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Immunoreactivity for a panel of 15 monoclonal antibodies (MAbs), which are known to react with different gastrointestinal tumor antigens, was assessed in formalin-fixed paraffin-embedded sections that were prepared from cholecystectomy specimens obtained from Mexican patients. Each case was classified histologically into one of the following groups: (1) invasive adenocarcinoma (N = 21), (2) high-grade dysplasia (carcinoma in situ) (N = 2), (3) low-grade dysplasia (N = 4), hyperplasia (4) (N = 15), and (5) chronic cholecystitis (N = 10). Significant differences (P < 0.05) were identified among the five histopathologic groups in the proportion of epithelial cells demonstrating immunoreactivity with MAbs to Lewisb; Lewis(a); sialylated Lewis(a); sialylated Lewis(a) and Lewis(a); Y antigen; H antigen; X antigen; X-like antigen; 200-kDa protein of CEA; 180-, 160-, 50-, 40-kDa proteins of CEA; 30- to 37-kDa protein; and an undefined antigen identified by MAb 99-57, with invasive carcinoma more frequently being positive as compared to nonneoplastic (hyperplasia, chronic cholecystitis) epithelium. Significant differences were also observed among the five histopathologic groups (P < or = 0.0005) in the proportion of epithelial cells demonstrating immunoreactivity with MAbs to Y antigen and the 20- to 50-kDa glycoprotein. However, with these two antibodies immunoreactivity was more frequently found in nonneoplastic epithelium rather than in invasive carcinomas. No significant differences in immunoreactivity were detected among the different histologic groups with MAb to blood group B antigen, types 1 and 2. This study demonstrates that cellular antigens are both developed and lost during the process of neoplastic transformation in the gallbladder.(ABSTRACT TRUNCATED AT 250 WORDS)

Seven human ocular melanoma cell lines were established in vitro and 3 of these, GU-4, LLN-40 and its subline C17-11, were characterized. Mice were immunized with these ocular melanoma cell lines, and 2 hybridomas producing monoclonal IgG1 antibodies (MAb) were produced. MAb 2/10SN recognizes a 44-kDa monomeric protein, whereas MAb 10/12SN reacts with an 83/65-kDa heterodimeric protein. These melanoma-associated antigens (MAA) are detected at high concentrations in the cytoplasm of ocular melanoma cells. However, cell-surface labelling techniques suggest that these MAA are also associated with the cell-surface membrane. These 2 ocular MAA are also expressed by several skin melanoma cell lines. Immunohistochemical studies have localized these antigens to ocular and skin melanomas, to sweat ducts and basal squamous cells in normal skin, with limited expression in several other normal tissues and some carcinomas. Biodistribution studies in nude mice with human ocular melanomas have demonstrated good localization of 125I-labeled MAb 2/10SN at the tumor sites. Therefore, these 2 MAbs, 2/10SN and 10/12SN, recognize MAA which appear to be unique and may prove useful for imaging purposes.

Two monoclonal antibodies (MAbs), IgG2a MAb ASHG4 and IgG2b MAb ASHE2, were produced in mice immunized with cultured human malignant glioma cells. Both MAbs bound strongly to the surfaces of long-term cultured glioma cells, and MAb ASHE2 also bound strongly to short-term cultured glioma cells. Sections of frozen glioma tissues bound both MAbs strongly, whereas normal brain tissues showed weaker reactivities, and tissues derived from carcinomas of various histological types were completely unreactive. Furthermore, the MAbs did not bind to peripheral blood cells or bone marrow cells. Although both MAbs bound to the same Mr 27,000-29,000 protein, they may detect different or overlapping epitopes on this antigen. Because MAbs ASHE2 and ASHG4 lysed cultured glioma cells with human peripheral blood lymphocytes as effector cells, they are promising reagents for approaches to immunotherapy of human malignant gliomas.

The binding specificities of monoclonal antibodies against human cutaneous malignant melanoma were analyzed using radioimmunoassay (RIA), mixed hemadsorption assay (MHA), and peroxidase-antiperoxidase assay on a variety of malignant and nonmalignant cells. Twenty-four of the 30 monoclonal antibodies bound to the majority of melanoma cell lines tested, and only two antibodies did not bind to any of the melanoma lines. Three antibodies bound to melanoma lines only, 13 antibodies reacted also with fetal cells, 21 antibodies bound to at least one carcinoma cell line and nine antibodies reacted with one or more cell lines of leukemic or lymphoid origin. The antibodies could be divided into seven groups based on their binding characteristics. These groups had been established by a panel of monoclonal antimelanoma antibodies produced and characterized at The Wistar Institute.

