Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:155 http://www.jeccr.com/content/29/1/155 Page 2 of 5 promoter for transcription [7-10]. Only a few germline single nucleotide polymorphisms (SNPs) in the D-loop have been shown to be prognostic of cancer risk and outcome, but their predictive values have not been fully determined [11-14]. The D-loop contains a length of 1122 bps (nucleotide 16024-16569 and 1-576) refers to mitochondria database (http://www.mitomap.org). In this study, we sequenced a region of about 1 kb in the mitochondrial D-loop from 60 patients with esophageal squamous cell carcinoma (ESCC), the main pathological type of esophageal cancer in China, to assess the rela- tionships between germline SNPs of the D-loop with cancer risk and gemline SNPs with disease outcome in ESCC patients. Methods Tissue specimens and DNA extraction Blood samples were collected at the Fourth Hospital of Hebei University from 66 ESCC patients who underwent esophageal cancer resection in the Department of Thor- acic Surgery between 2003 and 2004. The patients were selected when they received endoscopy examination and specimen were confirmed as ESCC by pathologist. All the patients comes from the Hebei Province of China a high risk area of ESCC. The tumor-free controls as determined per endoscopy, radiograph, and blood exam- ination, were randomly selected from the same area. Both patients and controls contain 42 males and 24 females with the mean age of 59.78 ± 8.32 in ESCC patients and 60.84 ± 8.77 in controls. Genomic DNA was extracted immediately with a Wizard Genomic DNA extraction kit (Promega, Madison, WI) from blood samples. The study was approved by the Human Tissue Research Committee of the Fourth Hospital of Hebei Medical University. All patients provided written informed consent for the collection of samples and sub- sequent analysis. PCR amplification and sequence analysis The forward primer 5’-CCCCATGCTTACAAGCA AGT-3’ (nucleotide 16190-16209) and reverse primer 5’- GCTTTGAGGAGGTAAGCTAC-3’ (nucleotide 602- 583) were used for amplification of a 982 bp product from mtDNA D-Loop region as described previously [15]. PCR was performed according to the protocol of PCR Master Mix Kit (Promega, Madison, WI) and puri- fied prior to sequencing. Cycle sequencing was carried out with the Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem, Foster City, CA) and the products were then separated on the ABIPRISM Genetic Analyzer 3100 (Applied Biosystem). Polymorph- isms were confirmed by repeated analyses from both strands. SNPs were identified directly from blood mitochondria. Statistical analysis The c2 test was used to analyze dichotomous values, such as the presence or absence of an individual SNP between ESCC patients and healthy controls. The survi- val curve was calculated using the Kaplan-Meier method, and compared with the log-rank test. Multivari- ate survival analysis was performed using a Cox propor- tional hazards model. All of the statistical analysis was done with the SPSS 11.5 software package (SPSS Com- pany, Chicago, IL). A p value of < 0.05 was considered statistically significant. Results A total of 66 patients were enrolled in this study. Six of these patients were lost to follow-up. A review was con- ducted every six months over a five-year period. Those patients lost to follow-up during this time period were as follows: 1 patient in Year 2; 1 patient in Year 3; 3 patients in Year 4; and, 1 patient in Year 5. Sixty patients shared the same performance status (ECOG Score: Zero). Those analyzed included: 12 patients at Stage I; 33 patients at Stage II, and 15 patients at Stage III. Thirty-six patients died during follow-up. None of these patients had received any adjuvant chemotherapy or radiation therapy after ESCC resection. Data for the 5 year follow-up period were analysed with clinical char- acteristics using the Kaplan-Meier method and were compared by the log-rank test. Sex, age and local lym- phatic metastasis were not statistically significant predic- tors of the length of post-operational survival, but TNM stage was correlated with survival in these patients (Table 1). As expected, patients at different stages had different 5 year survival rates: stage I, 75%, stage II, Table 1 Univariate analysis of clinical characteristics associated with post-operational survival in ESCC patients No. cases 5 years survival rate (%) p value Characteristics Gender Male Female Age (years) ≤ 55 > 55 TNM classificationa I II III Lymphatic metastases Yes No 37 23 17 43 12 33 15 12 48 aThe survival in each stage was compared as I versus II, I versus III and II versus III 35.10 47.80 23.50 46.50 75 36.40 20 33.30 41.70 0.129 0.282 0.012 0.418