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Erythrocyte polyagglutination antigens T and Tn are truncated O-glycan chains that are also carcinoma-associated antigens. We investigated whether Tk polyagglutination antigen could similarly be a carcinoma-associated marker and a target of immunotherapy. Monoclonal antibody LM389 was raised against Tk erythrocytes and tested by immunohistochemistry. LM389 strongly reacted with 48% human colorectal carcinomas. Labeling of normal tissues was visible on epithelial cells, mainly digestive, but was confined at a supranuclear level. Expression of the antigen on cloned human carcinoma cells correlated with sialosyl-Tn expression. O-Sialoglycoprotein endopeptidase treatment revealed that on carcinomas and cell lines, the epitope was present on O-glycans. Antibody specificity was determined using synthetic carbohydrates. Direct binding and inhibition studies indicated that LM389 best ligands were terminated by two branched N-acetylglucosamine units. Screening of murine cellular cell lines with LM389 allowed development of an experimental model with Tk-positive and -negative cells in syngeneic BDIX rats. Vaccination of rats with Tk erythrocytes provided a protection against growth of rat Tk-positive, but not of Tk-negative, tumor cells in association with the development of antibodies. Taken together, the results indicate that Tk polyagglutination antigen is a new colorectal carcinoma-associated antigen, absent from the normal cell surface, resulting from alteration of O-glycans biosynthesis and with potential as a target of immunotherapy.

A mouse IgM monoclonal antibody FW6 was established after immunization of mice with mucins from human amniotic fluid and was characterized with regard to its binding epitope. According to a series of biochemical criteria the epitope is located on O-linked neutral carbohydrates of M(r) 700,000 and M(r) 570,000 mucins in human amniotic fluid. The epitope is presumed to contain alpha 3/4-linked fucose and terminal beta 3/4-linked galactose that are labile to the fucosidase I from almond emulsion or to the galactosidase from bovine testes, respectively. Immunoreactive fractions of glycan alditols from human amniotic fluid mucins were partially characterized by fast atom bombardment-mass spectrometry and methylation analysis to be composed of monofucosylated polylactosamine-type deca- or nonasaccharides. According to antibody competition studies, inhibition assays with defined carbohydrates and binding assays on neoglycolipids monoclonal antibody FW6 are presumed to recognize a novel epitope that is distinct from known carbohydrate markers of the Lex/Ley family associated with colonic carcinomas. The selective reactivity of this monoclonal antibody to the majority of human colonic carcinomas and its nonreactivity to normal colonic mucosa may render this antibody as a valuable tool in cancer diagnosis or cancer treatment.

We report the characterization of an IgG2a monoclonal antibody, (MAb) 660, prepared against rat gastric high molecular weight glycoproteins. By immunoperoxidase staining, MAb 660 reacted only with the mucous cells of surface gastric epithelium and with a few duodenal goblet cells close to the pylorus in normal adult rats. In fetuses, it reacted with intestinal and colonic goblet cells. The adult colon was always negative. The MAb 660 stained 100% (30 of 30) of chemically induced colonic carcinomas and 100% (7 of 7) of duodenal carcinomas. Several weeks before the appearance of tumors, histologically normal glands, then hyperplasia and dysplasia were precociously stained with MAb 660. The tissue distribution was different from that of blood group related antigens and M1 fucomucins. The recognized antigen was not sensitive to neuraminidase treatment. After electrophoresis in polyacrylamide gel, staining with periodic acid-Schiff reagent and Western blotting showed that the MAb 660 recognized an epitope associated with high molecular weight glycoproteins. This epitope was unaffected by beta-mercaptoethanol reduction-periodate treatment and neuraminidase and trypsin digestion. However, trypsin digestion performed after beta-mercaptoethanol reduction destroyed the 660 epitope. These data suggest that the antibody could recognize the peptide moiety of the mucin rather than its carbohydrate moiety. Thus, the new antigen identified by MAb 660 is a mucin-type glycoprotein with an oncofetal behavior in the rat colon and is precociously expressed by precancerous colonic mucosa.

Apparently normal mucosae adjacent to colon adenocarcinomas were studied by cutting strips of mucosa from the entire length of 120 surgical specimens (94 located on the distal colon and 26 on the proximal colon). These mucosae were coiled into "Swiss rolls." Their mucus alterations were mapped by immunoperoxidase using antibodies against M1 antigens, oncofetal markers associated with precancerous colonic mucosa. We demonstrated mucus modifications in patches of mucosa at a distance from frank tumors. The extent of these alterations was not related to invasion by the adjacent carcinoma according to Dukes' classification. However, these mucus modifications were more frequently observed on the distal than on the proximal side, were more often found adjacent to mucinous hyperplasia or adenoma, and were observed in 8 of 10 mucosae bearing metachronous or synchronous distal colonic adenocarcinomas. Our results suggest that the M1 modifications characterizing an early stage of carcinogenesis could have a putative prognostic value in estimating the risk for metachronous distal colonic adenocarcinomas.

