Study On The Relationship Of EhpB4, EphrinB2 And The Formation, Development And Prognosis Of Esophageal Cancer

IntroductionIn the world wide, the high incidence areas of esophageal cancer include: China (21), South America (13), Western Europe (11), South Africa (10), Japan (9) and the former Soviet Union (8) (expressed as crude incidence per100,000)^[1]. From the 90's, due to the efficient intervention to the risk factors, the incidence and mortality of esophageal cancer have been decreased gradually in Henan Province , but are still higher than the average level of the whole country^[2]. It is important to have early diagnosis and treatment for increasing the 5-year survival rate of esophageal cancer. The methodology of molecular biology and other experimental technology has been well developed, such as PCR , western blotting and immuno-histochemistry methods, which can be applied for the investigation on esophageal cancer at the molecular level. The Ephs and Ephrins comprise the largest receptor subfamilies of tyrosine kinase, (with 14 receptors and 8 ligands) and could be subdivided into EphA and EphB sub-classes based on their sequence homologies and binding properties to Ephrin ligands^[3,4]. EphA receptors bind to glycosylphosphatidylinositol (GPI)-anchored Ephrin ligands (EphrinA subfamily), whereas EphB receptors bind Ephrin ligands that contain trans-membrane and cytoplasm domains (EphrinB subfamily)^[5]. It was found that gene expression and/or protein synthesis of Eph receptors were increased in human tumors^{[12-22,23,24]}. In particular, it was found by our and other labs that EphB4 was highly expressed in many tumor tissues including brain glioma^[23], head and neck^[20,21], breast ^[15,16], lung^[19,25], endometrium^[17,18], gastric cancer^[24] and colon^[13,14]. However, the report of the EphB4 and EphrinB2 expression levels in esophageal cancer is rear. In the present study, we applied real-time PCR and immunohistochemistry techniques to measure the mRNA levels of EphB4 and EphrinB2 genes in primary esophageal cancers and their matched normal esophageal tissues. The correlation of the gene expression and the clinical & pathological parameters was also analyzed by using statistical analysis models. The patients were followed up to study the relationship between the gene expression levels of EphB4 and EphrinB2 and the survival time.Materials and methods1. Subjects and sample collectingThe subjects were esophageal cancer patients hospitalized in The First Affiliated Hospital of ZhengZhou University, Henan Province, from year 2002 to 2005. The specimens of cancer tissues and matched normal esophageal tissues from the subjects were taken during surgery. The identification of normal and tumor tissues was based on the pathological analysis. The samples were snap frozen in liquid nitrogen and then stored at -80℃for future processing. The clinical data such as gender, age, family history, tumor location, size, stage and metastasis were collected and the patients were also followed-up. The data were statistically analyzed.2. Determination of EphB4 and EphrinB2 mRNA in samples Real-time PCR technique was used to determine the mRNA level of EphB4 and EphrinB2 in esophageal cancer tissues and matched normal esophageal tissues. Ct value represents the quantity of mRNA.3. Protein determination of EphB4 and EphrinB2 in samplesEphB4 and EphrinB2 protein expression were determined in 96 pairs of esophageal cancer tissues and corresponding non-cancer esophageal tissues. Three images at(y|¨)(y|¨)(y|¨)(y|¨)0 magnification were observed for each sample and the number of cells expressing target protein was calculated. The percentage of cells expressing target protein was estimated by dividing the number of positively stained cells by the number of total cells per high-power field area. It was established the index of protein expression level that the ratio was of percentage of positively immunostaining cells in esophageal cancer sample to that in matched normal esophagus sample.4. Statistical analysisFor each sample, aΔCt(Ct_{EphB4 or EphrinB2}-Ct_β-actin) value represents the difference between the target gene and the internal control gene,β-actin. Paired-sample t-test was used forΔCt analysis. x²-test was employed to analyze the EphB4 and EphrinB2 gene expression in esophageal cancer and matched normal tissues. The associations of EphB4 and EphrinB2 gene expression with individual clinical parameter (family history, metastasis, tumor stage, histology, gender, tumor size and patient's age) were also analyzed by x²-test. Independent t-test and one way ANOVA were used to analyze EphB4 and EphrinB2 protein expression with clinical parameters. Pearson's correlation analysis was used to estimate the correlation of the two gene expression. For each gene, Kaplan-Meier survival curve for sample with positive versus negative gene expression were plotted and log-rank test were used to compare the equality of the two survival curves. Multivariable analyses with the Cox proportional hazards model were used to estimate the effects of the clinical parameters and the two gene expression on survival time. The statistically significant level was set at a=0.05. All statistical analyses were performed using the program SPSS12.0 for Windows (SPSS, Chicago, IL). Results1 Real-time PCR results1.1 Expression of EphB4 and EphrinB2 genes in esophageal tumor tissue and the matched normal esophageal tissuesUp-regulation of EphB4 and EphrinB2 mRNA expression