Peanut lectin histochemistry of Barrett ' s esophagus

Barrett’s epithelium refers to the replacement of the normal stratified 
squamous epithelium with columnar epithelial cells, possibly as a consequence 
of chronic reflux of gastric content into the esophagus, and is thought to be a 
pre-neoplastic disorder with the later development of esophageal adenocarcinoma. 
In this study, biopsy specimens from patients with Barrett’s esophagus 
were examined with fluorescein-linked peanut lectin to determine if lectin reactivity, 
indicative of a previously reported colon cancer-associated mucin in colonic 
polyps, was present in the columnar lined esophagus. In all of the patients 
studied, positive but variable labelling with this lectin was present, providing 
additional evidence that Barrett’s epithelium represents a heterogeneous preneoplastic 
change of the esophagus.

B ARRETT'S ESOPHAGUS IS DEFINED AS one that is lined with columnar epithelium instead of typical stratified squamous epithelium ( l ,2).It is generally thought that in adults this disease results from ch ronic or recurrent reflux of gastric content into the esophagus with the progressive replacement of squamous epithelium by columnar epithelium in a cephalad direction.This epithelium has been well characterized using h istological (3), cell kinetic (4).histochemical (5-7) and immunocytochemical methods (8).Esophageal strictures and ulcers are frequently observed complications associated with Barrett's esophagus (2); in add ition, adenocarcinoma of the esophagus has been observed (9) with an estimated prevalence of 8.5% ( 10).C urren t treatment is aimed at preventing gastroesophageal reflux; endoscopic surveillance sho uld be considered because of the potential late r developme nt of carcinoma.
Previous studies have explored alterations in gastrointestinal mucins associated with the development o f cancer, particularly colonic adenocarcinoma, in chemically induced animal models ( 11 , 12) as well as in human colonic ade nomas and carcinomas ( 13, 14).Fluorescent lectins a re proteins that appear to identify specific carbohydrate structures on fixed tissue sections; one example is pea-nut lectin.Altered labe lling of goblet cell rnucins with peanut lectin has been reported as a cancer-associated mucin change in benign colonic polyps, which are precancerous lesions in the human colon ( 14).As Barrett's esophagus is purported to be a pre-neoplastic disorder of the esophagus (9).this study examined mucosa I biopsies in a se ries of patients with Barrett's esophagus to determine if labelling with this lectin was present.

PATIENTS AND METHODS
Patients and biopsies: A total of 12 5 endoscopic esophageal biopsies were examined from 22 patients including 13 males and nine females seen over a two year period.The number of biopsies and clinical details from each patient are summarized in Table I.The majority of patients (86%) were more than 50 years o ld .Most had radiographic and endoscopic evidence of esophageal stricture (64%).One patient (case 19) had a total gastrectomy with esophagojejunostomy for gastric antral adenocarcinoma, and subsequently developed stricture proximal to the surgical anastomosis.A second patient (case 3) had radiotherapy for squamous cell carcinoma of the esophagus.A third (case 9) had a coexistent colloid carcinoma of the esophagogastric junction.The clinical details of these 22 patients were previously defined (8).All endoscopic biopsies were obtained under direct vision from the esophagus with an Olympus fibreoptic gastroscope between 20 and 35 cm from the incisor teeth.Each biopsy was routinely fixed and processed, embedded in paraffin and serially sectioned at S µm.Serial sections were also examined by routine light microscopy fo llowing staining with hematoxylin and eosin, periodic acid-Schiff reagent and alcian blue, pH 2.5.Only biopsies positive to periodic acid-Schiff reagent were examined, and these were classified as specialized columnar type if the surface epithelium stained blue with alcian blue.Lectin labelling: Deparaffinization, sequential washes in 95.80 and 70% alcohols to 0.0 l M phosphate buffered saline, pH 7.4, was followed by lectin labelling as previously described (15).Fluorescein isothiocyanace conjugated peanut lectin 186 (FITC-PNA) derived from A rachis hypogaea (Vector Laboratories, Burlingame, California) was used as previously described ( 15).A solution of the same lectin concentration containing the sugar hap-ten inhibitor, D-galactose, at a concentration of 0.3 M was used on adjacent control sections ( 15).Following preparation, observations were recorded using a Zeiss epifluorescence photomicroscope.

