Mucin-Type Glycoproteins as Tumor Markers

Transcription

1 Special Feature Mucin-Type Glycoproteins as Tumor Markers Harry G. Rittenhouse, PhD; George L. Manderino, PhD; and G. Michael Hass, PhD T he advent of monoclonal antibody technology has dramatically improved the feasibility of developing immunoassays useful in diagnosing and monitoring cancer. Numerous investigators have injected mice and rats with various embryonic tissues, primary human cancer tissues, and human tumor cells in hope of generating monoclonal antibodies against tumorspecific antigens. To date, this goal has remained elusive. Although no tumor-specific antigens have yet been identified, several new monoclonal antibodies d e t e c t i n g tumor-associated antigens have been described. Tumor-associated antigens are not only present in the serum and tissues of cancer patients, but are also expressed in embryonic tissues and at low levels in the tissue and serum of healthy persons. Because they lack absolute tumor specificity, most of these monoclonal antibodies will be used clinically to improve monitoring of cancer patients, rather than for early diagnosis or screening. Mucins Antigens identified by many of these From the Dept of Experimental Biology, Abbott Diagnostics Division, Abbott Laboratories, North Chicago, IL 60064, recently obtained monoclonal antibodies belong to a complex, heterogeneous family of glycoproteins known as mucins. 1 Serologic immunoassays developed with these monoclonal antibodies h a v e shown c o n s i d e r a b l e promise in the detection or monitoring of certain cancer patients Table). These antibodies include CA 19-9,2"5 Ca50, 6 DuPan-2, 7 and sialylated Le x8 with primary utility for pancreatic and colorectal cancer; Ca " and MoV212 for ovarian cancer; and F36/ for breast cancer. Other monoclonal antibodies t h a t show t u m o r specificity by immunohistochemical techniques and, in some cases, by serologic a s s a y i n c l u d e B , 1 4, 1 5 DF3, 1 4, 6 115D8,17 M HMFG, and HMFG 2 20 for breast cancer; YPAN l 2 1 for pancreatic cancer; and CA l 2 2 for a wide variety of tumor types. Before a d d r e s s i n g t h e s e m a r k e r s individually, the properties of mucins and the carbohydrate nature of their epitopes detected in serum will be discussed. Structure and Function Mucins are glycoproteins of high m o l e c u l a r w e i g h t s e c r e t e d by t h e seroviscous tissues found in the mouth, lungs, cervix, and intestines. Functioning primarily as biological lubricants, mucins provide a barrier to protect cells against osmotic and ph gradients and physical trauma. Much of our understanding of the properties of mucins is derived from studies on such readily available m a t e r i a l as cervical or hog gastric mucin. 1 DuPan 2 23 and CA have been isolated from serum and ascites fluid of cancer patients, permitting further charac- Serolog c Assays For Mucins Antibody CA19-9 CA125 CA50 DU-PAN-2 CO-51.4/CA19-9 CSLEX 1 F 36/22 MoVj Isotype Main Cancer Specificity References gg, igg, igm IgGa* lgg3 Pancreatic, Gastrointestinal Ovarian Pancreatic, Gastrointestinal Pancreatic Gastrointestinal Breast, Colon Breast Ovarian 2, 3, 4, 5 9, 10, * Monoclonal antibody specific for the Lea antigen used as the catcher monoclonal antibody and soluble 19-9 monoclonal antibody used for a solid phase double determinant RIA LABORATORY MEDICINE VOL. 16, NO. 9, SEPTEMBER 1985

2 terization of cancer-associated mucins. The classification of an antigen as a mucin is usually based on molecular w e i g h t and c a r b o h y d r a t e content. Undissociated mucins typically have molecular weights in excess of 500 kilodaltons and are thus excluded from Sephacryl S-300 or equivalent gel filtration resins. The second property distinguishing mucins is their unusually high carbohydrate content of 60% to 80%.' A "typical" mucin molecule may possess as many as 200 oligosaccharide chains, each containing an a v e r a g e of t e n monosaccharide units linked to a polypeptide backbone of 800 amino acids. Since carbohydrate is more dense than protein, mucins sediment in cesium chloride gradients at densities 1.45 g/ml) intermediate between typical proteins 1.2 to 1.3 g/ml) and proteoglycans 1.5 g/ml). 