The only generally accepted marker for increased risk of esophageal adenocarcinoma is the presence of high-grade dysplasia (HGD) . Once a carcinoma has developed, lymph node metastasis can occur in the very early stages . According to the literature, lymph node metastases can be found in approximately 25% of patients with a T1-category. Lymph node metastases are a strong predictor for a dismal long-term survival, which is reflected in our survival analysis with a five-year survival rate of patients with involved lymph nodes of <30% compared to patients without lymph node metastasis of >50% (P = 0.01). In order to characterize specific molecular changes between Barrett’s epithelium and esophageal adenocarcinoma and the changes in higher tumor stages with lymph node involvement, we analyzed E-cadherin and Eph B3 in 141 patients with esophageal adenocarcinoma.
E-cadherin is a calcium dependent transmembrane adhesion protein . During embryonic development, E-cadherin is expressed in all cells, but it disappears in embryonic mesoderm cells . Epithelial-to-mesenchymal transformation is a process by which polarized epithelial cells convert to non-polarized and motile cells to allow histogenesis, organogenesis and morphogenesis in embryonic development . Epithelial tumors implement a similar process known as epithelial-to-mesenchymal transition (EMT), during dedifferentiation, invasion and metastasis in the process of tumor progression [25, 26]. Lack of E-cadherin-mediated adhesion in human tumors correlates with loss of epithelial morphology and acquisition of mesenchymal invasive characteristics. E-cadherin is believed to be the master programmer of EMT, because signaling pathways contributing to EMT converge on regulation of this molecule . In particular, E-cadherin has been implicated in the malignant progression of Barrett’s metaplasia to adenocarcinoma [19, 28–30]. In our patient group we show by quantitative PCR a significant reduction of E-cadherin expression in adenocarcinoma compared to Barrett’s metaplasia. This difference in mRNA expression could be clearly reconfirmed on the protein level with IHC in our patients. Corresponding to the international literature, a correlation could not be found between the E-cadherin PCR and the strength of IHC reactivity and the staging of the carcinoma [19, 20]. In order to further elucidate the tumor progression Eph B3 was analyzed in our patients.
In the mammalian gut, Eph B3 is expressed in cells at the bottom of the intestinal crypts near stem cell niches [31, 32]. Physiologically, Eph B3 is responsible for constructing and maintaining the architecture of the crypt-villus axis in the intestinal epithelium by repulsive interaction with its ligand (EFN B1) . A characteristic of the Eph B/EFN B signaling system is the ability to elicit bi-directional signaling that leads to the restriction of cell migration and cell intermingling across segmental boundaries . Pathologically, loss of the Eph B3 allele induces the development of colon adenomas as the first morphological step towards colon cancer, whereas Eph B-mediated compartmentalization could be demonstrated to be a mechanism suppressing colorectal cancer progression [16, 17, 33]. These findings suggest that Eph B3 functions as a tumor suppressor. Analogous to the findings in colon cancer, in our patients Eph B3/E-cadherin coexpression is significantly correlated with a favorable tumor stage. Although E-cadherin mRNA expression and IHC positivity did not show a significant difference with regard to tumor stage, coexpression of both proteins was significantly and inversely correlated with depth of invasion, lymph node involvement and lymph node coefficient (= number of involved lymph nodes/number of removed lymph nodes).
Recently in vitro analysis indicated that Eph B activation triggered redistribution of E-cadherin from the cytoplasm to the basolateral membrane without altering protein levels in a colon cancer cell line. Consequently, Eph B signaling couples cell contraction with cell-to-cell-adhesion by promoting the recruitment of E-cadherin in colon cancer . This mechanism may be equally present in esophageal cancer. In our study, simultaneous expression of E-cadherin and Eph B3 was accompanied by an intracellular E-cadherin distribution comparable to that in healthy esophageal mucosal cells. A strong membranous accentuated immunohistologic reaction was seen in 10% of all patients, while we detected a fairly strong cytoplasmatic and a faint membranous staining in carcinoma cells without Eph B3 expression.
Taking the in vitro analysis by Cortina into account, the above observations suggest that Eph B signaling seems to restrict the capacity of malignant cells for infiltrative growth by enforcing E-cadherin adhesion. In an Apc
Min/+ mouse model the EphB mediated compartmentalization was demonstrated to be a mechanism suppressing cancer progression . In a clinical study Eph B3 expression was significantly reduced in advanced Dukes’ stage tumor specimens . In vitro examinations of a colon cancer cell line (HT-29) demonstrated that Eph B3/EFN interaction potentiated junctional adhesion molecules ZO-1, E-cadherin and plakoglobin, which are representatives of tight junctions and desmosomes, respectively .
In regard to the literature concerning the interaction between Eph B3 and E-cadherin, both proteins together have a significant tumor suppressor function. Comparable to the tumorigenesis of colon cancer we could show an altered Eph B3 and E-cadherin IHC activity of esophageal carcinoma compared to the normal mucosa and a reduced E-cadherin mRNA expression rate in esophageal carcinoma compared to normal mucosa. In the dysplasia-carcinoma sequence Eph B3 activity is reduced and E-cadherin is dissolved in the cytoplasm. Lack of E-cadherin-mediated adhesion correlates with the loss of epithelial morphology and the acquisition of mesenchymal characteristics. In our patients with esophageal cancer we could find a significant inverse correlation between a persisting simultaneous expression of Eph B3 and E-cadherin and depth of invasion and lymph node metastasis as the strongest predictive factors for long-term survival. Corresponding to colon cancer we assume from our findings an impact of Eph B3 on E-cadherin and reinforcement of the cell-cell-junctions in esophageal cancer.