Lewis antigen alterations in gastric cancer precursors

GASTROENTEROLOGY 1992;102:424-430

Lewis Antigen Alterations in Gastric Cancer Precursors JULIO TORRADO, PELAYO CORREA, PAOLO BERNARDI, DIEGO ZAVALA,

BERNARD0

RUIZ,

and JACQUES BARA

Department of Anatomic Pathology, Hospital Nuestra Sra. Aranzazu, San SebastiBn, Spain; Department of Pathology, Louisiana State University Medical Center, New Orleans, Louisiana; and Mucin Immunochemistry Laboratory Institut des Recherches Scientifiques sur le Cancer, Villejuif, France

To explore the dynamics of the progressive loss of cell differentiation observed in the gastric precancerous process, the abnormal expression of Lea antigen in the gastric epithelium was investigated. Gastric biopsy specimens of 122 subjects with Le(a-bf) phenotype who had intestinal metaplasia of the gastric mucosa were studied. The subjects are residents of a rural area in the Colombian Andes with very high risk of gastric cancer. The abnormality was detected with increasing frequency in lesions with other markers of progression of the precancerous process, namely, colouic-type of morphology of the metaplastic cells, expression of sulfomucins, and dysplastic changes, The concomitant expression of the abnormal Lea antigen and sulfomucins was found to be a more reliable marker of more advanced lesions such as colonic metaplasia and dysplasia than either marker alone.

A

lthough multifocal chronic atrophic gastritis (MAG) and intestinal metaplasia of the gastric mucosa (IM)are considered precursors of gastric carcinoma,’ their elevated prevalence in high-risk populations makes these markers of little use in identifying individuals at the highest risk. It has been proposed that some types of IM represent a more advanced stage in the precancerous process, as represented by the colonic type of morphology and the secretion of sulfated mucins.2-5 Lewis blood group antigens are carbohydrate moieties structurally integrated in the mucous secretions. They are considered to play an important role in cell recognition, differentiation, and intercellular cohesion.68 They are controlled by the Lewis gene and the secretor gene, as shown diagrammatically in Figure 1. It has been speculated that during the process of carcinogenesis, in individuals of Le(a-b+) phenotype, the abnormal gastric epithelial cells develop a blockage of the synthesis of Leb antigen resulting in accumulation of its precursors, which then follow an anomalous pathway and express Lea antigen. This would be the case if al-2+fucosyltransfer-

ase becomes incapable of competing for the type 1 precursor oligosaccharide. In such case, the al-Z-Lfucosyltransferase would act on type 1 precursor and express Le” antigen. Another alternative pathway for the biosynthesis of Lewis antigens in human gastric mucosa has been proposed.“” Lewis antigen system alterations have been described in gastric carcinoma and associated lesions.‘1-‘4 In the present report, we report the anomalous expression of Le” antigen in subjects with Le(a-b+) phenotype in Nariiio, Colombia, a high-risk area for gastric cancer. Materials and Methods The study was conducted in 176 subjects (90 male, 86 female) with IM diagnosed in gastric biopsy specimens. Because no gender differences were detected, the data are presented for both sexes. These subjects belong to a cohort that is followed up to examine the natural history of precursor lesions in the rural Andean region of Narifio, Colombia, where gastric cancer rates are very high.15 Extensive studies using subjects participating in that cohort have provided information on the morphology, risk factors, and time trends of the different precursor lesions.“sl’ The morphology of the lesions has been documenfed.*“‘16 To better examine the prevalence of the alterations of the Lewis system, the most recent gastric biopsy specimen of the subjects was used for the tabulations. Additionally, the ABO and Lewis antigens were determined in this series and in a group of 115 volunteers chosen from a random household survey of the population of Tuquerres, Narifio. At least four biopsy specimens were taken: two from the antrum (midportion of antral lesser and greater curvatures), one from the incisura angularis, and one from the corpus (midanterior wall). They were immediately fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Histochemical identification of mucins was performed with the periodic acidSchiff-alcian blue stain, pH 2.5,” as well as the high-iron diamine-alcian blue sfain.lg The type of IM was independently determined in H&E stains. If all of the metaplastic epithelium consisted of ab0 1992 by the American Gastroenterological 0016-5065/92/$3.00

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LEWIS ANTIGENS AND GASTRIC CANCER 425

