Expression of Lewis3,Lewisb, Lewis, Lewisy,Sialyl-Lewis8 ......immunoperoxidase method using a panel of mouse moAbs.2 We simultaneously tested salivas using an ELISA for secretor status

[CANCER RESEARCH 49, 745-752, February I, 1989]

Expression of Lewis3, Lewisb, Lewis", Lewisy, Sialyl-Lewis8, and Sialyl-Lewis"

Blood Group Antigens in Human Gastric Carcinoma and in NormalGastric TissueJunichi Sakamoto,1 Tadashi Watanabe, Takahiko Tokumaru, Hiroshi Takagi, Hiroaki Nakazato, and

Kenneth O. LloydDepartment of Gastroenterological Surgery, Aichi Cancer Center, 1-1 Kanokoden Chikusaku, Nagoya, Japan fJ. S., H. N.]; the Second Department of Surgery, NagoyaUniversity Faculty of Medicine, 65 Tsurumaicho Showaku, Nagoya, Japan [T. W., T. T., H. T.]; and the Memorial Sloan-Kettering Cancer Center, New York,New York Â¡0021[K. O. L.]

ABSTRACT

A panel of 6 mouse monoclonal antibodies detecting blood groupantigens of the Lewis systems and their sialylated derivatives have beenused to define the immunoanatomic distribution of these antigenic structures within the normal human gastric mucosa and in gastric cancertissues. The reagents employed detect the following blood group specificities: Lewis', Lewisb, Lewis", Lewis', sialylated Lewis', and sialylatedLewis". We have analyzed the presence of these antigens in histologically

normal gastric mucosa and in gastric carcinoma from 61 patients by theimmunoperoxidase method. In addition, we simultaneously examined theblood group and secretor status in 31 of the 61 individuals studied.

Immunohistochemical analysis revealed that these antigenic systemsare differentially expressed in cell types and cell layers of the normalgastric epithelium. Major differences were observed in surface epitheliaand in deep glands including Brenner's gland of the gastroduodenaljunction, mainly in the pronounced expression of Lewis* and Lewis1'antigens in the former and the expression of Lewis" and I .CMis' in thelatter. In secretor individuals, Lewisb was the dominant antigen in thesurface epithelium, and in nonsecretors, Lewis' was observed in thesurface epithelium. Lewis" and Lewis' were both detected in the deepglands and in Brenner's glands regardless of the secretor status. The

expression of sialylated derivatives in normal gastric tissues was considerably reduced but was consistent with the expression of their precursorsin normal gastric epithelium.

In gastric cancers, more pronounced expression of Lewis* and sialylated Lewis* was observed in secretor individuals and acted as a tumor-associated antigen. Comparison of the plasma level of sialylated Lewis*

and its tissue expression demonstrated that the shedding of the antigeninto interstitial stroma correlated with the detection of the antigen inserum.

These studies confirmed the importance of blood group antigens asnormal differentiation antigens. Examination of secretor status clarifiedthe mechanism of Lewis*and Lewisbantigen expression in gastric surface

epithelium. Alterations in the expression of these antigens and an increaseof sialylated derivatives in gastric cancers demonstrated that these bloodgroup antigens are useful tools for the analysis of histogenesis andorganogÃ©nesisin the stomach and its neoplastic and nonneoplastic diseases.

INTRODUCTION

Blood group antigens are a group of carbohydrate determinants typically found on erythrocytes. Their specificities arealso expressed in many epithelial tissues and they act as majoralloantigenic systems in humans (1, 2). Those antigens arecarried by two types of backbone structures, type 1 chains[containing Gal(/31-3)GlcNAc] and type 2 chains [containingGal(/31-4)GlcNAc]. As schematically presented in Fig. 1, ABHand Lewis structures are formed by sequential addition of

Received 3/2/88; revised 9/19/88; accepted 11/2/88.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.

