cancer-associated alterations of blood group antigen ... · cancer-associated changes. the present...

[CANCER RESEARCH 46, 5976-5984, November 1986]

Cancer-associated Alterations of Blood Group Antigen Expression in HumanColorectal Polyps1

Steven H. Itzkowitz, Mei Yuan, Linda D. Ferrell, Alka Palekar, and Young S. Kim2

Gastrointestinal Research Laboratory, Veterans Administration Medical Center, and Departments of Medicine fS. H. I., M. Y., Y. S. K.J and PathologyÂ¡L.D. F., Y. S. K.], School of Medicine, University of California, San Francisco, California 94121; and Institute of Pathology, Shady Side Hospital PiliPennsylvania 15232 Â¡A.P.]

Pittsburgh,

ABSTRACT

Human colorectal carcinoma tissues may exhibit several patterns ofaltered blood group substance (BGS) expression: (a) reappearance of A,B, H, or Lewis0 antigens in distal colon; (/>) deletion of BGS in the

proximal colon with or without precursor substance accumulation; and(c) incompatible BGS expression in proximal or distal colon. The presentstudy evaluated these cancer-associated alterations in colorectal polypswith different malignant potential. With respect to ABH antigens, hy-perplastic polyps (HPs), considered to have no malignant potential, didnot exhibit incompatibility and only a few cases demonstrated BGSreappearance or deletion. Adenomatous polyps (APs) however, frequentlyreexpressed ABH antigens or expressed incompatible BG-A or B in 27%of polyps; one specimen demonstrated BG-B deletion. Precursor expression was not found in HPs but was frequently observed in APs. Reappearance of ABH in distal polyps was significantly correlated withincreasing grade of dysplasia, but was not significantly correlated withpolyp size or histolÃ³gica!type. With respect to Lewis antigen expression,It-Mis1'reappearance occurred in almost every distal polyp, and Lewis"-Lewisb coexpression was also quite common. Le* deletion was frequently

noted, especially in HP, but the significance of this finding is unclear.This study indicates that several antigenic alterations that occur incolorectal cancer tissues also appear in premalignant polyps, and oftenin early stages of the neoplastic process. The observation that incompatible expression of BG-A or B occurs only in AP and cancer tissues (aswell as mucosa adjacent to cancer) but not in fetal colonie mucosa, adultnormal colonie mucosa, or HP, suggests that this may be a cancer-specific phenomenon.

INTRODUCTION

Human colonie epithelium demonstrates rather unique alterations in BG3 antigen expression during normal developmentand in the setting of malignancy (1-10). BG-A, B, H, and Leb

are normally expressed throughout the entire colon of the fetus,but at parturition the mucosa assumes the normal "adult"

phenotype characterized by loss of these antigens in the distal(but not proximal) colon. The Le" antigen, also expressed

throughout the entire fetal colon, maintains this distributioninto adulthood. Colorectal carcinomas are capable of demonstrating a variety of patterns of altered BGS expression. Themost frequent finding is reappearance of BG-A, B, H, or Leb in

the distal colon. This phenomenon suggests reactivation orderepression of fetal gene function, thereby qualifying theseBGS as oncodevelopmental antigens in the human colon. Asecond pattern is the demonstration of unexpected or incompatible BGS expression by cancer cells. Although this phenom-

Received 10/4/85; revised 4/23/86; accepted 7/30/86.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.

1Supported by the Veterans Administration Medical Research Service [S. H.I., M. Y., Y. S. T.], the Veterans Administration Medical Investigator Award |Y.S. T.), and the Veterans Administration Associate Investigator Award [S. H. I.].

2To whom requests for reprints should be addressed, at G.I. Research Lab(151M2), Veterans Administration Medical Center, 4150 Clement St., San Francisco, CA 94121.

1The abbreviations used are: BG, blood group; BGS, blood group substance;Le", Lewis* antigen; Leb, Lewis0 antigen; HP, hyperplastic polyps; AP, adenom-atous polyps; ABC, avidin-biotin-complex; PBS, phosphate-buffered saline; RBC,red blood cell.

enon had been reported only sporadically in the past, we recently observed incompatible ABH expression in 60% of coloncancers and adjacent mucosa using a sensitive immunoperoxi-dase method (10). A third pattern is deletion of BGS, whichmay or may not be associated with accumulation of precursorsubstance. For A, B, H, and Leb antigens, deletion would onlyapply to proximal colonie cancers, whereas Lea deletion may

pertain to proximal or distal cancers. Because these 3 phenomena do not occur in normal colonie mucosa, they are consideredcancer-associated changes.

The present theory of colorectal cancer morphogenesis holdsthat cancer arises from a premalignant polyp rather than a denovo malignant transformation of normal-appearing mucosa(11-13). Not all colonie polyps are premalignant, however.HPs are considered to have no premalignant potential (12-14).APs have premalignant potential but not all will realize thispotential and become frankly malignant. Major "risk factors"

for premalignant change in AP are (a) larger polyp size (b)predominant villous component, and (c) severe cellular dysplasia (11).

