Gut 1997; 41: 37-42

Blood groups Lewisb and ABH expression ingastric mucosa: lack of inter-relation withHelicobacter pylori colonisation and occurrence ofgastric MALT lymphoma

G Oberhuber, A Kranz, C Dejaco, B Dragosics, I Mosberger, W Mayr, T Radaszkiewicz

Department of ClinicalPathologyG OberhuberI MosbergerT Radaszkiewicz

Department ofInternal Medicine IVA KranzC DejacoB Dragosics

Department ofBloodGroup Serology,University ofVienna,Medical School,W.ihringer Gurtel18-20, A-1090 Vienna,AustriaW MayrCorrespondence to:Dr Georg Oberhuber,Department of ClinicalPathology,University of Vienna,Medical School,W5hringer Gurtel 18-20,A-1090 Vienna, Austria.

Accepted for publication23 January 1997

AbstractBackground-Blood group Lewisb anti-gens mediate Helicobacter pyloni attach-ment to gastric mucosa with attachmentbeing particularly strong in subjects withABH blood group 0.Aims-To determine whether H pylonicolonisation or the occurrence of gastricmucosa associated lymphoid tissue(MALT) lymphomas might be related togastric Lewisb expression or occurrence ofparticular ABH blood groups on gastricmucosa.Patients-Gastric resection specimensfrom 89 cases with gastric MALT lym-phoma and gastric mucosal biopsy speci-mens from 95 patients undergoing upperendoscopy due to upper gastrointestinalcomplaints, including five cases withgastric MALT lymphoma, were studied.Methods-Hpylon was visualised with theWarthin-Starry stain. Immunostaining(Lewisb, Lewisa, A, B) was performed byapplying a three step immunoperoxidasetechnique and indirect immunofluores-cence staining on formalin fixed andparaffin wax embedded tissue. In 40patients red blood cell Lewis phenotypeand ABH blood groups were additionallydetermined by haemagglutination assay.Results-Gastric surface epithelial celisshowed an immunoreactivity to bloodgroups A, B, and AB in 80 (43-5%), 22(12%)/, and 11 (6%) cases respectively andno immunoreactivity to any of these bloodgroup substances (blood group 0) in 71(38.5%) patients. Lewisb expression of allgastric surface epithelial cells (secretorstatus) was found in 130 (707%) cases.Lewisa expression of all gastric surfaceepithelial cells (non-secretor status) wasfound in 36 (19-6%) cases, secretor statusremained unclassified in 18 (9.8%)patients. Colonisation with H pylon wasfound in 134 (72/8%) cases. The occurrenceof H pylon was neither significantlyassociated with secretor status nor withcertainABH blood groups. The infiltrationof gastric mucosa with MALT lymphomawas highly significantly associated with Hpyloni colonisation (p

Oberhuber, Kranz, Dejaco, Dragosics, Mosberger, Mayr, Radaszkiewicz

(table 1). Gastric mucosal biopsy specimensobtained through upper endoscopy wereavailable from 95 cases (including five caseswith MALT lymphoma) and gastric resectionspecimens from 89 cases with MALTlymphomas.

Tissue fixed in 8% phosphate bufferedformalin, pH 7 4, was processed for routinelight microscopy. All cases were stained withhaematoxylin and eosin. Hpylori was visualisedwith the Warthin-Starry stain. MALTlymphomas were additionally stained withGiemsa, Gomori's reticulin, and periodic acidSchiff stains.

Biopsy specimens of 90 consecutivelysubmitted non-lymphomatous cases wereobtained from patients subjected to upperendoscopy due to upper gastrointestinalcomplaints in the gastroenterological out-patient clinic of Internal Medicine IV, ViennaUniversity Hospital, between January andApril 1995. A mean of 2-3 biopsy specimenswas submitted for routine histology. Cases withgastric carcinoma and AIDS were excludedfrom this study.The 94 cases of primary B cell lymphomas

