Study Of Tumor Suppressor Gene Promoter Hypermethylation And Microsatellite Instability In Esophageal Squamous Cell Carcinoma

Study of tumor suppressor gene promoter hypermethylation and microsatellite instability in esophageal squamous cell carcinoma Esophageal squamou cell carcinoma is one of the most common cancer in China.Esophageal carcinogenesis is a multistep process involving genetic and epigenetic alterations.Epigenetic alterations attracts more and more attention recently.Promoter methylation of suppressor gene,another mechanism of gene inactivation,was studied widely.Its prognostic value has been suggested in several reports.But study of the correlation between methylation and clinicopathological features or prognosis is relatively less in esophageal squamou cell carcinoma.Microsatellite instability is another hot research topic in cancer which reflect genomic instability and play an important role in tumorigenesis.It was explored in various tumor.In ESCC,the role of MSI is not clear,less reports focus on the relationship between MSI and hypermethylation of hMLH1 gene in domestic.The current research explored the role of these genetic and epigenetic changes in ESCC tumorigenesis by detected the frequency of promoter hypermethylation in hMLH1,E-cadherin and p16^INK4a gene and the incidence of MSI.The current research is comprised of the following three parts.Part 1 Study of promoter hypermethylation of hMLH1,E-cadherin,and p16^INK4a in esophageal squamous cell carcinoma.Objectives:1.To detect the frequency of hypermethylation in the promoter region of hMLH1,E-cadherin and p16^INK4A gene.2.To explore the role of these epigenetic changes in ESCC tumorigenesis.Methods:Review the clinical data of 105 patients with squamous cell carcinoma of esophagus.The genomic DNA from these patients was obtained from the frozen tumor specimens and matched normal mucosal tissue specimens using phenol-chloroform extraction.Methylation changes in the promoter region of hMLH1,E-cadherin,and p16^INK4a genes were determined by using methylation-specific PCR(MSP).The results of MSP were confirmed by using general sequencing or clone sequencing of selected PCR products. Immunohistochemical staining of hMLH1,E-cadherin,and p16^INK4a protein was performed in the tumor tissues using Envision two-step method.The frequency of hypermethylation of hMLH1,E-cadherin and p16^INK4a in tumor tissues and normal mucosal tissue and its relationship with clinicopathological features was explored.Results:1.The positive rates of genes promoter hypermethylation of E-cadherin,hMLH1,and p16^INK4a were 57.1%(60/105),20.9%(22/105)å’Œ50.5% (53/105) in tumor tissues respectively,which was 10.5%(11/105),1.9% (2/105)å’Œ7.6%(8/105) in normal mucosal tissue respectively.The positive rates were significantly higher in tumor tissues than in normal mucosal tissue for each of three genes.2.The positive rates of protein expression of E-cadherin,hMLH1,and p16^INK4a were 51.4%(54/105), 78.1%(82/105),28.6%(30/105) respectively.Promoter hypermethylation of E-cadherin(P=0.021) or p16(INK4a)(P=0.026)gene was remarkably associated with the loss of protein.HMLH1 promoter hypermethylation was not significantly correlated with the loss of protein.3.ESCC with E-cadherin gene promoter hypermethylation was significantly correlated with lymph node metastasis(P=0.016),p16^INK4a gene promoter hypermethylation was more likely to happen in poorly-differentiated squamous cell carcinoma (P=0.024),hMLH1 promoter hypermethylation was not associated with any of the clinicopathological features.Conclusions:1.Promoter hypermethylation of p16^INK4a and E-cadherin genes was common in ESCC and occurs more frequent in tumor tissues than in normal mucosal tissues.They were associated with loss of protein,which might be involved in tumorigenic process of ESCC.2.P16^INK4a promoter hypermethylation was only correlated with poorly-differentiation.It may occur early in tumorigenic process of ESCC.E-cadherin promoter hypermethylation was correlated with expression deletion and lymph node metastasis,which may play an significant role in pathogenesis of ESCC.3.There were some hMLH1 promoter hypermethylation cases in ESCC,but not associated with expression deletion and clinicopathological features.It might not be involved in tumorigenesis of ESCC.Part 2 Study of risk factors for postoperative recurrence in esophageal squamous cell carcinomaObjectives:To analyze the recurrence pattern of esophageal squamous cell carcinoma and the correlation between