(CANCER RESEARCH 49, 197-204, January I, 1989]

Expression of Tn, Sialosyl-Tn, and T Antigens in Human Colon Cancer1

Steven H. Itzkowitz,2 Mei Yuan, Carolyn K. Montgomery, Thomas Kjeldsen, Helio K. Takahashi, William L. Bigbee,and Young S. Kim3

Gastrointestinal Research Laboratory fS. H. I., M. Y., Y. S. K,] and Pathology Department [C. K. M.J, Veterans Administration Medical Center, SanFrancisco, California 94121; Departments of Medicine [S. H. I., Y. S. K.] and Pathology [Y. S. K., C. K. MJ, University of California San Francisco, SanFrancisco, California 94143; The Biomembrane Institute and Department ofPathobiology, University of Washington, 201 Elliott Avenue West, Seattle, Washington 98119[T. K., H. K. T.J; and the Biomedicai Sciences Division, Lawrence Livermore National Laboratory, University of California, Livermore, California 94550 [W. L. B.J

ABSTRACT

Mucin glycoproteins are major secretory products of the colon andcontain 0-linked oligosaccharides synthesized on a polypeptide backbone. The initial step in the synthesis of 0-linked oligosaccharides is theaddition of ^V-acetylgalactosamine to serine or threonine residues formingthe Tn antigen. This substance can then receive additional carbohydrateresidues such as sialic acid to form sialosyl-Tn antigen, or galactose toform T antigen. In the colon, the T antigen is an oncodevelopmentalcancer-associated antigen but little is known about Tn and sialosyl-Tnexpression. The present comparative immunohistochemical study wasperformed to analyze the expression of these antigens in fetal, normaladult, and malignant colorectal tissues with an aim toward elucidatingwhether Tn and sialosyl-Tn are also oncodevelopmental colon cancer-associated antigens and to gain insight into the earliest steps of mucinglycosylation in colonocytes. We used three reagents to detect Tn antigen(two monoclonal antibodies Kin I.III and (I -I, and one lectin Viciavillosa), two reagents to detect sialosyl-Tn (monoclonal antibodies TKH2and B72.3) and one to detect T antigen (monoclonal antibody AH9-16).

Except for occasional reactivity with VVA and CU-1, cells of normalcolonie mucosa did not express Tn, sialosyl-Tn, or T antigens. However,in the transitional mucosa immediately adjacent to cancer, all threeantigens were expressed (ranging from 35 to 67% of cases dependingupon the reagent). In colon cancers, the percentage of cases expressingeach antigen were as follows: Tn 72-81%, sialosyl-Tn 93-96%, and T71%. Unlike T antigen, which was preferentially expressed by moderatelywell- and well-differentiated adenocarcinomas, both Tn and sialosyl-Tnantigens were expressed by most histológica!subsets of colon cancers,including poorly differentiated adenocarcinomas and murinoli* (colloidand signet ring cell type) carcinomas. The majority of cancers expressedboth Tn and sialosyl-Tn, usually in association with T antigen. Only onecancer lacked all three antigens. Fetal colonie mucosa! cells expressedall three antigens, particularly in goblet cell mucin.

These results indicate that like T antigen, Tn and sialosyl-Tn areoncodevelopmental cancer-associated antigens in the colon. Moreover,Tn and sialosyl-Tn antigens appear to be useful markers of poorlydifferentiated adenocarcinomas and mucinous carcinomas: two histológica! subsets that often fail to express other cancer-associated antigens andthat are often associated with a poor clinical outcome. Finally, theseresults provide additional evidence for incomplete glycosylation in coloncancer cells and suggest that the regulation of mucin oligosaccharidesynthesis is fundamentally different in normal and malignant colonocytes.

INTRODUCTION

Colonie epithelium is characterized by the production of

Received 4/27/88; revised 9/27/88; accepted 10/4/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by the Veterans Administration Medical Research Service [S. H.

I., M. Y., Y. S. K.]; VA Research Associate Award [S. H. I.]; VA MedicalInvestigator Award [Y. S. K]; NCI Grant CA42981 [S. H. I.]; NCI Grant CA47551[Y. S. K.]; a fellowship from the Danish Medical Research Council and Hjort's

Legat, Denmark [T. K.j; and International Fogarty Fellowship FOS TW03783[H. K. T.]. Dr. Itzkowitz is a recipient of a grant from the Aaron DiamondFoundation.

