Serum marker potential of placental alkaline phosphatase-like activity in testicular germ cell tumours evaluated by H17E2 monoclonal antibody assay

A monoclonal antibody (H17E2) was used in a solid-phase localisation of enzyme activity (ILEA) assay to evaluate placental-like alkaline phosphatase (PLAP) as a serum marker of testicular germ cell tumours. Single or repeated assays were performed on 213 normal blood donor and a smaller number of term pregnancy and testicular cancer sera. The detection limit of PLAP by this system was 0.14 O.D. units equivalent to 0.04iul-1. Of 50 patients with established metastatic disease tested before treatment, 88% of 16 with seminoma, 54% of 13 with mixed seminoma and malignant teratoma and 33% of 21 with malignant teratoma had serum PLAP greater than 0.2 O.D. units. This compared to an incidence of 2% in non-smokers and of 29% in smokers who had been free of disease for more than 12 months. In 15 of 22 successfully treated patients, pre-treatment serum PLAP exceeded 0.2 O.D. units (mean 0.69 O.D.) and varying (53-97%) reductions in the initial levels occurred with treatment. These results with monoclonal antibody ILEA assay suggest that measurement of PLAP levels will be useful in the management of patients with germ cell tumours, particularly seminoma.     

Levels of alkaline phosphatase isozymes in human seminoma tissue

The three human isozymes of alkaline phosphatases were quantitatively determined in normal testis and seminoma tissues. The highly selective assays were based on isozyme specific monoclonal antibodies. In the normal testis approximately 90% of the catalytic activity originates from the tissue unspecific alkaline phosphatase, and the remaining activity was due to trace expression of both intestinal (approximately 5%) and placental alkaline phosphatase (PLAP) or PLAP-like isozyme (approximately 5%). In homogenates of seminoma tissues, highly increased levels of all three isozymes were identified. Both the tissue unspecific alkaline phosphatase and PLAP-like enzymes displayed relative increases of 10- to 100-fold and intestinal alkaline phosphatase 2- to 10-fold compared with normal testis. This finding indicates that the entire genome coding for alkaline phosphatases may be activated in seminomas. The PLAP-like enzyme from seminoma cells comprises a heterogenous population of molecules demonstrating partial heat sensitivity and microheterogeneity upon starch gel electrophoresis in contrast to the pregnancy related PLAP. These findings have implications for the different PLAP assays used in the clinical monitoring of seminoma patients.     

Is placental alkaline phosphatase (PLAP) a useful marker for seminoma?

The usefulness of placental alkaline phosphatase (PLAP) as a tumour marker was assessed in 1578 serum samples from 236 patients with seminoma. Smoking habits were known for all but 7 patients (22 samples). Smoking was associated with significantly higher mean levels of PLAP in disease-free patients (28.8[S.E. 2.1]U/l vs. 15.9[1.3] U/l in non-smokers). Mean PLAP levels were higher in patients with active disease (78.6 [23.5] U/l in non-smokers and 47.2 [18.5] U/l in smokers). The median values showed a similar trend. However, there was considerable overlap between the various groups and differences between mean and median values indicated that PLAP values were distributed asymmetrically. The predictive value of PLAP as a tumour marker was consequently much less than superficial inspection of these values might suggest. In 97 patients on surveillance, only 2 out of 11 patients who relapsed had elevated PLAP at the time of clinically detectable relapse. With the upper limit of normal PLAP quoted by our laboratory (35 U/l), specificity and sensitivity were, respectively, 88% and 45% (all patients) and 96% and 47% (non-smokers). The sensitivity and specificity of PLAP were assessed in more detail for a series of threshold values (normal vs. abnormal) with a graphical method. Only in non-smokers did PLAP seem useful and even in this group the positive predictive value of an “abnormal” test may be low; less than 50% in clinically relevant circumstances. Serum PLAP assay cannot usefully stand alone as a marker for seminoma and its routine estimation contributes little to follow-up.     

Placental alkaline phosphatase as a tumour marker in seminoma using the H17 E2 monoclonal antibody assay

Serum samples from 62 patients with seminoma were assayed for placental alkaline phosphatase-like activity using the monoclonal antibody H17 E2, in order to evaluate its utility as a serum tumour marker. Fifteen of 16 patients (94%) with active seminoma had elevated serum PLAP levels. Sixteen of 46 (35%) of patients considered to be in remission had elevated PLAP levels (false positive rate 35%). Fifteen false positive results were considered attributable to concomitant smoking, and if these patients are excluded, only one false positive case was detected. In 7 out of 7 patients sequential PLAP assays reflected clinical response to treatment.     

