1.Microsatellite Instability And MLH1,MSH2 Gene Inactivations In Sporadic Insulinomas: Clinical Implications 2.Differential Expression Of α-internexin In Pancreatic Endocrine Tumors And Its Implications

Purpose The molecular pathogenesis of sporadic insulinomas is unknown.There is a lack of biomarker to distinguish benign and malignant insulinoma.The aim of the first part of this study is to identify if mismatch repair genes are inactivated in insulinomas,which induce the microsatellite instability, and if the genetic/epigenetic alterations of the gene(s) as well as microsatellite instability could be used to distinguish benign and malignant insulinoma and to predict the outcome of the patients.Experimental Design We detected microsatellite instability,allelic typing and promoter methylation by PCR.Denaturing high performance liquid chromatography(DHPLC) was used to analyze exon mutations which were further confirmed by sequencing.Expression of MLH1 and MSH2 gene in 61 insulinomas and paired normal tissues were tested by immunohistochemical staining.The data were correlated with clinical-pathological characteristics,and were analyzed by both univariate and multivariate statistical methods.Results We found expression of MLH1 and MSH2 protein was reduced in 38%and 16%of 58 sporadic insulinomas,respectively.Promoter methylation and loss of heterozygosity(LOH) of MLH1 gene was found in 32% and 50%of sporadic insulinomas,respectively.Promoter methylation or both methylation and LOH of MLH1 were significantly associated with reduced expression of MLH1(P=0.041 and P=0.027,respectively).LOH of MSH2 gene was not found in sporadic insulinomas.A high rate of microsatellite instability (MSI-H) was found in 33%of sporadic insulinomas,which was correlated with the reduction of MLH1 or MSH2(P=0.006 and P=0.049,respectively).Reduced expression of both MLH1 and MSH2 were significantly correlated with MSI-H (P=0.004).Reduced expression of MLH1 or both MLH1 and MSH2 were significantly associated with tumor malignancy(P=0.008 or P=0.018).Although the rate of reduced expression of MSH2 in malignant tumors was higher than in benign tumors,the statistic difference was not significant(P=0.072).MSI-H was significantly associated with tumor malignancy and incurable insulinoma(P=2.6×10^-5 and P=0.002,respectively).Conclusion Allelic loss and promoter methylation contribute to the inactivation of MLH1 gene in sporadic insulinomas.Assessing alterations of MLH1 and MSH2 gene,as well as MSI-H,could be used to distinguish benign from malignant insulinomas and to predict the outcome of patients.The genetic/epigenetic silencing of MLH1 gene may play an important role in tumorigenesis in a subset of the tumors. Purpose The molecular pathogenesis of pancreatic endocrine tumors is unknown.There is a lack of biomarker to distinguish benign and malignant pancreatic endocrine tumors.The aim of the second part of this study is to identify ifα-internexin expressed in pancreatic endocrine tumors,as well as analyzing the molecular mechanisms regulating the expression ofα-internexin; and to find if the alteration of the expression ofα-internexin could be used to distinguish benign and malignant pancreatic endocrine tumors and to predict the outcome of the patients.Experimental Design Expression ofα-internexin in 242 pancreatic endocrine tumors were tested by immunohistochemical staining,and part of the tumors were also tested the expression of mRNA and protein ofα-internexin by RT-PCR and western blot,respectively.We also detected the protein expression ofα-internexin in 17 cell lines by western blot.Before and after treated with 5-aza-dC,the mRNA,promoter methylation and histon modification ofα-internexin in cell lines were detected by RT-PCR,sequencing and chromatin immunoprecipitation analysis,respectively.The data were correlated with clinical-pathological characteristics,and were analyzed by both univariate and multivariate statistical methods.Kaplan-Meier was used to analyse the cumulative survival.Results We found 132 of 238(55.4%) pancreatic endocrine tumors expressedα-internexin,whereas only one of 53 pancreatic tissues expressed the protein(P = 9.97×10^-14).The percentage of the expression ofα-internexin was significantly higher in functional tumors than in non-functional ones(71.4%vs. 35.4%,P = 2.36×10^-8).Reduced expression ofα-internexin was significantly correlated with malignant tumors or tumors with metastasis(P=0.016 and P=0.003,respectively).Besides,reduced expression ofα-internexin was also significantly correlated with patients' outcome of dead with tumors or survival with tumors(P=0.025 and P=0.017,respectively).Promoter methylation was significantly associated with reduced expression ofα-internexin in 14 tumor cell lines(P=0.002).However,promoter unmethylation status was found in both pancreatic endocrine tumors and normal pancreatic tissues,which indicated that promoter methylation was not play a role in the regulation mechanism of the expression ofα-internexin in pancreatic endocrine tumors.Conclusion Reduced expression ofα-internexin could be used to distinguish benign from malignant pancreatic endocrine tumors and to predict the outcome of patients.Promoter methylation contributes to the inactivation ofα-internexin gene in many kinds of tumor cell lines,but it may not play an important role in pancreatic endocrine tumors.