Extracellular Vesicle-Based Bronchoalveolar Lavage Fluid Liquid Biopsy for EGFR Mutation Testing in Advanced Non-Squamous NSCLC

In Ae Kim, Jae Young Hur, Hee Joung Kim, Wan Seop Kim, Kye Young Lee, In Ae Kim, Jae Young Hur, Hee Joung Kim, Wan Seop Kim, Kye Young Lee

Abstract

To overcome the limitations of the tissue biopsy and plasma cfDNA liquid biopsy, we performed the EV-based BALF liquid biopsy of 224 newly diagnosed stage III-IV NSCLC patients and compared it with tissue genotyping and 110 plasma liquid biopsies. Isolation of EVs from BALF was performed by ultracentrifugation. EGFR genotyping was performed through peptide nucleic acid clamping-assisted fluorescence melting curve analysis. Compared with tissue-based genotyping, BALF liquid biopsy demonstrated a sensitivity, specificity, and concordance rates of 97.8%, 96.9%, and 97.7%, respectively. The performance of BALF liquid biopsy was almost identical to that of standard tissue-based genotyping. In contrast, plasma cfDNA-based liquid biopsy (n = 110) demonstrated sensitivity, specificity, and concordance rates of 48.5%, 86.3%, and 63.6%, respectively. The mean turn-around time of BALF liquid biopsy was significantly shorter (2.6 days) than that of tissue-based genotyping (13.9 days; p < 0.001). Therefore, the use of EV-based BALF shortens the time for confirmation of EGFR mutation status for starting EGFR-TKI treatment and can hence potentially improve clinical outcomes. As a result, we suggest that EV-based BALF EGFR testing in advanced lung NSCLC is a highly accurate rapid method and can be used as an alternative method for lung tissue biopsy.

Keywords: EGFR mutation testing; NSCLC; bronchoalveolar lavage (BAL); extracellular vesicles; liquid biopsy.

Conflict of interest statement

K.Y.L. is a cofounder of and owns stock in Exosignal, Inc. (Seoul, Korea). All the other authors have no conflicts of interest to declare. The funder (Chong Kun Dang Pharmaceutical Corp) had no role in study design, data collection, analysis and interpretation, or the decision to submit the work for publication.

Figures

Figure 1
Figure 1
Concordance rate for EGFR genotyping among tissue biopsy, BALF liquid and plasma liquid biopsy (n = 110) (double colors mean double EGFR mutation).
Figure 2
Figure 2
Comparison of sensitivity between BALF and plasma liquid biopsy depending on metastatic tumor stage. * Indicates statistically significant difference (p < 0.05). (M0: no metastasis; M1a: intrathoracic metastasis; M1b: single extrathoracic metastasis; M1c: multiple extrathoracic metastasis).

