(CANCER RESEARCH 49. 212-218. January I. 1989)

Blood Group-related Antigens in Human Kidney: Modulation of LewisDeterminants in Renal Cell Carcinoma1

Carlos Cordon-Cardo, Victor E. Reuter, Connie L. Finstad, Joel Sheinfeld, Kenneth O. Lloyd, William R. Fair, andMyron R. MelamedMemorial Sloan-Kellering Cancer Center, New York, New York 10021

ABSTRACT

Seven mouse monoclonal antibodies and the lectin t lex europaeus,detecting blood group related antigens of the ABH and Lewis systems,have been used to determine the immunophenotype of human renal cellcarcinomas. Immunohistochemical analyses have demonstrated thatthese antigenic systems are differentially expressed by distinct normalcell types and domains of the human nephron. In the present study weanalyzed the immunophenotype of 29 primary and 15 metastatic renalcarcinomas by the immunoperoxidase method. Blood type was known inall of the cases and secretor status in nine cases. ABH specificities werenot detected in tumor cells of the primary tumors studied, although twoof the métastasesshowed heterogeneous expression of H and A antigens,respectively. Lewis" (Le") determinant was detected in 76% of primaryrenal cell carcinomas; however, Le" was only expressed by occasionalcells in 20% of the metastatic tumors analyzed. Lewis*(Le*) was detected

with a heterogeneous pattern of expression in 31% of the primary and26% of the metastatic renal tumors studied. Lewis1(Ley) antigen expres

sion was found in 17% of the primary and 20% of the metastatic tumorsanalyzed. Detection of precursor type 1 structure was observed in 28%of primary and 20% of metastatic renal cell carcinomas.

The present study suggests the histogenesis of renal cell carcinoma inthe proximal nephron, based on the expression of Le" and Le* antigens.

It also shows: (a) an apparent deletion, downregulation or structuralmodification of Le" determinant in most of the metastatic tumors; (b)

undetectable levels of ABH specificities in tumor cells of primary renalcell carcinoma; and (c) enhanced expression and/or neosynthesis ofprecursor type 1 structure and Ley determinant in some renal cell carci

nomas.

INTRODUCTION

Antigenic determinants of the A, B, H, and Lewis bloodgroup-related molecules are carbohydrate structures carried onboth lipids and proteins (1-3). Initially found on erythrocytes(4-6), they have also been described on certain epithelial cellsand in body fluids, and have been mapped in normal fetal andadult tissues (7-10). Using antibodies detecting blood groupspecificities, distinct immunophenotypes were assigned to normal cell types and domains within the nephron (11-16). Secretor status has also been shown to influence the expression ofthese blood group related antigens in tissues (3, 17). Interest inthese antigens as differentiation markers has increased in recentyears because of modifications observed during transformation,including simplification of mature structures, accumulation ofprecursor molecules, deletion of A/B/H antigens, abnormal A/B specificities, and enhanced or modified expression of Lewisdeterminants in certain tumors (18-25). We now report on theexpression of these antigens in a series of 29 primary and 15metastatic renal cortical carcinomas using a well characterizedpanel of antibodies and the lectin Ulex europaeus detectingprecursor type 1 chain, H, A, B, Le", Le", Le*, and Ley blood

group related antigens.

Received 6/8/88; revised 9/6/88; accepted 10/5'88.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'Supported in part by NCI Grants CA-47538, CA-14134, CA-41021, CA-

08478, and CA33049.

MATERIALS AND METHODS

Tissues

Normal and neoplastic tissues were obtained from surgical specimenswithin 1-2 h of resection. Fresh tissues were fixed overnight in 10%formaldehyde in phosphate buffered saline, pH 7.5 (PBS,2 Difco Lab

oratories, Detroit, MI), and embedded in paraffin. Additional tissuefrom some cases was snap-frozen in isopentane precooled in liquidnitrogen, embedded in OCT compound (Miles Laboratories Inc., Na-perville, IL), and stored at —70°Cuntil needed.

Radical nephrectomy specimens from 29 patients with primary renalcortical carcinoma, and metastatic renal carcinomas from 15 patientswere obtained for the present study (39 different individuals representing 44 specimens). In the majority of primary tumors we were able toexamine adjacent normal kidney. Primary and metastatic tumors fromthe same individual were available for study in four cases. In sevencases we were able to compare immunoreactivities on frozen andparaffin-embedded tissue sections of the same tumor. Selection of caseswas based on excellence of tissue preservation. All tumors were classified according to histologie pattern(s) and cell type(s). The patientsincluded 13 group O, 18 group A, seven group B, and one group ABindividuals. Secretor status was determined in nine cases by eitherpresence of Lewis antigens in saliva and/or on red blood cells (10, 24).The blood group and secretor status of the patients from whom thespecimens were derived were correlated with the immunohistologicpatterns of reactivity.

Reagents

Purified agglutinin I from Ulex europaeus at 4 Mg/ml (Vector Laboratories, Burlingame, CA) served to identify the H-antigen. Mouse mAbHT 29-36 (T-36) recognizes A antigen (all variants), mAb S8 detectsB-antigen, mAbs T-174, T-218, P-12 and F-3 have specificities for Le*,Leb, Le", and Ley antigens, respectively. Finally, mAb K-21 detects

precursor type 1 chain antigen (Table 1). The antibodies were used asundiluted culture supernatants, or purified immunoglobulin preparation at an approximate concentration of 25-40 Mg/m' (17, 24).