We reasoned that if colon cancers demonstrate rather distinctive alterations of BGS expression, their premalignant precursor lesions might similarly demonstrate some or all of thesechanges. We therefore performed the present study on HPs andAPs to test this hypothesis and to determine whether anychanges in BGS expression might be correlated with the abovepremalignant risk factors.

MATERIALS AND METHODS

Tissues. All polyps were removed by endoscopie polypectomy, fixedimmediately in formalin, and embedded in paraffin after appropriateorientation of the polyp. Polyps from patients with a familial polyposissyndrome were excluded. Table 1 describes the location and size of the25 HPs and 52 APs used in this study. Proximal location refers to thececum, ascending colon, or transverse colon (to the splenic flexure)whereas distal location includes descending colon, sigmoid colon, andrectum. One patient provided 2 proximal APs, another patient provided2 distal APs, and a third provided 3 distal APs. All other APs weresolitary. Two APs were associated with a synchronous colon cancer.Three HPs were obtained from 1 patient but the remainder weresolitary. As expected, there were more distal polyps than proximalpolyps available for analysis in both the HP and AP groups. HPs tendto be small and indeed, all examined were <0.5 cm in diameter. Twenty-eight APs were <1 cm and 24 were >1 cm in size.

Adenomatous Polyps: Pathological Classification. Classification ofpolyps according to histolÃ³gica! type and grade of dysplasia was performed according to the criteria of Konishi and Morson ( 15) as detailedpreviously.4 Each polyp was classified independently by two patitolo

gists (M.Y., L.D.F.) using sections stained with hematoxylin and eosin.Differences were subsequently resolved by joint observation. Results ofall immunoperoxidase staining were scored prior to hematoxylin andeosin interpretations to prevent introduction of bias into the inuminohistochemical assessment.

* M. Yuan, S. H. Itzkowitz, L. D. Ferrell, Y. Fukushi, A. Palekar, S. Hakomori,and Y. S. Kim. Expression of Le*- and sialylated Le'-related antigens in humancolorectal polyps. J. Nail. Cancer Inst., in press.

5976

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BG ANTIGENS IN HUMAN COLORECTAL POLYPS

By the above criteria, 34 polyps were classified as tubular, 9 astubulovillous, and 9 as viIIons; 21 polyps presented with mild dysplasia,IS with moderate dysplasia, and 16 with severe dysplasia (includingcarcinoma in situ). Three polyps, all with carcinoma in situ, had a focusof invasive carcinoma and are included in the latter group.

Antisera. Monoclonal antibodies to BG-A, B, H, Le", and Leb were

generously supplied by Dr. Don Baker (Chembiomed Ltd., Edmonton,Alberta, Canada). The specificities of these mouse monoclonal antibodies have been described previously (10). Table 1 lists the antigenicdeterminants of the BGS.

Immunoperoxidase Staining. Serial sections, 5 Â¿tmthick, were stainedusing the ABC technique:Step 1. Deparaffinize, clear, and hydrate sections. Each subsequent stepis preceded by 3 washes with PBS (0.1 M phosphate and 1.5 M NaCl,pH 7.4).Step 2. Incubate sections with methanol/1% hydrogen peroxide for 30min.Step 3. Incubate with mouse primary anti-BGS, 20 /Â¿g/ml,diluted in

5% normal rabbit serum for 3 h.Step 4. Incubate with biotinylated rabbit anti-mouse IgA + IgG + IgMdiluted 1:20 in 1% normal rabbit serum for 30 min.Step 5. Incubate with ABC for 60 min.Step 6. Flood slide for 5 min with 0.05 mg/100 ml of 3-3'-diaminoben-

zidine in 1 ml of PBS with 0.1 % hydrogen peroxide added just prior touse.Step 7. Counterstain with methyl green 1% in acetic buffer for 2 s.Step 8. Dehydrate, clear, and mount.

Controls. With anti-A, B, H antibodies, positive staining of erythro-cytes and blood vessel endothelium served as useful internal controls.Substitution of PBS for specific antisera or secondary antibodies completely abolished any staining. In preliminary experiments, pretreatment with trypsin did not alter the intensity or distribution of antigenexpression.

Determination of Erythrocyte ABH and Lewis Phenotype. The patient's blood type was determined by ABH staining of erythrocytes and

endothelial cells within the tissue sections. Because saliva or gastricsecretions were not available, we were unable to confirm the secretorstatus of the patients. However, the Le" and Leb phenotype of RBCs

was determined by the staining of erythrocytes in the tissue sections.

