to: Dr. M.P. van Dieijen-Visser, Department of Clinical Chemistry, De Wever-Hospital, P.O. Box 4446, 6401 CX Heerlen, The Netherlands.
0009-8981/88/$03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
132
specificity of 97%. A literature survey is included to allow better comparison with data published elsewhere.
Introduction
Levels of the acid phosphatase isoenzyme, prostatic acid phosphatase (EC 3.1.3.2) in serum are frequently elevated in the presence of prostatic carcinoma and can be valuable in the follow-up of treatment. Methods for quantitation of serum PAP are either based on assay of enzyme activity, using specific substrates or inhibitors of the prostatic isoenzyme, or on immunochemical procedures. Various results with regard to detection of elevated serum PAP in prostatic cancer have been reported [l-20]. Due to improvement of reproducibility of the immunological assay, values close to the upper-normal limit can be interpreted with increased confidence [21,22]. However, convincing evidence that the immunological approach introduces a significant clinical benefit is still not available [21]. During the past few years prostatic specific antigen determination has been suggested as a specific test for monitoring carcinoma of the prostate [7,15,23-281. Commercial assays for PSA have recently been introduced. The Hybritech double monoclonal antibody assay was used in the present study. The purpose of this study is to evaluate and compare the diagnostic value of prostatic acid phosphatase (PAP) and/or prostatic specific antigen (PSA) for the detection of prostatic carcinoma. In common practice the accuracy of tumor markers to discriminate between benign and malignant samples is often evaluated using a single cutoff value. Different cutoff values have been used in different studies throughout the literature, making comparison on sensitivity and specificity of PAP and PSA difficult. Therefore in the present study receiver operating characteristic curves have been constructed to examine the influence of changes in cutoff values on the sensitivity and specificity and to select optimal cutoff values for diagnostic purposes. Patients and methods Patients
Blood samples, taken before rectal examination, were obtained from males attending the Urology Department of the De Wever Hospital in Heerlen. The patients consisted of 140 prostatic cancer patients (34 untreated and 106 previously treated) and 101 patients with benign prostatic hypertrophy (BPH). The diagnosis of prostatic carcinoma was established histologically, whereas the diagnosis BPH has been made on a clinical basis (history, rectal palpation, flowmetry, endoscopy). Bone metastases have been established by bone scan. The cancer group was classified according to the TNM system of the Union Intemationale Contre le Cancer: TO-4 describes the stage of the tumor; MO-3 describes the stage of the lymph nodes [l]; MO, no distant metastases; Ml, distant metastases present [29].
133
Serum was separated from the clot by centrifugation at 1500 X g for 15 min at 4O C and for the PAP-EA serum was immediately transferred to tubes containing sodium citrate buffer (0.2 mol/l, pH 5.2). Samples were then stored at - 20 o C until examination. Methods Enzymatic assay for prostatic acid phosphatase (PAP-EA) Prostatic acid phosphatase can be distinguished from other acid phosphatases by inhibition of its enzymatic activity in the presence of 1-tartrate, using p-nitrophenyl phosphate as a substrate. The assay was performed according to the manufacturer’s instructions (Boehringer Mannheim, FRG (cat. no. 125.008). Immunoenzymetric assay for prostatic acid phosphatase (PAP-IEMA) A test kit from Hybritech (Instruchemie, Hilversum, The Netherlands, Tandem-E-PAP, cat. no. 4073) was used and the assay was performed according to the manufacturer’s instructions. The assay appeared linear up to concentrations of 30 pg/l. In our laboratory the within-assay variation was ll-5% in the range of 3-29 pg/l with a between-assay variation of 14-10%. All samples were analysed in duplicate. Measurement of the zero standard (0.1 f 0.06, mean k SD, n = 10) resulted in a detection limit of 0.3 I*g/l. Immunoradiometric assay for prostatic specific antigen PSA-IRMA A testkit from Hybritech (Instruchemie, Tandem-R-PSA, cat. no. 3813) was used and the tests were performed according to the manufacturer’s instructions. The assay was linear up to concentrations of 100 pg/l. In our laboratory the within-assay variation was 7-6% in the range of 2.8-40 pg/l with a between assay variation of 15-78. All samples were assayed in duplicate. Measurement of the zero standard (0.2 k 0.04, mean f SD, n = 10) resulted in a detection limit of 0.4 pg/l. Alkaline phosphatase (EC 3.1.3.1) from Instruchemie (cat. no. 3141) was determined on a Cobas-Bio centrifugal analyser, using p-nitrophenylphosphate as a substrate at 37 o C. The upper limit of the reference interval is 130 U/l. Statistics
Receiver operating characteristic curves are graphical presentations of pairs of sensitivity and specificity, when taking different cutoff values to determine positivity of tumor marker test results. The ROC-curve farthest to the upper left comer belongs to the best discriminating test for the problem under investigation [30]. Results
Repeated freezing and thawing (n = 5) did not significantly alter the PAP and PSA concentrations. A good correlation has been observed between PAP-EA and
134
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Fig. 1. Distribution of PAP-IEMA values relation to tumor grade. Prostatic carcinoma
BPH
TO
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(a) and PSA-IRh4A values (b) in the different groups in untreated (0); prostatic carcinoma under treatment (k).