Immunohistochemistry

The method chosen for the present analysis was the avidin-biotincomplex immunoperoxidase technique (17, 24, 26). Formalin-fixed andparaffin-embedded tissue sections were deparaffinized, then treated for30 min in 1% hydrogen peroxide in PBS to remove endogenousperoxidase activity. Frozen tissue sections were fixed in cold acetonefor 10 min and treated with 0.3% hydrogen peroxide for 15 min.Sections were washed in PBS and then incubated with the blockingserum for 20 min. Blocking serum was drained off and deparaffinizedsections were incubated with mAb overnight at 4°C,whereas frozen

sections were incubated for l h at room temperature. The secondaryantibodies, biotinylated horse anti-mouse IgG or goat anti-mouse IgM(Vector Laboratories, Burlingame, CA), were incubated on both deparaffinized and frozen sections for l h and then washed and incubatedwith the avidin-biotin complex for 30 min. The peroxidase reactionwas performed by incubating tissue sections for 6 to 12 min with 5 mgof diaminobenzidine tetrahydrochloride (Sigma Chemicals, St. Louis,MO) in 100 ml of tris buffer containing 100 n\ of 0.3% hydrogenperoxide. Sections were washed with distilled water, counterstainedwith hematoxylin, and mounted with permount.

2The abbreviations used are: PBS, phosphate buffered saline; mAb, monoclonal antibody; SSEA-1, stage-specific embryonic antigen-1.

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BLOOD GROUP ANTIGENS IN RENAL CARCINOMA

Table 1 Derivation and specificity of mouse monoclonal antibodies identifyingblood group-related antigens

Antibody(immunoglobulin

subclass)K2I

(IgM)T36 (IgGj)S8 (IgM)T174(IgG,)T218(IgM)PI 2 (IgM)F3 (IgM)Immunizing

celltypeTera-1

HT-29SK-RC-7SK-CO-10SK-CO-10Human placentaSK-LU-3Blood

groupspecificity"Precursor

IABLe"Le"Le"Le'CRLdésignation''BG1

BG2BG3BG5BG6BG7BG8

" The antigenic determinant recognized by each monoclonal antibody is cited

elsewhere (22, 25, 31).* Antibodies are available from Cambridge Research Laboratories (CRL, Cam

bridge, MA). Purified agglutinin I from Utex europaeus (Vector Laboratories,Burlingame, CA) served to identify the H antigen.

Immunoperoxidase Analysis Using a Lectin. The lectin Ulex europaeus was incubated on deparaffinized tissue sections for 2 h at roomtemperature. Sections were washed with PBS and incubated with goatanti-l/fex lectin antibody (1:1000 dilution) (Vector Laboratories, Burlingame, CA) overnight at 4°C.The immunoperoxidase method was

performed as described above using biotinylated rabbit anti-goat im-munoglobulins as secondary reagent.

Neuraminidase Treatment of Tissue Sections. Frozen tissue sections,previously fixed with acetone, were incubated with neuraminidase (Vibrio cholerae, Calbiochem, Behring Diagnostics, San Diego, CA) containing 0.2 t'/ml in O.OSOM sodium acetate, 0.150 M sodium chloride,0.004 M calcium chloride, pH 5.5 for l h at 37°C.Sections were then

washed with PBS and immunostained as described above.Controls. Frozen and deparaffinized sections of tissues expressing

the appropriate blood group antigen were used for titration of thereagents as well as positive and negative controls. Negative controlsincluded substitution of the mAb by another ni.\b of the same speciesand subtype, or incubation with PBS alone.

RESULTS

Table 1 summarizes the derivation of the panel of antibodies,their immunoglobulin subtype, and their specificity for bloodgroup related antigens. Tables 2 and 3 summarize the clinicalinformation and immunoreactivities of these antibodies onsections of renal tumors, correlating blood type and secretorstatus of the individuals as well as histological pattern(s) andcell type(s) of the tumors. In seven cases we were able tocompare the immunostaining patterns in formalin-fixed paraffin-embedded and frozen tissue sections of the same tumor. Innine cases secretor status was determined and correlated withtumor immunophenotype.

Blood Group Antigen Expression in Normal Adult Kidney. Aspreviously reported, antibodies detecting blood group specificities were differently expressed in the human nephron of secretor and nonsecretor individuals (17).

Glomerular tufts were immunoreactive for A/B/H antigensin endothelial cells, according to the blood group type of theindividuals, regardless of secretor status. Parietal and visceralepithelial cells and mesangial cells of glomeruli were unreactivefor all antibodies tested.

Epithelial cells of proximal convoluted tubules in secretorindividuals expressed the Le" determinant. Epithelial cells ofHenle's loop showed marked reactivity for Lex determinant,with patchy expression of precursor type 1 chain, Le" and Leyantigens in some secretors. Nonsecretors expressed only Le"

determinant, while precursor type 1 and other Lewis determinants were not detected in the proximal domain of the nephron.

Collecting ducts in secretor individuals showed a gradient ofincreasing staining of the epithelial cells towards the deepmedulla for A/B/H antigens, as well as for Leb and Ley deter

minants. However, epithelial cells within collecting ducts innonsecretors showed complete lack of immunoreactivity forthese determinants. Precursor type 1 antigen was heteroge-neously expressed on epithelial cells of collecting ducts, regardless of secretor status.

Blood Group Antigen Expression in Primary Renal Carcinoma.Precursor type 1 chain was detected in eight of 29 cases with aheterogeneous or focal staining pattern of reactivity (Fig. 2, Dand F), showing strong immunostaining in only one case.