Table 2 Description of polyps used in this study

Hyperplastic polyps Adenomatous polyps(n = 52)

LocationProximalDistalSize<1

cm1-2cm>2cmHistological

type:TubularTubulovillousVillousGrade

ofdysplasiaMildModerateSeverePatient's

bloodtypeABHABLe'VLe"-Le-'/Le1"Le-/Le"-52025

(all <0.5cm)00NAÂ°NANANANANA8(1:7)*3(1:2)14(3:11)0021

(5:16)4(0:4)11412816g349921151624

(4:20)7(4:3)20(3:17)1

(0:1)4(1:3)28

(5:23)20(5:15)

" NA, not applicable.b Numbers in parentheses, proximal to distal ratio.

Table 2 lists the number of HPs and APs by erythrocyte ABH andLewis phenotype.

Scoring. An adenomatous polyp was considered positive if antigenstaining occurred in at least 5% of the optical fields using a low-power(xlO) objective, and a hyperplastic polyp was scored positive if at least5% of glands were stained.

Statistics. When applicable, comparison of antigen expression by APversus HP was performed using Fisher's exact test (2-taiIed). For APs,correlation of ABH reappearance and Leb reappearance with premalig-

nant parameters was tested using the Kendall rank correlation coefficient. Differences were considered significant at the P < 0.05 level.

Table 1 Antigenic structures of blood group substances'

HA01,3 Gal Â»GlcNAc-RÂ«1,2Fuc

Â«1,3 01,3GalNAc >â€¢Gal Â»GlcNAc-R

Fuc

Â«1,3BGal Â»Gal Â»GlcNAc-R+Â«1,2i

FucLe-01,3Gal

Â»3lcNAc-RÂ«1,4FucLe"01,3Gal

Â»GlcNAc-R[Â«1,2Â«1,4Fuc

Fuc* Fuc, fucose; Gal, galactose; GlcNAc, A'-acetylglucosamine; GalNAc, yV-ace-

tylgalactosamine; R, core glycolipid or glycoprotein.

RESULTS

BGS Reappearance. As mentioned above, the reappearanceof BG-A, B, H, and Leb in colon cancers applies only to the

distal colon where these BGS are usually absent in normal adultmucosa. Indeed, this cancer-associated phenomenon was themost common observation noted in colonie polyps (Table 3).Moreover, BGS reappearance applied almost exclusively toadenomatous polyps, although 3 HPs did reexpress BG-A (Fig.1). The frequency of BGS reappearance in APs was striking.Fully 95% (19 of 20) distal APs from blood type A patientsreexpressed BG-A. BG-B was reexpressed in 2 of 3 (67%)polyps from blood type B patients, and BG-H reappeared in 9of 17 (53%) of blood type H patients. Leb was reexpressed in

37 of 41 (90%) distal APs regardless of the Lewis phenotypeof the patients' RBCs. This considerable reexpression of Leb indistal APs, and the expected presence of Lea throughout thecolon resulted in the coexpression of Le" and Leb in 31 distal

APs and 8 proximal APs (Fig. 2).Although the reappearance of BGS in tissues of the distal

colon is most commonly observed in cancers and APs, we havenoted that BG-A may be reexpressed in a few cases of normalcolonie mucosa (10) and 3 of 7 (43%) distal HPs (presentstudy). These HPs were 0.1, 0.3, and 0.4 cm in size, and all 3were from erythrocyte Le"~b~ patients. Although BGS reap

pearance in these nonmalignant tissues is difficult to explain,curiously it seems to apply primarily to BG-A.

5977



.Table 3Reappearance ofBGSin distalpolyp?RBC

phenotypePolyp expressesHyperplastic

polyps

TotalAdenomatous

polypsTotal<1Size

(cm)

1â€”2>2Histological

typeDegree ofdysplasiaTubularTubulovillousVillousMild

ModerateSevereAA3/719/2ob107213516

67BB0/22/3110200011HH0/119/17333423234ABAorB0/1LCa_b+[@b0/1622/231282173211

65ha-b-@b0/413/15670454346Lea+b_j@b2/30021I00I 1

..-

-, 4@'@ @lbf!:@@!b t


a Applies only to BG-A, B, H, and Laba p < 0.01, compared to hyperplastic polyps; Fisher's exact test (2-tailed).

@d â€¢*

a@

I

-@ @.,

@ .. -4â€•@T@'@-?Ã¸@

â€˜@â€˜@s@,.

â€¢1

I

d I

p

.â€˜ t@, @, â€œ--

f-.-.@ .â€˜@ â€˜@*-

:--:@- â€¢ â€¢â€˜@.â€˜ - .@ C.- ,@

V0

S

â€˜,,

â€˜@o â€œâ€¢@ -

4@::@b!@@ .@i . .

â€˜.- -

t@

,@. @â€˜!

--â€˜â€˜I

-.â€” .. _ .. a

U

â€˜@1A0.

Fig. 1. BG-A reappearance in distal polyps. A, adenomatous polyp demonstrating staining in the supranuclear cytoplasm (solid arrow) and goblet cell mucin (openarrow). Note the positive staining ofendothelium (bottom right), x 125. B, hyperplastic polyp. Only one gland in this field is stained positive, with antigen expressionfound on cell membranes and cytoplasm. Note again the positively stained endothetium of numerous blood vessels, x 50.

