ESR1 Mutation Longitudinal Dynamics in RWD Cohort of HR+/HER2- Metastatic Breast Cancer Patients Treated With Standard of Care Hormonal Therapy

**Background
**ESR1 mutations (ESR1m) are known drivers of resistance to aromatase inhibitors. While ESR1m has been linked to worse survival outcomes, clonal complexity over time has not been well-evaluated in a real-world context. Here we characterized longitudinal dynamics of ESR1m, including rare point mutation emergence and polyclonality in hormone receptor- positive/HER2-negative metastatic breast cancer (HR+/HER2- mBC) patients (pts) treated with aromatase inhibitor (AI) plus CDK4/6 inhibitor (CDK4/6i).

**Methods
**We identified a cohort for longitudinal analysis from the Tempus real-world database (RWD). Eligible pts had HR+/HER2- mBC (diagnosed before 01/2023), received AI+CDK4/6i therapy in mBC, and had comprehensive genomic profiling from tissue/liquid biopsy (Tempus xT/Tempus xF) within 2 yrs of treatment, with ≥1 on-treatment xF tests. We analyzed ESR1 clonal dynamics, starting from first ESR1m detection in a subset of pts who had multiple xF tests. Polyclonality was also assessed, defined as presence of at least 2 distinct ESR1m in a sample.

**Results
**Of 301 HR+/HER2- mBC pts on AI+CDK4/6i therapy, 102 (33.9%) developed an ESR1m. In the ESR1m group, 72 pts (70.6%) had 2+ on-treatment liquid biopsies (Tempus xF/xF+), comprising the final cohort for observation. At ESR1m detection, 64.8% pts had a single detectable ESR1m, most commonly D538G (31.9%) and Y537S (12.5%). In contrast, 34.7% had polyclonal mutations (19.7% with 2 distinct mutations; 15.3% with 3+). Recurrent detection of D538G (52.8%), Y537S (20.8%), and Y537N (16.7%) was noted in repeat xF testing. Detection of additional ESR1m (26.4%) over time confirmed known common mutations such as Y537N (8.3%), D538G (5.6%), and Y537S (5.6%). However, longitudinal testing also found rare mutations such as V422del and H524L. Mutational dynamics shifts were also revealed by longitudinal testing. In 36.1% pts, a previously detected ESR1m was no longer found, often in those who initiated a SERD. D538G (5.6%) and Y537N (4.2%) were the most commonly lost mutations. Recurrent and new ESR1m detection occurred in pts who continued AI+CDK4/6i (44.4%). Many pts showed a combination of these dynamics.

**Conclusions
**Multimodal RWD analyses demonstrate valuable clinical insights on ESR1m patterns detected from longitudinal molecular surveillance testing in HR+/HER2- mBC pts treated with AI+CDK4/6i. We observed shifting dynamics in ESR1m burden, including detection of rare mutations and polyclonality. These findings show the value of monitoring ESR1m longitudinally over disease course and prompt further research into clinical impacts of clonal dynamics.