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TYPE 1 PRECURSOR

Figure 1. Diagrammatic representation of the biosynthesis of Lewis antigens. The bars intercepting the arrows represent the hypothetical points of blockage leading to abnormal expression of IX? in individuals of Lefa-b+) phenotype. Se gene, secretor gene; Le gene, Lewis gene.

sorptive cells with a well-defined brush border alternating with goblet cells, the lesion was classified as small intestinal type (SIM). If the metaplastic epithelium consisted entirely of columnar cells with abundant cytoplasmic mutins collected in multiple vacuoles, without a recognizable brush border, the lesion was classified as colonic metaplasia. In the current series, all specimens containing foci of colonic metaplasia also contained foci of SIM and were therefore called mixed intestinal metaplasia (MIM). The presence of sulfomucins was evaluated independently in goblet cells and columnar cells. All cases with sulfomucins in columnar cells also had them in goblet cells. Therefore, two types of positivity were recorded: goblet cells positive only (G+C-) or goblet and columnar cells positive (G+C+). The investigation of anomalies within the Lewis system was restricted to 122 subjects (63 male, 59 female) whose phenotype was Le(a-b+). The Lewis phenotype was determined in gastric biopsy specimens using segments of the mucosa with well-preserved surface (foveolar) epithelium and glands. This “tissular phenotype” has definite advantages over erythrocytic phenotype. There is a very good correlation between Lewis and secretor genotype and their corresponding phenotypes in red blood cells and gastric mucosa.‘3~20-23The foveolar gastric epithelium is under the influence of the secretor gene, whereas the deep gastric glands are independent of such gene. Subjects of secretor genotype express ABH antigens both in the foveolar epithelium and in the deep gastric glands. Subjects without the secretor genotype express ABH antigens only in the deep glands and not in the foveolar epithelium. Lewis antigens are expressed only in the foveolar epithelium and not in the deep glands. Individuals with both the secretor gene and the Lewis gene express Leb antigen. Individuals without the secretor gene who have the Lewis gene express the Le” antigen. Subjects without the Lewis gene (with or without the secretor gene) do not express the Lewis antigens.‘3~20-23 Formalin-fixed, paraffin-embedded tissues show excellent preservation of ABH and Lewis antigens. The anomalous presence of Le” antigen was categorized in the following three patterns: (a) Le” antigen restricted to

goblet cells (Figure 2); (b) Le” antigen abundant in goblet cells and weakly positive in columnar cells (Figure 3); and (c) Lea antigen abundant in columnar cells (in which case positive goblet cells were always found) (Figure 4). Five mouse monoclonal antibodies were used to identify blood groups A, B, H type 2, and Le”, and Leb specificities. Monoclonal antibody 2.25 LE (working dilution, 1:40; Institut des Researches Scientifiques, Villejuit, France) reacts strongly with Leb antigen and very weakly with Le” antigen.” Monoclonal antibody 7 LE (working dilution, 1:60) reacts with Le” antigen and does not react with Leb antigen.zz~24 Monoclonal antibodies 2521 B8 (working dilution, ~150) and 164 B5GlO (working dilution, 1:70; Diagast, Lille, France) have anti-A and anti-B specificities, respectively.25,2” Finally, monoclonal antibody A 583 from Dako (working dilution, 1:lO; Carpenteria, CA) was used to recognize H type 2 structures. The immunoperoxidase staining was performed on 3urn sections, after routine deparaffinization and rehydration. The endogenous peroxidase was abolished after 15-minute treatment with 3% hydrogen peroxide. The

Figure 2. Intestinal metaplasia of gastric mucosa showing positive staining of goblet cells (arrows) with anti-Lewis a antibody in a subject of Lefa-b+) phenotype. The columnar cells are not stained. Pattern 1.