Incompatible BGS Expression. The expression of incompatible BGS was limited exclusively to the AP group (Table 4). Ofthe 51 APs that were â€œeligibleâ€•to express incompatible BGS(i.e., the polyp from the AB individual was excluded) 14 demonstrated BG-A or B incompatibility, for an overall frequencyof 27%. This is less than the 60% incidence ofABH incompatibility noted previously in cancer tissues (10). Although incompatibility may occur anywhere in the colon, 10 of 14 cases ofincompatible expression were in distal polyps (Fig. 3). Interestingly, of the 4 patients whose RBC were@ 3 polypsexhibited â€œincompatibleâ€•Leb expression (1 proximal, 2 distal).The complete absence of incompatibility in HPs suggests thatthis phenomenon is cancer associated. This concept is supported by ot@rprevious observations that only colon cancer

tissue (and adjacent transitional mucosa), but not fetal or normal adult colonic mucosa, was capable of incompatible BGSexpression (10).

BGS Deletion. Deletion ofBGS, an event not found in normalcolonic mucosa, is seen only rarely in colon cancers and fetalcolonic mucosa (10). Table 5 indicates that ABH deletion isalso encountered in colorectal polyps, and rather infrequently.In APs, only one example of ABH deletion was noted and thisoccurred in a 1.1-cm tubular polyp with carcinoma in situ.Although this polyp did not express the expected BG-B, it didexpress precursor BG-H and also incompatible BG-A. Thus, inthe presence of adequate precursor, this unusual situation circumstantially suggests that the preferential appearance of BGA at the expense of BG-B could be related either to a loosening

5978




Fig. 2. Coexpression of Le1 and Lcb in distal APs. A, Staining with anti-Le*. Some apical

cell membranes and occasional cell cytoplasmare positive, x 125. B, Staining with anti-Leb.

Antigen is expressed primarily in the cytoplasm but some apical membrane staining isalso noted, x 12S.

2B

Table 4 Incompatible BGS expression in polyps

RBCphenotypeA

BHHHLe"Â»-Polyp

expressesBA

A onlyB onlyA and BLe"Hyperplastic

polypsTotal0/80/3

0/140/140/14Adenomatous

polypsLocationTotal4/24

4/74/200/202/203/4Proximal1

3001Distal3

1422Size

(cm)<11

310

01-22

132

0>21

000

3Histological

typeTA"14

20

1TVA2

002

1VA1

020

1Degree

ofdysplasiaMild01

20

0Moderate2

1202Severe2

202

1" TA, tubular adenoma; TVA, tubulovillous adenoma; VA, villous adenoma.

3A 13BFig. 3. Villous adenoma obtained from the rectum of blood type A patient demonstrating I, simultaneous re-expression of BG-A; B, incompatible BG-B; and C,

accumulation of BG-H. Staining is primarily in the cytoplasm of cells scattered throughout the neoplastic glands. Gland lumen is on the left in each picture, x 125.

5979




of substrate specificity so that the B enzyme mistakenly synthesizes A determinant or, alternatively, an A-like yV-acetylgalac-tosaminyltransferase might be active.

Interestingly, ABH deletion was also noted in HPs. Onepolyp failed to express BG-B, and 2 polyps failed to expressBG-H. In this study, no examples of BG-A deletion wereencountered in either HPs or APs, in contrast to 3 cases of BG-A deletion in colon cancer (10). The rarity of this event,however, makes it difficult to speculate upon its significance.

Deletion of Le" was a frequent finding in both APs and HPs

and occurred in both proximal and distal polyps (Table 5). Allof the HPs and all but one of the APs demonstrating Leadeletion came from patients whose RBCs were Lea negative. Innormal colonie mucosa, the Le" antigen is frequently expressedin goblet cell vacuoles (Fig. 44). Fig. 4B demonstrates Le"deletion in a distal AP. In 6 distal APs, Le" deletion wasaccompanied by Leb reappearance (Fig. 4C). Deletion of Lea

was even more prevalent in HPs than in APs (P < 0.0005;Table 5). Five of these HPs exhibited no Le" antigen at all,

Table 5 BGS deletion in polyps'

RBCphenotypeABH

ABLe'-"*Polyp

isnegativefor:A

BH

A or BLe"Hyperplastic

polyps012

2Adenomatous

polyps0

1 (1.1 cm; TA; severedysplasia)

01

(0.3 cm; TA; moderateLe'-"*

all patientsLe"Le'220/25 (80%)*

Proximal: 1Distal: 191

(0.3 cm; TA; moderatedysplasia)

ll/52(21%yProximal: 2Distal: 9

" For BG-A, B, H, and Leb this applies to proximal polyps. For BG-Le" this

applies to proximal and distal polyps.6 P < 0.0005 compared to adenomatous polyps; x2 test.c Size: <1 cm, n = 8; >2 cm, n = 3. HistolÃ³gica! type: TA, n = 9; TVA, n = 1;

