Hydrophobicity and lectin affinity of alkaline phosphatase isozymes in seminoma and normal testis

Isozymes of alkaline phosphatases (ALP) in seminoma and normal testis were separated by use of high-performance liquid chromatography and a TSK-gel phenyl-5PW column. The tissue-nonspecific (liver) ALP (LAP) was the dominating isozyme, consisting of more than 90% ALP activity. The placental ALP (PLAP)-like enzyme contributed to 4-8% of the total ALP activity. The intestinal isozyme (IAP) could not be identified. The glycosylation patterns of the isozymes were studied using concanavalin A (Con A) affinity chromatography and batch elution with competing sugar. All PLAP activity in placental extracts and LAP activity in liver extracts was bound to Con A-Sepharose. In the tumor extracts, only 50-70% of the PLAP-like enzyme and 20-50% of the LAP activity from seminomas were bound to Con A-Sepharose. A similar binding pattern of the PLAP-like enzyme and LAP was also seen in the normal testes. This variability in Con A reactivity with PLAP or the PLAP-like enzyme was also reflected in serum of seminoma patients and of pregnant women. Thus, ALP expressed in seminoma has different lectin affinity characteristics compared with the same isozyme from placenta and liver, but almost identical to ALP in the normal testes. These findings imply that the PLAP-like enzyme and LAP in the testis can be discriminated from PLAP of placenta and LAP of liver by carbohydrate lectin affinity. It also supports the concept that the increased amounts of ALP in seminomas result from the enhanced eutopic expression of enzymes normally expressed in the testis.     

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.     

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.     

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.     

Identification and characterization of alkaline phosphatase isozymes in human colorectal adenocarcinomas

Using alkaline phosphatase isozyme-specific immunocatalytical assays, the content of isozymes was determined in normal mucosas and adenocarcinomas from human colon or rectum. Tumor levels of both the tissue (liver)-unspecific and the placental-like alkaline phosphatase (PLAP-like) were elevated compared to normal mucosas of the same patients. Such elevations have been reported previously, particularly in seminomas and ovarian tumors. In several tumors, moreover, the intestinal isozyme was expressed in lesser amounts than in the adjacent mucosa. The present results indicate that the activation of two of the phosphatase isozymes, including expression of the typical germ cell line phosphatase (the PLAP-like isozyme), may occur even in nongonadal tumors. This may reflect an induction pattern of phosphatase isozymes, with implications for malignant transformation also in other tumors.     

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.     

Monoclonal antibody assay of serum placental alkaline phosphatase in the monitoring of testicular tumours

A monoclonal antibody (H17E2) recognising both placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase was incorporated in a solid phase immunoassay. This was used to measure levels of PLAP in 257 sera from 148 patients with germ cell neoplasms of the testis. High levels of PLAP were found in all patients with active seminomas (mean 0.85 O.D.) compared to those in clinical remission (mean 0.20 O.D.) (P less than 0.0001). More importantly, changing levels of PLAP correlated with the course of disease in 79 samples from 33 patients with seminoma (P less than 0.0001). Elevated PLAP levels were also noted in patients in remission who were smokers (mean 0.32 O.D.) compared to non-smokers (mean 0.15 O.D.) (P less than 0.001). These data demonstrate that determination of PLAP levels using this sensitive immunoassay is an important new adjunct in the monitoring of the response to treatment in patients with seminoma.     

Significance of alkaline phosphatase isozymes in the monitoring of patients with colorectal carcinoma

The clinical course of colorectal carcinoma may be monitored by tumor markers such as carcinoembryonic antigen (CEA), carcinoma antigen (CA) 19-9 and CA-50. Alkaline phosphatase isozymes were previously used to study the clinical course of testicular and gynecologic tumors. In this study we investigated 8 patients with advanced colorectal carcinoma. Their sera were analyzed for the tumor markers CEA, CA 19-9, CA-50 and three alkaline phosphatase isozymes: the nonspecific liver isozyme LAP, the intestinal isozyme IAP and the placental isozyme PLAP. Rising levels of CEA, CA 19-9 and CA-50 were seen as expected, and PLAP also showed rising levels during tumor progression. LAP remained elevated. This indicates an association between progression of colorectal carcinoma and a raised serum content of alkaline phosphatase isozymes.     

Characterization of the placental alkaline phosphatase-like (Nagao) isozyme on the surface of A431 human epidermoid carcinoma cells

A431 human epidermoid carcinoma cells monophenotypically express the placental alkaline phosphatase (PLAP)-like enzyme shown by its catalytic and antigenic characteristics, properties which are shared by the Nagao isozyme. More specifically, it is L-leucine sensitive just as is the rare placental D-variant of PLAP and the testicular heat-stable enzyme. Collectively, these are all referred to as PLAP-like enzymes. The enzyme was localized to the surface of the plasma membrane since it was released in an active form by bromelain treatment of cells. The number of molecules per A431 cell was estimated by radioimmunoassay at 7.5 X 10(5), a value significantly higher than that observed for HeLa TCRC-1 cells (5 X 10(4) which express the S-variant of PLAP, also referred to as the Regan isozyme. The quantity of the enzyme was increased significantly (10-fold) by treating the cells with modulating agents including sodium butyrate, prednisolone, and hyperosmolar sodium chloride. The identification of a cell line such as A431 with enhanced expression in the amount of the PLAP-like enzyme and which can be further enhanced by modulating agents will facilitate studies of the differences and the similarities between this protein and other variants of PLAP. The A431 cell line now takes its place with other cell lines which are phenotypically restricted in their expression of alkaline phosphatase. Finally, the A431 cell line is also shown here to be a suitable model system for in vivo tumor studies such as immunolocalization.     

