Performance of next-generation sequencing on small tumor specimens and/or low tumor content samples using a commercially available platform

BackgroundNext generation sequencing tests (NGS) are usually performed on relatively small core biopsy or fine needle aspiration (FNA) samples. Data is limited on what amount of tumor by volume or minimum number of FNA passes are needed to yield sufficient material for running NGS. We sought to identify the amount of tumor for running the PCDx NGS platform.Methods2,723 consecutive tumor tissues of all cancer types were queried and reviewed for inclusion. Information on tumor volume, success of performing NGS, and results of NGS were compiled. Assessment of sequence analysis, mutation calling and sensitivity, quality control, drug associations, and data aggregation and analysis were performed.Results6.4% of samples were rejected from all testing due to insufficient tumor quantity. The number of genes with insufficient sensitivity make definitive mutation calls increased as the percentage of tumor decreased, reaching statistical significance below 5% tumor content. The number of drug associations also decreased with a lower percentage of tumor, but this difference only became significant between 1–3%. The number of drug associations did decrease with smaller tissue size as expected. Neither specimen size or percentage of tumor affected the ability to pass mRNA quality control. A tumor area of 10 mm2 provides a good margin of error for specimens to yield adequate drug association results.ConclusionsSpecimen suitability remains a major obstacle to clinical NGS testing. We determined that PCR-based library creation methods allow the use of smaller specimens, and those with a lower percentage of tumor cells to be run on the PCDx NGS platform.背景：下一代测序（Next Generation Sequencing, NGS）检测通常依托相对少量的穿刺活检或细针抽吸活检（Fine Needle Aspiration, FNA）样本开展。目前针对需满足多少肿瘤体积阈值、或最少需要多少次细针抽吸操作才能获取足够样本以开展NGS检测的相关数据仍较为匮乏。本研究旨在明确适配PCDx NGS平台检测所需的肿瘤样本量。 方法：本研究纳入2723例连续入组的各类肿瘤组织样本。收集所有样本的肿瘤体积、NGS检测成功率及NGS检测结果等信息，并开展序列分析、突变检出与灵敏度评估、质量控制、药物相关性分析以及数据汇总与统计分析。 结果：6.4%的样本因肿瘤样本量不足而被全部检测流程拒收。随着肿瘤细胞占比降低，灵敏度不足、无法准确定性检出突变的基因数量随之增加，当肿瘤占比低于5%时，该差异达到统计学显著性。药物相关性关联数量同样随肿瘤细胞占比降低而减少，但该差异仅在1%~3%区间内达到统计学显著性。如预期所示，组织样本体积越小，药物相关性关联数量亦随之减少。样本体积与肿瘤细胞占比均不会影响mRNA质量控制的通过率。当肿瘤样本面积达到10 mm²时，可获得较为充足的误差余量，确保获取可靠的药物相关性分析结果。 结论：样本适配性仍是临床NGS检测面临的主要障碍。本研究证实，基于聚合酶链式反应（Polymerase Chain Reaction, PCR）的文库构建方法可支持使用体积更小、肿瘤细胞占比更低的样本在PCDx NGS平台上开展检测。