Lewisx- and sialylated Lewisx-related antigen expression in human malignant and nonmalignant colonic tissues

Biochemical studies have revealed that some normal cells express the LeX trisaccharide Gal beta 1----4(Fuc alpha 1----3)GlcNAc either on short-chain fucolipids or as a single immunodeterminant on glycolipid oligosaccharide side chains. Cancer cells, including those from colonic adenocarcinomas, express this antigen on longer type 2 blood group side chains as difucosylated or trifucosylated fucolipids. Moreover, sialylated forms of difucosylated LeX also accumulate in colon cancer but not in normal colonic mucosa. In the present study, six monoclonal antibodies which selectively recognize the various LeX-related antigens were used for immunohistochemical examination of these antigens in serial sections of human colonic tissue. All of these antigens were oncodevelopmental in human colon. Monoclonal antibodies anti-SSEA-1 and AH8-183, directed against short-chain, monofucosylated LeX, were unable to discriminate well between normal and malignant colonic tissue. However, the other four antibodies were much better at distinguishing cancer from normal tissue. FH6 was the most specific in that no normal tissues bound this antibody. However, FH6 failed to stain poorly differentiated cancers and some colloid-type carcinomas. FH4, which was also highly specific, stained almost all cancers, regardless of the degree of differentiation. FH4 primarily stained cancer cell cytoplasm, whereas the sialylated antigen defined by FH6 predominantly stained cell membranes. Differences were noted between the expression of LeX-related antigens in autopsied normal mucosa compared to mucosa of benign colonic diseases. Monoclonal antibodies recognizing long-chain polyfucosylated and sialylated LeX-related antigens appear to be useful tools for detection of colon cancer.     

In vitro and in vivo antitumor effects of murine monoclonal antibody NCC-ST-421 reacting with dimeric Le(a) (Le(a)/Le(a)) epitope.

A murine monoclonal antibody (MAb), NCC-ST-421 (IgG3), was raised by using a human gastric cancer xenograft St-4 as immunogen. Immunization was achieved by transferring immunocompetent normal BALB/c mouse spleen cells into BALB/c-nu/nu mice bearing St-4 tumors. Hybridomas were produced from spleen cells of the mice after rejection of the tumors and were screened for preferential reactivity with cancers on formalin-fixed paraffin sections, as described previously for establishment of MAb NCC-ST-439 (M. Watanabe et al., Jpn. J. Cancer Res., 76: 43-52, 1985). The immunobiological and immunochemical properties of the new MAb NCC-ST-421 are described here. The MAb is essentially directed to a structure with dimeric Le(a) (V4III4Fuc2Lc6Cer) epitope (Gal beta 1----3[Fuc alpha 1----4]GlcNAc beta 1----3Gal beta 1----3[Fuc alpha 1----4]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 Cer). It cross-reacts with Le(a) but does not show any effect on Le(a)-positive RBC in vitro or on Le(a)-positive tissue loci in vivo. ST-421 strongly induced antibody-dependent cellular cytotoxicity using human peripheral blood leukocytes as effector cells with a variety of human tumor cells, using the short-term 51Cr release assay. It also showed striking complement-dependent cytotoxicity with a human complement source and was able to produce lysis of a variety of human cancer cell lines, supporting its observed ability to cause cytotoxic suppression of tumor growth in nude mice. In another series of experiments, i.p. injection of ST-421 completely inhibited growth of human tumor xenografts in nu/nu mice, and this inhibitory activity was closely dependent on expression of the dimeric Le(a) antigen on the cell surface. While Le(a) antigen was expressed in the kidneys of nu/nu mice, infusion of ST-421 in these mice did not cause histological change in kidney tissue. This finding suggests that the MAb does not damage normal cells or tissues which contain cross-reacting Le(a) antigen. These results demonstrate that ST-421 exerts a significant antitumor effect in vitro as well as in vivo, does not affect Le(a) antigen expressed on normal tissues, and therefore has potential application in therapy of certain types of human cancer which express the dimeric Le(a) antigen.     