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sections were successively incubated with (a) the working dilution of primary antibodies for 30 minutes: (b) peroxidase-conjugated rabbit anti-mouse immunoglobulin (P 161, Dako; diluted 1:lOO) for 15 minutes; (c) peroxidaseconjugated swine anti-rabbit immunoglobulin (P 399, Dako; diluted 1:lOO) for 15 minutes; and (d) a freshly prepared solution of 0.05% 3-Y-diaminobenzidine tetrahydrochloride (D 5636; Sigma, St. Louis, MO) and 0.01% hydrogen peroxide in phosphate-buffered saline (PBS), 0.01 Mel/L phosphate and 0.15 Mel/L NaCl at pH 7.2, for 10 minutes. The slides were faintly counterstained with Lillie’s hematoxylin and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). Positive and negative controls were stained with every batch of samples. All incubation steps were performed at WC and followed by three washing steps with PBS. The ABH phenotype was determined on tissue samples by immunohistochemical staining of the gastric epithelium with anti-ABH monoclonal antibodies. The positive pattern of these reagents was used to establish the secretor status: cases showing positive stain in the foveolar epithe-

Figure 4. Intestinal metaplasia of gastric mucosa showing intense staining of goblet and columnar cells with anti-Lewis a antibodies in a subject of Le(a-b+) phenotype. Pattern 3.

lium as well as deep glands were considered secretors and cases with negative reaction in the superficial epithelium were considered nonsecretors. The Lewis phenotype was determined by positive staining of the gastric surface epithelium with anti-Lea or anti-Leb monoclonal antibodies. Positive cases showed a diffuse homogeneous staining restricted to the superficial gastric (foveolar) epithelium.‘3*20 The x2 statistics were calculated to test for the overall association between two parameters, Odds ratios and 95% confidence intervals were calculated.27 The one degree of freedom x2 test was used to test the linear trend of the odds ratios.*’

Results

Figure 3. Intestinal

metaplasia of gastric mucosa showing strongly positive staining of goblet cells (slrort arrows) and weakly positive staining of columnar cells (long arrows) with anti-Lewis a antibodies in a subject of Le(a-b+) phenotype. Pattern 2.

For descriptive purposes, ABO and Lewis phenotypes distributions were studied in the IM group and in the group of 115 volunteers. No significant differences between the two series were found in the ABO-Lewis phenotypes or secretor status, as seen in Table 1. The prevalences of Le(a+b-) and nonsecre-

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Table 1. Percentage

Distribution by ABO and Lewis Phenotypes and Secretor Status in 176 Subjects With Intestinal Metaplasia and 115 Volunteers From Narifio, Colombia ALe”

ALeb

ALe-

BLe”

BLeb

BLe-

OLe”

OLeb

OLe-

ABLe”

ABLeb

ABLe-

1.1 1.7

11.4 16.5

4.5 3.5

0.6 1.7

4.0 a.7

1.7 0.9

4.5 4.3

52.8 45.2

17.6 17.4

0 0

1.1 0

0.6 0

IM cases (%) Volunteers (%)

IM cases (%) Volunteers (%)

Le(a-b-)Se

Le(a-b+)

Le(a+b-) 6.3

69.3

7.8

70.5

Le(a-b-)se

24.4 19.1

0 2.6

Se, secretors; se, nonsecretors.

tor status are substantially lower in Narifio than in other populations.2g In the 122 Le(a-b+) individuals, the abnormal expression of the Le” antigen and the concomitant expression of sulfomucins in the gastric epithelium showed no significant difference by gender. Table 2 shows the age distribution of normal and abnormal expression of Le” antigen in 122 Le(a-b+) individuals with IM. A steady increase in prevalence of abnormalities with age is detected: 53% before age 40; 70% at ages 40-59, and 81% after age 60. This tendency is caused by pattern 3 abnormalities. Table 3 shows the distribution of sulfomucins in goblet and columnar cells by histological diagnosis. In both cell types, sulfomucins are more prevalent in more advanced lesions. Sulfomucins in columnar cells are seen only in colonic metaplasia or dysplasia. In the present series, dysplasia was always seen in a background of IM. Table 4 shows the anomalous Le” antigen expression in different types of gastric cancer precursors. The abnormal expression increases in prevalence as the degree of histological abnormality progresses: 42% for SIM, 77% for MIM, and 87% for dysplasia. This tendency is more marked for pattern 3. Table 5 shows a strong association of the anomalous expression of Le” antigen and the presence of sulfomucins in goblet and columnar cells (P < 0.001). All cases with sulfomucins in columnar cells had abnormal expression on Le” antigen. Ninety-six percent of the gastric biopsy specimens with positive

Table 2. Number

ofCases and

Proportional Distribution of of Le” in Individuals of

Abnormal

Expression

Lela-b+)

