Rapid growth and spontaneous metastasis of human germinal tumors ectopically transplanted into scid (severe combined immunodeficiency) and scid-nudestreaker mice.

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F2 hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c-nu/nu and CD1-nu/nu). The YST-2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude-streaker (AKR/J-nustr/nustr) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one-third (36.4%) of scid mice and very rapidly in all of scid-nustr (scid/scid; nustr/nustr) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid-nustr mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E.

A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 micrograms. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1-Tc1. The cytotoxic cell population was more than 90% CD4+. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.

SCID (severe combined immunodeficiency) mice as a new system to investigate metastasis of human tumors.

In severe combined immunodeficiency (scid) mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) grew rapidly to enormous sizes in all of the animals after both subcutaneous and intraperitoneal transplantation, while only half of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice. Furthermore, transplanted tumors metastasized spontaneously to distant organs such as lung, liver, kidney, pancreas, and spleen in scid mice, while metastases were not found in athymic nude mice. Similar results were observed in scid mice and scid-nude (streaker) double mutant mice with human classic (typical) seminoma which has been neither transplantable nor metastatic in athymic nude mice. Thus, scid mice provide an invaluable experimental system to investigate the mechanism of metastasis which is the most important and life-threatening problem in cancer patients.

Estrogen and progesterone receptors in endodermal sinus (yolk sac) tumor. Evaluation of immunocytochemical and biochemical methods.

Frozen tissue from four ovarian and two testicular endodermal sinus (yolk sac) tumors was studied with both biochemical and immunocytochemical methods to detect the presence of estrogen receptors (ERs) and progesterone receptors (PRs). Using the biochemical assay, one of the ovarian tumors was shown to contain low levels of ERs, whereas in the remaining five tumors no ERs were detected, and PRs were undetectable in all six tumors. In the frozen sections immunostained for ERs and PRs, no specific nuclear staining was detected in any of the six tumors. Since the immunocytochemical method provides a more tissue-specific evaluation of hormone receptors, the low level of ERs detected in one tumor using the biochemical assay is attributed to nonspecific binding. It appears that endodermal sinus tumor expresses neither ERs nor PRs.

Human yolk-sac tumor antigen 2G10: biochemical characterization and significance as a serum antigen.

The biochemical characterization of human yolk-sac tumor (YST) antigen 2G10, detected by monoclonal antibody (MAb) M912-2G10, was studied. Previous results indicated that glycolipids having a non-reducing terminal N-acetyllactosamine structure were the epitope of 2G10 on human erythrocytes. In this study, the glycoprotein nature of 2G10 on the infantile embryonal carcinoma line, MTE, was investigated. 2G10 activity, measured by a new enzyme-linked immunosorbent assay (2G10-ELISA), was recovered in residual fractions of MTE from which glycolipids were removed. Chromatographically, 3H-galactose-labelled 2G10 on MTE had a molecular weight (mw) of about 580 kDa, which decreased after pronase or alkaline-borohydride treatment. Our results indicate the glycoprotein nature of 2G10 on MTE. Furthermore, 2G10, both on erythrocytes and on MTE, was sensitive to galactosidase but not to neuraminidase and fucosidase, suggesting that terminal galactose is involved in the antigenic structure. It was also found by 2G10-ELISA that 2G10 sheds from tumor cells. Shedding occurs in nude mice transplanted with MTE as well as in patients with germ-cell tumors (GCTs). The serum level of 2G10 in non-tumor patients was low, but high levels were detected in patients with YSTs and with GCTs having YST components. Immunohistochemically, the presence of 2G10-positive YST components was shown in patients who had high serum levels of 2G10. Sera from other urogenital and childhood solid tumors did not have elevated 2G10. The mw of shed 2G10 was lower than that of 2G10 on the cell surfaces. Our results clearly indicate the usefulness of serum 2G10 as a tumor marker for GCTs having YST components.

[Monoclonal antibodies raised against cultured cells and their application in cancer diagnosis].