25 The oligosaccharides found on mucins are of particular interest. During the past several years, studies have indicated that certain tumors display altered production of mucins.26"30 More recent immunohistochemical studies using lectins, which bind selectively to mucins, have demonstrated differences in mucin structure and distribution among normal, colonic tumors, and preneoplastic colonic lesions.30"32 The epitopes recognized by many of the antibodies mentioned above are mucin-associated carbohydrates. Mucin-associated oligosaccharides are also commonly present on glycolipids associated with cell membranes. 1 Accordingly, monoclonal antibodies to specific glycolipid s t r u c t u r e s have shown promising specificity for certain tumors. An excellent discussion of glycolipids as tumor markers appears in a recent review by Hakom o r i. 3 3 F i n a l l y, t h e l i n k a g e of carbohydrate to the protein component of mucin is restricted to a min o r i t y of s e r u m g l y c o p r o t e i n s including m u c i n s a n d IgAi. 3 4 This linkage is termed 0-glycosidic since it involves the hydroxyl groups of serine or threonine on the polypeptide and the reducing ends of ^ a c e t y l g a l a c t o s a m i n e on t h e c a r b o h y d r a t e. 1 In contrast, the vast majority of serum glycoproteins contain oligosaccharide-protein linkages between the amide nitrogen of asparagine and the reducing end of N-acetylglucosamine, termed N-glycosidic. Traditional Protein Markers eg, CEA, AFP, PAP) Tumor-Associated Oligosaccharides on Mucins eg, CA 19-9, CA 125, Du-PAN-2) Genes for different mucins ) f Protein \ \ Glycosyltransferases O = N-linked oligosaccharides ;*. y <T"O~0 Glycoprotein =Antigeni genie determinant Antibodies are directed to the protein portion. N Protein backbones for different mucins Glycosyltransferases A =0-linked oligosaccharides O Q ^-7 r <^~y M ucin f' v v A =Ai Antigenic determinant Antibodies are directed to the carbohydrate portion. Fig 1. Biosynthetic origin of protein markers vs O-linked oligosaccharide tumor markers. Tumor-Associated Carbohydrate Epitopes Solid phase sandwich-type radioimmunoassays for some mucin markers using the identical monoclonal antibody both on the solid phase and as a tracer revealed that mucin in cancer patients' sera contained repeating antigenic epitopes. This result is not surprising because mucins are large molecules containing numerous carbohydrate chains with repeating sequences. Since amino acid sequences are not commonly duplicated within a molecule, the demonstration of repeating antigenic determinants on a given molecule is highly suggestive of a carbohydrate. The abrogation of the antigenic activity of CA 19-9,35 DuPan2, 23 CA l, 22 and YPAN l 2 1 after treatment with neuraminidase, an enzyme that selectively cleaves sialic acid from oligosaccharides, provides f u r t h e r evidence that the antigenic epitopes on these mucins are carbohydrates. Origin of Tumor-Associated Carbohydrate Epitopes The major differences in the biosynthesis of polypeptides and mucins carry important implications as to how tumor-associated antigens arise Fig 1). Clinically useful antibodies to relinked glycoprotein tumor markers such as carcinoembryonic a n t i g e n CEA), alpha-fetoprotein AFP), and prostatic acid phosphatase PAP) recognize polypeptide epitopes. Elevated levels of these markers in the serum of certain cancer patients may result from increased transcription of genes encoding for these polypeptides, as well as an increase in the number of tumor cells Fig 1). Elevated levels of tumorassociated mucin molecules may result from similar mechanisms. Mechanisms t h a t alter the structure, density, or organization of carbohydrate on mucins, however, can result in the generation of tumor-associated markers even without enhanced production of the polypeptide backbone Fig 1). Suppressed transcription of normally active glycosyltransferases or enhanced transcription of normally quiescent glycosyltransferases can result in aberrant oligosaccharide on mucins. 