Abstract 3867: Activating ESR1 mutations detection by single ddPCR assay

Abstract

Abstract Background: In metastatic breast cancer, most mutations of ESR1 (which encodes the estrogen receptor alpha ERα) occur in exon 5 (E380Q) and/or 8 (L536, Y537, D538). These mutations are responsible for acquired resistance to hormone deprivation but can be targeted by selective estrogen receptor degraders (SERD) such as fulvestrant. Circulating tumor DNA (ctDNA) allows a non-invasive assessment of ESR1 mutations, but implementing its routine use requires a sensitive, specific and fast technique, at a moderate cost. Methods: We designed a ddPCR assay scanning for all mutations targeting the 536-538 amino acid residues of ERα with a unique pair of probes and multiplexed it with other probes targeting E380Q. Sensitivity and specificity were assessed in vitro; this multiple hotspot scanning (MHS) ddPCR was used to detect ESR1 mutants in cohorts of patients with hormone deprivation-resistant metastatic breast cancer. Results: All the above-mentioned mutations were detected in a single MHS ddPCR assay with a high specificity and with a limit of detection of ~0.1% in mutant allele frequency, allowing ESR1 mutant detection in circulating tumor DNA. A perfect concordance with targeted next generation sequencing was observed in the first 31 clinical plasma samples tested. This technique also detected ESR1 polyclonal mutations. Conclusion: This technique allows for a sensitive, large scale and repeated screening of ESR1 mutants with a single ddPCR assay. The clinical utility of ESR1 mutation detection by this technique is now investigated in the randomized phase 3 PADA-1 trial (sponsors: Unicancer/Gineco), in which patients with rising levels of circulating ESR1 mutants during aromatase inhibitor / palbociclib therapy are eventually switched to fulvestrant / palbociclib (NCT03079011). ESR1 mutants detectable in a single ddPCR assayAA changesMutant frequency (as per Toy et al, Cancer Discov 2017).E380Q19%L536H4%L536P2%L536R1%Y537S11%Y537C6%Y537N5%Y537D1%D538G32%D538-L539ins1%total % of mutations covered~82% (Many of the 18% remaining mutations occur outside exon 5 & 8 and have no proven functional impact) Citation Format: Francois-Clement Bidard, Emmanuelle Jeannot, Luc Cabel, Nicolas Epaillard, Radouane El Ayachy, Aurelien Noret, Anne Vincent-Salomon, Jean-Yves Pierga, Ivan Bieche, Charlotte Proudhon, Marc-Henri Stern. Activating ESR1 mutations detection by single ddPCR assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3867.