2 Present address: Division of Gastroenterology, Mount Sinai Medical Center,l Gustave Levy Place, New York, NY 10029.

'To whom requests for reprints should be addressed, at Gastrointestinal

Research Laboratory (151M2), VA Medical Center, 4150 Clement Street. SanFrancisco, CA 94121.

mucus, the main component of which is mucin. Mucins arehigh molecular weight glycoproteins containing oligosaccharideside chains that are connected by O-glycosidic linkages to serineor threonine residues in the protein (apomucin) backbone.

The first step in 0-linked oligosaccharide synthesis is theaddition of /V-acetylgalactosamine to serine or threonine resulting in the formation of the Tn antigen (Fig. 1). Once synthesized, the further glycosylation of Tn antigen can proceed alongdifferent pathways. The factors that regulate the different glycosylation pathways are not well understood, but these reactionsdepend to a great extent upon the activity of specific glycosyl-transferase enzymes, the availability of precursor substances,and other factors such as divalent cation requirements andavailability of nucleotide sugar donors (1). The addition of sialicacid by an «2,6-sialyltransferase (Fig. 1, Pathway 1) forms thesialosyl-Tn antigen (SAa2,6GalNAca-O-Ser/Thr) and this

structure does not apparently undergo further glycosylation.The addition of galactose by a /31,3-galactosyltransferase (Pathway 2) results in the formation of the Thomsen-Friedenreichantigen (Gal/31,3GalNAca-O-Ser/Thr) also known as T antigen. This disaccharide forms the so-called Core 1 structurewhich can be further glycosylated. Individuals with the Tnsyndrome are deficient in this /31,3-galactosyltransferase resulting in the expression of Tn antigen on their hematopoietic cells(2). If 7V-acetylglucosamine is added to the Tn antigen (Pathway3), this forms the Core 3 structure which can also be furtherglycosylated. Other glycosylation reactions not shown in Fig. 1can occur and have been described in recent reviews (1,3).

Of the alterations in glycosylation that have been describedin cancer cells, incomplete glycosylation is a frequent event.This results in the appearance of precursor structures which innormal cells would remain cryptic or masked because of furtherglycosylation (4). For example, the T antigen is rarely expressedby normal colonocytes whereas cells of malignant, premalig-nant, and to some extent hyperplastic colonie mucosa, frequently express this antigen (5-10). Moreover, expression of Tantigen in premalignant adenomatous polyps seems to correlatewith histologie criteria of malignant potential (10).

Previous biochemical studies have characterized oligosaccharide structures of normal human colonie mucin as being primarily based on Core 3 structures (11, 12), and others havedemonstrated Core 3 GlcNAc transferase activity in human(and rat) colonie mucosa (13). This would suggest that innormal colonocytes, Pathway 3 of Fig. 1 might predominateover Pathways 1 and 2. Little is known about the oligosaccharide structures of mucin from colon cancer cells. We mightpostulate, based on the prevalence of T antigen expression, thatin contrast to normal colonocytes, cancer cells preferentiallysynthesize oligosaccharides by Pathway 2 and perhaps Pathway1, and that incomplete glycosylation beyond the initial stepsmight then result in the unmasking of these core-region structures.

The present immunohistochemical study was performed toinvestigate this hypothesis. This analysis was facilitated by the

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Apomucm (R)

GalNAc - R

Siaa2,6GalNAc-R Gal p 1,3 GalNAc - R GlcNAcp 1,3GalNAc - R

Elongation Elongation

Fig. I. Simplified scheme of the biosynthesis of O-linked mucin glycoproteins.The synthesis of Tn antigen is catalyzed by a polypeptidyl-GalNAc transferase.Once formed, the Tn antigen has several biosynthetic fates, of which the threebest characterized are indicated. Enzymes responsible for each of the threepathways are as follows: Pathway 1, «2,6-sialyltransferase; Pathway 2, /31,3-galactosyltransferase; and Pathway 3, /îl.S-A'-acetylglucosaminyltransferase. Synthesis along Pathways 2 and 3 may proceed to further oligosaccharide elongation,whereas Pathway 1 is a terminal step (indicated by interrupted arrow). See textfor details.

recent availability of reagents that can identify Tn and sialosyl-Tn structures, although as yet, no reagent specific for the Core3 structure is available. Our aims were threefold: (a) to definethe expression of Tn and sialosyl-Tn antigens in fetal, normaladult, and malignant colorectal tissues; (¿>)to compare theexpression of these antigens to T antigen in order to gaininsight into the initial steps of glycosylation; and (c) to comparedifferent anti-Tn and anti-sialosyl-Tn reagents to determinetheir relative reactivities.