RESULTS
All of the patients in this study had esophageal biopsies that stained posi• tively with FITC-PNA.Including patients with specialized columnar epithelium in the esophagus, the surface epithelium and/or underlying glandular ep• ithelium were labelled with FITC-PNA .Adjacent control sections with the inhibitor sugar were uniformly negative (Figure I).The degree of labelling, as re• fleeted in the intensity of fluorescence, was variable, even in the same biopsy sections (Figure 2).Although labelling appeared to localize to mucus primarily in the supranuclear regions within the epithelial cells, some, often adjacent, goblet cells in the same endoscopic biopsies were weakly labelled or failed to label (Figure 3).In some biopsies, mucus associated with the luminal membrane of the surface epithelium was also labelled (Figure 4).but it was not pos-sible in this study to determine if this fluorescence was due to secreted and adherent mucus or, in part, to exposed sugars on the epithelial cell luminal membrane.

DISCUSSION
Prior studies using a variety of histo• logical (3), histochemical (5• 7) and immunocytochemical techniques (8) have demonstrated that muco:;al biopsies from patients with Barrett's esophagus repre• sent a morphological epithelial mosaic apparently in response to chronic reflux of gastric content into the esophagus.This study of a series of patients with Barrett's esophagus u sing a specific fluorescein-linked lectin, peanut agglutin in, revealed positive labelling in all patients similar to positive peanut lectin reactivity of goblet cell mucins reported previously in benign and malignant neoplastic colonic disease ( 13, 14).

Lectln histochemistry of Barrett's esophagus
Although positive lectin staining was present in all patients, no differences were seen in Barrett's epithelium of pa• ticnts with or without different types of cancer.This suggests that the epithelial mucins in Barrett's epithelium arc altered or incompletely glycosylatcd, chis being possibly related to abnormal or impaired epithe lium differentiation .All of the biopsies used in this study, however, were examined in a retrospective manner.A prospective study in Barrett's patients with later ca ncer development is necessary to define the usefulness of these observations in biopsy surveillance and the detection of precancerous change, and chronological or sequential study is required prior to the development of frank carcinoma to define the value of biopsy surveillance in clinical practice.In addition, the natural history of this mucin change, the effects of therapy and its reversibility deserve careful evalua- ) have also explored in detail the pattern of lectin binding to normal and pathological gastric mucosa, including intestinal metaplasia and gastric cancer; epithelial mucus was characterized by an absence of peanut lectin binding but strong binding ro poorly differentiated cells was reported ( 19).Although not entirely novel, studies of the gastric mucosa in Barrett's esophagus might also be examined.
Although all of the patients in this study were positive, the variable nature of lectin labelling for individual epithc-lial cells, even in the same biopsy sec• tions under identical labelling conditions, implies that there are significant differences in goblet cell mucin reactivity, lending further support to the concept that Barrett's esophagus is a very heterogeneous mucosa!disorder.Previous studies in this group of patient biopsies (8) using a cytochemical si lver staining technique as well as fluorescence and peroxidaseantiperoxidase single and double staining immunocytochemical methods, have demonstrated significant differences in the peptide-containing cell types of

TABLE 1
Patients with Barrett's esophagus • Number ,n parentheses indicates total number of esophageal biopsies exam,ned:t Previous squamous cell carcinoma esophagus treated wtlh radiotherapy ftve years earlier:t Colloid type odenocorc,nomo involving esophagogasfrtc Junction.§ Previous gastric antral odenocarcinomo followed by total gos trectomyond esophogo}ejunostomy