The M1 antigens associated with gastric fucomucins, oncofetal markers of the distal colonic mucosa, were demonstrated to be more closely associated with adenomas [92 of 139 (66%)] than with invasive adenocarcinomas [27 of 218 (12%)]. They were always expressed in tumors containing the M3 antigen normally associated with the intestinal mucus. The M1 antigens, present in 100% of hyperplastic polyps (30 of 30), were not specific for a particular histological type of adenoma but were found to be more closely associated with those showing a villous differentiation [41 of 47 (87%)] than with those having a tubular pattern [51 of 92 (55%)]. The presence of these M1 antigens depended neither on the size nor on the degree of cytological atypia of the nodular adenomas. However, M1 antigens were found in 94% of the adenomas (35 of 37) concomitant with adenocarcinomas; in contrast, only 56% of adenomas (55 of 102) observed on noncancerous mucosa contained these M1 antigens. As already demonstrated during rat colonic carcinogenesis, mucus modification characterized by the presence of M1 antigens could represent early molecular changes occurring before malignant transformation related to a chemical carcinogen. These M1 antigens might be regarded as early precancerous markers of an oncofetal type, associated with human distal colonic mucosa.

We obtained seven monoclonal antibodies (MAb) against a gastric mucin of an ALeb patient. By immunoperoxidase on normal gastric mucosae, two MAbs (3-3A and 2-25 LE) reacted exclusively with the A and Lewis-positive individuals, respectively; the five other MAbs (1-13 M1, 2-11 M1, 2-12 M1, 9-13 M1, and 58 M1) stained the mucus cells of surface gastric epithelium independently of ABO or Lewis status. They did not stain normal colonic mucosae, but did stain fetal and precancerous colonic mucosae. Using serial sections, each anti-M1 MAb stained the same goblet cells in fetal and precancerous colon. Extensive search of other normal tissues showed that M1 antigens were restricted to the epithelium embryologically derived from the foregut (gastric and bronchial epithelium) and from Müllerian ducts (mucus cells of endocervix and prostatic utriculus). Some differences in the reactivities of the various anti-M1 MAb were observed in subesophageal, subtracheal, and endocervical mucus cells, suggesting that each anti-M1 MAb characterized a different M1 epitope. A mixture of these five anti-M1 MAbs allowed the estimation of M1 mucus modification in the precancerous colonic mucosae with a sensitivity near to that obtained with polyclonal anti-M1 antibodies. Papain and mercaptoethanol treatments destroyed the M1 epitopes, at variance with the A- or Lewis-related antigens. Our results therefore suggest that the expression of M1 epitopes in precancerous colonic mucosae cannot be due exclusively to alterations in mucin glycosylation but may be related to the reexpression of antigens associated with native gastric mucin which is normally produced by the fetal colon during the sixth month of gestation.

The present immunohistological study was performed to investigate the expression of intestinal mucus-associated antigens in the different histological types of gastric carcinoma according to the classification of Laurèn and the WHO classification. We used the following antigenic markers: M3, present in all the goblet cells of the whole small and large intestine; M3SI, expressed in all the goblet cells of the small intestine, but only in some of them of the large intestine; M3D, mainly produced in the upper small intestine; and M3C, specific for colonic mucus cells. All these antigens were found to be similarly expressed in both Laurèn's intestinal and diffuse types. Nevertheless, M3 and M3C appeared to be more largely produced in carcinomas showing well-differentiated cells (tubulopapillary, mucinous, and signet ring cells according to the WHO classification). Our results evidenced the occurrence of two main fetal antigenic patterns in gastric carcinomas, one of the gastric type (M3SI produced to a larger extent than M3 and M3C) and the other one of the small intestinal type (M3 and M3SI more largely expressed than M3C). On the basis of their similar antigenic pattern, the histogenesis of carcinomas showing the fetal small intestinal antigenic profile may be associated with intestinal metaplasia. On the other hand, carcinomas with the fetal gastric pattern may originate from undifferentiated stem cells of the gastric mucosa. Thus, such immunohistological studies could lead to a better understanding of the histogenesis of gastric carcinomas.

M1 antigens, associated with adult rat surface gastric epithelium and which are present in fetal but not adult distal colon, were investigated in this colonic mucosa during carcinogenesis. Fifty Wistar rats were given s.c. injections of 1,2-dimethylhydrazine for 28 weeks. Using an immunohistochemical method, M1 antigens associated with goblet cells were shown to be present after 2 weeks of 1,2-dimethylhydrazine treatment in histologically normal mucosa and then in 78% of mucinous hyperplasia and polypoid-like glands, in 54% of hyposecreting glands, in 58% of dysplasias, Grades 1 and 2, in two of 12 dysplasias, Grade 3, and in five of five transitional mucosas adjacent to carcinoma. The production of sialomucins associated with M1 antigens was often seen in the same histological lesion, although not always associated in the same goblet cells. The number of these histological lesions as well as the production of M1 antigens increased with the number of injections. Thus, these antigenic changes of an oncofetal nature can be regarded as early transformations of goblet cell differentiation in colonic mucosa subjected to chemical carcinogen.