33 Changes in the glycosylation patterns on mucins may alter oligosaccharide density, resulting in the unmasking of carbohydrate epitopes normally not accessible to antibodies. Altered glycosylation patterns might o r i e n t a t e c a r b o h y d r a t e epitopes in such a way t h a t monoclonal antibodies bind them with higher avidity, thus making them more detectable in sera from cancer patients. Similar alterations in oligosaccharides might result from altered activity of glycosidases. Enhancement of glycosidase activity may also account for the expression of tumor-associated carbohydrate epitopes on mucins. These and other m e c h a n i s m s in t h e complex t r a n scriptional a n d p o s t - t r a n s l a t i o n a l events required to synthesize mucins may lead to considerable diversity, resulting in numerous potential tumor-associated mucins. Mucin Markers For Pancreatic and Colorectal Cancers Although none of the markers discussed in this article are absolutely LABORATORY MEDICINE VOL. 16, NO. 9, SEPTEMBER

3 Lewis' LNF II) and sialylated Lewis' 19-9) Lewis' LNF III) and sialylated Lewis" Lewis" A>fl1,4 Lewis " As81,3 ^ A / «1, ^ A > ^ 1 ^ Sialylated Lewis" ^1,3 A \01,4 ^ > ^ A A a\a»l,3 = Galactose === N-Acetylglucosamine Fucose -= N-Acetylneuraminic acid Fig. 2. Structures of tumor-associated - carbohydrate specific for given cancers, the markers can be grouped according to their primary clinical utility. A common feature of the above monoclonal antibodies is that they recognize epitopes that appear to be selectively expressed in c e r t a i n epithelial-derived cancers of the gastrointestinal tract. Interestingly, all these markers are neuraminidase-sensitive, which highlights the critical role played by sialic acid in their antigenic determinants. Perhaps the best characterized tumor-associated oligosaccharide determinant in this group of markers is the sialylated Lewis 3 Lea) pentasaccharide Fig 2) recognized by the CA 19-9 monoclonal antibody. 3 5 This struct u r e, also t e r m e d g a s t r o i n t e s t i n a l cancer-associated antigen GICA), is found in glycolipid and mucin components of tumor cell membranes and on mucin in cancer serum. 24 An extensive clinical study 4 using an RIA for CA 19-9 demonstrated elevated levels of serum antigen in 67% of patients with advanced adenocarcinoma of the upper GI tract, including pancreatic cancer. This antigen in serum is a mucin containing multiple sialylated Le a structures as well as numerous other oligosaccharides including the appropriate Le a or Le b blood group determinants. 3 6 The CA 50 epitope appears to be similar, if not identical to that recognized by CA 199, since it also binds to sialylated Lea.6 Knowledge of the structure of the CA 19-9 epitope provides insight into genetic restrictions on its expression in patients with GI cancer. Approximately 5% to 10% of the U.S. popu- epitopes. lation is Leab~, t h a t is, they lack the fucosyltransferase responsible for adding fucose residues found in Le a and Le b structures Fig 2). Since this fucose residue is an integral part of the 19-9 epitope, Le ab " persons always test negative in the CA 19-9 assay. In addition, Le a b + persons may produce suboptimal levels of sialylated Lea due to competition for the substrate ie, Lea) between the putative 2-3 sialyltransferase that produces the 19-9 epitope and the 1-3) fucosyltransferase t h a t completes the Le b structure Fig 2).37 These genetic restrictions thus contribute to the false-negative rate seen in the CA 19-9 assay. The monoclonal antibodies DuPan2 and YPan 1 also recognize neuraminidase-sensitive determinants on mucin molecules and show promise for monitoring certain patients with GI cancer. More than 65% of tested patients with pancreatic cancer exhibited elevated DuPan 2 serum levels. 