Study On The Application Value Of TCGA And ACRG Classification In Gastric Cancer Population In Hebei Province

Gastric cancer(GC)is a malignant tumor originating from the stomach.It is one of the most common malignant tumors in the world,and also the leading cause of cancer-related death.As one of the most commonly diagnosed cancers,GC has an annual incidence of 18/100000 in men and 9/100000 in women.The underlying mechanism of GC is complex,mainly involving gene repair dysfunction,cell growth,death and apoptosis gene dysfunction and so on.Related studies have found that different types of GC have different mechanisms of development,markers for detection of GC and clinicopathological characteristics.It is of vital importance to investigate the molecular mechanism of GC classification,so as to clarify the development mechanism of GC,thereby providing new evidence and direction for the treatment of GC,and improving the prognosis of patients.TCGA classification and ACRG classification are new molecular classification methods related to GC put forward by scholars in recent years,which can overcome the shortcomings of traditional pathological classification and lay a foundation for individualized treatment of GC.TCGA classification mainly arises from European population,whereas ACRG classification is mainly based on Asian population,predominantly from Japan and South Korea.These two genotypes have significant values in scientific research with respect to gene amplification,gene mutation,clinical characteristics,and prognosis,and are of great significance for accurate diagnosis and individualized treatment in clinical practice.However,the clinical characteristics of TCGA classification and ACRG classification in GC population in Hebei Province and their relationship with clinical parameters and prognosis remain poorly understood.The purpose of this study was to explore the relationship of the two classification methods with clinicopathological characteristics and prognosis of GC after using immunohistochemistry(IHC)and next-generation sequencing(NGS)for tumor classification,to analyze their relationship with clinical prognosis by big data mining,and to gain insight into the molecular mechanism of the occurrence and development of GC,thereby providing a new direction and experimental theory for GC research.Part One Clinical characteristics and prognostic significance of ACRG classification in GC population in Hebei ProvinceObjective: To study the expression of ACRG classification in GC tissues and to explore the relationship between molecular classification,clinicopathological characteristics and prognosis of GC,to further clarify whether ACRG classification is appropriate for GC population of Hebei Province,and to explore the effect of ACRG classification on prognosis.Methods:1.Immunohistochemical staining was used to detect the expression level of MDM2,P21,E-cadherin and vimentin in 65 GC patients.2.The correlation of the expression level of related proteins with the clinicopathological characteristics of patients with GC was analyzed.Results:1.IHC staining showed that MLH1,MDM2 and P21 proteins were mainly located in the nucleus,while E-cadherin and vimentin were mainly expressed in the cytoplasm and membrane of tumor cells.2.Significant difference was found in the expression levels of MLH1 and MDM2 proteins in GC tissues of patients of different age groups(?60 vs.>60years of age)(P < 0.05).3.The expression levels of E-cadherin and vimentin in different locations were statistically different(P < 0.05).4.There was no significant difference in sex,age,tumor location,Lauren classification or postoperative adjuvant therapy among the four ACRG subtypes of GC patients(P > 0.05).5.MSS/TP53+esophagogastric junction(EGJ)tumor(10.3%)showed low frequency,while most MSS/TP53-subtype were present in the distal stomach(41.7%).6.Patients with MSS/EMT subtype mostly had diffuse GC(53.8%),while patients with MSS/TP53 subtype tended to develop intestinal GC(57.1%;patients with MSD/TP53-had intestinal GC).7.The prognosis was significantly different between MSI subtype and MSS/EMT subtype(P < 0.001),as well as between MSS/EMT subtype and MSS/TP53-subtype(P < 0.001).The prognosis of MSS/EMT subtype was the worst,followed by MSS/TP53-,MSS/TP53+ and MSI.8.ARCG molecular subtypes(P = 0.045),Lauren classification(P =0.001)and adjuvant therapy(P = 0.013)were associated with overall survival(OS)in GC.Conclusions:1.MLH1 and MDM2 proteins were significantly correlated with age.2.E-cadherin and vimentin were significantly correlated with the location of the tumor.3.Patients with