基于杂交技术平台的癌症患者液体活检中临床相关突变的多重检测。

Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform.

机构信息

Pangaea Oncology, Laboratory of Oncology, Quirón Dexeus University Hospital, Barcelona, Spain.

Universitat Autónoma de Barcelona, Barcelona, Spain.

出版信息

Clin Chem. 2021 Mar 1;67(3):554-563. doi: 10.1093/clinchem/hvaa248.

DOI:10.1093/clinchem/hvaa248

PMID:33439966

Abstract

BACKGROUND

With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids.

METHODS

Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes.

RESULTS

Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease.

CONCLUSIONS

The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

摘要

背景

随着精准肿瘤学的出现，液体活检在临床环境中迅速得到认可。然而，在某些情况下，分离出的 DNA 量不足以进行下一代测序（NGS）分析。nCounter 平台可能是一种替代方法，但它从未在液体中检测临床相关改变方面进行过探索。

方法

从癌症患者的血液、脑脊液和腹水中纯化循环游离 DNA（cfDNA），并使用 nCounter 3D 单核苷酸变异（SNV）固体肿瘤面板进行分析，该面板允许检测 24 个基因中的 97 个驱动突变。

结果

验证实验表明，nCounter SNV 面板可以在突变等位基因分数为 0.02-2%的样本中检测到突变，而这些样本中突变 DNA/µL 的浓度≥5pg。在对 70 例癌症患者 cfDNA 的回顾性分析中，与同一液体活检和配对肿瘤组织中的先前基因分型相比，该面板成功检测到 EGFR、KRAS、BRAF、PIK3CA 和 NRAS 突变[Cohen kappa 分别为 0.96（CI=0.92-1.00）和 0.90（CI=0.74-1.00）]。在一项包括 91 例不同恶性肿瘤患者液体活检的前瞻性研究中，90 例液体活检结果有效，在 SNV 面板中检测到 EGFR、KRAS、BRAF、PIK3CA、TP53、NFE2L2、CTNNB1、ALK、FBXW7 和 PTEN 突变。最后，一名 NSCLC 患者的连续液体活检显示，SNV 面板突变分析的半定量结果与疾病的演变相关。