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Cited by 165Lung Cancer Treatments and MutationsHER2/EGFR in Cancer ResearchCancer Genomics and Diagnostics

In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

Open access
Hirano, Toshiyuki, Yasuda, Hiroyuki, Tani, Tetsuo, Hamamoto, Junko, Oashi, Ayano, Ishioka, Kota, Arai, Daisuke, Nukaga, Shigenari +7 more·2015·Oncotarget
DOI 10.18632/oncotarget.5887PMC PMC4770737
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Abstract

// Toshiyuki Hirano 1, * , Hiroyuki Yasuda 1 , Tetsuo Tani 1, * , Junko Hamamoto 1 , Ayano Oashi 1 , Kota Ishioka 1 , Daisuke Arai 1 , Shigenari Nukaga 1 , Masayoshi Miyawaki 1 , Ichiro Kawada 1 , Katsuhiko Naoki 2 , Daniel B. Costa 3 , Susumu S. Kobayashi 3 , Tomoko Betsuyaku 1 , Kenzo Soejima 1 1 Division of Pulmonary Medicine, Department of Medicine, Keio University, School of Medicine 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan 2 Keio Cancer Center, Keio University, School of Medicine 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan 3 Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA * These authors have contributed equally to this work Correspondence to: Hiroyuki Yasuda, e-mail: hiroyukiyasuda@a8.keio.jp Kenzo Soejima, e-mail: ksoejima@cpnet.med.keio.ac.jp Keywords: EGFR mutation, EGFR tyrosine kinase inhibitors, EGFR exon 20 insertion mutations, in vitro modeling, lung cancer Received: August 18, 2015 Accepted: October 03, 2015 Published: October 15, 2015 ABSTRACT EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1 st (erlotinib), 2 nd (afatinib) and 3 rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR . An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1 st and 2 nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations.

AI snapshot

Written from the paper’s full text.

TL;DR

This study created an in vitro selectivity-index model comparing erlotinib, afatinib, osimertinib, and rociletinib across clinically relevant EGFR mutants in Ba/F3 and human NSCLC cell lines. It found wide therapeutic windows for afatinib in exon 19 deletion/L858R, for osimertinib and rociletinib in T790M-positive mutants, and unexpectedly for osimertinib in several EGFR exon 20 insertion mutants.

Key findings

  • In Ba/F3 and human lung cancer cell lines, afatinib was most potent for classic EGFR mutations: IC50 values for PC-9 (exon 19del) and H3255 (L858R) were 0.8 nM and 0.3 nM, respectively, versus erlotinib 7 nM and 12 nM, osimertinib 17 nM and 4 nM, and rociletinib 84 nM and 35 nM.
  • For T790M-positive mutants, erlotinib was ineffective (PC-9ER >10000 nM; H1975 1185 nM), while osimertinib and rociletinib were potent: osimertinib IC50 13 nM in PC-9ER and 5 nM in H1975; rociletinib 37 nM and 23 nM. The selectivity index indicated IC50 values for T790M more than 100-fold lower than wild-type EGFR for osimertinib and rociletinib.
  • For 1st-generation-TKI-resistant exon 20 insertion mutations, osimertinib showed a wide therapeutic window: IC50 values for Y764_V765insHH, A767_V769dupASV, and D770_N771insNPG were 237, 333, and 42 nM, respectively, with selectivity index around −1, whereas afatinib IC50 values were 134, 158, and 43 nM.
  • Apoptosis assays in Ba/F3 cells at 0.1 μM showed afatinib induced annexin-V positivity in 82.5% of wild-type EGFR cells versus 56.0% of D770_N771insNPG cells, while osimertinib induced 29.1% in wild-type EGFR cells versus 48.6% in D770_N771insNPG cells, supporting a wider therapeutic window for osimertinib.

Methods

  • In vitro study using Ba/F3 stable cell lines exogenously expressing wild-type or mutated EGFR, plus human NSCLC cell lines PC-9 (exon 19del), H3255 (L858R), PC-9ER (exon 19del+T790M), H1975 (L858R+T790M), and BID007 (A763_Y764insFQEA).
  • EGFR mutations examined included delL747_P753insS (exon 19del), L858R, exon 19del+T790M, L858R+T790M, A763_Y764insFQEA, Y764_V765insHH, A767_V769dupASV, and D770_N771insNPG.
  • Main analyses were MTS cell proliferation assays to calculate IC50 values, immunoblotting for phosphorylated EGFR/AKT/ERK, and annexin V/propidium iodide apoptosis assays; an in vitro selectivity index was calculated as the ratio of IC50 values between mutated and wild-type EGFR, with IC50 calculated using GraphPad Prism.

