Table2_Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.XLSX

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

肺癌是遗传驱动型肿瘤的典型范例。目前已有多款针对特定生物标志物的药物被开发，这类药物的应用需先对相关生物标志物的肿瘤遗传改变进行检测。当前可用于文库构建的下一代测序（next-generation sequencing，NGS）技术主要分为三类：1）锚定多重PCR技术、2）基于扩增子的PCR技术以及3）基于杂交捕获的PCR技术。锚定多重PCR测序技术被纳入国家肺癌基因组医学网络（national Network Genomic Medicine Lung Cancer，nNGM）的常规分子检测研究中。四家中心采用搭载锚定多重PCR技术的ArcherDX-Variantplex nNGMv2基因检测panel，对常规诊断阶段已完成预检测的样本开展重分析。各中心独立完成数据分析后，依据研究设计方案进行集中汇总。本研究采用预设的检测标准，并通过梯度稀释实验确定该基因检测panel的灵敏度。nNGMv2基因检测panel测序的样本成功率达98.9%（N=90）。采用默认过滤参数时，除2个潜在的MET外显子14跳变变异外，其余变异均在相近的等位基因频率下被成功检出；这2个MET变异均可通过调整后的变异检出过滤参数被识别。另有3个预检测分析未检出的变异（KEAP1、STK11、TP53）被成功检出。仅DNA总投入量与平均测序覆盖度呈显著相关，而基于qPCR的DNA质量评分则无此关联。即使DNA投入量低至6.25 ng，仍可成功完成测序分析。基于锚定多重PCR的nNGMv2测序技术搭配操作便捷的Archer-Analysis分析流程，是一种稳健且特异性优异的肿瘤基因突变检测技术，可用于肺癌患者的精准医学诊疗。