Annals of RSCB Vol. XV, Issue 2

IMUNOHISTOCHEMICAL STUDY ON BARRETT S OESOPHAGUS SEQUENCE DYSPLASIA GASTRO-ESOPHAGEAL ADENOCARCINOMA Doina-Carina Voinescu 1, Carmen Popescu 2, Sajjad Yousaf 3, Elena Cioboată 3, B. Pintilie 3, M. Cruce 3 1 - UNIVERSITY OF MEDECINE AND PHARMACY OF GALATI 2 - LABORATORY OF PATOLOGY, No. 1 EMERGENCY HOSPITAL OF CRAIOVA 3 - UNIVERSITY OF MEDECINE AND PHARMACY OF CRAIOVA Summary The etiology of gastro-esophageal adenocarcinomas is related to gastro-esophageal reflux disease and the development of Barrett s esophagus or cardia intestinal metaplasia, a pre-neoplasic condition which is diagnosed endoscopically with an histological confirmation, an incomplete intestinal metaplasia. The medical societies have recommanded endoscopic cancer surveillance routinely for pacients with Barrett s metaplasia or cardiac intestinal metaplasia. Earlier detection of the metaplasia-dysplasia-gastro-esophageal adenocarcinoma and distinction between Barrett s metaplasia and cardia intestinal metaplasia are essential for the aplication of the prophylaxis of malignant transformation. The rising incidence and poor prognosis of gastro-esophageal adenocarcinomas have intensified research into earlier methods of detection of this diseases and the relationship to Barrett s esophagus. In the present study we investigates several immunohistochemical features of gastro-esophageal adenocarcinomas, 87 resection specimens of Barrett s esophagus and adenocarcinoma junction were carefully selected. We chosed the immunohistochemical study for MUC1, MUC2 and MUC5AC mucins for evidence the lesions of Barrett's oesophagus sequence dysplasia adenocarcinoma and the potential of utilization in the early detection of gastroesophageal adenocarcinomas junction. Keywords: Barrett s oesophagus; cardiac intestinal metaplasia; dysplasia; adenocarcinoma junction; immunohistochimical methods. doina_voinescu@yahoo.com Introduction Barretts's oesophagus represents the replacement of the normal squamous epithelium of the lower oesophagus by specialised intestinal metaplasia, under the influence of chronic gastro-oesophageal reflux (Spechler et al., 1996; Weinstein et al., 1996; Kim et al., 1997; DeMeester et al., 2000). Barrett s oesophagus is a premalignant condition that predisposes to the development of oesophageal adenocarcinoma. The intestinal metaplasia of the gastroesophageal junction, as a result of a chronic inflammation, irrespective of etiology, is considered an important risk factor for the development of the lesion towards dysplasia and gastroesophageal carcinoma junction. Dysplazia is a histopatological marker for the early diagnosis of adenocarcinoma at the level of gastro-oesophageal junction, as evidenced by the simultaneous presence of the same resection pieces on both dysplasia and neoplasia (Sampliner 1998; Spechler, 2002). 252

According to the World Health Organization, the gastro-oesophageal adenocarcinoma's junction are represented by: the distal oesophagus adenocarcinoma, the gastric cardia adenocarcinoma and the undercardia adenocarcinoma. The distal oesophagus adenocarcinoma and gastric cardia adenocarcinoma seem to predominate in the case of Caucasian men (M:F=7:1) (Blot et al, 1991; Lagergren, 2005), with a similar average age (56-62 years), the diagnosis being late - in advanced stages of disease and the prognosis being unfavorable, with 5-year mortality rates exceeding 80% (Ruol et al, 2000). Surgical cure rates (subtotal esophagectomy and proximal gastrectomy) are compromised by the fact that most patients are diagnosed at the late stage of disease of the delayed onset of symptoms. In this study I have made a detailed analysis within 87 cases of Barretts's oesophagus sequence - dysplasia adenocarcinoma at the level of gastrooesophageal junction. The immunohistochimical study included the analysis for MUC1, MUC2 and MUC5AC mucins, the markers possible useful for the precocious diagnosis of Barrett's oesophagus sequence dysplasia junctional adenocarcinoma. Materials and methods Patients The material for this study is based on the oesophagian biopsy specimens collected from the gastro-oesophageal sections and on the surgical resection specimens; the material has been worked out in the Histopatological Laboratory from No. 1 Emergency Hospital of Craiova and within the framework of Victor Babes Institute of Bucharest. Between 2000-2009 from 587 of cases diagnosed with gastric cancer we selected 68 cases of gastro-oesophageal adenocarcinoma junction. Also from 154 cases diagnosticated in the same periode with oesophagian biopsy, we selected 19 cases of benign and malignant Barrett's oesophagus. The patients included in this study presented symptoms of gastro-esophageal reflux disease which included: heartburn and regurgitation at least twice within 1 week and persisted at least six months, all the symptoms could be relieved significantly with H2 receptor blocker or proton pump inhibitor, odynophagia with progresiv dysphagia addition decrease weight, occult haemorrhages (seldom). Endoscopy and biopsy protocol Endoscopic dates included macroscopic changes to the gastro-esophageal junction: a red velvety mucosa it can extend either circumferentially or as one or several tongues, polipoid lesions, ulcerations, presence or absence of hiatus hernia. For each resection specimen a sample of tumor tissue and normal tissue was available in paraffin embedded slides. The biopsy protocols included four quadrant biopsies at 2 cm levels in distal esophagus above and below the esophagogastric junction and sample of normal tissue. Specimens were fixed in 10% buffered formalin, embedded in paraffin wax and carefully microdisected. The paraffin sections used for the immunohistological exam were realized at 2 µm. They were mounted on glass slides treated with poly-l-lysine (SIGMA). Immunohistochemistry for MUC1, MUC2 and MUC5AC mucins We performed immunostaining on formalin fixed, paraffin embedded tissue sections using the immuno-enzyme polymer method name LSAB/HRP(Universal DAKO Labbeled Streptavidin Biotin 2 System Horseradish Peroxidase). Five-micrometer-thick serial sections were cut from each paraffinembedded block. The sections were deparaffinized in xylene and rehydrated through graded concentrations of alcohol slides were incubated in 3% hydrogen peroxide for 20 min to block nonspecific background staining due to endogenous peroxidase. Antigen retrieval was 253

