The Significance Of High-risk SNP In Esophageal Cancer In Screening For High-risk Group

1 Background and AimThe incidence of esophageal cancer(EC) ranks sixth in malignant tumors in all around the world, and ranks fourth in cancer-related fatal tumors. The prognosis of EC is extremely poor, and 5 years survival rate of patients with advanced EC is only15%-25% [1]. China is one of the countries with the highest incidence and mortality rate of esophageal cancer in the world. There are more than 500 thousands new cases of esophageal cancer in the world each year and more than half of them occur in China. But, the examinations used in population screening and diagnosis for early esophageal cancer are very limited. Variety of endoscopic techniques play important roles in the early diagnosis of esophageal cancer. There are many limitations of endoscopic survey. Then, the first reason is the limitation of physician experience and the patients’ own tolerance and other problems in gastroscopy census; the second is that blindness and abnormal phenomena in gastroscope biopsy operation of people without symptoms; the last is the lower detection rate of esophageal cancer in the screening of asymptomatic population[2]. All the limitations make the means of detection difficult to carry out in the high incidence of asymptomatic people. How to find a non-invasive means of detection of esophageal cancer, that Is the molecular targets with higher specificity and sensitivity for early esophageal cancer, has a very important significance for the early detection, warning, and accurate screening of People at high risk.The genome-wide association study(GWAS) is a study of the mechanism of esophageal cancer in the field of genetics. There were many single nucleotide polymorphism(SNPs) and related susceptible genes have been found in the GWAS about esophageal cancer, which is closely related to the high risk of esophageal squamous cell carcinoma(ESCC) so far[3-5]. But, a lot SNPs that have been discovered failed to get widely used in clinical practice. How to combine these susceptible SNPs to build a high accuracy of esophageal cancer risk prediction model is an important issue of the GWAS area. Form a genetic point of view, our group is trying to build a cohort of asymptomatic patients in high incidence area and detect SNP with high risk of esophageal cancer and calculate genetic risk score in all enrolled patients. Through regular endoscopy checks and long-term follow-up explore the relationships between GRS and EC and verify the value of SNP on esophageal cancer high-risk warning in order to provide a suitable molecular marker for early diagnosis and clinical survey of esophageal cancer.The main contents of this study is to establish hospital outpatient queue and asymptomatic population groups in high incidence area,completing the detection of SNP and GRS calculation of the group objects and then Preliminarily analyzing the distribution differences of the GRS between the two queues, explicating the role of SNP in the high-risk groups warning,aiming at providing a basis for the feasibility study of the follow-up issues.2 materials and methods2.1 the object of study2.1.1 Queue 1( Hospital outpatient queue)The subjects were derived from the Anyang Tumor Hospital of Henan Province,Heping Hospital affiliated to Changzhi Medical College of Shanxi and Hebei Ci County People’s Hospital outpatient endoscopy patients(2012-2014). All subjects were Han people, and are residents of high incidence of esophageal in China. We selected total 1834 cases, 879 cases of males and females 955 cases, male to female ratio was 0.92: 1;the average age was 52 ± 12 years, with a median age of 53 years( range from 18-86 years). There were only 1503 cases that detected to complete all of SNPs in genotyping data, including males 708 cases, females 795 cases, male to female ratio was 0.89: 1; and a average age was 53 ± 12 years old, the median age was 53 years( range from 23-86 years).2.1.2 Queue 2(Asymptomatic population cohort of high incidence area)This queue of esophageal cancer from Henan Province Key Laboratory countryside epidemiological findings were all asymptomatic population within Henan Xinxiang Huixian area. The total cases is 3157, 1484 cases of males and females1673 cases, male to female ratio was 0.89: 1;the average age was 56 ± 9 years, with a median age of 56 years( range from 23-86 years). There were only 3147 cases that detected to complete all of SNPs in genotyping data, including males 1477 cases,females 1670 cases, male to female ratio was 0.88: 1; and a average age was 56 ± 9years old, the median age was 56 years( range from 23-86 years). All subjects were Chinese Han population, and from the high incidence area of esophageal cancer.2.2 Collection and collation of data subjectsWe had a epidemiological survey questionnaire for every subject, including the information about their age, address, occupation, smoking, alcohol consumption and family history, et