recurrence and clinicopathological features.To explore the value of promoter hypermethylation of hMLH1,E-cadherin and p16^INK4a used as recurrence-associated prognostic indicators.Methods:Clinical data of 105 patients with squamous cell carcinoma of esophagus,who underwent esophagectomy with two-field lymph node dis section from Jan.2006 through Feb.2007,were reviewed.The recurrence pattern was investigated,along with promoter hypermethylation of hMLH1,E-cadherin and p16^INK4a.X² test(or the Fisher's exact test) were used to analyze the difference of recurrence rate.Multivariate logistic regression analysis was carried out in order to investigate the relationship between the development of recurrence and each of independent variables.Results:1.Recurrence was recognized in 41(39%) patients within 2 years after operation.Of the 41 cases of recurrence,21(51.2%) were locoregional,including 11 cases(52.4%) of mediastinal lymph node metastasis,and 5 cases(23.8%) of cervical lymph node metastasis,17 (41.5%) were hematogenous,and mainly located at lung,bone,and liver(14,82.4%).3 cases(17.3%)was simultaneous locoregional and hematogenous recurrence.2.Univariate analysis showed that greater lymph node metastasis(P＜0.001),depth of invasion(P=0.002) and lymphatic vessel invasion(P=0.001) significantly predicted recurrence.The two year recurrence rate of patients with number of positive lymph node≥3 was 69%.3.Logistic analysis showed that T3(P=0.023) and number of lymph node metastasis(P=0.001) independently predicted recurrence,but lymphatic vessel invasion was not an independent risk factor.4.E-cadherin promoter hypermethylation was the only recurrence-associated prognostic indicator in patients with lymph node-negative esophageal squamous cell carcinoma.Conclusions:1.About 39%patients would develop recurrent disease within 2 years after esophagectomy with two-field lymph node dissection.The main sites of recurrence were mediastinal lymph node,cervical lymph node, lung,bone,and liver,to which much attention should be paid after operation.2.T3 and number of lymph node metastasis were the important risk factors influencing recurrence.The two year recurrence rate of patients with lymph node metastases≥3 were very high,more aggressive therpy would be helpful for them.3.E-cadherin promoter hypermethylation was significantly associated with recurrence in patients with lymph node-negative esophageal squamous cell carcinoma.Our study suggests that E-cadherin promoter methylation may be a new prognostic biomarker in lymph node-negative esophageal squamous cell carcinoma.Part 3 Study of MSI and its correlation with abnormality of hMLH1 gene in esophageal squamous cell carcinomaObjective:1.To detect the frequency of MSI and LOH of D3S1611(an introgenic marker of hMLH1 gene) in ESCC.2.To explore the role of microsatellite instability(MSI) in carcinogenesis and the relationship between MSI and abnormality of hMLH1 gene in ESCC.Methods:The genomic DNA of tumor and matched normal mucosal were obtained from the first part of study.Four microsatellite loci were selected to test microsatellite alterations,which involved BAT-26,D16s265,D9S1748,and D3S1611(an introgenic marker of hMLH1 gene). Fluorescence polymerasechain reaction was used to amplify the microsatellite.Automatic capillary array elec-trophoresis DNA analysis system and Genemapper software was used to do the test.MSI of the four microsatellite loci and LOH of D3S1611 loci were recorded.Results:1.The incidence of MSI in esophageal squamous cellcarcinoma was 19%(20/105).MSI in D3s1611,D9s1748 and D16s265 were detected in 8,5,and 7cases,respectively.MSI was not detected in BAT-26 loci.All MSI positive cases were MSI-L,none of MSI-H was found.2.MSI-L was not correlated with any clinicopathological characteristics.3.LOH in D3S1611,a in trogenic marker of hMLH1,was observed in 58.6%of informative cases,but it did not correlate with expression deletion or promoter hypermethylation.4.MSI-L was not associated with LOH,expression deletion,and promoter hypermethylation of hMLH1 gene.Conclusions:1.MSI-H was rare in ESCC.Relatively low-levels of MSI-L was found in ESCC.2.MSI-L was not correlated with any clinicopathological characteristics.In ESCC,MSI-L was not considered as a major event in its tumorigenesis.3.MSI-L might not correlate with the abnormality of hMLH1 gene.4.LOH of hMLH1 was common in ESCC,but it was not correlated with expression deletion or promoter hypermethylation.HMLH1 may be a target gene,but it need further reaserach.