Skip to main content
SpringerLink
Log in

Digital PCR-Based Detection of EGFR Mutations in Paired Plasma and CSF Samples of Lung Adenocarcinoma Patients with Central Nervous System Metastases

  • Original Research Article
  • Published:
Targeted Oncology Aims and scope Submit manuscript

Abstract

Background

The presence of specific mutations in the EGFR gene informs the clinical pathway of therapy for patients with lung adenocarcinoma (LAC), including those with central nervous system (CNS) metastases. Plasma circulating cell-free DNA (cfDNA) has been demonstrated to carry the mutational information of LACs, which serves as a biomarker to guide treatment. However, whether the cerebrospinal fluid (CSF) enriches circulating tumor DNA (ctDNA) released from CNS metastatic lesions of LAC, and whether the CSF ctDNA can be used to characterize these lesions remains unknown.

Objective

To explore the EGFR status in CNS metastases of LAC patients, and to guide the treatment of intra- and extracranial tumors in these patients.

Patients and methods

The EGFR mutational status in the cfDNA from paired CSF and plasma samples from LAC patients with CNS metastases, including 20 brain metastases (BM) and 15 leptomeningeal metastases (LM), was assessed by droplet digital polymerase chain reaction (ddPCR). The clinical outcomes of the EGFR status-based intervention were investigated.

Results

EGFR mutations were detected in 23/35 LAC patients (65.7%). EGFR mutations in the plasma or CSF were detected in 6/11 (54.5%) and 5/10 (50%) BM patients, and in 4/11 (36.4%) and 9/12(75%) LM patients, respectively. The prevalence of the T790M mutation was significantly higher in plasma (9/23) than in CSF (3/23) samples. The sensitivity and specificity of the ddPCR-based EGFR mutation test in CSF or plasma samples versus the primary tumor samples were 56% and 89% versus 46% and 100%, respectively. Twelve patients received a first-generation EGFR TKI (tyrosine kinase inhibitor) after the detection of sensitive EGFR mutations in their CSF or plasma, and five patients were switched from a first-generation EGFR TKI to osimertinib after the detection of the T790M mutation.

Conclusions

The EGFR T790M mutation in plasma cfDNA is a sensitive marker for EGFR TKI resistance when CNS metastases progressed. CSF ctDNA increases the diagnostic validity for EGFR genotyping of lung cancer brain metastasis. ddPCR in CSF and plasma samples could provide less invasive and close monitoring of the EGFR status of LAC patients with CNS metastases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Kromer C, Xu J, Ostrom QT, Gittleman H, Kruchko C, Sawaya R, et al. Estimating the annual frequency of synchronous brain metastasis in the United States 2010-2013: a population-based study. J Neurooncol. 2017;134(1):55–64. https://doi.org/10.1007/s11060-017-2516-7.

    Article  PubMed  Google Scholar 

  2. Cheng H, Perez-Soler R. Leptomeningeal metastases in non-small-cell lung cancer. Lancet Oncol. 2018;19(1):e43–55. https://doi.org/10.1016/S1470-2045(17)30689-7.

    Article  PubMed  Google Scholar 

  3. Ge M, Zhuang Y, Zhou X, Huang R, Liang X, Zhan Q. High probability and frequency of EGFR mutations in non-small cell lung cancer with brain metastases. J Neurooncol. 2017;135(2):413–8. https://doi.org/10.1007/s11060-017-2590-x.

    Article  CAS  PubMed  Google Scholar 

  4. Rau KM, Chen HK, Shiu LY, Chao TL, Lo YP, Wang CC, et al. Discordance of mutation statuses of epidermal growth factor receptor and K-ras between primary adenocarcinoma of lung and brain metastasis. Int J Mol Sci. 2016;17(4):524. https://doi.org/10.3390/ijms17040524.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lee CK, Lord S, Marschner I, Wu YL, Sequist L, Rosell R, et al. The value of early depth of response in predicting long-term outcome in EGFR-mutant lung cancer. J Thorac Oncol. 2018;13(6):792–800. https://doi.org/10.1016/j.jtho.2018.03.010.