Since cultured cells have the possibility to produce tumor-associated substances in vitro, we generated several monoclonal antibodies using cultured cells as immunogen and applied them to cancer diagnosis. Two monoclonal antibodies (F602-1 and F602-6) were generated against a newly established ovarian mesonephroid cancer cell line (RMG-II), and a double determinants sandwich enzyme-immunoassay system was developed. Their epitopes were proved to exist on the CA125 molecules, but to be different from the one recognized by OC125, and almost the same result of serum assay as CA125 was obtained by this assay. A new monoclonal antibody (MSN-1) was produced by immunizing a new endometrial cancer cell line (SNG-II). MSN-1 recognized the Lewis-b carbohydrate moiety on the cell surface glycolipid, and seldom reacted immunohistochemically with normal endometrium, but reacted with about 90% of endometrial cancer cases. By application of MSN-1 to flow-cytometry, the possibility of differentiating endometrial normal cells from the cancer cells was demonstrated.

Immunohistochemical differences between intracranial germinomas and their gonadal equivalents. An immunoperoxidase study of germ cell tumors with epithelial membrane antigen, cytokeratin, and vimentin.

Twenty-six intracranial germ cell tumours (11 germinomas, 10 teratomas, 2 endodermal sinus tumours, 1 teratocarcinoma, and 2 undifferentiated embryonal carcinomas) and 26 gonadal germ cell tumours (13 testicular seminomas, 2 ovarian dysgerminomas, 9 ovarian teratomas, and 2 myometrial choriocarcinomas) were studied by immunoperoxidase with monoclonal antibodies (MAbs) against epithelial membrane antigen (EMA), cytokeratin, and vimentin. Typical tumour cells in three of the 11 germinomas (two of the latter being situated in the posterior fossa) expressed both EMA and cytokeratin, whereas those in the seminomas and dysgerminomas did not. In one seminoma, a few multinucleated giant cells expressed cytokeratin. In three of seven germinomas, vimentin-positive tumour cells were found, but all seminomas and dysgerminomas were negative. In the other forms of intracranial and gonadal germ cell tumours, epithelial and mesenchymal elements displayed the expected patterns of immunoreactivity to the respective determinants. The immunoperoxidase differences between the intracranial germinomas and their gonadal equivalents indicate that, in the former, early epithelial or mesenchymal differentiation of the primordial germ cells may be present. The findings draw attention to the heterogeneous cellular composition of these otherwise morphologically homogeneous-appearing tumours and, especially in the posterior fossa, to their transitional links to the immature teratomas.

Blood group-related antigens in human germ cell tumors.

With the use of immunohistochemical techniques, seven mouse monoclonal antibodies and the lectin from Ulex europaeus, detecting blood group antigens of the ABH and Lewis systems, have been used to define the distribution of these antigenic structures in germ cell tumors. The reagents used recognize the following blood group antigens: A, B, H, Lewisa, Lewisb, X (Lewisx), Y (Lewisy), and type I precursor antigen. Tumors from 29 patients were studied. Tumors studied consisted of pure embryonal carcinoma for eight patients, pure yolk sac tumor for two patients, embryonal carcinoma plus yolk sac tumor in one patient, and yolk sac tumor plus seminoma in one patient. Also studied were nine classic seminomas and a group of six patients with tumors classified as seminomas that exhibited atypical histological features. One patient had an anaplastic carcinoma arising from the mediastinum which could not be conclusively identified as a germ cell tumor morphologically and was analyzed separately. All embryonal carcinomas and yolk sac tumors exhibited strong positivity for type I precursor structure as detected by the K-21 monoclonal antibody. In marked contrast, there was non staining in classic seminomas but heterogeneous staining in five of six atypical seminomas. The majority of embryonal carcinomas and all yolk sac tumors studied demonstrated strong positivity for blood group antigen H. For seminoma, however, only one of the atypical cases and two of the classic cases (occasional cells) stained for H. Focal expression of the Y antigen was identified in 5 of 17 seminomas and in the majority of embryonal carcinomas and yolk sac tumors. Two yolk sac tumors and two classic seminomas expressed blood group X. The remaining blood group antigens were not expressed by seminomas while they were variably expressed by embryonal carcinoma and yolk sac tumors. These data suggest that K-21 and blood group antigen H may be distinguishing markers of nonseminomatous germ cell tumor versus seminoma. If so, it is possible that the heterogeneous expression of blood group substances in seminomas with atypical histologies is an indication of differentiation towards nonseminomatous germ cell tumor.