1To whom requests for reprints should be addressed, at Department ofGastroenterological Surgery, Aichi Cancer Center, 1-1 Kanokoden Chikusaku,Nagoya 464, Japan.

fucose, galactose and GalNAc to the backbone carbohydratechains of both lipids and proteins (3). In addition to the genescontrolling the synthesis of those structures, another gene locuscontrols the expression of the antigens in secretions and onvarious cell types of different organs. Thus the classical terms"secretor" and "nonsecretor" indicate the capacity of an indi

vidual to secrete or not secrete such substances in saliva (4).Secretor individuals produce ABH, Lewisb (Leb) and Lewis*(Ley), whereas nonsecretors produce Lewis' (Le") and Lewis"(Le") antigens in their saliva (5).

Interest in the secretor status and Lewis expression in normaltissues and in tumors has increased in recent years because ofseveral clinicopathological observations in gastrointestinal oncology. These include: (a) an apparent loss of ABH and enhanced expression of Le" in human gastric cancers (6). (b)Evidence that secretor individuals express Le" instead of Leb inthe distal colon, and the appearance of Leb or Ley in cancers of

distal colon (7). (c) Patients with certain epithelial tumors showelevated serum levels of Lewis antigens (8). (d) Sialylatedderivatives of the Lewis antigens, such as sialylated-Le" orsialylated-Le", may act as tumor-associated markers in certain

alimentary tract cancers (9).In this study, we focused on the expression of Le3, Leb, Le",

and Ley, sialylated Le" (Ã‡A19.9), and sialylated Le" (CSLEX1)

antigens in stomach. We have analyzed the presence of theseantigens in frozen and paraffin sections of normal stomach andgastric cancers obtained from 61 gastric cancer patients by theimmunoperoxidase method using a panel of mouse moAbs.2

We simultaneously tested salivas using an ELISA for secretorstatus in 31 of those patients to correlate immunohistologicaldistribution with secretor status. We also analyzed the serumlevels of sialylated-Le" antigen by RIA and compared them with

the results of immunopathology.

MATERIALS AND METHODS

Tissues. Primary gastric carcinoma tissue was obtained from 61patients undergoing surgical resection at Nagoya University Hospitaland at Aichi Cancer Center. 18 were from cardia, 20 were from body,and 23 were from nutrii in of the stomach. Five were well differentiated,23 were moderately differentiated, 29 were poorly differentiated, twowere mucinous, and two were signet ring cell carcinomas. In all casesnormal mucosa distant from the tumor lesion was obtained from thesame patient. The tissues were prepared from surgical pathology specimen within 1 to 2 h of resection and fresh tissues were fixed in 10%formaldehyde in phosphate buffered saline (PBS), pH 7.5, and embedded in paraffin. Alternatively, tissues were snap frozen in isopentaneprecooled in liquid nitrogen, were embedded in OCT compound andwere stored at â€”¿�70Â°Cuntil needed.

Reagents. moAbs T-174, T-218, P-12, and F-3 with specificities forLe", Leb, Le", and Ley antigens, respectively, were used (7, 10-12).

1The abbreviations used are: moAb, monoclonal antibody; ELISA, enzyme-

linked immunosorbent assay; RIA. radioimmunoassay.

745

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BLOOD GROUPS IN STOMACH TUMORS

Type 1 Chains Type 2 Chains

Gal(3l-Â»3GlcNAcGalÃŸMGlcNAc GalÃŸMGlcNAc Gal(3l-*3GlcNAc Gal(3H-4GlcNAc GalÃŸl-~4GlcNAc Gal(3l-*4GlcNAc Gal0 H-4GlcNAc

1,3Fuco

1.2 1,4NANA Fuca

11.4Fuea

11.1Fuea

1,2 | 1,4Fuea Fuea

1.3Fuca

1.2Fuca

1,2 | 1,3Fuea Fuea

Sialyl-Led Lea H-l LeD Sialyl-X H-2

GalNAcalâ€”-3GalÃŸ1â€”SGlcNAc Gala lâ€”-3GalÃŸlâ€”-3GlcNAc

1,2Fuca

GalNAcalâ€”-3GalÃŸ1â€”4GlcNAc

1.2Fuca

Gala lâ€”-3GalÃŸl-~4GlcNAc

jl.2Fuca

ILIFuca

A-l B-l A-2 B-2

Fig. 1. Structural relationship between A, B, H, Lewis, Le", Le', sialylated Le* and sialylated Le* blood group determinants. //-/ and H-2, monofucosyl type 1 andtype 2 H-determinants, respectively. Le* and Ley specificities are also known as X and Y, respectively. All sugars are in the D-configuration, except for fucose, whichis in the L-form.