A, B, and/or H specificities were not detected in any of theprimary tumors studied, regardless of histological pattern ofthe tumor or secretor status of the individual (Fig. \,A,D, G).Anomolous expression of A and/or B antigens was not observedin the present study. Endothelial cells and erythrocytes in thetumors were immunostained with the appropriate antibodies,regardless of secretor status.

Lewis3 was found in nine of 29 cases, with a heterogeneous

pattern of staining. These tumors were all acinotubular andpapillary. The pattern of reactivity was patchy with strongcytoplasmic staining and marked luminal positivity (Fig. l, Band E). Secretor status did not influence the results. The twosarcomatoid tumors were unreactive for Le" (Fig. \H). Expression of Leb was only found in one primary tumor, with heterogeneous immunoreactivity (20-30% positive tumor cells) (Table2, Case 15). None of the métastasesstudied showed positivereactivity for Leb determinant.

Lewis" determinant was expressed in 22 of 29 cases. 14

showed strong and homogeneous immunoreactivity (Fig. 1, Cand F); the others showed either heterogeneous staining (fourcases) (Fig. 2E) or focal staining with 5-10% tumor cells beingimmunoreactive (four cases) (Fig. 2C). Both acinotubular andpapillary tumors showed variable reactivities; however, sarcomatoid tumors v/ere unreactive for Le" antigen (Fig. 1/). Secretor status did not influence the expression of Le* specificity in

tumor cells. In some cases where patchy staining was notedclear cells showed diffuse staining (Fig. 2.4) whereas granularcells were unreactive (Fig. 2B) with PI2 antibody (Table 2,Case 6). Le1 determinant was absent in the majority of the cases

analyzed. It was detected in four of 29 cases with patchystaining; two of these cases showed only a few immunoreactivetumor cells, with punctate, cytoplasmic staining pattern.

Blood Group Antigen Expression in Metastatic Renal Carcinomas. 15 metastatic renal carcinomas were analyzed for thepresent study. In five cases we were able to compare the primaryand metastatic tumors from the same patients; one of them(Table 2, Case 15) had two métastasesat different sites (Table3, Cases 7 and 8).

Precursor type 1 chain was found in three of 15 metastatictumors, but with only occasional cells staining variably. Ingeneral, A, B, and/or H specificities were not detected (13 of15 cases). However, one of these metastatic tumors (Table 3,Case 11) showed heterogeneous expression of both A and Hantigens in approximately 10-20% of tumor cells, while anothertumor (Table 3, case 9) showed patchy staining for H specificityin approximately 50% of tumor cells (Fig. I/).

Lewis" antigen was expressed in four of 15 métastaseswith

patchy immunostaining; the remaining 11 cases were unreactive(Fig. \K). Lewisb was undetected in all metastatic tumorsstudied. Lewis" determinant was expressed in occasional cells

of three metastatic tumors. The remaining cases were unreactivewith anti-X antibody, regardless of histological pattern and celltype of the tumor (Figs. \L and 2G). Lewisy determinant was

detected in three of 15 cases. One of these showed diffuse213

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BLOOD GROUP ANTIGENS IN RENAL CARCINOMA

Table 2 Immunoreactivity of a panel of antibodies detecting blood group antigens in primary- renal carcinomas

Case1234567g91011121314151617181920212223242526272829Age4462517761676559725844645457624945507447513971586663746661SexFFFFMMMFMMMFFFMMFMMMMFMMMMMMMBloodgroupOAOOBAOAAOAOAAOBOAOAABOAAABBAASecretorHistology

statuspatternS

AcinarAcinarS

AcinarAcinarAcinarAcinarS

AcinarNSAcinarAcinarS

AcinarAcinarAcinotubularNS

AcinotubularAcinotubularAcinotubularTubularTubularPapillaryPapillaryPapillaryS

PapillaryNSPapillaryPapillaryPapillaryPapillaryPapillaryS

PapillarySarcomatoidSarcomatoidCell

type"CC9050701008050704050801006070808050701005050101020GC105030205030605040302020305050901008010080100100SC

PSooooooeO'o20

e0ooO'050

OOOO'o•o10

O'0ooo100

O1000Hoooooooooo0oo0ooooooooooooo0oAntigen

Expression*AOO0oooo0o0ooo0oooooooooooooooBo0ooo0ooo0oo0ooooooooo0ooooooLe'oooo0oeooeoooO'e0O'oo0e9oe9o0o0Le"

Le"Le'O

<O4o

i»

o»o»

oO•0o«»ooeoO9OOO'OOO'Oo

ooooo0oooooo0ooooo00oo0O'oo9o

eo'OO'Oo

oooo»oooODO000

" Percentage of each cell type: CC, clear cell; GC, granular cell; SC, Sarcomatoid cell; PS, precursor structure, type 1 chain; S, secretor; NS, nonsecretor.* Immunoreactivities: •,homogeneous staining (more than 75%); 0, heterogeneous staining (25%-75%); 3, occasional cells staining (10-25%); O', less than 5%

positive cells; O. unreactive.

