Prognostic Factors Analysis And Experimental Study On Aberrant DNA Methylation In Thymic Epithelial Tumors

Thymic epithelial tumors(TETs) are primary neoplasms of the anterior mediastinum with highly variable histopathological characteristics.This heterogeneity has long hindered the development of a standardized protocol for diagnostic and prognostic evaluation of TETs. The modified Masaoka staging system,is currently the most commonly employed scoring method,but its value is limited by the fact that it assigns scores only for specific macroscopic surgical,but not histological, characteristics,making it difficult to reliably assess tumor severity in patients.In 1999,the World Health Organization(WHO) published histologic criteria of TETs that was based on the morphology of epithelial cells and on the ratio of lymphocyte-to-epithelial cell,which stratified TETs into six entities:types A,AB,B1,B2,B3 and C,and provided a new means of predicting states of malignancy of TETs.However,its factual accuracy is disputed.The insufficiencies of current staging systems are confounded by the fact that no genetic or epigenetic prognostic indicators have been included.Previous studies showed that multiple genetic alterations involved in many kinds of carcinomas,but the molecular mechanisms of TETs are poorly understood.Recent data suggests that carcinomas appear to be a process that is caused by genetic alterations and by epigenetic mechanism.It mainly involves DNA methylation and histone modifications.DNA methylation is one of the major mechanisms of epigenetics.Aberrant methylation of promoter regions resulting in inactivation of human tumor suppressor gene expression has been proposed to be an important mechanism in cancer. Cancer cell DNA is frequently characterized by global DNA hypomethylation,localized gene-specific hypermethylation,and overexpression of DNA methyltransferases(DNMTs),which were so called "methylation imbalance",have already been reported in many kinds of tumors,such as lung cancer,colon cancer and oral carcinoma. Although multiple genetic and epigenetic changes have been detected in many kinds of cancers,the precise molecular mechanisms in the development and/or progression of TETs still remain unknown.In this study,we firstly evaluated the prognostic factors of TETs based on the clinic and pathologic data of the patients,then detected the expression and the methylation pattern of TSGs in TETs by using the real-time RT-PCR and the nested MSP,and the expressions of DNMTs were also measured by real-time RT-PCR.Finaly the global DNA methylation status in TETs were measured by Immunohistologic analysis and ELISA. PartⅠRetrospective study of prognostic factors of patients with thymic epithelial tumorsObjective:To investigate the prognostic factors of patients with thymic epithelial tumors(TETs).Methods:137 patients with TETs were surgically treated at our hospital from June 1997 and September 2007.The data of patients including age,gender,symptoms,histological types,stage and grade, pathological findings and operative reports were recorded.Follow-up was obtained via telephone and postal mail.The patients were divided into MasaokaⅠ/Ⅱgroup andⅢ/Ⅳgroup,and WHO A/AB/B1group and B2/B3/C group,and the Kaplan-Meier method,log-rank test and COX regression model were performed to analyze the prognostic factors of TETs.Results:Among the 137 patients,124(90.5%) received complete resection,9(6.6%) incomplete resection,and 4(2.9%) surgicat biopsy. The rate of complete resection was significantly higher in Masaoka stagesⅠ/Ⅱthan that in stagesⅢ/Ⅳ(P＜0.001).The overall survival rate of 5-year and 10-year were 71.4%and 50.1%,respectively.Patients in stagesⅠ/Ⅱhad a better long-term survival than those in stagesⅢ/Ⅳ(P＜0.001).According to WHO histologic criteria,the rate of 5-year and 10-year survival in patients with type A/AB/B1 TETs were significantly higher than those in patients with type B2/B3/C TETs(P＜0.001).The 5-year and 10-year survival rate in complete resection group were significantly better than those in incomplete resection and biopsy group (P＜0.001).Cox regression analysis showed that the prognosis of patients with TETs was related to the Masaoka stage,WHO histologic criteria, extent of resection and the age at operation.Conclusions:The Masaoka stage,WHO histologic criteria,extent of resection and the age at operation were the important prognostic factors in patients with TETs. PartⅡAbnormal DNA methylation and its clinical significance in TETsPart A The mRNA expression levels and promoter