A melanoma-associated proteoglycan antigen is expressed by primary cutaneous and ocular melanomas, metastatic melanomas, nevus cells, some astrocytomas, and fetal fibroblasts, and it is shed into culture supernatant by both melanoma and nevus cells. The antigen is also expressed by tumor cells in vivo. Melanoma and nevus cells, but not normal melanocytes, were specifically stained by the immunoperoxidase procedure. The proteoglycan antigen, purified by immunoaffinity chromatography using a monoclonal antibody that specifically detects this antigen, was used to immunize rabbits. The resulting serum was tested by sequential immunoprecipitation and found to react with the same population of molecules detected by the anti-proteoglycan monoclonal antibodies. Furthermore, the reactivity patterns of the rabbit serum and of the monoclonal antibodies with a variety of tumor and normal cells were the same. Based on the these data, we conclude that the entire proteoglycan molecule is a melanoma-associated antigen. The monoclonal antibodies and immunoglobulin from the rabbit serum were tested in a double determinant immunoassay for the detection of antigen in a total of 339 sera from patients with various diseases. Elevated levels of circulating proteoglycan antigen were found in 76% of patients with a high metastatic melanoma tumor burden compared to 2% of healthy donors. A fraction (22%) of patients with light tumor burden or nonmelanoma neoplastic disease also had elevated levels of circulating proteoglycan antigen. The source of the antigen for the latter patients may be collagenous connective tissue which, as judged by immunoperoxidase staining, expresses the antigen in both normal and transformed tissues.

Over 600 hybridomas were derived from the immunization of mice with live cells and aqueous extracts of the human prostatic carcinoma cell line PC3. A total of 26 hybridomas with restricted reactivities were selected, subcloned and antibodies tested on a variety of tumor and normal cells. Seven monoclonal antibodies showed reactivity for prostate cancer and other tumor cell lines, including breast carcinomas. Three of the antibodies obtained after immunization with live cells reacted with live cells only and three of the four antibodies obtained after immunization with cell extract reacted with cell extracts and spent culture media. The fourth antibody in the latter group was reactive only in the immunoperoxidase staining assay. Antibody PrS5 recognized a 90,000 molecular weight molecule from 125I-surface-labeled cells in immunoprecipitation analysis. Antibodies PrE3 and PrD8 detected a nonacid glycolipid pentasaccharide from PC3 cells and meconium, and a glycoprotein of 115,000 molecular weight from 125I-surface-labeled red blood cells. The similar patterns of reactivity in RIAs and antigen analysis suggest that antibodies PrE3 and PrD8 recognize the same molecule. The results emphasize the usefulness of immunohistochemistry in the testing of monoclonal antibodies and the impact of the form in which the antigen is presented on the resultant antibody specificity.

A monosialoganglioside antigen of gastrointestinal adenocarcinomas defined by murine monoclonal antibody was demonstrated by immunoperoxidase (IP) assay in fixed paraffin-embedded tumors in 59% of colonic adenocarcinomas, 86% of pancreatic adenocarcinomas, and 89% of all gastric adenocarcinomas. In all patients with detectable levels of antigen in circulation, the resected tumors also expressed the antigen in IP assay. Six of eight individuals with no detectable levels of antigen in their serum samples expressed the antigen in the tumor tissue. Removal of the sialic acid residue of the antigen abolished the IP reaction. The successful use of the IP assay on fixed tissue to demonstrate the specific sites of gastrointestinal cancer antigen localization in human tumors and normal tissues provides an important tool for the study of developing neoplasia.

Monoclonal antibodies made against gastrointestinal carcinoma antigen (GICA) and stage specific embryonic antigen (SSEA) were evaluated for their ability to distinguish normal mesothelial cells present in pleural and peritoneal fluids from adenocarcinoma cells in tissue and cytology specimens. The presence of GICA was documented in a high percentage of adenocarcinomas from the gastrointestinal tract (75/98) and in 52% of pulmonary (15/29) and 29% of ovarian (6/21) adenocarcinomas. GICA was found infrequently in breast carcinoma (1/18) and not in mesotheliomas (0/16). A similar pattern of GICA expression was seen in malignant effusions from adenocarcinomas (18/47) and mesotheliomas (0/6). SSEA was found in a high percentage of adenocarcinomas derived from the gastrointestinal tract (47/56) and the lung (26/29). SSEA was detected in breast carcinoma (8/15) more often than GICA. SSEA was detected rarely in mesotheliomas (1/16). Reactivities for epithelial membrane antigen, keratin, carcinoembryonic antigen, GICA and SSEA in adenocarcinoma and mesotheliomas were compared.

An anticolorectal carcinoma antibody, 17-IA, that is of gamma 2a isotype and has been used in previous immunotherapy trials, binds to an antigen on most human colon tumors. This antigen is destroyed by the fixative and embedding procedures employed for routine histologic evaluation of tissues. It can be demonstrated by the avidin/biotin-labelled peroxidase complex (ABC) immunoperoxidase technique on briefly fixed, frozen sections of fresh human tissues. With this assay technique, the antibody is found to bind in vitro to both human colonic carcinoma and normal gastrointestinal epithelia. Tissues removed from patients having received the 17-IA antibody intravenously in an immunotherapy trial show bound antibody 1-2 after administration. This is no longer evident 3 weeks later though the antigen is present on the cell surface and is still capable of binding antibody in vitro.