Patterns of seminoma tissue markers and deletions

Seminomas and control tissues were analyzed for several tumor markers. Very high levels of placental alkaline phosphatase (PLAP)-like enzyme levels were found in all 18 seminomas studied. The majority of the seminomas were of phenotype I, thus differing from palcental PLAP. The mean amount of enzyme protein as measured by monoclonal antibodies, was 100 times higher than in non-malignant tissues and 10 times lower than in placental tissue. The specific enzymatic activity in seminomas was about half of that observed in placenta. Similarly, the specific activity of PLAP-like enzymes in sera of patients with seminoma was only about half of that found in pregnancy sera. HCG was strongly elevated in 3 seminomas, but not obviously related to PLAP. Thirteen of the 17 pure seminomas had HCG over 100 IU/g, which was not seen in normal testes. Liver alkaline phosphatase (LAP) and intestinal alkaline phosphatase (IAP) were high in seminomatous tissues, the mean increases being 60-fold and 20-fold, respectively. The highest IAP levels were found in 2 yolk-sac tumors. Ferritin was moderately elevated in seminomas, but high in several control tissues. Carcinoembryonic antigen (CEA) was not elevated and alpha-fetoprotein (AFP) was not detected at all in pure seminomas. A decrease in carbohydrate antigen 50 (CA-50) content was noted in seminomas as compared to normal testes, yolk-sac tumors and choriocarcinomas. Defects in tumor-related enzymes may account for increase of PLAP and decrease of CA-50.     

Placental alkaline phosphatase, alphafetoprotein and carcinoembryonic antigen in testicular tumors. Tissue typing by means of cytologic smears.

Indirect immunofluorescence and radioimmunoassay with specific rabbit antisera demonstrated the occurrence of alphafetoprotein (AFP), carcinoembryonic antigen (CEA) and placental alkaline phosphatase (PLAP) in primary testicular tumor cells. Embryonal carcinomas had AFP- and CEA-containing cells, sometimes PLAP. PLAP and sometimes CEA were found in seminoma cells. Sera from patients with advanced non-seminomatous tumors could contain any of these antigens or any combination of them. Sera from patients with seminomas had raised PLAP or CEA. PLAP appears to be a new marker for seminoma.     

Electrophoretic heterogeneity of alkaline phosphatase isozymes in seminoma and normal testis

Electrophoretic patterns of seminoma- and normal-testis-derived alkaline phosphatase isozymes, the placental alkaline phosphatase (PLAP)-like enzyme and the tissue-nonspecific (liver) alkaline phosphatase (LAP), were studied on starch gel and isoelectric focusing (IEF). Different migration patterns of the PLAP-like enzyme were observed with respect to both seminomas and normal testes on starch gel electrophoresis. On IEF, seminomas showed different staining patterns among different tumors; however, a common main activity was focused at pIs of 4.3-4.6, corresponding to pIs of PLAP. Normal testes showed two enzyme-staining regions, at pIs of 4.1 and 5.0-5.2, which were discriminated from pIs of PLAP and the PLAP-like enzyme in seminoma. The PLAP-like enzyme in seminoma was differentiated from PLAP by digestion with neuraminidase. Neuraminidase treatment simplified the distribution patterns of the PLAP-like enzyme in normal testis, but did not alter the pattern of microheterogeneity in seminoma. Two factors other than sialylation, namely structural modification of the carbohydrate moiety and variation of hydrophobicity, were shown to contribute to the microheterogeneity of the PLAP-like enzyme in seminoma. LAP in seminoma and in normal testis also showed marked electrophoretic heterogeneity and differences in pI distributions from LAP of liver. However, the migration patterns after desialylation were very similar to each other. The findings imply that electrophoretic heterogeneity demonstrated in LAP in seminoma and in normal testis is caused by a difference in sialic acid content in the molecule, and the heterogeneity of the PLAP-like enzyme in seminoma is considerable.     

