Etiology And Somatic Mutations Of Esophageal Squamous Cell Carcinoma In A High-incidence Area

BackgroundBased on the lastest International Agency for Research on Cancer(IARC)statistics,455,800 new esophageal cancer cases(3%of all cancers)were estimated and 400,200 deaths as a result of esophageal cancer(5%of all cancer deaths)occurred worldwide in 2012.The incidence of esophageal cancer,one of the most lethal malignancies,shows significant regional differences worldwide,and about half of new cases of esophageal cancer occurring in China.Esophageal cancer has two main histopathological subtypes:esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In the high-risk regions,(i.e.,Central and East Asia as well as eastern Africa),90%of esophageal cancer cases are ESCC,compared with less than 30%in North America.The main risk factors for ESCC in western countries are smoking and alcohol drinking,which account for almost 90%of the population attributable fraction.But,the relative contribution of smoking and alcohol consumption in high-incidence areas is relatively weaker and the conclusion remains inconsistent,and the risk factors for ESCC are not yet clear.Genetics studies have shown that genetic and environmental factors play a key role in the carcinogenesis of ESCC.The initiation and progression of tumors usually originate from a series of somatic mutations.By analyzing the somatic mutations of ESCC,especially the function of significant mutation genes,we can explore the driving genes of esophageal squamous cell carcinoma,which will contribute to understanding the essence of initiation,promotion.and progression of ESCC.Analyzing the mutation characteristics of ESCC can reversely validate the risk factors of ESCC.In addition,exploring the somatic mutations of ESCC is essential and important for the early diagnosis,clinical targeted therapy and prognosis of ESCC.With the progress of next-generation Sequencing technology,whole exome sequencing(WES)is highly sensitive to identify common and rare mutations,and find most disease-related mutations in exon regions.Although it only sequences about 1%of the genome,it becomes the most cost-effective strategy to identify Mendelian disease-causing genes.However,the limitation of the current studies on the risk factors and somatic mutations of ESCC includes the following aspects:Firstly,although the incidence of ESCC has been reduced in recent years in China,its morbidity and mortality are still the fourth malignant tumors,which causes more serious disease burden,especially in high incidence areas.At present,the risk factors of ESCC is not yet clear.The relationships between some potential adjustable exogenous risk factors,such as smoking,alcohol drinking,teadrinking and individual weight,and the risk of ESCC have not been explored in detail.Reducing the exposure of these exogenous risk factors may help prevent the occurrence of ESCC,especially in high-incidence areas.Secondly,somatic mutations in tumor tissue usually is the initiating factor for tumor development.However,few researches have explored the somatic mutation of ESCC in in high-incidence areas of East China.The application of WES will greatly enhance our ability to explore genetic variants that have not yet been identified,recognize tumorigenesis at the whole genome level,and identify the pathogenesis of exogenous factors.To deal with the above-mentioned issues and and the feasibility of realistic research,this thesis is divided into four studies to explore the etiology and somatic mutations of ESCC in high incidence areas.In Study 1,2 and 3,a population-based case-control study was conducted in Taixing to investigate the effects of smoking and alcohol drinking,tea drinking and temperature,body mass index(BMI)and Stunkard body shape for the risk of ESCC.In Study 4,we applied WES technology to sequcense the cancer tissues and matched premalignant tissues from ESCC patitents collected in Taixing People's Hospital,and construct a preliminary somatic mutation profiling of ESCC.ObjectivesStudy 1 investigated the assocaitions between smoking,second-hand smoke exposure and alcohol consumption and the risk of ESCC.Study 2 investigated the assocaitions between green tea drinking,especially its temperature and the risk of ESCC.Study 3 investigated the assocaitions between adult height,BMI change and Stunkard body shape change and the risk of ESCC.Study 4 analyzed cancer tissues and matched premalignant tissues sequencing data and construct a preliminary somatic mutation profiling of ESCC in a high-risk area.Materials and MethodsIn Study 1,2 and 3Firstly,we conducted a rigorously designed population-based case-control study for upper gastrointestinal cancer from October 2010 to September 2013 in Taixing,a high-risk area in China.Our goal enrolled all possible newly-diagnosed esophageal cancer cases in the local Four large hospital gastroscopy room,and interviewed these cases before they were aware of their diagnosis.Moreover,we further supply missed esophageal cancer cases in endoscopy units by the local Cancer Registry.Controls were randomly selected from the general population by matching the by sex and age groups.Finally,1418 ESCC cases and 1992 controls participated in this study.General demographics,occupancy history,occupational history,family and socioeconomic status,personal health records,family history of cancer,smoking,alcohol and tea drinking,and dietary history.Secondly,the unconditional logistic regression models was applied to calculate odds ratios(ORs)with 95%confidence intervals(CIs)for ESCC risk in association with each exposure factor.To test the potential interaction,we used likelihood ratio tests for nested models with and without interaction terms.The non-linear relationship between continuous exposure factor and ESCC risk was further estimated using a restricted cubic spline regression with 5 knots.In