Table 3 Immunoreactivity of a panel of antibodies detecting blood group antigens in metastatic renal carcinomas

Case123456789101112131415Case

no.of

primary51415152129Age506161615457626245212359513761SexFMMFMFMMMFMFMMMBloodgroupAABBAAOOOBAOABOAHistologypatternAcinarAcinarAcinarAcinarAcinar/SolidAcinar/SolidAcinotubularAcinotubularTubular/SolidPapillaryPapillaryPapillaryPapillaryPapillary/SolidSarcomatoidCell

type"CC1001007050908050302010GC30100100501020705070808090SC

PSOoooooo030

OO'oO'20

0O'100

OHOooooo0oeo9ooooAntigen

expression*AOOOooooooo9OoooBOooooooooooo0ooLe*Oooooo9eo99OOOoLe"OOoooooooooooooLe"0oO'oooo9O0O'OoooLé>0oo0oO'ooO'o•oooo

" Percentage of each cell type: CC, clear cell; GC, granular cell; SC, Sarcomatoid cell; PS, precursor structure, type 1 chain.* Immunoreactivities: •,homogeneous staining (more than 75%); 0, heterogeneous staining (25-75%); 3, occasional cells staining (10-25%); O', less than 5%

positive cells; O, unreactive.

immunoreactivity (Fig. 2//), while the other two contained onlyoccasional tumor cells that were reactive.

In five cases we studied the primary and metastatic lesionsfrom the same patient. Four of five primary renal carcinomashad clear and/or granular cell features and expressed Le" anti

gen with strong and homogeneous immunoreactivity. However,three métastaseswere unreactive and two showed staining inonly a few tumor cells (less than 5%) with mAb PI2. The fifthcase had spindle cell features and showed no immunostainingin both primary and metastatic tumor cells.

Lewis" Antigen Expression in Normal Kidney and Renal Carcinomas after Neuraminidase Treatment. Expression of Le" an

tigen on normal kidney specimens showed a heterogeneousimmunoreactivity with variable staining intensity on the proximal tubules, as well as on the descending and thin loop ofHenle. After neuraminidase treatment, enhanced immunoreactivity and a more uniform, homogeneous staining pattern wasobserved on the proximal tubules. Lewis" immunoreactivity

detected along the thin loop was not altered after enzymedigestion; distal tubules and collecting ducts remained negative.

Neuraminidase treatment of primary and metastatic lesions,either unreactive or heterogeneously expressing Le" determi

nant, revealed no qualitative changes in immunostaining pattern; however, increased staining intensity in heterogeneouslyLe"-positive areas was observed following enzyme digestion.These results cannot distinguish between sialylation of the Le"antigen or masking of the Le" determinant by other sialylat cd

carbohydrate structures in the heterogeneous areas. Unreactivelesions remained negative after neuraminidase digestion.

Comparative Analysis of Formalin-fixed, Paraffin-embedded,and Frozen Tissue Sections. Determinants of the A, B, H, andLewis blood group related antigens are carbohydrate structurescarried on both glycoproteins and glycolipids. Since these determinants may reside on lipid moieties, processing of tissuesfor paraffin sections could alter the antigenic determinants by:solubilization through ethanol immersion, masking due to for-

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BLOOD GROUP ANTIGENS IN RENAL CARCINOMA

-.- t "r", -••>"•- A

B

TI "'1

. ' V

f.*»- •"• • •: Vv ^ "" •; ' *•̂

•^v."**•••'•'"̂ i^^WJLiL i'~'*' ••? >- «.fe^1''-'^*'•"'"' '^..J'":-•. ' •••'^-:\ *' ''•»;ts.. ,-.x^T ••••'•.w~" •'•'V *•-t:"-*^,,

» «¿r ' \V . >/ X' J-\~9 .' ' ' • *••T1J¿;' ""'i'

H

r ::-'

K

,

\hFig. 1. Localization of blood group related H, Lewis", and Lewis* antigens in primary renal cortical carcinomas (acinar, A-C; papillary, D-F; sarcomatoid, G-I)

and a metastatic renal carcinoma (.'/,). f. H blood group antigen expressed in normal endothelial cells but not detected in tumor cells; B, Lewis' antigenheterogeneously expressed in tumor cells: C, Lewis1 antigen homogeneously expressed in tumor cells; I), detection of H antigen in endothelial cells and absence intumor cells; /•.'.Lewis* antigen heterogeneously expressed in tumor cells; /•'.localization of Lewis* antigen in tumor cells; G. H antigen expressed in endothelial cellsbut not detected in tumor cells of a sarcomatoid renal cortical carcinoma; //, Lewis* antigen not detected in a sarcomatoid renal cortical carcinoma; /, Lewis" antigen

not detected in a sarcomatoid renal cortical carcinoma; J, H antigen heterogeneously expressed by the majority of tumor cells in a metastatic renal carcinoma; A.Lewis* antigen not detected in a metastatic renal carcinoma; /., Lewis" antigen not detected in a melastatic renal carcinoma, (x 200)

malin fixation, or denaturation by high temperature duringparaffin embedding. Several previously reported studies pointedout problems associated with the use of paraffin-embeddedtissues (27-30). We did not observe loss of antigen expressionin formalin-fixed and paraffin-embedded tissues compared withfrozen tissues of normal kidney/urothelium (17, 25) or normalcolonie mucosa/colonie carcinoma (24, 31). We found, however, that pretreatment of deparaffinized sections with hydrogenperoxide was necessary to obtain satisfactory immunostainingreactions.

We compared frozen and deparaffinized tissue sections fromthe same tumors in seven cases (Tables 2 and 3: primary tumors,4,7, 11, 13, 15; metastatic tumors, 8, 15). Both Le"-positiveand Le"-negative tumors were examined and no differences

were observed. Since paraffin embedding and deparaffinizationinvolve use of ethanol, it is likely that the blood group-relateddeterminants that remain to be stained by this panel of antibodies are on glycoproteins (mucins). These structures are probablythe dominant forms of blood group-related antigens in normalkidney and renal cell carcinoma.

