Citokeratina en Esofago de Barret

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Cytokeratin Subsets for DistinguishingBarretts Esophagus From Intestinal Metaplasia

in the Cardia Using Endoscopic Biopsy SpecimensHala M. T. El-Zimaity, M.D., and David Y. Graham, M.D.

Gastrointestinal Mucosa Pathology Laboratory and Departments of Medicine and Pathology, Veterans

Affairs Medical Center and Baylor College of Medicine, Houston, Texas

OBJECTIVES: It has been suggested that Barretts epithelium

and intestinal metaplasia in the gastric cardia have different

cyotokeratin (CK) staining patterns and that Barretts epi-

thelium can be distinguished by CK staining pattern. The

aim of this study was to test the utility of CK staining fordistinguishing Barretts esophagus from gastric intestinal

metaplasia.

METHODS: Topographically mapped gastric biopsy speci-

mens were obtained from patients without Barretts esoph-

agus, and esophageal biopsies were obtained from patients

with long-segment Barretts esophagus (3 cm). Serial

sections were stained with Genta or El-Zimaity triple stain,

and biopsies with intestinal metaplasia were stained with

antibodies against CK 4, 13, 7, and 20.

RESULTS: Sections from 33 biopsies with Barretts esopha-

gus, 23 with intestinal metaplasia of the gastric cardia, 27with intestinal metaplasia of the gastric body, and 33 with

intestinal metaplasia of the antrum were examined. CK 4

and CK 13 stained squamous epithelium only. The proposed

diagnostic CK Barretts 7/20 pattern was found in only

39% of long-segment Barretts compared to 35%, 4%, and

24% in intestinal metaplasia from the gastric cardia, body,

and antrum, respectively. The criteria proposed had a sen-

sitivity of 45% and a specificity of 65%.

CONCLUSIONS: These results do not support keratin pheno-

typing as a tool for differentiating intestinal metaplasia

originating in the cardia from intestinal metaplasia of

Barretts. (Am J Gastroenterol 2001;96:13781382. 2001by Am. Coll. of Gastroenterology)

INTRODUCTION

Intestinal metaplasia is a common finding in biopsy speci-

mens taken from just below the gastroesophageal junction in

patients with current or past Helicobacter pylori infection

(15). This finding has prompted considerable discussion

regarding whether it is a variant of gastroesophageal reflux

disease (GERD) (6 9), (e.g., short-segment Barretts), a

consequence ofH. pyloriof the stomach, or both (15).

Barretts esophagus is considered a premalignant condi-

tion such that the distinction between H. pylorirelated

intestinal metaplasia of the cardia and short-segments Bar-

retts esophagus is important. Recently, Ormsby et al.(10),

using antibodies to cytokeratin (CK) 7 and CK 20, found

that the staining pattern of gastric intestinal metaplasia wasentirely different from that of Barretts epithelium. The

Barretts CK 7/20 pattern was defined as staining of the

superficial epithelium with CK 20 and staining of both the

superficial and deep metaplastic epithelium with CK 7. This

pattern was present in 97% of specimens with long-segment

Barretts and was not observed in gastric intestinal meta-

plasia (10). They hypothesized that the Barretts CK 7/20

pattern was specific for Barretts epithelium.

The current study was designed to confirm their hypoth-

esis and to extend the observation to biopsies taken at

endoscopy by investigating the utility of CK 7, 20, 4, and 13

in the histological distinction of Barretts esophagus fromintestinal metaplasia in the cardia.

MATERIALS AND METHODS

Patients and Histology

Mucosal biopsy specimens were obtained from patients who

had previous upper GI endoscopy with gastric mapping

which typically involved taking 14 biopsies from specified

sites (11). The anatomic cardia was defined as the mucosa

immediately below the site of the junction of the mucosa of

the tubular esophagus and the stomach (Z-line) and above

the beginning of the first gastric fold. Biopsies of the gastric

cardia (mean and median of two biopsies) were always

taken antegrade (not retrograde with retroflexion of the

endoscope). None of the patients had tongues of gastric type

epithelium. Each biopsy was placed in a separate bottle of

10% buffered formalin. Biopsies were embedded on edge,

sectioned at 5 m with six sections per slide, and stained

with the Genta stain (12) or El-Zimaity triple stain (13). The

median size of biopsy specimens (fixed tissue measured on

a glass slide) was 8 4 mm. Each specimen was reviewed

by one pathologist and scored using a visual analog scale

from 0 (absent/normal) to 5 (maximal intensity) for intes-

tinal metaplasia (14).

