An immunohistological study of testicular germ cell tumours using two different monoclonal antibodies against placental alkaline phosphatase.

Using two monoclonal antibodies directed against placental alkaline phosphatase (H17E2 and D20L) the immunohistological staining of testicular germ cell tumours was compared with that of a wide range of normal and malignant tissues. All seminomas and malignant teratomas tested gave strong positive labelling with H17E2 but were either negative or only patchily positive with D20L. Neither antibody gave any positive reaction on the normal tissues tested. All other malignancies were negative with both antibodies apart from two cases of ovarian and one case of endometrical cancer (strongly stained by H17E2) and three cases of colonic carcinoma (weakly and patchily stained by both H17E2 and D20L). This indicates that germ cell neoplasms generally express a form of placental alkaline phosphatase recognised by antibody H17E2.     

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.     

Expression on cultured human tumour cells of placental trophoblast membrane antigens and placental alkaline phosphatase defined by monoclonal antibodies

The cellular reactivity of six monoclonal antibodies (McAbs) produced to isolated human placental syncytiotrophoblast microvillous plasma membranes has been examined using a variety of normal and malignant cell types. Two McAbs reacted with antigenic determinants common to most normal human cells. Two other McAbs (H310 and H316) reacted predominantly with normal placental trophoblast and with lymphocytic cells, as well as with most transformed or neoplastic cultured cell lines. Two further McAbs (H315 and H317) identified foetal differentiation antigens expressed only on the membranes of normal placental trophoblast and of certain tumor cell lines. H317 has been shown to be specific for the heatstable L-phenylalanine-inhibitable placental-type alkaline phosphatase isoenzyme. These latter McAbs (H315 and H317) may prove useful in monitoring of some human cancers.     

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.     

Expression of placental alkaline phosphatase in epithelial ovarian tumours

The expression of placental alkaline phosphatase in 116 ovarian epithelial tumours was examined in formalin-fixed tissues used for routine histopathologic examination. In the total material, 51% of the tumours displayed positive immunoreactivity, as described by the monoclonal anti-placental alkaline phosphatase antibody C2, with similar incidence (46-67%) in the four major groups of the adenocarcinomas, i.e., serous, mucinous, endometrioid and mesonephric tumours. By use of a histochemical staining index the mucinous and mesonephric tumours demonstrated a more intense staining (2.1 and 2.6) compared to the serous and endometrioid tumours (0.9 and 1.5). The relevance of the findings is discussed in relation to the use of monoclonal antibody technologies for radioimmunolocalization and radioimmunotherapy.     

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.     

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.     

The stimulation by methotrexate of human chorionic gonadotropin and placental alkaline phosphatase in cultured choriocarcinoma cells.

Treatment of the BeWo line of choriocarcinoma cells with methotrexate in doses that inhibit DNA synthesis causes a tenfold increase in synthesis of human chorionic gonadotropin and a threefold increase in activity of placental alkaline phosphatase. No concomitant increase in lactic dehydrogenase activity occurs under these conditions. This effect of methotrexate can be blocked by simultaneous addition of thymidine or folinic acid, neither of which alone increases human chorionic gonadotropin synthesis or placental alkaline phosphatase activity in BeWo cells.     

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.     

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.     

Elevation of serum placental alkaline phosphatase levels in cigarette smokers

In an ongoing longitudinal study for tumor markers in breast cancer patients, levels of placental alkaline phos-phatase (PAP) measured by RIA were found to be elevated in 34% of patients who smoked cigarettes as opposed to 5 % of non-smoking subjects. A normal range was established (0-2.14 ng/ml) using healthy non-smoking females. In an expanded analysis by disease state, PAP levels were found to be elevated significantly (p<0.02) in the smoking groups of each category. This isoenzyme, when detected at elevated serum levels by RIA, was enzymatically active in 48% of the patients as analyzed by immunoenzymatic assay. One smoker with high PAP levels discontinued smoking following a myocardial infarction. Her PAP levels returned to normal within 2 months.     

Demonstration of placental alkaline phosphatase in human breast cancer.

