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The T-antigen system and the mean nuclear volume have been proposed as risk variables in bladder tumors. This study includes 34 patients with initially noninvasive (Ta) transitional cell carcinomas who experienced different courses of disease. Tissue specimens of primary tumors were analyzed for the expression of T-antigen, Tn-antigen, and sialosyl-Tn-antigen using monoclonal antibodies (MoAb) and the lectin peanut agglutinin (PNA) in an indirect immunoperoxidase method. In addition, the mean nuclear volume was estimated by morphometry. Tissue from 7 of 13 patients (54%) who had invasive disease during a follow-up period of 5 years expressed T-antigen, as defined by MoAb HH8 in the primary tumor, whereas tissue of only 3 of 21 patients who did not have invasive disease expressed the antigen (P less than 0.02). No association was found between tumor progression to invasion and the expression of Tn-antigen or sialosyl-Tn-antigen. Tn-antigen expression was partially lost in invasive tumors (P less than 0.03) when compared with the expression in primary noninvasive tumors. A high mean nuclear volume in tissue specimens of primary tumors correlated with a progression to invasive disease (P less than 0.01). A significantly (P less than 0.003) higher mean nuclear volume was found in tumor areas that were positive for PNA compared with areas that were negative for PNA in primary tumors. A significantly lower mean nuclear volume was found in Tn-antigen-positive invasive Grade 3 tumor areas than in Tn-antigen-negative areas (P less than 0.005). The combined use of T-antigen expression and mean nuclear volume is of potential clinical interest for determining patients who are at high risk of disease progression.

Carbohydrate antigens representing some of the initial steps in mucin O-linked glycosylation were examined in specimens of normal pancreas, chronic pancreatitis, and pancreatic adenocarcinoma. Tn antigen, recognized by Vicia villosa lectin, was expressed by all specimens of normal pancreas (acinar cells) and pancreatic cancers and all but one case of chronic pancreatitis. Sialosyl Tn antigen, recognized by monoclonal antibody TKH2, was expressed in a cancer-associated fashion, being completely absent in normal pancreas but expressed by 56% of chronic pancreatitis and 97% of pancreatic cancers. T antigen, recognized by monoclonal antibody AH9-16, was expressed in 68% of normal pancreas (acinar cells), 67% of chronic pancreatitis, and 48% of pancreatic cancer tissues. These results indicate that normal acinar cells of the pancreas are capable of expressing selected carbohydrate structures associated with the initial steps of mucin glycosylation. The marked expression of sialosyl Tn compared with T antigen in pancreatic cancers suggests that with malignant transformation there is selective usage of glycosyltransferase enzymes involved in mucin oligosaccharide synthesis.

Recently, the ganglioside FucGM1 (Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]-Gal beta 1-4Glc beta 1-1 Cer) was identified as a small cell lung cancer (SCLC) marker both in chemical and histochemical studies. In order to further determine whether the FucGM1 ganglioside is shed from the tumor site and consequently is present in the serum of SCLC patients, we produced a series of new monoclonal antibodies raised against FucGM1 and related glycolipids. Shedding of the FucGM1 ganglioside was studied both in vitro and in vivo using SCLC cell lines and nude mice xenografts of SCLC cells as model systems, and finally immunochemical analyses were performed on serum samples from patients with SCLC. High-performance thin-layer chromatography immunostaining demonstrated the presence of FucGM1 in conditioned culture media obtained from FucGM1-positive SCLC cell lines. Furthermore, tumor extracts of SCLC cell line xenografts in nude mice were positive for the FucGM1 marker, and more importantly the marker was also present in serum samples from these mice. Twenty serum samples were obtained from patients with histologically verified SCLC. Eight patients had localized disease, and the remaining patients had disseminated cancer involving metastases to other organ sites. Sera from 4 of these patients were clearly positive, and 2 additional cases were found to be weakly positive. The positive patient sera were all from patients with extensive disease. Sera from 12 patients with non-SCLC and 20 healthy individuals were all found to be negative. These results clearly establish the FucGM1 glycolipid as a potential serum marker of SCLC for which a sensitive immunoassay should be developed and tested using a larger series of serum samples.

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.