Mouse moAb 19.9 was used to detect the sialylated Le" structure (9),and antibody CSLEX1 was used to define sialylated Lex structure (13).

Immunohistochemistry-Immunoperoxidase Staining Using moAb.Formalin-fixed and paraffin-embedded tissue sections were depuratimi/ed with xylene and ethanol for use in this technique. Tissue sectionswere incubated in supressor serum and then were incubated with moAbovernight at 4"( '. Both peroxidase-antiperoxidase and avidin-biotin

methods were used in our experiments. The secondary antibodies werehorse anti-mouse horseradish peroxidase conjugate (Vector Laboratories, Burlingame, California) incubated on sections for 1 h. The peroxidase reaction was performed by incubating tissue sections for 6 to12 min with 5 mg of diaminobenzene tetrahydrochloride (Sigma Chemical Company, St. Louis, MO) in 100 ml of Tris buffer, pH 7.6, plus100 Â¿ilof 0.3% hydrogen peroxide. Sections were washed with distilledwater counterstained with hematoxylin and mounted with paramount.Fresh frozen sections were also used for this method. For the negativecontrols, moAbs against melanoma antigens of the same species andsubtype were used.

Determination of Secretor Status. Saliva was collected without preliminary stimulation and glycoproteins were extracted. An ELISA procedure for reactivity with anti-Le* and anti-Leb antibodies were performed

as described previously (5).Serum Level of CA 19.9. Blood samples were obtained from 28

patients among 61 cases tested by immunopathology. The serum levelsof CA 19.9 antigen were determined by commercial radioimmunoassaykits (RIA; Centocor, SRL, Tokyo, Japan). The cut off level was set at37 unit/ml according to the study of Del Villano (14).

Serum Level of CSLEX1. Blood samples from 16 patients wereobtained. The serum levels of CSLEX1 antigen were tested by RIA kit(Midori Pharmaceutical Company, Tokyo, Japan) and the titer wasdetermined by ELISA assay using the sandwich method with peroxi-dase-labeled CSLEX1 antibody. The dilution of serum sample wasstarted from 1:4; reactivities below that titer were considered to benegative.

RESULTS

Blood Group Antigen Expression in Gastric Cancer and inNormal Gastric Epithelium. The expression of Le", Leb, Le",Ley, sialylated Le",and sialylated Le" antigens in gastric cancer

specimens and gastric epithelium from the same individuals,

was determined using the immunoperoxidase method (Table1).

In normal gastric mucosa, Le* was expressed in 30 and Le"was present in 49 out of 61 cases tested. Le" was present in 51and Ley was positive in 55 cases. Sialylated Le" was detectedonly in nine specimens of normal gastric mucosa and sialylatcd-Le" was positive in 31 cases.

In carcinoma tissues, Le" expression was found in 41/61 andLeb was detected in 42/61 tumors. Le" was expressed in 44/61and Ley was expressed in 43/61 cases. Sialylated Le" was foundin 30 cases of carcinoma tissues and sialylated Le" was expressed in 28 tumor specimens. Le" and sialylated Le" expres

sion was greatly increased in carcinoma tissues as compared totheir presence in the corresponding normal tissues. In someindividuals (19/61 for Le" and 23/61 for sialylated Le") the

antigen was detected in the tumor samples but not in normalgastric epithelium. In Fig. 2A, tumor-associated expression ofLe" is demonstrated. In Fig. 2B, expression of Le" antigen in aserial section is shown. Le" was present in the normal gastric

gland but not in tumor tissues.Immunostaining of Tumor and Normal Tissue from the Same

Individuals and Correlation with Secretor Status. 31 patientswere chosen for a more-detailed analysis in which Le", Leb, Le",Ley, sialylated Le", and sialylated Lex expression in tissue

sections was determined by immunoperoxidase staining of bothfrozen and paraffin-embedded samples and the saliva samplesfrom the same individual were analyzed to determine theirsecretor status (Table 2).