MATERIALS AND METHODS

Tissues. Primary colorectal carcinoma tissues were obtained from 29patients undergoing surgical resection. Nineteen were colonie adeno-carcinomas, of which 14 were well differentiated or moderately differentiated, and five were poorly differentiated. The other 10 were muci-nous (colloid) carcinomas in which more than 60% of tumor volumedemonstrated abundant mucin production. In 25 cancer specimens,well-oriented mucosa immediately adjacent to the cancer was available

for examination, and this region is referred to as TM. TM is grosslynormal but usually exhibits microscopic abnormalities such as elongated and sometimes branched crypts, but no cellular atypia. 22 specimens of normal colonie mucosa were obtained at immediate autopsyfrom individuals without any colonie disease, according to the protocolestablished at the Department of Pathology, University of MarylandSchool of Medicine and kindly provided by Drs. Benjamin Trump andAbulkalam Shamsuddin. 11 cases of normal colonie mucosa wereproximal to the splenic flexure, and 11 were distal. Fetal colonie tissuerepresenting both proximal and distal regions was obtained from 10second-trimester abortuses as approved by the Committee on HumanResearch, University of California San Francisco. All tissues used inthis study were fixed in formalin, embedded in paraffin and cut into 5-¿imserial sections for immunohistochemical staining.

Antibodies and Lectins. For the detection of Tn antigen, 2 Mabs4 and

one lectin were used. Mab ETn 1.01 (IgM isotype) was developed byimmunizing a Biozzi mouse with erythrocytes from an individual withthe Tn syndrome.5 This antibody requires the Tn antigen GalNAc

4The abbreviations used are: Mab, monoclonal antibody; VVA, Vicia villosa

agglutinin; TM, transitional mucosa; PBS. phosphate buffered saline.' W. L. Bigbee, R. G. Langlois. L. H. Stanker. M. Vanderlaan, and R. H.

Jensen, manuscript submitted.

monosaccharide and does not bind to normal cells presenting MN orT antigens. The immunogen for the CU-1 Mab was a high-molecularweight glycoprotein Tn antigen secreted by a human squamous lungcarcinoma cell line LU-65 (14). This IgG3 Mab does not cross-reactwith blood group A antigen, unlike other Tn Mabs that have also beenraised against the LU-65 glycoprotein Tn antigen (15). The binding ofCU-1 to Tn antigen can be inhibited by monosaccharide GalNAc andspecifically by /»-nitrophenyl-a-D-GalNAc but not by p-nitrophenyl-/3-D-GalNAc. The B4 lectin from Vicia villosa seeds specifically agglutinates Tn-exposed erythrocytes, a reaction which can be inhibited bysaccharides or glycoproteins containing GalNAc (16).

Two Mabs were used to detect sialosyl-Tn. TKH2 (IgGi) was raisedagainst ovine submaxillary mucin as immunogen (17). The B72.3 Mab(IgG]) was developed several years ago using membranes from a humanbreast carcinoma metastasis as immunogen (18). This antibody hasundergone considerable testing both as a diagnostic and therapeuticreagent (19). The high molecular weight mucin-like glycoprotein recognized by B72.3 has been termed TAG-72, and recent biochemicalstudies suggest that the epitope involved is sialosyl-Tn (17, 20).

Monoclonal anti-T antibody (AH9-16) was obtained from Chem-biomed, Ltd. (Edmonton, Canada) and kindly provided by Dr. R.Murray Ratcliffe. We have used this IgM antibody in previous studiesof colonie tissues (10).

