| 1.Background Gastric cardia adenocarcinoma(GCA)is one of the most common malignant tumors of digestive tract in north China.According to the recent epidemiological data in China,high incidence area of GCA is similar to the esophageal cancer and linzhou is one of the highest incidence areas of cardia adenocarcinoma in the world.Most patients with early gastric cardia adenocarcinoma are difficult to have obvious clinical specific symptoms,and more than 95% of the patients who are diagnosed with GCA are in the middle or late stage,with relatively low quality of life,and the overall 5-year survival rate is not ideal.The cause of gastric cardia adenocarcinoma in China is the result of multiple mechanisms,the most important of which are environmental and genetic factors.Previous studies have demonstrated that differences in environmental and heritage factors have different degrees of impact on patient survival.In addition,the tumor stage,patient gender,age,smoking,alcohol genetic susceptibility,pathological type,general type,and tumor location may also affect the survival of patients with gastric cardia adenocarcinoma.Until today,research on cardiac adenocarcinoma is less,and the sample size of epidemiology of GCA is very small,no system and comprehensive comparative study of molecular biology.The aim of this study is to analyze the influencing factors of survival on cardia adenocarcinoma through the large database of clinical,pathological and follow-up information of cardia adenocarcinoma in our laboratory.We also apply Molecular experiments,like Whole genome Sequencing exons(Whole Exome Sequencing,WES)and immunohistochemical to test the expression of protein,we hope to explore the clinical and molecular mechanisms affecting the survival of gastric cardia adenocarcinoma.Furthermore,we try to find nova genes those affect GCA's prognosis,and provide support for earlier diagnosis,high-risk population screening and targeted therapy of GCA.2.Materials and methods2.1 Subjects All patients were from the database of the Henan Key lab for esophageal cancer research in the First Affiliated Hospital of Zhengzhou University.All of them were pathologically confirmed with primary gastric cardia adenocarcinoma.They did not merge with other tumors.The survival time was followed up.The diagnosis time was1973-2018.Accurate medical record number and pathology number,complete post-clinical pathology records,including TNM staging,differentiation degree,etc.The contents were comprehensive,detailed,authentic,reliable,and includes large-scale survival follow-up.2.2 Methods2.2.1 Epidemiological research The general condition and clinical pathological information of 35744 patients with gastric cardia adenocarcinoma from Henan Key Lab for Esophageal Cancer research were analyzed by chi-square test,Kaplan-Meier survival analysis and cox single factor and multivariate regression analysis.2.2.2 Whole exon sequencing,target gene sequencing Whole Exome Sequencing(WES): DNA extraction kit was used to extract gastric cardia adenocarcinoma and adjacent normal tissues.After quality inspection,a DNA library was established.After the library quality inspection,the second generation sequencing platform was used.Machine sequencing.The standardized data was used to analyze the data,and finally the high frequency mutation genes and mutation types associated with gastric cardia adenocarcinoma were screened.At the same time,the survival-related genes were selected for protein validation analysis.2.2.3 Immunohistochemistry For the sequencing of partial mutations and survival-associated significant genes,3333 cases of generalized information,clinical pathology and survival follow-up of gastric cardia adenocarcinoma patients with cancer and adjacent control tissues were detected by immunohistochemistry,and their expression was analyzed.Prognosis survival analysis of patients with gastric cardia adenocarcinoma.2.2.4 Statistical analysis.SPSS21.0 statistical software was used,including chi-square test,Kaplan-Meier,and survival analysis of Cox regression model.3.Results3.1 Epidemiological investigation and survival analysis of patients with gastric cardia adenocarcinoma3.1.1 Compositional ratio of clinical phenotype in the early and late stages Among the patients with early cardiac adenocarcinoma,there were 1261 male patients(78.7%),and with 342 females(21.3%).In the middle and late stage of gastric cardia adenocarcinoma,there were 14,546 male patients(77%),while female patients had 4,334(23%),there was no significant difference between gender(P=0.073).The number of patients with early cardiac cancer gland greater than or equal to 60 years old was 980(61.3%),and those younger than 60 were 620(38.8%).Among patients with advanced gastric cardia adenocarcinoma,the number of people over 60 years old was 10,881(57.7%),and the number of people younger than 60 years old was 7,968,(42.3%).The difference was significant(P=0.003).There were1247 people in high incidence area of early stage adenocarcinoma,accounting for78.1% of the total number,and 329 patients in low incidence area,and accounting for21.9% of the total.There were 12,722 people(67.9%)in the high-incidence area of? ? patients with advanced gastric cardia adenocarcinoma,and 6,026 patients in the low-incidence area,accounting for 32.1%.The difference was significant(p =1.26E-17).Among the patients with early-stage gastric adenocarcinoma,219 were