Detection Of DNMT1 And HDAC1 Proteins Expression And Analysis Of Their Role In Pancreatic Cancer And Precancerous Lesions

Pancreatic ductal adenocarcinoma(PDAC) is an almost uniformly lethal disease, and its prevalence has been increasing in recent years.However,the early lesions of pancreas can not be effectively detected by images due to its retroperitoneal location. Moreover,there are currently no ideal markers with sufficient sensitivity and specificity to detect this kind of cancer early enough to effect cures.Recently, pathologists raised the concept of pancreatic intraepithelial neoplasias(PanINs) and pancreatic intraductal papillary mucinous neoplasms(IMPNs),which were acknowledged as the precancerous lesions.Mount of studies on PanINs and IPMNs in respect to molecular biology,histopathology and clinicopathology showed that they were important phrases during the carcinogenesis of PDAC.Therefore,it may reveal the molecular mechanism of pancreatic cancer to explore the molecular aberrations harbored in the progression of PanINs or IPMNs toward PDAC.Epigenetic modifications have been justified to playing an important role during carcinogenesis.Aberrant methylation,an overall decrease in DNA methylation associated with increased methylation of CpG islands which can yield to chromosome instability and regression of tumor suppressor genes respectively,is an early carcinogenetic molecular event;as well as,high levels of histone acetylation results in genic transcriptional activation;however,low levels of histone acetylation can contribute to genic transcriptional repression.It was evidence that DNA methylation initiated gene repression,then,the histone modifications execute the final gene silencing.Moreover,it was identified that DNA methyltransferase-1(DNMT1) and histone deacetylases-1(HDAC1),as two critical enzymes in epigenetic modifications, corporately resulted in tumor gene expression pattern.In this study,we carried out an immunohistochemical analysis of DNMT1 and HDAC1 expression in a large series of PanINs,IPMNs and PDAC lesions in order to evaluate their role in multistage pancreatic carcinogenesis and providing new clues for the diagnosis and treatment of PDAC.1.Diagnosis and classification of PanINs,IPMNs and PDAC lesionsObjective:To collect surgical resection specimens of pancreas,and find enough PanINs,IPMNs and PDAC lesions of different grades,according to the diagnostic criteria,so as to proceed to the next study of molecular pathology.Materials and Methods:200 cases of pancreatic surgical resection specimens between April 2005 and October 2007 were collected from Changhai Hospital.10 cases of normal pancreatic tissues were obtained from autopsy specimens.48 cases of paraffin-embedded IPMNs specimens were from the Department of Pathology.HE staining was performed on four-micrometer-thick serial paraffin sections.The diagnosis and classification of each case were evaluated by two pathological doctors.Results:In total,20 cases of normal pancreatic ductal,48 cases of PanIN-1A, 103 cases of PanIN-1B,99 cases of PanIN-2,30 cases of PanIN-3,18 cases of IPMA, 10 cases of IPMB,20 cases of IPMC,and 54 cases of PDAC were found.Conclusions:The tissue atypia increased gradually along the progression of normal pancreatic ductal→PanIN-1A→PanIN-1B→PanIN-2→PanIN-3→PDAC or normal pancreatic ductal→IPMA→IPMB→IPMC→PDAC.The multistage progression of PDAC was shown vividly.2.Expression of DNMT1 protein in different pancreatic tissuesObjective:To detect the expression of DNMT1 protein in different pancreatic tissues(normal,PanINs,IPMNs and PDAC) and evaluate its significance during the carcinogenesis of PDAC,in order to provide new clues for the diagnosis and treatment of pancreatic cancer.Materials and Methods:Immunohistochemical detection of DNMT1 protein in the 20 cases of normal pancreatic ductal,48 cases of PanIN-1A,103 cases of PanIN-1B,99 cases of PanIN-2,30 cases of PanIN-3,18 cases of IPMA,10 cases of IPMB and 20 cases of IPMC and 54 cases of PDAC with SP method.Results:The positive expression rates of DNMT proteins escalated with the severe tissue atypia along the progressive multistages:normal pancreatic ductal→PanIN-1A→PanIN-1B→PanIN-2→PanIN-3→PDAC,or normal pancreatic ductal→IPMA,IPMB→IPMC→PDAC.The difference is statistically significant.Conclusion:High expression of DNMT1 is an early event in PDAC.The increased expression of DNMT1 with the severe tissue atypia suggests its contribution to drive the progression of PDAC.3.Expression of HDAC1 protein in different pancreatic tissuesObjective:To detect the expression of HDAC1 protein in different pancreatic tissues(normal,PanINs,IPMNs and PDAC) and evaluate its significance during the carcinogenesis of PDAC,in order to provide new clues for the diagnosis and treatment of pancreatic cancer.Materials and Methods:Immunohistochemical detection of HDAC1 protein in the 20 cases of normal pancreatic ductal,48 cases of PanIN-1A,103 cases of PanIN-1B,99 cases of PanIN-2,30 cases of PanIN-3,18 cases of IPMA,10 cases of IPMB and 20 cases of IPMC and 54 cases of PDAC with SP method.Results:The positive expression rates of HDAC1 proteins escalated with the severe tissue atypia along the progressive multistages:normal pancreatic ductal→PanIN-1A→PanIN-1B→PanIN-2→PanIN-3→PDAC,or normal pancreatic ductal→IPMA,IPMB→IPMC→PDAC.The difference is statistically significant.Conclusion:High expression of HDAC1 is an early event in PDAC.The increased expression of DNMT1 with the severe tissue atypia suggests its contribution to drive the progression of PDAC.4.Correlations of the expression of DNMT1 and HDAC1 in PDAC with the clinicopathologic variablesObjective:To analyze the correlations of the expression of DNMT1 and HDAC1 in PDAC with the clinicopathologic variables and evaluate its biological and prognostic value.Materials and Methods:To collect the overall clinicopathologic reference of the 54 cases of PDAC,including the patient's age,sex,smoking and drinking history, tumor size,tumor location,perineural invasion,venous invasion,grade of differentiation,proliferation activity and TNM staging.Results:High-level DNMT1 protein expression was statistically significantly correlated with perineural involvement,degree of differentiation and TNM staging. While high-level HDAC1 protein expression was statistically correlated with degree of differentiation,proliferative activity,and TNM staging.No significant correlation was observed between the levels of DNMT1 and HDAC1 protein expression and patient age,gender,smoking,alcohol,tumor size,tumor location,venous involvement. The overall survival rates of high-level of DNMT1 or HDAC1 groups were less than the low-level groups respectively.Conclusion:The increased expressions of DNMT1 and HDAC1 reflected the malignant biological behavior of PDAC.DNMT1 and HDAC1 may serve as two new prognosticators of PDAC.5.SummaryThis study preceded the immunohistochemical detection of DNMT1 and HDAC1,two critical enzymes in the epigenetic modification,in different pancreatic tissues(normal,PanINs,IPMNs and PDAC),and analyzed the correlations of their expression with clinicopathologic variables of PDAC patients.The results showed that:the expression of DNMT1 and HDAC1 increased with the severe tissue atypia.High-level DNMT1 protein expression was statistically significantly correlated with perineural involvement,degree of differentiation and TNM staging.While high-level HDAC1 protein expression was statistically correlated with degree of differentiation,proliferative activity,and TNM staging.High-level expressions of DNMT1 or HDAC1 both suggested poor prognosis.On the whole,it is shown that the increased expression of DNMT1 and HDAC1 was an early event and promoted the progression of PDAC.DNMT1 and HDAC1 may serve as two new prognosticators of PDAC.