al. All selected objects are drawn 3-5ml peripheral venous blood in EDTA anticoagulated or sodium citrate anticoagulant tube, then quickly placed in-20 ℃ refrigerator to prepare for subsequent DNA extraction and SNP detection.2.3 screening and genotyping of SNP in this study2.3.1 SNP screeningWe first selected 9 SNP loci from our present research findings. Meantime, we screened these reported SNPs during 2010.1-2013.12 based GWAS of esophageal cancer in Chinese population. Then, we removed these SNP locus that were clearly and highly correlated. Finally, we finalized the 34 SNPs sites to the study of esophageal cancer risk prediction models.2.3.2 SNP genotypingIn this study, 34 SNPs of 4991 samples were detected by using Sequenom Mass array SNP technology for these SNPs genotyping and allele frequency calculation.Due to the detection rate of rs1045485, rs2244438 and rs3769823 was pretty low,eventually the full results of gastroscopy and the remaining SNP genotyping data are detailed in the 4650 cases of esophageal cancer risk prediction model into the sample analysis.2.4 esophageal cancer genetic risk score(GRS) calculationWe will not consider intergenic chain reaction, that the risk of every SNP locus to esophageal cancer is independent of each other. According to the algorithm of genetic risk score(GRS) in Che’s study [6]. Through literature search risk ratio(OR),and minor allele frequency(MAF), we calculated the explained variance weighted genetic risk score(EV-GRS).2.5 statistical analysisIn this study, we used PLINK statistical software and SPSS 21.0 software for remaining statistical analysis to screen SNP sites and calculate EV-GRS. Independent samples t-test was used to compare EV-GRS distribution among the queues and X2 test was used to analyze the differences between EV-GRS groups. Significance levelα = 0.05.3 the results3.1 to analysis the esophageal endoscopy and pathology results in asymptomatic populations from the high incidence areaThe data were collected from the high incidence area of esophageal endoscopy screening asymptomatic population, 616 cases were detected with all types of esophageal disease, the detection rate of 33.6%. Among the detection of various types of esophageal diseases, inflammation of the esophagus accounted for the vast majority(33.6%, 535/1834), in which chronic esophagitis highest detection rate(24.6%, 452/1834), followed by reflux esophagitis(2.9 %, 53/1834); There was a higher detection rate of esophageal cancer(1.5%, 27/1834). This study detected 616 cases of various types of esophageal diseases there are differences(P <0.05) among different age groups in the 60-69 age group of esophageal cancer detection rate was the highest(51.9%), chronic esophagitis was highest in the period of 50-59 years old(35.2%), and reflux esophagitis and esophageal leiomyoma in the 40-49 age group got the highest detection rate(32.1%, 36.4%).3.2 The distribution and difference of EV-GRS in queue1 and queue23.2.1 The distribution of EV-GRS in queue1 and queue2In queue 1, calculated EV-GRS of 1503 cases of SNP genotyping data were com plete,the average value is 2.65 + 0.55, median 2.65, range 0.51-5.05. The K-S test sho wed EV-GRS obey normal distribution(Z value =0.964, P value(SIG 2-tailed) = 0.310); In queue 2,calculated EV-GRS of 3147 patients whose gene SNP genotype data w ere complete, average value 2.57±0.52, median value 2.58, ranging 0.94-4.36. K-S tes t shows that the EV-GRS obey normal distribution(Z value=0.804,P value(sig 2-taile d)=0.537).3.2.2 The distribution difference of EV-GRS in queue1 and queue2EV-GRS in the double group queues are subject to normal distribution, and two overall variances are uniform(F = 1.727, P = 0.189); Using two independent samples t-test showed the patients in array 1 is higher than the average EV-GRS queue 2subjects(t = 3.755, P = 0.000,95% CI = 0.09-0.03)。In this study, 5% and 95% based on the EV-GRS divided into three groups,Respectively are the low risk groups(EV-GRS <1.77), the middle-low risk groups (EV-GRS is 1.77-3.48) and high-risk groups(EV-GRS> 3.48)。Queue 1 is higher than the proportion of high-risk patient cohort 2, while low-risk patients is higher than in the population cohort 2, the difference was statistically significant(X2 = 8.5, P =0.014)。4 conclusions1) Of the endoscopy screening of asymptomatic people in high incidence area of EC, the detection of various types of esophageal diseases is pretty high, which is the majority of esophageal inflammation. There are significant differences among different types of esophageal diseases.2) Genetic risk score has a differences in the distribution of the high incidence of hospital outpatient and asymptomatic population. The high incidence of the population can be divided into different subgroups by Genetic risk score. This study plays a role for the follow-up work of this topic. And this study provides an important basis for further evaluation of the significance of high-risk SNP in esophageal cancer.