performed by using 10 mm citrate buffer, ph 6.0 for 10-20 min in microwave. Using the standard streptavidin-biotin peroxidase complex method were performed on all slides, were incubated for 30 minutes in secondary antibody solution. Diaminobenzidine (DAB) was used as a chromogen and developed a brown precipitate, very fine, localizing the antigen searched in the cytoplasm. The slides were counterstained with Mayer s hematoxylin. Negative controls were counterstained with the same tumor samples and staining methods by omitting the primary antibodies. The antibodies used in immunohistological studies have identified the molecular biomarkers followed for the research of Barretts's oesophagus sequence dysplasia adenocarcinoma junction; the positive expression was represented by the membranar, cytoplasmic and nuclear brown precipitate. The antibodies and the working parameters are described in Table 1. Table 1: The selected polyclonal antibodies and sera and working parameters was : ANTIBODY PRODUCER DILUTION CLONE PECULIARITY MUC 1 Novocastra 1:100 Ma 695 mucus epithelial tissue MUC 2 Novocastra 1:100 Ccp 58 mucus epithelial tissue MUC 5AC Neomarkers 1:50 45M1 mucus epithelial tissue Mucins, intense glycosylated proteins, represent the major components of mucus covering the luminal epithelial tissues. Synthesis and secretion of mucins are characteristic of glandular epithelial tissues. In our study we used monoclonal antibodies like MUC-1, MUC-2 and MUC-5AC. MUC1 glycoproteine is a type of breast apomucin also known as DF3 antigen. We used a monoclonal antibody-type of MUC1 glycoprotein, clone Ma695, using LSAB + technique in 1:100 dilution. For positive control of this reaction we included in the study a fragment of the endometrium. Immunopositivation showed a cytoplasmic and membrane pattern. MUC 2 glycoprotein is an intestinal apomucin which is not synthesized by normal gastric mucosa. I worked with the antibody-type glycoprotein MUC-2, clone ccp58 in 1:100 dilution. Primary antibody incubation time was 60 minutes at room temperature. The immunohistology reactions showed a cytoplasmic and membrane staining pattern. Mucin 5AC (MUC5AC) / B-1 gastric mucin outlined in mucosecretant columnar cells of surface gastric epithelium and in caliciform cells of fetal colon or in cells of colon that presents various precancerous lesions. In our study we used monoclonal antibody MUC5AC, clone 45M1 in dilution 1:50. I got positive imunoreacţii cytoplasm and the cell surface. Results From 87 cases of the current study it was identified 19 cases of Barrett s oesophagus with benign (16) and malignant lesions (3). Also, there were 68 cases of adenocarcinoma junction divided topographically: at the distal esophagus (3 cases), at the junction (48 cases) and subcardial (17 cases). To monitor the degree of dysplasia was preferred the classification defined by Riddell et al., (1983) and the distribution of 19 cases is shown in Table 2. 254