In normal gastric epithelium of the secretor individuals, Le"

Table 1 Reactivity of anti-blood group-related antibodies with normal stomachand stomach carcinoma tested with immunoperoxidase method

Le' Le" Le"

Sialyl- Sialylated ated

Le' Le' Le"

Normal (+)/carcinoma(+)Normal(â€”(/carcinoma(+)Normal(+)/carcinoma (â€”)Normal(-)/carcinoma (-)22"198123841184131074121447232292171023

Â°Number of cases out of 61 cases tested.

746




Sis

B

Fig. 2. Immunohistological analysis of normal gastric epithelium and gastriccarcinoma with anti-Le* and anti-Le* blood group antibodies using the inumimi

peroxidase technique. A, section has normal gastric epithelium on the right handside, which is negative with anti-Le* and gastric carcinoma tissue in the lower leftpart, which is positive with anti-Le'; B, sequential section of the same specimenstained with anti-Le" antibody. The normal gastric glands show positive staining

and the cancer tissue is negative. Magnification: x 40.

was positive in 10 out of 22 patients and Leb was positive in allsamples. The expression of Lea and Leb in secretors was mainly

confined to the foveolar epithelium of the stomach but in threecases, Le" expression was extended down into the deep gastricpits. Le" was positive in deep gastric glands in only one case of

normal gastric epithelium in secretors and in this case, thefoveolar epithelium was strongly positive. No case was notedin which either Le" or Leb was expressed exclusively in deep

gastric pits and not in foveolar epithelium. The expression ofLe" antigen in normal epithelium was different from Le" andLeb. Le* was detected in deep gastric glands in 18 out of 22

secretor individuals and was not expressed in the foveolarepithelium. Ley antigen expression was similar to Le", however,Ley was occasionally detected in the foveolar epithelium in 6/22 secretor cases. In all these six cases, Ley was positive in the

deep gastric glands as well as in the foveolar epithelium. Fig. 3shows the typical staining pattern of Le", Leb, Le", and Ley

antigen in normal gastric epithelium of a secretor individual.In nonsecretors, Le" was expressed in seven out of nine cases

and Leb was weak and heterogenously positive in two cases, inwhich Le" was clearly positive. There were two cases of nonsecretors, who expressed neither Le" nor Leb in their gastric

epithelium. In these cases, analysis of saliva revealed that theglycoprotein has no reaction with Le" or Leb but they werepositive with anti-Le" or anti-Ley antibodies. Le" and Ley were

positive also in the deep gastric glands of nonsecretors. Leyexpression was weaker in nonsecretors than was Le", and either

faint or heterogenous antibody staining was observed in thedeep gastric pits with no extension to the foveolar epithelium.

In noncancerous pathological conditions such as chronicgastritis, intestinal metaplasia or mucosa adjacent to cancers,these patterns of antigen distribution were more variable andsometimes, Le" and Leb was detected deep in gastric mucosa.

The expression of sialylated Le" and sialylated Le" in normalgastric epithelium was consistent with the expression of Le"and Le", respectively. That in Le"-positive specimens, sialylatedLe", was also positive, although the staining pattern was moreheterogenous and weaker. Sialylated Le" was positive in 17

normal gastric epithelium and its expression was mainly in thedeep gastric glands.