Reagents. Biotinylated B4lectin from Vicia villosa was obtained fromSigma Chemical Co. (St. Louis, MO). Histostain SP, provided in a kitwith biotinylated rabbit anti-mouse IgG + IgA + IgM and streptavidin-peroxidase conjugate, was obtained from Zymed Laboratories (SouthSan Francisco, CA). The buffer used was PBS (0.1 M phosphate, 1.5 MNaCl, pH 7.4) containing 1% bovine serum albumin.

Tissue Staining. The Mabs CU-1, TKH2, and B72.3 were used asundiluted hybridoma supernatant. Other working concentrations wereas follows: Mab ETn 1.01, 1.3 Mg/ml; VVA, 5 Mg/ml; Mab AH9-16, 2iig/ml.

The streptavidin-peroxidase technique of immunohistochemistry wasperformed as described previously (21), with all steps conducted atroom temperature. Briefly, after deparaffmization and rehydration,sections were incubated with fresh 3% hydrogen peroxide in methanolfor 10 min and then washed three times with buffer. Next, 5% normalrabbit serum in PBS was applied for 20 min and removed by blotting.Primary antibody or biotinylated-VVA were incubated for 90 min andwashed three times with buffer. Then, for antibody-stained slides,biotinylated secondary antibody (1:75 in PBS) was added for 20 minand washed three times with buffer. All sections then received streptavidin-peroxidase conjugate (10 Mg/mOfor 30 min and were washed withbuffer three times. Finally, slides were reacted with diaminobenzidinesubstrate for 10 minutes, rinsed with tap water, and mounted.

Negative controls consisted of substituting normal mouse serum forMabs and PBS for VVA, which resulted in negative staining.

Scoring. Staining intensity was graded as follows: absent (0), weak(1+), moderate (2+), strong (3+). For carcinomas, the number of lowpower (xlO) optical fields in a specimen which were positively stainedwas expressed as a percentage of the total number of optical fieldscontaining tumor. For normal mucosa and TM, the percentage of totalcrypts which stained positive were scored.

RESULTS

Normal Adult Colon. In normal adult colonie epithelium, Tnexpression was essentially absent (Table 1). Only three specimens reacted weakly with VVA in some of the goblet cells, andwith CU-1 in the supranuclear cytoplasm (Fig. 2). The lack ofstaining of normal colonie mucosa with anti-Tn reagents isconsistent with the lack of any cross-reactivity with blood groupA antigen.

In these same tissues, sialosyl-Tn and T antigens were notexpressed at all.

Transitional Mucosa. Unlike normal mucosa, colonocytes ofthe mucosa immediately next to cancer often expressed Tn andsialosyl-Tn (Table 1, Fig. 3). All three anti-Tn reagents stained

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Table 1 Expression ofTn, sialosyl-Tn, and T antigens in human colonie tissues

Numbers represent percentage of positive cases.

Tn Sialosyl Tn

Normal colon (n =22)Transitionalmucosa (n =25)Colon

cancer (n =29)Welldifferentiated (n =4)Moderately

differentiated(n=10)Poorly

differentiated (n=5)Mucinous

(n =10)FetalColon (n = 10)1.01035727560808010VVA14677275508090100CU-114618175707510050TKH2038961001007510050B72.3038931001006010040AH9-160477110078336730

"V* >:•y

\ •<-

%

f.

'•

<

• BFig. 2. Tn expression in normal colonie mucosa. In general, Tn was not

expressed in normal colonie mucosa. However, in a few cases, VVA (A) boundweakly to some goblet cells (arrow) and Mab CU-1 (B) bound weakly to supra-nuclear cytoplasm (arrow). (Original magnification, x 50)

cell cytoplasm (primarily the supranuclear region) but in addition, VVA reacted with goblet cell vacuoles.

Sialosyl-Tn was also found mainly in supranuclear cytoplasm,but in a few specimens of moderately-well differentiated cancer,the goblet cells of TM stained strongly with TKH2. Curiously,capillaries in TM reacted with TKH2 (but not B72.3). Thisstaining was found only in mucosa!, not submucosal capillaries,was strongest in TM closest to the tumor, and occurred even ifepithelial cells of TM did not react with TKH2.