PAP-IEMA for all samples (r = 0.99). Between samples with maximum concentration of 30 pg/l a correlation coefficient of 0.61 has been found. Figures la and b represent the distribution of PAP-IEMA and PSA-IRMA levels in the different groups (BPH, treated and newly diagnosed prostate cancer patients) in relation to tumor stage. Figure 2 presents ROC-curves for PAP-EA, PAP-IEMA and PSA-IRMA. At equal specificity, a higher sensitivity has been observed for PSA compared to PAP. Table I presents the sensitivities, specificities and predictive values of the different tests using cutoff values as selected from the ROC-curves. Although less specific for prostatic carcinoma alkaline phosphatase concentrations were measured routinely as a possible marker for metastasis. Eight of the patients with BPH had increased alkaline phosphatase concentrations. For the treated patients 36, of the 106 (sensitivity 36%) and for the newly diagnosed patients 9 of the 34 (sensitivity 35%, specificity 92%) had increased alkaline phosphatase concentrations. For the 9 newly diagnosed patients with metastases 8 had increased
I
specificity
’ b ’ *
4.0 4.0 5.0 5.0 2.1 2.1 10.0 10.0 5.0 5.0
u/I U/l
7/100 7/100 lO/lOl lO/lOl 40/96 40/96 12/96 12/96 3/96 3/96
BPH ’
*/9
2/9 7/16
3/9 8/16
l/9 12/16
l/9 7/16
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TO-1
214 9/38
l/l 22/39
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4/9 6/16 l/9 5/16
l/l 22/39
4/4 22/38
7/9 11/16
l/l 18/39
l/l 22/40
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2/9 6/18
T3
carcinoma
3/8 9/39
b
prostatic
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Stage of carcinoma
values of PAP and PSA to detect
positive test/patients with BPH. positive test/patients with prostatic cancer. positive test/patients with metastases. patients and for treated patients.
+ 10.0 + 10.0
@S/l)
Cutoff value
and predictive
Number of patients with a Number of patients with a Number of patients with a Values for newly diagnosed
PAP-EA PAP-EA * PAP-IEMA PAP-IEMA *** PSA-IRMA PSA-IRMA * .* PSA-IRMA PSA-IRMA *** PAP-IEMA + PSA PAP-IEMA + PSA *
Tumormarker
Sensitivity,
TABLE
6/9 36/11
7/9 45/71
5/9 33/13
7/9 23/13
Metastases ’
29 25 38 38 80 60 51 43 31 32
Sensitivity
93 93 90 90 58 58 88 88 91 97
Spccificity
59 79 51 80 38 60 59 79 19 92
PVpos
19 54 81 58 90 58 87 59 83 57
PVneg
E
Reference
1 2 3 3 4 5 6 7 8 5 1 1 2 3 9 10 10 10
Tumor marker
PAP-EA PAP-EA PAP-EA PAP-EA PAP-EA PAP-EIA PAP-EIA PAP-IEMA PAP-IEMA PAP-MA PAP-MA PAP-MA PAP-RIA PAP-WA PAP-RIA PAP-RIA PAP-RIA PAP-MA
4/68 l/48 2/29 17/84 6/68 5/36 2/36 S/129 19/213 4/36 3/14 2/16 3/16
