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Updated: Mar 23, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing
Ali Saber1, Anthonie J van der Wekken2, Klaas Kok3
1Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands.
Abstract:
ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.
Insights
Resistance to crizotinib in ALK-positive non-small cell lung cancer is common. New ALK mutations, not fusion genes, were identified as the likely cause of resistance in this study.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Anaplastic Lymphoma Kinase (ALK)-positive non-small cell lung cancer (NSCLC) patients initially benefit from crizotinib treatment.
- Acquired resistance to crizotinib is a significant clinical challenge, necessitating further investigation into its underlying mechanisms.
Purpose of the Study:
- To identify novel fusion genes in tumor samples from patients who developed resistance to crizotinib.
- To investigate the role of these fusion genes and known resistance-associated mutations in acquired crizotinib resistance.
Main Methods:
- Paired-end RNA sequencing was performed on re-biopsied tumor samples from three crizotinib-resistant NSCLC patients.
- Bioinformatic tools (deFuse, EricScript) were used for fusion gene identification, and the IGV browser for mutation analysis.
- Sanger sequencing and digital droplet PCR were employed for validation of identified fusion genes and mutations.
Main Results:
- ALK fusion genes, with EML4 as the common partner, were detected in all three patients' post-treatment samples and were also present in their pre-treatment biopsies.
- Additional fusion genes were identified in two patients, but only CLIP4-VSNL1 possessed a predicted open reading frame (ORF).
- Acquired ALK mutations (p.C1156Y and p.G1269A) were detected in two patients' post-treatment samples but were absent in their pre-treatment biopsies.
Conclusions:
- The identified fusion genes are unlikely to be the primary drivers of crizotinib resistance, as they were present before treatment initiation.
- Acquired mutations in ALK are strongly implicated as the mechanism conferring resistance to crizotinib in NSCLC patients.