Expression of placental alkaline phosphatase in gastric and colorectal cancers. An immunohistochemical study using the prepared monoclonal antibody

The authors developed monoclonal antibodies (MoAb) against human placental alkaline phosphatase (PLAP). Four specific MoAb reacting only with PLAP and two nonspecific MoAb reacting equally with isozymes of alkaline phosphatase (hepatic, intestinal, and placental) were obtained. Immunohistochemical staining with the specific MoAb showed that the cell membrane and cytoplasm of cancer cells were stained in gastric and colorectal carcinoma. The incidence of PLAP positivity was 23% (25 of 107) of all gastric carcinomas. Among gastric carcinomas, the 42% (13 of 31) positivity of highly differentiated carcinoma (papillary adenocarcinoma and well-differentiated tubular adenocarcinoma) was a significantly higher rate than that found in poorly differentiated carcinoma (poorly differentiated adenocarcinoma and signet-ring cell carcinoma, five of 41, 12%). The incidence of PLAP positivity was 11% (four of 35) in colorectal carcinoma. In contrast, gastric adenoma, intestinal metaplasia, and noncancerous tissue adjacent to cancer did not show staining. These results indicated that expression of PLAP was apt to occur in more highly differentiated gastric carcinoma and was highly specific for carcinoma in the gastrointestinal tract, although its incidence was not high.     

Production of placental alkaline phosphatase (PLAP) and PLAP-like material by epithelial germ cell and non-germ cell tumours in vitro.

Placental and placental-like alkaline phosphatase (PLAP) levels in the culture media of 87 cell lines of neoplastic and ''normal'' origin were measured by a conventional immunosorbent enzymatic assay (IAEA) and by a new immunoradiometric assay (IRMA). The IRMA detected immunoreactive PLAP in 37 of 80 (46%) human epithelial and germ cell cultures, while the IAEA detected PLAP in only 25 (33%). Of the 52 non-germ cell tumour cultures, the IRMA detected expression in 24 (46%) and the IAEA in only 16 (31%). In 17 cases (21%) the IRMA recorded levels double that of the IAEA, while in five cultures (6%) the reverse was true. The IRMA was much more robust than the IAEA and had considerably lower inter- and intra-assay coefficients of variation (3.75-8.5% vs 5.2-46%). Detection of PLAP(-like) expression by IAEA is dependent on neoplastic expression of enzymatically functional molecules and quantification assumes constant enzyme kinetics. PLAP-like material has a higher catalytic rate constant than PLAP and thus will give higher values on a stoichiometric basis in an IAEA. The higher detection rate and levels of PLAP-like material in neoplastic cultures when measured by the IRMA clearly demonstrate ectopic expression of non-enzymatic PLAP and PLAP-like genes. The incidence of PLAP(-like) expression by non-germ cell and possible germ cell tumours has been underestimated and its utility as a tumour marker should be re-examined using assays which measure antigen mass rather than phosphatase activity.     

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.     

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.     

Evidence for homology of normal and neoplastic human placental alkaline phosphatases as determined by monoclonal antibodies to the cancer-associated enzyme

The HeLa TCRC-1 human adenocarcinoma cell line expresses a form of alkaline phosphatase that is similar to the common S-variant of placental alkaline phosphatase (PLAP) on the basis of electrophoretic mobility, catalytic properties, and reactivity with polyclonal antibodies. More sensitive probes of changes in protein structure than polyclonal antibodies are monoclonal antibodies (MAbs) which recognize individual antigenic sites on molecules. Therefore, we produced MAbs to HeLa TCRC-1 cells and selected those which bound to the alkaline phosphatase expressed by the cancer cells. Seven MAbs were obtained and characterized by (a) fine specificity analysis using allelic variants of PLAP and other human alkaline phosphatase isozymes, (b) immunoglobulin isotype, and (c) relative binding affinities to PLAP from two sources, placental tissue and HeLa TCRC-1 cells. The seven MAbs bind the enzymes from both sources with equal affinity indicating a high degree of structural homology if not identity between the normal S-variant of PLAP and its cancer-associated counterpart. We note that most of the MAbs to cancer cell surface-bound PLAP express either Ig (immunoglobulin) G2a or IgG2b heavy-chain isotypes, a higher incidence of these classes of IgG than has been observed with the purified and soluble PLAP immunogen which yields MAbs predominantly of the IgG1 isotype. Finally, some of these antibodies, like the ones prepared from purified PLAP, recognize differences between allelic variants.     

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.     

Phenotypes of placental-type alkaline phosphatase in seminoma sera as defined by monoclonal antibodies

PLAP-like enzymes could be detected in serum of patients with primary testicular tumors, in particular seminomas. The use of a panel of monoclonal antibodies (MAbs) permitted typing into 6 different testicular serum phenotypes, of which one appeared similar to a placental type (II) and 2 have not been previously described. Most tumor sera belonged to type I, as described for seminoma tissues. With a more advanced tumor the mean serum PLAP-like levels increased. After operation, after radiotherapy or with no evidence of disease lower or non-detectable enzyme levels were found. In typing the tissue PLAP-like antigen in serum of the same patient or sera from recurrences of a seminoma in the same patient, the same phenotypes of PLAP-like antigen were usually but not always found. None of the 6 phenotypes appeared to confer a poorer prognosis. We conclude that the expression of PLAP-like antigen is eutopic and is enhanced by testicular malignancy, especially in seminoma cells.     

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.     

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.     

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.