Multiple biochemical tumor markers in seminoma. A double-blind study

The value of certain biochemical tumor markers have been well established in nonseminomatous testicular cancer. However, the lack of frequent tumor markers in the sera of patients with seminoma has prompted us to embark on this double blind study. The authors studied 89 patients with the histologic diagnosis of seminoma utilizing placental alkaline phosphatase (PLAP), gamma-glutamyl transpeptidase (gamma GT), human chorionic gonadotropin (hCG), and alpha-fetoprotein (AFP). It was found that 12/30 patients (40%) with active tumor had elevated serum PLAP and 10/30 (33%) of these patients had elevated serum levels of GGT. Eighty percent of the patients with clinically active tumors had detectable serum levels of one or more of these biochemical markers. Since the frequency of the previous tumor markers have been scarce in seminoma, these serial utilization of these biochemical markers should assist the clinician to detect and monitor seminoma patients more efficaciously. However, the false-positive, false-negative, rates, and biologic half lifes of these markers should be taken in account.     

Alkaline phosphatase isozymes in human testicular germ cell tumors, their precancerous stage, and three related cell lines

The four known isozymes of the human alkaline phosphatase (ALP) were detected by isoelectric focusing in extracts of various types of germ cell tumors, three related cell lines, and their precancerous elements (atypical germ cells). In seminoma, placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (PLAP-like) could be separated by isoelectric focusing following isolation by immunoaffinity. The occurrence of both isozymes in seminoma could explain partial heat sensitivity and variation in electrophoretic patterns of the seminoma isozyme frequently observed upon starch gels, in comparison to the normal placental phenotype. The four ALP isozymes are produced not only in germ cell tumors, but already in precancerous tissues. Quantitative analysis showed that the amount of the four isozymes varies in parallel in the tumors tested. Maximal expression was found in seminoma. The relation between ALP gene overexpression and gene amplification by polyploidy of chromosomes 1 and 2 in these lesions is discussed. On the other hand, the ectopic expression of intestinal alkaline phosphatase and PLAP associated with overexpression of PLAP-like in tumor cells as well as in their precancerous stage indicates gene activation by some unknown mechanisms, probably a regulatory process affecting the three tissue-specific ALP genes simultaneously.     

An assessment of combined tumour markers in patients with seminoma: placental alkaline phosphatase (PLAP), lactate dehydrogenase (LD) and beta human chorionic gonadotrophin (beta HCG).

We have assessed the tumour markers placental alkaline phosphatase (PLAP), lactate dehydrogenase (LD), and human chorionic gonadotrophin (beta HCG) using 2,000 serum samples from 286 patients with seminoma. The ROC curves show that no one marker performs adequately for the detection of disease either at initial staging or during follow-up. We used a Markov model heuristically to devise strategies, in which marker results were assessed in combination, which might be useful in clinical practice. We found that the best strategy was to consider a test result abnormal only if either the beta HCG was greater than 6 Ul-1 or the LD was greater than 400 U l-1 and the PLAP level was greater than 60 U l-1. This will detect about 50% of patients with disease and the false-positive rate is 2%. In practical terms this means that PLAP need only be estimated in patients whose beta HCG is less than 6 IU l-1 and whose LD is greater than 400 U l-1.     

Eutopic expression of placental-like alkaline phosphatase in testicular tumors

Very high levels of placental-like alkaline phosphatases (PLAP-like enzymes) were observed in tissues from 13 typical seminomas. Four tumors with seminoma components contained these enzymes to varying degrees, and other testicular tumors had them in smaller or non-detectable amounts. Analysis using monoclonal antibodies produced against the common placental alkaline phosphatase (PLAP) phenotypes and enzyme inhibition studies with amino acids and peptides showed the PLAP-like enzymes present in seminoma to be similar to those PLAP-like enzymes which are expressed in lower amounts in two embryonal carcinomas and in trace amounts in normal testicular tissue. These similarities suggest that the increased expression of PLAP-like enzymes in seminomas results from enhanced eutopic expression of enzymes found in normal testis.     

Placental-like alkaline phosphatase and DNA flow cytometry in spermatocytic seminoma.