Appearance of serum antibodies to rat yolk-sac carcinomas during the latent period prior to primary tumor development.

Rat yolk-sac tumors were induced by intraperitoneal (i.p.) displacement of the visceral yolk sac in fetectomized W/Fu rats. Serum was obtained from each female rat prior to the pregnancy preceding the tumor-inducing procedure and then once a month during the induction period. The sera were analyzed for the presence of antibodies binding to cultured cells of one of the yolk-sac tumors. Sera were also assayed for complement-dependent cytotoxic antibodies on tumor cells. In rats that developed tumors, antibodies reacting specifically with the target tumor cells could be detected in all of 10 rats. Antibodies appeared before tumor detection in all animals but one, and in 6 rats as early as 11 to 25 weeks prior to tumor detection. Nine rats developed antibodies demonstrable in the binding assay and in 6 of those the antibodies appeared 8 to 25 weeks before the tumor became palpable. Analysis of the isotypes of the Ig that bound to tumor cells showed that IgG1 and IgG2b were most frequently present. In one rat IgG2a antibodies appeared one month before tumor detection followed by IgG1 and IgG2b antibodies detectable 4 weeks later. IgG2c and IgM antibodies were not detected in any of the rats. At dilution 1/10, sera of all 10 rats showed specific cytotoxicity to the tumor cells in the presence of added rabbit complement. In 9 of these animals antibodies were demonstrated 1 to 4 months prior to tumor detection.

Cervical adenocarcinoma arising in florid mesonephric hyperplasia: report of a case with immunocytochemical studies.

A case of mesonephric adenocarcinoma of the uterine cervix arising in florid mesonephric hyperplasia is reported. The reviewed literature contained many cases of cervical "mesonephroma" but only a few of these were considered to be demonstrably of mesonephric origin. These tumors were usually associated with proliferating mesonephric remnants in the cervix. Similar tubuloglandular mesonephric proliferations without formation of a frankly malignant tumor have also been described in the cervix, most recently as florid mesonephric hyperplasia. This latter entity appears to be benign. Carcinoembryonic antigen (CEA) was focally positive in this cervical adenocarcinoma, suggesting that CEA may not be useful in distinguishing this variant from the more common mullerian adenocarcinoma of the cervix.

Monoclonal antibodies with fine specificities distinguishing alpha-fetoproteins of hepatoma and yolk sac tumor origin.

Monoclonal antibodies with fine specificities distinguishing alpha-fetoproteins of hepatoma (HEP-AFP) and yolk sac tumor (YST-AFP) origin have been obtained. These murine antibodies were produced by hybridomas made by fusion of X63-Ag8.653 myeloma cells with BALB/c spleen cells immunized with either HEP-AFP or YST-AFP and selected for their differential association with these antigens on the basis of Scatchard plot analysis. Three monoclonals (MA120, MA132 and MA136) selectively reacted with HEP-AFP. Their reactivity with YST-AFP was low. One monoclonal (MA122) reacted strongly with YST-AFP, whereas the reaction with HEP-AFP was significantly less strong. The difference in the association constants of these antibodies for the two AFPs appeared to be due to their specificity for the carbohydrate portions of the AFPs, which are different, at least in part. Indirect immunoperoxidase staining confirmed that MA122 was able to stain sections of an infantile embryonal carcinoma, but not of hepatoma.

Forssman antigen on yolk sac derived rat tumor cells.