In gastric cancers, the expression of Le" was increased and in10 secretor specimens, Le" was expressed only in gastric cancerwhile normal gastric epithelium was unreactive. Sialylated Le*

expression was also increased in gastric cancer of secretors suchthat in 11 secretor patients, sialylated Le" acted as a tumorassociated antigen. Sialylated Lex expression was variable in

cancers.In nonsecretors, blood group antigen expression was en

hanced in cancers as compared to normals but no prominenttumor-related antigens, such as Le" or sialylated Le" in secre

tors, were noticed.Correlation between Immunoperoxidase Staining and Serum

Level of Sialylated Le* Detected by Monoclonal Antibody

CA19.9. In 28 cases, blood samples were collected to determinethe serum level of sialylated Le" and the results were correlated

with the immunoperoxidase staining of tissue specimens fromthe same patient (Fig. 4). In 16 patients, sialylated Le" was not

expressed in gastric cancer tissues and in all these cases, serumCAI9.9 levels were under 20 U/ml and in negative range. In12 patients, sialylated Le" was expressed in gastric cancer

tissues and in those cases, serum CAI9.9 level ranged from 10to 170 U/ml and five cases were over 37 U/ml, which is thecut-off level in the radioimmunoassay. In these 12 cases, theimmunopathology findings showed a typical positive stainingpattern. As shown in Fig. 5, the antigens were shed from thecytoplasm to the surrounding stroma and interstitial tissueswere stained. In cases with negative serum levels, especiallyunder 20 U/ml, this shedding of the sialylated Le" antigen was

not observed, and the antigen was confined to the cytoplasm ofcancer cells.

Correlation between Immunoperoxidase Staining and SerumLevel of Sialylated-Le" Detected by Monoclonal Antibody

CSLEX1. Blood samples were collected from 16 cases to testthe serum level of sialylated Le", and the results were compared

with the immunoperoxidase staining of the corresponding cancer tissues (Fig. 6). In six cases, sialylated Le" was not expressed

in cancer sections and in 12 cases the staining was positive withCSLEX1 antibody. As shown in Fig. 6, two cases of negativeimmunoperoxidase staining had high serum titer of sialylatedLe" antigen. In the cases which showed positive immunoper

oxidase staining, one had a serum titer of 1:16 and 3 had titersof 1:8. No significant differences in the serum CSLEX1 titerswere observed between immunohistochemistry-positive patientsand -negative patients.

DISCUSSION

Glycolipids and glycoproteins with blood group specificitieshave been regarded as differentiation antigens, defining partic-

747




Table 2 Immunoreactivity of antibodies detecting blood group antigens in adult gastric foveolar epithelium, deep gland cells, and in gastric carcinoma

Bloodtype

Case ABOno.(RhD)1

O(+)2

O(+)3

A(+)4

O(+)5

A(+)6

A(+)7

A(+)8

B(+)9

A(+)10

O(+)1

1 AB(+)12

B(+)13

O(+)14

O(+)15

O(+)16

A (+)Blood

group-relatedantigens'Secretor

status"SSSSssssssssssssLocus*FEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCLe10O0ooeooâ€¢ooâ€¢oo0ooo0oâ€¢oo0ooâ€¢ooâ€¢oooooâ€¢0oo9o0â€¢oo0o0Le"â€¢OOâ€¢o99o90O00O00o00eâ€¢0o00o00oâ€¢â€¢oâ€¢0oâ€¢00oâ€¢o00o00o0Le"O0ooo0ooeoâ€¢0o00o00o00o00o00000o0ooooo0oo00o00o0oLe"e0oo00o00000o00e00O00000o00o00000000o0oo00000o0â€¢Sialyl-atedLe"OOoooooo9oo0oo0ooooo0oo0oo0oo0ooooo0ooooo0ooooooSialyl-atedLe"e0oo09O00O0eoeooooooo990O90000Oooo00e0oo00o900oeBloodtypeCase ABOno.(RhD)17