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BLOOD GROUP ANTIGENS IN RENAL CARCINOMA

B

Fig. 2. Localization of blood group relatedantigens Lewis", Lewis', and Precursor type 1

determinant in primary renal cortical carcinomas (acinar, A-D: papillary, E and F) and ametastatic renal carcinoma (G and H). A, Lewis" antigen strongly expressed in tumor cells ofclear type (Case 6, Table 2); B. Lewis" antigen

not detected in tumor cells of granular type(Case 6, Table 2); C, Lewis" antigen expressed

in occasional tumor cells (Case 7, Table 2); D,precursor type 1 determinant heterogeneouslyexpressed in tumor cells (Case 7, Table 2); E,Lewis" antigen expressed in tumor cells of a

papillary renal cortical carcinoma (Case 20,Table 2); F, precursor type 1 determinant expressed by most of the tumor cells in a papillary renal cortical carcinoma (Case 20, Table2); G, Lewis" antigen not detected in a met

astatic renal carcinoma (Case II, Table 3). H,Lewis" antigen expressed by the majority oftumor cells in a metastatic renal carcinoma(Case II, Table 3). (X 200)

> t*.

*.

•?*• -^

>•- a ''•* %J.*,' ^ •.. ^ !*" '/* â̂€¢*

* -»-v»M T .

DISCUSSION

The expression and/or modulation of blood group-relatedantigens in the human urinary tract has been extensively discussed (11-17). However, most of the studies concerned either

normal kidney development or changes in blood group antigensin neoplastic urothelium. These studies revealed deletion of A,B and/or H antigens in transitional cell carcinomas (18-20,

25). They also showed correlation of abnormal patterns ofLewis determinants with histological grade of urothelial carcinomas (21, 23).

Previous studies on the expression of A, B, and H antigensin kidney tumors produced conflicting results. Sarodsky et al.(32) analyzed the expression of A, B, and H specificities in 42

H

renal carcinomas by the red cell adherence test on tissue sections. They reported that agglutination was observed in 39 ofthe 42 cases studied, with only three tumors being negative.More recently, Ghazizadeh et al. (33) analyzed the expressionof A, B, and H specificities, as well as T-antigen and carcinoem-bryonic antigen, in 30 cases of renal carcinoma by immunohis-tochemical methods. They reported that none of the 30 tumorsstudied showed immunoreactivity for A, B and/or H. Our dataagree with that of Ghazizadeh and collaborators since none ofthe 29 primary tumors showed positive staining for either A,B, or H antigens. Differences in results may be due to theenhanced specificity of immunohistochemistry over the red celladherence test (34, 35), or to the difficulty in interpreting thesite of red cell agglutination in highly vascularized renal tumors.

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BLOOD GROUP ANTIGENS IN RENAL CARCINOMA

Expression of Le" and Leb determinants was analyzed in eight

renal carcinomas by Ernst et al. (12) using immunoperoxidasetechniques on paraffin-embedded tissue sections. They reporteddetection of Le" in six of eight cases; Leb was undetected in alltheir cases. Similarly, we have identified Lea expression in a

subset of our cases (nine of 29 primary tumors), and lack ofexpression of Leb determinant in the majority of tumors. Only

one of the primary renal cell carcinomas showed heterogeneousimmunoreactivity for Leb determinant in our study. In thisparticular case, we were able to analyze frozen and deparaffin-ized tissue sections, observing similar patterns of staining andthe same immunophenotype in both situations.

Expression of Le" was demonstrated in 12 of 19 primary

renal carcinomas by Liebert et al. (36) using an antibody detecting SSEA-1. Anti-SSEA-1 antibody is directed against thecarbohydrate determinant as anti-Le" antibodies (lacto-A'-fu-copentaose III) (37, 38). Expression of 3-fucosylated-yV-acetyl-lactosamine carbohydrate determinants was reported in 12 of30 renal carcinomas by Fleming et al. (39) in a recent study.The two antibodies used, AGE 4.36 and AGE 4.48, detectepitopes on the Le" hapten. Our data on renal tumors showedexpression of Le" in 22 of 29 primary (76%) and in only three

of 15 metastatic (20%) renal cell carcinomas. The studies ofLiebert et al. (36) and Fleming et al. (39) did not distinguishbetween primary and metastatic tumors. We have observed asimilar degree of tumor heterogeneity in the staining pattern tothat reported for anti-SSEA-1. In addition, in our study sarco-matoid tumors were found to be unreactive for mAb P12.Variations observed among these studies could be explained bydifferences in the fine specificities of the antibodies used for theLe" structure.

Lewis" determinant was observed in normal human kidney

of both secretor and nonsecretor individuals in epithelial cellsof proximal tubules and segments of Henle's loop (17). We

observed that the majority of primary renal carcinomas expressLe" and to a lesser degree Lea determinants, regardless of

secretor status. Moreover, ABH specificities, which are knownmarkers of the distal nephron and absent in the proximaltubules, were not detected in tumor cells of renal carcinomas.These findings are consistent with suggestions that renal carcinoma arises from the proximal nephron (40-43). However,most of the metastatic tumors showed low expression or absence of Le" determinant. Consequently, deletion, down-regulation, or structural modifications of Le" determinant (e.g.,

sialylation) may be regarded as possible markers for poor prognosis in renal cell carcinoma (2). Intriguing as these observations may be, more cases with clinicopathological correlationmust be analyzed in order to draw firm conclusions.