Fig. 3. Tn and sialosyl-Tn expression in transitional mucosa. A, in contrast tonormal mucosa (see Fig. 2/4), goblet cell binding of VVA was more prevalent andmore intense. B, binding of Mab TKH2 occurred in supranuclear cytoplasm(arrow) and goblet cells (not shown). Note the reactivity of capillaries in transitional mucosa with this antibody. (Original magnification. A, x 125; B, x 50)

The T antigen was found only in the supranuclear cytoplasmof cells in TM.

Colon Cancer. The majority of colon cancers expressed Tnantigen although the percentage of total positive cases varieddepending upon the reagent used (Table 1). With each of thethree anti-Tn reagents, the negative cases were mostly amongthe well- and moderately well-differentiated adenocarcinomas.All mucinous carcinomas expressed Tn antigen except for onecase not stained with VVA and two cases negative with Mab1.01. There were some differences at the cellular level betweenthe three anti-Tn reagents. In general, the staining intensitywas moderate to strong with Mab CU-1 and VVA, but wasweak to moderate with Mab 1.01. In well- and moderatelydifferentiated adenocarcinomas, all three reagents stained cellcytoplasm and cancer gland luminal contents, whereas cellmembranes were stained with Mabs CU-1 and 1.01 but onlyrarely with VVA (Fig. 44). All three reagents stained cellcytoplasm in poorly differentiated adenocarcinomas (Fig. 5). Inmucinous colon cancers, both Mab CU-1 and VVA bindingwere particularly prevalent and intense in the mucin within thecells and in extracellular mucin lakes (Fig. 6A). However, Mab1.01 staining was often weaker and rarely found in extracellularmucin.

The expression of sialosyl-Tn was nearly ubiquitous in coloncancer. Only one poorly differentiated cancer specimen did notbind TKH2 and two were negative with B72.3. The prevalence,

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B

Fig. 4. Well-differentiated colonie adenocarcinoma stained with Mah CU-1(A) and TKH2 (B). CU-1 reacted mostly with cell cytoplasm and gland luminalcontents, and less with apical cell membranes. TKH2 bound to cell membranesand luminal contents but not to cell cytoplasm. (Original magnification, x 25).

cellular location, and staining intensity of TKH2 and B72.3were strikingly similar. In well-differentiated adenocarcinomas,both Mabs reacted strongly, and preferentially stained cellmembranes and cancer gland contents, with only occasionalcytoplasmic staining (Fig. 4B). In poorly differentiated adenocarcinomas, cytoplasmic staining could be found (Fig. 5B). Inmucinous carcinomas, practically all of the extracellular mucinlakes were markedly positive, with weaker staining of intracel-lular mucin and virtually no membrane staining (Fig. 6Ä).

T antigen was expressed in 71 % of colon cancers, usuallyamong the moderate- and well-differentiated adenocarcinomas.Compared to Tn and sialosyl-Tn, T antigen was found in fewermucinous carcinomas. Anti-T staining of adenocarcinomas occurred mainly on cell membranes and glandular contents, butin mucinous carcinomas staining was mostly intracellular withrare expression in mucin lakes.

Coexpression of Tn, Sialosyl-Tn, and T Antigens in ColonCancer. Table 2 summarizes the patterns of simultaneousexpression of Tn, sialosyl-Tn, and T antigens that were found.Most often, tumors expressed all three antigens although therewas heterogeneity in cellular location and staining intensitybetween antigens. In six tumors (25%), T antigen was absentbut Tn and sialosyl-Tn were expressed. This suggests that either

B

Fig. 5. Poorly-differentiated colonie adenocarcinoma stained with VVA(A)and Mab TKH2(B). Cell cytoplasm is intensely stained in both cases. (Originalmagnification, x SO)

the T antigen is not synthesized (due to insufficient galactosyl-

transferase activity), or it is masked by additional carbohydrateresidues. Two cancers did not express Tn but did express bothsialosyl-Tn and T suggesting that all of the Tn precursorsubstance was further glycosylated. In one poorly differentiatedadenocarcinoma, all three antigens were absent, representing inall likelihood, the inability of these tumor cells to synthesizeGalNAc onto the protein backbone or possibly a lack of synthesis of the mucin polypeptide. In no case was Tn expressedwithout concomitant sialosyl-Tn or T expression.

