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Introduction: Accurate staging of patients with locally advanced breast cancer or locoregional recurrent (LRR) breast cancer is essential for providing a tailored treatment. According to current guidelines, staging is preferably done with positron emission tomography (PET) using 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), combined with diagnostic computed tomography (CT). Previous studies suggest that [18F]FDG PET might underperform in patients with grade 1-2, estrogen receptor positive (ER+) breast cancer as these tumors are often metabolically less active than other subtypes of breast cancer. Thus, there is a need to improve current diagnostics for this population. 16α-[18F]-fluoro-17β-estradiol ([18F]FES), radioactive labeled estradiol, has emerged as a powerful imaging tool for in vivo visualization of ER+ lesions. The aim of this study was to investigate whether [18F]FES PET/CT improves staging of patients with clinical stage II/III or LRR ER+ breast cancer, compared to [18F]FDG PET/CT. Methods: Patients with clinical stage II/III or LRR 1-2, ER+ breast cancer were included in this prospective multicenter clinical trial (NCT03726931). All patients underwent a [18F]FES PET/CT and a [18F]FDG PET/CT scan. Both PET scans were visually assessed, independently of each other. Visually suspect lesions for malignancy were verified pathologically (by biopsy or fine needle aspiration) or by conventional imaging (mammography, ultrasound, magnetic resonance imaging, CT). The stage of the disease was determined twice in a multidisciplinary team, based on conventional diagnostics (conventional imaging and pathology) and [18F]FDG PET/CT or [18F]FES PET/CT, respectively. Semi-quantitative PET parameters, standardized uptake values (SUVs) and tumor-to-blood ratios (TBRs), were obtained by defining volumes of interest in the aorta ascendens and in suspect tumor lesions. These PET parameters were correlated with various pathological features, such as histological subtype, grade and ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. Results: A total of 40 patients were included. On patient level, the sensitivity of [18F]FES PET/CT and [18F]FDG PET/CT to detect ER+ disease was 87.5% and 82.5%, respectively. [18F]FES PET/CT correctly determined the stage in 34/40 patients (85.0%) whereas [18F]FDG PET/CT correctly determined the stage in 29/40 patients (72.5%). [18F]FES PET/CT correctly changed the stage in 8/40 patients (20.0%) with upstaging in 6 patients and downstaging in 2 patients, compared to [18F]FDG PET/CT. On lesion level, a total of 140 suspect lesions were analyzed: 82 lesions were pathologically verified whereas 58 were radiographically verified. The sensitivity for lesion detection on [18F]FES PET/CT and [18F]FDG PET/CT was 90.6% and 77.3%, respectively. [18F]FES PET/CT identified more lesions correctly: 120/140 (85.7%) lesions vs 103/140 (73.6%) on [18F]FES PET/CT vs [18F]FDG PET/CT, respectively. [18F]FES PET/CT also missed less lesions (12/140, 8.6%) compared to [18F]FDG PET/CT (29/140, 20.7%). [18F]FES SUVmax, SUVmean, SUVpeak and TBR correlated with ductal subtype and percentage of PR expression (p Citation Format: Ramsha Iqbal, Emma C Bonjer, Annette van Zweeden, Lemonitsa H Mammatas, Jaap JM Teunissen, Jan Booij, Gwen MP Diepenhorst, Laura Schijf, Susanne van der Velde, Katya Duvivier, Ellis Barbé, Daniela E Oprea-Lager, Catharina W Menke-van der Houven van Oordt. Towards a new standard for staging: A comparative study of [18F]FES PET/CT vs [18F]FDG PET/CT in patients with clinical stage II/III and locoregional recurrent estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-02-11.