Abstract Introduction: Breast cancer is a heterogeneous disease, clinically defined by expression of the biomarkers ER, PR, and HER2, with 15-30% of breast cancers overexpressing the HER2 protein. Antibodies to tumor antigens that are present in patient sera are potential biomarkers for early detection and prognosis of breast cancer. The purpose of this study is to identify a panel of novel serum biomarkers for the early diagnosis of HER2+ breast cancer. Methods: We screened sera from breast cancer patients for autoantibodies using custom nucleic acid programmable protein microarrays (NAPPA). Plasmid cDNAs encoding 10,000 full-length human proteins with c-terminal GST or FLAG tags were printed on arrays with anti-GST or anti-FLAG antibodies, and transcribed and translated in situ using HeLa expression lysate. Serum samples were obtained at diagnosis from patients with stage I-III HER2+ breast cancer classified by IHC and/or FISH testing (HER2+/ER-/PR-, 47%; HER2+/ER+/PR+, 48%; HER2+/ER-/PR+, 5%). Control sera were age-matched and obtained from women undergoing screening mammography or diagnostic mammography for benign breast disease. The microarrays were screened with sera from 29 cases and 29 controls. 850 antigens were selected, displayed in microarray format, and screened with an independent set of sera 27 cases, 29 benign breast disease controls, and 25 healthy controls. Bound IgG was detected by fluorescence. Results: From the initial 10,000 proteins, we identified 850 proteins using cutoff values of AUC (0.68), sensitivity (0.29), and pAUC (0.0075) at 94% specificity for further analysis. From these, 50 candidate autoantibody biomarkers for the detection of HER2+ breast cancer were identified based on visual analysis and statistical cutoff values of 15% sensitivity at 98% specificity and K-statistic (≥ 0.9). Conclusion: We identified a 50-autoantibody serum signature for the detection of HER2+ breast cancer. Blinded validation studies are ongoing. Citation Format: Benjamin A. Katchman, Rizwan Alam, Matthew S. Field, Jennifer Viloria, Garrick Wallstrom, Joshua LaBaer, Paul Engstrom, Karen S. Anderson. A protein microarray signature of autoantibody biomarkers for the detection of HER2+ breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2015-1318