Even though most of the antigenic markers of the distalnephron were undetected in renal carcinoma, we have identifiedprecursor type 1 chain and/or Ley determinant in a subset of

renal tumors. This is not surprising since increased expressionof blood group precursor structures have been previously reported in other types of epithelial tumors and their derived celllines, especially gastrointestinal tumors (24, 31). Since thesemolecules are the backbone structure for the synthesis of otherblood group-related antigens, accumulation of precursor maybe due to inactivation of glycosyltransferases and/or down-regulation of fucosyltransferases. Enhanced expression andneosynthesis of Ley determinant has also been reported in

studies of gastrointestinal tumors (31, 44), but it is not asignificant occurrence in kidney tumors. Modulation and neosynthesis of Lewis antigens, including Le" and Ley determi

nants, in tumor cells may be due to activation of suppressed

genes (i.e., Hh and Se) and/or upregulation of fucosyltransferases. Activation of these enzymes has been previously reportedin other epithelial tumors (44,45), and their modulation relatedto possible oncogene-coded products (2).

Differentiation in renal carcinoma may be assessed by histológica!pattern, cell type, or nuclear grading (46, 47). Immu-nophenotypic characteristics which are indicators of malignanttransformation (19, 20, 25, 41-43, 48, 49) may also reflecttumor cell behavior. In a recent study of renal tumors, forexample, Ulrich and coinvestigators correlated histológica!growth pattern, nuclear grade, and lectin-binding. They showedthat nine of 10 low grade renal carcinomas bind Lotus tetragon-olobus agglutinin, but none of eight poorly differentiated tumorsshowed binding activity for Lotus tetragonolobus agglutinin(43). Similarly, detection of SSEA-1 (Le") antigen was enhanced

in renal cell carcinomas of the clear cell type, but a sarcomatoidtumor was negative (36). In the present analysis, we haveexamined histológica! pattern and predominant cell types, andobserved the expression of Le" determinant in 76% of the

primary renal carcinomas, with a heterogeneous pattern in somecases. However, sarcomatoid tumors and 80% of the metastatictumors studied were found to be unreactive.

The variable pattern of Le"-antigen reactivity observed be

tween primary tumors of the same morphological type andwithin lesions of some individual tumor specimens could resultfrom structural modifications or masking of the Le" epitope.

For example, Fukushi et al. (50) showed that well differentiatedrenal carcinomas had a higher incidence of positive staining forpolyfucosylated and sialylated Le"-determinants than did un-

differentiated tumor cells. In the present study, primary andmetastatic lesions from the same patient were examined in fivecases. Lewis" antigen was not expressed in the metastatic lesions

from three cases. One case, an acinotubular lesion with predominantly clear cell features, showed Le" antigen reactivity in the

primary tumor and in one of two métastaseswith a heterogeneous staining pattern. The remaining case, a sarcomatoidtumor, failed to express Le" antigen in both primary and met

astatic tumors. Neuraminidase treatment of primary and metastatic lesions, either unreactive or heterogeneously expressingLe" determinant, revealed minor changes in some immunostain-

ing patterns; these were, mainly, increased staining intensityand homogeneous staining in heterogeneously Le"-positive

areas after neuraminidase digestion. However, unreactive lesions remained negative after neuraminidase treatment. Thesepreliminary results cannot distinguish between sialylation ofthe Le" antigen or masking of the Le"-determinant by other

sialylated carbohydrate structures in the heterogeneous stainingareas.

This study yields additional evidence favoring the origin ofrenal cell carcinoma from the proximal nephron. The apparentloss of Le" antigen reactivities found between primary and

metastatic tumors is significant (24% primary versus 80% metastatic) and may be clinically relevant. The absence of Le*

antigen in metastatic and some primary tumors may representan obstacle to clinical applications using monoclonal antibodiesfor immunolocalization. However, our data points toward thepossibility of an immunophenotypic grading system potentiallycapable of predicting tumor cell behavior or response to therapy. Further analyses to correlate the immunophenotype andclinicopathological data in a large series of patients with renaltumors are presently underway.

ACKNOWLEDGMENTS

The authors would like to acknowledge Cambridge Research Laboratories for kindly providing purified monoclonal antibodies. We thank

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BLOOD GROUP ANTIGENS IN RENAL CARCINOMA

Mary Ellen McGroarty and Dolors Casals for expert technical assistance.

REFERENCES

1. Feizi, T., and Childs, R. A. Carbohydrate structures of glycoproteins andglycolipids as differentiation antigens, tumour-associated antigens and components of receptor systems. Trends. Biochem. Sci., IO: 24-29, 1985.

2. Hakomori, S. Aberrant glycosylation in cancer cell membranes as focused onglycolipids: Overview and perspectives. Cancer Res., 45: 2405-2414, 1985.

3. Lloyd, K. O. Blood group antigens as markers for normal and abnormaldifferentiation and malignant change in human tissues. Am. J. Clin. Pal hol..87: 129-139, 1987.

4. Mourant, A. E. A new blood group antigen of frequent occurrence. Nature(Lond.), 158: 237-239, 1946.

5. Andrersen, P. H. The blood group system L. A new blood group L2. A caseof epistasy within the blood groups. Acta Pathol. Microbiol. Scand., 25:728-735, 1948.

