Abstract Introduction: Notwithstanding the increasing efficacy of systemic therapy, metastatic breast cancer (MBC) is still an incurable disease. The prolonged exposure to endocrine therapy will result in acquired resistance with consequent disease progression. Understanding the underlying mechanisms of resistance is therefore crucial for early resistance detection and for treatment choice optimization. The aim of this study was a comprehensive characterization through circulating tumor DNA analysis (ctDNA) for hypothesis generation on endocrine resistance. Methods: This retrospective study analyzed a pilot cohort of 35 metastatic breast cancer (MBC) patients (pts) treated and evaluated for ctDNA at Northwestern University (Chicago, IL). ctDNA was analyzed using the PredicinePLUS™ NGS 180-gene panel (Predicine Inc, CA). Endocrine resistance was defined as a relapse during the first 2 years of adjuvant endocrine therapy or progressive disease within endocrine therapy (ET) for MBC. Associations between clinico-pathological characteristics and gene variants were tested though Fisher’s exact test. Results: The study included 27 hormone receptor positive MBC (HR+) pts, 5 HER2 positive and 1 Triple Negative MBC (TNBC) patient. Among HR+ pts, 24 received ET in previous lines, including 21 cases treated with an aromatase inhibitor (AI)-based backbone, while 14 received an ET association with CDK4/6 inhibitors. Fifteen were classified as endocrine resistant according to clinical criteria. In the subgroup of pts previously treated with AI, the main detectable gene variants wereTP53 (48%), PIK3CA (26%), GNAS (30%), ESR1 (25%), CDH1 (22%), BRCA2(22%), ARID1A (41%) and AR (52%). Notably, TP53 and ESR1 aberrations were mainly polygenic (ESR1:c.1138G>C, c.1261A>C, c.1551G>A c.1607T>C, c.1609T>A, c.1610A>C, c.1610A>G, c.1613A>G, c.172G>A; TP53:c.1024C>T, c.473G>A, c.524G>A, c.536A>G, c.559G>A, c.586C>T, c.587G>C, c.638G>A, c.659A>G, c.713G>A, c.772del), while the main genes showing copy number variations were in BRCA2 (Loss), JAK2 (Loss), PPP2R2A (Loss), RB1 (Loss), SERPINB3 and SERPINB4 (Gain). Moreover, among the totally detected 448 gene variants, only ESR1 mutations were associated with previous AI prescription (P=0.030). Conclusion:The present study offers an insight on the mutational landscape of MBC patients treated with endocrine therapy alone or associated with CDK4/6 inhibitors. ESR1 mutations were confirmed as the predominant resistance factor and were mainly polygenic. New promising targets such as SERPINB3, SERPINB4, ARID1A and AR add new intriguing clues on the potential role of Epithelial to Mesenchymal transition in endocrine resistance and warrant further investigation on a larger, prospective, cohort. Citation Format: Lorenzo Gerratana, Qiang Zhang, Andrew A Davis, Ami N Shah, Jianjun Yu, Shidong Jia, Youbin Zhang, Firas Wehbe, Amir Behdad, Leonidas C Platanias, William J Gradishar, Massimo Cristofanilli. Characterization of metastatic breast cancer through a novel next generation sequencing platform for hypothesis generation on endocrine resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-09.