A Analysis On TP53,HER-2 And MRP-1 Mutation In ESCC,And Their Protein Expression And The Clinical Pathological Characteristics Effect On The Prognosis

1 Background and objective of researchEsophageal cancer is one of the 8 most common malignant tumor in the world,the death rate ranked sixth. China is a high incidence area of esophageal cancer,with the main pathological types of esophageal squamous cell carcinoma(ESCC),accounting for about 95%. At present, researchers views is consistent of the pathogenesis of esophageal cancer, believe that cancer is a gene environment interaction results, evolution process involving multiple genes and multiple factors and stages of the variation of accumulation. With the development of human understanding of cancer genetics and the emerging of the second and third generation high-throughput sequencing technologies, the research of genomics has entered a stage of vigorous development. China researchers through independent GWAS carried out a number of ESCC gene research, has achieved remarkable success.Tumor invasion, differentiation, lymph node metastasis and distant organ metastasis can affect ESCC patients prognosis; phenomenon of drug resistance is the main effect factors on the choices of treatment methods and the therapeutic effect in cancer patients receiving chemotherapy. Study on genes related pathogenesis and progression and chemoresistance of ESCC for preventing the progression of early stage, and conquering drug resistance has important significance. And oncogene and anti oncogene activation must by its encoding product or affecting other gene encoding product induced malignant transformation of normal cells, using immunohistochemical method analysis related protein expression of the susceptible sites or pathogenic gene which impacted on tumor has important significance.This study combined with P53, HER-2 and MRP-1gene exon sequencing results in ESCC tissues to analyze the gene mutation type and mutation rate, combined three protein immunized group, to explore three genes protein expression and clinical pathological features effect on prognosis in patients ESCC, further analysis the expression of P53, HER-2, MRP-1 to explore whether there is correlation between three protein independent or combined expression on prognosis of value judgment.So that we can further understanding the genome changes have occurred in the development in ESCC; access to efficient, sensitive and specific protein markers,guide groups to implement effective prevention and intervention therapy and gene therapy.2 Materials and methods2.1 Research objectsThis study divided into whole-exome sequencing(WES) group of esophageal carcinoma from high incidence area of ESCC patients 116 cases and carcinoma tissue protein expression of 728 cases of ESCC patients. All the sample data and information used in this research are from Henan Province, the First Affiliated Hospital of Zhengzhou University, Key Laboratory for esophageal cancer research,with 50 million cases of esophageal cardiac cancer and biological samples information library information between 1973 to 2015.From the database according to the pathology after surgery for ESCC, with immunohistochemical results of successful patient follow-up, verified and complemented pathology and clinical information, protein expression analysis group of 728 cases. And collected corresponding cancer tissues and adjacent normal tissue paraffin blocks of 116 cases for WES from hospital, putted in- 20 degrees refrigerator for preservation.2.2 Experimental methodsExtracted DNA from paraffin embedded tissue of 116 ESCC cases for Exon sequencing, And then Conducted DNA electrophoresis, measured and standardized the DNA concentration, Sequencing.2.5 Statistical methodsSPSS21.0 was used for statistical analysis of data. Differences between groups were compared by using the x2 test, The correlation between variables were analyized by using Spearman rank. Kaplan Meier plot survival curve is used to estimate the survival rate, and log rank test for differences in survival; Cox proportional hazards regression model screening of factors affecting the prognosis of patients with ESCC. The test standards: ?=0.05.3 Results3.1 Whole-exome sequencing results3.1.1 Overall mutation116 patients with ESCC had a different degree of gene mutation, a total of166945 mutations. The SNP mutations were dominant, accounting for 94.96%,Insertion and Deletion, which accounted for 2.29% and 2.74%. 