目的:肿瘤组织的下一代测序(NGS)是提供个性化药物和靶向治疗的组成部分。液体活检的NGS,一种侵入性小得多的技术,是一种新兴的临床工具,已迅速扩大临床效用。在血液中循环的无细胞总核酸(cfTNA)中的基因突变代表了整个肿瘤生物学,并且可以揭示来自不同肿瘤部位的不同突变。从而解决肿瘤异质性挑战。
方法:具有自动样品制备功能的新型IonTorrentGenexusNGS系统,机载图书馆准备,模板,测序,测序使用数据分析和OncomineReporter软件。从血浆中提取的cfTNA用靶向泛癌症(~50个基因)Oncomine精密度测定(OPA)进行验证。评估标准包括分析灵敏度,特异性,检测限(LOD),准确度,重复性,可重复性和绩效指标的建立。
结果:获得ISO15189认证,已实施微创cfTNANGS诊断服务。观察到血浆和组织之间的高灵敏度(>83%)和特异性。当覆盖深度>22000×时,测序LOD为1.2%。液体活检结果的周转时间(TAT)减少(>68%):与参考实验室的>15天相比,从样品接收到向肿瘤学家发布的最终报告进行内部分析的TAT为5天。
结论:现在可以从血浆中可靠地评估肿瘤衍生的体细胞变异,以提供微创肿瘤特征。这项认可服务的成功实施导致:对肿瘤组织不可用或无法访问的患者进行适当的分子谱分析。血浆NGS的快速TAT结果。
OBJECTIVE: Next generation sequencing (NGS) on tumour tissue is integral to the delivery of personalised medicine and targeted therapy. NGS on liquid biopsy, a much less invasive technology, is an emerging clinical tool that has rapidly expanded clinical utility. Gene mutations in cell-free total nucleic acids (cfTNA) circulating in the blood are representative of whole tumour biology and can reveal different mutations from different tumour sites, thus addressing tumour heterogeneity challenges.
METHODS: The novel Ion Torrent Genexus NGS system with automated sample preparation, onboard library preparation, templating, sequencing, data analysis and Oncomine Reporter software was used. cfTNA extracted from plasma was verified with the targeted pan-cancer (~50 genes) Oncomine Precision Assay (OPA). Assessment criteria included analytical sensitivity, specificity, limits of detection (LOD), accuracy, repeatability, reproducibility and the establishment of performance metrics.
RESULTS: An ISO 15189 accredited, minimally invasive cfTNA NGS diagnostic service has been implemented. High sensitivity (>83%) and specificity between plasma and tissue were observed. A sequencing LOD of 1.2% was achieved when the depth of coverage was >22 000×. A reduction (>68%) in turnaround time (TAT) of liquid biopsy results was achieved: 5 days TAT for in-house analysis from sample receipt to a final report issued to oncologists as compared with >15 days from reference laboratories.
CONCLUSIONS: Tumour-derived somatic variants can now be reliably assessed from plasma to provide minimally invasive tumour profiling. Successful implementation of this accredited service resulted in:Appropriate molecular profiling of patients where tumour tissue is unavailable or inaccessible.Rapid TAT of plasma NGS results.