6. Cartron, J. P., Mulet, C., Bauvois, B., Rahuel, C., and Salmon, C. ABH andLewis glycosyltransferases in human red cells, lymphocytes and platelets.Blood Trans. Immunohematol., 23: 271-280, 1980.

7. Szulman, A. E. The histológica! distribution of blood group substances Aand B in man. J. Exp. Med., ///: 785-792, 1960.

8. Szulman, A. E. The histologie distribution of the blood group substances inman as disclosed by immunofluorescence. III. The A, B and H antigens inembryos and fetuses from 18 mm in length. J. Exp. Med., 119: 503-511,1964.

9. Watanabe, K., Hakomori, S. Status of blood group carbohydrate chains inontogenesis and oncogenesis. J. Exp. Med., 144: 644-656, 1976.

10. Sakamoto, J., Yin, B. T., and Lloyd, K. O. Analysis of the expression of H,Lewis, X, Y, and precursor blood group determinants in saliva and red cellsusing a panel of mouse monoclonal antibodies. Mol. Iinmuno!.. 21: 1093-1098, 1984.

11. Bariety, J., Oriol, R., Hinglais, N., Zanetti, M., Bretton, R., Dalix, A. M.,and Mandel, C. Distribution of blood group antigen A in normal andpathologic human kidneys. Kidney Int., 17: 820-827, 1980.

12. Ernst, C., Atkinson, B., Wysocka, M., Blaszczyk, M., Herlyn, M., Sears, H.,Steplewski, /... and Koprowski, H. Monoclonal antibody localization ofLewis antigens in fixed tissue. Lab. Invest., 50: 394-400, 1984.

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

14. Combs, S. G., Marder, R. J., Minna, J. D., Mulshine, J. L., Polovina, M.R., and Rosen, S. T. Immunohistochemical localization of the immunodom-inant differentiation antigen lacto-AAfucopentaose III in normal adult andfetal tissues. J. Histochem. Cytochem., 32: 982-990, 1984.

15. Fukushi, Y., Hakomori, S., and Shepard, T. Localization and alteration ofmono-, di-, and trifucosyl al—»3type 2 chain structures during humanembryogenesis and in human cancer, j. Exp. Med., 160: 506-518, 1984.

16. Hawkins, P., Anderson, S. E., McKenzie, J. L., McLoughlin, K., Beard, M.E., and Hart, D. N. Localization of MN blood group antigens in kidney.Transplant. Proc., 17:1697-1708, 1985.

17. Cordon-Cardo, C., Lloyd, K. O., Finstad, C. L., McGroarty, M. E., Reuter,V. E., Bander, N. H., Old, L. J., Melamed, M. R. Immunoanatomic distribution of blood group antigens in the human urinary tract: Influence ofsecretor status. Lab. Invest., 55: 444-454, 1986.

18. Bergman, S., Javadpour, N. The cell surface antigen A, B or O(H) as anindicator of malignant potential in stage A bladder carcinoma: preliminaryreport. J. Urol., 119: 49-54, 1978.

19. Limas, C., Lange, P., Fraley, E. E., and Vessella, R. L. A, B, H antigens intransitional cell tumors of the urinary bladder: correlation with clinicalcourse. Cancer (Phila.), 44: 2099-2107, 1979.

20. Coon, J. S., McCall, A., Miller, A. W., Farrow, G. M., and Weinstein, R. S.Expression of blood group related antigens in carcinoma "in situ" of theurinary bladder. Cancer (Phila.), 56: 797-804, 1985.

21. Limas, C., and Lange, P. H. Lewis antigens in normal and neoplasticurothelium. Am. J. Pathol., ¡21:176-182, 1985.

22. Rettig, W. J., Cordon-Cardo, C., Ng, J. S. C., Oettgen, H. F., Old, L. J., andLloyd, K. O. High molecular weight glycoproteins of human teratocarcinomadefined by monoclonal antibodies to carbohydrate determinants. Cancer Res.,45:815-821,1985.

23. .lulil. B. R., Hartzen, S. H., and Hainau, B. A, B, H antigen expression intransitional cell carcinomas of the urinary bladder. Cancer (Phila.), 57:1768-1775, 1986.

24. Cordon-Cardo, C., Lloyd, K. O., Sakamoto, J., McGroarty, M. E., Old, L.J., and Melamed, M. R. Immunohistologic expression of blood-group antigens in normal human gastrointestinal tract and colonie carcinoma. Int. J.Cancer, 37: 667-676, 1986.

25. Cordon-Cardo, C., Reuter, V. E., Lloyd, K. O., Sheinfeld, J., Fair, W. R.,Old, L. J., and Melamed, M. R. Blood group-related antigens in human

urothelium: enhanced expression of precursor. Le" and Le' determinants inurothelial carcinoma. Cancer Res., 48:4113-4120, 1988.

26. Hsu, S. M., Raine, L., and Fauger, H. Comparative study of the peroxidase-antiperoxidase and avidin-biotin complex method for studying polypeptidehormones with radioimmunoassay antibodies. Am. J. Pathol., 75: 734-740,1981.

27. Juhl, B. R., Norgaard, T., and Bjerrum, O. J. The effect of Tween 20 onindirect immunoperoxidase staining of blood group antigen A in humanurothelium. J. Histochem. Cytochem., 32: 935-941, 1984.

28. Lin, C.-W., Fujime, M., Kirley, S. D., Prout, G. R., Jr. Visualization ofurothelial blood group isoantigens A and B using direct biotin labeled antibodies and avidin-biotin-peroxidase complex. J. Histochem. Cytochem., 32:1339-1345,1984.