Human IgG3 monoclonal antibody directed to an unbranched repeating type 2 chain (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----R) which is highly expressed in colonic and hepatocellular carcinoma

Previously established human monoclonal antibodies (MAbs) directed to carbohydrate antigens are essentially all IgM class, and show relatively low affinity and low reactivity at 37 degrees C. We report here the establishment of a human IgG3 MAb displaying high affinity antigen-binding activity at 37 degrees C and efficiently activating cellular cytotoxicity directed to human tumor cell lines expressing the polylactosamine antigen. The IgG3 MAb (MH21-134) reacted with the repeated unbranched polylactosamine structure Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----R, i.e., nLc6, nLc8, etc., but did not react with sialyl 2----3 or 2----6 substituted derivatives at the terminal Gal. This specificity differs from that of several anti-i antibodies, or human anti-i-like MAbs which react with sialyl 2----3 substituted structures. Directly biotinylated MH21-134 antibody was used in immunohistochemical staining of 154 formalin-fixed, paraffin-embedded tissue sections to study distribution of the antigen. High incidence of positive staining was found in colon cancer (11/17; 65%) and hepatocellular carcinoma (8/12; 67%), followed by large cell and squamous cell carcinoma of lung cancer (10/13; 59%, and 14/26; 54%, respectively). TLC immunostaining of glycolipid extracts from a variety of tumor tissues showed the presence of nLc6 and/or nLc8 in over 50% of cases. The antigens nLc6 and nLc8 were found to be absent from normal colonic epithelia, kidney, and pancreas. Only a weak band corresponding to nLc8 and one corresponding to nLc6 were found in liver and spleen, although all these normal tissues, including gastrointestinal epithelia, lung, liver, spleen, erythrocytes, and lymphocytes, were essentially negative on immunohistology. However, the antigen was found to be highly expressed in myelocytes and weakly in bronchial glands of lung and pancreatic duct epithelia. Nevertheless, expression of unsubstituted, unbranched polylactosamine antigen could be an important basis for induction of humoral immune response against certain types of human cancer, despite its limited expression in normal cells.     

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.     

Structural studies of the carbohydrate moieties of carcinoembryonic antigens

Three samples of carcinoembryonic antigens were purified from liver metastases of primary colon cancer. The asparagine-linked sugar chains of carcinoembryonic antigens (CEA) were released as oligosaccharides by hydrazinolysis and the structures of oligosaccharides, thus obtained, was studied in combination with methylation analysis and several limited exoglycosidase digestions. All three CEAs contain approximately 25 asparagine-linked sugar chains in one molecule and about 10% of them was high mannose type. However, structural features of the outer chain moieties of the remaining complex-type sugar chains were different by CEA samples. The complex-type sugar chains were mono-, bi-, tri-, and tetraantennary with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores, half of which were bisected; 86% of their proximal N-acetylglucosamine was fucosylated. The major outer chains in two samples were N-acetyllactosamine and Gal beta 1----4(Fuc alpha 1----3)GlcNAc (X-antigenic determinant) and the remaining one sample contained Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc (Y-antigenic determinant) as an additional major outer chain. Furthermore, small amounts of type 1 chain and Lea antigenic determinant were found in some samples. Acidic oligosaccharides consisted of sialic acid containing fractions and sialidase-resistant fractions, and their contents seemed to be in a reciprocal relationship. Sialic acid was linked at the C-3 and C-6 positions of the nonreducing terminal galactose residues of the outer chains.     

Structure of the sugar chain of alphafetoprotein purified from a human yolk sac tumor and its reactivity with concanavalin A

We have attempted to determine the carbohydrate moiety of human alphafetoprotein (AFP) produced by a yolk sac tumor. AFP was obtained from the cystic fluid of human yolk sac tumors grown in nude mice and was purified using an immunoadsorbent column coupled with monoclonal anti-AFP antibody. Then, the carbohydrate chain of the purified AFP was quantitatively released from the polypeptide chain. The resulting oligosaccharide was labeled and, by sequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation, the structure was determined to be: Sia alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 (GlcNAc beta 1----4) (Sia alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3) Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6) GlcNAcOT Compared with the known structure of the sugar, chain of human hepatic AFP, it was found that the sugar chain of yolk sac AFP contained an additional sugar, N-acetylglucosamine (bisect GlcNAc) linked to the beta-mannose. In the light of recent knowledge, this result indicates that the Concanavalin A (Con-A) binding site of the sugar chain is blocked by this GlcNAc in human yolk sac AFP. This fact forms the basis for the clinical use of the Con-A binding test to determine the origin of AFP in patients.     

