基于寡核核苷酸干扰PCR（ORNI-PCR）方法对ALK-TKI抗性突变高度敏感，准确地检测
Highly sensitive and accurate detection of ALK-TKI resistance mutations by oligoribonucleotide interference-PCR (ORNi-PCR)-based methods
影响因子:4.40000
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
摘要
使用ALK酪氨酸激酶抑制剂（TKIS）治疗，患有变性淋巴瘤激酶（ALK）阳性非小细胞肺癌（NSCLC）的患者。尽管大多数患者受益于ALK-TKIS，但耐药突变的发展很常见，导致NSCLC复发。要在早期复发阶段识别耐ALK-TKI的NSCLC，对于检测突变的高度敏感和准确的方法至关重要。这项研究的目的是建立高度敏感，准确，成本效益和临床实用的方法，以检测两个频繁的ALK-TKI抗性突变，ALK G1202R和LIFISIS BILISICE，ALK G1202R和LIFISE L1196M，ALK G1202R和LIFISE;通过首先优化实验条件以特异性扩增与ALK G1202R和L1196M突变相对应的ALK-TKI抗性突变体DNA，从而检查了寡核苷酸干扰PCR（ORNI-PCR）。然后将ORNI-PCR与液滴数字PCR（DDPCR）或实时PCR结合使用，以检测从NSCLC患者中提取的无细胞DNA（CFDNA）中的这些突变。ERNI-PCR.ORNI-PCR，然后在DDPCR/实时PCR中检测到1-10拷贝在G1202R和L1196M DNA中，在模型CFDNA中均采用了1-10拷贝。使用基于ORNI-PCR的方法鉴定出患者CFDNA中的这些突变，而常规DDPCR未能检测到它们。NORNI-PCR，然后是DDPCR/实时PCR，可以通过液体活检高度敏感，准确地检测ALK突变。尽管临床数据受到限制，但我们的结果表明，这些方法可能在早期复发阶段识别耐ALK-TKI-TKI耐药的NSCLC有用。
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.