Abstract BACKGROUND: A majority of the 270,000 women diagnosed with breast cancer in the United States annually are candidates for breast conserving surgery (lumpectomy), however, up to 30% of patients require reoperation due to positive margins. The ideal resolution is to make residual in-breast disease visible at the time of surgery. The goal of this research is to develop a systemically administered fluorescent molecular imaging agent that is targeted to breast tumors for intraoperative margin assessment using fluorescence guided surgery (FGS). METHODS: To identify targetable cell-surface markers for use in FGS, gene expression profiling of publicly available Affymetrix mRNA microarray data from patients with invasive ductal or invasive lobular carcinoma tumors (n=34) and surrounding normal breast tissues (n=32) was performed. Protein expression of select targets was determined by immunohistochemistry (IHC) staining of patient tissue specimens. A humanized CEACAM6 (carcinoembryonic cell adhesion molecule 6) specific antibody fragment (scFv-Fc [IgG4]) was conjugated to the near-infrared IRDye800CW fluorescent dye (LiCor) by NHS-ester linkage to create CEACAM6-800. The conjugate was characterized for in vitro specificity by immunocytochemistry (ICC) and live cell uptake studies. As proof-of-principle, the conjugate (15 µg) was intravenously injected into nude mice bearing orthotopic human MCF-7 breast cancer mammary fat pad (MFP) xenograft tumors with endogenous expression of CEACAM6. FGS was performed using the “SurgVision” clinical imaging platform (approved for clinical use in Europe) at 24 h post-injection.RESULTS: We identified 263 genes with higher mRNA expression in tumor relative to normal. From this list, we selected 9 genes for confirmation of protein expression by IHC. Of the 9 targets, only CEACAM6 had no protein expression in normal breast tissues and robust expression in ~50% of tumors. ICC staining using the CEACAM6-800 conjugate and multiple human ductal carcinoma tumor cell lines confirmed the cell surface localization of CEACAM6. In live-cell uptake studies, CEACAM6-800 accumulated on the cell surface as early as 10 min after addition to the media and was internalized after 90 min. Blocking studies demonstrated specificity. In the proof-of-principal FGS study, we observed high fluorescence in MCF-7 MFP tumors relative to background and the tumors and their residuals were completely removed in the first attempt. CONCLUSION: We have confirmed CEACAM6 protein as a cell-surface adhesion receptor target for breast cancer FGS. The CEACAM6-800, fluorescent-dye to antibody-fragment conjugate was prepared and tested in vitro for sub-cellular localization and specific uptake. In vivo studies demonstrated specific uptake into MFP tumors and FGS for tumor resection. We are currently performing studies to determine the in vivo pharmacokinetics of CEACAM6-800 uptake and clearance in tumor and normal tissues and pre-clinical post-surgery survival using our pre-clinical models. These studies will enable future translational studies. Citation Format: Narges Tafreshi, Mikalai Budzevich, Weihong Sun, Daruka Mahadevan, Marie Catherine Lee, David L Morse. Breast tumor targeted fluorescence guidance for intraoperative margin assessment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-04.