Differential expression of difucosyl type 2 chain (LeY) defined by monoclonal antibody AH6 in different locations of colonic epithelia, various histological types of colonic polyps, and adenocarcinomas

The expression of LeY (Fuc alpha 1----2Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----R) (in which Fuc is fucose, Gal is galactose, and GlcNAc is N-acetylglucosamine) defined by monoclonal antibody AH6 in various locations of human colonic epithelia, colonic polyps, and adenocarcinomas has been studied. In normal colonic mucosa, strong staining by AH6 was observed in the proximal regions such as the terminal ileum and cecum. The staining was, however, greatly reduced in the epithelia of the ascending colon and became very weak in the epithelia of transverse, descending, and sigmoidal colon as well as the rectum. Of 481 crypts in 40 biopsy samples of the epithelia of normal sigmoidal colon, 421 crypts did not show any staining, and only a weak staining of the lowest crypt area was observed in 60 crypts (12%); of 474 crypts in 40 biopsy samples of normal rectal epithelia, 110 crypts (26%) showed a weak staining. At the fetal stage, the LeY staining was much more intense in all locations of the colon than in corresponding locations of adult epithelia, and staining was observed in the epithelia of the sigmoidal colon and rectum. A strong staining was observed in 24 of 25 cases of colorectal adenocarcinomas, irrespective of their original location. The expression of LeY in polyps was correlated with histological type as well as the degree of dysplasia of the polyps. Of various adenomas examined, tubular adenomas, many of which showed mild or moderate dysplasia and less malignant potentials, displayed the least LeY expression. Tubulovillous and villous adenomas, which have higher malignant potential and showed a higher incidence of severe dysplasia, showed a greater area and intensity of LeY expression. No LeY was detectable in juvenile polyps, and only a very weak staining was observed in the dysplastic area of hyperplastic polyps. The extent and intensity of staining in various adenocarcinomas and adenomas could not be correlated with blood group ABO status of the hosts nor with location of the tumors. These results suggest that LeY in colonic adenocarcinomas and polyps at the distal region of the colon and rectum is a typical oncofetal antigen and is a useful marker for diagnosis of colonic cancer. Its expression in colonic polyps can be correlated with the degree of dysplasia and may indicate the degree of malignant potential of the polyp. Thus, LeY expression in polyps may have prognostic value.     

Expression of LewisX and sialylated LewisX antigens in human colorectal polyps

The LewisX (LeX) antigen [characterized by trisaccharide Gal beta 1----4 (Fuc alpha 1----3)N-acetylglucosamine] is an oncodevelopmental antigen in the human colon. Monoclonal antibodies (MoAbs), anti-SSEA-1 and AH8-183, which recognize LeX antigen either on short oligosaccharide side chains or as a terminal immunodeterminant on longer carbohydrate side chains of glycoconjugates, bind to most colon cancer tissues but also to some normal colon mucosae. However, the monoclonal antibodies FH1, FH4, FH6, and IB9, which recognize extended difucosylated and trifucosylated LeX structures or their sialylated derivatives, are more cancer-associated because they rarely bind to normal colon mucosa. In the present study, these MoAbs were used to compare the expression of various LeX-related antigens in premalignant (adenomatous) and nonpremalignant (hyperplastic) colorectal polyps. Antigen expression in polyps was also compared to antigen expressions of normal colon mucosa and colon cancer tissues. The four MoAbs recognizing extended LeX antigens bound to adenomatous polyps (APs) significantly more than to hyperplastic polyps (HPs). In contrast, anti-SSEA-1 and AH8-183 recognizing monofucosyl LeX were less able to distinguish between APs and HPs. In APs, staining with the four MoAbs recognizing extended LeX antigens correlated with the premalignant parameters of larger polyp size, more severe dysplasia, and increased villose component. However, staining with AH8-183 correlated only with polyp size, and anti-SSEA-1 correlated only with polyp size and degree of dysplasia. In general, the staining frequency of HPs was similar to that of normal colon mucosa, although FH6, which did not stain any specimens of normal mucosa, stained a few HPs. The staining frequency of APs was less than that of colon cancer tissues, but these differences were generally not statistically significant. In conclusion, extended LeX antigens and their sialylated derivatives are cancer-associated antigens that are expressed preferentially in premalignant colon polyps, that tend to correlate with malignant potential in these polyps, and that may eventually help to define mechanisms involved in the polyp-to-cancer sequence.     

