Associations Of Functional Single Nucleotide Polymorphisms In DNA Repair Genes With Susceptibility, Radiation-induced Injury And Survival In Esophageal Cancer Patients

Esophageal cancer is one of the most common human malignancies, and China is the country with the highest incidence and mortality of esophageal cancer in the world. Esophageal cancer is ranked the fourth among the top ten cancers in China and esophageal squamous cell carcinomas (ESCC) account for90%of all the cases. Radiation therapy is one of the main treatments for esophageal cancer, and the prognosis remains poor, with a5-year survival rate of less than20%in patients with advanced stage. Although cigarette smoking, alcohol intake, nutritional deficiencies and dietary carcinogen exposure may contribute to the etiology of esophageal cancer, only a small fraction of exposed individuals develop esophageal cancer. In addition, there are huge differences in side effects and survival rates amomg the patients, even though they have had the same type of pathology and staging and received the same treatment. These suggest that genetic variation may also play a vital role in susceptibility to and treatment efficacy of esophageal cancer.Internal factors and external environmental factors, including physical and chemical, biological carcinogens, can damage cellular DNA, resulting in changes of DNA structure and sequences and thus increasing genomic instability of proliferating cells. There are several DNA repair pathways in humans for repairing DNA damage, and reduced DNA repair capacity (DRC) may confer the difference in susceptibility to and treatment efficacy of esophageal cancer. It is recognized that the variation of individual DRC is determined by genetic factors, possibly functional single nucleotide polymorphisms (SNPs) of core genes in DNA repair pathways, which may be the underlying molecular mechanisms of the inter-individual variation of DRC.Current studies of the association of SNPs in DNA repair genes with susceptibility, radiation-induced-injury and survival in esophageal cancer patients have the following characteristics:First, the results for the role of SNPs of DNA repair genes as a genetic marker for cancer risk are conflicting, partially because of the small sample size and limited statistical power or no consideration of gene-environment interactions. Second, few studies have investigated the biological mechanisms of SNPs. Third, clinical translational studies on the associations of functional SNPs in DNA repair genes with side effects and survival in esophageal cancer treated with radiotherapy are scare and the results are inconsistent.To further explore these unsolved research questions, we conducted a hospital-based case-control study by enrolling more than1000ESCC cases and cancer-free controls, respectively. We selected12polymorphisms in6representive DNA repair genes (XPC, ERCC2, ERCC5, FEN1, hOGGl and NEIL1) using candidate-gene and functional SNP approaches, genotyped them in these subjects using Taqman real-time PCR method, and tested their associations with ESCC risk. We also evaluated gene-environment interactions. Meanwhile, we explored the molecular mechanisms underlying the positive associations both in vivo and vitro by correlating the variant genotypes with mRNA expression levels in cell lines and adjacent tissues. Finally, we enrolled187ESCC patients treated with definitive radiotherapy or in combination with chemotherapy and evaluated the associations between those12functional SNPs and acute radiation-induced esophageal toxicity (RIET), radiation pneumonitis (RP) and overall survival.This study provides the data that help us in our understaning of biological mechanisms of functional SNPs in the DNA repair genes in the etiology of esophageal cancer and outcomes of the patients. The results, once validated, will also improve our ability to implement effective, individualized prevention, diagnosis, treatment and prognosis of esophageal cancer. Part Ⅰ:Function Exploration of Functional Single Nucleotide Polymorphisms in DNA Repair Genes and Theie Associations with Susceptibility to Esophageal CancerObjective:DNA repair genes play a crucial role in the development of esophageal cancer, and its potentially functional SNPs may affect individual susceptibility to esophageal cancer by altering DNA repair capacity (DRC). However, few studies have explored their associations with esophageal cancer susceptibility and underlying biological mechanisms.Materials and methods:In this hospital-based case-control study, we enrolled1126esophageal squamous cell carcinoma (ESCC) cases and1131cancer-free controls frequency-matched to the cases on age and sex. We selected12SNPs in6DNA repair genes(XPC, ERCC2, ERCC5, FEN1, hOGGl and NEIL1) using candidate-gene and functional SNP approaches and genotyped them in these subjects using Taqman real-time PCR method. We used multivariate logistic regression model and multi-factor dimensionality reduction (MDR) method to analysis the main effects, combined effects of different genotype and gene-environment interactions. We also calculated false-positive report probabilities (FPRPs) for significant findings. We then detected genotype-associated mRNA expression levels using ESCC adjacent normal tissues from125patients and also used additional data on genotypes from270individuals from4ethnic populations and transcript expression levels available online for the genotype-phenotype association analysis.Results:We found that overall three SNPs in three genes (ERCC2rs238406、ERCC5rs2296147and FEN1rs4246215) were significantly associated with ESCC risk in the single locus analysis. We also found that individuals exhibitting an increased risk as the number of ERCC2, ERCC5at-risk genotypes increased in the combined analysis, and such a cumulative effect was dose-dependent for ERCC2(Ptrend=0.007). In the stratification analysis, we found a1.58-fold