THE AMERICANJOURNAL OF GASTROENTEROLOGY Vol. 96, No. 5, 2001 2001 by Am. Coll. of Gastroenterology ISSN 0002-9270/01/$20.00Published by Elsevier Science Inc. PII S0002-9270(01)02355-3


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Cases were selected because of previously documented

intestinal metaplasia. Indications for upper GI endoscopy

included duodenal ulcer, gastric ulcer, and previous diag-nosis of intestinal metaplasia. Cases with previously docu-

mented long-segment Barretts metaplasia were obtained

from the files of the Veterans Affairs Medical Center.

High IronDiamine Staining

Biopsies with intestinal metaplasia were stained with high

irondiamine/Alcian blue (HID/AB) to identify neutral,

sialo-, and sulfomucins. Briefly, slides were immersed in

HID solution for more than 18 h, at 2325C. Slides were

then rinsed with deionized water and stained with 1% Alcian

blue, pH 2.5 for 2 min (15). Subtyping intestinal metaplasia

was done according to the system used by Jass and Filipe

(16, 17). Type I is classified as complete intestinal metapla-sia and type II and III are grouped incomplete metaplasia.

Immunohistochemical Studies

For immunophenotyping, 5-m thick sections were stained

using a modified streptavidin-biotin complex method with

antigen retrieval as required. Briefly, the following reagents

were used in sequential steps at 36C: inhibitor for endog-

enous peroxidase, primary antibody for 12 h, biotinylated

secondary antibody, avidin-biotin complex with horseradish

peroxidase, 3,3-diaminobenzidine tetrahydrochloride

(DAB). When indicated, slides were pretreated for antigen

retrieval by steam for 15 min in a Black and Decker(Schaumburg, IL) steamer in 10 mmol/L citrate buffer (pH

6.0), followed by cooling for 20 min. Slides were counter-

stained with hematoxylin. The antibody panel included CK

7, 20, 4, and 13 from Dako (Carpinteria, CA). After grading

slides for intensity of stain, intestinal metaplasia glands

were visually divided into thirds (upper, middle, and lower).

The staining pattern was considered superficial if the upper

third or two thirds was positive. Slides were then analyzed

for the different patterns as defined by Ormsby et al.(10),

e.g., superficial staining with CK 20 and strong CK 7 stain-

ing of both superficial and deep glands was defined as a

Barretts CK7/20 profile. The whole area of metaplasia was

also screened for patchy or diffuse staining.

Statistical Analyses

Scores were analyzed using Sigma Stat (Jandel Scientific

Software, San Rafael, CA). Fishers exact test or, whenappropriate, the 2 test (both two-tailed) were used for

comparison of proportions.

RESULTS

Intestinal Metaplasia

A total of 116 biopsies with intestinal metaplasia from 102

patients were examined (33 Barretts esophagus, 23 cardia,

27 body, and 33 antral). CK 4 and CK 13 stained squamous

epithelium only; areas with intestinal metaplasia remained

unstained. Intestinal metaplasia was incomplete in 79% and

65% of patients presenting with intestinal metaplasia in the

esophagus and cardia, respectively.

Intestinal Metaplasia in Barretts

A total of 33 biopsies from 29 patients with Barretts esoph-

agus were examined. The proposed diagnostic CK Barretts

7/20 as defined by Ormsbyet al.(10) was found in only 39%

of biopsies (Table 1) (Fig. 1).

Incomplete intestinal metaplasia was present in 27 biop-

sies from 23 patients; 11 biopsies had the esophageal pat-

tern, 12 biopsies had a gastric pattern, and four had other

patterns. Two patients had multiple biopsies. The first pa-

tient had two biopsies and both had the esophageal pattern.

The second patient had four biopsies, one had an esophagealpattern, and three had a gastric pattern.

Six biopsies from six patients had complete intestinal

metaplasia. Only three (50%) had the diagnostic Barrett

pattern.

