Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation

  • Atsushi Osoegawa
  • Takafumi Hashimoto
  • Yohei Takumi
  • Miyuki Abe
  • Tomonori Yamada
  • Ryoji Kobayashi
  • Michiyo Miyawaki
  • Hideya Takeuchi
  • Tatsuro Okamoto
  • Kenji Sugio


Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.


EGFR Acquired resistance EGFR G719X Osimertinib Torin-1 



tyrosine kinase inhibitor


epidermal growth factor receptor


acquired resistance


malignant pleural effusion


Rho-associated coiled-coil forming kinase


multiplex ligation-dependent probe amplification


gefitinib resistant


Lethal Concentration, 50%



The authors would like to thank Mr. Brian Quinn for his critical comments on the manuscript.


This work was supported by KAKENHI (Grant Number JP25462181), Japan Society for the Promotion of Science, Tokyo, Japan.

Compliance with ethical standards

Conflict of interest

AO declares that he has no conflict of interest. TH declares that he has no conflict of interest. YT declares that he has no conflict of interest. MA declares that she has no conflict of interest. TY declares that he has no conflict of interest. RK declares that he has no conflict of interest. MM declares that she has no conflict of interest. HT declares that he has no conflict of interest. TO declares that he has no conflict of interest. KS declares that he has no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

10637_2018_592_Fig4_ESM.gif (203 kb)
Supplemental Figure 1

Genomic alteration in G719S-GR cells. Copy number variations were estimated by comparing with NCC oncopanel data obtained from four normal tissues as a control. The amplification of EGFR, IL7R, MYC and the FGFR1 locus was observed. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. (GIF 203 kb)

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High resolution image (TIFF 2765 kb)
10637_2018_592_MOESM2_ESM.docx (27 kb)
Supplemental Table 1 (DOCX 27 kb)


  1. 1.
    Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, Seto T, Satouchi M, Tada H, Hirashima T, Asami K, Katakami N, Takada M, Yoshioka H, Shibata K, Kudoh S, Shimizu E, Saito H, Toyooka S, Nakagawa K, Fukuoka M, West Japan Oncology G (2010) Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 11(2):121–128. CrossRefPubMedGoogle Scholar
  2. 2.
    Park K, Tan EH, O'Byrne K, Zhang L, Boyer M, Mok T, Hirsh V, Yang JC, Lee KH, Lu S, Shi Y, Kim SW, Laskin J, Kim DW, Arvis CD, Kolbeck K, Laurie SA, Tsai CM, Shahidi M, Kim M, Massey D, Zazulina V, Paz-Ares L (2016) Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-lung 7): a phase 2B, open-label, randomised controlled trial. Lancet Oncol 17(5):577–589. CrossRefPubMedGoogle Scholar
  3. 3.
    Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG, Halmos B (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352(8):786–792. CrossRefPubMedGoogle Scholar
  4. 4.
    Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X, Christensen J, Kosaka T, Holmes AJ, Rogers AM, Cappuzzo F, Mok T, Lee C, Johnson BE, Cantley LC, Janne PA (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316(5827):1039–1043. CrossRefPubMedGoogle Scholar
  5. 5.
    Yano S, Wang W, Li Q, Matsumoto K, Sakurama H, Nakamura T, Ogino H, Kakiuchi S, Hanibuchi M, Nishioka Y, Uehara H, Mitsudomi T, Yatabe Y, Nakamura T, Sone S (2008) Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res 68(22):9479–9487. CrossRefPubMedGoogle Scholar
  6. 6.
    Uramoto H, Shimokawa H, Hanagiri T, Kuwano M, Ono M (2011) Expression of selected gene for acquired drug resistance to EGFR-TKI in lung adenocarcinoma. Lung Cancer 73(3):361–365. CrossRefPubMedGoogle Scholar
  7. 7.
    Sugio K, Uramoto H, Ono K, Oyama T, Hanagiri T, Sugaya M, Ichiki Y, So T, Nakata S, Morita M, Yasumoto K (2006) Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking. Br J Cancer 94(6):896–903. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Yang JC, Sequist LV, Geater SL, Tsai CM, Mok TS, Schuler M, Yamamoto N, Yu CJ, Ou SH, Zhou C, Massey D, Zazulina V, Wu YL (2015) Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-lung 2, LUX-lung 3, and LUX-lung 6. Lancet Oncol 16(7):830–838. CrossRefPubMedGoogle Scholar
  9. 9.
    Jiang J, Greulich H, Janne PA, Sellers WR, Meyerson M, Griffin JD (2005) Epidermal growth factor-independent transformation of Ba/F3 cells with cancer-derived epidermal growth factor receptor mutants induces gefitinib-sensitive cell cycle progression. Cancer Res 65(19):8968–8974. CrossRefPubMedGoogle Scholar
  10. 10.
    Kobayashi Y, Togashi Y, Yatabe Y, Mizuuchi H, Jangchul P, Kondo C, Shimoji M, Sato K, Suda K, Tomizawa K, Takemoto T, Hida T, Nishio K, Mitsudomi T (2015) EGFR exon 18 mutations in lung Cancer: molecular predictors of augmented sensitivity to Afatinib or Neratinib as compared with first- or third-generation TKIs. Clin Cancer Res 21(23):5305–5313. CrossRefPubMedGoogle Scholar
  11. 11.
    Kondo J, Endo H, Okuyama H, Ishikawa O, Iishi H, Tsujii M, Ohue M, Inoue M (2011) Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc Natl Acad Sci U S A 108(15):6235–6240. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Homig-Holzel C, Savola S (2012) Multiplex ligation-dependent probe amplification (MLPA) in tumor diagnostics and prognostics. Diagn Mol Pathol 21(4):189–206. CrossRefPubMedGoogle Scholar
  13. 13.
    Osoegawa A, Gills JJ, Kawabata S, Dennis PA (2017) Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib. Oncotarget 8(50):87044–87053. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Crystal AS, Shaw AT, Sequist LV, Friboulet L, Niederst MJ, Lockerman EL, Frias RL, Gainor JF, Amzallag A, Greninger P, Lee D, Kalsy A, Gomez-Caraballo M, Elamine L, Howe E, Hur W, Lifshits E, Robinson HE, Katayama R, Faber AC, Awad MM, Ramaswamy S, Mino-Kenudson M, Iafrate AJ, Benes CH, Engelman JA (2014) Patient-derived models of acquired resistance can identify effective drug combinations for cancer. Science 346(6216):1480–1486. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ma L, Teruya-Feldstein J, Behrendt N, Chen Z, Noda T, Hino O, Cordon-Cardo C, Pandolfi PP (2005) Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression. Genes Dev 19(15):1779–1786. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Liu Q, Kirubakaran S, Hur W, Niepel M, Westover K, Thoreen CC, Wang J, Ni J, Patricelli MP, Vogel K, Riddle S, Waller DL, Traynor R, Sanda T, Zhao Z, Kang SA, Zhao J, Look AT, Sorger PK, Sabatini DM, Gray NS (2012) Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics. J Biol Chem 287(13):9742–9752. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Atsushi Osoegawa
    • 1
  • Takafumi Hashimoto
    • 1
  • Yohei Takumi
    • 1
  • Miyuki Abe
    • 1
  • Tomonori Yamada
    • 1
  • Ryoji Kobayashi
    • 1
  • Michiyo Miyawaki
    • 1
  • Hideya Takeuchi
    • 1
  • Tatsuro Okamoto
    • 1
  • Kenji Sugio
    • 1
  1. 1.Department of Thoracic and Breast SurgeryOita University Faculty of MedicineYufuJapan

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