TP53 And MUC6 Mutation In Gastric Cardia Adenocarcinoma And Survival Analysis

Backgrounds and ObjectsGastric cardiac adenocarcinoma(GCA) is a common Gastrointestinal carcinoma in north China, with poor progrnosis and low five year survival. In China, GCA and esophageal cancer(EC) have similar epidemiological characteristics, which are reflected in the significant regional distribution and obvious familial aggregation, that is to say, in the area of high incidence of EC, GCA also has high incidence. And both esophageal and gastric cardia carcinoma presents family aggregation phenomenon, therefore, also known as "sister cancer".Whole-exome sequence(WES), known as targeting exon capture, to detect disease-related genes mainly for the coding region of the human genome. Since the target gene accounts for only 1% of the human genome, the WES has a higher coverage and depth. Currently, the WES is an effective method in founding causative gene in the academia. In the former study, scientists have found pathogenic gene of EC. But the pathogenesis of GCA is not clear. Therefore, the present study was performed to summarize some of the results of the whole genome exon sequencing of GCA in our group. TP53 is an important human tumor suppressor gene, located on human chromosome 17P 13.1, the full length is 20kb, consisting of 11 exons and 10 introns, encoding 393 amino acid protein with molecular weight of 50KD, occuring mutations in malignant tumors patients and leading to protein functional changes. Human MUC6 located in the chromosome 11p15.5, with 33 exons, encoding stomach mucin, and often present abnormal expression in tumor tissue.In summary, this study mainly analyzed the variability of TP53 and MUC6 in GCA, and the relationship between GCA clinicopathological characteristics and prognosis and TP53 and MUC6 mutation, to provide the molecular basis for the precise clinical treatment of GCA..Materials and Methods2.1 TP53 and MUC6 mutation and the survival analysis in GCA2.1.1 PopluationA total of 69 GCA cases were enrolled from 500 thousands esophageal and cardiac tumor database of Henan Key Laboratory for Esophageal Cancer Research (1973-2015) in the First Affiliated Hospital of Zhengzhou University. Male 56 cases,with an average age of 62.5±6.7, female 13 cases, with an average age of 62.5 ±4.3.2.1.2 MethodsCollecting the post-operation specimen of GCA in hospital, and stored in liquid nitrogen, then transferred in -80 ℃ refrigerator to store. At the same, checking the name, agent, age, contact information and address of the patients, and supplementing the clinical and pathological information.Telephone and home interview were used in regular following-up, recording the survival status, death time and the reasons, etc.QIAGEN kits were used to extract genomic DNA from fresh frozen tissue. DNA concentration were detected precisely by NanoDrop 2000, not less than the desired concentration 50ng/μl, and the OD value was 1.7±2.0. DNA purity was detected by Agarose gel electrophoresis, and asked clearly bands.WES was adopted in detecting TP53 and MUC6 mutation in GCA, and BWA(Burrows-Wheeler Aligner) and GATK(The Genome Analysis Toolkit 1.6) software were for gene sequence analysis.SPSS21.0 software was choose in statistical analysis. Rate and constituent ratio were for data description, Spearman rank correlation was used for correlation analysis, Fisher exact probability method was for group comparison, Kappa test was used to detect the consistency. P<0.05 was considered with statisticall difference.2.2 Correlation and survival analysis of TP53 and MUC6 in GCA2.2.1 PopulationThe cases were the same as the first part, including 56 males, mean age 62.5+ 6.7 years old, female 13 cases, mean age 62.5+4.3 years old.2.2.2 MethodsThe same methods of data collection and follow-up were used. The SPSS21.0 software was for statistical analysis. Rate and constituent ratio data were used for describing, the difference of group were checked by Chi-quest test and Fisher’s exact test and the Kaplan Meier and log rank test and Cox regression model was used for survival analysis. P<0.05 was considered with statisticall difference.Results3.1 TP53 and MUC6 mutation in GCA3.1.1 Gene mutation of 69 cases of GCAWES detection of 69 cases of GCA showed that 80016 somatic mutations were found, including 70458 SNP (88.05%),5394 Deletion (6.74%) and 4164 Insertion (5.21%). In the 70458 SNP, missense mutation accounts for the most, with the rate of 22.01%(15507/70458); In the 5394 Deletion and 4164 Insertion,3’UTR mutations both account for the most, with a rate of 29.61%(1597/5394) and 40.25%(1676/4164) respectively.3.1.2 TP53 mutation in GCA29 cases of 69 GCA occurred TP53 mutation, with a rate of 42.0%.30 mutations were found, including 24 SNPs