EphB2 Down-regulation In Gastric Cancer And Its Clinical Significance

PurposeEph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands, the ephrins, show diverse expression patenrs during development. Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network, paterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfinding, and vascula formation. Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis. lt is therefore of clinical importance to further analyze the function of these molecules, particularly their function in tumor cell growth, since manipulation of the activities of these molecules may have therapeutic applications. EPHB2 is a member of the Eph receptor tyrosine kinase family and plays an important role in cell growth, differentiation, and signal transduction. EphB2 was isolated as a new member of Eph subfamily in 1994 by Kiyokawa from a human gastric cancer cDNA library. A loss of EPHB2 expression has been observed in human tumors, particularly in colonic adenomas and carcinomas. However, the role of EPHB2 in the development and/or progression of gastric cancer are still unclear. To determine the possible role of EPHB2 gene in gastric cancer, the differential expression of EPHB2 between the primary gastric cancer and related metastatic sites were investigated in the present study. The relationship between EPHB2 and clinicopathological parameters was analyzed, and the prognostic significance of EPHB2 was evaluated by survival analysis. In addition, gene mutation and allelic loss of EPHB2 was also measured to clarify the development or progress of gastric cancer.MethodA tissue array including 337 gastric cancer patients was established and the expression of EPHB2 was measured using the immunohistochemistry method. The differential expression of EPHB2 was compared among normal gastric mucosa (n = 337), primary tumors (n = 337) and metastatic lymph nodes (n = 50).The allelic loss of EPHB2 on chromosome in gastric cancer patients (n = 13) without EPHB2 expression was determined using loss of heterozygosity.The gene mutation of EPHB2 in gastric cancer patients (n = 13) without EPHB2 expression was determined using polymerase chain reaction-single strand conformation polymorphism.Results1. A tissue array including six slides was successfully established, which enrolled 337 samples of normal gastric mucosa, 337 samples of primary tumors and 50 samples of metastatic lymph nodes. An evident decreasing expression pattern of EPHB2 was found consistent with the development and progress of gastric cancer. The positive expression rates of EPHB2 in normal gastric mucosa, primary tumors and metastatic lymph nodes were 100%(337/337), 47.5% (160/337) and 18%(9/50), respectively. The expression level of EPHB2 in normal gastric mucosa was significantly higher than that in primary gastric tumor tissues (P﹤0.01) and the expression level of EPHB2 in primary gastric tumors was significantly higher than that of metastatic lymph nodes (P = 0.003). The expression of EPHB2 was correlated with tumor size, depth of invasion, lymph nodes status, differentiation of tumor, and TNM stage significantly (P < 0.01).2. Survival analysis showed that loss of EPHB2 correlated with the median survival time significantly. Additionally, the multivariate Cox proportional hazards model suggested that the expression of EPHB2, site of the primary gastric tumor, depth of invasion, and lymph nodes status were independent prognostic factors for gastric cancer.3. The results of polymerase chain reaction-single strand conformation polymorphism indicated that no fragments with large deletion or insertion allelic of EPHB2 were found in gastric cancer. Single strand conformation polymorphism showed that no significant gene mobility shift was observed in normal gastric mucosa, primary tumors and metastatic lymph nodes, which suggested that the gene mutation of EPHB2 was not the key reason for dysfunction of EPHB2 in gastric cancer.4. There existed an evident loss of heterozygosity of EPHB2. In the samples of primary gastric tumors (n = 13), the lost rate of D1S2864, D1S512, D1S3214, D1S2698, and D1S458 was 15.4% (2/13), 7.7% (1/13), 15.4% (2/13), 7.7% (1/13), and 30.8% (4/13), and the overall lost rate was 61.5%. In the metastatic lymph nodes of the same patients above-mentioned, the lost rate of D1S2864, D1S512, D1S3214, D1S2698, and D1S458 was 46.2% (6/13), 30.8% (4/13), 30.8% (4/13), 30.8% (4/13), and 30.8% (4/13), and the overall lost rate was 84.6%. The results suggested that the loss of heterozygosity of EPHB2 might be the main cause of dysfunction of EPHB2 in gastric cancer.Conclusion1. Results of IHC showed that the expression of EPHB2 in gastric cancer was lower than the normal gastric mucosa, which suggested that EPHB2 might play an important suppressor in the progress of gastric cancer.2. Survival analysis showed that loss of EPHB2 correlated with the median survival time significantly, which suggested that EPHB2 might be an important prognostic indicator for gastric cancer.3. Loss of heterozygosity for the point 35-36.1 of the short arm on chromosome 1 might be main cause of dysfunction of EPHB2 in gastric cancer.4. Gene mutation of EPHB2 was not the key reason for dysfunction of EPHB2 in gastric cancer.5. The metastasis of gastric cancer is an accumulating result of mutation and alteration of genes. Our results suggested that the lower expression of EPHB2 might play an important role in the development and progress of gastric cancer.