Phenotype

by Age

21-39

40-59

60-84

All ages

22 (29.7) 35 (47.3) 17 (23.0) 74

6 (19.4) 13 (41.9) 12 (38.7) 31

36 (29.5) 56 (45.9) 30 (24.6) 122

sulfomucin staining showed the Lewis abnormality (48/53), compared with 55% (38/69) of biopsy samples negative for sulfomucin. The prevalence of pattern 3 Lewis abnormality increased considerably as the degree of sulfomucin expression became more advanced. Table 6 shows the odds ratios of colonic metaplasia and dysplasia, given the independent or concomitant expression of sulfomucins and Lewis abnormalities. Patients with IM have increased risk of expressing colonic type (MIM) cell morphology if they have either sulfomucins or Lewis abnormalities; having both markers substantially increases the risk. This measure, however, is somewhat unstable, because only one patient with SIM had both markers. In the case of dysplasia, the increased relative risk (11.0) associated with the presence of both markers, in comparison with either marker alone, appears more stable. Three of the five subjects negative for Lewis alterations and positive for sulfomucins had only minimal (borderline) staining for sulfomucins. The above results are not altered after adjusting for sex. Discussion The relevance of mucosal alterations of Lewis antigens in individuals at high risk for gastric cancer resides in their potential role in the precancerous process. Lewis antigens are prominent in the surface of tumor cells and have in the past been described as “oncofetal” antigens, markers of the neoplastic

Table 3. Number of Cases and Percentage Distribution Sulfomucins in Goblet and Columnar Cells Histological diagnosis Sulfomucins

Le” anomaly Negative (%) Patterns 1 and 2 (%) Pattern 3 (%) Total

a (47.1) a (47.1) 1 (5.9) 17

of

G-CG+CG+C+ Total

(%) (%) (%)

SIM

MIM

Dysplasia

Total

35 (92.1) 3 (7.9) 0 (0) 38

17 (54.8) 9 (29.0) 5 (16.1) 31

7 (32.1) 22 (41.5) 14 (26.4) 53

69 (56.6) 34 (27.9) 19 (15.6) 122

G, goblet cells; C, columnar

cells.