elevated risk associated with the ERCC2rs238406TG/TT genotypes in subjects with the cumulative smoking dose of more than16pack-years, so was in those with one or more unfavorable genotypes of ESCC2; further homogeneity tests suggested that there were statistically significant differences in the risk estimates between some strata. Multivariat logistic regression model and MDR consistently found that there existed an interaction between ERCC2rs238406variants and cumulative smoking dose (P=0.018). FPRPs confirmed the above significant findings were reliable. In the genotype-phenotype correlation analysis using the lymphoblastoid cell lines derived from peripheral lymphocytes from270people, we found that the trend for the rs238406allele G�'T effect on ERCC2mRNA expression reached borderline significance for Asians (Ptrend=0.098), statistical significance for Europeans (Ptrend=0.011), but no statistical significance for CHB (Ptrend=0.960). Other SNPs associated with ESCC susceptibility had no impact on mRNA expression levels.Conclusion:Our large study is among the few that investigated the associations between potentially functional SNPs in DNA repair genes and esophageal cancer susceptibility. These findings provide further evidence to support the notion that genetic variations in DNA repair genes contribute to ESCC susceptibility. Further studies with larger samples and functional evaluation of the SNPs are warranted to validate these findings. Part II:Association Study of DNA Repair Gene Polymorphisms with Radiation-induced Injury and Overall Survival in Esophageal Cancer PatientsCharpter I:Association Study of DNA Repair Gene Polymorphisms with Radiation-induced Injury in Esophageal Cancer PatientsObjective:DNA repair genes play an important role in radiation-induced DNA damage; its functional single nucleotide polymorphisms (SNPs) may alter DNA repair capacity and thus contribute to the variation in response to radiotherapy. Few studies have investigated their associations with acute radiation-induced-pneumonitis (RP) and radiation-induced esophageal toxicity (RIET) in esophageal cancer patients.Materials and methods:We enrolled187patients with esophageal squamous cell carcinoma (ESCC), who underwent definitive radiotherapy or in combination with chemotherapy at Fudan University Shanghai Cancer Center between January2008and December2011. We genotyped the12SNPs in6DNA repair genes (XRCC1, hOGGl, FEN1, NEIL1, RAD51and WRN) using Taqman real-time PCR method. The primary endpoints were≥grade2RP and≥grade2RIET according to Common Terminology Criteria for Adverse Events (CTCAE) version4.0criteria. We performed univariate and multivariate Cox proportion hazards regression analyses to calculate the hazards ratio (HR) and confidence interval (CI) and evaluated the effects of genotypes on risk of RP and RIET.Results:The187ESCC patients had a median age of64years-old, with138males and49females. The median occurrence time was35and24days for RIET and RP, respectively, from the first day of irradiation. Twenty patients (15.0%) exhibited≥grade2acute RP and42patients (22.5%) exhibited≥grade2acute RIET. We found that the volume of lung receiving at least20Gy (V20) was significantly associated with≥grade2acute RP, and the length of esophagus receiving at least50Gy (LE50) and age were significantly associated with≥grade2acute RIET as well. NEIL1rs4462560GC/CC genotypes statistically significantly reduced risk of both≥grade2acute RIET (adjusted HR=0.42,95%CI=0.21-0.86, P=0.017) and≥grade2acute RP (adjusted HR=0.39,95%CI=0.16-0.95, P=0.037), compared with those with the GG genotype.Conclusion:Genetic polymorphisms in the NEIL1gene may be an independent predictive factor for the risk of RP and RIET in esophageal cancer patients treated with radiotherapy. The findings need further validation in studies with larger samples and different ethnic populations. Charpter II:Association Study of DNA Repair Gene Polymorphisms with Overall Survival in Esophageal Cancer Patients Treated with Radiation RadiotherapyObjective: Functional single nucleotide polymorphisms (SNPs) in DNA repair genes may alter DNA repair capacity and thus contribute to the survival of radiotherapy. However, few studies have investigated the associations of DNA repair gene polymorphisms with overall survival (OS) in esophageal cancer patients treated with radiation radiotherapy.Materials and methods:We enrolled187ESCC patients who underwent definitive radiotherapy or in combination with chemotherapy at Fudan University Shanghai Cancer Center between January2008and December2011. We used the Taqman real-time PCR method to genotype12SNPs in6DNA repair genes (XRCC1, hOGG1, FEN1, NEIL1, RAD51and WRN). The primary endpoint was OS. We performed Kaplan-Meier analysis to investigate the association of genotypes with OS and Log-rank to test for the difference in survival curves. We also used multivariate Cox proportion hazards regression analyses to evaluate the effects of genotypes on OS.Results:At the time of the last follow-up (January2013),97(55.1%) patients died with a median survival time (MST) of11months, and79(44.9%) patients still alive for MST of27months. The overall survival at1-,2-and3-year was70.5%,49.9%and43.4%, respectively. Both tumor stage and the length of gross tumor volume of primary esophageal cancer (LGTV-P) were significantly associated OS. Patients carrying WRN rs2725335GA/GG genotypes had a better OS than those with the AA genotype (adjusted HR=0.43,95%CI=0.22-0.86, P=0.017), while patients carrying the HOGG1rs1052133CC genotype had a worse OS than those with the GG genotype with statistical borderline significance (adjusted HR=1.79,95%CI=0.98-3.27, P=0.057).Conclusion:Genetic polymorphisms in the WRN gene may be an independent, predictive factor for OS in esophageal cancer patients treated with radiotherapy. The results need to be further validated in larger studies in the future.