基于寡核糖核苷酸干扰PCR（ORNi-PCR）方法的高敏感性与高准确性ALK-TKI耐药突变检测技术

Highly sensitive and accurate detection of ALK-TKI resistance mutations by oligoribonucleotide interference-PCR (ORNi-PCR)-based methods

LUNG CANCER

影响因子:4.4

分区:医学2区 / 肿瘤学3区呼吸系统3区

发表日期:2024 Nov

作者: Chiori Tabe, Toshitsugu Fujita, Kageaki Taima, Hisashi Tanaka, Tomonori Makiguchi, Masamichi Itoga, Yoshiko Ishioka, Sadatomo Tasaka, Hodaka Fujii

DOI: 10.1016/j.lungcan.2024.107969

摘要

间变性淋巴瘤激酶（ALK）阳性非小细胞肺癌（NSCLC）患者通常接受ALK酪氨酸激酶抑制剂（TKIs）治疗。尽管大多数患者从ALK-TKIs中获益，但耐药突变的发生很常见，导致NSCLC复发。为了在早期复发阶段识别ALK-TKI耐药NSCLC，开发高敏感性和高准确性、成本效益高、临床实用的突变检测方法尤为关键。本研究旨在建立一种针对两种常见ALK-TKI耐药突变（ALK G1202R和L1196M）的高敏感性、准确性、经济实用的液体活检检测方法。首先优化实验条件，以特异性扩增对应G1202R和L1196M突变的ALK-TKI耐药突变DNA。随后，将ORNi-PCR与数字滴度PCR（ddPCR）或实时PCR结合，用于检测从NSCLC患者血浆中提取的游离DNA（cfDNA）中的突变。结果显示，ORNi-PCR结合ddPCR/实时PCR能够检测到模型cfDNA中1-10拷贝的G1202R和L1196M突变DNA。临床患者的cfDNA中，这些突变通过ORNi-PCR方法得以检测，而传统的ddPCR未能检测到。采用ORNi-PCR结合ddPCR/实时PCR的方法，实现了对ALK突变的高敏感性和高准确性的液体活检检测。虽然临床数据有限，但我们的结果表明这些方法在早期复发阶段识别ALK-TKIs耐药NSCLC方面具有潜在应用价值。

Abstract

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.