Intestinal Metaplasia in the Stomach

A total of 83 gastric biopsies with intestinal metaplasia were

examined (23 cardia, 27 body, and 33 antral).

Intestinal Metaplasia in the Cardia

In all, 23 biopsies from 18 patients were examined. The

proposed diagnostic CK Barretts 7/20 pattern as defined

by Ormsby et al. (10) was found in eight of 23 biopsies

Table 1. Cytokeratin 7/20 With Barretts Esophagus and Gastric Intestinal Metaplasia, by Site

DiseaseN

(biopsies)Barretts CK7/20

PatternGastric CK7/20

PatternOther

Patterns

Barretts esophagus 33 13 (39%) 15 (15%) 5 (15%)6 complete 2 (33%) 3 (50%) 1 (17%)

27 incomplete 11 (41%) 12 (44%) 4 (15%)

Gastric intestinal metaplasiaCardia 23 8 (35%) 7 (30%) 8 (35%)

8 complete 2 (25%) 1 (13%) 5 (63%)15 incomplete 6 (40%) 6 (40%) 3 (20%)

Body 27 1 (4%) 7 (26%) 17 (63%)12 complete 0 5 (42%) 7 (58%)11 incomplete 1 (9%) 3 (27%) 7 (64%)

Antrum 33 8 (24%) 5 (15%) 20 (61%)14 complete 1 (7%) 2 (14%) 11 (79%)19 incomplete 7 (37%) 3 (16%) 9 (47%)

1379AJG May, 2001 Cytokeratin Subsets in Barretts Versus Stomach


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(35%), (Table 1). Incomplete intestinal metaplasia was

present in 15 biopsies from 11 patients. The esophageal

pattern was present in six (40%) (Fig. 2). Four patients had

two biopsies each. An esophageal pattern was seen in both

biopsies in one patient; the second patient had a gastric

pattern in both biopsies; and two patients had a mixed

pattern (i.e., one esophageal and one gastric in one patient,

and one gastric and one other pattern in the other patient).

Complete intestinal metaplasia was present in eight bi-

opsies from seven patients. The diagnostic pattern was

present in two of eight biopsies with complete intestinal

metaplasia (25%). One patient had two biopsies, one biopsy

had an esophageal pattern and the other biopsy had super-ficial staining of the glands with CK 7 and CK 20 (other

pattern).

Intestinal Metaplasia in the Corpus

The proposed diagnostic CK Barretts 7/20 pattern as de-

fined by Ormsby et al. (10) was found in only one of 27

biopsies (3%) (95% C.I. 0% to 20%) (p 0.0036 com-

pared to Barretts) (Table 1). The proposed diagnostic CK

Barretts 7/20 pattern was found in only one of 11 biopsies

(11 patients) with incomplete intestinal metaplasia. Al-

though CK 7 stained two cases with incomplete intestinal

metaplasia in the body, the characteristic Barretts stain-

ing of the entire length of the gland was observed in one

patient only. Similarly, complete intestinal metaplasia was

present in 12 biopsies from 11 patients; none had the Bar-

retts pattern.

Intestinal Metaplasia of the Antrum

The proposed diagnostic CK Barretts 7/20 pattern as de-fined by Ormsbyet al.(10) was found in nine of 33 biopsies

(27%) (Table 1). Incomplete intestinal metaplasia was

present in 19 biopsies from 16 patients. Seven biopsies

(37%) had the esophageal pattern. Three patients had two

biopsies each. One patient had an esophageal pattern and a

gastric pattern. The other patient had an esophageal pattern,

and superficial staining with CK 20 with no staining with

Figure 1. Representative section for cytokeratins 20 (A) and 7(B) in the esophagus. The proposed diagnostic pattern was observed in only39% of biopsies. Contrary to the proposed diagnostic pattern, CK 20 was expressed in the entire length of the gland in 17 biopsies (59%),and CK 7 was superficial or mixed, with areas of no staining in eight cases (28%).

Figure 2. Representative section for cytokeratins 20 (A)and 7 (B) in the cardia. The diagnostic cytokeratin Barretts 7/20 was found in35% of biopsies. Superficial staining with CK 20 was observed in 65% of biopsies, and CK 7 stained the entire length of the gland in 52%.

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