Fetal Colon. In fetal colonie mucosa, Tn, sialosyl-Tn, and Twere all expressed but with considerable variation dependingupon the reagent used (Table 1). In the case of the anti-Tnreagents, Mab 1.01 staining was essentially absent from mu-cosal cells and meconium (luminal secretions), Mab CU-1

reacted with half of the cases in the cells but not in meconium,and VVA bound strongly to both cells and meconium in allcases. A striking finding was the strong and prevalent gobletcell staining with both VVA and Mab CU-1 (Fig. 7, A and B).In terms of sialosyl-Tn expression, both TKH2 and B72.3reacted strongly with goblet cells of the mucosa in about halfthe cases (Fig. 1C). Meconium staining was frequently positivewith TKH2 but weak or negative with B72.3. Three fetal

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Ju£;Fig. 6. Mucinous carcinoma, colloid type, stained with VVA (A) and Mab

B72.3 (B). VVA strongly stains mucin in both intracellular (arrow) and extracellular (arrowhead) locations. B72.3 preferentially stains extracellular mucin withonly weak staining of intracellular mucin. (Original magnification, x SO)

Table 2 Simultaneous expression ofTn, sialosyl-Tn and T antigens in coloncancer tissues

°In five of the 29 cancers, sections were unavailable for staining with one of

the antibodies, so these cases are omitted from this simultaneous comparison.

specimens expressed T antigen weakly in cell cytoplasm andsecretions.

The five fetal specimens that expressed CU-1-defined Tn alsoexpressed sialosyl-Tn and two of them expressed T. However,since VVA stained all of the fetal colonie specimens, there werefour cases where only VVA-defined Tn occurred without sialosyl-Tn or T.

DISCUSSION

A common feature of colon cancer is the production of mucin.However, it is not known if mucin expression simply reflectsthe lineage of the cells that give rise to the cancer, or whetherit may play a role in the biological behavior of the cancer cell.

t 'V* '**

.»'* f* i fu <&.^^A^V'"t ,

B

Tn SialosylTn+

++ +- +T+ +No.

(total,24)°15

62

11H t•

«••';•

•»•

Fig. 7. Fetal colonie mucosa stained with CU-1 (A), VVA (B), and TKH2(C). All three reagents react strongly with goblet cells, although VVA binding ismore prevalent. (Original magnification, x 50)

In support of the latter concept, patients with mucinous carcinomas of the colon (a histológica! subset in which most of thetumor volume consists of mucin) have been found to have aparticularly poor outcome in some reports (22, 23). However,since other series have not confirmed this poor prognosis (24,25), it raises the possibility as to whether qualitative rather than

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Tn, SIALOSYL-Tn, AND T IN COLON CANCER

quantitative mucin alterations might be important in the biologyof colon cancer.

To gain insight into qualitative mucin alterations that mightoccur in colon cancer, we have examined the expression ofcarbohydrate antigens associated with the earliest steps of mucin glycosylation. By simultaneously comparing the expressionof three antigens (Tn, sialosyl-Tn, T) in normal and malignantcolonie tissues, several observations emerged. Normal colono-cytes did not express sialosyl-Tn or T and only rarely expressedTn antigen. The occasional expression of Tn suggests that insome cases this structure may be in the process of being furtherglycosylated. In contrast to normal mucosa, three-quarters ofthe colon cancers expressed Tn and T antigens, and practicallyall of them expressed sialosyl-Tn. Therefore the appearance ofthese core-region antigens provides further evidence for incomplete glycosylation in colon cancer cells. The considerableexpression of Tn antigen represents another example of precursor accumulation in colon cancer much like the expressionof blood group H substance represents accumulation of precursor for the A and B substances (26), and T antigen representsaccumulation of precursor for the MN blood group antigens(27). While Tn expression in some colon cancer cells mightsuggest that glycosylation is blocked just after the addition ofthe initial GalNAc residue, the fact that there is frequentcoexpression of the sialosyl-Tn and T along with Tn (Table 2)indicates that the biosynthetic machinery necessary for sialyla-tion and further glycosylation of Tn exists in these tissues.

