间变性淋巴瘤激酶 (ALK) 阳性非小细胞肺癌 (NSCLC) 患者接受 ALK 酪氨酸激酶抑制剂 (TKI) 治疗。尽管大多数患者受益于 ALK-TKI，但耐药突变的发生很常见并导致 NSCLC 复发。为了在早期复发阶段识别 ALK-TKI 耐药的 NSCLC，高灵敏度和准确的突变检测方法至关重要。本研究的目的是建立高灵敏度、准确、经济有效且临床实用的突变检测方法。通过液体活检检测两种常见的 ALK-TKI 耐药突变 ALK G1202R 和 L1196M。通过首先优化实验条件以特异性扩增与ALK G1202R 和 L1196M 突变。然后将 ORNi-PCR 与微滴式数字 PCR (ddPCR) 或实时 PCR 相结合，检测从 NSCLC 患者中提取的游离 DNA (cfDNA) 中的这些突变。ORNi-PCR 随后进行 ddPCR/实时 PCR 检测到 1-10模型 cfDNA 中 G1202R 和 L1196M DNA 的副本。使用基于 ORNi-PCR 的方法鉴定了患者 cfDNA 中的这些突变，而传统的 ddPCR 无法检测到它们。 ORNi-PCR 后进行 ddPCR/实时 PCR 可以通过液体活检高度灵敏且准确地检测 ALK 突变。尽管临床数据有限，但我们的结果表明这些方法可能有助于识别早期复发期的 ALK-TKI 耐药 NSCLC。版权所有 © 2024 作者。由 Elsevier B.V. 出版。保留所有权利。

Patients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) are treated with ALK tyrosine kinase inhibitors (TKIs). Although most patients benefit from ALK-TKIs, the development of resistance mutations is common and results in NSCLC recurrence. To identify ALK-TKI-resistant NSCLC at the early recurrent phase, highly sensitive and accurate methods for the detection of mutations are essential.The aim of this study was to establish highly sensitive, accurate, cost-effective, and clinically practical methods for the detection of two frequent ALK-TKI resistance mutations, ALK G1202R and L1196M, by liquid biopsy.The efficacy of oligoribonucleotide interference-PCR (ORNi-PCR) was examined by first optimizing experimental conditions to specifically amplify the ALK-TKI resistance mutant DNA corresponding to ALK G1202R and L1196M mutations. ORNi-PCR was then combined with droplet digital PCR (ddPCR) or real-time PCR to detect these mutations in cell-free DNA (cfDNA) extracted from NSCLC patients.ORNi-PCR followed by ddPCR/real-time PCR detected 1-10 copy(s) of G1202R and L1196M DNA in model cfDNA. These mutations in patients' cfDNA were identified using ORNi-PCR-based methods, whereas conventional ddPCR failed to detect them.ORNi-PCR followed by ddPCR/real-time PCR enables highly sensitive and accurate detection of ALK mutations by liquid biopsy. Although the clinical data are limited, our results show that these methods are potentially useful for identifying ALK-TKI-resistant NSCLC at the early recurrent phase.Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.