First establishment of a human monoclonal antibody directed to sulfated glycosphingolipids SM4s-Gal and SM4g, from a patient with lung cancer.

Several human monoclonal antibodies directed to tumor-associated glycolipid antigens have been established, but more than one-half of them react with gangliosides and the others react with neutral glycolipids. We report here the first establishment of a human IgM monoclonal antibody directed to the sulfated glycolipid. This monoclonal antibody, M14-376, did not react with SM3 and SB1a which have a terminal HSO3----3Gal beta 1----R1, but with the simple sulfolipids SM4s-Gal and SM4g which contain a terminal HSO3----3Gal beta 1----O----CH2----R2; however, lyso-SM4s-Gal and lyso-SM4g did not bind M14-376. These results suggest that terminal HSO3----3Gal and part of the hydrophobic region of the glycolipid are recognized by M14-376. Directly biotinylated M14-376 was used for immunohistochemical staining of 140 formalin-fixed, paraffin-embedded lung cancer tissue sections to study the distribution of the antigen. A high incidence of positive staining was found in adenocarcinoma (39.5%, 17 of 43), followed by large cell carcinoma (20.0%, 5 of 25), while this antigen was rarely detected in small cell carcinoma (4.7%, 1 of 21) and squamous cell carcinoma (3.9%, 2 of 51). Thin layer chromatography immunostaining of glycolipids extracted from lung cancer tissues showed the presence of only SM4s-Gal in adenocarcinoma, but SM4g was not found in any subtype of lung cancer. Immunohistochemical staining revealed that this antigen was expressed in normal kidney, testis, and brain, but erythrocytes, granulocytes, and lymphocytes were negative in cytofluorometric analysis.     

Profiles of Lewisx-containing glycoproteins and glycolipids in sera of patients with adenocarcinoma

Oligosaccharides with Lex determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) are accumulated in large quantities in various adenocarcinomas. Monoclonal antibodies recognizing mono-, di-, or trimeric Lex showed a preferential staining of specific stages of human fetal tissues and various human adenocarcinomas. Thus, these carbohydrate epitopes are typical of oncodevelopmental antigens. The present study investigated the presence of Lex epitope in sera of normal individuals and cancer patients, utilizing two high-affinity monoclonal antibodies, SH1 and SH2, directed to mono- and dimeric Lex structures, respectively. The Lex antigen in serum was eluted in the void volume fraction of a gel filtration column, determined by using monoclonal antibody SH1, and found to be carried on a glycoprotein with a molecular weight of approximately 200,000. The Lex antigen was present in the void volume fraction of the majority (85%) of sera from adenocarcinoma patients. Although the Lex epitope was also detected in a smaller proportion (33%) of normal sera, its levels were significantly lower than in cancer sera. Lex antigen was also detected in serum glycolipid fraction; however, no significant differences were observed in normal and cancer sera. A double determinant solid phase immunoassay utilizing SH2 as the capture antibody and SH1 as the detecting antibody allowed direct determination of Lex levels in sera. By the use of this direct assay, the levels of serum Lex were found to increase in association with the progression of colorectal cancer (Dukes A to D). The percentage of detectability in sera from colon cancer patients was as follows: Dukes A, 20%; Dukes B, 45%; Dukes C, 67%; and Dukes D, 74%. The levels of serum Lex were also of prognostic value in Dukes C cancer patients after surgery and during postoperative follow-up.     

