Placental Alkaline Phosphatase As a Tumor Marker for Seminoma1

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[CANCER RESEARCH 42, 3244-3247, August 1982] 0008-5472/82/0042-OOOOS02.00 Placental Alkaline Phosphatase as a Tumor Marker for Seminoma1

Paul H. Lange,2 Jose L. Millan, Torgny Stigbrand, Robert L. Vessella, Erkki Ruoslahti, and William H. Fishman

Departments of Urologie Surgery, University oÃMinnesota College of Health Sciences, and Veterans Administration Medical Center, Minneapolis, Minnesota 55455 [P. H. L, P. L. V.I La Jolla Cancer Research Foundation, La Jolla, California 92037 [J. L. M., E. P., W. H. FJ; and Department of Physiological Chemistry, University of Umea, Umea, Sweden [T. S.J

ABSTRACT pathologically confirmed, untreated (active) seminoma and from 22 with active nonseminomatous cancer. When a specimen was found to A sensitive and specific enzyme-linked immunoabsorbent have an elevated PLAP level, we retrieved other samples obtained from assay was used in a retrospective study of serum levels of the same patient to study changes during the clinical course. (Appro placental alkaline phosphatase (PLAP) in testicular cancer. priate samples were available in 14 cases.) In 27 patients, seminoma Sixteen of 28 men with active seminoma had elevated PLAP was thought to be cured (inactive) when the serum was obtained, and in 6, inactive nonseminomatous cancer was present. Six further control levels, and 71% had elevated levels of either PLAP, human specimens came from patients with benign intrascrotal masses. All chorionic gonadotropin, or both. Only four of 22 men with sera were tested for PLAP as coded specimens in La Jolla and later active nonseminomatous cancer had elevated PLAP levels, and decoded in Minneapolis for evaluation. the levels were normal in all control patients, including 33 men Staging. In this article, the stages of testicular cancer are defined apparently cured of testicular cancer. In six of ten serial stud as follows: Stage I, tumor limited to the testis, epididymis, and spermatic ies, PLAP levels provided information not otherwise available cord; Stage II, small retroperitoneal mÃ©tastases (by bipedal lymphan- that would have been useful clinically, and the levels never giography or lymphadenectomy); Stage IIC, bulky retroperitoneal mÃ© were elevated inappropriately. Our data suggest that PLAP is tastases; Stage III, mÃ©tastasesoutside the retroperitoneum. a clinically useful serum tumor marker for seminoma. ELISA. PLAP serum levels were determined by a sandwich ELISA developed recently which is described elsewhere (4, 16). The lower limit of detection of this assay is 0.4 ng/ml. The upper limit of normal, INTRODUCTION defined in 126 men and nonpregnant women, is 1.85 ng/ml. In patients with NSGCT,3 the serum markers AFP and HCG often improve staging accuracy, signal recurrences weeks RESULTS before they are detectable by anatomical methods, aad may Chart 1 illustrates the PLAP levels in our patients with active show whether a chemotherapy regimen will be effective (11, testicular tumor. Serum PLAP levels were elevated in 16 of 28 14,18). This success contrasts with the relative lack of markers men with active seminoma (average, 10.6 ng/ml; range, 1.9 to for seminoma, which accounts for 40% of testicular cancers. 33.2 ng/ml). Of these 16, 10 also had elevated HCG levels. Between 10 and 30% of men with seminoma have elevated An additional 4 had only HCG elevated. Thus, in 20, the level HCG levels (12,15), but the AFP levels are always normal, and of at least one of the proteins was elevated. There were no no other marker has been proven reliable. serum PLAP elevations among patients with an inactive testic PLAP (phosphomonoesterase, alkaline optimum, EC 3.1.3.1) ular tumor or among any patients who had benign scrotal is an oncodevelopmental protein which is expressed by pla masses at the time serum was obtained. cental syncytiotrophoblastic cells by the 12th week of preg Serial studies were performed in 10 seminoma cases in nancy but is also produced ectopically by a variety of malignant which appropriate specimens were available. In 4 cases (one tumors including seminoma (5, 6, 9, 17, 20, 21, 23). Stage I, 3 Stage III), the HCG levels were elevated also; and We found that, when serum PLAP levels were measured with the PLAP levels, although reflecting the clinical course, were a new sensitive ELISA (4, 16), PLAP appears to be a clinically either not as good as or no better than HCG levels in this useful marker for seminoma. regard. In 3 cases (Stages I, II, and IIC), the HCG levels were elevated, but PLAP reflected the clinical course more accu MATERIALS AND METHODS rately at some point. In 3 cases (Stages I, II, and III), HCG levels Serum. From among the serum specimens preserved in our liquid were normal throughout, and the changing PLAP levels would nitrogen tumor resources bank at the University of Minnesota, we have provided clinically useful information. In no instance in selected 209 specimens representing various stages and clinical cir our serial studies was the PLAP level elevated inappropriately. cumstances of testicular cancer, particularly seminoma. These sera Four cases are illustrative. came from 88 patients, and in all, the AFP and HCG levels had been Case 1. This 40-year-old man had a radical orchiectomy determined with reliable RIA. Sera were obtained from 28 patients with which on pathological analysis was pure seminoma. Postop- eratively, he was classified as clinical Stage I disease on the 1 Supported by Grants CA 21967 and Cancer Center Support Grant CA 30199 basis of bipedal lymphangiography, full lung tomography, and from the National Cancer Institute and by research funds from the Veterans Administration. abdominal computerized axial tomography. Accordingly, 3 2 To whom requests for reprints should be addressed, at Department of weeks after surgery, the patient began a course of 2500 R of Urologie Surgery, University of Minnesota Health Sciences, Box 394, Mayo radiation therapy to the abdomen and remains free of disease Memorial Building, Minneapolis, Minn. 55455. 3 The abbreviations used are: NSGCT, nonseminomatous germ cell tumor; 3 years later. AFP, a-fetoprotein; HCG, human chorionic gonadotropin; PLAP, placental alka Analysis of sera obtained before surgery and subsequently line phosphatase; ELISA, enzyme-linked immunoabsorbent assay; RIA, radioim mune assay. revealed normal AFP and HCG levels as determined by RIA Received October 6, 1981 ; accepted April 28. 1982. throughout his clinical course. However, PLAP determinations