Study 445 qualified pairs of cancer tissues and matched premalignant tissues from ESCC patients were collected in Taixing People's Hospital from July to December 2015.The extracted DNA was sequenced by WES and the sequencing data was analyzed using bioinformatics methods to indentify somatic mutations and construct the ESCC somatic mutation profiling.Results.1.Smoking,especially tobacco exposure at a young age,or with a high intensity or accumulative consumption,had a marginally adverse effect on ESCC risk(OR?1.50).However,the smoking-ESCC association was not found among women.Similarly,exposure to passive smoking was not associated with ESCC risk among either male or female never smokers.Alternatively,alcohol drinking showed a monotonic dose-responserelationship with the increased risk of ESCC in men(OR from 1.50 to 2.60),which was pronounced among current-smokers.Alcohol drinking is not common among Chinese women,and we did not observe any association between ESCC risk and alcohol drinking among the female study population.2.We observed an inverse association between green tea consumption and the risk of ESCC risk among men,which is mainly derived from hot temperature of green tea beverage(ORs of drinking warm tea,hot tea and very hot tea were 1.29,1.47 and 2.15 respectively).In general,a monotonic dose-response risks on ESCC occurrence were found with earlier,longer and more green tea drinking exposure,except that consuming a little warm tea drinking marginally prevented ESCC development.In addition,alcohol drinking could intensify the harmful effect of hot green tea drinking on ESCC occurrence.3.We found that before adult height reached 170cm among men and 157cm among women,the ESCC risk shapely rose by about 25%with height increased by 5cm,then the ascending trend of ESCC risk slowed down.Although BMI and body shape at age 20 years did not affect the ESCC incidence,BMI and Stunkard body shape at 10 years ago showed a monotonic reverse dose-response relationship with ESCC risk.Among participants who were underweight,normal weight or learner than Stunkard body shape 4,loss of body weight would promote the ESCC occurrence and gain(OR from 1.50 to 3.68)of body weight could prevent ESCC occurrence(OR from 0.18 to 1.02).However,the change ofbody size did not significantly affect the ESCC risk among participants who were overweight,obesity or fatter than Stunkard body shape 3.4.The WES data of 45 ESCC cases showed that the types of base substitutions ESCC were mainly C>T conversion(close to 50%)and T>C and C>A transversion.Rainfall plot illustated five local hypermuted.loci.The association between smoking exposure and C>A mutation frequency of ESCC patients was not significant(P = 0.525).MutSigCV algorithm identified five possible mutation-driven genes that may be really associated with ESCC development,which are NOTCH1,ZNF814,TPRX1,TP53 and HLA-C,respectively.In addition,54 recurrent somatic mutations were found,which were licated in ZNF717,ZNF814,FRG1,CLASRP,CTBP2,EVPL,GXYLT1,IFITM3,NBPF16,OR4C5,POU6F2,SLC9B1P1,TEKT4,TPSAB1 and so on.We constructed a preliminary somatic mutation profiling of ESCC.Conclusions1.The effect of smoking on the risk of ESCC is relatively weak.alcohol drinking,especially in combination with tobacco smoking,significantly increases the risk of ESCC among Chinese men.Moreover,alcohol drinking showed a monotonic dose-response relationship with the increased risk of ESCC in men.2.Drinking high-temperature green tea will significantly increase the risk of ESCC in Chinese men,and the tea temperature and risk of ESCC showed a monotonous increase dose-response relationship in Chinese men.In addition,alcohol consumption will significantly strengthen the harmness of high-temperature drinking tea on ESCC.3.Before adult height reached 170cm among men and 157cm among women,the ESCC risk shapely rose,then the ascending trend of ESCC risk slowed down.To our best knowledge,we firstly expound individual body weight loss would promote the ESCC occurrence and individual body weight gain could prevent ESCC occurrence among population with underweight and normal weight.But individual body weight change could not significantly affect the ESCC risk among overweight and obesity population.4.We constructed a preliminary somatic mutation profiling of ESCC in a high-incidence area using WES technology.We indentified various potential genetic variations of ESCC,which will contribute to the early diagnosis and targeted clinical treatment of ESCC.Analyzing the types of base mutation with smoking,we validated the association smoking and the risk of ESCC is relatively weak.Research SignificanceThe findings of the above-mentioned adjustable risk factor emphasize that the importance of integrating the elimination of these modifiable lifestyle risk factors into primary prevention strategies for reducing the ESCC incidence in high-incidence areas.Preliminary construction of ESCC somatic mutations,on one hand,can validated the case-control study results.On the other hand,it hints we can validate the causal relationship between other exposures and ESCC risk from somatic mutation level with large samples,and the somatic mutations of ESCC is essential and important for the early diagnosis and clinical targeted therapy of ESCC.Innovations1.We conducted an arigorous,population-based case-control study and found various targeted results,which are conducive to personalized etiological intervention for ESCC.2.We establish a preliminary somatic mutation profiling of ESCC in a high-incidence area in East China,which will be an important supplement for somatic mutations of ESCC in China.In addition,these results will contribute to explore the early diagnosis and targeted therapy of ESCC in future3.Through the correlation between base mutation of ESCC and smoking,the validated association between smoking and ESCC risk will be credible.