Abstract Introduction The ESR1 gene encodes the estrogen receptor alpha (ERα), a transcription factor that regulates gene expression in response to estrogen. Mutations in ESR1 can lead to constitutive activation of ERα, allowing estrogen receptor-positive (ER+) breast cancer cells to proliferate even in the absence of estrogen; this proliferation contributes to resistance against standard hormone therapies such as treatment with aromatase inhibitors (AI), which function by reducing estrogen levels. ESR1 mutations are typically acquired during treatment and are most frequently observed in metastatic breast cancer. Detecting ESR1 mutations, often through circulating tumor DNA (ctDNA) in liquid biopsies, is critical for tracking disease progression and optimizing therapeutic strategies. As reported at the recent ASCO 2025 meeting, treatment switch before clinical progression based on the detection of ESR1 mutations in ctDNA can potentially improve outcomes in HR+/HER2- advanced breast cancer. Thus, identifying ESR1 mutations enables clinicians to make informed treatment decisions and supports the development of targeted approaches to overcome endocrine resistance. Molecular assays such as digital PCR (dPCR), real-time PCR (RT-qPCR), and next-generation sequencing (NGS) are commonly employed for detection of ESR1 mutations, with each platform offering distinct advantages in sensitivity and mutation coverage. However, a common limitation of these technologies is their sensitivity, which is compounded by the limited quantity of ctDNA that can be isolated from human specimens. To address these limitations, we developed a novel assay specifically designed for the detection of ESR1 mutations; the assay leverages the high specificity and sensitivity of the SuperRCA® technology developed by Rarity to enable accurate identification of low-frequency ESR1 variants. Methods The SuperRCA® uses 2 consecutive rolling circle amplification (RCA) reactions, which enable highly specific genotyping and accurate quantification of low-frequency variants. The sensitivity of the assay for ESR1 was assessed by testing a quantitative, serial dilution of synthetic controls of commonly found resistant variants and numerical positive signals detected using flow cytometry. Assay specificity was assessed by the number of positive signals from cfDNA extracted from normal individuals. Results The assay was capable of detecting rare ESR1 mutations at a sensitivity of 0.01% VAF down to 0.001% VAF. The number of positive signals was essentially zero for normal individuals , indicating high specificity. Conclusions These results demonstrate the SuperRCA® can be used to detect clinically relevant ESR1 variants, which may offer a powerful tool for improving the management of endocrine-resistant breast cancer. References: 1 ESR1 mutations in HR+/HER2-metastatic breast cancer: Enhancing the accuracy of ctDNA testing. Konstantinos Venetis et al. Cancer Treat Rev. 2023. PMID: 378649562 ESR1 mutations and therapeutic resistance in metastatic breast cancer: progress and remaining challenges. Sarah K Herzog et al. Br J Cancer. 2022. PMID: 34621045 3 ESR1 mutations-a mechanism for acquired endocrine resistance in breast cancer. Rinath Jeselsohn et al. Nat Rev Clin Oncol. 2015. PMID: 26122181 4 Camizestrant + CDK4/6 inhibitor (CDK4/6i) for the treatment of emergent ESR1 mutations during first-line (1L) endocrine-based therapy (ET) and ahead of disease progression in patients (pts) with HR+/HER2 - advanced breast cancer (ABC): Phase 3, double-blind ctDNA-guided SERENA-6 trial. Nicholas C. Turner et al. ASCO 2025. Abstract LBA4 Citation Format: D. Nguyen, L. Chen, V. Radic, C. Ma, F. Racke. Detection of Treatment-Resistant ESR1 Mutations by Ultra-sensitive SuperRCA Technology abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-03-23.
Nguyen et al. (Tue,) studied this question.