O/36 8/129 9/213 II/213
BPH a
l/6 lo/23 7/12 15/36 11/25 11/23 26/33 26/33 6/21 13/41 l/11 9/24 7/20 5/18
2/8 7/31 l/26 4/12 6/25 8/31 12/24 8/24 4/18 7/58
O/9
O/7 o/12 l/10
5/33 2/21 6/41 S/41
B
3/24 l/18 3/58 3/58
A
Stage of carcinoma b
5/7 5/10 5/10 5/10
3/8 26/58 9/20 18/28 13/25 27/58 25/31 22/31 15/29 17/36
9/31 11/29 11/36 13/36
C
d
d
24/25 23/25 26/30 62/72 14/14 12/14 9/11 lO/ll
13/15 d 85/116 21/25
15/25 24/30 53/72 58/72 15/18
D
Sensitivity and specificity of different tumor markers to detect prostatic carcinoma
TABLE II
28 39 35 40 53 38 41 64 51 41 71 70 52 48 51 43 43 42
Sensitivity
100 94 96 95 97 94 98 93 80 91 86 94 94 91 89 79 88 81
Specificity
100 83 89 88 88 91 97 98 15 88 95 98 86 84 83 90 92 87
PVpos
31 67 60 62 81 48 52 28 58 48 54 50 72 64 63 24 33 32
PVneg
0.2 u/ml 1.5 u;1 4.0 u/1 0.8 U/1 0.85 U/l 0.65 U/l 2.4 15.5 2.0 3.0 66.0 80.0 3.2 1.2 2.5 3.3 2.0 2.0
(h%/l) c
Cutoff value
a b ’ d
10 11 8 12 4 13 14 4 6 15 14 23 7 7 7 15 15 28 27
70/352
l/16 13/19 12/29 3/29 O/29
l/48
5/8 8/12 7/12 o/12 16/26 8/26 44/70 12/45
4/9 O/26 4/26 6/28
3/28
64/90 11/16
O/9 27/34 25/36 17/36 4/36 37/45 16/45
O/9 4/14 7/12 9/45
8/28 5/10
O/l 219
17/84
5/9 15/19 5/10 10/20 27,‘61 17/19 43/56 22/28 lo/28 9/28 55/61 48/61 104/128 29/30
11/25
l/7 3/14
l/6 4/25
25/762 O/16
4/10
5/18 4/8 lo/25
l/11
2/15
Number of patients with positive test/patients with BPH. Number of patients with positive test/patients with prostatic cancer. Cutoff value dete rmining pathology, upper-normal limit. TNM classification: (15): TO-l; R; T3; T4 (11): TO-2; T3-4 (14,27): Tl-ZNOMO; Tl-3Nl-3MO; Tl-4NXMx; T2-4N x Ml.
PAP-RIA PAP-RIA PAP-WA PAP-RIA PAP-RIA PAP-RlA PAP-RlA PAP-IRMA PAP-IRMA PAP-IRMA PAP-IRMA PSA-IEMA PSA-IEMA PSA/PAP PSA + PAP PSA-IRMA PSA-IRMA PSA-IRMA ySM-IEMA 27/34 12/15 d 16/19 d
5/9 27/34
19/25
10/14
38 36 44 31 55 56 32 60 40 37 41 84 79 45 34 84 60 80 57 86
80
72
31 8 29 20 19
75 99 30 83 52
54
28
(5): TO-ZNXMO; T3-4NXMo;
96 97 93 97 100
94 32 59 90 100
72
91
73 17 100 85 97
80
87
90 97 100 95 98
TO-2MO; T3-4MO; T2-4Ml
d 296/344 96/116 53/116 53/116 19/19 d 19/19 d 233/265 d
d
d
Tl-4NXMl
4.0 3.1 2.2 3.1 3.0 2.2 4.7 2.0 3.0 2.0 1.8 1.8 2.5 7.5/15.5 7.5 + 15.5 2.0 10.0 4.0 10.0
(6):
138
20
40
false
d0 positives
40
I%l
Fig. 2. Specificity-sensitivity diagram (ROC-curve) for PAP-EA (0), PAP-IEMA (*) and PSA-IRMA (0) for untreated newly diagnosed patients. Cutoff values for the different methods (gg/l for PAP-IEMA and PSA-IRMA and U/l for PAP-EA) are: a = 0.5; b = 1.0; c = 1.5; d = 2.0; e = 2.7; f = 3.0; g = 4.0; h = 5.0; i = 7.5; j = 10.0 and k =15.0.