By means of indirect immunofluorescence (IFF) tests with rabbit antiserum to Forssman (F) glycolipid, expression of F antigen was investigated on 16 rat tumor cell lines; 14 chemical carcinogen-induced tumors, one yolk sac-derived (AT-1) tumor and another spontaneous tumor. Four lines of the carcinogen-induced tumors gave weakly positive reactions in the IIF tests and the AT-1 cells a strongly positive reaction. Flow fluorocytometric analyses on the AT-1 cell line revealed that some of the major and the vast majority of the minor population of the AT-1 express F antigen. Specificity of the F antigen demonstrated on AT-1 cells was determined by absorption experiments, in which absorption of the F antiserum with guinea pig kidney sediment, sheep red blood cells and F-liposome but not bovine red blood cells or G-liposome abolished the reaction. Results of this study together with those of our previous studies demonstrated that F antigen appears as a result of malignant transformation of F negative rat cells induced by chemical carcinogens, apparently "distorted differentiation" and the viral oncogenes.

[Anti-tumor activity of an anti-human alpha-fetoprotein antibody-human serum albumin-mitomycin C conjugate against human yolk sac tumor maintained in vitro and in nude mice in vivo].

The anti-cancer drug, mitomycin C (MMC), was conjugated to an affinity-purified horse antibody to human alpha-fetoprotein (aAFP) via purified human serum albumin (HSA) as an intermediate carrier. The conjugate (aAFP immunoglobulin (IgG): HSA: MMC, molar ratio 1: 1.10:29.8) was 21 or 38 times as cytotoxic as free MMC or PBS at the MMC concentration of 100 ng/ml, respectively, against alpha-fetoprotein-producing human yolk sac tumor in vitro. The in vivo effects of the conjugate and various controls were also tested against human yolk sac tumor growing in nude mice. The conjugate over a total of 6 injections retarded the initial tumor growth at the concentration of MMC, containing equivalent amounts of 2 micrograms/mouse (0.1 mg/kg)/injection in the conjugate, whereas free MMC and normal horse IgG-HSA-MMC showed only slight inhibitory effects alone at non-toxic levels. These results suggest that the specific antibody-conjugate was considerably more effective than free MMC against the tumor maintained in nude mice as well as in vitro cultures.

Evaluation of a conjugate of purified antibodies against human AFP-dextran-daunorubicin to human AFP-producing yolk sac tumor cell lines.

The anticancer drug, DNR, was conjugated to an affinity-purified horse antibody to human AFP (aAFP) via a dextran bridge. The conjugate (immunoglobulin: DNR molar ratio, 1:50) was twice as potent as free DNR in an in vitro cytotoxicity assay against an AFP-producing human yolk sac tumor. The in vivo effect of aAFP, DNR, and the conjugate was tested against the human yolk sac tumor growing in nude mice. The conjugate, at a concentration of DNR containing the equivalent amount of 20 micrograms or 70 micrograms/mouse significantly retarded tumor growth whereas free aAFP showed only a slight inhibition of tumor growth compared to the PBS-treated control. Mice which received 20 micrograms/mouse of free DNR showed a moderate retardation of tumor growth whereas those which received 70 micrograms/mouse of DNR or a mixture of DNR and aAFP showed emaciation and early death due to acute toxicity of the drug. These results suggest that the anti-body-drug conjugate accumulated preferentially on the AFP-producing tumor cells and that cytotoxicity occurred.

Role of immunohistochemistry in diagnosis of nasopharyngeal tumours.

To evaluate the usefulness of immunocytochemistry in the differential diagnosis of nasopharyngeal tumours, 35 undifferentiated nasal carcinomas were examined with a panel of monoclonal antibodies against a wide variety of epithelial and non-epithelial antigens. The results were compared with those obtained from a series of nasopharyngeal tumours comprising three squamous cell carcinomas, six lymphomas, one rhabdomyosarcoma, and one yolk sac tumour. All of the carcinomas were positive with at least one of the antiepithelial markers and negative for the leucocyte common antigen and were clearly distinguishable from the nasopharyngeal lymphomas, which gave the opposite staining pattern. It was concluded that such a panel of monoclonal antibodies would be extremely useful for the differential diagnosis of nasopharyngeal tumours, particularly with small or technically inadequate biopsies.