References

    1. Mok T.S., Wu Y.-L., Thongprasert S., Yang C.-H., Chu D.-T., Saijo N., Sunpaweravong P., Han B., Margono B., Ichinose Y., et al. Gefitinib or Carboplatin–Paclitaxel in Pulmonary Adenocarcinoma. N. Engl. J. Med. 2009;361:947–957. doi: 10.1056/NEJMoa0810699.
    1. Wu Y.-L., Zhou C., Hu C.-P., Feng J., Lu S., Huang Y., Li W., Hou M., Shi J.H., Lee K.Y. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): An open-label, randomised phase 3 trial. Lancet Oncol. 2014;15:213–222. doi: 10.1016/S1470-2045(13)70604-1.
    1. Soria J.-C., Ohe Y., Vansteenkiste J., Reungwetwattana T., Chewaskulyong B., Lee K.H., Dechaphunkul A., Imamura F., Nogami N., Kurata T., et al. Osimertinib in Untreated EGFR-Mutated Advanced Non–Small-Cell Lung Cancer. N. Engl. J. Med. 2017;378:113–125. doi: 10.1056/NEJMoa1713137.
    1. Rosell R., Carcereny E., Gervais R., Vergnenegre A., Massuti B., Felip E., Palmero R., Garcia-Gomez R., Pallares C., Sanchez J.M. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): A multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13:239–246. doi: 10.1016/S1470-2045(11)70393-X.
    1. Loong H.H., Kwan S.-c.S., Mok T.S.-k., Lau Y.-m. Therapeutic Strategies in EGFR Mutant Non-Small Cell Lung Cancer. Curr. Treat. Options Oncol. 2018;19:58. doi: 10.1007/s11864-018-0570-9.
    1. Nour-Eldin N.-E.A., Alsubhi M., Emam A., Lehnert T., Beeres M., Jacobi V., Gruber-Rouh T., Scholtz J.-E., Vogl T.J., Naguib N.N. Pneumothorax complicating coaxial and non-coaxial CT-guided lung biopsy: Comparative analysis of determining risk factors and management of pneumothorax in a retrospective review of 650 patients. Cardio Interv. Radiol. 2016;39:261–270. doi: 10.1007/s00270-015-1167-3.
    1. Liu H.E., Vuppalapaty M., Wilkerson C., Renier C., Chiu M., Lemaire C., Che J., Matsumoto M., Carroll J., Crouse S., et al. Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients. Front. Oncol. 2020;10:572895. doi: 10.3389/fonc.2020.572895.
    1. Diaz L.A., Jr., Bardelli A. Liquid biopsies: Genotyping circulating tumor DNA. J. Clin. Oncol. 2014;32:579. doi: 10.1200/JCO.2012.45.2011.
    1. Möhrmann L., Huang H.J., Hong D.S., Tsimberidou A.M., Fu S., Piha-Paul S., Subbiah V., Karp D.D., Naing A., Krug A., et al. Liquid biopsies using plasma exosomal nucleic acids and plasma cell-free DNA compared with clinical outcomes of patients with advanced cancers. J. Clin. Oncol. 2018;24:181–188. doi: 10.1158/1078-0432.CCR-17-2007.
    1. Wu Y.-L., Sequist L.V., Hu C.-P., Feng J., Lu S., Huang Y., Li W., Hou M., Schuler M., Mok T., et al. EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: Analysis of LUX-Lung 3 and 6. Br. J. Cancer. 2017;116:175–185. doi: 10.1038/bjc.2016.420.
    1. Mok T., Wu Y.-L., Lee J.S., Yu C.-J., Sriuranpong V., Ladrera G., Fuerte F., Margono B., Wu L., Tsai J., et al. Detection and Dynamic Changes of EGFR Mutations from Circulating Tumor DNA as a Predictor of Survival Outcomes in NSCLC Patients Treated with First-line Intercalated Erlotinib and Chemotherapy. Clin. Cancer Res. 2015;21:3196–3203.
    1. Shah R., Patel T., Freedman J.E. Circulating extracellular vesicles in human disease. N. Engl. J. Med. 2018;379:958–966. doi: 10.1056/NEJMra1704286.
    1. Cui S., Cheng Z., Qin W., Jiang L. Exosomes as a liquid biopsy for lung cancer. Lung Cancer. 2018;116:46–54.
    1. Yu W., Hurley J., Roberts D., Chakrabortty S., Enderle D., Noerholm M., Breakefield X., Skog J. Exosome-based liquid biopsies in cancer: Opportunities and challenges. Ann. Oncol. 2021;32:466–477. doi: 10.1016/j.annonc.2021.01.074.
    1. Castellanos-Rizaldos E., Grimm D.G., Tadigotla V., Hurley J., Healy J., Neal P.L., Sher M., Venkatesan R., Karlovich C., Raponi M., et al. Exosome-Based Detection of EGFR T790M in Plasma from Non–Small Cell Lung Cancer Patients. Clin. Cancer Res. 2018;24:2944–2950.
    1. Wan Y., Liu B., Lei H., Zhang B., Wang Y., Huang H., Chen S., Feng Y., Zhu L., Gu Y. 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;29:2379–2383.
    1. Park J., Lee C., Eom J.S., Kim M.-H., Cho Y.-K. Detection of EGFR Mutations Using Bronchial Washing-Derived Extracellular Vesicles in Patients with Non-Small-Cell Lung Carcinoma. Cancers. 2020;12:2822.
    1. Purcell E., Owen S., Prantzalos E., Radomski A., Carman N., Lo T.-W., Zeinali M., Subramanian C., Ramnath N., Nagrath S. Epidermal Growth Factor Receptor Mutations Carried in Extracellular Vesicle-Derived Cargo Mirror Disease Status in Metastatic Non-small Cell Lung Cancer. Front. Cell Dev. Biol. 2021;9:389. doi: 10.3389/fcell.2021.724389.
    1. Castellanos-Rizaldos E., Zhang X., Tadigotla V.R., Grimm D.G., Karlovich C., Raez L.E., Skog J.K. Exosome-based detection of activating and resistance EGFR mutations from plasma of non-small cell lung cancer patients. Oncotarget. 2019;10:2911.