Limitations

  • The paper states that the study includes only in vitro experiments, that pharmacokinetics and pharmacodynamics may vary among EGFR-TKIs, and that in vivo concentrations may vary; it concludes that further in vivo and human clinical trials are mandatory.
  • Evident from the text: the therapeutic-window model is based on IC50 ratios in cell lines rather than direct in vivo measurements, and the exon 20 insertion conclusions rely largely on Ba/F3 cells, with only one human cell line (BID007) available for the A763_Y764insFQEA insertion.
  • Evident from the text: the paper reports no clinical outcomes for osimertinib in exon 20 insertion mutant NSCLC; it cites pharmacokinetic data from a phase I study and calls for dose-adjusted clinical trials.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.18632/oncotarget.5887

Cites

42 of 42 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 42 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    IC50 values of afatinib, osimertinib, and rociletinib for H1975 were 57 nM, 5 nM, and 23 nM, respectively.

    “The calculated IC50 values of afatinib, osimertinib, and rociletinib for H1975 were 57 nM, 5 nM, and 23 nM, respectively.”
    Quote in the paperSubstantial claim
  • Findingown data

    Erlotinib did not inhibit proliferation of PC-9ER and H1975 cells harboring EGFR T790M at low concentrations.

    “Erlotinib did not inhibit the proliferation of cell lines harboring EGFR T790M, PC-9ER and H1975, at low concentrations.”
    Quote in the paperSubstantial claim
  • Findingown data

    In Ba/F3 cells harboring classic EGFR mutations, afatinib dramatically inhibited phosphorylation of EGFR, AKT, and ERK.

    “In Ba/F3 cells harboring classic EGFR mutations, exon 19 deletions and L858R, afatinib dramatically inhibited the phosphorylation of EGFR, AKT, and ERK.”
    Quote in the paperSubstantial claim
  • Findingown data

    Erlotinib did not inhibit phosphorylation of EGFR, AKT, and ERK in Ba/F3 cells harboring EGFR T790M mutations.

    “Erlotinib did not inhibit the phosphorylation of EGFR, AKT, and ERK in Ba/F3 cells harboring EGFR T790M mutations, exon 19del+T790M, and L858R+T790M.”
    Quote in the paperSubstantial claim
  • Findingown data

    IC50 values of afatinib for PC-9 and H3255 were 0.8 nM and 0.3 nM, respectively.

    “The calculated IC50 values of afatinib for PC-9 and H3255 were 0.8 nM and 0.3 nM, respectively.”
    Quote in the paperSubstantial claim
  • May combine two claims. 32
  • The quote only partly states this claim. 5
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    IC50 values of rociletinib for PC-9 and H3255 were 84 nM and 35 nM, respectively.

    “The calculated IC50 values of rociletinib for PC-9 and H3255 were 84 nM and 35 nM, respectively.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Osimertinib potency was comparable to erlotinib in PC-9 and H3255.

    “The potency of osimertinib was comparable to that of erlotinib.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For Ba/F3 cells harboring classic EGFR mutations, osimertinib showed mutation specificity similar to afatinib, with SI values around -2, indicating IC50 values about 100-fold lower than wild-type EGFR.

    “Interestingly, for Ba/F3 cells harboring classic EGFR mutations, osimertinib showed mutation specificity similar to that of afatinib, and the SI values were around −2, indicating IC50 values for these classic mutations were about 100-fold lower than those for the wild-type EGFR.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

  • May combine two claims.

    Findingown data

    Osimertinib potency was comparable to erlotinib, while rociletinib was less potent than erlotinib and osimertinib in Ba/F3 cells harboring classic EGFR mutations.

    “The potency of osimertinib was comparable to that of erlotinib, while rociletinib was less potent than erlotinib and osimertinib.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Erlotinib effectively inhibited proliferation of Ba/F3 cells harboring classic EGFR mutations, though less potent than afatinib.

    “Although less potent than afatinib, erlotinib effectively inhibited the proliferation of Ba/F3 cells.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Afatinib, osimertinib, and rociletinib effectively inhibited proliferation of PC-9ER and H1975 cells.

    “However, afatinib and 3rd generation EGFR-TKIs, osimertinib and rociletinib, effectively inhibited the proliferation of lung cancer cells.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Erlotinib effectively inhibited proliferation of PC-9 and H3255 cells, though less potent than afatinib.

    “Although less potent than afatinib, erlotinib effectively inhibited the proliferation of lung cancer cells as previously described [20].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Sequist, 2013, Structural, Biochemical, and Clinical Characterization of Epidermal Growth Factor Receptor (EGFR) Exon 20 Insertion Mutations in Lung Cancer, Sci Transl Med

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The in vitro model indicated the difference of erlotinib efficacy for classic EGFR mutations and its inefficacy for T790M mutations.

    “Our in vitro model clearly indicated the difference of erlotinib efficacy for classic EGFR mutations and its inefficacy for T790M mutations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IC50 values of osimertinib for PC-9 and H3255 were 17 nM and 4 nM, respectively.