A rat yolk-sac antigen defined by monoclonal antibodies.

The distribution of a rat yolk-sac antigen (Rat YSA-I) as defined by a monoclonal antibody raised against rat yolk-sac carcinoma cells is described. The antigen is present on rat yolk-sac carcinomas, on visceral yolk-sac endoderm and on embryonal endoderm of 9-day-old embryos. It is not present on a variety of rat tumors other than yolk-sac carcinomas and not detectable on pre-implantation embryos, inner cell mass and fetal endoderm. Rat YSA-I differs from alpha-fetoprotein and Forssman antigen and is species-specific. In adult rats the only cells displaying this antigen are the spermatozoa and certain cells of the spermatogenic lineage.

Antigenic markers in extragonadal germ cell tumors.

Using an immunoperoxidase technique, a series of 13 extragonadal germ cell tumors were screened for the presence of 7 different antigens: human chorionic gonadotropin, beta-subunit (beta-hCG), pregnancy-specific beta 1-glycoprotein (SP1), human placental lactogen (hPL), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), alpha 1-antitrypsin (alpha-AT) and ferritin. Syncytial giant cells in embryonal carcinoma and choriocarcinoma were positive for beta-hCG and SP1, while isolated foci of mononuclear cells in the embryonal carcinoma stained for AFP, alpha-AT and ferritin. Yolk sac tumor components showed immunoreactivity for AFP, alpha-AT and ferritin. In seminomas, a positive reaction for ferritin was found only in isolated cells of 2 cases. One seminoma was positive for alpha-AT. Teratomas were negative for all antigens, except for CEA and SP1 in duct-lining cells of sweat glands in one teratoma. Germ cell tumors of extragonadal sites appear to exhibit the same antigenic markers as their gonadal counterparts. Such similarities lend support to the hypothesis of a common cell origin of these neoplasms.

An ovarian tumour specific mucin antigen--immunohistological and biochemical studies.

A new mucin antigen was detected in a mucinous cystadenoma of human ovary. An antiserum produced in rabbits against the crude cyst fluid, following appropriate absorptions, showed immunoreactivity exclusively with tumour epithelium, particularly endocervical type epithelium. The antigen responsible for the immunoreactivity was isolated in the fraction with density 1.45g/ml following density gradient ultracentrifugation in cesium chloride, which indicated that it was indeed a glycoprotein. The native glycoprotein was very large since it was excluded from Sepharose CL-2B. The complex could be dissociated following reduction with dithiothreitol. The subunits were included in Sepharose CL-2B and the position of elution would indicate a molecular weight in the order of 1-5 x 10(6). This suggests the native antigen was a complex made up of subunits held together by disulphide bonds. Amino acid analysis of the new antigen showed a resemblance with intestinal mucins, as two-thirds of the protein consists of threonine, serine, proline, alanine and glycine. This similarity in peptide core may explain the potential of the epithelium in ovarian mucinous cystadenoma to produce inappropriate intestinal mucins during the process of malignant transformation.

[Human yolk sac tumor of the thymus heterotransplanted into nude mice].

A human yolk sac tumor of the thymus (YSK-1) was transplanted into athymic nude mice. Histological and ultrastructural investigations revealed that the YSK-1 tumor, even after serial transplantation in nude mice, characteristically displayed Schiller-Duval bodies, endodermal sinus structures and ultrastructural profiles, numerous microvilli, desmosome-like cell attachments, annulate lamellar structures, many lipid droplets and interspersed glycogen particles. Serological study by radioimmunoassay demonstrated AFP, CEA, HCG, alpha 1-AT and transferrin in the sera and cyst fluid of the tumor-bearing nude mice. Immunohistochemical investigation using the PAP method showed that the YSK-1 cells produced CEA, HCG, alpha 1-AT, transferrin as well as AFP. The increase in the level of AFP paralleled the increase in tumor size.