of the stomach, which were retrieved from ourfiles, were classified according to the REALclassification3 and the classification of Isaacsonet al,4 which, in general, are based on theupdated Kiel classification.5 Use of theWorking Formulation6 turned out to beinappropriate. In all cases, the T and B cellnature of the infiltrations was tested with apanel of antibodies applicable on paraffin waxsections as described previously.6 Primarygastrointestinal tract lymphoma was definedaccording to the criteria of Dawson et a17 aspredominant confinement to the alimentarytract excluding generalisation within threemonths after diagnosis. For staging, anadaption of the Ann Arbor system forextranodal lymphomas8 and its modification byMusshoff9 were used. Survival times of caseswith MALT lymphomas were determined in apreviously performed study.6 Details on theevaluation of data have been published.6

IMMUNOHISTOCHEMISTRY

Immunostaining was performed by applying asensitive three step immunoperoxidasetechniquel' and indirect immunofluorescencestaining on formalin fixed and paraffin waxembedded tissue sections. From 10 casesfrozen sections were available as well and wereused both untreated and treated with 100%ethanol for 30 minutes.For visualisation of Leb and Le', sections

were incubated at 37°C for five minutes in0 05% protease, type XXIV (Sigma, St Louis,MO, USA) in phosphate buffered saline (PBS)

TABLE 1 Study population

Male FemaleAge (SD) n (%.) n (%.)

Overall 52-7 (20 8) 88 (47 8) 96 (52-2)Gastric tissue, non-lymphomatous 47-6 (17-9) 44 (48-9) 46 (51-1)MALT lymphoma 62-8 (13) 44 (46 8) 50 (53 2)

before the application of the primary mono-clonal antibody. Controls included omittingthe primary antibody and the use of irrelevantisotype matched primary antibodies. All im-munostained slides were graded systematicallyand blindly.

MONOCLONAL ANTIBODIESFor immunohistochemistry murine mono-clonal antibodies with Leb antigen specificitywere obtained from Signet Laboratories(Dedham, MA, USA), DiaMed (Cressier SurMarat, SUI), Ortho Diagnostic Systems(Raritan, NJ, USA), and Biotest Diagnos-tics (Dreieich, FRG). A monoclonal antibodywith Lea antigen specificity was obtainedfrom Signet Laboratories (Dedham, MA,USA).For the immunohistochemical evaluation of

blood groups A and B monoclonal antibodieswere obtained from Zymed (Zymed Labora-tories Inc, SF, CA, USA).For haemagglutination assays monoclonal

antibodies with Leb and Lea specificity wereobtained from DiaMed (Cressier Sur Marat,SUI), Ortho Diagnostic Systems (Raritan, NJ,USA), and Biotest (Dreieich, FRG). Anti-bodies with A, B, or AB specificity wereobtained from the same sources.

HAEMAGGLUTINATION ASSAYLewis phenotype and ABO blood groups weredetermined in 40 consecutive cases whounderwent upper endoscopy by a standardhaemagglutination assay performed with a3% suspension of peripheral blood. Thisgroup included 10 cases from whom freshfrozen gastric mucosal biopsy samples werealso available. Before blood donation, subjectsgave written informed consent to participate inthis study.

BLOCKING STUDIES

To prove further the specificity of Leb and Lealabelling, blocking assays were performed witha panel of highly purified oligosaccharides(lacto-N-difucohexaose I (Leb), lacto-N-fucopentaose I (H substance), lacto-N-fuco-pentaose II (Lea), Oxford Glyco Systems,Abingdon, UK). Two protocols were applied:(1) the primary antibodies were absorbed witholigosaccharides for two hours at roomtemperature before applying them in immuno-histochemistry; (2) prebound primary mono-clonal antibodies were dissociated from theirbinding sites by incubating sections with oligo-saccharides for two hours at room temperature.Each experiment was performed in four caseseach with diffuse and focal Leb and Leastaining. Concentrations of 0 05 up to 2 mg/mloligosaccharides were used for blocking. Ina second set of experiments dissociationof prebound Leb antibodies was assayed infour cases with focal Leb staining at aconcentration of 1 mg/ml with an additionalpanel of oligosaccharides (3-fucosyllactose,lacto-N-neotetraose, lacto-N-fucopentaose II,

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Blood groups Lewisb andABH expression in gastric mucosa

lacto-N-fucopentaose III, 3'sialyl-N-acetyllactosamine, 6'sialyl-N-acetyl lactosamine,3'-sialyl-3-fucosyl lactose, Lewisx, 3-sialylLewisX, 3-sialyl Lea (Glycoset III, OxfordGlyco Systems, Abingdon, UK)) for two hoursat room temperature.