Immunohistochemical analysis was done on 7 testicular tumors classified as spermatocytic seminoma (SS) and 25 classic seminomas. Except for a few scattered cells, the spermatocytic seminomas were negative for placental-like alkaline phosphatase (PLAP); the classic seminomas were all positive for this enzyme. The SS also were negative for alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (hCG), and leukocyte common antigen (LCA). The ploidy of the seven tumors of SS was as follows: two, diploid; two, near-diploid; one, tetraploid; one, aneuploid; and one, uninterpretable. The essentially negative staining of SS for PLAP was strikingly different from the pattern in classic seminoma. Thus, staining for this enzyme is useful for making the differential diagnosis between classic seminoma and SS. To differentiate between malignant lymphoma and SS, staining for leukocyte common antigen is helpful.     

Tumor Markers in Colorectal Carcinoma An Evaluation of Carcinoembryonic Antigen, Tissue Polypeptide Antigen, Placental Alkaline Phosphatase and Pseudouridine

The biologic markers carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), placental alkaline phosphatase (PLAP) and pseudouridine were analysed in 37 patients with colorectal carcinoma. CEA, TPA and PLAP were derived from the serum and pseudouridine from the urine. The incidence of all four markers increased with advancing stages of the disease. Patients with distant metastases had elevated levels of CEA, TPA, PLAP and pseudouridine in 85, 27, 18 and 33 per cent of the total cases, respectively. When survival was compared, patients with 2 to 4 elevated markers had shorter survival than those with none or only one elevated marker.     

Placental-like alkaline phosphatase. Re-evaluation of the tumor marker with exclusion of smokers

This report demonstrates that smoking is a major factor of nonspecific elevation of the tumor marker placental-like alkaline phosphatase (PLAP). In 98 healthy nonsmokers the mean of the enzyme activity was determined as 0.068 U/L (range, +/- 2 SD 0-0.144 U/L) compared to a mean of 0.378 U/L (range, +/- 2 SD 0-1.02 U/L) in 65 smokers. In view of this finding the usefulness of PLAP as a tumor marker was re-evaluated in 286 patients with various neoplasms and a negative smoking history. Of these patients, 23% and 50% had elevated values for PLAP and carcinoembryonic antigen, respectively. When compared to the range of PLAP in normal smokers only 4.1% of the patients showed elevated values. An increased incidence of elevated PLAP was found in patients with tumors of the lung, pancreas, stomach, colon/rectum, ovaries, and in 2 of 3 seminomas. It was concluded from the data that PLAP is a useful tumor marker for selected neoplasms provided its use is confined to nonsmokers.     

Tumor markers in mammary carcinoma. An evaluation of carcinoembryonic antigen, placental alkaline phosphatase, pseudouridine and CA-50

In 104 patients with breast cancer, carcinoembryonic antigen (CEA), placental alkaline phosphatase (PLAP) and the carbohydrate antigen CA-50 were analysed in serum. Excretion of the modified nucleoside, pseudouridine, was analysed in urine. The patients were subdivided in three different clinical stages according to disease manifestations. Levels of CEA and pseudouridine correlated to clinical stage and 58 per cent of the patients with distant metastases had elevated levels of CEA, compared with 36 per cent for pseudouridine. For PLAP and CA-50, the levels did not show any clear correlation to clinical stage. Increased activity of PLAP correlated strongly to tobacco smoking. A decrease in the level of CEA was observed following radical mastectomy. Increase in CEA levels predicted relapse in 5 out of 14 patients within about 3 to 6 months. In patients with tumor manifestations, elevated CEA levels predicted an inferior prognosis compared to those with ordinary levels.     

Prognosis of primary testicular seminoma: a report on 57 new cases

Most urologists perform adjuvant radiation therapy for stage 1 (TxN0M0) testicular seminoma after orchiectomy, although the majority of patients with clinical stage 1 seminoma do not have occult metastases and therefore do not require elective nodal irradiation. However, there are currently no clinical or histological parameters that can be used to distinguish patients who need radiation therapy from those who do not. We reported previously that estimates of volume-weighted mean nuclear volume (MNV) were a better predictor of the prognosis of prostate cancer and renal cell carcinoma than subjective histological grading. Here, we examined the usefulness of estimation of MNV for predicting the prognosis of primary testicular seminoma. A retrospective study of 57 patients with testicular seminoma diagnosed between April 1981 and March 1997 at Kobe City General Hospital was performed. Unbiased estimates of MNV data were compared for prognostic value with the level of beta-human chorionic gonadotropin (beta-HCG), alpha-fetoprotein (AFP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). Fifty patients were stage 1 (TxNoMo), and 7 patients were stage 2 (TxN1-2M0). All patients received orchiectomy, followed by radiation therapy. Estimates of MNV of stage 2 patients were significantly larger than that of stage 1 patients (P = 0.0142). Although the LDH level was also significantly higher in stage 2 (P = 0.001), there were no significant differences between stages 1 and 2 with respect to beta-HCG (P = 0.997), ALP (P = 0.226), and AFP (P = 0.467). Multivariate logistic regression analysis revealed that the estimate of MNV was the only variable predicting lymph node metastasis (P = 0.0315). In stage 1 patients, only the estimate of MNV was significantly correlated with progression-free survival (P = 0.0118). These findings indicate that the estimate of MNV may be an important prognostic indicator for testicular seminoma. Estimates of MNV may also be useful for excluding patients from surveillance protocols.     