3'Flank, 5'Flank, RNA mutation rate is higher, respectively, 15.36%, 15.32%, 42.81%.3.1.2 Mutation results of TP53 geneThe cases of occured mutations in the TP53 exon region was 80, and so the frequency of mutation was 68.6%. A total of 100 mutations, including 83 SNP, 9Insertion and 8 Deletion. The highest incidence of 58% missense mutations, nonsense mutation rate of 20%, then the frameshift insertion rate was 9%. TP53 mutation rate of SNP was significantly lower than that of WES, and the mutation rate of Insertion and Deletion was significantly higher than that of whole genome exon sequencing.3.1.3 Invariant results of HER2 and MRP-1 geneHER2 gene mutation frequency was 1.72%(2/116), a total of two mutations;MRP-1 gene mutation frequency was 3.45%(4/116), a total of 10 mutation events.3.2 Immunohistochemical protein expression group3.2.1 The expression of P53, HER-2 and MRP-1 of carcinoma tissue and its relationship with clinicopathological features in ESCC(1) The positive expression rate of P53 protein was 44.1%(321/728). The positive rate of P53 protein expression in no lymph node metastasis group was significantly lower than that of lymph node metastasis; P53 expression rate in pathologic stage III, IV was significantly higher in the class I, II group; P53 protein in patients with advanced stage is more than 2 times than that of early stage.(2) The positive expression rate of HER-2 protein is 46.0%(280/728). With the poorly differentiated, lymph node metastisis, The distant organ metastasis, the pathological grade increased, the clinical staging increased, the positive expression of HER-2 in ESCC was gradually increased.(3) Multidrug resistance associated protein(MRP-1) protein expression rate is51.5.8%(375/728). With the pathological grading increase positive rate in order to rise, I, II, III, IV, the MRP-1 positive rate followed by 34.4%, 50.8%, 52.4% and72.7%; MRP-1 in clinical stage increased, the positive rate increased in the order of34.4%, 51.6%, 72.7%; MRP-1 expression of organ metastasis group positive rate of53.5% was significantly higher than that of no organ metastasis group the positive rate of 48.9%.(4) The has no correlation between the expression of P53, HER-2 and MRP-1.3.2.2 ESCC P53, HER-2 expression of MRP-1 three protein differences in the effects on the prognosis of the patients(1)The expression of P53 protein positive group survival was poor compared with negative group( c2=6.522, P = 0.01), the median survival time were for 2.638,4.016, by 1.5 times; In no distant metastasis group(M0) the P53 protein positive group than those in the negative group, the prognosis is poor, the median survival time were 273.2 and 4.197.(2) HER-2 protein positive group than negative group has a poor prognosis( c 2=8.199, p=0.004). The median survival was 2.732, 4.170. HER-2 negative expression group was than positive group, the prognosis is good no matter in Lymph node metastasis(N1); In no distant metastasis group(M0) or no distant metastasis group(M1) the expression of HER-2 negative group has a better prognosis than positive group.(3)The survival analysis results showed that there was no correlation between MRP-1and tumor survival.(4)The combination expression group of HER-2 and P53 survival analysis showed that P53, HER-2 double positive expression group and single positive group and no positive group, the prognosis of the double positive group was the worst.4 Conclusions4.1 Exon sequencing group(1) 116 patients with ESCC had a different degree of gene mutation, a total of166945 mutations, including SNP mutations, accounting for 94.96%.(2) The cases of occured mutations in the TP53 exon region was 80, and the frequency of mutation was 68.6%. A total of 100 mutations, missense mutation had the highest incidence was 58%.(3) TP53 exon SNP gene mutation rate was significantly lower than that of WES,Deletion and the mutation rate of Insertion was significantly higher than that of whole exome sequencing.(4) This study shows that Henan Province, Linzhou and other places in high incidence area the HER2 and MRP-1 exon mutation rates were 1.72% and 3.45% of ESCC.4.2 Protein expression analysis group Conclusion(1)The expression of P53 protein is one of the independent factors affecting the survival of patients with ESCC, compared with that in the negative control group,positive group survival were poor, and regardless whether there are metastasis to distant organs or not.(2) The expression of HER-2 protein is one of the independent factors affecting the survival of patients with ESCC. HER-2 over expression in patients with ESCC is associated with poor prognosis regardless whether there are lymph node metastasis and distant organ metastasis or not.(3)There is no correlation between MRP-1 and ESCC survival.(4)P53 and HER-2 two protein expression group prognosis is the poorest than simple expression of a protein group.