The Impact Of CDH1 Gene Mutation And Protein Expression On Survival For The Patients With Esophageal Squamous Cell Carcinoma

1 Background and objective of researchEsophageal cancer is a common malignant tumor with very poor prognosis,with a 5-year survival rate of only about 15% in middle and late stage patients.Although the 5-year survival rate of early esophageal cancer can reach 90%,early cancer accounted for only about 5% of the first clinically diagnosed patients.In the 24 years of this study,500,000 cases of esophageal cancer patients with clinical diagnosis and treatment,pathology and follow-up information database and 15,000 cases of advanced patients after the postoperative specimen tissue chip.A preliminary analysis of these databases found that the survival of patients with a complete match between age,sex,treatment,TNM staging and other clinical phenotypes was significantly different.The authors believe that the heterogeneity of tumor tissue and related molecular changes may be one of the main causes of this difference in survival.Recent studies have suggested that many tumor-related proteins and abnormal gene expression or mutations can affect the survival of patients,and thus establish candidate molecular targets for targeted therapy.At present,there are no targeted drugs for esophageal cancer.It is clear that elucidating the molecular mechanism of the occurrence and development of esophageal cancer and its effect on survival is the key to achieve early detection and targeted precision therapy to further reduce mortality.This study group has found that the expression of E-cadherin(CDH1)is related to the prognosis of esophageal cancer.But the molecular basis for the difference in the expression of E-cadherin is unclear.In addition,due to the small sample size,the difference between the expression of E-cadherin and the clinical phenotype is not very clear.Using the established large database of esophageal cancer and large sample Bank,the molecular mechanism of CDH1 protein expression difference and its relationship with clinical phenotype,especially survival,were further elucidated by whole genome targeting sequencing and protein expression technique.2 Materials and methods 2.1 Research objectsIn this study,5,935 patients with esophageal squamous cell carcinoma,3,864 cases in men,the average age of diagnosis was 60.55±8.40 years,2,071 cases in women,the average age of diagnosis was 61.55±8.09 years,All patients were from the first affiliated Hospital of Zhengzhou University,Henan province,the key open laboratory of esophageal cancer after 24 years of 500,000 cases of esophageal and cardiac cancer clinical diagnosis,pathology,follow-up database.The basic information of patients with esophageal squamous cell carcinoma was obtained by household survey,field flow adjustment and Population survey,and its clinical diagnosis,pathology information and tumor tissue specimens all came from the patient treatment hospital.Among them,2,095 patients from low-incidence areas,3,840 cases in the region,(the area of low incidence: =1.83:1).There were 5,881 cases of tumor family history,1,974 cases of tumor family history positive,3,837 cases of family history negative patients.The pathological staging of TNM in patients with esophageal squamous cell carcinoma was based on the pathological staging standard of the sixth edition of Esophageal squamous cell carcinoma in UICC.Among them,the degree of infiltration: 148 cases,Stage II 3,625 cases,Stage III 1,990 cases,172 cases in Stage IV,infiltration degree: 216 cases of mucosal layer,1,391 cases of muscle layer,4,214 cases of fiber membrane,114 cases of fiber membrane;lymph node metastasis: Positive 2,674 cases,negative 3,261 cases;Degree of tissue differentiation: 485 cases with high differentiation,3,231 cases of middle differentiation,1,928 cases of low differentiation,172 cases of organ metastasis,5,763 cases without organ metastasis.The tumor site was located in 912 cases in the neck section and thoracic segment,4,025 cases in the middle chest,883 cases in the lower thoracic section,and 115 cases did not clearly record the tumor site.There were 2,691 cases of tumor long diameter ≤4cm and 2,832 cases of >4cm.In 69 cases,genome-wide sequencing was performed,and in 199 cases,genome-wide targeted sequencing was carried out.The CDH1 protein was determined by immunohistochemistry in the sample tissues of 5,935 patients.Selection criteria: After radical surgical treatment of esophageal cancer,clear pathological diagnosis of esophageal squamous cell carcinoma patients,before surgery without any specific treatment for the tumor(radiotherapy,chemotherapy,etc.),address information can be carried out in detail survival follow-up,the tumor tissue samples complete and clinicopathological information complete people into the selection of experimental samples.2.2 Collection and preservation of tumor tissue samplesThe tumor tissue samples involved in this study are from the patient’s hospital,this laboratory and the relevant hospitals to reach a cooperative intention,the collection of tissue samples at room temperature preservation,standardized treatment of the use of tumor tissue samples 2.3 Survival Follow-upThrough telephone follow-up,village doctors village cadres inquiries,household surveys and other ways to contact and address information of a full range of patients regularly(March-June)to carry out survival follow-up,when the end event died,detailed record of the patient’s survival information.2.4 Experimental methodsIn 69 cases of genome-wide sequencing,the group found mutations in the CDH1 gene,and then targeted sequencing for mutation analysis.The DNA in the paraffin samples of tumor tissue was extracted and handed over to the ≥20ng/ul source Novogene company After the concentration was determined to reach the standard concentration,and the sequencing analysis was carried out.Paraffin tissue slices were examined by he staining,and the paraffin tissue samples of qualified patients with esophageal squamous cell carcinoma were selected and sent to Seville