Abstract P4-12-04: Association of single-strand breaks (SSBs) in normal breast DNA with estimates of breast cancer risk

Abstract

Abstract Background: Formation of single strand breaks in DNA is a constant process, estimated to occur 10,000 times per day in each cell, either from endogenous biosynthetic errors or from interference by endogenous or exogenous agents. Base excision repair in some cases may be impaired or may be exceeded by the rate of DNA damage, leading to cancer. Nick translation of with labeled nucleotides can be used as a quantitative indicator of SSBs. Methods: Healthy women were recruited through the Love-Avon Army of Women and breast clinics of Northwestern and John Hopkins Universities. Digital or digitized mammograms were evaluated for percent density using Cumulus software. The medians (ranges) were age 51 (36 to 60); BMI 28.2 (18.7 to 51.3); life-time Gail estimate 12.5 (5.6 to 28.3); % breast density 16.5 (2.4 to 52.5); Masood score 13 (0 to 18). Breast tissue was obtained by random fine needle aspiration (rFNA). Specimens were rinsed into ice-cold phosphate buffered saline and were stored at −80°C. Thawed samples were centrifuged at 2200 g for 60 min. A kit from Norgen Inc., was used to separate DNA, RNA, and protein from the pellet. The lipids were extracted with ethyl acetate-hexane (3:2) from the supernatant fluid, and triglycerides were precipitated from cold 90% methanol, leaving a purified lipid fraction containing the steroids. Steroids were then fractionated by HPLC on a C18 column, and estradiol was analyzed by a radioimmunoassay. Blood was obtained at the same time as the rFNA samples and was frozen prior to analysis. An aliquot containing 200 ng of DNA (260/280 ratio 1.3 to 2.3) was taken for the nick translation assay. Incorporation of free nucleotides at SSBs with dCTP labeled with3H was catalyzed by Polymerase I from E. coli. A quality control preparation prepared from calf thymus DNA and a reagent blank without DNA was included with each set of 6 samples. Incorporation of3H-dCTP was linear with dose of DNA and reached a maximal at 30 min. Separation of free from incorporated3H-dCTP was accomplished by gel chromatography on an 80 × 15 mm column. The concordance of interassay values was 0.96. Results: Incorporation of nucleotides into DNA ranged from 0.03 to 8.59 pmol/μg, median 0.63 pmol/μg DNA. The association with other factors associated with breast cancer is shown in the Table. A significant correlation, was found with % breast density, life-time risk by the Gail model, but not serum estradiol concentrations. Conclusions: Assessment of SSBs by the nick translation procedure may be a useful indicator of breast cancer risk. Future studies will relate this method with actual risk as assessed by analysis of pre-diagnosis specimens with subsequent occurrence of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-12-04.