| Backgrounds:Esophageal cancer has become the sixth leading cause of cancer death worldwide,and its incidence rate continues to increase in the world.As the esophageal cancer patients tend to be diagnosed at an advanced stage and are thus often ineligible for radical surgery,the 5-year survival rate of esophageal cancer patients was demonstrated about 10%to 40%.In recent years,concurrent chemoradiation therapy(CCRT)has been widely implemented in an attempt to improve the long-term survival rate of patients with locally advanced esophageal cancer or who refused the curative surgical resection.With the advancement of radiation therapy technology,the dose distribution in the target area is more ideal and the radiation dose of normal tissue is controlled strictly.However,there are still part of patients who are difficult to achieve objective remission or show serious radioactive damage after clinical treatment.Of which the complications of radiation-induced lung injury,the acute radiation pneumonitis and radiation-induced pulmonary fibrosis,are radiation dose limiting factors for esophageal cancer,which have a great impact on the quality of life of the patients.Recently,it has been found that single nucleotide polymorphisms(SNPs)play an important role in predicting the treatment outcomes of complex human diseases.However,up to now,no reliable biomarkers have been well investigated to predict the response to CCRT and the risk of radiation-induced lung injury.In this study,a two-stage study design with a candidate gene strategy were used to improve the statistical power and minimize cost.The main purpose of this study was to investigate the genetic variation of chemoradiotherapy sensitivity and risk of radiation-induced lung injury in esophageal squamous cell carcinoma in order to find useful biomarkers to predict the response to CCRT and thus can guide the individualized treatment of patients with esophageal squamous cell carcinoma.Methods:A total of 371 patients with esophageal cancer received radiotherapy at Shandong Cancer Hospital Affiliated to Shandong University between January 2016 and April 2017 were enrolled in this study.Histopathological examination was used to diagnose squamous cell carcinoma.Peripheral blood samples were collected from patients before treatment,and peripheral blood whole genome DNA was extracted.A total of 17 SNPs of 11 genes involved in radiosensitivity and radioactive injury related pathways were screened by candidate SNPs strategy.This study was divided into two phases.In the first phase,163 candidate samples were screened out to be statistically significant among candidate SNPs for clinical radiotherapy efficacy or radiation-induced lung injury.In the second phase,verifying the SNPs in 208 independent samples.Finally,the two parts of information of 371 patients were integrated to analyze the correlation between the single nucleotide polymorphisms and the response to radiotherapy.All samples were genotyped using the Sequenom MassArray system.lung injury was evaluated and graded according to the patient's symptoms and imaging features(NCI-CTCAE 3.0).The primary endpoint was>2 radiation-induced lung injury.Patients are assessed for the curative effect 1-2 months after completion of treatment(RECIST 1.1).This study defined complete response(CR)or partial response(PR)as responders,defined stable disease(SD)or progressive disease(PD)as non-responders.SPSS 19.0 software was used for all the statistical analyses.The cumulative incidence of Kaplan-Meier was used to assess the risk of radiation-induced lung injury.Cox proportional hazards model was used to evaluate the correlation between clinical factors and genotypes and radiation-induced lung injury.Logistic regression was performed to assess the association between treatment response and each genotype while adjusting for other covariates.Results:Of the 371 esophageal squamous cell carcinoma patients,294 were male and 77 were female,with a mean age of 65 years.73 patients(19.68%)in stage ?,226 patients(60.92%)in stage III,and 72 patients(19.40%)in stage ?.107 patients received radiotherapy alone,264 patients received chemoradiotherapy,with the median dose of 60Gy(37.8-70Gy),sub-dose of 1.8-2,0Gy.233(62.8%)had objective responses and 138(37.2%)did not respond.Patients with radiotherapy dose>55Gy got higher clinical response rate than those treated with lower than 55Gy(66.30%verus 52.63%,P = 0.015).The objective response rates of patients treated with chemoradiotherapy and radiotherapy alone were 66.29%and 54.21%,respectively,with significant differences between the two groups(P = 0.042).The other clinical factors such as age,stage,KPS,tumor sites and other factors in multivariate analysis did not find significant correlation with radiosensitivity.In addition,we found that NOS3 rs 11771443 located on chromosome 7 was associated with radiosensitivity to patients with esophageal squamous cell carcinoma.Patients with rs11771443 TT genotype were more sensitive to chemoradiation than patients with the CC/CT genotype(P= 0.001).Multivariate analysis showed that the correlation between the two groups was still statistically significant(OR = 3.483,95%CI:1.705-7.116,P = 0.001)after adjusting for gender,age,pathological types,smoking and physical status,staging,tumor location,with or without chemotherapy.Hierarchical analysis showed that both in the radiotherapy alone group and chemoradiotherapy group,patients with TT genotype had better curative effect than patients with CC/CT genotype,and the correlation was more obvious in the radiotherapy alone group(P = 0.009 vs P = 0.012).64 patients(17.25%)had grade 2 or higher radiation-induced lung injury at the end of follow-up.The risk of radiation pneumonitis in MLD?10Gy patients was higher than that in MLD<10Gy patients(HR = 1.857,P = 0.022,95%CI:1.095-3.151)in multivariate analysis.Other factors such as gender,age,staging,tumor location,radiotherapy dose were not significant in multivariate analysis.In addition,we found that the genetic variation of ALDH2 rs441 located on chromosome 12 had a significant correlation with the occurrence of radiation-induced lung injury.Compared with ALDH2 rs441 CC genotype,patients with both CT and TT genotypes had a significantly lower risk of developing radiation-induced lung injury(P = 0.001,HR = 3.589,95%CI = 1.632-7.892).Of the 371 patients,97 had ?2 grades of radio-induced esophagitis,and 19 had ?3 grades of esophagitis.Logistic univariate analysis showed that the lesion location and V20 were closely related to the occurrence of grade 2 or more radiation esophagitis(P =0.031,P=0.026).The incidence of grade 2 esophagitis was highest in patients with thoracic esophageal cancer during and after radiotherapy.Patients carrying rs1805388 TT/CT genotype had a 2.28-fold higher risk of grade 2 esophagitis than rs1805388 CC genotype(OR = 2.280,95%CI:1.360-3.820,P = 0.002).Patients carrying the rs441 CC locus were 4.812 times more likely to have grade 2 or more radiation esophagitis(OR=4.812,95%CI:1.124-20.610,P=0.034).Conclusion:1.rs11771443 of NOS3 gene can predict radio-chemotherapy sensitivity in esophageal squamous cell carcinoma patients,patients with TT genotype were more sensitive to chemoradiation.2.There was a significant correlation between rs441 of ALDH2 gene and the risk of grade 2 or more radiation-induced lung injury in patients with esophageal squamous cell carcinoma.The risk of radiation-induced lung injury was significantly higher in patients with CC genotype.3.The ALDH2 gene rs441 locus and LIG4 gene rs1805388 locus polymorphism were significantly associated with the occurrence of>2 grades of esophagitis during esophageal squamous cell carcinoma. |
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