3244 CANCER RESEARCH VOL. 42

STAGE:32165ni was found in the resected lymph nodes. The HCG level was normal for more than 5 months before a rapid rise was noted (Chart 4). No tumor was evident clinically, but 1 month later, there was a mass in the abdominal wall above the orchiectomy scar. It was excised and found to be anaplastic seminoma. PIAR 84i-â€¢-

â€¢n*â€¢â€¢,m.NORMALI----LIMITâ€¢ Rt and leti orchieciomy for i12832tÃ® seminoma 35 and 25 yrs previous ?now with stage Ã•Ã•csemmoma-biopsy < proven ~J\ 2 Radialion therapy to abdomen II '" IIÂ«U::.mâ€¢â€¢i... seminoma â€¢biopsyproven _ / \ 5 Radiation therapy to chest Seminoma NSGCT NED//' l 6 Chart 1. PLAP (PLAP) values in patients with active seminoma and active '60 NSGCT separated according to stage of disease. i3) li 18Ãœ Ã¨420fÂ¥\\\\ /'1 'S1 k1t* r^t'' \ of stored samples revealed a significantly elevated level of 33.2 Y'* \ /o^. \\"* ng/ml preoperatively, which returned to normal levels 8 days after surgery and remained normal as revealed in 4 serum t*N.|^r'*^2i samples obtained over the next 3 years. N0i v ... i3 i i i i i .. i Case 2. This 50-year-old man underwent right and left or- 4 5 6 7 8 v/ 15 273D.U7.06.05.04.03.01.0 chiectomies 35 and 25 years previously and had received Months extensive abdominal and chest irradiation for seminoma. He Chart 2. Serial HCG(hCG)and PLAPserum values in a patient with seminoma came to us with a large retroperitoneal mass which was found (Case 2; see text). NED, no evidence of disease. at exploratory laparotomy to be unresectable seminoma. There

was no evidence of other mÃ©tastases. He received 3000 R to 1 Qrchiectomy 4 yrs previou IODOrr NSGCT now with abdominal matt the upper abdomen, and the mass shrank dramatically. How 2 Lymphadenectomy- pur* Â«Â«minomi 3 Considered F 00 ever, 4 months postoperatively, he returned with lung, medias- 4. NED tinal, and left-neck masses found to be pure seminoma. He 5. Elevated nCG lint noted 6 Chemotherapy was given 3000 R to the chest, and the masses disappeared. He has had no evidence of tumor in the ensuing 4 years. Except for a borderline abnormal level immediately after laparotomy, this man's HCG level was normal throughout (Chart 2). However, the PLAP level clearly reflected the disease process, beginning to rise 3 months before the chest and neck mÃ©tastases were discovered and declining to normal during irradiation. Case 3. Four years earlier, this 55-year-old man had had a left radical orchiectomy for teratocarcinoma and seminoma and

