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Updated: May 29, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Direct sequencing in cytological specimens as a useful strategy for detecting EGFR mutations in non-small cell lung
Min-Chul Cho1, Chang-Min Choi, Sollip Kim
1Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Reblic of Korea.
Background:
New therapeutics targeting epidermal growth factor receptor (EGFR) have significantly improved tumor responses to therapy in non-small cell lung cancer (NSCLC) patients. Molecular testing for EGFR mutations informs important therapeutic decisions in clinical practice. In this study, we sought to validate the clinical relevance of sequencing-based EGFR mutation testing combined with cytological analysis using body fluid specimens.
Methods:
Two NSCLC cell lines were used in sensitivity analyses. In addition, we performed cytological analyses and directly sequencing of exons 18-21, for 32 specimens. The absence of EGFR mutations determined by direct sequencing in 14 specimens was confirmed by real-time PCR. Changes made to patients' therapeutic strategies after reports of EGFR mutation status were investigated by querying electronic medical records.
Results:
Sensitivity studies showed that detection of in-frame deletions in exon 19 and point mutations in exon 21 was possible in specimens containing 10% and 5% mutant DNA, respectively. In clinical practice, EGFR mutations were detected in 18 of 32 specimens (56.3%). Twelve patients with EGFR mutations detected by direct sequencing were started on treatment with EGFR tyrosine kinase inhibitor (TKI) after reports of EGFR mutation. EGFR-TKI therapy was discontinued for two patients with TKI-resistant T790M mutation. The results of real-time PCR were consistent with those of direct sequencing in 13 of 14 specimens (92.9%) in which no mutation was detected by direct sequencing.
Conclusions:
Combined direct sequencing and cytological analysis of body fluid specimen might be clinically useful and sensitive test for the detection of EGFR mutations in NSCLC patients.
Insights
Direct sequencing combined with cytology effectively detects epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) body fluid specimens. This approach aids therapeutic decisions for NSCLC patients receiving targeted therapies.
Area of Science:
- Oncology
- Molecular Diagnostics
Background:
- Targeted therapies for epidermal growth factor receptor (EGFR) mutations have improved outcomes in non-small cell lung cancer (NSCLC).
- Accurate EGFR mutation testing is crucial for guiding clinical treatment decisions in NSCLC patients.
Purpose of the Study:
- To validate the clinical utility and sensitivity of sequencing-based EGFR mutation testing.
- To assess the combined approach of direct sequencing and cytological analysis on body fluid specimens for EGFR mutation detection.
Main Methods:
- Sensitivity analyses were performed on NSCLC cell lines.
- Direct sequencing of EGFR exons 18-21 and cytological analysis were conducted on 32 clinical specimens.
- Real-time PCR confirmed negative results from direct sequencing, and electronic medical records tracked therapeutic strategy changes.
Main Results:
- Direct sequencing detected EGFR mutations in 56.3% (18/32) of specimens, with high sensitivity for exon 19 deletions and exon 21 mutations.
- Twelve patients initiated EGFR tyrosine kinase inhibitor (TKI) therapy based on positive mutation results.
- Real-time PCR results were consistent with direct sequencing in 92.9% of mutation-negative cases.
Conclusions:
- The combination of direct sequencing and cytological analysis of body fluid specimens offers a sensitive and clinically relevant method for detecting EGFR mutations in NSCLC.
- This approach supports informed therapeutic strategies for NSCLC patients, including the use of EGFR-TKIs.