smokers,accounting for 40.9%,and 317 were denied smoking,accounting for 59.1%.Among the patients with advanced gastric cardia adenocarcinoma,2,071 were smokers,accounting for 46%,and 2,430 were denied smoking,accounting for 54%.,the difference is significant p=0.013.There were 169 patients(28.9%)with alcohol history and 415 patients(71.1%)with no alcohol history in early stage.There were1629 patients(34.4%)with alcoholic history,and 3106 patients(65.6%)with no alcohol history in the middle and late stage.The difference was significant(P=0.004).In the early stage,the proportion of patients with well differentiation was243(20.9%).The number of patients with moderate differentiation was 564,accounting for 48.7% of the total.The number of poorly differentiated patients was352(30.4%).In the mid and late stage,The number of well differentiated patients was1116,accounting for 9.3%,and the number of patients with moderate differentiation was 5605(45.4%),5346 people with poor differentiation(45.3%),the degree of tumor differentiation was significant in the early stage compare to middle and late stages of gastric cardia adenocarcinoma(P= 5.36071E-42).There were 477 patients(87.4%)with positive family history and 69 patients(12.6%)with negative patients in the early stage.3254(81.7%)patients with positive family history and 728 patients(18.3%)were negative in the middle and late stage,and the difference was significant(P=0.001).3.1.2 Univariate survival analysis Women's survival is significantly longer than men's.The survival of patients older than 60 years old was significantly lower than that of patients younger than 60 years old,and the race had no effect on survival.The survival period of smoking and drinking patients was significantly lower than that of patients without smoking or drinking history;the survival time of patients with high incidence was significantly longer than that of patients with low incidence.The survival time of early cancer was significantly longer than that of patients with advanced gastric cardia adenocarcinoma.3.1.3 Multivariate survival analysis Gender,age of diagnosis,high and low incidence area,degree of tumor differentiation,TNM stage,smoking,drinking,and tumor stage were independent prognostic factors for patients with gastric adenocarcinoma.3.2 WES and Immunohistochemistry3.2.1 WES3.2.1.1 High frequency mutant gene A total of 114 high-frequency mutated genes were detected,and two new genes ERICH3 and IVL related to the survival of gastric cardia adenocarcinoma were detected.3.2.1.2 Signatures of mutation A total of two mutation characteristics were detected: signature.1 and signature.17,positive by family history,both of which decomposed Signature.1,the difference between the two was that the negative decomposition of Signature.C,close to Signature.18.Positive decomposition of Signature.A,with Signature.4.3.2.1.3 Mutations of ERICH3 and IVL Among them,IVL had significant difference in single-gene survival analysis,and Q=0 in the MUSic test,p value was 0,the number of mutation samples was 33,SNVs was 33,and indels was equal to 0.The mutation detection rate in patients with gastric cardia adenocarcinoma 5.54%.ERICH3 was significantly different in single-gene survival analysis,while MUSic tested Q= 1.72E-06,p value was 6.29E-09,mutation sample number was 20,SNVs was 19,and indels was equal to 1.Tubal adenocarcinoma the mutation detection rate in patients was 3.63%.3.2.2 Expression and survival analysis of ERICH3 and IVL in gastric cardia adenocarcinoma3.2.2.1 Expression of ERICH3 and IVL negative-positive proteins in gastric cardia adenocarcinoma ERICH3 and IVL proteins were expressed in gastric cardia adenocarcinoma.IVL was significant in age,high and low incidence area,smoking,and family history chi-square test.ERICH 3 was only significant relate with the degree of differentiation3.3.2.2 Survival analysis of ERICH3 and IVL protein expression and prognosis in patients with cardiac cancer In univariate analysis,the positive expression of ERICH3 and IVL were significant risk factors for the survival of patients with cardiac adenocarcinoma.In cox multivariate analysis,IVL was an independent risk factor for survival.4.Conclusions4.1 Age,high and low incidence area,smoking,drinking,differentiation degree and family histories are significantly different between early and middle-late stage patients.4.2 Gender,age of diagnosis,high and low incidence area,degree of tumor differentiation,TNM stage,smoking,drinking,and tumor stage are independent prognostic factors for patients with gastric cardia adenocarcinoma.4.3 A total of 114 high frequency mutant genes associated with gastric cardia adenocarcinoma are detected.Two key mutations related to the survival of cardiac cancer are screened for ERICH3 and IVL.4.4 Two total mutant signs of gastric cardia adenocarcinoma are detected: Signature.C and Signature.17.4.5 IVL protein expression is negatively correlated with survival outcome in patients with gastric cardia adenocarcinoma,and is significantly different in single factor survival analysis,and is an independent risk factor in multivariate survival analysis;4.6 ERICH3 protein expression is negatively correlated with survival outcome in patients with gastric cardia adenocarcinoma.It is significant difference in single factor analysis,and there is no significant difference in multiple factors. |
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