STATISTICSFor statistical evaluation the x2 test was used.For analyses stratified by lymphoma status theMantel-Haenzsel test was used. In cases withMALT lymphoma product limit survivalestimates for censored survival times werecalculated, and a comparison of survival curvesaccording to the log rank test was performed."Survival times were calculated from alldeaths.

Results

IMMUNOSTAINING OF GASTRIC TISSUESecretor status was found in 130 (70 7%) casesand was disclosed by labelling of all gastric

Figure 1: Labelling ofgastric surface epithelial cells by the Leb monoclonal antibody (brownstaining). Gastric glands remain unstained. Immunoperoxidase, original magnificationX200.

c;

-i.E Z- 1|l

e. I

I

P::r

Fb...;A.."' .-I0' -A

Figure 2: Labelling ofgastric surface epithelial cells by the Le monocloncstaining). Immunoperoxidase, original magnification X 200.

surface epithelial cells by the Leb monoclonalantibodies (fig 1). Patients showing decorationof all gastric surface epithelial cells by the Leamonoclonal antibody (fig 2) were classified asnon-secretors (36 (19-6%) cases). In this groupfocal Leb staining was also found in all but onecase. Finally, patients showing focal Leblabelling and no or only focal Lea stainingremained unclassified with respect to secretorstatus (18 cases (9-8%)).

Immunoreactivity to blood groups A B, andA and B was found in 80 (43.5%), 22 (12%),and 11 (6%) cases, respectively. No stainingwith any of these two antibodies was seen in71 (38-5%) patients. Blood group A or Bimmunoreactivity was detected on surfaceepithelial cells and on endothelial cells with theexception of three cases in which blood groupB antigen was only found on endothelial cellsbut not on gastric surface cells. In one of thesecases Lewis phenotype was determined by anhaemagglutination assay and disclosed a Le(a+b-) phenotype.Tumour cells of MALT lymphomas were

negative for blood groups A, B, Lea and Leb.Gastric epithelial cells in close proximity anddistant to areas of lymphomatous infiltrationshowed a similar staining pattern for Leb, Lea,and ABH blood groups.

RED BLOOD CELL PHENOTYPE AND LEbIMMUNOREACTIVITY

Subjects with Le (a-b+) RBC phenotype(secretors) showed Leb staining of all gastricsurface epithelial cells in all 21 cases. InLe (a+b-) subjects, considered as non-secretors, Lea staining of all gastric epithel-ial cells was found in all nine cases. In theLe (a-b-) cases Leb or Lea staining of allgastric epithelial cells was found in four andtwo of 10 cases, respectively. In the remaindereither focal or no staining of Leb or Lea wasfound.

Alcohol fixation of frozen sections orexamination of untreated frozen sections didnot alter the result obtained in paraffin waxembedded material as shown in 10 cases.ABH blood groups determined by im-

munohistochemistry were identical to thosedetermined by haemagglutination.

geps BLOCKING STUDIES

Reactivity of the Leb and Lea monoclonala-.;< antibodies was blocked with lacto-N-

difucohexaose (Leb) or lacto-N-fucopentaoseII (Lea), respectively. Absorption of the

t;- " primary antibodies with I mg/ml of therespective oligosaccharides completely inhib-ited staining in all cases. Additionally,incubation of the slides with 1 mg/ml lacto-N-difucohexaose (Leb) or lacto-N-fucopen-taose II (Lea) resulted in dissociation ofprebound Leb and Lea antibodies.

