Liquid Biopsy for EGFR Mutation Detection in NSCLC: Evaluation of Plasma ctDNA and Comparison with Plasma exoDNA

Authors
Parisa Mashayekhi* 1
Mir Davood Omrani* 2
Ali Dehghanifard 1
Adnan Khosravi 3
Mohammad Mehdi Jahani 2
Negin Khosravi 4
1 Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
2 Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Research Center of Thoracic Oncology (RCTO), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Molecular Genetics, Ahar Branch, Islamic Azad University, Ahar, Iran
10.66224/ibj.5018
Abstract
Background: Accurate detection of actionable epidermal growth factor receptor ( EGFR) mutations is essential for guiding targeted therapy in non-small cell lung cancer (NSCLC). Liquid biopsy approaches using circulating tumor DNA (ctDNA) and exosomal DNA (exoDNA) offer noninvasive alternatives for molecular profiling. This study evaluated the diagnostic performance of nested PCR combined with sanger sequencing for detecting common EGFR mutations (exon 19 deletions and the L858R point mutation) in plasma samples from Iranian NSCLC patients.

Methods: In this retrospective observational study, blood samples were collected from 30 NSCLC patients with confirmed EGFR mutations. ctDNA was extracted from plasma and analyzed using nested PCR followed by sanger sequencing. Specificity was assessed in 20 EGFR–wild‑type NSCLC patients serving as controls. Diagnostic performance was further evaluated in relation to clinicopathological factors.

Results: EGFR mutations were detected in plasma ctDNA in 63.3% of patients. Detection sensitivity was significantly associated with tumor stage but was independent of mutation subtype, age, sex, or smoking status. The assay showed high specificity, with no false‑positive results in control samples (95% CI: 83.9–100.0%). Although exoDNA analysis demonstrated a higher sensitivity than ctDNA (76.6% vs. 63.3%), this difference was not statistically significant. Notably, the combined analysis of ctDNA and exoDNA increased overall detection sensitivity to 80%.

Conclusions: Nested PCR with sanger sequencing represents a reliable rule‑in strategy for EGFR mutation detection in plasma. Integrating ctDNA and exoDNA analyses substantially improves sensitivity and may enhance noninvasive molecular diagnostics in NSCLC.
Keywords
Exome sequencing
FKBP10 protein
Frameshift mutation
Osteogenesis imperfecta