Department of Endoscopy and Pulmonary Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
Burning Rock Biotech, Guangzhou, People's Republic of China.
J Thorac Oncol. 2017 Apr;12(4):663-672. doi: 10.1016/j.jtho.2016.11.2235. Epub 2016 Dec 19.
Circulating tumor DNA (ctDNA), which represents an unbiased way to assess tumor genetic profile noninvasively, facilitates studying intratumor heterogeneity. Although intratumor heterogeneity has been elucidated substantially in a few cancer types, including NSCLC, how it influences the ability of tumor cells harboring different genetic abnormalities in releasing their DNA remains elusive. We designed a capture-based panel targeting NSCLC to detect and quantify genetic alterations from plasma samples by using deep sequencing. By applying the panel to paired biopsy and plasma samples, we imputed and compared the ctDNA-releasing efficiency in subclones harboring distinct genetic variants.
We collected 40 pairs of matched biopsy and plasma samples from patients with advanced lung cancer and applied capture-based sequencing using our LungPlasma panel, which consists of critical exons and introns of 168 genes. We derived a normalized relative allelic fraction score (NRAFS) to reflect ctDNA-releasing efficiency.
By using mutations detected in biopsy samples as a reference, we achieved 87.2% by-variant sensitivity, including for single-nucleotide variants, insertions or deletions, and gene fusions. Furthermore, the by-variant sensitivity for the seven most critical and actionable genes was 96.2%. The average NRAFS for subclones carrying mutations from seven actionable genes was 0.877; in contrast, the average NRAFS for other mutations was 0.658. Mutations from four genes involved in cell cycle pathways had a particularly low NRAFS (0.480) compared with the other two groups (p = 0.07).
We have demonstrated that subclones carrying driver mutations are more prone to release DNA. We have also demonstrated the quantitative ability of capture-based sequencing, paving its way for routine utilization in clinical settings.
循环肿瘤 DNA（ctDNA）是一种非侵入性评估肿瘤遗传特征的方法，能够促进肿瘤异质性的研究。虽然在一些癌症类型中，包括 NSCLC，已经深入阐明了肿瘤内异质性，但肿瘤细胞携带不同遗传异常释放其 DNA 的能力如何影响尚不清楚。我们设计了一个基于捕获的 NSCLC 靶向panel，通过深度测序从血浆样本中检测和量化遗传改变。通过将该 panel 应用于配对活检和血浆样本，我们推断并比较了携带不同遗传变异的亚克隆的 ctDNA 释放效率。
我们收集了 40 对来自晚期肺癌患者的配对活检和血浆样本，并应用我们的 LungPlasma panel 进行基于捕获的测序，该 panel 由 168 个基因的关键外显子和内含子组成。我们得出了一个标准化相对等位基因分数（NRAFS）来反映 ctDNA 释放效率。
我们将活检样本中检测到的突变作为参考，实现了 87.2%的变异体检测灵敏度，包括单核苷酸变异、插入或缺失和基因融合。此外，七个最关键和可操作基因的变异体检测灵敏度为 96.2%。携带七个可操作基因突变的亚克隆的平均 NRAFS 为 0.877；相比之下，其他突变的平均 NRAFS 为 0.658。涉及细胞周期途径的四个基因的突变具有特别低的 NRAFS（0.480），与其他两组相比（p=0.07）。
我们已经证明携带驱动突变的亚克隆更倾向于释放 DNA。我们还证明了基于捕获的测序的定量能力，为其在临床环境中的常规应用铺平了道路。