    Article  PubMed  Google Scholar 

  6. Heon S, Yeap BY, Lindeman NI, Joshi VA, Butaney M, Britt GJ, et al. The impact of initial gefitinib or erlotinib versus chemotherapy on central nervous system progression in advanced non-small cell lung cancer with EGFR mutations. Clin Cancer Res. 2012;18(16):4406–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hasegawa T, Sawa T, Futamura Y, Horiba A, Ishiguro T, Marui T, et al. Feasibility of rebiopsy in non-small cell lung cancer treated with epidermal growth factor receptor-tyrosine kinase inhibitors. Intern Med. 2015;54(16):1977–80. https://doi.org/10.2169/internalmedicine.54.4394.

    Article  CAS  PubMed  Google Scholar 

  8. 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):224ra24. https://doi.org/10.1126/scitranslmed.3007094.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang Y, Liu Z, Yin H, Hu J, Zhong S, Chen W, et al. Improved detection of EGFR mutations in the tumor cells enriched from the malignant pleural effusion of non-small cell lung cancer patient. Gene. 2018;644:87–92. https://doi.org/10.1016/j.gene.2017.10.073.

    Article  CAS  PubMed  Google Scholar 

  10. Connolly ID, Li Y, Pan W, Johnson E, You L, Vogel H, et al. A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma. J Neurooncol. 2017;135(1):29–36. https://doi.org/10.1007/s11060-017-2557-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ballester LY, Glitza Oliva IC, Douse DY, Chen MM, Lan C, Haydu LE, et al. Evaluating circulating tumor DNA from the cerebrospinal fluid of patients with melanoma and leptomeningeal disease. J Neuropathol Exp Neurol. 2018;77(7):628–35. https://doi.org/10.1093/jnen/nly046.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84(2):1003–11. https://doi.org/10.1021/ac202578x.

    Article  CAS  PubMed  Google Scholar 

  13. Minari R, Bordi P, Del Re M, Facchinetti F, Mazzoni F, Barbieri F, et al. Primary resistance to osimertinib due to SCLC transformation: issue of T790M determination on liquid re-biopsy. Lung Cancer. 2018;115:21–7. https://doi.org/10.1016/j.lungcan.2017.11.011.

    Article  CAS  PubMed  Google Scholar 

  14. Martinez-Ricarte F, Mayor R, Martinez-Saez E, Rubio-Perez C, Pineda E, Cordero E, et al. Molecular diagnosis of diffuse gliomas through sequencing of cell-free circulating tumor DNA from cerebrospinal fluid. Clin Cancer Res. 2018;24(12):2812–9. https://doi.org/10.1158/1078-0432.CCR-17-3800.

    Article  CAS  PubMed  Google Scholar 

  15. Liang S, Zhe L, Jungfang T, Hongbo W, Lili G, Mingzhi L, et al. EGFR mutation status analysis in cerebrospinal fluid and plasma of advanced lung adenocarcinoma with brain metastases. J Thorac Oncol. 2017;12(1):S240.

    Article  Google Scholar 

  16. Joo JW, Hong MH, Shim HS. Clinical characteristics of T790M-positive lung adenocarcinoma after resistance to epidermal growth factor receptor-tyrosine kinase inhibitors with an emphasis on brain metastasis and survival. Lung Cancer. 2018;121:12–7. https://doi.org/10.1016/j.lungcan.2018.04.013.

    Article  PubMed  Google Scholar 

  17. Hata A, Katakami N, Yoshioka H, Takeshita J, Tanaka K, Masago K, et al. Prognostic impact of central nervous system metastases after acquired resistance to EGFR-TKI: poorer prognosis associated with T790M-negative status and leptomeningeal metastases. Anticancer Res. 2015;35(2):1025–31.

    PubMed  Google Scholar 

  18. Hata A, Katakami N, Yoshioka H, Takeshita J, Tanaka K, Nanjo S, et al. Rebiopsy of non-small cell lung cancer patients with acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitor: comparison between T790M mutation-positive and mutation-negative populations. Cancer. 2013;119(24):4325–32. https://doi.org/10.1002/cncr.28364.

    Article  CAS  PubMed  Google Scholar 

  19. Jiang BY, Li YS, Guo WB, Zhang XC, Chen ZH, Su J, et al. Detection of driver and resistance mutations in leptomeningeal metastases of NSCLC by next-generation sequencing of cerebrospinal fluid circulating tumor cells. Clin Cancer Res. 2017;23(18):5480–8. https://doi.org/10.1158/1078-0432.CCR-17-0047.