Reactivity of neuraminidase-treated colorectal carcinoma cells with antibodies that detect the X-carbohydrate structure was greater than the reactivity of untreated cells. The same results were obtained with glycolipid extracts of meconium, a colorectal carcinoma cell line, three freshly excised human adenocarcinomas, and normal bronchial mucosa. The glycolipid was either not expressed or expressed in smaller quantities on the corresponding normal colon tissue. Further study showed that the major sialo-X glycolipid has six sugars including a single sialic acid which blocks X-antigenicity. These glycolipids were further analyzed by ion-exchange high-pressure liquid chromatography and thin-layer chromatography. These monosialo-X glycolipid antigens might serve as potential tumor markers.

A highly glycosylated protein with a molecular weight of 30,000 to 60,000 and a protein core of 20,000 daltons has been identified by antimelanoma monoclonal antibodies. The antigenicity of this melanoma-associated glycoprotein (MAG) was not destroyed in fixed paraffin-embedded melanoma tissue, and was present in malignant cells of cutaneous superficial spreading melanomas in skin (31/33) and in half of all metastatic melanomas examined (5/10). The antigen was not expressed by normal melanocytes. The strong reactivity of dysplastic nevi with the anti-MAG antibodies was comparable to that seen in radial growth phase melanoma. Antigen expression was much weaker in compound nevi where reactivity ranged from moderate in the junctional component and the upper dermis to absent at the base of the nevus.

A method is described for selection early after fusion for hybridomas that secrete IgG2a monoclonal antibodies (MAbs) with binding specificity for antigens on the human tumor cells used to immunize mice. By combining this preselection method with antibody-dependent macrophage-mediated cytotoxicity assays, it was possible to select those MAbs mediating a tumoricidal effect against tumor cells in culture and inhibiting the growth of tumor xenografts in nude mice. Two such MAbs, GA733 and CO441, inhibited the growth of colorectal carcinoma cells, and one of them (GA733) was effective even when administered 6 days after implantation of tumor cells. These results suggest the potential usefulness of the 2 MAbs in immunodiagnosis and immunotherapy of human tumors.

The expression of human blood group A and B isoantigens in normal and malignant tissues from stomach, colon, and pancreas was analyzed in an immunoperoxidase assay using monoclonal antibodies specific for these isoantigens. Appropriate isoantigen expression was demonstrated in the normal epithelium from the stomach, pancreas, and proximal but not distal colon of blood group A, AB, or B patients. Half of all gastric carcinomas and of proximal colon carcinomas showed complete loss of isoantigen, whereas the adjacent mucosa in these cases continued to express appropriate isoantigen. Isoantigen expression was completely lost in only 13% of pancreatic carcinomas tested. Neither A nor B isoantigen was detected in normal epithelium from the distal colon. By contrast, 85% of carcinomas derived from this site showed reexpression of isoantigen. Inappropriate expression of A isoantigen was detected in pancreatic carcinomas (2/5) but not in gastric or colon carcinomas (0/21). Inappropriate expression of B substance was not detected in any tissue (0/38). Interestingly, differential binding of antibodies to Type 1 versus Type 2 and/or difucosyl versus monofucosyl blood group B substances was manifested by differences in intensity of staining for endothelium and red blood cells.

The cell surface antigen distribution on traumatic neuroma Schwann cells and neurofibroma Schwann-like cells was characterized using monoclonal antibodies that define melanoma-associated antigens. Immunofluorescence staining of cultured cells, immunoprecipitation of radioiodinated antigens from cells placed in short-term cultures, and immunoperoxidase staining of frozen tissue sections revealed most of the melanoma-associated antigens tested on traumatic neuroma and neurofibroma Schwann cells and on fetal and adult femoral nerve. The cross-reactivity of the antibodies with neural cells may reflect the common neural crest embryological origin of Schwann cells and melanocytes. Cell sorter analysis of neurofibroma cells using a monoclonal antibody directed against the melanoma nerve growth factor receptor resulted in cell cultures highly enriched for Schwann-like cells which may bear the genetic defect responsible for neurofibromatosis. The antigen detected by this monoclonal antibody is the neurofibroma nerve growth factor receptor and the antibody was a potent inhibitor of nerve growth factor binding to neurofibroma cells.

To assess the change in concentrations of circulating gastrointestinal cancer-associated antigens in response to therapy, we analyzed the sera of patients with hepatic metastasis from colorectal carcinoma who were treated with intrahepatic arterial chemotherapy. Serial serum samples were assessed for the tumor-associated antigens, carcinoembryonic antigen (CEA) and the gastrointestinal cancer antigen CA 19-9. Computed axial tomographic (CAT) scans were made to assess the size of the hepatic metastasis. In 9/10 of these patients the CEA predicted tumor response within 2-6 weeks after initiation of treatment, and in 7/10 the information was supported or more dramatically demonstrated by the CA 19-9. Combining data from both tumor markers may provide a more accurate assessment of the clinical response than one antigen alone. Recurrence of hepatic metastatic growth or extrahepatic tumor also was identified by elevation of one or both circulating tumor-associated antigens prior to other laboratory or clinical evidence of tumor growth.