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

BACKGROUND

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.

OBJECTIVE

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

Informations de copyright

Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [T.F. and H.F. hold the following patents and patent applications for ORNi-PCR: (1) name: Method for suppressing amplification of specific nucleic acid sequences; patent numbers: Japan 6,928,323 and 6,964,900; (2) name: Method for detecting differences in target nucleic acid region; patent publication number: WO 2019/203350; patent numbers: Japan 6,653,932, Canada 3,096,462, Korea 10-2,285,085, Israel 277,764, the US 11,155,873, and Singapore 11,202,009,878 V. Wisteriagen, LLC. owns the rights to the commercial use of ORNi-PCR. T.F. and H.F. are founders and directors of Wisteriagen, LLC. and own equity in the company].