    “The calculated IC50 values of osimertinib for PC-9 and H3255 were 17 nM and 4 nM, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IC50 values of erlotinib for PC-9 and H3255 were 7 nM and 12 nM, respectively.

    “The calculated IC50 values of erlotinib for PC-9 and H3255 were 7 nM and 12 nM, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Data from the in vitro model were in agreement with reported efficacy of afatinib for classic EGFR mutations and inefficacy for T790M mutations.

    “Data from our in vitro model were in agreement with these results.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Erlotinib and afatinib did not demonstrate mutation specificity for EGFR T790M mutations; their SI values were more than zero, indicating IC50 values for T790M higher than wild-type EGFR.

    “Unfortunately, erlotinib and afatinib did not demonstrate mutation specificity for EGFR T790M mutations. The SI values were more than zero, indicating that the IC50 values of erlotinib and afatinib for EGFR T790M mutations were higher than those for the wild-type EGFR.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

  • May combine two claims.

    Findingown data

    The proportion of annexin-V positive cells after afatinib treatment was 82.5% for wild type EGFR and 56.0% for EGFR D770_N771insNPG.

    “The proportion of annexin-V positive cells was 82.5% for the wild type EGFR and 56.0% for EGFR D770_N771insNPG.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Rociletinib was less potent than erlotinib and osimertinib in BID007.

    “However, rociletinib was less potent than erlotinib and osimertinib.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For EGFR exon 20 insertion mutations, most of which are known resistant to 1st and 2nd generation EGFR-TKIs, osimertinib was potent and presented a wide therapeutic window.

    “Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

  • May combine two claims.

    Findingown data

    Osimertinib had low effect on apoptosis in Ba/F3 cells harboring wild type EGFR and high effect in Ba/F3 cells harboring EGFR D770_N771insNPG.

    “the effect of osimertinib on apoptosis in Ba/F3 cells harboring wild type EGFR was low, while it was high in Ba/F3 cells harboring EGFR D770_N771insNPG.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Of erlotinib, osimertinib, and rociletinib, osimertinib most effectively inhibited Ba/F3 cell growth for other exon 20 insertion mutations.

    “Of the other three EGFR-TKIs, osimertinib most effectively inhibited Ba/F3 cell growth.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IC50 values of erlotinib and osimertinib for BID007 were 45 nM and 40 nM, respectively.

    “The calculated IC50 value of erlotinib and osimertinib for BID007 was 45 nM and 40 nM, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IC50 values of osimertinib for Y764_V765insHH, A767_V769dupASV, and D770_N771insNPG were 237, 333, and 42 nM, respectively.

    “The IC50 values of osimertinib for Y764_V765insHH, A767_V769dupASV, and D770_N771insNPG were 237, 333, and 42 nM, respectively (Figure 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The proportion of annexin-V positive cells after osimertinib treatment was 29.1% for wild type EGFR and 48.6% for EGFR D770_N771insNPG.

    “The proportion of annexin-V positive cells was 29.1% for the wild type EGFR and 48.6% for EGFR D770_N771insNPG.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Osimertinib presented the lowest SI values of around -1 for EGFR exon 20 insertion mutations, indicating IC50 values about 10-fold lower than wild type EGFR.

    “Surprisingly, we found that osimertinib presented the lowest SI values of around −1, indicating IC50 values for EGFR exon 20 insertion mutations about 10 fold lower than those for the wild type EGFR.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

  • May combine two claims.

    Findingown data

    For 1st generation EGFR-TKIs resistant exon 20 insertion mutations Y764_V765insHH, A767V769dupASV, and D770_N771insNPG, osimertinib showed similar potency and higher mutation specificity than afatinib.

    “for 1st generation EGFR-TKIs resistant exon 20 insertion mutations, Y764_V765insHH, A767V769dupASV, and D770_N771insNPG, osimertinib showed a similar potency and higher mutation specificity than afatinib.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Erlotinib is effective for EGFR exon 19 deletions, L858R, and A763_Y764insFQEA.

    “erlotinib is effective for EGFR exon 19 deletions, L858R, and A763_Y764insFQEA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Erlotinib and osimertinib effectively inhibited phosphorylation of EGFR, AKT, and ERK in PC-9 and H3255, though less potent than afatinib.

    “Although less potent than afatinib, erlotinib and osimertinib effectively inhibited the phosphorylation of EGFR, AKT, and ERK in PC-9 and H3255.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations.

    “The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

  • May combine two claims.

    Findingown data

    Erlotinib and osimertinib effectively inhibited proliferation of BID007 cells, though less potent than afatinib.

    “Although less potent than afatinib, erlotinib and osimertinib effectively inhibited the proliferation of BID007 cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Afatinib is potent against EGFR mutated lung cancer cells in vitro and in vivo.

