br Four potentially functional variants of TGFB and HSPB gen
Four potentially functional variants of TGFB1 and HSPB1 genes were selected from the gene SNP database of the National Institute of Environmental Health Sciences Genome Program and related lit-erature using the LD TAG SNP Selection tool (https://snpinfo.niehs. nih.gov/snpinfo/snptag.html). The selected SNPs met at least two of the following criteria: (1) a minor allele frequency of at least 5%, (2) to be located in the promoter, regulatory or untranslated region of the gene, (3) TaqMan SNP Genotyping Assay available. Additionally, three previously described SNPs, rs1800469 rs2868371 and rs2868370, associated with RIET development have been selected for validation.
Statistical analyses were performed using SPSS (version 19.0). The end points of analysis (development of RIET acute and late) were estimated and scored using Common Terminology Criteria for Adverse Events version 4.0. The RT starting point was consid-ered to calculate the time to different RIET development; patients who did not experience the end points were censored at 3 months since RT started or at the time of death or last contact. Kaplan– Meier analysis was performed to estimate the cumulative RIET incidence. Cox proportional hazards analysis was performed to cal-culate hazard ratio (HR) and confidence interval (CI). Multivariate Cox regression analysis with a stepwise backward elimination pro-cedure was used to adjust for factors significant on univariate anal-ysis, as well as any other factors that might have misled genotypes univariate analysis. A p-value 0.05 was considered significant. In addition, the pointwise P value was adjusted for multiple testing using a Bonferroni approach by multiplying the pointwise P value by the number of tests.
Table 1 (continued)
Characteristic No. of Patients (%)
Median (range) 60 (2–70) Radiation fractionation
Abbreviations: KPS, Karnofsky Performance Status; NSCLC, NOS, non-small-cell lung cancer, not otherwise specified; Dmin, minimum dose; Dmax, maximum dose; MED, mean BMS 986120 dose; Vx, volume of normal esophagus receiving Gy or more radiation.
SNPs in silico analysis
In order to identify functional effects, they were analyzed by RegulomeDB (www.regulomedb.org/), and SNPinfo (FuncPred). Additionally, differential expression analysis was performed using the data from the Genotype-Tissue Expression project (https:// www.gtexportal.org/home/).
164 SNPs associated with esophagitis in lung cancer
Associations between patient-, tumor-, and therapy-related characteristics and radiation esophagitis toxicity.
Parameter Acute Esophagitis (n = 247)
HR 95% CI p value HR 95% CI p value Gender
No. of cigarettes per day
No. of packs yearsa
History of alcoholism
History of dysphagia
History of weight loss
Weight before RT, Kg
Radiation treatment, days
Radiation total dose, Gy
Table 2 (continued)
Parameter Acute Esophagitis (n = 247)
Abbreviations: KPS, Karnofsky performance status; NSCLC, non-small-cell lung carcinoma; SCLC, small-cell lung carcinoma; RT, radiation therapy; CRT, chemoradiation; GTV, gross tumor volume; CTV, clinical target volume; PTV, planning target volume; MED, mean esophagus dose; Dmin of esophagus, the minimum dose; Dmax of esophagus, the maximum dose; V(x), volume of normal esophagus receiving X Gy or more radiation. a Number of pack years = (packs smoked per day) (years as a smoker).
b Dosimetric parameters were analyzed as continuos variables.
Correlation between esophageal toxicity, clinical–pathological characteristics and the selected SNPs
The genotype distributions of TGFB1 and HSPB1 SNPs among LC patients are displayed in Supplementary Table 1. Univariate analy-ses in order to identify associations between patient-, tumor-, and therapy-related characteristics and acute and late RIET are shown in Table 2. Concerning patient characteristics, age, smoking status and weight loss history were found to be associated with grade 2 acute RIET in univariate analysis. The PTV and the GTV were also significantly associated with acute grade 2 esophagitis, whereas CTV was associated with both acute grade 2 and 3 esophagitis. Mean and median esophagus dose (MED) and dosimetric parame-ters from volume of esophagus (in percent) exposed to 5 Gy (V5) to V55, were likewise associated with acute RIET grade 2 risk in the univariate analysis. Regarding the type of treatment, radiation frac-tionation and concurrent radiochemotherapy, were associated with acute grade 2 esophagitis. Concerning late esophageal dam-age, median time for development after completion of RT was 9 months (3–18 months). Irradiation dose and fractionation were both correlated with grade 2 late RIET. Across the SNP selected for validation, there were no statistically significant connections between the rs2868371 CC genotype and higher risk of acute or late esophagitis (acute grade 2 esophagitis: p value = 0.758; acute grade 3 esophagitis: p value = 0.489; late grade 1 esophagitis: p