Abstract 7063: Monoclonal antibody evaluation of oxidative DNA base damage in normal and neoplastic breast tissues: development of antibodies specific for 8-OH-adenine and 4,6-diamino-5-formamidopyrimidine (FAPY-adenine)

Abstract Breast cancer affects 1 in 8 women and often results from mutations caused by oxidative DNA damage from reactive oxygen species. Two key lesions, 8-hydroxy-adenine (8-OH-Ade) and 4, 6-diamino-5-formamidopyrimidine (FAPY-Ade), exhibit two distinct patterns: FAPY-Ade lesions are predominant in normal tissues, while 8-OH-Ade lesions are abundant in cancerous tissues. These shifts reflect tissue redox status, which may provide a pathway to assess future breast cancer risk. Quantitation of these lesions by chemical detection methods is precise but time-intensive and impractical for high-throughput clinical applications. To address this, monoclonal antibodies have been developed and offer a rapid, scalable, and clinically practical alternative for efficiently detecting and quantifying these biomarkers. This innovation can potentially support personalized breast cancer screening strategies. BALB/c mice were immunized with 8-OH-Ade or FAPY-Ade haptens coupled to Keyhole Limpet Hemocyanin (KLH). After three monthly immunizations, the serums of IgG and IgM titers were evaluated. The mice having the highest IgG titer for each antigen were given an intravenous boost, and the spleens were removed three days later for fusions according to standard methods. Using hapten-conjugated BSA-coated 96-well Pro-Bind plates, the ELISA results demonstrated 30-40 positive wells for each antigen. Up to ten positive wells for each antigen were selected and cloned to yield monoclonal antibody-producing hybridomas. The ELISA of the anti-FAPY-Ade antibody, FA5, showed concentration-dependent reduced binding to immobilized BSA-FAPY-Ade hapten conjugate in increasing FAPY-Ade, confirming its FAPY-Ade binding specificity. Immunohistochemistry using normal and cancerous breast tissue revealed significant staining of normal breast tissue compared to cancerous breast tissue using the FA5 antibody. Conversely, the anti-8-OH-Ade antibody, 8A6, displayed weak staining of normal breast tissue with higher staining of cancerous breast tissues. The results demonstrate that monoclonal antibodies specific for 8-OH-Ade and a novel antibody specific for the ring-opening reactive oxygen product, FAPY-Ade, are practical and rapid alternatives to chemical detection methods for their analysis. Immunohistochemistry mirrored past results using chemical detection methods for these oxidized bases, with high FAPY-Ade compared to 8-OH-Ade in normal tissues and the opposite result in cancer-derived tissues. The potential for high-throughput analysis of 8-OH-Ade and FAPY-Ade levels in the DNA of breast tissues may provide information on future breast cancer risk. Further research is underway to evaluate this hypothesis. Citation Format: Zenzeale A. Hudson, Sarah McLaughlin, Alfredo A. Paredes, Gary K. Ostrander, Eric H. Holmes. Monoclonal antibody evaluation of oxidative DNA base damage in normal and neoplastic breast tissues: development of antibodies specific for 8-OH-adenine and 4, 6-diamino-5-formamidopyrimidine (FAPY-adenine) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7063.