Guan et al. BMC Cancer 2010, 10:431 http://www.biomedcentral.com/1471-2407/10/431 it is not known if VEGF polymorphisms are associated with prognosis within this particular population. Previous studies primarily focused on three common functional single nucleotide polymorphisms of the VEGF gene, including the -460 T > C, -634 G > C (also assigned as +405 G > C) and +936 C > T (minor allele frequency = 0.422, 0.431 and 0.222 in Caucasians, respectively, according to the Hapmap database). The -460 T > C SNP is located in the promoter region and may influence the promoter activity [10]; the -634 G > C SNP lies within the 5′-untranslated region and may affect the transcriptional factor binding affinity [11]; the +936 C > T SNP is located in the 3′-untranslated region and has been associated with lower VEGF plasma levels [12]. In the present study, we evaluated the association of these three potentially functional VEGF SNPs (i.e., -460 T > C, -634 G > C [also assigned as +405 G > C] and +936 C > T) with overall survival (OS) of LA- NSCLC patients. Methods Study populations Clinical data were derived from a large dataset of 576 NSCLC patients established at The University of Texas M. D. Anderson Cancer Center (Houston, TX), in which patients were recruited and histologically con- firmed between Oct. 1998 and Nov. 2006. Details of this study population have been described previously [13]. Briefly, this analysis consisted of 124 Caucasian patients with stage IIIa or IIIb NSCLC according to the TNM staging system, a relatively homogenous group that was treated by chemoradiotherapy. Those patients who had surgical resection, or had been treated elsewhere before coming to M. D. Anderson were excluded from the ana- lysis. The study protocol was approved by the M. D. Anderson Cancer Center institutional review board and informed consents were waived. We complied with Health Insurance Portability and Accountability Act (HIPAA) regulations. Genotyping Genomic DNA was extracted from the buffy coat frac- tion of each blood sample by using a Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. DNA purity and concentrations were deter- mined by spectrophotometric measurement of absor- bance at 260 and 280 nm by UV spectrophotometer. The selected three VEGF SNPs (-460 T > C/rs833061, -634 G > C/rs2010963, and +936 C > T/rs3025039) were genotyped using the polymerase chain reaction (PCR) -restriction fragment length polymorphism (RFLP) method. The PCR primers used for -460 T > C, -634 G > C, and +936 C > T polymorphisms were 5′-CTCTTTAGCCAGAGCCGGGG-3′ (forward) and Page 2 of 9 5′-TGGCCTTCTCCCCGCTCCGAC-3′ (reverse); 5′-CG ACGGCTTGGGGAGATTGC-3′ (forward) and 5′-GGG CGGTGTCTGTCTGTCTG-3′ (reverse); and 5′-AGGG TTCGGGAACCAGATC-3′ (forward) and 5′-CTCGG TGATTTAGCAGCAAG-3′ (reverse), respectively. The following PCR conditions were performed: one cycle at 95°C for 5 min; 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. The PCR products were studied after digestion with BsaHI, BsmFI, and NlaIII restriction enzymes. Genotypes of these VEGF SNPs were determined as pre- viously reported [14]. For the PCR-RFLP-based genotyp- ing assay, two research assistants independently read the gel pictures, and the genotyping was repeated if there was a disagreement of the result. We selected 10% of the samples for replication, and the results were 100% concordant. Statistical analysis The two-sided c2 and Student t tests were performed to determine any statistically significant differences in the distributions of the VEGF genotypes by the demographic variables and clinical features. We used the Kaplan- Meier estimates to evaluate OS among three genotype groups, and the log-rank test to test for equality of the survival distributions. We conducted univariate analysis and used multivariate Cox proportional hazard models to estimate the effect of each SNP on survival with or without other confounding factors. Haplotype frequen- cies and individual haplotypes were generated using SAS PROC HAPLOTYPE. The associations between haplo- type and overall survival (OS) were determined using a dominant genetic model to preserve statistical power. All analyses were performed using SAS software (version 9.1; SAS Institute, Cary, NC). Results Population characteristics Clinical and pathological characteristics of the patients included in current study are shown in Table 1. Among the 124 patients, there were 67 males (54%) and 57 females (46%), whose ages ranged from 35 to 88 years. There were 40 (32.3%) adenocarcinoma, 38 (31.6%) squamous cell carcinoma and 46 (37.1%) large cell carci- noma. All patients received radiotherapy, delivered as 1.8 to 2 Gy per fraction once a day with a total median radiation dose of 66 Gy (ranging between 50 and 72 Gy). A number of 112 (90.3%) patients also received platinum plus taxane or etoposide-based chemotherapy. At the end of follow-up, 92 (74.2%) patients died, and the MST was 17 months (ranging between 1 and 97 months) in the overall study subjects. To determine if there was any confounding factor influencing patients’ death or survival time, we