Study Of Sex Difference And Molecular Basis On The Survival Of Patients With Esophageal Cancer

1.Background Esophageal cancer(EC)is one of the most common malignant tumors in the world.According to the world cancer research report of World Health Organization(WHO)in 2014,the incidence of EC in China accounts for about 50% of the world’,and it is the country with the highest incidence of EC in the world.Patients with early EC have no specific symptoms in clinical practice.Most of the patients were found to be in the advanced stage.The patient’s quality of life is low,and the 5-year survival rate is less than 20%.The two prominent characteristics of EC in China are obvious regional characteristics and family aggregation.In different regions,different family history patients have significant differences in survival.Furthermore,tumor stage,age,smoking and drinking,eating habits,genetic susceptibility,pathological type,general type,tumor location,treatment methods can also affect the survival of patients with EC,howerer,clinically it is found that patients with the same factors have different survival period between gender disparity.Previous studies on gender differences in EC have been conducted for a comparative analysis of one or clinical features.Currently there is no systematic or larger sample comparative study to be printed.So,in this study,we will compare all clinicalpathological parameters and survival of different gender in EC with large sample,the effect of sex hormone receptors: estrogen receptor(ER),including ERα and ERβ,progesterone receptor(PR)and androgen receptor(AR)on survival and expression in different gender tissues with EC,and go to the whole exome sequencing(WES)and the partly related protein P53,E-calcium mucins1(E-Cadherin1,CDH1)immunohistochemical examinations were done;In order to observe a mechanism affects the difference in survival between men and women with EC.We hope to find strong data support and theoretical basis for the subsequent gender differences in EC and provide a new method for the treatment of EC.2.Materials and methods2.1 Subjects All the subjects were come from the database of Henan Key Lab for Esophageal Cancer Research,the First Affiliated Hospital of Zhengzhou University.All the patients’ tissue was pathologically confirmed as primary EC.They didn’t combine with other tumors.The survival time was definite.The diagnosis time was1973-2017.We have all the patients’ accurate records,medical record number and pathology number,with a detailed home address or a valid telephone contact which can be followed up to the patient or immediate family member.2.2 Research methods2.2.1 Epidemiological investigation We compare the gender differences of patients’ general information,clinicopathological information and survival period of 23,269 patients with EC from Anyang Cancer Hospital.2.2.2 Immunohistochemistry The specimens were subjected to conventional immunohistochemistry and HE staining.We detect the expression levels of ERα,ERβ,PR and AR in EC and adjacent control tissues were measured by immunohistochemistry and compare the positive expression of the ERα,ERβ,PR and AR proteins and the effection on the survival in the 1,035 patients(711 males and 324 females)with ESCC.2.2.3 WES and partial related protein validation WES: ESCC cancer tissue and normal tissue adjacent to carcinoma were extracted with DNA extraction kit.After quality contral,a DNA library was established.After the library quality inspection,WES was performed using a second-generation sequencing platform.Analyze the data and finally screene the high frequency mutant genes and mutation types associated with ESCC.Using the Welcome Trust Sanger Institute mutational signatures framework and Non-negative Matrix Factorization(NMF)methods to decompose point mutations into multiple different mutation characteristics,and then cluster analysis was performed with the known 30 mutation features,and the sample mutation process was explained using annotation information of known features similar to the sample mutationcharacteristics.To compare the high-frequency mutations and mutation characteristics between male and female patients with ESCC,and to analyze the expression of some related proteins in ESCC and the prognosis.The number of samples entering the sequencing part was 523,and 375 of the sequencing samples were subjected to immunohistochemistry.Except for 375 cases of immunohistochemical samples,the sample number was increased to 5,736 cases2.2.4 Statistical methods SPSS 21.0 statistical software was used.It was mainly including Chi-square test,Kaplan-Meier,and Cox regression model survival analysis.3 Results3.1 Epidemiological investigation and survival analysis of male and female EC patients3.1.1 General conditions,lifestyle,incidence and survival differences between male and female patients In total,there were 23,269 patients(males: 14,408 cases,61.0%;females: 8,861 cases,39.0%)with EC.The incidence of male was significantly higher than that of women,male/female =1.63.The proportion of the male patients were higher than females patients in diagnostic age less than or equal to 55 years old,older than 55 years old,patients with history of smoking,drinking,married,married age less than or equal to 20 years old and older than 20 years old patients;The overall survival of the females patients were longer than the male patients in the same condition one of the above factors,and the difference had statistical significance.3.1.2 Differences in clinical pathological features and overall survival between male and female patients The incidence rate of clinical pathological features in male patients were higher than female patients in family history,high/low incidence area,location,pathological type,early middle and late stage,lymph node metastasis,T stage,N stage,TNM stage,pathological grade had no statistical significance;The incidence rate of the degree of differentiation and M stages in male patients and female patients had no statistical significance;The overall survival of the females patients were longer than the male patients in the same condition one of the most above factors,and the difference had statistical significance.3.1.3 Differences in treatment methods and overall survival between male patients and female patients The surgery alone is the main treatment method both male patients and female patients,secondly is radiation alone.The proportion of male patients with surgery alone,radiation alone and chemotherapy radiation