The screening of a series of 11 metastatic breast tumors for the presence of the placental isoenzyme of alkaline phosphatase (EC3.1.3.1) by RIA revealed one strong producer. The alkaline phosphatase of this tumor was characterized with respect to its immunochemical cross-reactivity, inhibition by L-phenylalanine and levamisole, subunit molecular weight (Mr) and isoelectric point (pl) in two-dimensional electrophoresis, and one-dimensional peptide map. In all parameters of the characterization, the tumor alkaline phosphatase, except for subunit molecular weight which was slightly lower (60,000 versus 64,000 for the placental isoenzyme). No strong placental alkaline phosphatase producers were found among 16 primary tumors examined by RIA. The screening of patients' sera for the placental alkaline phosphatase using RIA indicated elevated levels over post-menopausal controls in 20% of the metastatic patients. Only 3% of the primary patients had elevated serum levels. These results suggest that the placental isoenzyme of alkaline phosphatase may be a useful tumor marker for recurrent breast cancer.     

Residual retroperitoneal tumour tissue in patients treated for metastatic non-seminomatous testicular germ cell tumours: an immunohistochemical investigation

The morphology of resected residual retroperitoneal tumour tissue from 18 patients treated with a combined chemotherapy regime for advanced testicular non-seminomatous germ cell tumours was studied. In five cases (28%) the resected tissue comprised only fibrous tissue and in ten cases (56%) only mature teratoma (T) was present. Embryonal carcinoma (EC) with yolk sac tumour (YST) differentiation was found in addition to T in one case and in two cases the resected tissue comprised pure EC. In all patients with residual T, T had also been present in the primary tumour. Resected tissue containing T was investigated for the presence of various marker proteins, including alpha-1-antitrypsin (A1 AT), carcino-embryonic antigen (CEA), ferritin (FER), lactoferrin (LF), and pregnancy-specific beta-1-glycoprotein (SP1), in addition to the well-established markers for germ cell tumours, alphafetoprotein (AFP) and human chorionic gonadotropin (HCG). AFP and HCG were present in only two cases. A1 AT and CEA were demonstrated in various amounts in epithelial structures in 11 out of 11 cases with T, while FER was found in ten and LF and SP1 in seven cases. Since A1 AT, CEA and LF were also found in the secreted material within the lumen of the teratoid structures, aspiration of cystic fluid for demonstration of these proteins in addition to AFP and HCG is recommended for diagnostic assessment. CEA and SP1 are suggested for localization and treatment of tumour tissue with the recently-developed methods using specific antibodies which are either radiolabelled or conjugated to anti-neoplastic drugs.     

Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.     

Tumour and cellular localization by use of monoclonal and polyclonal antibodies to placental alkaline phosphatase

Monoclonal and polyclonal antibodies against placental alkaline phosphatase (PLAP) were evaluated for tumour immunolocalization of human PLAP-producing Hep 2 tumours in nude mice. The antibodies were labelled with 125I and injected i.p. in mice with developing Hep 2 tumours. The distribution of 125I-anti PLAP in various tissues showed that the labelled antibody was enriched in the tumour, the mean concentration ratio being 7.1 and 6.8 for polyclonal and monoclonal antibodies, respectively. A PLAP negative tumour (RD) showed a mean ratio of 1.2. There was a positive correlation between PLAP content and uptake of labelled antibody in the tumours. Hep 2 tumour cells in tissue culture showed 100% positivity for PLAP, while imprints of the tumour after passage in nude mice showed 40-50% positivity. PLAP offers potential as a useful marker for localizing tumours in humans.     

Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors.

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.     

EXPRESSION OF PLACENTAL ALKALINE PHOSPHATASE IN ESOPHAGEAL CANCER CELL LINE Eca109

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Comparison of CA 125 and placental alkaline phosphatase as ovarian tumor markers

The serum concentrations of CA 125 and placental alkaline phosphatase were analyzed in 16 patients with ovarian cancer. Increased serum levels of CA 125 and placental alkaline phosphatase were observed in 75% and 50% of the cancer patients, respectively. The serum levels of these tumor markers were not correlated, supporting their distinct antigenic nature. CA 125 seems to be a more promising tumor marker for ovarian cancer than placental alkaline phosphatase.