428 TORRADO ET AL.

Table

4. Number of Cases and Percentage Distribution of Abnormal Expression of Le” in Biopsy Specimens

GASTROENTEROLOGY Vol. 102,No. 2

neoantigens. 31The abnormal expression of Lea antigen in subjects of Le(a-b+) phenotype is frequent in With Metaplasia and Dysplasia gastric carcinomas1’~‘3~‘4 and in nontumoral gastric mucosa suspected to play a premalignant role.” Histological diagnosis The prevalence of Le(a-b-) phenotype in Nariiio Le” anomaly SIM MIM Dysplasia Total is somewhat higher than in Japanese (1.8%) and (5%-18%) and is comparable with Negative (%) 22 (57.9) 7 (22.6) 7 (13.2) 36 (29.5) white populations Patterns 1 and 2 (%) 14 (36.8)16 (51.6) 26 (49.1) 56 (45.9) the prevalence in some black populations (around Pattern 3 (%) 2 (5.3) 8 (25.8) 20 (37.7) 30 (24.6) 22%).29 These findings are consistent with the Total 38 31 53 122 marked interpopulation variability in the prevalence of Lewis phenotypes. The local population in the present study has a predominantly Indian somatic phenotype. Because the same high prevalence transformation of tissues. Further research has of Le(a-b-) phenotype was found in volunteers from pointed out that they undergo specific prenatal and a random household survey of the town of Tuquerres postnatal changes during the development of tissues (Table l), we do not favor an explanation based on a and organs. Similar, but less predictable, changes are generalized loss of Lewis antigen expression as part observed during malignant transformation. They of an early precancerous process affecting a large have been linked to cell growth, control, adhesion, proportion of the population.3g Such explanation, and cell recognition. 30,31The population of Nariiio, however, is not ruled out. This phenomenon has Colombia, is appropriate for this type of study bebeen observed in erythrocytes of cancer patients cause of its documented high risk of gastric cancer with high levels of CA 19.9.3g In our extensive experiand precancerous lesions.“‘5-‘7 The subjects of this ence with advanced gastric carcinoma, the loss of report represent adequately the population at large; Lewis antigens has never been detected in the nonthe phenotypic distribution of Lewis antigens and tumoral mucosa.‘3,14 secretor status in the study group did not show signifIt has been postulated that the precancerous proicant differences compared with the local voluncess represents a sequence of cellular alterations inteers. Nevertheless, there are significant differences volving at least seven stages.’ Two such stages, when these groups are compared with the white popnamely, colonic metaplasia and dysplasia, are of speulation of Europe and the United States”; the people cial importance because they represent advanced from Nariiio show a higher percentage of Le(a-b-) changes and, therefore, could be used to identify individuals (24.4%), associated with a lower prevahigh-risk individuals. Both are statistically assolence of Le” phenotype (6%7% vs. >20% in most ciated with the expression of sulfomucins. Our repopulations). Another important fact to emphasize is sults indicate that the abnormal expression of Le” the absence of individuals with Le(a-b-) nonsecreantigens correlates positively with such histological tor phenotype in the study group. and histochemical alterations. The concomitant exAlterations in the expression of blood group antipression of both markers is a better indicator of cogens of the Lewis system are currently used for the ionic metaplasia and dysplasia. diagnosis of cancer and precancerous lesions: These The above results support the notion of a continantigens are especially abundant in mucosecretory uum of phenotypic alterations in the precancerous epithelia. ABH antigens are secreted in the saliva, process. The cellular and architectural alterations gastric juice, milk, and other fluids in approximately identifiable with H&E stains (colonic metaplasia and 70%-80% of subjects of European extraction”; their dysplasia) are intimately associated with alterations secretion is dependent on the presence of the secretor gene. In our study, ABH expression in the gastric epithelium was 100% correlated with their expresTable 5. Number ofCases and Percentage Distribution of sion in the red blood cells and the vascular endoAbnormal Expression of Le” by Expression of thelium, as seen in histological sections (internal Sulfomucins in Goblet and Columnar Cells control). A and B antigens have been reported abnorSulfomucin expression mally expressed in blood group 0 patients in neoplastic and preneoplastic tissues.32-35 No ABH alterG+C+ Total G-CG+CLe” anomalv ations were detected in our series. Negative (%) 31 (44.9) 5 (14.7) 0 (0) 36 (29.5) Alterations in the expression of Lewis antigens Patterns 1 and 2 (%) 30 (43.5)19 (55.9) 7 (36.8) 56 (45.9) have been described in gastrointestinal neoPattern 3 (%) 8 (11.6) 10 (29.4) 12 (63.2) 30 (24.6) plasms.36-38 These alterations consist of the loss of Total 69 34 19 122 isoantigens, caused by incomplete synthesis and G, goblet cell; C, columnar cell. resulting in synthesis of aberrant glycosilation,

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Table 6. Odds Ratios for Intestinal

Metaplasia

With a

429

Colonic Component and Dysplasia According to Abnormal Patterns

of Sulfomucins and Lewis Antigens No. of cases

Odds ratios (95% CI)

Le

S,,”

SIM

MIM

D

Total

MIM/SIM

D/M

_

_

_ + +

+ _ +

20 2 15 1

5 2 12 12

6 1 11 35

31 5 38 48

1.0 4.0 (0.5-35.8) 3.2 (0.9-11.1) 48.0 (5.0-461.7)

1.0 1.04 (0.1-11.1) 1.7 (0.5-5.3) 11.2 (3.7-33.5) x2, b = 20.53; P < 0.0001

xzl

M, metaplasia, all types; CI, confidence “Abnormal patterns of sulfomucins. bxz for linear trend (1 df).

intervals.

in the mucin secretions that can be identified by histochemical (sulfomucins) or immunochemical (Lewis antigens) methods. The severity of the Lewis alterations (patterns 1-3) correlate with the severity of the morphological alterations (SIM-MIM-dysplasia) and with the severity of sulfomucin expression (G+C-, G+C+). The expression of abnormal Le” antigen in Le(a-b+) subjects (who constitute approximately 70%-80% of the population) is a highly sensitive tool that may prove useful in identifying individuals at highest risk for developing gastric cancer. Its usefulness is enhanced when sulfomucin stains are also available. References 1. Correa P. A human 2.

3.

4. 5.

6.

7.

8.

9.

10.

b = 13.81; P = 0.0002

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Received May 31, 1991. Accepted June 24,199l. Address requests for reprints to: Pelayo Correa, M.D., Department of Pathology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, Louisiana 70112. Supported by a grant from Consejeria de Salud de1 Gobierno Vasco and grant POl-CA28842 from the National Cancer Institute.