VA, n = l. Dysplasia: mild, n = 6; moderate, n = 3; severe, n = 2.

whereas in the other 15 Le" was deleted from the goblet cell

vacuole but was present in the cell cytoplasm and on the apicalmembrane. The deletion of Lea is an unusual event that we

have not noted in colon cancer although others have (16). Thus,the finding of Le" deletion by colorectal polyps is an intriguing

yet unexplained phenomenon.Precursor Expression. BG-H serves as the precursor for BG-

A and B. In HPs, BG-H was never found in polyps of bloodtype A or B patients. However, in APs, the BG-H percursorfrequently accompanied BG-A or B expression, so we analyzedthis further (Table 6). Of 31 patients who were either bloodtype A or B, 18 expressed precursor substance in addition tothe appropriate BGS. In the 5 cases from the proximal colon,this is interpreted as precursor accumulation, whereas in the 13cases from the distal colon, this represents reappearance of boththe precursor and the A or B antigen. Only one patient (bloodtype B) expressed precursor substance without the appropriate"completed" antigen. This case, also described above under BG-

B deletion, was a proximal polyp, and therefore, BG-H presencemay be expected to occur in that region.

In 12 APs from blood type A or B individuals, the BG-Hprecursor was absent. If this occurred in the distal colon, theabsence of BG-H is to be expected. Such was the case in 10 of12 of these polyps. However, in 2 proximal polyps (one from ablood type A and one from a blood type B patient), precursorwas absent. Since this occurred together with the appropriateexpression of A or B antigen, it suggests that all precursor wasconverted to the appropriate "completed" antigen.

BGS Expression by Multiple Polyps. Three HPs were obtained from one patient of blood type H. All 3 of these distalpolyps appropriately did not express BG-A, B, H, or Leb.However, 2 of the 3 demonstrated Le" deletion.

Multiple APs were obtained from 3 patients. One blood typeA patient provided 3 distal tubular adenomas, ranging in sizefrom 1.0-2.0 cm, and with mild or moderate dysplasia. All 3

4A 4B 4CFig. 4. I. Le* expression in normal colonie mucoa. Note positive staining of goblet cell vacuoles in this crypt of the distal colon, x 50. lÃ¬,two glands in a distal

AP demonstrating the absence of Le* expression, x 125. C, two glands from the same polyp as B demonstrating reappearance of Leb. Staining is primarily incytoplasm although some membranes are also stained, x 125.

5980




polyps reexpressed BG-A as well as BG-H, and reexpressed Lebresulting in Lea/Leb coexpression. A second patient of blood

type A had 2 distal tubular AP; one measured 1.1 cm and hada focus of severe dysplasia and the other was 0.7 cm with afocus of carcinoma in situ. Both polyps reexpressed BG-A, BG-H, and Leb (with Lea/Leb coexpression) but interestingly thelatter polyp also expressed incompatible BG-B. The third patient was also blood type A and provided 2 proximal, tubular,0.2-cm polyps with mild dysplasia. Both showed the expectedpresence of BG-A, Le", and Leb but also an accumulation ofBG-H.

BGS Expression by a Simultaneous Cancer and Polyp. Twoof the APs examined in this study were obtained from individuals who had a simultaneous colon cancer reported in ourprevious study (10). One patient was blood type H, Lea~b+whohad a well-differentiated Duke's C carcinoma of the ascending

colon with a 0.4-cm, mildly dysplastic tubular adenoma in thececum. The cancer, but not the polyp, demonstrated incompatible ABH expression and both the cancer and polyp demonstrated coexpression of Le" and Leb. The other patient wasblood type A, Le"~b+ who had a moderately differentiatedDuke's A carcinoma of the rectum associated with a 1.1-cm

tubular adenoma with severe dysplasia in the sigmoid. Both thecancer and the polyp demonstrated BG-A reexpression, BG-Hprecursor expression, and Lea/Leb coexpression, but only thecancer exhibited incompatible BG-B expression.

Comparison of Altered BGS with Premalignant Parameters.Table 7 compares each pattern of altered BGS expression withpolyp size, histological type, and grade of dysplasia. The reap

pearance of BG-A, B, or H in distal polyps seemed to correlatewith polyp size and dysplasia but not with histological type.However, using Kendall rank correlation, ABH reappearancewas significantly correlated only with the grade of dysplasia (P< 0.01). As noted above (Table 3), Leb reappearance in distal

polyps was quite frequent. In fact, only 4 polyps did not exhibitthis pattern. Therefore, because of this marked prevalence, Leb

reappearance was not significantly correlated with any of the 3premalignant parameters.

Although incompatible BGS expression showed some correlation with 3 premalignant parameters, it did not reach statistical significance. This probably reflects the uncommon occurrence of this phenomenon and the relativey small number ofpolyps examined. Likewise, deletion of BGS, even less common, did not show statistically significant correlation with anyof the 3 premalignant parameters.

