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 a...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.展开更多
Estrogen and estrogen receptor-alpha(ER)signaling are important factors in the pathogenesis and treatment of ER-positive breast cancers.Therefore targeted therapies against ER,known as endocrine therapies,are widely u...Estrogen and estrogen receptor-alpha(ER)signaling are important factors in the pathogenesis and treatment of ER-positive breast cancers.Therefore targeted therapies against ER,known as endocrine therapies,are widely used in the treatment of ER-positive breast cancers.While these therapies have shown great success,de novo and acquired resistance are common.The approach to the problem of endocrine therapy resistance is twofold:identify the mechanisms of resistance and develop alternative treatments to overcome these mechanisms.This review focuses on the progress and integration of these two aspects of resolving endocrine therapy resistance in ER-positive breast cancer patients.展开更多
基金funded by the National Natural Science Foundation of China(81673390 and 61871403)Jiangsu Provincial key research and development program(BE2016745)+7 种基金The Open Project Program of MOE Key Laboratory of Drug Quality Control and Pharmacovigilance(DQCP2015MS02)sponsored by Qing Lan ProjectJiangsu Provincial Natural Science Foundation(BK20191322 and BK20180292)"Double First-Class"University project(CPU2018GY05 and CPU2018GY34)National Key R&D Program of China(2016YFA0201204)Jiangsu Provincial Science Fund for Distinguished Young Scholars(BK20180005)Jiangsu Provincial Medical Youth Talent Program(QNRC2016889)Innovation and Entrepreneurship Training Program for Undergraduate(202010316032S)
文摘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.
基金This work was supported by:The Avon Foundation(BHP)and the Canney Foundation.We would also like to thank and acknowledge the support of National Institutes of Health P30 CA006973the Sandy Garcia Charitable Foundation,the Commonwealth Foundation,the Santa Fe Foundation,the Marcie Ellen Foundation,The Helen Golde Trust and The Robin Page/Lebor Foundation.
文摘Estrogen and estrogen receptor-alpha(ER)signaling are important factors in the pathogenesis and treatment of ER-positive breast cancers.Therefore targeted therapies against ER,known as endocrine therapies,are widely used in the treatment of ER-positive breast cancers.While these therapies have shown great success,de novo and acquired resistance are common.The approach to the problem of endocrine therapy resistance is twofold:identify the mechanisms of resistance and develop alternative treatments to overcome these mechanisms.This review focuses on the progress and integration of these two aspects of resolving endocrine therapy resistance in ER-positive breast cancer patients.