refused further treatment. He then later presented with a ret 23~T"6ÃŽI i I /A. l l8 l. .9 1_ 10 1 /A 15 I IT 1 19 I _/A 20 I 21 i 22 I 23 I 24 1 roperitoneal mass and elevated HCG levels. At lymphadenec-

tomy, the mass, a pure seminoma, was excised, apparently Chart 3. Serial HCG(ftCG)and PLAPserum values in a patient with seminoma completely, and the HCG level fell to normal values. The patient (Case 3; see text). FOD, free of disease; NED, no evidence of disease. declined further treatment but agreed to have serial determi 1000 35.0 nations of his serum AFP and HCG levels. Seven months later, Radiation therapy the HCG level became abnormal, but when the tumor could not FOD FOD be located, the patient again refused treatment. The HCG level Elevated hCG noted, NED continued to rise, and when the patient was finally persuaded C to return for rÃ©Ã©valuation8months later, he had retroperitoneal to. Chemotherapy ends and pulmonary mÃ©tastases. He received vinblastine, bleomycin, and c/s-platinum (5), and the masses shrank. He discontin ued chemotherapy in midcourse and was lost to'follow-up with 50 t ! a rising HCG level. Our retrospective analysis showed that the PLAP levels closely paralleled the HCG levels throughout much of this patient's course (Chart 3). However, after lymphadenectomy, 5 months before the HCG levels became abnormal, the PLAP levels were elevated, thereby indicating residual tumor. Case 4. This 36-year-old man had a right radical orchiectomy at another hospital. Because the tumor was an anaplastic I 25 1 9 13 17 M 38 Â«245 47 Â« 50 51 52 Months seminoma and the HCG level had been elevated preoperatively, Chart 4. Serial HCG(/)CG)and PLAPserum values in a patient with seminoma we performed a retroperitoneal lymphadenectomy. No tumor (Case 4; see text). FOD, free of disease; NED, no evidence of disease.