Servicebio biotech company to make tissue chips,and then the expression of CDH1 protein was determined by immunohistochemical technique.2.5 Evaluation criteria of 2.5 CDH1 protein immunohistochemistryThe immunohistochemical results of tissue chip were placed at 400 times times high magnification,according to the degree of staining of tumor immune positive cells: the cell membrane was not coloring to 0 points,the cell membrane showed yellowish granules,the color was obviously 1 points compared with the background,the number of brown and yellow granules was 2 points,and there were a lot of dark brown granules with 3 points.According to the proportion of tumor positive expression cells(B)in all tumor cells,the proportion is less than 5%,0 points,1 points between 5%~25%,2 points between 25%~75%,and 75% points greater than 3.A+B 0-1 was negative(-),more than 2 were positive: 2-3 were weakly positive(+)and 4 were strong positive(+ +).2.6 Statistical methodsStatistics using SPSS22.0 to analyze the data,using 2 tests to analyze CDH1 gene mutations and clinical features,using Spearman rank and test to compare CDH1 protein expression and clinicopathologic features,using Kaplan-meier and Log-rank method to draw survival map and poor survival Test of the differences.Cox risk proportional regression model was used to analyze the independent influencing factors affecting the prognosis survival of patients with esophageal squamous cell carcinoma.3 Results 3.1 Whole-exome sequencing results of CDH1In 69 whole genome sequencing samples,8 cases of gene mutations occurred in 69 cases of CDH1 whole genome sequencing,and there were 4 cases of definite mutation loci,namely rs561600796,rs199844049,rs1006266313 and rs980743065.In 199 cases,2 cases of gene mutations in target sequencing and 1 cases identified mutant loci as rs760701558.A total of 268 cases of sequencing samples,10 cases of gene mutations occurred,CDH1 gene mutation frequency of 3.7%.The mutations of CDH1 gene were independent of sex,age,high and low incidence area,family history of tumor,degree of differentiation,degree of infiltration,lymph node metastasis,pathological staging,etc.The mutations of CDH1 gene were independent of sex,age,high and low incidence area,family history of tumor,degree of differentiation,degree of infiltration,lymph node metastasis,pathological staging,etc.Gene mutation group CDH1 protein expression,1 cases of tissue chip stripping,the remaining 9 cases of protein expression positive rate of 88.9%(8/9).The survival period of negative patients was 7.8 years,and the median survival period of positive patients was 3.7 years.In 258 cases in non-mutant group,the protein positive expression rate was 97.7%(252/258),and the median survival period of positive expression patients was 4.49 years.There were more truncated data in negative patients,which failed to calculate the median survival period.There was no significant difference in survival between the two groups(P>0.05)between the CDH1 gene mutation and the non-mutant group.3.2 Immunohistomechemical protein expression of CDH1The positive rate of CDH1 protein expression was 80.4%.CDH1 protein expression is related to sex,age,smoking and drinking,family history of tumor,tumor location,infiltration degree,absence of organ metastasis(P>0.05),and high and low hair area,tumor length,differentiation degree,lymph node metastasis positive and negative symptoms,pathological staging(P<0.05).There were significant differences in survival between CDH1 protein expression groups(P<0.001)in different degrees.CDH1 strong positive expression survival is worse than the weakly positive group,negative patients survival is the best.Analysis of survival single factors affecting patients with esophageal squamous cell carcinoma tip: Gender age,high and low incidence area,smoking,drinking,tumor location,differentiation degree,infiltration degree,lymph node metastasis positive and negative symptoms,pathological staging and so on are the important factors to survive the survival of patients with esophageal squamous cell carcinoma.Single factor stratification analysis of the survival of esophageal squamous cell carcinoma suggested that there was no significant difference in survival between women,smoking group and Alcohol expression Difference Group,and the correlation analysis showed that sex was related to smoking and drinking.There was no significant difference in survival between different levels of protein expression groups in the positive group of tumor family history,the middle cervical segment + thoracic segment group,infiltration to mucosal layer and fibrous membrane group,organ transfer group,low differentiation group,pathological staging stage I,Stage III and IV Group(P>0.05).Multi-Factor regression analysis: Age,high and low incidence area,smoking history,tumor location,differentiation degree,infiltration degree,lymph node metastasis positive and negative symptoms,CDH1 expression are independent factors affecting the survival of patients with esophageal squamous cell carcinoma.4 Conclusions 4.1 CDH1 exon sequencingCDH1 gene mutation rate is low.At present,there is not enough evidence to prove its relationship with clinicopathologic features and has a significant impact on survival.4.2 Results of immunohistochemical CDH1 protein expressionThe expression of CDH1 protein is an independent influencing factor of survival.CDH1 strong positive expression survival is the worst,weak positive person is the second,negative person survival is the best.CDH1 protein is expected to be a molecular target for prognosis assessment in patients with esophageal squamous cell carcinoma.The expression of CDH1 is related to the degree of differentiation and lymph node metastasis,and the detection of CDH1 can be a molecular marker to judge the degree of differentiation of tumor cells and an early intervention therapy to guide lymph node metastasis.The expression of CDH1 protein is related to high and low incidence area,and environmental factors may affect the survival of patients by influencing the expression of CDH1 protein.