were found in 74 of 96 (77%) and 59 of 96(61%) of esophagus tissues compared with the paired normal esophagus tissues respectively.EphB4 mRNA level had significant difference between cancer and normal tissues (t=6.411, P = 0.000). Pearson's correlation analysis showed that expression levels of EphB4 and EphrinB2 genes in esophageal cancer was highly positively correlated (r= 0.520, P = 0.000).1.2 Relationships between mRNA levels of EphB4 and EphrinB2 in esophageal cancer and the clinical and pathological parameters.Univariate analysis demonstrated that EphB4 expression was significantly associated with family history, metastasis, tumor size, tumor location and tumor stage. EphB4 gene level was increased significantly in poor differentiated tumors compared to the well differentiated tumor tissue (x²=6.294, P=0.043). EphB4 level was significantly higher in the patients with positive family history of cancer compared to the ones without cancer family history (x²=53.94, P=0.000), and so was EphrinB2 (x²=15.668,P=0.000) . Expression of EphB4 in esophagus tumor tissues with metastasis was significant higher than in the ones without metastasis (x²=11.039, P=0.001). Lager tumor size had significantly higher EphB4 level than the smaller ones (x²=14.415, P=0.001). Moreover, lower segment esophageal tumor had significantly higher levels of EphB4 and EphrinB2 than the upper segment ones (x² =9.167,P=0.010 and x²=6.075,P=0.048).2 Immunohistochemistry result2.1 EphB4 and EphrinB2 protein expressions in esophageal cancer tissues and the paired normal tissuesThe expression of EphB4 (P=0.000) and EphrinB2 (P=0.000) was up-regulated at protein level in cancer tissues compared with the normal counterparts, which was similar to the results of real-time PCR.2.2 Relationship between protein expression of EphB4 and EphrinB2 and the clinical and pathological parametersThe statistical result presented that the up-regulation of EphB4 and EphrinB2 proteins in metastatic cancers was much more obvious than non-metastatic cancers (t=-2.862, P=0.005 and t=-2.987, P=0.004 respectively). It also indicated that protein expressions of EphB4 and EphrinB2 were positively correlated with tumor size (F=13.129, P=0.000 and F=4.174, P=0.018 respectively). EphB4 protein expression level in patients with cancer family history was also significantly higher than the ones without family histroy (t=-3.181, P=0.002). EphB4 protein was highly expressed in esophageal cancer and positively related with tumor stages (F=5.882, P=0.004). However, EphrinB2 protein expressed in esophageal cancer was not significantly associated with tumor stages (F=0.82, P=0.443).Survival analysisThe Univariate survival analysis showed that EphB4 and EphrinB2 expression tumor metastasis, patient age and family history significantly associated with patient survival time. Patients with the higher expression of EphB4 and EphrinB2 showed a significantly decreased average survival time (P=0.000, P=0.019). Similarly, Patients with cancer family history showed a significantly decreased survival time in comparison with those without family history (P=0.000). For age, younger patients had decreased survival time than the elder ones (P=0.000). Moreover, patients with metastasis showed also a significantly decreased mean survival time comparing with those without metastasis (P=0.009).Cox regression analysisThe multivariate (Cox regression) survival analysis showed that EphB4, Ephrinb2 and metastasis significantly associated with patient survival time. The relative risk are 4.899(confidence interval, 1.804～to 13.300), 3.310(confidence interval, 1.685～to 6.505) and 2.000(confidence interval, 1.046～to 3.822) respectively. Conclusion1. Up-regulation of EphB4 and EphrinB2 mRNA expression were found in esophageal tumor tissues. EphB4 mRNA expression is significantly different between cancer and normal tissues. It was highly positively correlated with EphrinB2 mRNA level. Significant association was found between EphB4 gene expression and family history, metastasis, tumor size, tumor location and tumor stage. EphrinB2 mRNA expression in tumor tissue was strongly associated with some clinical parameters, including tumor location and family history.2. EphB4 and EphrinB2 protein were highly expressed in cancer tissues compared with the normal counterparts, which was similar to the results of real-time PCR. Up-regulation of EphB4 and EphrinB2 proteins in metastatic cancers was more significant than non-metastatic cancers. EphB4 and EphrinB2 protein expressions were positively correlated with tumor size. EphB4 protein expression was significantly higher in the patients with cancer family history compared with the ones without family history. EphB4 protein was highly expressed in esophageal cancer and positively related with tumor stages.3. Patients with the higher expression of EphB4 and EphrinB2 showed a significantly decreased average survival time. The multivariate (Cox regression) survival analysis showed that the expression of EphB4 and Ephrinb2 and metastasis were independent risk factors for esophageal cancer patient survival time.4. EphB4 and EphrinB2 expression were up-regulated in the tumor tissue compared with the normal tissue. The two genes might play the important roles in tumor formation and development. The gene high expression correlates with the poor prognosis of esophageal squamous cell carcinoma. However they may be considered as a novel prognostic indicator and a potential therapeutic target.