Annals of RSCB Vol. XV, Issue 2 Table 2. The correlation between the degree of dysplasia and the number of cases in the study The degree of dysplasia Nondisplasic Barrett oesophagus Low- grade dysplasia Barrett (LGD) High- grade dysplasia Barrett (HGD) Oesophageal adenocarcinoma Indefinite for dysplasia Number of cases 5 6 2 3 3 In the 68 cases of esophageal adenocarcinoma junction subject in the current study we identified the histopathologic type in accord to Lauren P classification (table 3). According to the World Health Organization, 68 cases of junctional adenocarcinoma were divided into three groups : G1 well differentiated adenocarcinoma (29 cases), G2- moderate differentiated adenocarcinoma (23 cases) and G3weak differentiated adenocarcinoma (16 cases). Mucinous adenocarcinomas, the undifferentiated frome those with "signet ring" with a tumoral component greater than 5% were included in G3. Table 3. Histopathological type of gastro-oesophageal adenocarcinoma junction Histopathological type Nr. of cases 52 7 Tubulopapillary Signet ring Mucinos 4 Undifferentiated 5 The results of immunohistochimical study of MUC1, MUC2 and MUC5AC mucins revealed that : MUC5AC expression is strong diffuse in Barrett's oesophagus. The MUC2 expression is constantly negative and MUC1 expression is focal positive and inconstant in Barrett's oesophagus (Figure 1; 2). Also, the MUC1 expression is strong positive in G2 (3 cases) and G3 (16 cases) adenocarcinoma (Figure 3; 4) and MUC2 expression is positive in mucinos adenocarcinoma (4 cases). Figure 1. Positive MUC1 immunostaining in glandular Barrett epithelium and absent MUC1 immunostaining in squamous epithelium. Cytoplasmic and membranar brown immunostaining that localize the antigen. IHC staining, ABC complex, counterstain by Mayer s haematoxylin. Ob. 40x 255

Annals of RSCB Vol. XV, Issue 2 Figura 2. Positive MUC5AC immunostaining in glandular Barrett epithelium. Cytoplasmic and membranar brown immunostaining that localize the antigen. IHC staining, ABC complex, counterstain by Mayer s haematoxylin. Ob. 40x Figure 3. Strong positive MUC1 immunostaining in moderate differentiated adenocarcinoma G2 (săgeată). Cytoplasmic and membranar brown immunostaining that localize the antigen. IHC staining, ABC complex, counterstain by Mayer s haematoxylin. Ob. 40x 256

Figure 4. Positive MUC1 immunostaining in weak differentiated adenocarcinoma G3. Cytoplasmic and membranar brown immunostaining that localize the antigen. IHC staining, ABC complex, counterstain by Mayer s haematoxylin. Ob. 100x Immunohistochimical expression of mucins is shown in Table 4. Table 4. Immunohistochimical expression of MUC1, MUC2 and MUC5AC mucins. Total cases adenocarcinoma Localisation Nr. cases MUC1 MUC2 MUC5AC 68 ++ -/+ + Distal oesophagus 3 ++ -/+ + Gastro-esophageal junction 48 ++ + ++ Undercardial 17 ++ -/+ + Barrett's Metaplasia Present 7 ++ -/+ + Missing/absent 61 ++ -/+ + histopathological type Tubulopapillary 52 ++ -/+ ++ Signet ring 7 -/+ -/+ ++ Mucinos 4 ++ ++ ++ Undifferentiated 5 ++ - ++ -/+ weak positive or absent ;+ weak positive; ++ moderate ; +++strong positive 257

