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 e fficiency 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.