AUGUST 1982 3245

Intraoperative palpation of the retroperitoneum revealed no The importance of PLAP in testicular tumor has therefore tumor, but the HCG level did not return to normal until the been unclear. Early studies of testicular tumor sera revealed abdomen was irradiated (3000 R). Forty-five months after detectable PLAP levels infrequently (5, 9, 17, 21). However, orchiectomy, the HCG level began rising, although whole-lung Fishman et al. (7), using a sensitive catalytic assay, studied the tomography, computerized tomography, and abdominal ech- sera of 27 treated and untreated seminoma patients and found ography revealed no tumor. Our experience has shown that, elevated PLAP levels in 4, all of whom also had elevated HCG provided other causes of elevated marker levels are eliminated, levels. Subsequently, Wahren era/. (23) studied PLAP levels in rising marker levels are always a sign of recurrent tumor (1 4); the tumor and sera of testicular tumor patients using an RIA and therefore, we gave the patient vinblastine, bleomycin, and with a minimum detectable level of 12 ng/ml. They found that c/s-diamminedichloroplatinum. After 3 courses, the HCG level seminoma tumors contain more PLAP than do nonseminoma was normal, but 2 months later, it was again elevated, although tous tumors and that serum values were more frequently ele no tumor was detectable. He is now receiving cyclophospha- vated in seminoma patients than in nonseminoma patients mide. Retrospective study of the sera showed that the PLAP unless the latter disease was far advanced. They reported levels were normal early in the course but warned of the second elevated PLAP levels in 10 of 19 seminoma patients, but recurrence 6 months before the HCG level did (Chart 4). horizontal studies were not presented. Finally, Uchida ef al. PLAP appeared less useful in patients with active NSGCT, (20) analyzed recently a variety of testicular tumor specimens being elevated in only 4 of 22 (Chart 1). One of these men had using indirect Â¡mmunoperoxidase techniques and reported the an elevated HCG level, one had an elevated AFP level, and the oncodevelopmental enzyme to be present in 8 of 9 seminomas other 2 had elevated AFP and HCG levels. Serial PLAP data in but in none of the 9 NSGCTs examined. these 4 patients did not provide new information. Four of the In the present clinical study, we measured serum PLAP levels 16 men had seminomatous elements in their tumors, and 3 had with a sensitive sandwich ELISA which has a minimal detecta falsely negative AFP and HCG levels. PLAP levels were normal ble level of 0.4 ng/ml. We found PLAP elevations in 16 of 28 in all these patients. active seminoma patients (57%) but in only 4 of 22 patients with nonseminomatous cancer. Moreover, PLAP was the only DISCUSSION marker elevated in 6 of 16 seminoma patients, and in all 4 of these cases in which serial studies were possible, PLAP levels The survival rates of men with NSGCT improved dramatically provided clinical information of importance. In these serial in the last decade because of improved chemotherapy and the studies, it is noteworthy that, although PLAP levels were high better monitoring made possible by the RIA for the markers in some instances, other clinically relevant abnormal levels AFP and HCG. At several medical centers, including ours, long- were found in the range of 2 to 10 ng/ml, stressing the need term survival rates are now > 90% in Stages I and II and > for a very sensitive assay if PLAP determinations are to be of 60% in Stage III (2). The rates often are lower in seminoma, clinical value. which was once considered the most curable type of testicular In the 14 seminoma patients with elevated HCG levels, serum cancer. In Stages IIC and III, the rate is < 40% (15, 18). PLAP was also elevated in 10. While PLAP often paralleled the Sensitive tumor markers might help improve the prognosis of HCG level, in 3 of the 7 cases where both markers were these patients. elevated and serial studies were possible, PLAP levels were at The value of AFP and HCG as markers of pure seminoma certain times better than HCG at either reflecting or predicting has been much debated. Now, it is believed that elevated AFP tumor behavior, while at other times in these same patients, levels always indicate nonseminomatous cancer, whereas ele HCG levels were more valuable. Thus, the simultaneous mea vated HCG levels, although they also may indicate nonsemi surement of both HCG and PLAP would have been necessary nomatous cancer, can accompany pure seminoma (12, 15). for accurate serum monitoring. Such a situation is reminiscent The frequency with which this occurs is being explored; the of nonseminomatous cancer where discordance between HCG estimates are 15 to 20% of cases in all stages combined. In and AFP is a common occurrence, and both of these markers these few patients, HCG is a clinically useful marker for semi must be measured together for optimal accuracy (14). Discord noma. ance between HCG and PLAP has been encountered in ovarian PLAP has been distinguished from the common tissue alka cancer also (5). Finally, among our seminoma patients, either line phosphatases by its heat resistance, its inhibition by L- HCG and/or PLAP was elevated in 68%. Again, this is similar phenylalanine, and its immunological properties (1 7). It resem to the prevalence of AFP and/or HCG elevations in nonsemi bles in many properties the alkaline phosphatase called Regan nomatous cancer (14). Hence, the impression that seminoma isoenzyme found by Fishman ef al. (5) in nontrophoblastic is usually a marker-negative tumor may need to be revised. tumors. PLAP has been reported in the sera of about 20% of Obviously, further study is needed before PLAP can be patients with various cancers, although some have reported a accepted as a clinical marker for seminoma with the same faith prevalence as high as 95% (1, 5, 8-10, 17, 19, 21, 22). one has in AFP and HCG as markers for nonseminomatous Substantial evidence of clinical utility has been lacking. testicular cancer. It is perhaps reassuring that, in previous One problem in developing PLAP as a tumor marker has studies using the sandwich ELISA, PLAP was never greater been variations in the sensitivity and specificity among the RIA than 1.8 ng/ml among over 100 men and nonpregnant women. and catalytic assays used in these studies (9, 21, 22). Also, Furthermore, in this study, there were no PLAP elevations several PLAP variants have been recognized recently. For among 39 men (including 27 with seminoma), who were con example, a heat-sensitive PLAP is present in patients with sidered cured of their testicular tumor or had benign scrotal Hodgkin's disease (3). The extent to which certain assays masses. Moreover, among our seminoma patients who had detect these variants is unknown. elevated PLAP levels and serial studies, PLAP was never

3246 CANCER RESEARCH VOL. 42

elevated inappropriately. 518, 1978. 10. Inglis, N. R., Kirley, S., Stolbach, L. L., and Fishman, W. H. Phenotypes of Our data provide evidence that PLAP levels can give useful the Regan isoenzyme and identity between the placental o-variant and the clinical information in patients with seminoma and suggest that Nagao isoenzyme. Cancer Res., 33: 1657-1661, 1973. both PLAP and HCG should be monitored in these patients. 11. Javadpour, N. The role of biologic tumor markers in testicular cancer. Cancer (Phila.), 45: 1755-1761. 1980. This may be particularly true for men with bulky or widespread 12. Javadpour, N., Mclntire, K. R., and Waldmann, T. A. Human chorionic mÃ©tastases, as PLAP and HCG levels may provide a much- gonadotropin (HCG) and alpha-fetoprotein (AFP) in sera and tumor cells of needed guide to appropriate use of the various treatment patients with testicular seminoma: a prospective study. Cancer (Phila.), 42: 2768-2772, 1978. options (15). 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Paul H. Lange, Jose L. Millan, Torgny Stigbrand, et al.

Cancer Res 1982;42:3244-3247.

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