The Study Of Fibroblast Growth Factor Receptor 4,Her2 And Estrogen Receptor In Hepatitis B Virus-associated Hepatocellular Carcinoma

Background: Primary liver canceris a malignant tumor occurring in hepatocytes and / or intrahepatic bile duct epithelial cells,the most common histological type is hepatocellular carcinoma(HCC).In clinic,liver cancer is mainly treated by surgery,but its onset is hidden and it is not easy to be detected in early.Most of the patients have lost the opportunity of operation when they see doctors.Currently,molecular targeted therapy is one of the hot topics in the field of cancer therapy,many specific targeted drugs have achieved good results in the treatment of related tumors.However,the occurrence of HCC is related to many factors.Up to now,no specific target has been found for HCC.At present,tyrosine kinase inhibitors are the majority of targeted drugs used in the treatment of tumors.Fibroblast growth factor receptor(FGFRs)is a kind of tyrosine kinase receptor.There are five identified members of the family(FGFR1?FGFR2?FGFR3? FGFR4 and FGFR5),among which FGFR4 is the most widely distributed member of the FGFRs family.FGFR4 is mainly expressed in mature hepatocytes,but few studies have been reported in HCC.Her2 protein is a transmembrane protein encoded by Her2 proto-oncogene and belongs to tyrosine kinase receptor like FGFR4 protein.Normally,Her2 protein participates in normal physiological functions such as cell proliferation and differentiation.When exposed to carcinogenic factors,the Her2 gene is abnormally activated.The main manifestation is that gene amplification leads to overexpression of protein and stimulates cell proliferation.Her2 monoclonal antibody(tratozumab et al.)has achieved significant therapeutic effect in the treatment of advanced breast cancerand gastric cancer,but there are few reports on the relationship between Her2 and hepatocellular carcinoma.Estrogen receptors mainly include ER? and ER? subtypes,and ER? subtype is the main subtype in human body.The molecular weight of ER? protein is 66 KDa,so it is also called ER? 66.Through selective splicing,ER? 66 gene can produce splicing variants such as ER? 46,ER? 36 and so on.It is reported that ER? 66 protein is mainly expressed in tumor nucleus,while ER? 36 protein is mainly expressed in tumor cytoplasm and / or cell membrane.It has been reported that ER? 36 may up-regulate the expression of Her2 protein through some mechanism,and promote the proliferation of tumor cells.However,there are few reports on the relationship between ER? and the occurrence and development of hepatocellular carcinoma.Hepatitis B virus infection is an important factor of human chronic liver disease.About 80% patients with HCC in China are caused by HBV infection.However,the relationship between FGFR4,Her2 and ER? protein in hepatocellular carcinoma has not been reported.The purpose of this study was to study the expression of FGFR4,Her2,ER? and HBsAg protein,amplification of Her2 gene and change of HBV DNA content in hepatitis B virus-associated hepatocellular carcinoma by immunohistochemistry,fluorescence in situ hybridization and PCR-fluorescence probe method;To analyze the correlation between the indexes and the clinicopathological parameters,to provide a new idea for the further study of the occurrence and development mechanism of hepatocellular carcinoma,and provide a basis for the molecular therapy of hepatocellular carcinoma.Method:1.103 cases of liver tissue archived wax mass after operation with complete clinicopathological data were selected from the second affiliated Hospital of Dalian Medical University from January 2014 to March 2017.Among them,50 cases of hepatocellular carcinoma with HBV infection,13 cases of hepatocellular carcinoma without HBV infection,20 cases of cirrhosis with HBV infection and 20 cases of normal liver tissue.2.Using immunohistochemical staining to detect the expression of FGFR4,Her2,ER? and HBsAg protein in the samples.The amplification of Her2 gene was detectedby fluorescence in situ hybridization,and the content of HBV DNA in cancer and adjacent tissues was detected by PCR-fluorescence probe method.3.SPSS21.0 software was used to analyze the experimental data,c 2 test and Fisher's exact probability were used to analysis the counting data;independent-sample t test and single factor analysis of variance were used to measure the differences between groups;Spearman correlation coefficient was used for the correlation between variables.Values of P<0.05 were considered significant.Result:1.Immunohistochemical results(1)FGFR4 protein1)The expression of FGFR4 in hepatocellular carcinoma(including HCC+HBV group and HCC group)was lower than that in paracancerous tissues(P<0.05);2)In HCC+HBV group,the expression of FGFR4 protein was correlated with the maximum diameter of the tumor.The expression of FGFR4 protein in the tumor with maximum diameter < 3 cm was higher than that in the group with the maximum diameter ? 