alkaline phosphatase concentrations. For the 71 treated patients with metastases 35 had increased alkaline phosphatase concentrations. Discussion Table II presents a review of the literature available on sensitivities and specificities of PSA and different PAP procedures for detection of prostatic carcinoma and in different staging of the carcinoma. Differences in sample populations, methods and cutoff values used, makes comparison of the different studies difficult. Foti et al [l] described a very low sensitivity for the PAP-EA and a high sensitivity for the PAP-RIA. However, in their study a very low cutoff value has been chosen for PAP-EA (0.2 U/ml) compared to other studies [2-41, whereas an
139
extremely high cutoff value has been taken for the PAP-RIA (80 pg/l) compared to other investigations [2-5,8-141. The use of a high cutoff value (80 pg/l) compared to all other investigations should result in a lower sensitivity, instead of in a higher sensitivity. The extremely encouraging results the authors presented on the sensitivity of the PAP-RIA method compared to the PAP-EA have not been confirmed by other authors. This illustrates that the conclusions in the different studies are very much dependent on the populations under investigation and the cutoff values chosen. Therefore in our study ROC-curves have been constructed to compare paired sensitivities and specificities of the different tumor markers at different cutoff values in a group of newly diagnosed patients. Comparable results have been observed for the patients receiving treatment. Table II shows that sensitivities to detect prostatic carcinoma using PAP-EA or PAP-IEMA were very much comparable. In the present study higher sensitivities have been observed for PSA-IRMA compared to PAP-EA and PAP-IEMA, independent of the cutoff value chosen. However, the upper-normal limit of 2.7 pg/l as suggested by the manufacturer introduces too many false positives and also explains the low specificity observed by others [15,23]. Therefore, a cutoff value of 10 pg/l has been choosen in our investigation, as has also been suggested by Siddall et al [27]. PAP and PSA are two biochemically and immunologically distinct proteins of prostate tissue. The value of the combined use of the different test has also been suggested by Kuriyama et al [7] and our results confirm this conclusion. Stamey et al [31] recently suggested PSA to be a more useful parameter in monitoring responses and recurrence after therapy. For the detection of metastasis alkaline phosphatase appears a more reliable parameter, especially in the untreated group. Although over the years significant technical improvements of the different procedures have been made we conclude that, neither PAP nor PSA as such appear to be effective in screening for prostatic carcinoma in a general population or to determine whether a patient has or does not have metastases. However, both parameters are suitable in monitoring patients receiving treatment, especially in evaluating new treatments where it is important to have early warning of progression of cancer. Although PSA seems to be a preferable parameter, combined use of PAP and PSA can also be of clinical significance, since PAP and PSA are supposed to be expressed independently by cancer cells. References 1 Foti AG, Cooper JF, Hers&man H, Malvaez PR. Detection of prostatic cancer by solid-phase radioimmunoassay of serum prostatic acid phosphatase. N Engl J Med 1977;297:1357-1361. 2 Lindholm GR, Stirton MS, Liedtke RJ, Batjer JD. Prostatic acid phosphatase by radioimmunoassay. Sensitivity compared with enzymatic assay. J Am Med Assoc 1980;244:2071-2073. 3 Griffiths JC. Prostate-specific acid phosphatase; re-evaluation of radioimmunoassay in diagnosing prostatic disease. Clin Chem 1980;26:433-436. 4 Davies SN, Go&man N. Evaluation of a monoclonal antibody-based immunoradiometric assay for prostatic acid phosphatase. Am Sot Clin Pathol 1983;79:114-119. 5 Davies SN, Griffiths JC. Prostate-specific acid phosphase: further studies with immunological techniques. Clin Chim Acta 1982;122:29-38. 6 Writh MP, Gomes de Ohveira J, Frohmuller HGW. Monoclonal antibody-based radioimmunoassay
140
