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

• Published in: Lung cancer (Amsterdam, Netherlands) Vol. 197; p. 107969
• Main Authors: Tabe, Chiori, Fujita, Toshitsugu, Taima, Kageaki, Tanaka, Hisashi, Makiguchi, Tomonori, Itoga, Masamichi, Ishioka, Yoshiko, Tasaka, Sadatomo, Fujii, Hodaka
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 01.11.2024
• Subjects: ALK; ddPCR; Gene mutation; NSCLC; ORNi-PCR; Real-time PCR; ddPCR; ALK; ORNi-PCR; Real-time PCR; NSCLC; Gene mutation
• ISSN: 0169-5002; 1872-8332; 1872-8332
• DOI: 10.1016/j.lungcan.2024.107969

[Display omitted] •We established ORNi-PCR-based methods to detect ALK-TKI resistance mutations.•ORNi-PCR-based methods but not conventional ddPCR detected the mutations.•ORNi-PCR-based methods enable highly sensitive and accurate mutation detection.•Early recurrent ALK-TKI-resistant NSCLC can be identified by ORNi-PCR-based methods. 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.