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

Lung Cancer. 2024 Nov:197:107969. doi: 10.1016/j.lungcan.2024.107969. Epub 2024 Sep 27.

Abstract

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.

Keywords: ALK; Gene mutation; NSCLC; ORNi-PCR; Real-time PCR; ddPCR.

MeSH terms

  • Aged
  • Anaplastic Lymphoma Kinase* / antagonists & inhibitors
  • Anaplastic Lymphoma Kinase* / genetics
  • Carcinoma, Non-Small-Cell Lung* / drug therapy
  • Carcinoma, Non-Small-Cell Lung* / genetics
  • Cell-Free Nucleic Acids / genetics
  • Drug Resistance, Neoplasm* / genetics
  • Female
  • Humans
  • Liquid Biopsy / methods
  • Lung Neoplasms* / diagnosis
  • Lung Neoplasms* / drug therapy
  • Lung Neoplasms* / genetics
  • Lung Neoplasms* / pathology
  • Male
  • Middle Aged
  • Mutation*
  • Polymerase Chain Reaction / methods
  • Protein Kinase Inhibitors* / pharmacology
  • Protein Kinase Inhibitors* / therapeutic use
  • Sensitivity and Specificity

Substances

  • Anaplastic Lymphoma Kinase
  • Protein Kinase Inhibitors
  • ALK protein, human
  • Cell-Free Nucleic Acids