Cell surface asparagine-linked sugar chains of human early myeloblastic leukemic cells (KG-1a)

The asparagine-linked sugar chains obtained from total cell surface membrane glycoproteins of human early myeloblastic leukemic cells (KG-1a cells) were studied. The sugar chains liberated by hydrazinolysis were purified by paper electrophoresis, paper chromatography, and Bio-Gel P-4 chromatography followed by analysis of exoglycosidase digestion and methylation study. Neutral oligosaccharides were all composed of high mannose type sugar chains. Acidic oligosaccharides were chiefly composed of typical bi-, tri-, and tetraantennary complex type sugar chains with Gal beta 1----4GlcNAc beta 1----groups and Neu-Ac alpha 2----3 or 6Gal beta 1----4GlcNAc beta 1----groups (in which Gal is galactosyl, GlcNac is N-acetylglucosamine, and NeuAc is N-acetylneuraminic acid) as side chains. Moreover the following two structures were identified in (in which Fuc is fucosyl): monosialyl bi- and triantennary sugar chains with a Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----group (X determinant) as one of the side chains; and monosialyl tetraantennary sugar chains with a Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc group (repeating N-acetyllactosamine unit) as one of the side chains. These data together with our previous studies on sugar chains of K562 cells [early erythroblast], adult erythrocytes [H. Yoshima, N. Shiraishi, A. Matsumoto, S. Maeda, T. Sugiyama, and A. Kobata, J. Biochem. (Tokyo), 91: 233-246, 1982], and HL-60 cells [promyelocyte] [A. Mizoguchi, S. Takasaki, S. Maeda, and A. Kobata, J. Biol. Chem., 259: 11943-11957, 1984] strongly suggest that the cell surface asparagine-linked sugar chains alter in an orderly fashion, systematically in association with lineage and maturation stages during hematopoietic cell differentiation.     

Specificity and immunobiological properties of monoclonal antibody IMH2, established after immunization with Le(b)/Le(a) glycosphingolipid, a novel extended type 1 chain antigen.

Extended lacto-series type 1 chain antigens lacking type 2 chain core have recently been shown to comprise a new type of tumor-associated carbohydrate antigen. Examples are Le(a)/Le(a) (IV3Gal beta 1----3[Fuc alpha 1----4]Glc-NAcLc4Cer) and Le(b)/Le(a) (IV3Fuc alpha 1----2Gal beta 1----3[Fuc alpha 1----4]Glc-NAcLc4Cer) (M. R. Stroud, et al., J. Biol. Chem., 266: 8439-8446, 1991; Eur. J. Biochem., 203: 577-586, 1992). We have now established an IgG3 mouse monoclonal antibody (IMH2) after immunization of mice with Le(b)/Le(a) antigen; however, monoclonal antibody (MAb) IMH2 reacted not only with the immunogen used but also with Le(y)/Le(x) and to a lesser degree with short-chain Le(y) or Le(b) with hexasaccharide ceramide (i.e., IV2FucIII3FucnLc4Cer or IV2FucIII4FucLc4Cer). It showed a high incidence of staining and strong reactivity with carcinomas of colon, rectum, liver, pancreas, and endometrium, but no reactivity with normal colonic mucosa at various loci, and minimal reactivity with normal liver, pancreas, or uterine endometrium. On the other hand, it reacted with normal gastric mucosa, cecal mucosa, urothelium, adrenal glands, and thymus. Its expression in colorectal tumors and normal cecal tissue was independent of secretor status, whereas that in normal urothelium was dependent on secretor status. MAb IMH2 displayed strong lymphocyte-activated or complement-dependent killing of human colonic cancer Colo205 cells in vitro, and inhibition of Colo205 growth in vivo; this inhibition was comparable to that by MAb NCC-ST-421, which is directed to Le(a)/Le(a) epitope (M. Watanabe, et al., Cancer Res., 51:2199, 1991). These results indicate that a new extended type 1 chain structure, Le(b)/Le(a), is a useful tumor marker associated with carcinomas of colon, rectum, pancreas, liver, and endometrium and that MAb IMH2 has potential diagnostic or therapeutic applicability for these carcinomas.     

