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Updated: Jan 28, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Application of amplicon-based targeted sequencing with the molecular barcoding system to detect uncommon minor EGFR
Kei Namba1, Shuta Tomida2, Takehiro Matsubara3
1Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama City, 700-8558, Japan.
Background:
In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations.
Methods:
We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh-frozen lung cancer samples.
Results:
Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018).
Conclusions:
Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities.
Insights
Molecular barcoding with high-throughput sequencing accurately identifies epidermal growth factor receptor (EGFR) compound mutations in non-small cell lung cancer (NSCLC). This method improves genomic heterogeneity analysis and aids in understanding resistance mechanisms for developing new treatments.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Compound mutations in EGFR, involving sensitizing and minor mutations, are crucial in non-small cell lung cancer (NSCLC) development and acquired resistance to tyrosine kinase inhibitors.
- Accurate determination of EGFR mutation status is vital for effective patient treatment and management.
- Traditional sequencing methods can be prone to errors, necessitating improved techniques for precise mutation detection.
Purpose of the Study:
- To assess the utility of molecular barcoding combined with high-throughput sequencing for investigating genomic heterogeneity in treatment-naïve NSCLC patients with EGFR mutations.
- To improve the accuracy of detecting both common and rare EGFR mutations, including compound mutations.
Main Methods:
- Amplicon-based targeted sequencing utilizing a molecular barcoding system (MBS) was employed.
- The study focused on detecting major common and uncommon minor EGFR mutations with an allele frequency as low as 0.5% in fresh-frozen NSCLC samples.
Main Results:
- The MBS demonstrated high concordance (98.4%) with clinical testing for common EGFR mutations.
- Uncommon EGFR mutations were identified in 10.9% of cases.
- Patients with the G719X mutation showed a significantly higher incidence of compound mutations compared to those with L858R or exon 19 deletion mutations, a finding validated in an independent cohort.
Conclusions:
- The molecular barcoding system (MBS) is a feasible and accurate method for establishing NSCLC patient genotypes.
- This approach enhances the understanding of the molecular underpinnings of EGFR compound mutations in NSCLC.
- The findings support the development of novel therapeutic strategies targeting EGFR mutations in lung cancer.
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