7 Despite several similarities, evidence has been presented showing that the DuPan-2, YPan 1, and CA 19-9 epitopes are distinct. 721,23 The sialylated derivative of lacton-fucopentaose III, Lewis" Lex) antigen, represents a structure differing from sialylated Lewis" carbohydrate only in t h e glycosidic l i n k a g e between galactose, N-acetylglucosamine and fucose moieties. Sialylated Le* Fig 2) is expressed in certain fetal tissue and on adenocarcinomas of the colon.38 A recently described monoclonal antibody, CSLEX1, is directed against sialylated Le\ 8-39 Competitive antibody binding experiments demonstrate no inhibition of CSLEX LABORATORY MEDICINE VOL. 16, NO. 9, SEPTEMBER 1985 by 19-9 antibody, indicating that these epitopes are distinct. A solid phase RIA employing the CSLEX1 antibody detected antigen, presumably on mucins, in patient sera. 8 In a limited study, the CSLEX1 assay detected 16 of 31 breast cancer, 17 of 48 colon cancer, and five of 27 of stomach cancer specimens tested. In contrast, only one of 37 specimens from normal, healthy controls was elevated. 8 The authors speculated that a panel assay employing antibodies detecting both sialyl a t e d Le a a n d s i a l y l a t e d Le* m a y provide a more sensitive assay for monitoring breast and GI cancer than either marker used alone. Another member of this family of neuraminidase-sensitive mucin tumor markers, CA l, 22 has been detected on the surface of a variety of tumor cells by immunohistochemistry. Available evidence, however, does not support a close structural relationship between the epitopes recognized by CA-1 and those recognized by 19-9, YPan 1, Ca 50, and DuPan 2. Ovarian Cancer The CA 125 monoclonal antibody, generated from a mouse immunized with a human ovarian carcinoma cell line, 40 recognizes a repeating epitope on a high molecular weight mucin-type glycoprotein.41 An RIA developed with this antibody detects elevated CA 125 levels in more than 80% of epithelial ovarian carcinomas including serous, endometrial, and cellular types. 9 Using a cutoff value of 65 units/ml, more than 70% of ovarian cancer patients had positive values, whereas only 0.2% of healthy controls and 2% of patients with benign diseases were positive. A promising aspect of this marker is its potential for detecting early ovarian cancer. Bast and Knapp 4 2 review the use of CA 125 in monitoring epithelial ovarian cancer. Two other monoclonal antibodies specific for ovarian cancer, MoVi and MoV 2, h a v e r e c e n t l y b e e n developed.12 Both antibodies appear to recognize carbohydrate determinants on high molecular weight mucin-type glycoproteins. 43 Using an immunoradiometric assay employing the MoV2 monoclonal antibody, effusions from eight of ten patients with well-differ-

4 entiated ovarian cancer and five of 11 patients with poorly differentiated ovarian tumors were positive. 43 In contrast, only one of seven effusions from patients with mammary carcinoma and none of ten effusions from patients with other diseases were positive. Further clinical studies are necessary to better understand the clinical utility of the MoV2 marker. Mucin-Reactive Monoclonal Antibodies in Breast Cancer Antibodies directed to mucin epitopes associated with breast cancer were obtained from mice immunized with human milk fat globule HMFG),17"20 breast carcinoma cell lines,13 and fractions of metastatic breast cell membranes.14"16 Two of these antibodies, HMFG-1 and HMFG2, are directed to oligosaccharides present on high molecular weight molecules believed to be mucins. 