Abstract A19: Detection of ESR1 mutations in plasma cell-free DNA from metastatic ER-positive breast cancer patients resistant to hormone therapy
Authors: Masotti Cibele , Knebel Franciele H. , Linck Rudinei D. M. , Lima Julianne , Shimada Andrea K. , Alessi João Victor , Zucchetti Bruna M. , Kleftogiannis Dimitrios , Barber Louise , Gerlinger Marco , Katz Artur , Mano Max S. , Camargo Anamaria A. , Bettoni Fabiana 
Published: June 2020

Abstract
About 70% of breast cancers (BC) express estrogen receptor α (ER) and most of these are sensitive to endocrine therapies (ET). However, a significant fraction of patients with ER-positive (ER+) BC who benefit from adjuvant ET recur with metastatic disease after prolonged exposure to these therapies. Even patients with ER+ metastatic BC who respond to ET will eventually progress with antiestrogen-resistant hormone-independent disease. Activating mutations in the estrogen receptor gene ESR1 have been described in advanced ER+ BC resistant to ET, particularly in metastatic patients exposed to therapy with aromatase inhibitors (AI). ESR1 mutations frequently occur in a hotspot region within the ligand-binding domain of ER, constitutively activating the receptor in a ligand-independent manner and thus explaining the acquired resistance to hormonal therapy. Recent studies have reported the detection of ESR1 mutations in codons 536, 537, and 538 in circulating tumor DNA (ctDNA) of metastatic BC patients, ensuring the establishment of a causal relationship among ESR1 mutations and acquired resistance to AI therapy. Thereby we evaluated the presence of ESR1 mutations in plasma cell-free DNA (cfDNA) of metastatic ER-positive BC patients who progressed on AI therapy. Among patients with no ESR1 mutations detected, we questioned the presence of any other mutated gene that could be related to acquired resistance to ET. To answer these questions, we first investigated the presence of ESR1 recurrent mutations (L536R, Y537C/N/S, D538G) by Droplet Digital PCR (ddPCR) in ctDNA of 24 ER+ metastatic BC patients after acquired resistance to therapy with AI. ESR1 mutations were detected in 25% (6/24) of the patients, with ESR1 D538G and Y537N being the most frequent, and four patients showed polyclonal disease with more than one ESR1 mutation. The longest interval between the end of aromatase inhibitor treatment and the detection of ESR1 mutations was 2.3 years, corroborating with some studies that showed that these mutations remain readily detectable in ctDNA many months and years after stopping therapy. cfDNA from patients with no ESR1 mutations detected were analyzed by targeted cfDNA sequencing (cfDNA seq) of a 151-cancer gene panel. We were able to detect mutations in genes already known to be associated to ET resistance such as PIK3CA and AKT. We found one patient with AKT E17K mutation and four patients (4/12, 33%) with detectable PIK3CA-mutated ctDNA, with three patients with E545K mutation and one with H1047L mutation. Mutations in PIK3CA are frequently observed in BC and have been suggested as a predictive biomarker for PI3K-selective inhibitor treatment, alpelisib. Based in our results, we can conclude that detection of tumor genetic alterations in plasma cfDNA may have important clinical implications for personalized medicine, including monitoring of recurrence and therapy resistance and therapeutic guidance.
Citation Format: Cibele Masotti, Franciele H. Knebel, Rudinei D. M. Linck, Julianne Lima, Andrea K. Shimada, João Victor Alessi, Bruna M. Zucchetti, Dimitrios Kleftogiannis, Louise Barber, Marco Gerlinger, Artur Katz, Max S. Mano, Anamaria A. Camargo, Fabiana Bettoni. Detection of ESR1 mutations in plasma cell-free DNA from metastatic ER-positive breast cancer patients resistant to hormone therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A19.