Structure of the asparagine-linked sugar chains of alpha-fetoprotein purified from human ascites fluid

alpha-Fetoprotein purified from human serum was found to contain an asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively released from the polypeptide moiety by hydrazinolysis and recovered as oligosaccharides after N-acetylation. The oligosaccharide mixture was separated into a neutral (N) and two acidic (A-1 and A-2) fractions by paper electrophoresis. By combination of sequential exoglycosidase digestion, methylation analysis, and concanavalin A-Sepharose column chromatography, the structures of these fractions were determined to be: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GLcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc; Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6(GlcNAc; and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc.     

Comparative study of the N-linked oligosaccharides released from normal human esophageal epithelium and esophageal squamous carcinoma

N-Linked sugar chains of normal human esophageal epithelium and esophageal squamous carcinoma were quantitatively released as oligosaccharides from their membrane preparations by hydrazinolysis. After being fractionated by serial lectin column chromatography using concanavalin A-Sepharose and Datura stramonium agglutinin-Sepharose, their structures were elucidated by exoglycosidase digestion in combination with methylation analysis. Both normal epithelium and esophageal carcinoma contained bi-, tri- and tetraantennary oligosaccharides as well as high mannose-type oligosaccharides. Interestingly, carcinoma had about 1.6 times larger amounts of tri- and tetraantennary oligosaccharides with the GlcNAc beta 1----4Man alpha 1----and/or the GlcNAc beta 1----6Man alpha 1----linkages than normal epithelium. Tri- and tetraantennary oligosaccharides with N-acetyllactosamine repeating units (the Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc group) were also increased in carcinoma. These data indicated that the altered glycosylation of proteins previously found in transformed rodent cells also occurs widely in human esophageal carcinoma.     

A carbohydrate epitope associated with human squamous lung cancer

A monoclonal antibody, 43-9F, specifically recognizes a tumor-associated antigen expressed both on surface membrane glycoproteins and on secreted soluble mucins of human squamous lung carcinoma (SLC) cells, and the corresponding antigen can be detected as a circulating tumor marker in plasma of SLC patients. Thin-layer chromatography immunostaining of neutral glycolipids extracted from SLC cells reveals a 43-9F-reactive glycolipid whose carbohydrate structure, as determined by fast atom bombardment-mass spectrometry, is identical with that of an Lea-active pentaglycosylceramide described previously: Gal beta 1-3[Fuc alpha 1-4]-GlcNAc beta 1-3Gal beta 1-4Glc-Cer. However, the Lea-active oligosaccharide hapten, lacto-N-fucopentaose II, with the same carbohydrate structure, fails to inhibit binding of 43-9F, and a well-characterized anti-Lea monoclonal antibody blocks only 40% of 43-9F binding sites on SLC cells, suggesting that the major epitope recognized by 43-9F is more complex than the Lea epitope. To search for a higher affinity 43-9F epitope among more complex oligosaccharides, a mixture of tritiated neutral oligosaccharide alditols from pooled human milk was passed through a 43-9F affinity column. A major retarded oligosaccharide was purified by high-performance liquid chromatography and shown by fast atom bombardment-mass spectrometry to have the following structure: Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4[Fuc alpha 1-3] GlcNAc beta 1-3Gal beta 1-4Glc. Oligosaccharides containing this sugar sequence are at least 100-fold more active than lacto-N-fucopentaose II as competitive inhibitors of 43-9F. Thus, antibody 43-9F binds to the above difucosyl Lea-X determinant with high affinity and weakly cross-reacts with the Lea antigen under some conditions such as occurs in thin-layer chromatography and enzyme-linked immunosorbent assay where multiple weak interactions of the decavalent IgM antibody may occur.     

Involvement of cell surface glycans in adhesion of human colon carcinoma cells to liver tissue in a frozen section assay: role of endo-beta-galactosidase-sensitive structures

