Iranian
Biomedical Journal
Volume 29, Issue 6 (11-2025)
IBJ 2025, 29(6): 405-412 |
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Mashayekhi P, Omrani* M D, Dehghanifard A, Khosravi A, Jahani M M, Khosravi N. Liquid Biopsy for EGFR Mutation Detection in NSCLC: Evaluation of Plasma ctDNA and Comparison with Plasma exoDNA. IBJ 2025; 29 (6) :405-412
URL: http://ibj.pasteur.ac.ir/article-1-4700-en.html
URL: http://ibj.pasteur.ac.ir/article-1-4700-en.html
Parisa Mashayekhi * 
, Mir Davood Omrani* , Ali Dehghanifard , Adnan Khosravi , Mohammad Mehdi Jahani , Negin Khosravi
, Mir Davood Omrani* , Ali Dehghanifard , Adnan Khosravi , Mohammad Mehdi Jahani , Negin Khosravi
Abstract:
Background: Accurate detection of actionable EGFR mutations is essential for guiding targeted therapy in NSCLC. Liquid biopsy approaches using ctDNA and 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, 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.
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, 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.
Type of Study: Full Length/Original Article |
Subject:
Cancer Biology
References
1. Kahlert C. Liquid biopsy: Is there an advantage to analyzing circulating exosomal DNA compared to cfDNA or are they the same? Cancer Res. 2019;79(10):2462-5. [DOI:10.1158/0008-5472.CAN-19-0019]
2. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer F O, Hesch R-D, et al. DNA fragments in the blood plasma of cancer patients: Quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001;61(4):1659-65.
3. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early-and late-stage human malignancies. Sci Transl Med. 2014;6(224):224. [DOI:10.1126/scitranslmed.3007094]
4. Gorgannezhad L, Umer M, Islam MN, et al. Circulating tumor DNA and liquid biopsy: Opportunities, challenges, and recent advances in detection technologies. Lab Chip. 2018;18(8):1174-96. [DOI:10.1039/C8LC00100F]
5. Makler A, Asghar W. Exosomal biomarkers for cancer diagnosis and patient monitoring. Expert Rev Mol Diagn. 2020;20(4):387-400. [DOI:10.1080/14737159.2020.1731308]
6. Sharma A, Johnson A. Exosome DNA: Critical regulator of tumor immunity and a diagnostic biomarker. J Cell Physiol. 2020;235(3):1921-32. [DOI:10.1002/jcp.29153]
7. Bamodu OA, Chung C-C, Pisanic TR. Harnessing liquid biopsies: Exosomes and ctDNA as minimally invasive biomarkers for precision cancer medicine. J Liq Biopsy. 2023:2:100126. [DOI:10.1016/j.jlb.2023.100126]
8. Franczak C, Filhine-Tresarrieu P, Gilson P, Merlin J-L, Au L, Harle A. Technical considerations for circulating tumor DNA detection in oncology. Expert Rev Mol Diagn. 2019;19(2):121-35. [DOI:10.1080/14737159.2019.1568873]
9. Jahani MM, Mashayekhi P, Omrani MD, Khosravi A, Dehghanifard A, Azad Manjiri S, et al. Assessing the sensitivity of nested PCR followed by direct sequencing on exosomal DNA for EGFR mutation detection in NSCLC. Iran Biomed J. 2024;28(4):208-15. [DOI:10.61186/ibj.4289]
10. Noor J, Chaudhry A, Noor R, Batool S. Advancements and applications of liquid biopsies in oncology: A narrative review. Cureus. 2023;15(7):42731. [DOI:10.7759/cureus.42731]
11. de Melo AMD, da Silva Filho MS, Alves BT, Aguilera KC, Correia Alencar AM, Carneiro de Andrade AML, et al. The innovations, advances and outlooks of liquid biopsy in oncology: A literature review. Amadeus Int Multidiscip J. 2018;2(4):117-28. [DOI:10.14295/aimj.v2i4.38]
12. Raez LE, Brice K, Dumais K, Lopez-Cohen A, Wietecha D, Izquierdo PA, et al. Liquid biopsy versus tissue biopsy to determine front line therapy in metastatic non-small cell lung cancer (NSCLC). Clin Lung Cancer. 2023;24(2):120-9. [DOI:10.1016/j.cllc.2022.11.007]