Discussion The current immunohistochemical study of mucins allowed the characterization of Barrett intestinal metaplasia based on the content of MUC-1, MUC-2 and MUC-5AC mucins. Characteristic for these mucins is the fact that these are not normally expressed in oesophagus, MUC-1 being a breast type apomucin, MUC 2 - an intestinal apomucin unexpressed in normal gastric mucosa and MUC-5AC is a mucin of surface gastric epithelium. MUC-1, MUC-2 and MUC-5AC immunostaining showed cytoplasmic and membrane staining. MUC-1 positive immunostaining was focal and inconsistently present in Barrett's epithelium and was missing in the squamos neighborhood epithelium. It was unable to determine a direct correlation between MUC-1 immunostaining and the degree of associated dysplasia. In cases of junctional adenocarcinoma MUC-1 expression was highly positive in 3 cases of moderately differentiated adenocarcinoma - G2 (13%) and in 16 cases of poorly differentiated adenocarcinoma - G3 (100%). The results of this study showed that MUC- 1 immunohistochemical expression absent in squamous epithelium near Barrett's epithelium and constant present in all junctional adenocarcinomas G-3 describes a marker of aggressiveness of the lesion studied. Inconsistent presence of MUC-1 positive expression in Barrett's epithelium was not allowed his assession as a malignisation marker. Data from the literature describes the absence of MUC-1 expression in metaplasic and dysplasic Barrett's epithelium and his presence in adenocarcinoma fact that suggest suggest its usefulness in distinguishing dysplasia from adenocarcinoma (Chinyama et al., 1999, Guillem et al., 2000). MUC-2 immunostaining was consistently negative in Barrett's epithelium and near squamous epithelium and with positive 258 expression in all 4 cases of mucosecretant adenocarcinoma presented in the study. These results have not allowed to evaluate the clinical usefulness of MUC-2 immunostaining the precocious diagnosis of Barrett's esophagus-sequence-dysplasiaadenocarcinoma. It was noticed only the clear expression of MUC-2, an intestinal mucin, in mucinous adenocarcinoma, poorly differentiated. Our data are inconsistent with the literature, Chinyama et al. (1999), Guillem et al., (2000), describing the aberrant expression of MUC-2 in Barrett's intestinal mucosa, expressions that are lost when the epithelium becomes neoplasic. MUC-5AC mmunostaining (mucin of gastric surface epithelium) was diffusely and weakly positive in cases of chronic esophagitis and Barrett's epithelium. We could not establish a correlation between the intensity of MUC-5AC immunostaining and the degree of Barrett dysplasia. MUC-5AC expression was weakly positive in distal esophageal adenocarcinoma and moderately positive in the gastroesophageal adenocarcinoma junction. Moderately positive expression MUC-5AC was expressed in all gastro-oesophageal junction adenocarcinomas regardless of histopathological type of neoplasia. The results obtained have not allowed to evaluate the usefulness of MUC-5AC immunostaining in the precocious diagnosis of Barrett's oesophagus-sequencedysplasia-junction adenocarcinoma, however immunostaining could be useful for the differentiation of oesophageal adenocarcinomas from those of gastric cardia adenocarcinoma, the last one expressing more intense reactions of MUC- 5AC. However, we can not consider immunostaining MUC-5AC as a reliable marker in differentiating tumors according to location, since we observed positive expression in adenocarcinomas withhout associated Barrett's metaplasia and also in adenocarcinomas with associated Barrett's epithelium.

Conclusions We concluded that molecular biomarkers evolved in this study: MUC1, MUC 2 and MUC5AC mucins have just described already diagnosed aggressive neoplasia, showing no value in the precocious diagnosis of premalignant stages during Barrett's oesophagus sequence dysplasia adenocarcinoma junction. The current marker of the esophagogastric adenocarcinoma precocious diagnosis is dysplasia, and the mucins immunohistochemistry with cell cycle abnormalities and genetic changes studies may constitute the future preneoplastic screening methods. References Blot W.J., Devesa S.S., Kneller R.W., Fraumeni JF. Rising incidence of adenocarcinoma of the esophagus and gastric cardia JAMA, 265:1287 9, 1991. Chinyama C.N., Marshall R.E., Owen W.J., Expression of MUC1 and MUC2 mucin gene products in Barrett s metaplasia, dysplasia and adenocarcinoma: an immunopathological study with clinical correlation. Histopathology, 35:517 24, 1999. DeMeester S.R., DeMeester T.R. Columnar mucosa and intestinal metaplasia of the esophagus: fifty years of controversy. Ann Surg; 231: 303 21, 2000. Guillem P., Billeret V., Buisine M.P., Mucin gene expression and cell differentiation in human normal, premalignant and malignant esophagus. Int. J. Cancer, 88: 856 61, 2000. Kim. R., Weissfeld J.L., Reyonalds J. C. and Kuller L.H. Etiology of Barrett s metaplasia and esophageal adenocarcinoma. Cancer Epidemiol. Biomarkers Prev., 6: 369-377, 1997. Lagergren J. Adenocarcinoma of oesophagus: what exactly is the size of the problem and who is at risk? Gut; 54 (Suppl. I):i1-i5, 2005. Riddell R.H., Goldman H., Ransohoff D.F.,. Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications. Hum. Patho.l;14: 931 68, 1983. Roul A.,Parenti A.,Zaninotto G., Merigliano S., Constantini M.,Cagol M. Intestinal Metaplasia Is the Probable Common Precursor of Adenocarcinoma in Barrett Esophagus and Adenocarcinoma of the Gastric Cardia. American Cancer Society: 2520-2528, 2000. Sampliner RE. Practice guidelines on the diagnosis, surveillance, and therapy of Barrett s esophagus. The Practice Parameters Committee of the American College of Gastroenterology. Am. J.Gastroenterol. ;93:1028 32, 1998. Spechler S.J., Goyal R.K. The columnar-lined esophagus, intestinal metaplasia, and Norman Barrett. Gastroenterology;110: 614 21, 1996. Spechler S.J. Columnar-lined esophagus. Definitions. Chest Surg. Clin. N. Am.;12: 1 13, 2002. Weinstein WM, Ippoliti AF. The diagnosis of Barrett s esophagus: goblets, goblets, goblets. Gastrointest. Endosc. 1996;44: 91 5, 1996. 259