Evaluation of endocrine resistance using ESR1 genotyping of circulating tumor cells and plasma DNA



Therapeutic efficacy of hormonal therapies to target estrogen receptor (ER)-positive breast cancer is limited by the acquisition of ligand-independent ESR1 mutations, which confer treatment resistance to aromatase inhibitors (AIs). Monitoring for the emergence of such mutations may enable individualized therapy. We thus assessed CTC- and ctDNA-based detection of ESR1 mutations with the aim of evaluating non-invasive approaches for the determination of endocrine resistance.

Patients and methods

In a prospective cohort of 55 women with hormone receptor-positive metastatic breast cancer, we isolated circulating tumor cells (CTCs) and developed a high-sensitivity method for the detection of ESR1 mutations in these CTCs. In patients with sufficient plasma for the simultaneous extraction of circulating tumor DNA (ctDNA), we performed a parallel analysis of ESR1 mutations using multiplex droplet digital PCR (ddPCR) and examined the agreement between these two platforms. Finally, we isolated single CTCs from a subset of these patients and reviewed RNA expression to explore alternate methods of evaluating endocrine responsiveness.


High-sensitivity ESR1 sequencing from CTCs revealed mono- and oligoclonal mutations in 22% of patients. These were concordant with plasma DNA sequencing in 95% of cases. Emergence of ESR1 mutations was correlated both with time to metastatic relapse and duration of AI therapy following such recurrence. The Presence of an ESR1 mutation, compared to ESR1 wild type, was associated with markedly shorter Progression-Free Survival on AI-based therapies (p = 0.0006), but unaltered to other non-AI-based therapies (p = 0.73). Compared with ESR1 mutant cases, AI-resistant CTCs with wild-type ESR1 showed an elevated ER-coactivator RNA signature, consistent with their predicted response to second-line hormonal therapies.


Blood-based serial monitoring may guide the selection of precision therapeutics for women with AI-resistant ER-positive breast cancer.

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Data availability

Additional data available on request from the authors.


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The authors are grateful to all the patients who participated in this study. We thank L. Libby for administrative support.


This work was supported by Grants from National Institute of Health (2RO1CA129933 to D.A.H, 2U01EB012493 to M.T, D.A.H, S.M, 5U01EB012493 to M.T, 5P41EB002503 to M.T), Susan G Komen Foundation (CCR15224703 to A.B), Howard Hughes Medical Institute (to D.A.H), ESSCO Breast Cancer Research Fund (to S.M), National Foundation for Cancer Research (to D.A.H), and the MGH-Johnson & Johnson Center for Excellence in CTCs (to M. Toner, S. Maheswaran). T. Sundaresan was supported by a Federal Share Research Career Development Award (K12CA087723) and a Santander Cancer Research Fellowship. N. Aceto was supported by the Human Frontiers Science Program, the Swiss National Science Foundation, and the Swiss Foundation for Grants in Biology and Medicine.

Author information




TKS, SM, and DAH conceived the project and provided leadership for the project. TKS, ZZ, TDD, NA, EJS, DBS, ML, RK, JI, SM, and DAH carried out experiments and/or analyzed data. BSW provided guidance and help in computational and statistical analyses. AB and JI provided clinical annotation and guidance. MT developed CTC-iChip technology for CTC isolation. TKS, TDD, SM, and DAH wrote the manuscript. All authors discussed results and provided input and edits on the manuscript.

Corresponding authors

Correspondence to Shyamala Maheswaran or Daniel A. Haber.

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Conflict of interests

Massachusetts General Hospital (MGH) has applied for patents regarding the CTCi-Chip technology, CTC detection signatures, and Enrich-seq technology. M.T, S.M, and D.A.H are cofounders and have equity in Tell-Bio, which is not related to this work. J.I and Z.Z hold equity in ArcherDx. A.B holds Consultant/advisory board position: Genentech/Roche, Immunomedics, Novartis, Pfizer, Merck, Radius Health, Spectrum Pharma, Taiho Pharm, Diiachi-Astra Zeneca, Eli Lilly, Sanofi, Puma, Foundation Medicine, and Phillips. Contracted research: Genentech/Roche, Immunomedics, Novartis, Pfizer, Merck, Radius Health, Sanofi, and Biothernostics. All authors’ interests were reviewed and are managed by MGH and Partners HealthCare in accordance with their conflict of interest policies.

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Patients provided written informed consent to an Institutional Review Board approved blood collection protocol (DF/HCC 05-300).

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Sundaresan, T.K., Dubash, T.D., Zheng, Z. et al. Evaluation of endocrine resistance using ESR1 genotyping of circulating tumor cells and plasma DNA. Breast Cancer Res Treat (2021). https://doi.org/10.1007/s10549-021-06270-z

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  • AI resistance ER + breast cancer
  • Acquired ESR1 mutations
  • High-sensitivity ESR1 sequencing
  • Circulating tumor cells (CTCs)
  • Circulating tumor DNA (ctDNA)