An Alternative Low-Cost Strategy for Simultaneous Sensitive Detection of Adjacent ESR1 Mutations in Single Circulating Tumor Cell

ESR1 mutation is of great clinical significance and being promoted as a marker of resistance to endocrine therapy in breast cancers. However, it is a challenging task to detect ESR1 mutations from traditional biopsies and cell-free DNA (cfDNA), especially for polyclonal mutations. This is mainly attributed to massive wild-type background and the low-abundance of the mutations. Here, using one-step single-cell amplification coupling with pyrosequencing, we developed and validated an original strategy for simultaneous sensitive detection of adjacent ESR1 mutations in single circulating tumor cell (CTC) from breast cancers. Unlike expensive single-cell sequencing used in previous studies, the strategy does not require complicated two-step amplification or high-cost single-cell amplification kits. Three pivotal parts involved in the strategy are collection of CTCs from whole blood of breast cancer patients, one-step single-cell amplification and pyrosequencing of single-cell amplicons. To achieve better efficiency of one-step single-cell amplification for pyrosequencing, a set of experimental conditions were thoroughly trialed. The developed strategy enabled a highly specific detection of the ESR1 hotspot mutations from large wild-type background with the specificity as low as 1% and a high sensitivity of two copies of artificial samples or single-cell level and pretty good quantitative accuracy ( R 2 ≥ 0.9888). Using this strategy, 141 single CTCs from 11 cases of breast cancer patients were identified and collected, 126 of which were successfully analyzed with a high rate of 89.4%. These results indicate that the cost-effective and reliable strategy could be used for clinical management, showing promising application in the treatment decision-making of breast cancer patients.