Adhesion of human colon carcinoma variant cell lines expressing different levels of the cell surface sialyl Lewis X (sLeX) antigen to frozen sections of mouse liver was examined. KM12-HX cells that bound the monoclonal antibody (mAb) FH6 (anti-sLeX) and thus expressed a high level of sLeX demonstrated a greater degree of adhesion to liver sections than their low-binding counterparts, KM12-LX cells. The adhesion of KM12-HX cells to liver sections was partially blocked by mAb FH6, but not by another anti-sLeX mAb, KM93. The adhesion was Ca2+ dependent but was not inhibited by anti-E-selectin. Endo-beta-galactosidase treatment significantly reduced adhesion and resulted in the loss of cell surface binding sites for mAb FH6. O-linked oligosaccharides from KM12-HX cells incubated in the presence of p-nitrophenyl-N-acetylgalactosaminide were fractionated by a combination of gel filtration, anion exchange chromatography, and normal phase high-performance liquid chromatography. The structure of a mAb FH6-reactive and endo-beta-galactosidase-sensitive glycan was estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in a post source decay mode and by glycosidase digestions to be NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc-NAc beta1-3Gal beta1-4(+/-Fuc alpha1-3)GlcNAc beta1-6(NeuAc alpha2-3Gal beta1-3)GalNAc-pNP. Mild detergent lysates of mouse liver surface-labeled with sulfo-NHS biotin were incubated with glutaraldehyde-fixed monolayers of KM12-HX cells, and bound components were isolated after EDTA treatment. A Mr 49,000 component that bound only to KM12-HX cells and not to KM12-LX cells was identified.     

Glycoproteins associated with metastatic potential of cancer

Carbohydrate moieties of glycoproteins have been implicated to be involved in cellular adhesion. Therefore, certain carbohydrate structures in glycoproteins are expected to be associated with metastatic behaviour of cancer cells; such carbohydrate structure can be used as an indicator for the treatment of cancer patients based on the predicted metastatic potential of the cancer cells. The following results recently reported is interesting from the above view point. In certain cancer cells of the mouse, beta 1----6 branching of asparagine-linked oligosaccharides, which can be detected by the reactivity with PHA-L lectin, associates with increased metastatic potential. In human bladder carcinomas, reactivity with Lotus tetragonolobus agglutinin (LTA) correlates with increased metastatic potential. LTA appears to react with Lex structure [Gal beta 1----4(Fuc alpha 1----3)GlcNAc] in the cancer cells. Expression of sialilated dimeric Lex increases in the metastatic nests of human colon carcinomas. On the other hand, sialyl antigen MGl has the tendency to be expressed in human gastric adenocarcinoma of low metastatic potential. MGl is defined by a monoclonal antibody raised against Ricinus communis agglutinin receptors isolated from human gastric adenocarcinoma xenografted in nude mice. Continued efforts using monoclonal antibodies and lectins may yield arrays of carbohydrate markers of clinical value to predict the metastatic potential.     

Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2----6 alpha-N-acetylgalactosaminyl (sialosyl-Tn) epitope

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.     

Tumor-associated antigen 43-9F is of prognostic value in squamous cell carcinoma of the lung. A retrospective immunohistochemical study.

BACKGROUND. Squamous cell lung carcinoma (SLC), the most frequent type of lung cancer, generally is treated surgically and its prognosis is poor. The only current clinically useful prognostic criterion is lymph node staging (TNM classification). Expression of a novel tumor-associated carbohydrate epitope Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3 Gal beta 1-4Glc identified by the 43-9F monoclonal antibody (MoAb) is associated with the growth pattern of SLC cell lines in athymic mice and in vitro. This implies that the 43-9F epitope may be related to tumor progression in patients with SLC and that, as such, it could be of prognostic value. METHODS. Primary tumor specimens from 231 patients with lung carcinoma (130 with SLC, 64 with adenocarcinoma, 10 with small cell carcinoma, 16 with large cell carcinoma, and 11 with adenosquamous carcinoma) were examined by immunohistochemical studies on formalin-fixed, paraffin-embedded tissue samples for immunoreactivity with an MoAb to the 43-9F antigen. Univariate and step-wise Cox regression analyses were used to compare survival time by histopathologic diagnosis, smoker status, TNM classification, and type of surgical treatment. RESULTS AND CONCLUSIONS. Patients with 43-9F epitope-positive SLC tumors had a significantly (P less than 0.01) better prognosis than patients with epitope-negative tumors. In contrast, no association was seen between 43-9F epitope expression and survival time for patients with lung adenocarcinomas. Further, the prognostic value of 43-9F expression in SLC was found to be superior to the N-classification with the added advantage that it requires access only to primary tumor tissue and thus is available before therapy.