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Updated: Jul 15, 2026
Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Enhanced detection of actionable mutations in NSCLC through pleural effusion cell-free DNA sequencing: A prospective
Hsin-Yi Wang1, Wei-Yu Liao2, Chao-Chi Ho2
1Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, National Taiwan University Hospital Yunlin Branch, No. 579, Section 2, Yunlin Rd., Yunlin County, Douliu City 640, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, No. 1, Section 4, Roosevelt Rd., Taipei, Taiwan.
Background:
Inadequate tumour samples often hinder molecular testing in non-small cell lung cancer (NSCLC). Plasma-based cell-free DNA (cfDNA) sequencing has shown promise in bypassing these tissue limitations. Nevertheless, pleural effusion (PE) samples may offer a richer cfDNA source for mutation detection in patients with malignant PE.
Methods:
This prospective study enrolled newly diagnosed advanced NSCLC patients with malignant PE. PE samples were collected for cfDNA NGS analysis. Meanwhile, PE cell pellet RNA was extracted for reverse transcription polymerase chain reaction (RT-PCR) for clinically relevant actionable mutations and then confirmed by Sanger sequencing. The concordance between PE cell pellet RT-PCR and PE cfDNA NGS analyses was analysed.
Results:
Fifty patients were enrolled. The median age was 68.5 years, and the female-to-male ratio was 29:21. Most patients (74 %) were non-smokers. Notably, 45/50 patients (90 %) had actionable mutations, including EGFR exon 19 deletions (24 %), EGFR L858R mutations (36 %), HER2 exon20 insertions (10 %), ROS1 rearrangements (4 %), EGFR exon20 insertions (2 %), ALK rearrangements (4 %), RET rearrangements (2 %), KRAS G12C mutations (2 %), and CD74-NRG1 fusions (2 %). Among the 50 enrolled patients, actionable mutations were detected in 44 (88 %) by PE cfDNA NGS, 39 (78 %) by PE cell pellet Sanger sequencing, and 33 (66 %) by clinical tissue genetic testing (P = 0.031). The detection of actionable mutations from PE cfDNA NGS remained consistently high across M1a to M1c stages.
Conclusions:
PE cfDNA genotyping has clinical applicability for NSCLC patients and can serve as an additional source for molecular testing. Incorporating PE NGS cfDNA analysis into genetic testing enhances diagnostic yield and aids in identifying actionable mutations in clinical practice.