A(+)18

O(+)19

B(+)20

AB(+)21

O(+)22

A(+)23

A(+)24

A(+)25

B(+)26

O(+)27

O(+)28

O(+)29

B(+)30

AB(+)31

A(+)Blood

group-relatedantigens'Secretor

status"SSSSSSNSNSNSNSNSNSNSNSNSLocus*FEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCFEDGGCLe"0O00O00oo0o00o00o00o00o00o09Ooooooo00o00oo0o0Leb0O00oe0oo0O00oo0o0ooooo0oo0oo000oooo9o9ooo9O0Le"O00O00o00oooo00o00o00o0o900o90O0Oo0o009O00o00Le>900O00Oe0o0000o900Oeo0o0990Oo0o0ooeo09Oo0ooo0Sialyl-atedLe10O0Oo9Ooo0o09Ooooo9O00ooo0oe90o0oooo0oo9oooo0Sialyl-atedLe"Oe0900O0Ooe0ooooeoo00oo0eee0e9o0oo0oo00oo00oo

Â°S, secretor; NS, nonsecretor.* FE, antigen positive in foveolar epithelium; DG, antigen positive in deep gastric glands; GC, antigen positive in gastric cancer.' Immunoreactivities: â€¢¿�,homogenous staining; 0, heterogenous staining; O, unreactive.

ular cell or tissue types representing developmental pathwaysand specific functions. These structures are formed by thesequential addition of different saccharides to the side chainsof both lipids and proteins (3, 15-17). It has been suggestedthat development and differentiation could be mediated bycontinuous changes of these cell surface glycoconjugatesthrough which cell-cell interactions may take place (18). Studies

in the murine embryo (19, 20) suggested that these structuresmight be the signals for cell recognition.

Interest in the blood group antigen expression in normaltissues and tumors has increased a great deal in recent yearsbecause of several observations demonstrating changes in theseantigenic systems following tumorigenesis (21-23). In the gastrointestinal system, expression of the antigens in colorectalcancers have been extensively discussed in the literature. Anapparent deletion of A and B antigens (6, 24); incompatibleexpression of A, B, and H antigens (25, 26); and expression ofnovel blood group-related antigens (9, 27, 28) were reported.Also, several studies on Lewis system antigens have been carriedout. Increased expression of Ley (4, 29) and incompatible

expression of Leb (4, 31) in nonsecretor individuals were ob

served in colorectal cancers.Concerning stomach tissues and tumors, the pioneering work

of Ernst et al. (31) demonstrated the presence of Lea antigen innonsecretors and Leb in secretors using an immunoperoxidasemethod on paraffin-embedded normal stomach tissues. Theyalso observed two Lea-positive cases in secretors and one Leb-positive case in a nonsecretor. In gastric cancers, Le" antigen

expression was more frequent and 80% of cancers were positivewith Le". These findings are also supported by the study of

Hirohashi et al. (32) using gastric cancer specimens. In anotherreport, Ernst et al. (6) demonstrated the deletion of A and Bantigens in gastric carcinomas. Mollicone et al. (33) demonstrated specific antigen localization in pyloric and duodenalmucosa using polyclonal and monoclonal antibodies againstLewis blood-group-related antigens. These reports (31, 33, 34)were not in complete agreement on the localization of Lewisantigens in normal gastric mucosa.

Our present results are basically consistent with these ofMollicone et al. (33) in terms of antigen expression in normal

748




Fig. 3. Localization of Lewis blood group-related antigens in normal adult stomach of asecretor individual detected by immunoperox-idase method. A, Le* antigen was not detected

in gastric mucosa! cells from this secretor individual. B, Leb antigen was present in the

foveolar epithelium but not detected in thedeep gastric glands. C, Lex antigen was de

tected only in the deep gastric glands and notpresent in the foveolar epithelium. D, distribution of Le" antigen was very similar to thelocalization of Le" antigen. Magnification: x

40.

