Single Nucleotide Polymorphisms In The Mitochondrial Displacement Loop Region And Outcome Of Malignant Fibrous Histiocytoma

Objective:Single nucleotide polymorphisms（SNPs） in the mitochondrial D-loop has been identified to be associated with the development of various types of tumor.In a previous study, we investigated the relationship between the SNPs and malignant fibrous histiocytoma（MFH）,and identified that SNPs in the D-loop increased MFH risk. In this study, we further studied the relationship between the SNPs and the clinic characteristics of MFH, and we aim to identify patient subgroups at high risk of a poor outcome, thereby helping to make therapeutic decisions for these patients.Material and methods:Blood samples were collected from 80 MFH patients who received treatment at the Fourth Hospital of Hebei Medical University between 2002 and 2011,blood samples were also collected from 100 healthy controls who had not had MFH as well as other tumors. The clinical data were completed for these people. The patients were followed up for 3 years. The mitochondria DNA of MFH patients and healthy controls were extracted using the GENMED Whole Blood Mitochondrial DNA extraction kit,and stored them at-20℃ for later use. PCR was performed to amplificate the mitochondrial DNA with the forward primer 5 ’-CCCCATGCTTACAAGCAAGT-3 ’ and the reverse primer 5 ’-GCTTTGAGGAGGT AAGCTAC-3’. The PCR conditions were: initial denaturation at 95℃ for 2 min, followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃for 1 min, and then final extension at 72℃for 5 min.SNPs were identified from the 982 bp mitochondrial D-loop of the MFH patients according to the gene bank AC₀00021 data. The relationship between the 3-year-survival rate and the SNPs as well as the clinical characteristics. The survival curve was calculated using the Kaplan–Meier method and was compared with the log-rank test. Multivariate survival analysis was performed using a Cox proportional hazards model. All of the statistical analysis was done with the SPSS 13.0 software package（SPSS Company, Chicago, IL）. A p-value of P≤0.05 was considered statistically significant.Results:Data for the 3 year follow-up period were analyzed with clinical characteristics using the Kaplan–Meier method and were compared by the log-rank test. These clinical characteristics include: sex, tumor location, age, tumor size, lymphatic metastasis. The later three were identified to be associated with the outcome of MFH with the P-value were 0.017,0.001,0.015 respectively.71 SNPs were found in the 982 bp mitochondria D-loop region from the blood samples of the healthy controls and MFH patients.28 of the 71 SNPs whose frequency were higher the 5% were used to analyze the association with the outcome of MFH patients. Five SNPs were identified to be associated with outcome of MFH patients. The five SNPs sites were 152、16390、16290、16304、523-524. SNPs of 152 C, 16,390 A, 16,290 T were associated with a shorter period of survival whereas the SNPs of 16,304 C, AC deletion at sites 523 and 524 were associated with a longer period of survival, with P=0.047, 0.016, 0.043, 0.043, 0.029 respectively.We performed multivariate analysis with Cox proportional hazards model including the factors of five SNPs and age, tumor size and lymphatic metastasis. Lymphatic metastasis status, tumor size, 16390G/A and 16290C/T were identified as independent predictors for MFH outcome.Conclusion:The mitochondrial D-loop is a highly variable polymorphic region, the D-loop is associated with various types of cancer. Single nucleotide polymorphism is statistically correlated the outcome of MFH patients. Five SNP sites were identified to be associated with outcome of MFH patients. The five SNPs sites were 152、16390、16290、16304、523-524.Two SNP sites were identified to be independent prognostic factors for MFH patients, the two SNPs were 16290,16390. SNPs in the mitochondrial D-loop may be viewed as the molecular indicators for cancer risk and outcome, The evaluation of genetic polymorphisms in the D-loop may be of great help to make therapeutic decisions for these patients.