    Article  CAS  PubMed  Google Scholar 

  20. Girard N. Optimizing outcomes in EGFR mutation-positive NSCLC: which tyrosine kinase inhibitor and when? Future Oncol. 2018;14(11):1117–32. https://doi.org/10.2217/fon-2017-0636.

    Article  CAS  PubMed  Google Scholar 

  21. Togashi Y, Masago K, Masuda S, Mizuno T, Fukudo M, Ikemi Y, et al. Cerebrospinal fluid concentration of gefitinib and erlotinib in patients with non-small cell lung cancer. Cancer Chemother Pharmacol. 2012;70(3):399–405. https://doi.org/10.1007/s00280-012-1929-4.

    Article  CAS  PubMed  Google Scholar 

  22. Sakai K, Horiike A, Irwin DL, Kudo K, Fujita Y, Tanimoto A, et al. Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor. Cancer Sci. 2013;104(9):1198–204. https://doi.org/10.1111/cas.12211.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Watanabe M, Kawaguchi T, Isa S, Ando M, Tamiya A, Kubo A, et al. Ultra-sensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using droplet digital PCR. Clin Cancer Res. 2015;21(15):3552–60. https://doi.org/10.1158/1078-0432.CCR-14-2151.

    Article  CAS  PubMed  Google Scholar 

  24. Taniguchi K, Okami J, Kodama K, Higashiyama M, Kato K. Intratumor heterogeneity of epidermal growth factor receptor mutations in lung cancer and its correlation to the response to gefitinib. Cancer Sci. 2008;99(5):929–35. https://doi.org/10.1111/j.1349-7006.2008.00782.x.

    Article  CAS  PubMed  Google Scholar 

  25. Cabanero M, Tsao MS. Circulating tumour DNA in EGFR-mutant non-small-cell lung cancer. Curr Oncol. 2018;25(Suppl 1):S38–44. https://doi.org/10.3747/co.25.3761.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ge M, Zhan Q, Zhang Z, Ji X, Zhou X, Huang R, et al. Different next-generation sequencing pipelines based detection of tumor DNA in cerebrospinal fluid of lung adenocarcinoma cancer patients with leptomeningeal metastases. BMC Cancer. 2019;19(1):143. https://doi.org/10.1186/s12885-019-5348-3.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Ruofan Huang and Xiao Xu contributed equally to the work.

Authors and Affiliations

  1. Department of Oncology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, 200040, China

    Ruofan Huang, Qiong Zhan, Mengxi Ge, Xinli Zhou & Xiaohua Liang

  2. Department of Central Laboratory, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, 200040, China

    Xiao Xu & Ming Guan

  3. Department of Clinical Laboratory, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China

    Dong Li

  4. Department of Laboratory Medicine, Huashan Hospital North, Fudan University, Shanghai, China

    Kun Chen

Authors
  1. Ruofan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qiong Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mengxi Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinli Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiaohua Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ming Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RH, XX, and KC performed ddPCR. QZ and MG collected patient data. DL analyzed the data. RH, MG, and XL contributed to writing the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xiaohua Liang or Ming Guan.

Ethics declarations

Funding

This work was supported by Shanghai Municipal Committee of Health and Family planning [201440584], Shanghai Municipal Natural Science Foundation [16ZR1404300], Scientific Research Foundation Huashan Hospital [787, 2017], National Natural Science Foundation of China [81672105], and the Clinical Research and Cultivation Project of Shanghai Tongji Hospital [ITJ(ZD)1803].

Conflict of interest

Ruofan Huang, Xiao Xu, Dong Li, Kun Chen, Qiong Zhan, Mengxi Ge, Xinli Zhou, Xiaohua Liang, and Ming Guan declare that they have no conflicts of interest that might be relevant to the contents of this article.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 496 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huang, R., Xu, X., Li, D. et al. Digital PCR-Based Detection of EGFR Mutations in Paired Plasma and CSF Samples of Lung Adenocarcinoma Patients with Central Nervous System Metastases. Targ Oncol 14, 343–350 (2019). https://doi.org/10.1007/s11523-019-00645-5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11523-019-00645-5

Search

Navigation