There were 3 polyps (one proximal and 2 distal) in which afocal area of invasive carcinoma was identified. All 3 polypsalso exhibited foci of carcinoma in situ and were included inthe severe dysplasia group. One was tubular, one was tubulov-illous, and the third was predominantly villous. Sizes rangedfrom 0.8-1.8 cm. Of the 3 polyps, 2 demonstrated incompatibleBGSâ€”one expressed BG-A in a blood type B patient, and oneexpressed BG-A and B in a blood type H patient. Both distalpolyps reexpressed the appropriate BGS and also reexpressedLeb resulting in coexpression of Lea/Leb.

Cellular Distribution of BGS in Adenomatous Polyps. Thecellular location of each BGS is presented in Table 8. Thenumber of polyps that expressed an individual BGS on the

Table 6 Precursor expression in adenomatous polyps

InterpretationRBC

phenotype PolypphenotypeAH+ A+(14)Â°H+

A- (0)H- A+(9)H-A-(l)B

H+ B+(4)H+B-0)

H-B+U)H-B-(l)AB

H- AB-LocationProximal

(3)Distal(11)Proximal

(1)Distal (8)DistalProximal

(2)Distal (2)ProximalProximalDistalDistalPrecursor

(BG-H)Accumulation

ReappearanceConverted

to BG-A(Expected absence)(Expected)Accumulation

Reappearance(Expected presence)Converted to BG-B(Expected)(Expected)"Finished"

antigen(BG-A.B)(Expected)

BG-Areappearance(Expected)

BG-A reappearance(Expected)(Expected)

BG-B reappearanceBG-B deletion(Expected)(Expected)(Expected)

" Numbers in parentheses, n.

Table 7 Altered BGS expression in adenomatous polypsComparison with polyp size, histological type, and grade of dysplasia

Reappearance"Polyp

size<1 cm1-2 cm>2cmHistological

typeTubularTubulovillous

VillousGrade

of dysplasiaMildModerateSevereA,B,H'%,

(67)""/is (73)'A(IOO)"/26

(73)%<78)

%(67)Vie

(50)10/i2(83)'Vu (92)'Le""h,

(86)"/l!(100)'A

(80)2%6

(85)%(100)V6(100)u/i6

(88)"/,2(92)12/n(92)Incompatibility*A,B,H%.(18)

â€¢/i6(50)'/Â«(13)7/34<21)%

(44)%(33)3/2l

(14)V,s

(33)Vu (38)Le"

BG-BC%â€¢

(7)'/Â«(Ã³) V, (100)%(38)2/w(6)

v.(i3%)%(33)

'/,(!!)%i

(10)2/,5(13)2/i6(13) Vi3(8)DeletionLeb<2/7

(29)

'/s(33)%

(25)

V,(33)'/I6

(6)Vu

(17)Le"Va

(29)y.

(38)Vu

(26)V, (11)V,(11)Vji

(29)Vis (20)V,. (13)

* Distal polyps only." Proximal and distal polyps.c Proximal polyps only.d Numbers in parentheses, percentage.' P < 0.01, Kendall rank correlation.

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Table 8 Cellular distribution of BGS in adenomatous polyps

Goblet cell mucin

(1)A(33)

B(ll)H (31)Le' (41)Le" (45)Membrane17/33(52%)

6/11 (55%)18/31 (58%)38/41 (93%)28/45 (62%)Cytoplasm31/33(94%)

11/11 (100%)25/31 (81%)34/41 (83%)44/45 (98%)Proximal2/7

(29%)1/4 (25%)4/9 (44%)5/9 (56%)6/8 (75%)Distal14/26

(54%)1/7(14%)2/22 (9%)7/32 (22%)

17/37(40%)

apical membrane, cytoplasm or goblet cell vacuole in at least5% of the polyp were counted and expressed as a percentage ofthe total number of polyps that were positive for that BGS. Ingeneral, if a BGS was expressed, it was present in cytoplasmmore so than on cell membranes. With the exception of BG-A,proximal goblet cell mucin stained more frequently than distalgoblet cell mucin.

DISCUSSION

The evaluation of BGS in human colon to date has mainlyfocused on patterns of expression in fetal, normal adult, andmalignant colorectal tissue. These studies, using a variety ofhistochemical and biochemical techniques, have all led to thefollowing observations and concepts about A, B, H expression:(a) BG-A, B, and H are oncofetal antigens in the distal humancolon; (b) in distal colon cancers, the reappearance of BGSappropriate for the patient's blood type occurs in a majority of

instances; (c) proximal cancers may occasionally exhibit deletion of the appropriate BGS, either with or without precursoraccumulation; and (d) cancers anywhere in the colon maydemonstrate incompatible BGS expression. Thus, distal reappearance, proximal deletion, and incompatible expression ofBGS are considered cancer-associated phenomena.