methylation patterns of TSGs in TETsObjective:To investigate the expression and the promoter hypermethylation of TSGs and potential clinical significance in patients with TETs.Methods:Real-time RT-PCR and methylation-specific PCR(MSP) were performed for evaluated the expression and promoter region methylation patterns of TSGs in TETs.The correlation between TSGs mRNA,TSGs methylation and clinicopathological data were analyzed.Results:According to WHO criteria,The mRNA levels of six of these genes,E-cad,RARβ,hMLH1,RASSF1A,APC1A and FHIT, were significantly reduced in WHO type B2/B3/C tumors relative to WHO type A/AB/B1 tumors(E-cad,p=0.036;RARβ,p=0.002;hMLH1, p=0.024;RASSF1A,p=0.042;APC1A,p=0.02;FHIT,p=0.035).The mRNA levels of APC1A,FHIT,RARβ,E-cad and hMLH1 were significantly reduced in stageⅢ/Ⅳrelative to stageⅠ/Ⅱ(APC1A,p= 0.005;FHIT,p=0.015;RARβ,p=0.047;E-cad,p=0.003;hMLH1, p=0.003).Of all 65 TETs samples,the promoter region of all 9 genes in all type A group samples displayed an unmethylated phenotype,whereas 60%of type AB samples,66.7%of type B1 samples,and all of WHO type B2/B3/C samples showed promoter hypermethylation in at least one gene.The frequency of hypermethylation of individual genes varied between 15.4%(RASSF1A) and 46.2%(FHIT).With the exception of RASSF1A and P16^INK4a,the frequency of promoter hypermethylation for each gene correlated with Masaoka stage.DAPK methylation linked to gender(p=0.034) and P16^INK4a promoter methylation correlated with the age of the patient when operated(p=0.011).No correlations were found between TSG promoter hypermethylation and the occurrence of myasthenia gravis(MG) symptoms(p＞0.05).Conclusion:Our findings suggest that epigenetic silencing of TSGs expression by promoter hypermethylation could play an important role in TETs,and may closely related to the Masaoka stage and WHO criteria.Part B Global DNA methylation status and its clinic significance in TETsObjective:To evaluate the global DNA methylation status in patients with TETs.Methods:Immunohistochemistry and ELISA with an antibody against 5-methylcytosine(5-MC) were performed to observe the global DNA methylation status in TETs.The results were compared with the clinicopathological data.Results:Tumors from primary stage(stageⅠ/Ⅱ) were more darkly stained compared to tumors from advanced stage(stageⅢ/Ⅳ)(p＜0.05). We compared the stain intensity amongst different subgroups.In WHO type A,AB,B1,B2,B3 and C,significant differences were detected amongst all the six groups,except between AB and B1(p=0.455),B3 and C(p=0.779).We then set WHO type A and AB to group A,WHO type B1,B2 and B3 to group B,WHO type C to group C.Statistic difference were found among these three groups(group A and B,p＜0.01; group A and C,p＜0.01;group B and C,p＜0.01).According to group A/AB/B1 and group B2/B3/C,the tumors from A/AB/B1 group were more darkly stained than that from B2/B3/C group(p=0.001).The similar results in fresh frozen TETs tissue were found by ELISA test. Correlations between global DNA methylation levels and gender or MG symptoms were also evaluated,but no statistically significant differences were found.Conclusion:Global DNA hypomethylation is associated with increasing severity and may be a determining factor in the development of TETs. Part C The expressions of DNMTs and clinic significance in TETsObjective:To investigate the expression of DNMTs gene and its potential clinical significance in TETs.Methods:The real-time RT-PCR was performed to evaluate the expression of the DNMTs.The correlations between DNMTs mRNA and clinicopathological data were analyzed.Results:All three DNMTs were found to be significantly up-regulated in WHO type B2/B3/C tumors relative to type A/AB/B1 (DNMT1,p＜0.05;DNMT3a,p＜0.05;DNMT3b,p＜0.01).Similarly,the expression of each DNMT was significantly increased in Masaoka stageⅢ/Ⅳtumors relative to stageⅠ/Ⅱtumors(DNMT1,p＜0.01;DNMT3a, p＜0.01;DNMT3b,p＜0.05).Moreover,a significant negative correlation was found between DNMT3b mRNA levels and the OD value of global methylation levels(r=-0.522,p=0.006).This correlation did not occur between OD values and DNMT1 or DNMT3a expression levels(r=-0.215, p=0.291,for DNMT1;r=-0.337,p=0.057,for DNMT3a),although the expression of the three genes correlated with each other across all TETs samples(DNMT1 vs.DNMT3a,r=0.592,p=0.001;DNMT1 vs. DNMT3b,r=0.419,p=0.033;DNMT3a vs.DNMT3b,r=0.777,p=0.001).Conclusion:DNMTs genes were overexpressed in TETs tissue,and overexpression of DNMTs was correlated with Masaoka pathologic system and WHO histologic criteria.