Winer-Jones Jessamine P, Vahidi Behrad, Arquilevich Norma, Fang Cong, Ferguson Samuel, Harkins Darren, Hill Cory, Klem Erich, Pagano Paul C, Peasley Chrissy, Romero Juan, Shartle Robert, Vasko Robert C, Strauss William M, Dempsey Paul W
Research and Development, Cynvenio Biosystems, Westlake Village, California, United States of America.
Engineering, Cynvenio Biosystems, Westlake Village, California, United States of America.
PLoS One. 2014 Jan 29;9(1):e86717. doi: 10.1371/journal.pone.0086717. eCollection 2014.
Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.
METHODOLOGY/PRINCIPAL FINDINGS: Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL.
CONCLUSIONS/SIGNIFICANCE: The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.
当代癌症诊断越来越依赖于用于分析遗传信息的复杂新分子方法。这些新技术的局限性在于缺乏足够的实体瘤组织样本。患者可能出现无法进行活检或不足以进行纵向监测的肿瘤。一个有吸引力的替代来源是外周血中的癌细胞。这些罕见的循环肿瘤细胞（CTC）在进行分子分析之前需要富集和分离。目前的CTC平台要么缺乏在临床环境中使用的通量或可靠性，要么提供的CTC样本纯度限制了可用分子工具的分子分析。
方法/主要发现：磁泳和微流控技术的最新进展已被用于生产一种名为LiquidBiopsy®的自动化平台。该平台使用高通量鞘流微流控技术对CTC群体进行阳性选择。此外，该平台定量分离可用于分子方法（如突变检测）的细胞。CTC回收率经过表征和验证，准确度（误差<20%）和精密度（CV<25%）至少低至9个CTC/ml。使用抗EpCAM抗体作为捕获剂，该平台在线性范围内可回收78%的MCF7细胞。背景细胞的非特异性回收与靶细胞密度无关，平均为55个细胞/mL。低至6个CTC/mL时可实现10%的纯度，1个CTC/mL时可实现优于1%的纯度。
结论/意义：LiquidBiopsy平台是一个经过验证的自动化平台，可提供对CTC群体的高通量分子分析。它可以经过验证并整合到实验室流程中进行CTC计数，并回收用于分子分析（如定量PCR和下一代测序）的始终保持高纯度的样本。该工具为CTC的临床相关基因谱分析开辟了道路。