Focal Leb immunoreactivity found in non-secretors could be blocked neither by 2 mg/mllacto-N-fucopentaose II (Lea) nor by 2 mg/ml

al antibody (brown lacto-N-fucopentaose I (H substance) nor bythe panel of oligosaccharides described in

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Oberhuber, Kranz, Dejaco, Dragosics, Mosberger, Mayr, Radaszkiewicz

Methods (Glycoset III, Oxford Glyco Systems,Abingdon, UK).

COLONISATION WITH H PYLORI

Colonisation with H pylori was found in 134(7288%) patients, including 55 (61-1%) ofMALT lymphoma negative and 79 (84%) ofMALT lymphoma positive cases. In two casesH pylori status remained unclear. Theoccurrence of MALT lymphomas was highlysignificantly associated with H pylori col-onisation (p

Blood groups Lewisb andABH expression in gastric mucosa

particularly interesting as those with bloodgroup O'1-17 and non-secretors12 151718 werefound to show increased susceptibility togastrointestinal diseases, such as gastric orduodenal ulcer. The occurrence of gastric andduodenal ulcer, however, is itself highlysignificantly associated with H pyloniinfection.' The increased susceptiblity togastric and duodenal ulcer in patients withblood group 0 and in non-secretors cannot beexplained with increased susceptibility to Hpylori infection. Mechanisms causing thisinter-relation have therefore still to bedetermined.

Expression of Leb and Lea antigens in gastricepithelia as related to secretor or non-secretorstatus and Lewis status of red blood cells hasnot been extensively studied. In this series focalstaining with Leb monoclonal antibodies wasfound in almost all non-secretors. According tothe literature immunhistochemical visualisa-tion of Lewis substances on tissues of variousorigins resulted in varied findings. Somegroups showed that gastric mucosa of non-secretors does not express Leb,ls 20 othersfound aberrant expression of Leb ongastric,2-23 small intestinal,24 and urothelial25epithelium in non-secretors. Concerning theassociation of secretor status with H pyloricolonisation focal expression of Leb in non-secretors could be of clinical relevance, as itmight suffice to allow attachment of thebacteria to gastric mucosa. We thereforeverified the correctness of our findings by anumber of controls including blocking studieswith oligosaccharides. Binding of themonoclonal Leb antibodies seemed to bespecific in all cases. However, owing to thedifference in results obtained by various groupsadditional studies are needed to clarify furtherthe association of secretor status and Lebexpression in gastric mucosa.The hypothesis that the availability of H

pyloni receptors might influence the de-velopment of MALT lymphomas was alsotested in this study. The origin of MALTlymphomas has been closely linked toinfestation with Hpylori. Because the stomachnormally contains no lymphoid tissue, theonset of B cell lymphoma of MALT type is tobe preceded by the acquisition of lymphoidtissue resembling intestinal Peyer's patchesinduced by H pyloni colonisation.2627 Histo-logical studies showed that the organism waspresent in gastric mucosa in almost all peoplewith gastric lymphoma.25 A nested case-controlstudy involving two large cohorts showed thatnon-Hodgkin's lymphoma affecting thestomach is associated with previous H pyloniinfection.29 Finally, eradication ofH pylori mayresult in complete remission in somepatients.3>32 In our study H pylori was alsosignificantly over-represented in patients withMALT lymphomas. According to our studyhypothesis attachment of these bacteria togastric epithelium via Leb might elicit a morevigorous immune response and thus be onefactor supporting the development of MALTlymphomas. However, no inter-relation be-tween the infiltration with gastric MALT

lymphomas, their grade, stage, and prognosisand secretor status or specific ABH bloodgroups expressed on gastric epithelia wasdetected. We therefore propose that secretorstatus does not contribute to the formation ofMALT lymphomas. A possible explanationmight be the availability of Leb receptors innon-secretors or of other factors mediatingH pylori attachment' such as phospha-tidylethanolamine and GM3 ganglioside insubjects developing MALT lymphomas.Alternatively, strong adhesion of bacteria togastric surface epithelial cells may not be aprerequisite for the formation of MALTlymphomas.We conclude that secretor status and specific

ABH blood groups were neither inter-relatedwith H pylori status nor with occurrence ofMALT lymphomas.

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