While the absence of exposed T, sialosyl-Tn, and Tn antigensin normal mucosa can be interpreted to mean that these structures are not synthesized, there is some evidence that indeedthese determinants are present in normal colonocytes. Forexample, peanut lectin binding sites (T antigen) can be observedin normal colonie mucosa after neuraminidase treatment (5, 7).In addition, oligosaccharides of normal colonie mucin oftenhave sialic acid conjugated to the linkage GalNAc (11, 12).However, although sialosyl-Tn was found as such, theSiaa2,6GalNAc structure was most frequently found as aninternal structure of longer oligosaccharides based on a Core 3structure. This would therefore suggest that the linkage GalNAcof normal colonie mucin preferentially receives a GlcNAc residue first (forming the Core 3 structure), because adding a sialicacid first would prevent further oligosaccharide elongation (Reference 1; Fig. 1).

Based on the present observations and available literature,we would propose the following: in normal colonocytes, the/31,3 GlcNAc-transferase (Fig. 1, Pathway 3) is highly active,converting the Tn substance down the Core 3 glycosylationpathway. This is performed efficiently since very little Tnantigen remains exposed in normal cells. In contrast, coloncancer cells have highly active ßl,3Gal transferase (Pathway2) and «2,6sialyltransferase (Pathway 1) enzymes and/or insufficient /31,3 GlcNAc transferase activity. This would allowthe galactosyltransferase and sialyltransferase enzymes to compete favorably with the GlcNAc transferase for the Tn precursor, but perhaps the efficiency of these reactions is suboptimalsince considerable Tn antigen remains exposed in cancer cells.In rectal carcinoma, other investigators have identified aGalNAc-GalNAc linkage (28) which has been referred to asCore 5 (3) and implies the activity of an a 1,3GalN Ac transferasethat might be different from the blood group A GalNAc transferase. Clearly, therefore, more work is needed to further clarifyglycosylation pathways in colon cancer, and to substantiate theabove hypothesis.

The observation that T and Tn antigens are carcinoma-

associated was made by Springer and coworkers over a decadeago (29). Much of that work involved the use of human anti-Tand anti-Tn antisera by immunochemical and some immuno-histochemical methods primarily in nongastrointestinal malignancies. The present immunohistochemical study, using murineMabs and a lectin, extends these observations to colon cancertissues, confirming that T, Tn, and sialosyl-Tn are carcinoma-associated antigens in this organ. The cancer-associated natureof T antigen in the colon has been known for several yearsbased on studies using peanut agglutinin, as well as polyclonaland monoclonal antibodies (5-10). A recent immunohistochemical report used Mabs raised against synthetic T antigenin its a or ßconfiguration on frozen sections (30). Those resultssuggested that the carrier molecule of the Gal/Si,3GalNAcdisaccharide of colon cancers might by glycolipid (ßconfiguration) rather than glycoprotein (a configuration). While wecannot completely exclude the possibility that we too are detecting T antigen on glycolipid instead of glycoprotein, the factthat we still observe T antigen after dewaxing our paraffin-embedded tissues, coupled with the knowledge that the expression of many carbohydrate antigens is shared by both glycopro-teins and glycolipids (31), supports our above hypothesis concerning core-region antigen expression in colonie mucin glycoprotein.

Only recently has Tn expression been reported in colon.Hirohashi et al. developed two Mabs that recognize Tn butcross-react with blood group A antigen, and these reagentsstained most colon cancers but not normal colonie mucosa (15).The CU-1 antibody used in the present study has previouslybeen shown not to cross-react with the A antigen, and in thatstudy, 88% of colon cancers but also 33% of normal mucosastained positive (14). Longenecker et al. (30) developed murineMabs to synthetic Tn antigen and found that by using immu-noperoxidase staining of frozen sections, all of seven coloncancers expressed Tn whereas normal tissues (including colon)did not. In terms of sialosyl-Tn expression in the colon, Kurosaka and coworkers first described this structure on glycopro-teins of a human rectal adenocarcinoma (28) and later developed a Mab specific for this carbohydrate structure using LS180 colon cancer cells (32). Recently, Kjeldsen et al. (17) andGold and Mattes (20) observed that Mab B72.3 recognizessialosyl-Tn. Therefore, based on previous immunohistochemical reports using the B72.3 antibody, sialosyl-Tn is absent innormal colonie mucosa but quite prevalent in colon cancer (33,34). Our study confirms this finding not only using B72.3 butalso a new Mab TKH2 developed against ovine submaxillarymucin known to express sialosyl-Tn. Moreover, the strength ofthe present study is the simultaneous comparison of Tn, sialosyl-Tn, and T in the same tissues, allowing at least tentativeconclusions to be drawn concerning the biosynthesis of mucin-associated antigen expression in the colon.