13. Jung A, Kirchner T. Liquid biopsy in tumor genetic diagnosis. Dtsch Arztebl Int. 2018;115(10):169-174. [DOI:10.3238/arztebl.2018.0169]
14. Zhu G, Ye X, Dong Z, Lu YC, Sun Y, Liu Y, et al. Highly sensitive droplet digital PCR method for detection of EGFR-activating mutations in plasma cell-free DNA from patients with advanced non-small cell lung cancer. J Mol Diagn. 2015;17(3):265-72. [DOI:10.1016/j.jmoldx.2015.01.004]
15. Yao Y, Liu J, Li L, Yuan Y, Nan K, Wu X, et al. Detection of circulating tumor DNA in patients with advanced non-small cell lung cancer. Oncotarget. 2016;8(2):2130-40. [DOI:10.18632/oncotarget.12883]
16. Ohira T, Sakai K, Matsubayashi J, Kajiwara N, Kakihana M, Hagiwara M, et al. Tumor volume determines the feasibility of cell-free DNA sequencing for mutation detection in non-small cell lung cancer. Cancer Sci.2016;107(11):1660-66. [DOI:10.1111/cas.13068]
17. Wang H, Zhang Y, Zhang H, Cao H, Mao J, Chen X, et al. Liquid biopsy for human cancer: Cancer screening, monitoring, and treatment. MedComm .2024;5(6):564. [DOI:10.1002/mco2.564]
18. Shegekar T, Vodithala S, Juganavar A. The emerging role of liquid biopsies in revolutionising cancer diagnosis and therapy. Cureus .2023;15(8):43650. [DOI:10.7759/cureus.43650]
19. Tran VT, Phan TT, Nguyen ST, Tran B-T, Ho TT, Pho SP, et al. Smoking habit and chemo-radiotherapy and/or surgery affect the sensitivity of EGFR plasma test in non-small cell lung cancer. BMC Res Notes. 2020;13(1):367. [DOI:10.1186/s13104-020-05209-9]
20. Guo K, Zhang Z, Han L, Han J, Wang J, Zhou Y, et al. Detection of epidermal growth factor receptor mutation in plasma as a biomarker in Chinese patients with early-stage non-small cell lung cancer. Onco Targets Ther. 2015:8:3289-96. [DOI:10.2147/OTT.S94297]
21. Ianza A, Di Chicco A, Biagi C, Giudici F, Dicorato A, Guglielmi A, et al. EGFR mutation analysis on circulating free DNA in NSCLC: A single-center experience. J Cancer Res Clin Oncol. 2021;147(8):2301-7. [DOI:10.1007/s00432-021-03658-8]
22. Luo J, Shen L, Zheng D. Diagnostic value of circulating free DNA for the detection of EGFR mutation status in NSCLC: A systematic review and meta-analysis. Sci Rep. 2014;4(1):6269. [DOI:10.1038/srep06269]
23. Ni J, Weng L, Liu Y, Sun Z, Bai C, Wang Y. Dynamic monitoring of EGFR mutations in circulating cell-free DNA for EGFR-mutant metastatic patients with lung cancer: Early detection of drug resistance and prognostic significance. Oncol Lett .2017;13(6):4549-57. [DOI:10.3892/ol.2017.6022]
24. Kim Y, Shin S, Kim B, Lee K-A. Selecting short length nucleic acids localized in exosomes improves plasma EGFR mutation detection in NSCLC patients. Cancer Cell Int .2019:19:251. [DOI:10.1186/s12935-019-0978-8]
25. Wan Y, Liu B, Lei H, Zhang B, Wang Y, Huang H, et al. Nanoscale extracellular vesicle-derived DNA is superior to circulating cell-free DNA for mutation detection in early-stage non-small-cell lung cancer. Ann Oncol .2018;29(12):2379-83. [DOI:10.1093/annonc/mdy458]
26. Castellanos-Rizaldos E, Zhang X, Tadigotla VR, Grimm DG, Karlovich C, Raez LE, et al. Exosome-based detection of activating and resistance EGFR mutations from plasma of non-small cell lung cancer patients. Oncotarget. 2019;10(30):2911-20. [DOI:10.18632/oncotarget.26885]
27. Krug A, Enderle D, Karlovich C, Priewasser T, Bentink S, Spiel A, et al. Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma. Ann Oncol. 2018;29(3):700-6. [DOI:10.1093/annonc/mdx765]
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