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Volume: Barrett's Esophagus
Chapter: Markers

Is impaired apoptosis as detected by immunohistochemistry sequence a potential marker for the development of dysplasia and carcinoma?

U. Halm, K. Caca, A. Tannapfel, J. Mössner (Leipzig)

According to the molecular model of tumorigenesis, development of malignant tumors may be considered as a disturbed balance of cellular replication, growth, differentiation, and apoptosis (programmed cell death) [1-3]. Several molecular changes have been identified in the transition of squamous esophageal epithelium through specialized intestinal metaplasia to adenocarcinoma. The most important genetic changes include altered function of oncogenes and tumor suppressor genes, impaired regulation of the cell cycle, aneuploidy, changes in cell adhesion molecules and apoptosis [4]. There have been extensive studies concerning mutations of tumor suppressor genes as p53 and p16, overexpression of the proliferating cell nuclear antigen, Ki67, and others, suggesting a pathogenetic role in the metaplasia-dysplasia-carcinoma-sequence of Barrett's esophagus [4-6]. In contrast data on the role of apoptosis in this sequence, which might help to assess the risk for development of malignancy are still limited.

Every cell in the body can undergo a stereotypic cell suicide termed apoptosis. Several morphological features distinguish apoptotic cells from cells that die in response to trauma or hypoxia. Apoptotic cell death is characterized by cell shrinkage, blebbing of the cytoplasmatic membrane, nuclear condensation and pathognomonic autodigestion of the genome into fragments that correspond in size to multiples of the amount of DNA found in individual nucleosomes [1-3].

Immunohistochemical detection of apoptosis in Barrett's esophagus

The in situ end labeling of fragmented DNA is a rapid and widely used method for the detection of apoptosis. It has the advantage that both the in situ end labeling and histologic features can be assessed simultaneously. For example, in this procedure free 3'-OH ends of DNA are elongated with digoxigenin-marked dUTP by terminal deoxynucleotidyl transferase, allowing the immunohistochemical detection of digoxigenin by antidigoxigenin antibodies [7]. The exclusion of necrosis, inflammation, and mitosis by histology confirmes the presence of apoptosis. Using this method we observed an increased rate of apoptosis in non-dysplastic intestinal metaplasia (IM) in 22 patients with Barrett's esophagus (6.8 ± 1.3%) compared with normal fundic epithelium of 9 patients without metaplasia (0.6 ± 0.4%). Moreover, a significantly lower rate of apoptosis in dysplastic tissue (3.1 ± 0.2%, n = 5) or adenocarcinoma (1.0 ± 0.3%, n = 9) was found (p < 0.01) [8]. Similar results were obtained by Hughes et al., who observed a decreasing rate of apoptosis during the metaplasia-dysplasia-carcinoma sequence with statistically significant differences between adenocarcinoma and dysplastic tissue or IM [9]. Katada et al. found a low apoptotic index determined by the in situ end labeling method for IM, but found nearly no apoptotic cells in dysplastic tissue or adenocarcinoma [10]. Other groups identified apoptotic bodies after labeling intraepithelial leucocytes with anti CD45 antibodies and counter-staining with haematoxylin and eosin and reported this method to be more reliable than DNA-laddering or the in situ end labeling. However, apoptotic cell death was lower in 13 patients with dysplastic tissue and 43 patients with adenocarcinoma compared to the compartment 4 of 12 patients with IM. The glandular proliferation to apoptosis index, which is calculated by dividing the values for proliferation by the apoptotic values was significantly increased during the metaplasia-dysplasia-carcinoma sequence [11]. The observation of decreased apoptosis has also been shown during the development of other gastrointestinal tumors [12].

Bcl-2 is a mitochondrial proto-oncogene of the Bcl-2 family that encodes an apoptosis blocking protein [13, 14]. The expression of bcl-2 protein can be detected by immunohistochemistry. In different gastrointestinal neoplastic lesions including adenomas and carcinomas of the colon and the stomach overexpression of bcl-2 protein was found [15, 16]. In Barrett's esophagus, however, results are conflicting. Using a monoclonal antibody to the bcl-2 protein Goldblum et al. studied bcl-2 expression in 36 esophageal resection specimens of patients with esophageal adenocarcinoma. Barrett's mucosa was present in each specimen: low-grade dysplasia (LGD) in 35, high-grade dysplasia (HGD) in 34, intramucosal carcinoma in 23 and submucosal carcinoma in 13 patients. Bcl-2 expression was absent in any of the cases of Barrett's mucosa with or without dysplasia or carcinoma. As a control, immunostaining was found in the proliferative zone of nonneoplastic tissue and in adjacent lymph follicles [17]. In contrast to Goldblum findings Katada et al. found positive bcl-2 , immunostaining in 13 (72%) patients with Barrett's metaplasia, 13 (100%) with LGD, 1 (25%) with HGD, 4 (40%) with well- or moderately differentiated adenocarcinoma and 2 (20%) with poorly differentiated adenocarcinoma [10].

Apoptosis may be triggered by the the cell membrane death receptor Fas (Apo1, CD95) after binding of Fas ligand (FasL). An immunohistochemical study found a decreased expression of Fas protein on the cellular surface in Barrett's esophagus with HGD or adenocarcinoma compared with normal squamous epithelium or Barrett's IM without metaplasia. In accordance an esophageal adenocarcinoma cell line (Seg1) showed a decreased membranous Fas expression, while retaining the wild type Fas protein in the cytoplasm, thereby rendering this cell line resistent to Fas mediated apoptosis [9].

The value of a diagnostic test depends on its sensitivity, specificity, positive and negative predictive values. All studies published so far comprised only small numbers of patients and did not evaluate the diagnostic efficacy in comparison to other markers. Moreover, different methods have been used to determine the frequency of apoptosis. The variability of apoptotic cell death seems high in different patients [8, 11]. Although evasion of apoptosis seems to be important in esophageal adenocarcinogenesis, at present, immunohistochemical staining of factors of the apoptotic pathway cannot predict the risk for development of adenocarcinoma in Barrett's esophagus. Before it can be used as a diagnostic tool, further studies are needed that evaluate these markers in comparison with established prognostic markers in a large group of patients.


Current studies indicate a decrease of apoptosis during the metaplasia-dysplasiaadenocarcinoma sequence of Barrett's esophagus. Because the diagnostic efficacy has not been evaluated, impaired apoptosis as determined by immunohistochemistry can not by used at present as a marker for development of malignancy in Barrett's esophagus. Further investigations will determine more precisely the role of apoptosis as a potential marker for the development of maligancy in Barrett's esophagus.


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Publication date: August 2003 OESO©2015