ESR1 mutations in breast cancer are known as one of the mechanisms of resistance to aromatase inhibitors. These mutations often occur in the hotspot regions in the ligand binding domain (LBD), but comprehensive mutational analysis has shown that mutations are observed throughout the whole LBD. We previously developed a molecular barcode sequencing (MB-NGS) technique to detect ESR1 hotspot mutations in plasma with high sensitivity. In this study, we have developed a multiplex MB-NGS assay that covers the whole LBD of ESR1. The assay demonstrated that the background errors in the plasma DNA of 10 healthy controls were below 0.1%; thus, the limit of detection was set at 0.1%. We analyzed the plasma DNA of 54 patients with estrogen receptor–positive metastatic breast cancer. Seventeen mutations were detected in 13 patients (24%), with variant allele frequencies ranging from 0.13% to 10.67%, including six rare mutations with a variant allele frequency <1.0% and a novel nonhotspot mutation (A312V). Three patients had double mutations located in the same amplicons, and it was revealed that the double mutations were located in different alleles. ESR1 hotspot mutations were associated with a longer duration of aromatase inhibitor treatment under metastatic conditions and to liver metastasis. The multiplex MB-NGS assay is useful for the sensitive and comprehensive detection of mutations throughout the whole LBD of ESR1. Our assay can be applied to any specific target region of interest using tailor-made primers and can result in minimized sequencing volume and cost.

乳腺癌中的ESR1突变是对芳香化酶抑制剂产生抗性的机制之一。这些突变通常发生在配体结合结构域（LBD）的热点区域，但是全面的突变分析表明，在整个LBD中都观察到了突变。我们之前开发了分子条形码测序（MB-NGS）技术，以高灵敏度检测血浆中的ESR1热点突变。在这项研究中，我们开发了一种多重MB-NGS分析方法，涵盖了ESR1的整个LBD。该测定表明10个健康对照的血浆DNA的背景误差低于0.1％；因此，将检出限设定为0.1％。我们分析了54例雌激素受体阳性转移性乳腺癌患者的血浆DNA。在13例患者中检测到17个突变（24％），等位基因频率变异范围为0.13％至10.67％，其中包括6个罕见的突变，等位基因频率<1.0％，以及一个新的非热点突变（A312V）。三名患者在同一扩增子中有双重突变，并且发现该双重突变位于不同的等位基因。ESR1热点突变与转移条件下芳香酶抑制剂治疗的持续时间延长和肝转移有关。多重MB-NGS分析可用于敏感和全面检测ESR1整个LBD中的突变。我们的检测方法可以使用量身定制的引物应用于任何特定的目标靶标区域，并可以使测序体积和成本降至最低。