29. Limas, C., and Lange, P. H. A, B, H, antigen detectability in normal andneoplastic urothelium: influence of méthodologiefactors. Cancer (Phila.),49: 2476-2484, 1982.

30. Seal, G. M., Rowland, R. G., Thomalla, J. V., Rudolph, R. A., Pfaff, D. S.,Kamer, M., and Eble, J. N. A, B and H antigens in normal urothelium: animmunohistochemical study using monoclonal antibodies with avidin-biotincomplex techniques. J. Urol., 133: 530-538, 1985.

31. Sakamoto, J., Furukawa, K., Cordon-Cardo, C., Old, L. J., and Lloyd, K. O.Expression of Lewis', Lewisb, X, and Y blood group antigens in humancolonie tumors and normal tissue and in human tumor-derived cell lines.Cancer Res., 46: 1553-1561, 1986.

32. Sarodsky, M. F., and Lamm, D. L. Application of the red cell agglutinationtest for cell surface antigens to renal cell carcinoma. J. Urol., 128:693-702,1982.

33. Ghazizadeh, M., Kagawa, S., and Kurokawa, K. Immunohistochemical studies of human renal cell carcinoma for ABO(H) blood group antigens, Tantigen-like substance and carcinoembryonic antigen. J. Urol., 133: 762-769, 1985.

34. Coon, J. S., and Weinstein, R. S. Detection of ABH tissue isoantigens byimmunoperoxidase methods in normal and neoplastic urothelium. Comparison with the erythrocyte adherence method. Am. J. Clin. Pathol., 76: 163-169, 1981.

35. Weinstein, R. S., Coon, J. S., Alroy, J., and Davidsohn, I. Tissue-associatedblood group antigens in human tumors. In: R. A. DeLellis (ed.), DiagnosticImmunohistochemistry, pp. 239-261. New York: Masson Publishing Inc.,1981.

36. Liebert, M., Jaffe, R., Taylor, R. J., Ballou, B. T., Solter, D., and Hakala, T.R. Detection of SSEA-1 on human renal tumors. Cancer (Phila.), 59: 1404-1412, 1987.

37. Solter, D., and Knowles, B. B. Monoclonal antibody defining a stage-specificmouse embryonic antigen (SSEA-1). Proc. Nati. Acad. Sci. USA, 75: 5565-5569, 1978.

38. Gooi, H. C, Hounsell, E. F., Edward, S. A., Majdic, O., Knapp, W., andFeizi, T. Differences in the fine specificity of monoclonal (class A) antibodiesto human myeloid cells. Clin. Exp. Immunol., 60: 151-158, 1985.

39. Fleming, S., and Brown, G. The expression of 3-fucosylated-./V-acetyl lacto-samine carbohydrate determinants in renal tumors. Histopathology, 11:171-182, 1987.

40. Wallace, A. C., and Nairn, R. C. Renal tubular antigens in kidney tumors.Cancer (Phila.), 29: 977-981, 1972.

41. Finstad, C. L., Cordon-Cardo, C., Bander, N. H., Whitmore, W. F., Melamed, M. R., and Old, L. J. Specificity analysis of mouse monoclonalantibodies defining cell surface antigens of human renal cancer. Proc. Nati.Acad. Sci. USA, 82: 2955-2959, 1985.

42. Holthofer, H., Virtanen, L, Pettersson, E., Tornroth, T., Alfthan, O., Linder,E., and Miettinen, A. Lectins as fluorescence microscopic markers for sac-charides in the human kidney. Lab. Invest., 45:391-398, 1981.

43. Ulrich, W., Horvat, R., Krisch, K. Lectin histochemistry of kidney tumoursand its pathomorphological relevance. Histopathology, 9:1037-1050, 1985.

44. Brown, A., Ellis, I. O., Embleton, M. J., Baldwin, R. W., Turner, D. R., andHardcastle, J. D. Immunohistochemical localization of Y hapten and thestructurally related H type-2 blood antigens on large bowel tumors andnormal adult tissues. Int. J. Cancer, 33: 727-735, 1984.

45. Holmes, E. H., Ostrander, G. K., Hakomori, S. Enzymatic basis for theaccumulation of glycolipids with X and dimeric X determinants in humanlung cancer cells (NC1-H69). J. Biol. Chem., 260: 7619-7624, 1985.

46. Hermanek, P., Siegel, A., and Chelpas, A. Histológica! grading of renal cellcarcinomas. Eur. J. Urol., 2: 189-197, 1976.

47. Mostofi, M. F. (ed.) Histológica! typing of kidney tumours. Int. Histol.Classification of Tumours, Vol. 25, World Health Organization, 1979.

48. Moon, T. D., Vessella, R. L., Palme, D. F., Nowak, J. A., Lange, P. H. Ahighly restricted antigen for renal cell carcinoma defined by a monoclonalantibody. Hybridoma, 4:163-169, 1985.

49. Oosterwijk, E., Ruiter, J., Wakka, J. C., Huiskens, J. W., Jonas, U., Fleuren,G. J., Zwartendijk, J., Hoedemaeker, P., and Warnaar, S. O. Immunohistochemical analysis of monoclonal antibodies to renal antigens. Am. J. Pathol.,725:301-309,1986.

50. Fukushi, Y., Orikasa, S., Shepard, T., and Hakomori, S. Changes of LeXand dimeric LeX haptens and their sialylated antigens during developmentof human kidney and kidney tumors. J. Urol., 135:1048-1054, 1986.

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