The occurrence of these phenomena in neoplastic colorectalpolyps, however, has been relatively unexplored. In the fewstudies that have looked at BGS expression in polyps, onlyABH antigens have been examined, and HPs were often under-represented. The present investigation is therefore the firstsystematic study of 5 BGS in a sizable series of colorectalpolyps. By evaluating polyps with presumably little or no malignant potential (HPs) and those with possible malignant potential (APs), several striking observations emerged. First, thereappearance of BGS in the distal colon, which is the mostfrequent pattern seen in cancer tissue, was also the most common blood group alteration found in polyps. Furthermore, inAP, reappearance of A, B, or H significantly correlated withdegree of dysplasia. These results therefore indicate that the"cancer"-associated reappearance phenomenon applies to pre-

malignant colorectal polyps as well. Reappearance of BGS inAPs has been described previously in a few studies. Denk et al.(5) initially reported that 5 distal colonie APs did not expressBG-A or B. However, in a subsequent study (17), this groupanalyzed BG-A and B expression in a larger number of distal(only) polyps and found that BGS reappearance occurred in 35of 79 (44%) APs and in 22 of 26 (85%) villous "papillomas."

Of the 6 HPs examined, none demonstrated BGS reappearance.Moreover, reappearance tended to occur in polyps with moresevere degrees of "dedifferentiation." Another study by Cooper

et al. (18), which looked at BG-A and B expression in distalcolonie polyps, found that 41% (18 of 44) of APs (includingpolyps with cancer) and none of 4 HPs demonstrated BGSreappearance. Villous adenomas reexpressed BGS slightly moreoften than tubular adenomas (54 versus 39%), and, as in ourstudy, Cooper et al. ( 18) noted more frequent reexpression of

BGS in larger polyps (2 cm). However, unlike the present studyand that of Denk et al., Cooper's group noted no correlation

between BGS reappearance and degree of cytological atypia.Cooper noted that BGS reexpression was more prevalent inpolyps with focal cancer compared to APs without focal cancer,and that BGS reexpression was found more often in polypsfrom patients with colon cancer than in polyps from patientswithout cancer or a history of cancer. Other investigators havenoted the reappearance of Ulex europeas binding sites in 12 of18 rectal adenomas, suggesting that BG-H is reexpressed inAPs (9).

The above observations indicate that BGS reexpression is arelatively common phenomenon in APs, but the significance ofthis is unclear. Although BGS reappearance may or may notdirectly correlate with each of the three premalignant parameters, it is noteworthy that in this study at least half of the small,tubular, and mildly dysplastic polyps already demonstrated thisevent. If this phenomenon represents oncodevelopmental genereactivation, it occurs quite early in premalignant lesions. Wecannot explain why 3 HPs which presumably have no premalignant potential also demonstrated BG-A reexpression. However, the ability of HP to express other cancer-associated antigens, such as the Thomsen-Friedenreich antigen (19-21) andthe M l antigen (22), has been known for several years.

The most remarkable observation in the present study wasthe expression of incompatible BGS. This was seen exclusivelyin the AP group with a slight predominance in the distal colon.To our knowledge, only one other study noted BGS incompatibility in a colonie polyp (18). In that study, one patient whowas blood type B had 3 distal polyps, 2 of which were BG-Bpositive and one of which revealed BG-A incompatibility. In asecond patient, the polyp did not demonstrate incompatibilitybut a synchronous cancer expressed incompatible BG-A.

To date, incompatible BGS expression has been observedalmost exclusively in cancers (primarily of gastrointestinal origin). The present study now extends this observation to premalignant neoplasms of the colon. This information, coupledwith the fact that incompatible BGS expression has not beenobserved in normal tissues or HPs, strongly suggests that BGSincompatibility is related to malignant transformation. Themolecular mechanism(s) involved in cancer-associated ABHincompatibility can only be speculated upon at present. Onepossibility is that transformed cells may synthesize A-like or fluke oligosaccharides rather than the exact BG-A or BG-Bimmunodeterminant usually found on RBC membranes. In thisregard, Knuth et al. (23) recently reported that 2 members ofcolon cancer-prone families had circulating antibodies that werenot absorbed by RBC but could be absorbed by cancer cell A-or B-like determinants. Hakomori et al. (24) implicated cross-reactivity with the Forssman antigen, which has an A-liketerminal A'-acetylgalactosamine, as being responsible for "neo-A" incompatibility. However, our BG-A antibody does notcross-react with Forssman-positive sheep RBC. More recently,the Tn antigen, an epitope found on mucin-like glycoproteins,has been implicated as an incompatible A-like antigen in severaltypes of cancer, including those of colonie origin (25). A secondexplanation for incompatible BGS expression might be alteredspecificity of the glycosyltransferase enzymes responsible forsynthesizing these carbohydrate antigens. For example, the BG-A glycosyltransferase might mistakenly add a terminal galactose rather than jV-acetylgalactosamine, resulting in the BG-Bantigen. This has been demonstrated under certain experimental conditions using erythrocytes (26). Third, and perhaps mostintriguing, is the possibility that transformed cells possess the

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genetic machinery to synthesize a BGS that is foreign to thehost. This hypothesis, contradicting classical immunogeneticteaching, has recently been supported by the observation thatcolon cancer cells from a type O individual possessed A-trans-ferase activity (27).

