Takano Shinichi, Fukasawa Mitsuharu, Shindo Hiroko, Takahashi Ei, Fukasawa Yoshimitsu, Kawakami Satoshi, Hayakawa Hiroshi, Kuratomi Natsuhiko, Kadokura Makoto, Maekawa Shinya, Enomoto Nobuyuki
First Department of Internal Medicine, Faculty of Medicine University of Yamanashi Chuo Japan.
JGH Open. 2021 Mar 22;5(4):508-516. doi: 10.1002/jgh3.12530. eCollection 2021 Apr.
The clinical applicability of digital next-generation sequencing (dNGS), which eliminates polymerase chain reaction (PCR) and sequencing error-derived noise by using molecular barcodes (MBs), has not been fully evaluated. We evaluated the utility of dNGS of cell-free DNA (cfDNA) in liquid biopsies obtained from patients with pancreatic cancer.
Fifty-eight patients with pancreatic cancer undergoing endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) were included. Samples were subjected to sequencing of 50 cancer-related genes using next-generation sequencing (NGS). The results were used as reference gene alterations. NGS of cfDNA from plasma was performed for patients with a mutant allele frequency (MAF) >1% and an absolute mutant number > 10 copies/plasma mL in or by digital PCR. Sequence readings with and without MBs were compared with reference to EUS-FNA-derived gene alterations.
The concordance rate between dNGS of cfDNA and EUS-FNA-derived gene alterations was higher with than without MBs ( = 0.039), and MAF cut-off values in dNGS could be decreased to 0.2%. dNGS using MBs eliminated PCR and sequencing error by 74% and 68% for and all genes, respectively. Overall, dNGS detected mutations in (45%) and (26%) and copy number alterations in , , , , and , which are targets of molecular-targeted drugs.
dNGS of cfDNA using MBs is useful for accurate detection of gene alterations even with low levels of MAFs. These results may be used to inform the development of diagnostics and therapeutics that can improve the prognosis of pancreatic cancer.
数字下一代测序（dNGS）通过使用分子条形码（MB）消除了聚合酶链反应（PCR）和测序错误衍生的噪声，其临床适用性尚未得到充分评估。我们评估了dNGS在胰腺癌患者液体活检中对游离DNA（cfDNA）的应用价值。
纳入58例接受内镜超声引导下细针穿刺活检（EUS-FNA）的胰腺癌患者。样本采用下一代测序（NGS）对50个癌症相关基因进行测序。结果用作参考基因改变。对血浆cfDNA进行NGS检测，筛选出突变等位基因频率（MAF）>1%且绝对突变数>10拷贝/血浆毫升的患者，通过数字PCR进一步验证。将有无MB的测序读数与EUS-FNA衍生的基因改变进行比较。
cfDNA的dNGS与EUS-FNA衍生的基因改变之间的一致性率，有MB时高于无MB时（P = 0.039），dNGS中的MAF临界值可降至0.2%。使用MB的dNGS分别将 和所有基因的PCR和测序错误消除了74%和68%。总体而言，dNGS检测到 （45%）和 （26%）中的突变，以及 、 、 、 和 中的拷贝数改变，这些都是分子靶向药物的靶点。
使用MB的cfDNA的dNGS即使在MAF水平较低时也有助于准确检测基因改变。这些结果可用于指导诊断和治疗方法的开发，以改善胰腺癌的预后。