3 cm(P<0.05);3)In HCC+HBV group,the expression of FGFR4 protein in paracancerous tissues was related to clinical stage.The expression of FGFR4 protein in paracancerous tissues of T1+T2 stage was lower than that in paracancerous tissues of T3+T4 stage(P<0.05).(2)Her2 proteinThe expression of Her2 protein in cancer tissue was scored as follows: 46 cases of(0),8 cases of(1+),3 cases of(2+)and 0 case of(3+).There was no expression of Her2 protein in paracancerous tissues.(3)ER? protein1)Expression of ER? protein in nuclear(Nuclear positive)The nuclear positive rate of ER? protein expression in cancer tissues(including HCC+HBV group and HCC group)was significantly lower than that in paracancerous tissues(P<0.05).In HCC+HBV group,the positive rate of ER? protein expression in paracancerous tissues was higher than that in cirrhosis group and normal group(P<0.05);The nucleus positive rate of ER? protein in cancer tissue is correlated with sex,the positive rate of ER? protein in female hepatocellular carcinoma is higher than that in male hepatocellular carcinoma(P<0.05).2)Expression of ER? protein in cytoplasm/membrane(Cytoplasm/membrane positive)In HCC+HBV group,the cytoplasm/membrane positive rate of ER? in cancer tissue was significantly higher than that in paracancerous tissue,liver cirrhosis group and normal group(P<0.05).(4)HBsAg proteinIn HCC+HBV group,the positive rate of HBsAg protein in paracancerous tissues was higher than that in cancer tissues(P<0.05).2.Detection of Her2 gene amplification by fluorescence in situ hybridizationFor samples with immunohistochemical score of Her2(2+),detection of Her2 gene amplification in cancer tissues by FISH,the results showed that no amplification of Her2 gene was detected.3.Detection of HBV DNA in tissue by PCR-fluorescence probe method(1)HBV DNA was detected in both cancer and paracancerous tissues,there was no significant difference in HBV DNA content(Ct value)between cancer and paracancerous tissues..(P>0.05);(2)The content of HBV DNA in cancer tissues and paracancerous tissues in HBsAg positive group was higher than that in cancer tissue and paracancerous tissue in HBsAg negative group(P<0.05).Conclusion:1.The expression of FGFR4 protein in hepatocellular carcinoma is lower than that in paracancerous tissue;The expression of FGFR4 protein in small tumor tissues was higher than that in larger tumor tissues;The expression of FGFR4 protein in paracancerous tissues with lower tumor stage was lower than that in paracancerous tissues with higher tumor stage,suggesting that FGFR4 protein may play an inhibitory role in the development of hepatocellular carcinoma.2.The expression of Her2 protein could be detected in hepatocellular carcinoma,but no amplification of Her2 gene was detected,suggesting that the role of Her2 protein in the carcinogenesis and development of HCC needs to be further studied.3.The nuclear positive rate of ER? protein in hepatocellular carcinoma was lower than that in paracancerous tissue,and the cytoplasm/membrane positive rate was higher than that of paracancerous tissue;The nuclear positive rate of ER? protein in female HCC was higher than that in male,suggesting that ER? protein in the nucleus might inhibit the occurrence of HCC,while ER? expression in cytoplasm/membrane might promote the development of HCC.4.In HBsAg positive group,the content of HBV DNA in cancer and paracancerous tissues were higher than that in HBsAg negative group,suggesting that the expression of HBsAg protein could reflect the relative content of HBV DNA in liver tissue and HBsAg positive is one of the poor prognostic factors in hepatocellular carcinoma.5.The expression of HBsAg protein in HCC tissues was lower than that in paracancerous tissues,but the content of HBV DNA was no different from or higher than that in paracancerous tissues,suggesting that the replication level of HBV in HCC tissues was lower than that in paracancerous tissues.The integration ability of HBV DNA was higher than that of paracancerous tissues.