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A single droplet digital PCR for ESR1 activating mutations detection in plasma

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Abstract

Activating mutations in the estrogen receptor 1 (ESR1) gene confer resistance to aromatase inhibitors (AI), and may be targeted by selective estrogen receptor downregulators. We designed a multiplex droplet digital PCR (ddPCR), which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with an unconventional assay interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S, or D538G mutations. Further validation was performed on plasma samples from a prospective study and compared with next generation sequencing (NGS) data. The multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them, all mutations being confirmed by NGS. In addition, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of circulating tumor DNA using this technique during treatment follow-up predicts the clinical benefit of palbociclib–fulvestrant. The multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations with good sensitivity. This method allows real-time liquid biopsy for ESR1 mutation monitoring in large cohorts of patients.

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Fig. 1: ESR1 exons 5 and 8 assay design.
Fig. 2: In vitro performance of the multiplex ESR1-ddPCR compared with simplex and conventional assays.
Fig. 3: Monitoring of circulating ESR1 mutant copies to predict response to palbociclib–fulvestrant therapy.

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Acknowledgements

We would like to thank members of the Circulating Tumor Biomarkers laboratory for helpful discussions and especially Amanda Silveira for critical reading of the manuscript. High-throughput sequencing has been performed by the ICGex NGS platform of the Institut Curie supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09–08 (France Génomique Consortium) from the Agence Nationale de la Recherche (“Investissements d’Avenir” program), by the Canceropole Ile-de-France and by the SiRIC 2 Curie program - SiRIC Grant « INCa-DGOS-Inserm_12554 ».

Funding

SIRIC 2 Curie (grant INCa-DGOS-Inserm_12554) and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115749 (project Cancer-ID). The funding sources of the study had no role in the design of the study, collection, analysis, or interpretation of the data or in the writing of this report.

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  1. These authors contributed equally: François-Clément Bidard, Charlotte Proudhon

Authors and Affiliations

  1. Circulating tumor biomarkers laboratory, Inserm CIC 1428, Institut Curie, PSL Research University, Paris, France

    Emmanuelle Jeannot, Lauren Darrigues, Marc Michel, Jean-Yves Pierga, Aurore Rampanou, François-Clément Bidard & Charlotte Proudhon

  2. Department of Biopathology and Genetics, Institut Curie, PSL Research University, Paris, France

    Emmanuelle Jeannot, Samia Melaabi, Camille Benoist, Ivan Bièche & Anne Vincent-Salomon

  3. INSERM U830, Institut Curie, PSL Research University, Paris, France

    Marc-Henri Stern

  4. Department of Medical Oncology, Institut Curie, PSL Research University, Paris & Saint Cloud, France

    Jean-Yves Pierga, Radouane El Ayachy, Aurélien Noret, Nicolas Epaillard, Luc Cabel & François-Clément Bidard

  5. Université de Paris, Paris, France

    Jean-Yves Pierga

  6. Medical Oncology, Department of the Institut Curie, Paris, France

    Jean-Yves Pierga

  7. Versailles Saint Quentin University, Paris Saclay University, Saint Cloud, France

    Luc Cabel & François-Clément Bidard

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  1. Emmanuelle Jeannot
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Correspondence to Charlotte Proudhon.

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Conflict of interest

CP, EJ, MHS, JYP, and FCB have ongoing patent applications relating to circulating tumor DNA analysis.

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Jeannot, E., Darrigues, L., Michel, M. et al. A single droplet digital PCR for ESR1 activating mutations detection in plasma. Oncogene 39, 2987–2995 (2020). https://doi.org/10.1038/s41388-020-1174-y

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