Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:155 http://www.jeccr.com/content/29/1/155 Page 4 of 5 Table 2 Multivariate analysis of prognostic factors associated with post-operational survival in ESCC patients with Cox proportional hazards model Factors Stage of tumor 16274(G/A) 16278(C/T) 16399((A/G) Relative risk 1.328 0 3.001 3.483 95% C.I. 0.955-1.848 0 1.029-8.756 1.068-11.359 p value 0.092 0.975 0.044 0.039 16278C/T and 16399A/G, were identified for their asso- ciation with post-operational survival at statistically sig- nificant levels by the log-rank test. Multivariate survival analysis identified 16278C/T and 16399A/G to be inde- pendent prediction markers for ESCC outcome. We suggest for the first time that SNPs in the D-loop is a prognostic factor in ESCC patients. The relative risk (RR) of death in patients was significantly higher (16278C versus 16278T, RR, 3.001; 95% CI, 1.029 - 8.756; p = 0.044. 16399A versus 16399G, RR, 3.483; 95% CI, 1.068 - 11.359; p = 0.039). Nucleotides 16278 and 16399 are located in hypervariable segment 1 (HV1), which is asso- ciated with high rates of mutation [16], but the functional significance of these SNPs in HV1 is not known. Minor alleles of 16278T and 16399G are associated with drama- tically shorter period of postoperative survival; the survi- val curve decreased rapidly in patients carrying these alleles (Figure 1). We compared the distribution fre- quency of these two SNPs between ESCC patients and normal controls; among 60 age-matched controls, only one carried the 16278T allele and none carried the 16399G allele. These two minor alleles appear to be cor- related with ESCC risk, but a much larger sample size will be needed to obtain a statistically significant differ- ence of distribution frequency for these alleles between ESCC patients and controls. It is very interesting if a nucleotide substitution can render an individual suscepti- ble to a tumour but subsequently modulates prognosis of that tumour. The 5 year survival rate displays a trend for distinction on the basis of sex (35.1% male versus 47.8% female), age (46.5% older than 55 versus 23.5% younger than 55) and lymphatic metastasis (33.3% yes versus 41.7% no). The prediction value of these factors need to be investigated by further study. The D-loop region of mtDNA is important for regula- tion of mitochondrial genome replication and expression. SNPs in this region might affect mtDNA replication and lead to alteration of the electron transport chain, which is responsible for the release of highly reactive oxygen spe- cies (ROS) and could contribute to nuclear genome damage as well as cancer initiation and promotion [17-19]. These three SNPs may altered transcription of mitochondrial genome, and that the production of ROS is enhanced when the mitochondrial transcription is altered [20], these ROS-mediated mechanism may accel- erate the tumor development. In conclusion, SNPs in the D-loop were found to be independent prognostic markers for ESCC outcome. The analysis of genetic polymorphisms in the D-loop might help to identify patient subgroups at high risk for a disease outcome, thereby helping to refine therapeutic decisions in ESCC cancers. Additional material Additional file 1: Distribution of 88 SNPs in 66 ESCC patients and controls. The data provided represent all the SNPs identified in the ESCC patients and controls. Acknowledgements This work was supported by National Natural Science Foundation of PR China No. 30801384. The research was supported in part by Natural Science Foundation of Hebei Province No. C2008000958. Author details 1Department of Gastroenterology and Hepatology, The Fourth Hospital of Hebei Medical University. Shijiazhuang, PR China. 2Department of Thoracic Sugery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, PR China. 3Department of Molecular Biology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, PR China. 4Department of Gynecology Ultrasound, The Fourth Hospital of Hebei Medical University, Shijiazhuang, PR China. 5Hebei Key Lab of Laboratory Animal Science, Hebei Medical University, Shijiazhuang, PR China. Authors’ contributions RZ and RW contributed to experimental design, data acquisition and analyses. FZ, CW and FHY contributed to experimental design, specimen collection, and data acquisition. YL and CW participated in data analyses. 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