MSS/EMT subtype tended to have diffuse GC,while those with MSS/TP53-subtype tended to have intestinal GC.Most of the MSS/TP53-subtype was present in the distal stomach.4.Among all ACRG subtypes,MSI subtype has the best OS and the lowest recurrence ratePart Two Clinical characteristics and prognostic significance of TCGA classification in GC population in Hebei ProvinceObjective: To make a comprehensive analysis of GC by NGS and classify 65 patients with GC according to TCGA classification,and to explore the relationship between molecular classification,clinicopathological characteristics and prognosis of GC,further determining whether TCGA classification is appropriate for GC population in Hebei Province,thereby evaluating the effect of TCGA molecular classification of GC on prognosis.Methods:1.The gene mutations in the GC tissues of 65 patients were screened and annotated by the NGS method.2.The relationship between TCGA and OS in patients with GC was analyzed using correlation statistics.Results:1.NGS showed that among 65 GC cases,there were 6(9.2%)with EBV+subtype,15(23.1%)with MSI subtype,14(21.5%)with GS subtype,and 30(46.2%)with CIN subtype.2.MSI subtype was more common in women(53.3%),whereas EBV+subtype was more common in men(83.3%).3.In 65 cases of GC,the mutation rate of TP53 was 80.0%,and percentage of CCNE1 amplification was 20.0%.4.Patients with diffuse type,molecular subtype of GC,MSS/EMT subtype and XELOX adjuvant chemotherapy had the worst prognosis.5.Kaplan Meier graphs showed that patients with multiple gene mutations had poor prognosis.Univariate and multivariate analysis indicated that patients with TP53 mutations(risk ratio = 4.193,95% CI = 1.260-13.945)had poor prognosis.Conclusions:1.There were gender differences in the occurrence probability of MSI and EBV+subtypes.2.The occurrence probability of GS subtype and MSI subtype was different in different age groups.3.CIN subtype of GC were more common at EGJ.4.Patients with diffuse type,GS molecular subtype,MSS/EMT subtype and XELOX adjuvant chemotherapy had the worst prognosis.5.TP53 had the highest mutation rate,and CCNE1 had the highest percentage of amplification.6.Patients with multiple gene mutations had poor prognosis.7.TP53 mutation was an independent prognostic indicator of GC.Part Three Study on the molecular mechanism underlying prognostic difference between MSI and EMT subtypes of ACRGObjective: To explore the clinical significance of ACRG classification in GC after the bioinformatics analysis by using the gene expression database(GEO)from NCBI and the large public dataset database(TCGA)of cancer genome,to explore the differentially expressed genes related to MSI and EMT by analyzing the mutation frequency of MSI and EMT,and to classify MSI and EMT related genes and pathways by GO enrichment analysis and KEGG analysis,so as to explore the influencing factors of MSI and EMT subtypes of GC at the genetic level.Methods:1.The difference of expression level between GSE62254 and TCGA data set was analyzed and compared using GEO data from NCBI and data from TCGA,to explore the prognosis of GC patients.2.The differentially expressed genes related to MSI and EMT were analyzed by GO enrichment analysis and KEGG analysis.3.Univariate Cox risk regression analysis was used to explore the relationship between differential genes and prognosis of patients with GCResults:1.GSE62254 and TCGA GC data set and comprehensive analysis showed that there was a significant correlation between EMT-positive tumor and poor OS,and the OS of MSI subtype was significantly better than that of EMT.2.The mutation frequency of MSI was significantly higher than that of EMT.3.Eight genes were significantly correlated with EMT and MSI classification ation in GC patients,including THBS4,CPE,RSPO3,APOD,CRISPLD1,GHR,SFRP4 and NAP1L3.4.APOD was poorly expressed in MSI but highly expressed in EMT.The expression of CPE was low in MSI.CRISPLD1 was highly expressed in EMT.The expression of GHR was low in MSI.The expression of NAP1L3 was low in MSI and high in EMT.The expression of RSPO3 was low in MSI and high in EMT.SFRP4 was highly expressed in both MSI and EMT,and was higher in EMT than in MSI.THBS4 was highly expressed in EMT.Conclusions:1.In this study,the OS of MSI was significantly better than that of EMT.3.Bioinformatics analysis showed that THBS4,CPE,RSPO3,APOD,CRISPLD1,GHR,SFRP4,and NAP1L3 were significantly correlated with OS of GC patients.4.APOD,CPE,GHR,NAP1L3 and RSPO3 were poorly expressed in MSI,but SFRP4 was highly expressed.APOD,CRISPLD1,NAP1L3,RSPO3,SFRP4 and THBS4 were highly expressed in MSI.