Radioimmunodetection of human choriocarcinoma xenografts by monoclonal antibody to placental alkaline phosphatase

Placental alkaline phosphatase (PLAP)-specific monoclonal antibody (MAb) 11-D-10, which did not react with other isoenzymes of alkaline phosphatase (AP), was raised by a hybridoma technique. MAb 11-D-10 was radiolabeled and administered to athymic mice bearing human choriocarcinoma containing PLAP. This antibody was found to be more specifically localized in tumor tissue as compared to normal tissues. The tissue-to-blood ratio (T/B ratio) of MAb 11-D-10 in tumor tissue increased from 1.38 at 2 days to 2.51 at 5 days after administration. On the other hand, the T/B ratios of isotype control non-immunized IgM in tumor tissue were 0.72 and 0.87 at 2 days and 5 days after administration, respectively. 131I-labeled MAb 11-D-10 was administered to athymic mice bearing choriocarcinomas of various sizes and various PLAP contents to examine the effect on the radioimage of the differences in tumor size and PLAP content. Tumors less than 0.3 cm in diameter could be imaged clearly by gamma-scintigraphy without blood pool image subtraction. The strength of the radioimage correlated fairly well with PLAP content.     

Serum placental-type alkaline-phosphatase levels in patients with epithelial ovarian-carcinoma

Serum placental alkaline phosphatase (PLAP)-type immunoreactivity was measured in 190 women with epithelial ovarian malignancy, 27 women with borderline ovarian cancer and 334 control subjects with non-neoplastic or benign gynaecological disease. Smoking, ABO blood group and menopausal status affect serum concentrations of PLAP and results were corrected for these. Circulating levels were elevated in patients with cancer and increased with stage. Levels were unaltered in borderline ovarian disease. Two-year stage corrected survival analysis demonstrated a significant worsening of prognosis in patients with serum PLAP-type levels greater than the 100th centile for controls.     

Maintenance chlorambucil after CVP in the management of advanced stage, low-grade histologic type non-Hodgkin's lymphoma. A randomized prospective study with an assessment of prognostic factors

One hundred sixty-two patients with Stages III and IV non-Hodgkin's lymphoma of low-grade histologic type were treated with combination chemotherapy using cyclophosphamide, vincristine, and prednisolone (CVP) followed by radiotherapy to sites of previous bulk disease. The patients were randomized to receive either follow-up alone or "maintenance" chemotherapy with 2 years of intermittent chlorambucil. A complete remission was obtained in 56% of patients and the median survival was 64 months (median follow-up, 74 months). Multivariate analysis revealed stage (P less than 0.0001) and Karnofsky performance status (P = 0.021) to predict complete response (CR) and the achievement of a CR (P less than 0.0001), female sex (P = 0.008), the absence of bulk disease (P = 0.038) and low serum alkaline phosphatase (P = 0.002) to predict prolonged survival. The median relapse-free survival (RFS) of the complete responders was 41 months. A prolonged RFS was predicted by low stage (P = 0.014), low serum lactic dehydrogenase (LDH) (P = 0.045) levels, and by the administration of maintenance chlorambucil (P = 0.045). A prolonged survival of the complete responders was predicted by a low number of nodal sites of involvement with lymphoma at presentation (P = 0.022) and lack of liver involvement (P = 0.011). The administration of oral maintenance therapy with chlorambucil for a full 2 years was only possible in 38% of patients, mainly because of progression of disease and the induction of thrombocytopaenia, but despite this it prolonged the median RFS by 38 months and its use could be considered when future studies are being designed.