Department of Respiratory Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562 Aomori, Japan.
Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562 Aomori, Japan.
Lung Cancer. 2024 Nov;197:107969. doi: 10.1016/j.lungcan.2024.107969. Epub 2024 Sep 27.
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.
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。虽然大多数患者从 ALK-TKI 中获益，但耐药突变的发展很常见，导致 NSCLC 复发。为了在早期复发阶段识别 ALK-TKI 耐药的 NSCLC，需要高度敏感和准确的检测突变的方法。
本研究旨在通过液体活检建立高度敏感、准确、具有成本效益且临床实用的检测两种常见 ALK-TKI 耐药突变（ALK G1202R 和 L1196M）的方法。
首先通过优化实验条件，特异性扩增与 ALK G1202R 和 L1196M 突变对应的 ALK-TKI 耐药突变 DNA，来检验寡核苷酸干扰-PCR（ORNi-PCR）的疗效。然后将 ORNi-PCR 与液滴数字 PCR（ddPCR）或实时 PCR 结合，检测 NSCLC 患者提取的循环游离 DNA（cfDNA）中的这些突变。
ORNi-PCR 后采用 ddPCR/实时 PCR 可检测到模型 cfDNA 中 1-10 个拷贝的 G1202R 和 L1196M DNA。通过基于 ORNi-PCR 的方法可在患者 cfDNA 中检测到这些突变，而传统的 ddPCR 则无法检测到。
ORNi-PCR 后采用 ddPCR/实时 PCR 可通过液体活检实现 ALK 突变的高度敏感和准确检测。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在早期复发阶段识别 ALK-TKI 耐药的 NSCLC。
