Relationship Of Her-2, Slug And E-cadherin Expression With Histological Classification And Prognosis Of Gastric Carcinoma

Gastric carcinoma(GC) still remains the fourth most common cancer worldwide and is the second cause of cancer related death. Clinically more than 90% of GC cases are in advanced stage. Even though radical gastrectomy is performed, the prognosis is still dismal with a low 5-year survival rate. Histopathological diagnosis and classification is the foundation of subsequent treatment. However, prognosis differs even in cases with identical histological type and uniform therapy. Great efforts were made to correlate the clinicopathological parameters with prognosis and molecular markers have been searching in recent years to predict the prognosis.Ras/Raf/MAPK and PI3K/Akt signaling pathways play important roles in GC. Receptor tyrosine kinases (RTKs) can activate these two pathways. Her-2, as a member of epidermal growth factor receptors, belongs to RTKs which regulate many basic cellular processes through Ras/Raf/MAPK and PI3K/Akt. In the case of breast carcinoma, overexpression of Her-2 protein or amplification of Her-2 gene was associated with recurrence and poor prognosis. A new therapeutic agent trastuzumab as a monoclonal antibody to Her-2 has been shown to be effective in Her-2 positive breast carcinoma patients. Data also showed that Her-2 played an important role in GC development. Recently, a global clinical study was conducted to determine response to trastuzumab therapy in Her-2 positive GC patients. However the frequency of Her-2 positivity varied from different studies and its clinical significance in gastric cancer is yet not clear. A large-sample clinicopathological observation and analysis concerning Her-2 status is still needed.On the other hand, Ras/Raf/MAPK pathway may finally affect the transcription factor-Snail family which induce epithelial mesenchymal transition(EMT) by downregulating E-cd. Slug/Snai2 is a member of Snail family. According to the gene expression profiling of Beijing Institute of Cancer Research, transcription factor Slug mRNA level was increased in poorly differentiated GC, but decreased in well differentiated one. E-cd mRNA expression level was the reverse. So we hypothesized that Slug and E-cd may be correlated with differentiation and prognosis in GC.Thus firstly we re-evaluated prognostic significance of WHO and Lauren classification by reviewing HE sections of 486 gastric cancer patients who underwent curative gastrectomy from January 2001 to December 2004 in PLA General Hospital. Statistical analysis demonstrated that age, lymphatic/vascular invasion, lymph node metastasis (including pN stage and N ratio grade) and pTNM stage were independent prognostic factors. Although WHO and Lauren classification had significant influence on overall survival in univariate analysis, they lost their independent value in multivariate analysis.Subsequently, to clarify the potential clinical relevance of Her-2 status in gastric cancer, we evaluated Her-2 status in 486 cases of GC by applying immunohistochemistry(IHC) and Fluorescence in situ hybridiza-tion(FISH), then correlated its positivity with clinicopathological parameters and follow-up data. Her-2 positivity was found in 10.93% of GC cases. Her-2 positive status were common in cases with papillary and glandular adenocarcinomas according to WHO classification (P=0.011), in cases with a well or moderately differentiated GC by WHO histological grade (P=0.000) and in cases of intestinal type by Lauren classification (P=0.024). Survival analysis revealed that the overall survival between Her-2 positive and negative cases showed no significant difference (P=0.709). Yet in signet ring cell carcinoma or diffuse GC, the 5-year survival rate of Her-2 positive cases was 0%, indicating worse prognosis. However, the difference was not statistically significant probably due to the fewer Her-2 positive cases.In the third part of the study, we explored the significance of Slug and E-cd in GC cell lines as well as in 486 GC cases. Initially we examined their expression in 6 GC cell lines at mRNA and protein level respectively, analyzed the role of Slug in relation with E-cd through silencing of Slug by short hairpin RNA. Then we investigated Slug and E-cd expression using tissue array and IHC method. Meanwhile, we analyzed the association of Slug and E-cd expression with clinicopathological parameters and prognosis. An increased Slug expression was observed in BGC823,MGC803 and SGC7901 GC cell lines which had greater capacity of tumorgenesis, while Slug expression level was low in AGS and N87 at both mRNA and protein level. Inversely, E-cd expression was low in BGC823,MGC803 and SGC7901 GC cell lines, while it was increased in AGS and N87 GC cell lines at both mRNA and protein level. In MKN45 GC cell line, a sequence of 12 bases was found missing by gene sequencing. Transient Slug silencing in SGC7901 cell line induced an increase in E-cd mRNA level up to 2.9-fold. Positive expression of Slug and E-cd was observed in 56.2% and 69.1% GC cases, respectively, which was positively correlated. The expression of Slug correlated with age, tumor location, depth of invasion, WHO classification, WHO histological grade, Lauren classification, lymph node metastasis status and pTNM stage. Though univariate analysis revealed Slug expression was unrelated to survival, multivariate analysis indicated that Slug expression was an independent prognostic factor in addition to pN, pTNM stage. The expression of E-cd correlated with gender, age, tumor location, WHO classification, WHO histological grade and Lauren classification, but unrelated with tumor size, depth of invasion, lymphatic/vascular invasion, lymph node metastasis or pTNM stage. Neither univariate nor multivariate analysis showed E-cd expression had a significant impact on patient's overall survival.In conclusion, precise WHO and Lauren classification remains the baseline study of GC. Her-2 status is not related to the prognosis.Slug expression is an independent prognostic factor in addition to pN, pTNM stage. E-cd tends to be a differentiation marker rather than a prognostic one.