C D

gastric epithelium. Le" was present in the surface foveolar

epithelium of nonsecretors but was also detected in 50% of thesecretors, which was not noticed in their study. Leb was positive

in secretors and mostly negative in nonsecretors. Both type 1antigens were almost exclusively detected in the foveolar epithelium of intact gastric mucosa. In epithelial tissues adjacentto cancer or in the concomittant inflammatory lesions, theexpression of type 1 antigens often extended deep into thegastric gland tissues and the distinct pattern of type 1 and type2 antigens in normal gastric mucosa disappeared. In contrast,Le" and Ley antigens were detected in deep gastric glands bothin secretors and in nonsecretors, although the expression of Ley

in nonsecretors was either weak or heterogenous. The patternof type 2 antigen distribution was also changed in pathologicalconditions, where the extension of antigen expression to foveolar epithelium or the loss of antigens was observed. Ourfindings on antigen expression in noncancerous pathologicalconditions might explain the dominant expression of Leb anti

gens in deep glands (1, 31). Another possible cause of thedivergent results observed by different investigators could becross-reactions of some of the reagents with type 2 structures.In fact, cross-reactivity of caprin anti-Leb and monoclonal anti-Leb antibodies with a Ley-like tetrasaccharide has been reported

(35, 36).The expressions of sialylated Le" and sialylated Le" antigens

in normal mucosa were weaker and heterogenous in most ofthe specimens. Sialylated Le" was negative in most of the

secretors but weakly positive in nonsecretors. In the report ofBara et al. (37), the antigen was positive in only 7% of thepatients. In our results, the antigen was often present in normal

gastric mucosa of the nonsecretors. Sialylated-Lex was claimed

to be absent in normal stomach (13), however we found theantigen in nearly 50% of the cases tested.

To define the immunoanatomic distribution of blood groupantigens in an organ, in our opinion, at least 40 to 50 specimensfrom different individuals should be examined to obtain anappropriate perspective. Such a large number of specimensmight be needed to eliminate biases caused by allospecificitiesof the individuals.

The conclusions of our study are summarized schematicallyin Fig. 7. We have noticed that secretor individuals could besubdivided into two categories based on their expression of Le"antigen. In one group, Le" was expressed in foveolar epithelium,as was Leb; in the other group, Le" was not present in any part

of the normal gastric mucosa. A possible explanation for thesedifferent patterns may reside in whether an individual is heterozygous or homozygous for the dimorphic genes Se and se (38,39). Cordon-Cardo et al. (22) found a similar phenomenon withLewis antigen expression in urothelium and they also claimedthat the difference of genotype in the secretor gene may be thecause of different antigen expression among the secretor individuals.

Alterations in blood group expression in malignancy havebeen classified (40) into five groups: (a) simplification of maturecarbohydrate structures; (b) enhanced expression of normalblood group antigens; (c) oncofetal or inappropriate tissueexpression; (


1000 n

a- 100

10

â€¢¿�â€¢¿�

U n re a c t ivc with

Immunoperox idase

Staining

Positive Staining

with

Immunoperox i dase

Fig. 4. Serum level of sialylated Le" (CA 19.9) antigen of patients with gastric

carcinoma compared with tumor tissue distribution. 16 patients were sialylatedLe* negative in cancer tissues by immunoperoxidase method (left). Serum titersof sialylated Le* antigen in these patients were all negative. 12 patients weresialylated Le" antigen positive in tissue immunostaining (right). Serum titers ofsialylated Le" antigen among these patients were positive Â¡nfive cases (>37 U/

ml).

Fig. 5. Sialylated Le* antigen distribution in gastric cancer tissue of a case

with positive antigen in serum. The antigen was detected in the cytoplasm ofmalignant cells and was also diffusely distributed in the surrounding stroma.Magnification: x 100.

Decrease of Leb and appearance of Le" may be the simplification

of mature carbohydrate structure, and enhanced expression ofsialylated Le" may be categorized as enhanced expression of

normal blood group antigen.The serum level of the sialylated Le" and sialylated Le"

antigens were evaluated in our study to determine whether thereis any significant correlation between tissue expression and theserum titer of the antigens. Serum level of sialylated Le wasextensively studied in colorectal cancers (8) and tested in 11specimens in gastric cancers (41) by Koprowski et al.. In theirreport, 72% of the patients with gastric cancers expressedsialylated Le" in their serum whereas all the patients expressed