and 6 Deletions. In the 24 SNP, missense mutation accounted for the most, with the rate of 66.67%(16/24). Frameshift mutations were the main type in the 6 Deletions, with a rate of 83.33%(5/6). In these GCA, TP53 mutation occurred in the 7th,5th,6th,8th,4th and 9th exon, the missense mutation occurred at 5,6,7 and 8 exons son; nonsense mutation occurred in exon 6; frameshift mutations occurred in the 5th,6th,8th,9th exons.3.1.3 MUC6 mutations in GCA52 cases of 69 GCA occurred MUC6 mutation, with a rate of 75.4%. A total of 175 mutations were found, including 167 the SNPs,4 Deletions and 4 Insertions. In the 167 SNPs, missense mutation accounts for the most, with a rate of 61.68% (103/167). Frameshift deletion was the main type in the 4 Deletions, with a rate of 75.00% (3/4).4 Insertions were all frameshift insertion. In these GCA, MUC6 mutation mainly occurred in the 31th exon,103 missense mutations,3 frameshift deletions,4 frameshift insertions, a non-frameshift deletion, and a silent mutation all occurred in the 31th exon. In addition, one case of silent mutation occurred in the 24th exon.3.1.4 Correlation analysis of TP53 and MUC6In these GCA, TP53 and MUC6 both mutated 19 cases(27.5%), TP53 mutated 10cases(14.5%), MUC6 mutated 33cases(47.8%), both normal 7 cases(10.1%). Speraman rank correlation show no significance correlation (r=-0.195, P=0.109); Kappa detection presented there was poor consistency in two gene mutation.3.2 Survival analysis of TP53 and MUC6 genes in GCA3.2.1 TP53 mutation and clinical pathological characteristics in GCAIn the 69 cases, a total of 29 cases occurred TP53 mutation, with a rate of 42.0%(29/69). The TP53 mutation in positive lymph nodes was significantly higher than the negative(51.0%vsl6.7%, P=0.013).3.2.2 MUC6 mutation and clinical pathological characteristics in GCA52 cases of 69 GCA occurred MUC6 mutation, with a rate of 75.4% (52/69). MUC6 in various clinical and pathological features of GCA presented high mutation rate, but the difference between the groups was not significant.3.2.3 MUC6 and TP53 mutation and clinicopathological features analysis in GCAIn these GCA, the group of MUC6 mutation was higher than the other three groups in the clinicopathological features. Different age group (P= 0.050) and different lymph node metastasis group were significantly different (P= 0.017).3.2.4 TP53 mutation and survival in GCA62 cases of the 69 GCA were followed up successfully, the success rate was 89.9%. The 5 year survival rate was 40% in the mutated group and 54% in non-mutated group respectively. Kaplan-Meier analysis showed a slightly higher survival status in non-mutated group of TP53.3.2.5 MUC6 mutation and survival in GCA52 cases of 69 GCA occurred MUC6 mutation, with the rate of 75.4%(52/69). The 5 year survival rate was 46% in the mutated group and 57% in non-mutated group. Kaplan-Meier analysis showed that the survival rate of MUC6 gene in the non-mutated group was higher than that in the mutation group, but there was no significant difference.3.2.6 TP53 and MUC6 and survival in GCAIn the 62 successfully follow up cases, the survival rates had no significant difference (x2= 1.912, P= 0.591) among the four groups of TP53 and MUC6 mutation, TP53 mutation, MUC6 mutation and both non-mutation, pairwise comparisons of each group had no significant difference in survival.3.2.7 Clinicopathological characteristics and survival in GCAAmong the 62 successfully follow up cases, survival rate had no statistically significant difference in the clinicopatholigical characteristics, concluding gender, age, height-prone areas, urban rural, gross type, tumor through the longest, differentiation, T stage, N stage, TNM stage and family history of carcinoma.3.2.8 Multivariate Cox regression analysis GCAThe data showed that TP53 mutation (OR= 1.326, P= 0.600,95% CI= (0.462-3.809)) and MUC6 mutation (OR= 1.851, P= 0.345,95% CI= (0.515-6.650)) in GCA may be associated with the risk of death of GCA patients, but this difference was not statistically significant.Conclusion4.1 TP53 and MUC6 mutation in GCATP53 and MUC6 mutation in these GCA were all with high mutation rate, and mainly was SNP mutation. TP53 mutation mainly occurred in 7th,5th,6th,8th,4th and 9th exons. MUC6 mutation mainly occurred in the 31th exons. Spearman rank correlation showed that both mutations had no significant correlation; Kappa consistency test showed two mutations poor consistency4.2 Clinicopathological characteristics and survival analysis with TP53 and MUC6 mutation in GCATP53 and MUC6 mutation were significantly associated with lymph node metastasis in these GCA patients, the survival rate of non-mutated group was superior than the mutated group, TP53 and MUC6 might be associated with risk of death of GCA patients.