20 Immunohistologic analysis of these antibodies demonstrates reactivity with a variety of cells of epithelial origin. Another antibody, M18,1819 also binds to a carbohydrate epitope found on a mucin present on certain cells of epithelial origin. Despite many similarities, these monoclonal antibodies can be distinguished in their reactivities to a variety of clinical specimens. The 115 D8 monoclonal antibody, which was also generated from mice immunized with HMFG, reacts with a mucin antigen termed MAM-6.17 This antigen has been found in many tumor tissues, as well as in a few normal tissues including lung epithelium.1744 Although its reactivity with normal tissue limits its clinical utility, this monoclonal antibody may be useful for monitoring certain patients with mammary carcinoma. Using human breast cancer cells as the immunogen, Papsidero and colleagues13 obtained a monoclonal antibody designated F36/22, directed to a mucin-associated antigen. This monoclonal antibody shows specificity to adenocarcinomas of the breast and ovaries. In immunohistochemical assays, the F36/22 epitope has been found in 19 of 19 ovarian carcinomas, three of 16 patients with benign ovarian disease, and none of 20 normal ovarian tissue specimens.45 In a serum immunoassay using the F36/22 mon- oclonal antibody, 61 of 116 of serum samples from patients with breast cancer were positive,13 whereas only five of 40 specimens from patients with benign mammary disease and two of 64 healthy volunteers were F36/22 antigen-positive. Membranes obtained from breast carcinoma cells that had metastasized to the liver were used to immunize mice in hope of raising monoclonal antibodies capable of discerning metastatic breast tumor lesions. Although no such antibodies have yet been definitively described, two interesting monoclonal antibodies, B and DF3,16 have been generated. These appear to bind distinct mucin-associated determinants. By immunohistochemical analysis, the DF3 monoclonal antibody has been shown to distinguish malignant from benign tissue sections from breast cancer patients. The B72.3 antigen is expressed in nearly all tested patients with colon carcinomas as well as in a majority of breast cancer patients. No detectable B72.3 activity has been found to date in normal adult tissues. In a study of 20 patients with breast carcinoma, no correlation was found between the B72.3 and DF3 antigens.15 One distinguishing feature of the B72.3 antibody is its highly specific reactivity to adenocarcinoma and lack of reactivity to mesothelial cells in effusions.14 It should be noted that none of the above antibodies react exclusively with breast cancer specimens. Most react extensively with other adenocarcinomas. This suggests that these antibodies recognize epitopes on mucins commonly found in a variety of epithelial tissues, particularly adenocarcinomas. Discussion A number of monoclonal antibodies directed toward carbohydrate epitopes on mucins have been shown to have potential clinical utility for monitoring certain cancer patients. Different monoclonal antibodies with distinct binding profiles in serum or tissue panels appear to bind to similar, if not identical, epitopes. Both CA 19-9 and Ca 50 monoclonal antibodies, for example, show variation in cancer serum profiles, yet both antibodies are inhibited from binding tu- mor specimens by sialylated Lea antigen. Similarly, monoclonal antibodies PMN 6, PMN 29, and PM 81 differentially bind to various myeloid cell types. But all three are inhibited by lacto-n-fucopentaose III, which suggests that they bind to distinct sites on the same oligosaccharide chain or to identical sites with different affinities.46 Many other monoclonal antibodies generated from mice immunized with human cell lines are directed toward the lacto-n-fucopentaose III structure, also termed the Lewis" Lie*) antigen Fig 2) Not only is this epitope commonly found on the surface of many tumor cell lines, but it appears to be highly immunogenic in mice and rats. Variations in density, frequency, or organization of oligosaccharide antigenic sites on mucins, coupled with varying affinities among monoclonal antibodies directed to these sites, may account for differences in the clinical profiles of antibodies directed toward the same binding site. Suppression of normally active glycosyltransferases and enhanced expression of normally quiescent glycosyltransferases and glycosidases may play an important role in synthesis or unmasking otherwise cryptic or aberrant epitopes. The