compared with conventional enzyme immunoassay in the detection of prostatic acid phosphatase. Eur Urol 1984;10:326-330. 7 Kuriyama M, Wang MC, Lee CL, et al. Multiple marker evaluation in human prostate cancer with the use of tissue-specific antigens. JNCI 1982;68:99-105. 8 Griffiths J, Rippe DF, Panfile PR. Comparison of enzyme-linked immunosorbent assay and radioimmunoassay for prostate-specific acid phosphatase in prostatic disease. Clin Chem 1982;28:183-186. 9 Klein LA, Shapiro P. Role of acid phosphatase measurement in management of prostatic cancer Urology 1981;17:550-553. 10 Wright GL, ScheBhammer PF, Brasil DN, Sieg SM, Leffell MS. Comparison of countercurrent immunoelectrophoretic assay with commercial radioimmunoassay. Kits for measuring prostatic acid phosphatase. Clin Chem 1981;27:1747-1752. 11 Huber PR, Zaugg T, Lmder E, Hagmaier V, Rutishauser G. Creatine kinase isoenqme (CK-BB) in combination to prostatic acid phosphatase measured by RIA in the diagnosis of prostatic cancer. Urol Res 1982;10:75-80. 12 Huber PR, Rist M, Hering F, Biedermann C, Rutishauser G. Tissue polypeptide antigen (TPA) and prostatic acid phosphatase in serum of prostatic cancer patients. Urol Res 1983;11:223-226. 13 V&ho P, Kontturi M, Lukkarinen 0, Ervasti J, Vikho R. Screening for cancer of the prostate. Cancer 1985;56:173-177. 14 Sturgeon CM, Hussey AJ, Beynon L, et al. Comparison of radioimmunoassay and immunoradiornettic assay for serum prostatic acid phosphatase. Chn Chim Acta 1986;161:47-57. 15 Siddah JK, Cooper EH, Newling DWW, Robinson MRG, Whelan P. An evaluation of the immunochemical measurement of prostatic acid phosphatase and prostatic specific antigen in carcinoma of the prostate. Eur Urol 1986;12:123-130. 16 Choe BK, Pontes ET, Dong MK, Rose NR. Doubleantibody immuno-enzyme-assay for human prostatic-acid phosphatase. Chn Chem 1980;26:1854-1859. 17 Maatman TJ, Gupta MK, Montie JE. The role of serum prostatic acid phosphatase as a tumor marker in men with advances adenocarcinoma of the prostate. J Urol 1984;132:58-60. 18 V&ho P, Kontturi M, Lukkarinen 0, Ervasti J, Viio R. Immunoreactive prostatic acid phosphatase in prostatic cancer; diagnosis and follow-up of patients. J Urol 1985;133:979-982. 19 Kaplan L, Chen I, Sperhng M, Bracken B, Stein EA. Clinical utility of serum prostatic acid phosphatase measurements for detection, diagnosis and therapeutic monitoring of prostatic carcinoma assessment and radioimmunoassays Am J Clin Path01 1985;84:334-339. 20 Stromme JH, Haffner F, Johannessen NB, Talseth T, Frederichsen P, Theodorsen L. Diagnostic efficiency of biological markers in blood serum on prostate cancer: a comparison of four different markers and 12 different methods. Stand J Chn Lab Invest 1986;46:443-450. 21 Griffiths JC. The laboratory diagnosis of prostatic adenocarcinoma. Crit Rev Clin Lab Sci 1983;19:187-204. 22 Pontes JE. Biological markers in prostate cancer. J Urol 1983;130:1037-1047. 23 Kuriyama M, Wang MC, Papsidero LD, et al. Quantitation of prostate-specific antigen in serum by a sensitive enzyme immunoassay. Cancer Res 1980;40:4658-4662. 24 Takeuchi T, Kuriyama M, Fujihiro S, Fujimoto Y, Okano M, Nishiura T. Evaluation of serum prostate-specific antigen in urologic cancers. J Surg Oncol 1983;24:157-160. 25 Liedte JR, Batjer JD. Measurement of prostate-specific antigen by radio-immuno-assay. Clin Chem 1984;30:649-652. 26 Killian C, Yang N, Emrich LJ, et al. Prognostic importance of prostate-specific antigen for monitoring patients with stages B2 to Dl prostate cancer. Cancer Res 1985;45:886-891. 27 Siddall JK, Shetty SD, Cooper EH. Measurements of serum seminoprotein and prostate specific antigen evaluation for monitoring carcinoma of the prostate. Chn Chem 1986;32:2040-2043. 28 Tumormarkers in prostate cancer. Symposium Proceedings, May 30, 1986, Hybritech. 29 UICC, Union International Contre le Cancer. TNM classification of malignant tumors, 3rd ed. Geneva: International Union Against Cancer, 1978. 30 Sackett DL, Haynes RB, Tugwell P. Clinical epidemiology (1985) Boston/Toronto: Little Brown Co., 1985. 31 Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine BA. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 1987;317:909-916.