Â«1:16ad

a:â€¢¿�JMU

1:4 -

Negat ive

Unreactive withImmunoperoxidaseStaining

Positive StainingwithTmmunoperoxidase

Fig. 6. Serum level of sialylated Le* antigen of gastric cancer patients compared with tumor tissue expression. Right, tumor antigen-positive cases (N â€”¿�10);left, negative cases (N = 6). No significant difference in the presence of serumantigen between the two groups was observed.

the antigen in tissues. In our present study, we detected theantigens in the serum of the five out of 12 patients whose tumorwas antigen positive by immunoperoxidase staining. In fiveother patients who were positive in tissues but negative inserum, the staining pattern of the antigen was restricted to thecell cytoplasm, whereas in both positive cases, shedding of thesialylated Le" antigen into stromal tissues was observed. Cor

relation between this pattern of tissue expression and serumlevel of the antigens has been discussed in colorectal cancers(42) and in pancreatic cancers (43). Although the serum titerof the antigen does not always correspond to the presence andprogression of gastric cancers in tissues, testing of sialylatedLea antigen or maybe Le" antigen itself, might to a certain

extent, be exploitable in the diagnosis of advanced or recurrentgastric cancers.

Another aspect of blood-group-related antigens in gastriccancers is the change in secretor gene-control expression. Insome nonsecretors, anomalous Leb expression was observed incancers. In colorectal cancers, anomalous Leb and Ley antigen

expression has been reported (7, 44). This observation is ofmuch interest in terms of ABO antigen expression because therelated H antigen could be a precursor of A or B structures. In

750




SECRETORSTATUS

SECRETOR NONSECRETOR

ANTIGENS, a b x y SIALYL SIALYLLe Le Le Le , a x"* â€”¿�Le-Lef

v a b x y SIALYL SIALYLLe Le Le Le' _Lea _L(;x

M TJ-O OI-H <

H MaeoM rr->i-iPO

d/CÃ–

o oc->> WZ Ho soW h-i

o ln o> >SO WO H-i POZ h-1o n

Fig. 7. Schematic representation of the immunoanatomic dissection of the blood group-related antigens within the human adult gastric epithelium and in gastriccancer and their pattern of expression in secretor versus nonsecretor individuals. Lines and bars, positive immunoreactivity with each monoclonal antibody; bar widths,relative intensity of an immunostaining pattern.

many cases of gastric cancers, expression of Lea and the deletionof Leb was observed. This means that the H antigen present in

normal gastric mucosa was lost in cancers and that might leadto the deletion of A and B antigens reported by several investigators (6, 24). From our present data, it seems possible thatenhanced expression of Lea antigen, accompanied by the changeof sialylated Le" could be indicators of malignant transforma

tion of gastric tissues. A panel of these reagents could be usefulin the immunopathological analysis of gastric cancers, particularly when the secretor status of the patient is available.

Although attempts have made to produce monoclonal antibodies against gastric cancer-related antigens (45, 46) or togastric mucosa! cells (47), this has been difficult because bloodgroup-related antigens seem to be major components of gastrointestinal tissues. However, with proper selection of antigen,together with the determination of secretor status, those anti-blood group monoclonal antibodies might be exploitable in theclinical and immunopathological diagnosis of gastrointestinalcancers.

REFERENCES

1. Szulman, A. E., and Marcus, D. M. The histologie distribution of the bloodgroup substances in man as disclosed by immunofluorescence. VI: The Le"and Leb antigens during fetal development. Lab. Invest.. 28: 565-574, 1973.

2. Marcus, D. M., and Cass, L. E. Glycosphingolipids with Lewis blood groupactivity. Uptake by human erythrocytes. Science (Wash. DC), 164: 553-555,1969.

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1989;49:745-752. Cancer Res Junichi Sakamoto, Tadashi Watanabe, Takahiko Tokumaru, et al. Carcinoma and in Normal Gastric Tissue

Blood Group Antigens in Human Gastricxand Sialyl-Lewis,a, Sialyl-Lewisy, Lewisx, Lewisb, LewisaExpression of Lewis

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