PO.CL01.06 · 临床研究
一种新型超灵敏基于qPCR的液体活检检测：外周血浆来源cfDNA中ESR1突变的检测
A novel ultra-sensitive qPCR-based liquid biopsy assay: ESR1 mutation detection in peripheral plasma derived cfDNA
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摘要 Abstract
中文摘要
背景：ESR1突变是HR+/HER2-转移性乳腺癌（mBC）对内分泌治疗获得性耐药的主要原因。监测ESR1突变的动态积累对患者迅速切换至有效的选择性雌激素受体降解剂（SERD）治疗至关重要。因此，临床实践中迫切需要一种快速且经济高效的ESR1突变状态监测检测。
方法：具有连续突变的ESR1序列对传统qPCR检测的开发构成重大障碍。在此设计了一种创新的抑制探针PCR并成功开发了检测（Eq-PCR）。将从ESR1突变乳腺癌细胞系（n=47）和患者来源的外周血样本（n=25）中提取的基因组无细胞DNA样本进行Eq-PCR、ddPCR（数字微滴PCR）和二代测序（NGS，覆盖78个基因）。
结果：我们的Eq-PCR检测对主要ESR1突变的检测限为0.5%突变频率。对于47个细胞系样本（10ng DNA输入），Eq-PCR与ddPCR之间的总体一致性为100%。对于从mBC患者收集的25个样本（cfDNA浓度范围为0.2ng-0.8ng/μl），Eq-PCR与NGS之间的总体一致性为PPV 100.0%（5个阳性样本）和NPV 85.0%（20个阴性样本）。
结论：开发了一种新型、经济高效的基于qPCR的主要ESR1突变检测，具有极高的准确性和快速的周转时间。该检测提供了一种迫切需要的临床工具，以便捷、及时和经济高效的方式监测HR+/HER2- mBC患者的关键ESR1突变。
查看英文原文 English abstract
Background: ESR1 mutations are a leading cause of acquired resistance to endocrine therapy in HR+/HER2- metastatic breast cancer (mBC). Monitoring of ESR1 mutations' dynamic accumulations is critical for patients to quickly switch to effective selective estrogen receptor degrader (SERD) treatment. Therefore, a rapid and cost-effective assay for ESR1 mutations status monitoring is in urgent demand in clinical practice.
Methods: ESR1 sequence with consecutive mutations poses a major hurdle for conventional qPCR assay development. Here an Innovative Suppression Probe PCR was designed and assay was successfully developed (Eq-PCR). Genomic cell-free DNA samples which were extracted from ESR1 mutated breast cancer cell lines (n=47) and patient-derived peripheral blood samples (n=25) were subjected to Eq-PCR, ddPCR (digital droplet PCR) and Next generation sequencing (NGS covering 78 genes).
Results: Our Eq-PCR assay has a limit of detection of 0.5% mutation frequency for major ESR1 mutations. For 47 cell lines samples (10ng DNA input), overall agreement between Eq-PCR and ddPCR is 100%. For 25 samples collected from mBC patients(cfDNA concentration ranged from 0.2ng-0.8ng/μl), overall agreement between Eq-PCR and NGS is 100.0% of PPV (5 positive samples) and 85.0% of NPV (20 negative samples).
Conclusion: A novel, cost-effective qPCR-based assay for major ESR1 mutations was developed with top accuracy and quick turn-around time. This assay provides an urgently needed clinical tool to monitor critical ESR1 mutation in HR+/HER2- mBC patients in a convenient, timely and cost-effective fashion.
利益披露 Disclosure
F. Ma, None..
H. Qian, None..
W. Wang, None..
Y. Yan, None..
B. Ji, None..
Y. Pei, None.