Extreme ESR1 Polyclonality, Mutation Dynamics, and Effects on Treatment Outcomes in Patients with ER+ Metastatic Breast Cancer

<title>Abstract</title> <p>Purpose ESR1 mutations are commonly acquired on endocrine therapy (ET) and contribute to therapeutic resistance. The impact of increasing degrees of polyclonality - including dynamics of ESR1-mutant clones, co-occurring mutations, and treatment outcomes - is not well understood. Methods We conducted a retrospective, descriptive review of patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) and &gt;10 concurrent ESR1 variants on circulating tumor DNA (ctDNA) using either Guardant360 or Tempus xF between August, 2020 and July, 2025, defined as extreme ESR1 polyclonality. Patient and tumor characteristics, frequency of ESR1 variants, co-occurring mutations, and clonal changes in ESR1 in response to various treatments were explored. Results Nine patients with ER+ MBC and extreme ESR1 polyclonality were identified; 5 were post-menopausal at diagnosis. Five had received adjuvant chemotherapy and 6 received adjuvant ET for a minimum of 3 years (range, 3-7). Patients received a median of 1 line of ET in the metastatic setting before their first ctDNA test, which showed 0-31 ESR1 clones on initial ctDNA (mean, 6). The most frequent ESR1 clones were Y537N, Y537S, and D538G. The most frequent co-alterations included TP53 and RB1 mutations and CCND1 amplification. Tumor mutational burden (TMB) at the time extreme ESR1 polyclonality emerged ranged from 6.7-56.5 mutations per megabase (mut/Mb); all assessable tumors were microsatellite stable (MSS). Capecitabine and olaparib were associated with a decline in ESR1 polyclonality on ctDNA after radiographic progression. Other cytotoxic agents did not have the same observed effect. Increased ESR1 polyclonality was observed after progression on the oral selective estrogen receptor degrader (SERDs) elacestrant and imlunestrant. Among the 4 patients treated with elacestrant, the median progression-free survival was 6 months (range, 4-6). At data cutoff, 3 patients had died from progressive disease, 1 had transferred care, 1 was in hospice, and 4 remained on active treatment. Conclusions This is the first case series to explore extreme ESR1 polyclonality. Characterizing the mutational dynamics associated with this phenomenon may improve our general understanding of ESR1 mutations and related impact on treatment resistance.</p>