sialylated Lewis" epitope, for example, appears to be synthesized by an as yet uncharacterized glycosyltransferase. Monoclonal antibodies directed to such glycosyltransferases might eventually be developed and may prove to be extremely useful in cancer patient management. Since few substances are shed in large quantities by tumors, many potentially useful, tumor-associated oligosaccharide antigens may not be detectable in serum in the ng/ml range. Assays of other mucin-rich fluids such as urine and sputum, as well as immunohistochemical assays, may offer useful alternatives to serum assays. In contrast to antibodies specific to the polypeptide portion of CE A, AFP, PAP, etc, repeating antigenic epitopes are often found on single mucin molecules. Repeating epitopes permit the development of "sandwich" immunoassays, in which a single monoclonal antibody can be used on both the solid phase and as the probe. The RIA developed for detecting the CA 125 epitope in ovarian cancer sera ex- LABORATORY MEDICINE VOL. 16, NO. 9, SEPTEMBER

5 emplifies such an assay. Further, the large molecular size and structural diversity of mucins present the possibility of finding two or more distinct tumor-associated antigens on the same molecule. Immunoassays can be developed using a combination of two monoclonal antibodies reactive to distinct epitopes. Such assays might provide better specificity and sensitivity than assays using either antibody alone.52 Summary Considerable effort continues to be expended searching for new and potentially useful monoclonal antibodies of clinical benefit in cancer management. It is likely that the heterogeneous, highly glycosylated mucin-type glycoproteins represent a new class of tumor markers that will play an increasingly important role in this field. Although the goal of generating monoclonal antibodies to unique tumor antigens may remain elusive if not impossible, more highly cancerselective antibodies especially if used in combination may lead to new and improved assays. The emerging application of monoclonal antibodies for tumor imaging and therapy in cancer patients will also likely benefit from monoclonal antibodies directed to mucin tumor markers. References 1. Feizi T, Gooi HC, Childs RA, et al: Tumor-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins. Bioch Society Trans; Carbohydrate Group Colloquium T. Feizi ed) Vol 12, 607th Meeting 1984, Koprowski H, Herlyn M, Steplewski Z, et al: Specific antigen in serum of patients with colon carcinoma. Science 1981;212: Del Villano BC, Brennan S, Brock P, et al: Radioimmunometric assay for a monoclonal antibody defined tumor marker, CA Clin Chem 1983;29: Ritts RE Jr, Del Villano BC, Go VLW, et al: Initial clinical evaluation of an immunoradiometric assay for CA 19-9 using the NCI serum bank. Int J Cancer 1984;33: Kuusela P, Jalanko H, Roberts P, et al: Comparison of CA 19-9 and carcinoembryonic antigen CEA) levels in the serum of patients with colorectal diseases, Br J Cancer 1984;49: Holmgren J, Lindholm L, Persson B, et al: Detection by monoclonal antibody of carbohydrate antigen CA 50 in serum of patients with carcinoma. Br Med J 1984;288: Metzgar RS, Rodriguez N, Finn OJ, et al: Detection of a pancreatic cancer-associated antigen {DU-PAN-2 antigen) in serum and ascites of patients with adenocarcinoma. Proc Natl Acad Sci USA 1984;81: Chia D, Terasaki PI, Suyama N, et al: Use of monoclonal antibodies to sialylated Lewis" and sialylated Lewis a for serological tests of cancer. Cancer Res 1985:45:435^ Klug TL, Bast RC Jr, Niloff JM, et al: Monoclonal antibody immunoradiometric assay for an antigenic determinant CA 125) associated with human epithelial ovarian carcinomas. 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Acta Cytologica 1984;28: Schlom J, Greiner J, Horan Hand P, et al: Monoclonal antibodies to breast cancer-associated antigens as potential reagents in the management of breast cancer. Cancer 1984;54: Kufe DW, Inghirami G. Abe M, et al: Differential reactivity of a novel monoclonal antibody DF3) with human malignant versus benign breast tumors. Hybridoma 1984;3: Hilkens J, Hilgers J, Buijs F, et al: Monoclonal antibodies against human milk fat globule membranes useful in carcinoma research. Protides of the Biological Fluids Peeters ed) Vol 31 pp ). 