Although ABH deletion has been correlated to invasivenessin bladder cancer (28), ABH deletion is less common in colorectal cancer. Two studies suggested that rÃ©currences/mÃ©tastases of colon cancers frequently lose ABH expression (7, 29),but another study noted preserved ABH expression in mÃ©tastases (5). We are aware of only one previous study that analyzedABH deletion in colonie polyps (29). Of 39 adenomatous polypsof the proximal colon, 31 (79%) exhibited ABH deletion.However, we noted only one example (BG-B) of ABH deletionamong 11 proximal APs. In HPs ABH deletion has not beenreported, but we found 2 cases of BG-H deletion and one caseof BG-B deletion among 5 proximal HPs. Deletion of ABH isprobably attributable to a deficiency of glycosyltransferase activity (30, 31), although degradation of antigen by bacterialglycosidases (32) or masking of antigen by other sugars cannotbe ruled out. In our preliminary experiments, however, pretreatment with trypsin did not unmask antigenic expression.

The accumulation of precursor blood group substances incolon cancer tissues has been noted by some investigators (8,9,33,34) but not by others (31,35). Differences in experimentalapproach may account for some of the discrepancies. Moreover,when considering BG-H expression as a "precursor" substance,it is important to know the patient's blood type (because in a

blood type H patient, BG-H expression is not a true precursor)and to simultaneously examine for BG-A and B expression(because "disappearance" of BG-H may indicate either chain

elongation to BG-A or B, or inability to synthesize H substance). The present findings in adenomatous polyps (Table 6)demonstrate that in 31 patients of blood type A or B, the polypsof 18 patients re-expressed BG-H elisially or accumulated BG-H proximally. Two other studies have examined BG-H expression in adenomatous polyps taken only from the distal colon.One study used monoclonal anti-H type-2 (identical to thereagent we used) and noted antigen (re)expression in 9 of 28(32%) polyps, 4 of which came from blood type H patients(33). The second study used Ulex europeas agglutinin in patientwith unspecified blood type and noted positive staining in 12of 18 (67%) adenomas (9).

Biochemical and immunohistochemical investigations of Le"and Leb expression by colonie epithelium have revealed severalinteresting observations relevant to cancer-associated antigenexpression. In normal colonie mucosa, Le" is expressedthroughout the colon whereas Leb is expressed proximally but

not elisially (4, 10). In colon cancers, regardless of the sitewithin the colon, Lea and Leb antigens are frequently expressed(4, 10). Thus, in distal colon cancer, the (re)expression of Lebantigen, by itself, is interpreted as a cancer-associated event (4,10, 36). In addition, coexpression of Le" and Leb is tumor-associated (36, 37). Furthermore, the Leb antigen may be con

sidered an incompatible BGS in the cancer of a person who isa nonsecretor (erythrocyte Lea+b~)and a few such cases have

been reported (10, 16). To date, no information is availableconcerning Lea or Leb expression in colonie polyps. The present

study demonstrated that many of these cancer-associated alterations in Lewis antigen expression may also occur in APs butnot in HPs. First, we noted considerable reexpression of Leb indistal APs but not in distal HPs (Table 3). This supports Lebdistal reappearance as a cancer-associated event. Second, 31 of41 distal APs, but no distal HPs, exhibited coexpression of Le"

and Leb (Fig. 2). Third, in 3 of 4 APs, incompatible Leb wasexpressed in patients whose RBC phenotype was Le"*b~(Table4). Finally, an unexpected observation was the deletion of Le",

occurring in a surprisingly high number of polyps; especiallyHPs (Table 5). However, in HPs, as noted above, completeabsence of Le" occurred in only 5 cases with the other 15demonstrating some Le" expression but not in goblet cell vacuoles. Although we have not observed Lea deletion in coloncancers, others have reported Le" disappearance in poorly dif

ferentiated or metastatic colon cancers (16). In some of ourAPs, an explanation for Lea deletion might be that Le" antigenwas converted to Leb possible reflecting the enhanced activityof an al,2-fucosyltransferase. However, this would not explainthe considerable Le" deletion in HPs, which could reflect adeficiency or repression of Â«1,4-fucosyltransferase. More workis needed to confirm and explain these findings.

ACKNOWLEDGMENTS

We would like to thank Dr. James Byrd for helpful advice and IrishHarrington for preparation of the manuscript.

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1986;46:5976-5984. Cancer Res Steven H. Itzkowitz, Mei Yuan, Linda D. Ferrell, et al. Expression in Human Colorectal PolypsCancer-associated Alterations of Blood Group Antigen

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