Expression And Significance Of Survivin, Smac And Ki-67 In Extrahepatic Biliary Tract Dysplasia And Carcinoma

Objective: Carcinoma of the extrahepatic biliary tract is relatively rare and is associated with a poor prognosis. Its pathogenic mechanisms remain unknown. But dysplasia– carcinoma sequence was regarded as a usual pathway in the development of invasive carcinoma of extrahepatic biliary tract. We examined the expression of Survivin, Smac and Ki-67 in normal epithelium, dysplasia lesions and invasive carcinoma to evaluate whether they play an important role in the progression of extrahepatic biliary tract carcinoma. To analyze whether the correlation of Survivin, Smac and Ki-67 protein expression play an important role in the development of extrahepatic biliary tract carcinoma. And to assess whether the expression of Survivin, Smac and Ki-67 is related to histopathological features and clinical prognosis.Methods: Specimens: 27 Specimens surgically removed extrahepatic biliary tract carcinoma including 24 cases well-moderately differentiation grade and 3 cases poorly differentiation grade; 10 cases dysplasia lesions; and 5 cases normal epithelia were used as no-neoplastic controls. Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin and performed on 4-μm sections for ten pieces. The slides stained with hemaloxylin and esosin(H & E) were reviewed to perform histodiagnosis by two pathological physician. Histological criteria for diagnosis of dysplasia have been described, that is, dysplasia is characterized by cuboidal or columnar cells showing mild to moderate nuclear dysplasia, with or without pseudostratification, loss of polarity, and occasional mitotic figure. The expressions of Survivin, Smac and Ki-67 were examined immunohistochemically in all specimens. A semiquantitative scoring system was used to assess the positive expression of Survivin, Smac and Ki-67. The stained sections were observed in continued five areas begaining with highest intensity of 400 magnification. The expression of Survivin, Smac and Ki-67 were defined four grades according to the percentage of positive cells: The percentage of positive cells ranged at less than 5%,it was defined as negative(-); The percentage of positive cells ranged at 6% to 25%, it was defined as weak positive(+); the percentage of positive cells ranged at 26% to 50%, it was defined as moderately positive(++); the percentage of positive cells ranged at more than 50%, it was defined as strong positive(+++). All statistical analyses were performed using SPSS 10.0 software. The difference of the expression was analyzed byχ2 test and Fisher's exact test. Spearman grading correlation analysis was used to analyze correlation.Results: 1. Features of the expression of Survivin, Smac and Ki-67: Significant Survivin protein expression was observed in dysplasia lesions and invasive carcinoma, the positive cells showed a light yellow or yellow granular Cytoplasmic staining mostly, and only four cases brown nuclear staining. In dysplasia lesions, 3 cases were defined as weak positive staining, 1 case was defined as moderately positive staining and 1 case was defined as strong positive staining. In invasive carcinoma, 8 cases were defined as weak positive, 5 cases were defined as moderately positive and 7 cases ware defined as strong positive. For Smac expression, positive expression was observed in normal epithelium, dysplasia lesions and invasive carcinoma. The positive cells showed a yellow or brown granular Cytoplasmic staining, nuclear reaction was interpreted to be nonspecific staining. In normal epithelium, 1 case moderately positive and 1 case strong positive were observed. In dysplasia lesions, 2 cases were defined as weak positive, 3 cases were defined as moderately positive. In invasive carcinoma, 7 cases were defined as weak positive, 8 cases were defined as moderately positive and 8 cases were defined as strong positive. Ki-67 positive expression was observed in dysplasia lesions and invasive carcinoma, the positive cells showed a brown nuclear staining. In dysplasia lesions, 1 case was defined as weak positive, 1 case was defined as moderately positive and 2 cases were defined as strong positive. In invasive carcinoma, 7 cases were defined as weak positive, 10 cases were defined as moderately positive and 5 cases were defined as strong positive. 2. Comparison of Survivin, Smac and Ki-67 expression in normal epithelium, dysplasia lesions and invasive carcinoma: Significant Survivin protein expression was observed in 1 out of 5(20%) normal epithelium, 5 out of 10(50%) dysplasia lesions and 20 out of 27(74.1%) carcinoma. The difference of Survivin expression between invasive carcinoma and normal epithelium was significant (p=0.037<0.05). Significant Smac protein expression was observed 2 out of 5(40%) normal epithelium, 5 out of 10(50%) dysplasia lesions and 23 out of 27(85.2%) carcinoma. The difference of Smac expression between dysplasia lesions and invasive carcinoma was significant (p=0.041<0.05). Significant Ki-67 protein expression was observed in 0 out of 5(0) normal epithelium, 4 out of 10(40%) dysplasia lesions and 22 out of 27(81.5%) carcinoma. The difference of Ki-67 expression between invasive carcinoma and normal epithelium was significant (p=0.001<0.05). The differ- ence of Ki-67 expression between dysplasia lesions and invasive carcinoma was significant (p=0.038<0.05).3. The correlation between Survivin and Smac, between Survivin and Ki-67 and between Smac and Ki-67 in dysplasia lesions and invasive carcinoma: 1 out of 10 cases dysplasia lesions was observed Survivin, Smac and Ki-67 positive staining in common, and positive staining almost lied in the same position of tissue. 1 out of 10 cases dysplasia lesions was observed Survivin, Smac and Ki-67 negative staining in common. 15 out of 27 cases carcinomas were observed Survivin, Smac and Ki-67 positive staining in common, and 4 out of 15 cases positive staining almost lied in the same position of the same tissue. 2 out of 27 cases carcinomas were observed Survivin, Smac and Ki-67 negative staining in common. The correlation between Survivin and Smac in dysplasia lesions and invasive carcinoma was no statistical significance. The correlation between Survivin and Ki-67 was no statistical significance in dysplasia lesions and invasive carcinoma. Expression of Ki-67 had a significant positive correlation with that of Smac in dysplasia lesions(r=0.688,p=0.028﹤0.05). The correlation between Smac and Ki-67 in invasive carcinoma was no statistical significance(r=0.371, p=0.057>0.05).4. Comparison of Survivin, Smac and Ki-67 expression with histopathological features or clinical stages in invasive carci- noma: Out of 27 specimens with invasive carcinoma, the positive rate of the Survivin, Smac and Ki-67 expression was higher in well-moderately differentiated type than in poorly differentiated one, and higher in cases without invasion of vessels, perineuriums and lymph nodes than in those with tumor invasion, and higher in cases at stagesⅠandⅡthan in those at stagesⅢandⅣ. Statistical analysis was no significance.Conclusions: 1. The increased tendency of Survivin, Smac and Ki-67 expression in dysplasia-carcinoma sequence of extrahepatic bilitry tract indicates they may be involved in neoplastic development. And the disturbance in the balance between cell apoptosis and proliferation may play an important role in the progression of extrahepatic biliary tract carcinoma.2. The gradually increased expression of Survivin, Smac and Ki-67 in dysplasia-carcinoma sequence of extrahepatic biliary tract indicates normal cell has a potencial of transition to dysplasia lesions, their over-expression may suggest the malignant transformation from dysplasia lesions to invasive cacinoma.3. The regulation of several oncogenes are involved in the development of extrahepatic biliary tract carcinoma, and the variation of single oncogene may not interpret tumor biologic behavior.