Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer
Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer
复制标题
DOI：
10.1093/annonc/mdw037
复制
发表时间：
2016-05-01
影响因子：
50.5
通讯作者：
Swanton, C.
中科院分区：
文献类型：
--
作者：
Jamal-Hanjani, M.;Wilson, G. A.;Swanton, C.
在早期NSCLCs的这项初步研究中，在肿瘤切除前，在来自患者的cfDNA中鉴定了普遍存在的和异质的变体。这种非侵入性方法可以揭示肿瘤的潜在基因组景观，本初步研究的目的是评估早期非小细胞肺癌（NSCLC）患者的无细胞DNA（cfDNA）中是否可以检测到普遍存在和异质性体细胞突变对三个I期和一个II期原发性NSCLC肿瘤进行多区域全外显子组测序（WES）并用AmpliSeq验证。选择普遍存在的和异质的单核苷酸变体（SNV）的子集。使用与下一代测序（mPCR-NGS）偶联的使用定制设计的引物的多重PCR来检测肿瘤DNA和从手术切除肿瘤之前获得的血浆分离的cfDNA两者中的这些SNV。使用来自48名假定正常的健康志愿者的cfDNA确定每个测定的检测限。使用mPCR-NGS方法成功扩增肿瘤DNA和血浆来源的cfDNA，并对37/50（74%）的SNV进行测序。25个（68%）是普遍存在的，12个（32%）是异质性SNV。肿瘤组织中mPCR-NGS和WES-AmpliSeq的变异检测具有良好的相关性（R-2 = 0.8722，P < 0.0001）。在cfDNA中检测到37个SNV中的16个（43%）。其中12个是普遍存在的SNV，变异等位基因频率（VAF）范围为0.15- 23.25%，其中4个是异质SNV，VAF范围为0.28- 1.71%。肿瘤的VAF与cfDNA之间存在统计学显著的线性关系（R-2 = 0.5144; P = 0.0018）。对于所有四名患者，在血浆中检测到至少两种变体。每个样品中存在的变异DNA的估计拷贝数范围为5至524。检测所需的变异拷贝数（VCRD）的平均值为3.16（范围：0.2-7.6拷贝）JPCR-NGS方法揭示了早期NSCLC肿瘤中的肿瘤内异质性，并且能够检测cfDNA中普遍存在的和异质性的SNV。需要进一步验证cfDNA中的mPCR-NGS，以确定其在临床实践中的潜在用途。
In this pilot study of early-stage NSCLCs, both ubiquitous and heterogeneous variants were identified in cfDNA from patients prior to tumor resection. Such non-invasive methods can reveal the underlying genomic landscape of tumors, and help determine their clonal evolution throughout the disease course with potential implications for clinical practice.The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC).Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers.Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R-2 = 0.8722, P < 0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R-2 = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies).The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.