Abstract 3277: A low plasma mitchondrial DNA copy number is associated with increased breast cancer risk

Previous studies have shown that plasma mtDNA can be separated into two fractions; a supernatant form that is cell-free and a pelleted form that is thought to be platelet associated. These fractions of plasma mtDNA should be measured in studies evaluating the role of plasma mtDNA as biomarkers for early detection or susceptibility to various cancers. Though one case-control study has shown lower plasma mitochondrial DNA (mtDNA) copy number to be associated with increased breast cancer risk this study did not evaluate the two fractions of plasma mtDNA. Hence, the role of plasma mtDNA in determining breast cancer risk was investigated further. We conducted a case-control study of 29 breast cancer cases and 28 cancer free controls to evaluate the association between (a) plasma mtDNA copy number and (b) peripheral blood leucocyte mtDNA copy number and breast cancer risk. Plasma obtained from the breast cancer cases and controls was centrifuged further at 18,000 g for 10 minutes to separate the plasma into the supernatant and pelleted fractions. Using flow cytometry, we stained the pelleted fraction of the plasma with Annexin V and CD61 to determine the cellular source of the plasma pellet. DNA from the two plasma fractions and the buffy coat was extracted using the Qiagen DNAesy DNA extraction kit. Nuclear DNA (18s) and mitochondrial DNA (ND1) copy numbers were measured using quantitative real time PCR. We used a two-sample independent t test to evaluate the difference in mtDNA in the two plasma fractions and mtDNA copy number in peripheral blood leucocytes among breast cancer cases and controls. The plasma pellet fraction predominantly contained particles that were 1-3 microns in size that stained positively for both Annexin V and CD61 indicating that these particles were platelet derived microparticles. The mtDNA in the plasma pellet accounted for 96% of all the plasma mtDNA seen in both cases and controls. Both the plasma supernatant mtDNA (2601034 mtDNA copies/ml of plasma vs. 4700446 mtDNA copies/ml of plasma; p = 0.004) and plasma pellet mtDNA (67787069 mtDNA copies/ml of plasma vs. 112883929 mtDNA copies/ml of plasma; p = 0.001) were significantly lower in the breast cancer cases as compared to controls. Peripheral blood leucocytes mtDNA copy number was not associated with breast cancer risk (4.27 vs. 3.73; p=0.14). There was no correlation between mtDNA in peripheral blood and the various fractions of plasma mtDNA (r= -0.08 - 0.07; p = 0.55 - 0.63). This is the first study to demonstrate platelet derived microparticles as the source of the plasma pellet mtDNA. This study shows that both the supernatant and pelleted fractions of plasma mtDNA are associated with breast cancer risk. Peripheral blood leucocytes mtDNA was not associated with breast cancer risk and was not correlated with plasma mtDNA. Future studies are needed to confirm these associations and evaluate possible mechanisms through which plasma mtDNA copy number is associated with breast cancer risk. Citation Format: Bharat Thyagarajan, Helene Barcelo, Kristin E. Anderson, Karen Swenson, Heather Nelson, Myron D. Gross. A low plasma mitchondrial DNA copy number is associated with increased breast cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3277. doi:10.1158/1538-7445.AM2014-3277