As far as the histological type of colon cancer is concerned,T antigen expression was more frequent in the well- and moderately differentiated tumors than in the poorly differentiatedadenocarcinomas or mucinous carcinomas. However, with Tnantigen, the inverse relationship was found. This supports earlier observations by Springer that anaplastic carcinomas oftenhave a greater Tn/T ratio than well-differentiated adenocarcinomas (29). In mucinous carcinomas, the strong staining intensity and high proportion of cells expressing Tn and sialosyl-Tnwere striking. Thor et al. have also described B72.3 staining ofmucinous colon carcinomas (33). Thus, these antigens appearto be good markers of colon cancer mucin elaborated both bysignet ring cell carcinomas (a rare histological subset character-

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ized by intracellular mucin accumulation) and colloid carcinomas (characterized by large extracellular lakes of mucin). Sincepatients with mucinous carcinomas (particularly signet ring celltype) and poorly differentiated adenocarcinomas often have apoor prognosis, the Tn and sialosyl-Tn antigens may be usefulmarkers for these subsets of patients.

The phenomenon of enhanced expression of all three antigensin TM is one that is shared by many other tumor-associatedantigens that are not usually expressed in "normal" colonie

mucosa. In this study, when antigens were expressed in TM,they were usually found in the supranuclear cytoplasm, butseveral cases showed strong goblet cell staining with VVA andTKH2, suggesting perhaps a premalignant mucin alteration inthese cells. Filipe and coworkers have noted enhanced sialo-

inucin expression in goblet cells of TM using histochemicaltechniques, so perhaps sialosyl-Tn contributes to sialomucinexpression. The observation of sialosyl-Tn expression definedby Mab TKH2 in mucosa! capillaries of TM is intriguing. Thismight represent blood-borne sialosyl-Tn antigen since mucosalcapillaries furthest from the cancer and those in truly normalcolonie mucosa did not express the antigen.

Fetal colonie mucosa was examined with an aim towarddetermining if Tn and sialosyl-Tn are oncodevelopmental antigens. Indeed, both of these antigens were expressed by second-trimester fetuses and usually in goblet cells. This suggests thatfetal colonocytes, like cancer cells, possess the enzymatic machinery to synthesize structures of Pathways 1 and 2. Otherstudies of core-region oligosaccharides found on human meco-nium glycoproteins have identified these antigens and severalothers, indicating a diverse repertoire of mucin oligosaccharidesynthesis in the fetus (35). Although all of the fetal specimensbound VVA, only half bound CU-1, TKH2, and B72.3. Thismay reflect subtle differences in epitope structure recognizedby the different reagents.

We purposely chose three different anti-Tn and two anti-sialosyl-Tn reagents for this study to examine their relativereactivities. The two sialosyl-Tn reagents were quite similar intheir staining pattern and intensity in all types of colonie tissues.However, there were some differences between the Tn reagentssuch that, in general, Mab 1.01 stained fewer cells and withweaker intensity than Mab CU-1 or VVA. The latter tworeagents exhibited rather comparable staining patterns, although three cancers were CU-1-positive/VVA-negative, andfive fetal specimens were VVA-positive/CU-1-negative. Themost likely explanation for the disparate staining is that lectinsand antibodies (raised with different immunogens) frequentlyhave somewhat different epitope specificities depending uponantigenic conformation and other factors such as affinity andantibody isotype. We have also noted differences between peanut lectin, a polyclonal antibody and a Mab for recognizing Tantigen in colonie tissues (10). The considerable binding of MabCU-1 to colon cancers is interesting in that the immunogen forthis Mab was the secreted mucin from a lung carcinoma cellline, indicating that mucin-producing tumors of different organsshare common epitopes.

ACKNOWLEDGMENTS

We wish to thank Annabelle Friera for technical assistance and RitaBurns for manuscript preparation.

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1989;49:197-204. Cancer Res   Steven H. Itzkowitz, Mei Yuan, Carolyn K. Montgomery, et al.   CancerExpression of Tn, Sialosyl-Tn, and T Antigens in Human Colon

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