alone was higher than that of female;The overall survival of the female patients were longer than the male patients in the three therapy methods one of the above factors,and the difference had statistical significance.3.1.4 Multifactor survival analysis(COX regression)Sex,age,family history,differentiation degree,pathological stage,early and late stage were independent risk factors for prognosis of patients with esophageal cancer.3.2 Expression and survival analysis of ERα,ERβ,PR and AR protein in ESCC3.2.1 The expression of ERα,ERβ,PR and AR protein in male and female ESCC tissues3.2.1.1 ERα、ERβ、PR and AR were expressed in the cytoplasm and nucleus of cancer cells.3.2.1.2 The positive expression rates of ERα,ERβ,and PR of female ESCC patients were higher than that of male patients.The positive expression rate of AR in male patients was higher than female patients.The differences were statistically significant.3.2.1.3 The positive expression rates of ERα,ERβ,PR and AR protein in cancer tissues of male and female ESCC patients were different in diagnosis age,family history,high and low incidence area,lymph node metastasis and pathological grade.The difference was statistically significant3.2.2 The survival analysis of sex hormone receptor and the patients of ESCC3.2.2.1 Single factor survival analysis.ERα、ERβ、PR and AR were the risk factors for the patients of ESCCpatients.The overall survival of patients whose tissue were positive expression of ERα、ERβ 、 PR were longer than negative expression;AR is opposite.There was no statistically significant difference in overall survival between the male and female patients whose tissue had the positive expression of ERα and PR.For the ERβ and AR,the males’ survival was longer than the female.3.2.2.2 Multifactor survival analysis.ERβ and AR were independent risk factors for the patients of ESCC patients.3.3 WES of ESCC cancer tissues,the expression of partly related protein and survival analysis.3.3.1 WES3.3.1.1 Somatic mutation The number of somatic cell mutations in male was significantly higher than that in female.The rate of somatic cell mutations between male and femal patients was no statical significance.3.3.1.2 High frequency mutant gene114 high frequency mutated genes were detected,13 of them were key related genes to ESCC,including P53,CDH1,etc.None of them had significant difference between male and female patients.3.3.1.3 Base replacement mutation type and mutation characteristics The percentage of three kinds base replacement mutation types(C>T/G>A,T>C/A>G,T>G/A>C)were difference between male and female patients,and the it had statistical difference;We detected Signature.1 and Signature.17,which had statistical difference between male and female ESCC tissue.3.3.1.4 The mutation of P53 and CDH1 There was no statistical significance in the proportion of gene mutation rate,point mutation rate and six kinds base replacement types of P53 and CDH1 in male and female patients of ESCC.There was no significant difference in gene mutation rate between the patients whose P53 protein was positive(67.5%)or negative(58.8%).There was no significant difference in overall survival between patients with P53 gene mutation or not.The overall survival was different among the different mutation types in the positive expression of P53 patients.There were statistically significant differences in the ratio of gene mutation types in patients with positive and negative P53 protein expression.The rate of missense mutation was hightest in the patients with positive P53 protein expression.There was significant difference in gene mutation rate between the patients whose CDH1 protein was positive(1.0%)or negative(9.7%).3.3.2 The expression of P53 and CDH1 protein in male and femal patients of ESCC and survival analysis.3.3.2.1 The expression of P53 and CDH1 protein in ESCC.Both P53 and CDH1 proteins were expressed in ESCC,P53 protein was mainly expressed in the cell nucleus,and CDH1 was mainly expressed in the cell membrane.There was no statistically significant difference of the positive expression rate between male and female ESCC.3.3.2.2 Survival analysis The overall survival of the patients with positive expression P53 was shorter than the negative ones,and the overall survival of the females’ was longer than the males’,and the differences had statistical difference;So it was CDH1.4.Conclusion4.1 the percentage of the male and female EC were statistacil difference in diagnostic age,history of smoking,drinking,married or not,married age,marital status,family history,high/low incidence area,location,pathological type,early middle and late stage,lymph node metastasis,differentiated degree,pathological grade and therapy method;Single factor survival anysis showed: diagnostic age,history of smoking,married or not,married age,marital status,family history,high/low incidence area,location,differentiated degree,lymph node metastasis,T stage,N stage,M stage,TNM stage,early middle and late stage and therapy method,which were the factors of influenceing the overall survival of EC patients;Cox mutifactor survival analysis showed: sex was an independent factor affecting the prognosis of esophageal cancer.Female patients were the protective factors for the prognosis of esophageal cancer.4.2 The expression percentage of ERα,ERβ,PR and AR in the diagnostic age,high/low incidence area,family history,lymph node metastas and pathological grade between male and female ESCC patients had statistical difference;Single factor survival anysis showed: ERα,ERβ,PR and AR are the factors affecting the prognosis of patients with ESCC;Cox mutifactor survival analysis showed: ERb and AR were independent factor affecting the prognosis of ESCC4.3 WES showed: The rate of somatic cell mutations between male and femal patients was no statical significance;The difference of the percentage of three mutation types(C>T/G>A,T>C/A>G,T>G/A>C)may affect the overall survival of the male and female patients.The different of Signature.1 and Signature.17 between male and female patients may be the molecular basis that lead to the male and the female’ different overall survival;There was no significant difference between P53 and CDH1 gene in the rate of gene mutation,point mutation and 6 base replacement types in patients with ESCC.The overall survival was no significant difference between the mutation of P53 gene or not.The different mutation types of P53 gene may have the relation with the P53 protein expression and the overall survival.