18. Foster CS, Neville AM: Monoclonal antibodies to the human mammary gland. Hum Pathol 1984;15: Gooi HC, Kei-Ichi LD, Edwards PAW, et al: Two mouse hybridoma antibodies against human milk fat globules recognize the I MA) antigenic determinant B-D-Galp- 1-4 )-B-D-GlcpNAc- 1-6). Carb Res 1983;120: Koldovsky U, Wargalla U, Hilkens J, et al: Reactions of monoclonal antibodies against human milk fat globule membranes with embryonal tissue. 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Cancer 1982;50: Gold D, Miller F: Chemical and immunological differences between normal and tumoral colonic muroprotein antigens. Nature 1975;255: Boland CR, Montgomery CK, Kim YS: Alterations in human colonic mucin occurring with cellular differentiation and malignant transformation. Proc Natl Acad Sci USA 1982;79: Nairn RC, Fothergill JE, McEntegart MG: Loss of gastrointestinal-specific antigen in neoplasia. Br Med J 1962;1: Yonezaqa S, Nakamura T, Tanaka S, et al: Glycoconjugate with Ulex Europaeus agglutinin-i LABORATORY MEDICINE VOL. 16, NO. 9, SEPTEMBER binding sites in normal mucosa, adenoma and carcinoma of the large bowel. J Natl Cancer Instit 1982;69: Boland CR, Montgomery CK, Kim YS: A cancer-associated mucin alteration in benign colonic polyps. Gastroenterology 1982;82: Listinsky CM, Riddell RH: Patterns of mucin secretion in neoplastic and non-neoplastic disease of the colon. Hum Pathol 1981;12: Hakomori S: Tumor-associated carbohydrate antigens. Ann Rev Immunol 1984;2: Baenziger J, Kornfield S: Structure of the carbohydrate u n i t s of IgAp J Biol Chem 1974;249: Magnani JL, Nilsson B, Brockhaus M, et al: A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-n-fucopentaose II. J Biol Chem 1982;257: Pak KY, Blaszczyk M, Herlyn M, et al: Identification and isolation of Lewis blood group antigens from human saliva using monoclonal antibodies. Hybridoma 1984;3:1-9. Brockhaus M, Wysocka M, Magnani JL, et al: Normal salivary mucin contains the gastrointestinal cancer-associated antigen detected by monoclonal antibody 19-9 in the serum mucin of patients. Vox Sang 1985;48: Blaszczyk M, Ross AH, Ernst CS, et al: A fetal glycolipid expressed on adenocarcinomas of the colon. Int J Cancer 1984;33: Fukushima K, Hirata M, Terasaki PI, et al: Characterization of sialylated Lewis" as a new tumor-associated a n t i g e n. 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Cancer Res 1984;44: Magnani JL, Ball ED, Fanger MW, et al: Monoclonal antibodies PMN 6, PMN 29 and PM-81 bind differently to glycolipids containing a sugar sequence occurring in lacto-n-fucopentaose III. Arch Biochem Biophys 1984;233: Huang LC, Brockhaus M, Magnani JL, et al: Many monoclonal antibodies with an apparent specificity for certain lung cancers are directed against a sugar sequence found in lacto-nfucopentaose III. Arch Biochem Biophys 1983;220: Huang LC, Civin CI, Magnani JL, et al: My-1, the human myeloid-specific antigen detected by mouse monoclonal antibodies, is a sugar sequence found in lacto-n-fucopentaose III. Blood 1983;61: Brockhaus M, Magnani JL, Blaszczyk M, et al: Monoclonal antibodies directed against the sugar sequence of lacto-n-fucopentaose III are obtained from mice immunized with human tumors. Arch Biochem Biophys 1982;217: Rettig WJ, Cordon-Cardo C, Ng JSC, et al: Highmolecular weight glycoproteins of human teratocarcinoma defined by monoclonal antibodies to carbohydrate determinants. Cancer Res 1985;45: Rosen ST, Mulshine JL, Cuttitta F, et al: Analysis of human small cell lung cancer differentiation antigens using a panel of rat monoclonal antibodies. Cancer Res 1984;44: Herlyn M, Sean HF, Verill H, et al: Increased sensitivity in detecting tumor-associated antigens in sera of patients with colorectal carcinoma. J Immunol Methods 1985;75:15-21.