Mucin glycoproteins are major secretory products of the colon and contain O-linked oligosaccharides synthesized on a polypeptide backbone. The initial step in the synthesis of O-linked oligosaccharides is the addition of N-acetylgalactosamine to serine or threonine residues forming the Tn antigen. This substance can then receive additional carbohydrate residues such as sialic acid to form sialosyl-Tn antigen, or galactose to form T antigen. In the colon, the T antigen is an oncodevelopmental cancer-associated antigen but little is known about Tn and sialosyl-Tn expression. The present comparative immunohistochemical study was performed to analyze the expression of these antigens in fetal, normal adult, and malignant colorectal tissues with an aim toward elucidating whether Tn and sialosyl-Tn are also oncodevelopmental colon cancer-associated antigens and to gain insight into the earliest steps of mucin glycosylation in colonocytes. We used three reagents to detect Tn antigen (two monoclonal antibodies ETn1.01 and CU-1, and one lectin Vicia villosa), two reagents to detect sialosyl-Tn (monoclonal antibodies TKH2 and B72.3) and one to detect T antigen (monoclonal antibody AH9-16). Except for occasional reactivity with VVA and CU-1, cells of normal colonic mucosa did not express Tn, sialosyl-Tn, or T antigens. However, in the transitional mucosa immediately adjacent to cancer, all three antigens were expressed (ranging from 35 to 67% of cases depending upon the reagent). In colon cancers, the percentage of cases expressing each antigen were as follows: Tn 72-81%, sialosyl-Tn 93-96%, and T 71%. Unlike T antigen, which was preferentially expressed by moderately well- and well-differentiated adenocarcinomas, both Tn and sialosyl-Tn antigens were expressed by most histological subsets of colon cancers, including poorly differentiated adenocarcinomas and mucinous (colloid and signet ring cell type) carcinomas. The majority of cancers expressed both Tn and sialosyl-Tn, usually in association with T antigen. Only one cancer lacked all three antigens. Fetal colonic mucosal cells expressed all three antigens, particularly in goblet cell mucin. These results indicate that like T antigen, Tn and sialosyl-Tn are oncodevelopmental cancer-associated antigens in the colon. Moreover, Tn and sialosyl-Tn antigens appear to be useful markers of poorly differentiated adenocarcinomas and mucinous carcinomas: two histological subsets that often fail to express other cancer-associated antigens and that are often associated with a poor clinical outcome.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyagglutinable erythrocytes expressing the rare human blood group phenotype Tn were tested for expression of both Tn (GalNAc alpha 1----O-Ser/Thr) and sialosyl-Tn (NeuAc alpha 2----6GalNAc alpha 1----O-Ser/Thr) antigens by agglutination with a panel of well-characterized monoclonal antibodies specifically directed to Tn and sialosyl-Tn antigens, respectively. Tn erythrocytes from 4 patients were strongly agglutinated by both sialosyl-Tn and Tn-specific monoclonal antibodies, indicating coexpression of sialosyl-Tn and Tn antigens on the cell membrane surface of Tn polyagglutinable erythrocytes. Human polyclonal anti-Tn antisera were found to express weak and variable anti-sialosyl-Tn antibodies in conjunction with the anti-Tn antibodies. The Tn phenotype thus involves not only the classically known Tn antigens but also sialosyl-Tn antigens. However, the human anti-Tn antibodies may be directed mainly toward the Tn antigen (GalNAc alpha 1----O-Ser/Thr).

Two monoclonal antibodies, TKH1 and TKH2, directed toward the sialosyl-Tn structure (NeuAc alpha 2----6GalNAc alpha 1----O-Ser or Thr), which display a remarkable immunohistological tumor specificity, were generated by immunization with ovine submaxillary mucin. The reactivity of these antibodies was monitored by solid phase enzyme-linked immunosorbent assay with different native and glycosidase-treated mucins and glycoproteins. Binding of the antibody to ovine submaxillary mucin glycoprotein was strongly inhibited by the O-linked disaccharide NeuAc alpha 2----6GalNAc alpha 1----O-serine, less strongly by NeuAc alpha 2----6GalNAc beta 1----O-propyl, and weakly by the monosaccharide GalNac. The reactivity was compared with previously established anti-Tn antibodies B72.3, NCC-Lu-35, and NCC-Lu-81. The antibody B72.3 was prepared previously after immunization with metastatic breast adenocarcinoma and its epitope was claimed to be GalNAc alpha 1----O-Ser (or - Thr) by Springer and associates [Springer, G.F., et al. In: T. Dao, et al. (eds.), Tumor Markers and Their Significance in the Management of Breast Cancer, pp. 47-70. New York: A.R. Liss, 1986]. The antibody was found to show very similar reactivity as that of TKH1/TKH2, and its reactivity to ovine submaxillary mucin was inhibited specifically by NeuAc alpha 2----6GalNAc alpha 1----O-serine, indicating that the antibody is clearly directed to sialosyl-Tn antigen. Immunohistological study of the distribution of this antigen in various normal human tissues and carcinomas by TKH1/TKH2 antibodies, as well as B72.3 and monoclonal antibodies NCC-Lu-35/81, which are directed to GalNAc alpha 1----O-Ser or Thr (Tn), was performed. The sialosyl-Tn antigen was not found in normal tissue except for a weak expression in Leydig cells of the testis, goblet cells of the colon, and parietal cells of the stomach. In contrast, the sialosyl-Tn antigen was strongly expressed in a large number of adenocarcinomas. As expected from the specificity studies, B72.3 shows the same reactivity as TKH1 and TKH2. Thus, both sialosyl-Tn (NeuAc alpha 2----6GalNAc alpha 1----O-Ser/Thr) and Tn (GalNAc alpha 1----O-Ser/Thr) are good tumor markers, and combined use of antibodies directed to these structures might be useful in the screening and classification of cancer.