    1. Krug A., Enderle D., Karlovich C., Priewasser T., Bentink S., Spiel A., Brinkmann K., Emenegger J., Grimm D., Castellanos-Rizaldos E. Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma. Ann. Oncol. 2018;29:700–706.
    1. Hur J.Y., Kim H.J., Lee J.S., Choi C.-M., Lee J.C., Jung M.K., Pack C.G., Lee K.Y. Extracellular vesicle-derived DNA for performing EGFR genotyping of NSCLC patients. Mol. Cancer. 2018;17:15. doi: 10.1186/s12943-018-0772-6.
    1. Hur J.Y., Lee J.S., Kim I.A., Kim H.J., Kim W.S., Lee K.Y. Extracellular vesicle-based EGFR genotyping in bronchoalveolar lavage fluid from treatment-naive non-small cell lung cancer patients. Transl. Lung Cancer Res. 2019;8:1051–1060. doi: 10.21037/tlcr.2019.12.16.
    1. Goldstraw P., Chansky K., Crowley J., Rami-Porta R., Asamura H., Eberhardt W.E., Nicholson A.G., Groome P., Mitchell A., Bolejack V., et al. The IASLC Lung Cancer Staging Project: Proposals for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Lung Cancer. J. Thorac. Oncol. 2016;11:39–51. doi: 10.1016/j.jtho.2015.09.009.
    1. Han H.-S., Lim S.-n., An J.Y., Lee K.-M., Choe K.H., Le K.H., Kim S.T., Son S.M., Choi S.Y., Le O.-J., et al. Detection of EGFR Mutation Status in Lung Adenocarcinoma Specimens with Different Proportions of Tumor Cells Using Two Methods of Differential Sensitivity. J. Thorac. Oncol. 2012;7:355–364. doi: 10.1097/JTO.0b013e31823c4c1b.
    1. Han J.-Y., Choi J.-J., Kim J.Y., Han Y.L., Lee G.K. PNA clamping-assisted fluorescence melting curve analysis for detecting EGFR and KRAS mutations in the circulating tumor DNA of patients with advanced non-small cell lung cancer. BMC Cancer. 2016;16:627.
    1. Rantakokko-Jalava K., Jalava J. Optimal DNA Isolation Method for Detection of Bacteria in Clinical Specimens by Broad-Range PCR. J. Clin. Mic. 2002;40:4211–4217. doi: 10.1128/JCM.40.11.4211-4217.2002.
    1. Thakur B.K., Zhang H., Becker A., Matei I., Huang Y., Costa-Silva B., Zheng Y., Hoshino A., Brazier H., Xiang J., et al. Double-stranded DNA in exosomes: A novel biomarker in cancer detection. Cell Res. 2014;24:766–769. doi: 10.1038/cr.2014.44.
    1. Miller R.J., Casal R.F., Lazarus D.R., Ost D.E., Eapen G.A. Flexible bronchoscopy. Clin. Chest Med. 2018;39:1–16. doi: 10.1016/j.ccm.2017.09.002.
    1. Doyle L.M., Wang M.Z. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 2019;8:727. doi: 10.3390/cells8070727.
    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;79:2462–2465. doi: 10.1158/0008-5472.CAN-19-0019.
    1. Jin Y., Chen K., Wang Z., Wang Y., Liu J., Lin L., Shao Y., Gao L., Yin H., Cui C., et al. DNA in serum extracellular vesicles is stable under different storage conditions. BMC Cancer. 2016;16:1–9. doi: 10.1186/s12885-016-2783-2.
    1. Hur J.Y., Lee K.Y. Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA. Cancers. 2021;13:3827. doi: 10.3390/cancers13153827.
    1. San Lucas F.A., Allenson K., Bernard V., Castillo J., Kim D.U., Ellis K., Ehli E.A., Davies G.E., Petersen J.L., Wistuba I., et al. Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes. Ann. Oncol. 2016;27:635–641. doi: 10.1093/annonc/mdv604.
    1. Lee S.E., Park H.Y., Hur J.Y., Kim H.J., Kim I.A., Kim W.S., Lee K.Y. Genomic profiling of extracellular vesicle-derived DNA from bronchoalveolar lavage fluid of patients with lung adenocarcinoma. Transl. Lung Cancer Res. 2021;10:104–116. doi: 10.21037/tlcr-20-888.
    1. Buttitta F., Felicioni L., Del Grammastro M., Filice G., Di Lorito A., Malatesta S., Viola P., Centi I., D’Antuono T., Zappacosta R., et al. Effective Assessment of egfr Mutation Status in Bronchoalveolar Lavage and Pleural Fluids by Next-Generation Sequencing. Clin. Cancer Res. 2013;19:691–698. doi: 10.1158/1078-0432.CCR-12-1958.
    1. Zhang H., Cai W., Wang Y., Liao M., Tian S. CT and clinical characteristics that predict risk of EGFR mutation in non-small cell lung cancer: A systematic review and meta-analysis. Int. J. Clin. Oncol. 2019;24:649–659. doi: 10.1007/s10147-019-01403-3.
    1. Kim N., Cho D., Kim H., Kim S., Cha Y.-J., Greulich H., Bass A., Cho H.-S., Cho J. Colorectal adenocarcinoma-derived EGFR mutants are oncogenic and sensitive to EGFR-targeted monoclonal antibodies, cetuximab and panitumumab. Int. J. Cancer. 2020;146:2194–2200. doi: 10.1002/ijc.32499.
    1. Kim I., Lee J.S., Kim H.J., Kim W.S., Lee K.Y. Cumulative smoking dose affects the clinical outcomes of EGFR-mutated lung adenocarcinoma patients treated with EGFR-TKIs: A retrospective study. BMC Cancer. 2018;18:1–10. doi: 10.1186/s12885-018-4691-0.

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