    “Afatinib, a clinically available 2nd generation EGFR-TKI, is potent against EGFR mutated lung cancer cells in vitro [26] and in vivo [34, 35].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [26] Wong, 2008, BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models, Oncogene source

      Not checked in the free run.

    • cites [34] Shah, 2013, Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations, J Clin Oncol source

      Not checked in the free run.

    • cites [35] Geater, 2014, 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 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Rociletinib response rate for EGFR T790M positive patients was 59%.

    “Similarly, the response rate of rociletinib for EGFR T790M positive patients was 59% [39].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Logan, 2015, Rociletinib in EGFR-Mutated Non-Small-Cell Lung Cancer, New Engl J Med source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Response rates to gefitinib or erlotinib are around 60-80%.

    “The response rates to gefitinib or erlotinib are around 60–80% [14, 17].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14] Johnson, 2006, Effect of epidermal growth factor receptor tyrosine kinase domain mutations on the outcome of patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors, Clin Cancer Res source

      Not checked in the free run.

    • cites [17] Yatabe, 2007, Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer, Cancer Sci source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Afatinib is reported to be ineffective against lung cancer harboring EGFR T790M positive mutations because concentrations needed are not achievable in humans due to dose limiting toxicities, indicating a narrow therapeutic window.

    “Afatinib is reported to be ineffective against lung cancer harboring EGFR T790M positive mutations, because concentrations at which afatinib inhibits lung cancer cells harboring EGFR T790M are not achievable in humans due to dose limiting toxicities such as skin rash and diarrhea, indicating a narrow therapeutic window of afatinib for EGFR T790M [40].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] de Vries, 2008, A phase I dose escalation study of BIBW 2992, an irreversible dual inhibitor of epidermal growth factor receptor 1 (EGFR) and 2 (HER2) tyrosine kinase in a 2-week on, 2-week off schedule in patients with advanced solid tumours, Br J Cancer source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Osimertinib and rociletinib are effective in lung cancer cells harboring EGFR T790M in preclinical models.

    “Osimertinib and rociletinib are 3rd generation EGFR-TKIs, both of which are reported to be effective in lung cancer cells harboring EGFR T790M in preclinical models [30, 32].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [30] Sun, 2014, AZD9291, an Irreversible EGFR, TKI, Overcomes T790M-Mediated Resistance to EGFR Inhibitors in Lung Cancer, Cancer Discov source

      Not checked in the free run.

    • cites [32] Niu, 2013, Discovery of a Mutant-Selective Covalent Inhibitor of EGFR that Overcomes T790M-Mediated Resistance in NSCLC, Cancer Discov source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Activated EGFR induces pro-survival and anti-apoptotic signals through ERK/MAPK and PI3K/AKT.

    “Activated EGFR induces EGFR-mediated pro-survival and anti-apoptotic signals through downstream targets such as extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinases (PI3K)/protein kinase B (AKT) [10, 11].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] Haber, 2007, Epidermal growth factor receptor mutations in lung cancer, Nat Rev Cancer source

      Not checked in the free run.

    • cites [11] Lawrence, 2006, Integration of EGFR inhibitors with radiochemotherapy (vol 6, pg 876, 2006), Nat Rev Cancer

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Inhibition of the EGFR pathway down-regulates pro-survival signals and up-regulates pro-apoptotic molecules, by which EGFR-TKIs exert their effects in patients with EGFR mutated lung cancer.

    “Inhibition of the EGFR pathway leads to the down-regulation of pro-survival signals and up-regulation of pro-apoptotic molecules [12], by which EGFR tyrosine kinase inhibitors (EGFR-TKIs) exert their dramatic effects in patients with EGFR mutated lung cancer.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Kobayashi, 2007, BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations, Plos Med

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Osimertinib response rate for EGFR T790M positive patients was 61% and progression free survival was 9.6 months.

    “Osimertinib showed a promising safety and efficacy, the response rate and progression free survival for EGFR T790M positive patients was 61% and 9.6 months, respectively [38].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Brown, 2015, AZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer, New Engl J Med source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Of 23 NSCLC patients harboring EGFR exon 20 insertion mutations, only two (8.7%) presented an objective response to afatinib, indicating limited efficacy of afatinib for exon 20 insertion mutations.

    “Of the 23 NSCLC patients harboring EGFR exon 20 insertion mutations, only two (8.7%) patients presented an objective response, indicating the limited efficacy of afatinib for exon 20 insertion mutations.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [44] Wu, 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

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    No dose-limiting toxicity was observed during the 28-day evaluation period at any dose levels.

    “No dose-limiting toxicity was observed during the 28-day evaluation period at any dose levels.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] Brown, 2015, AZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer, New Engl J Med source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    BID007 is the only cell line harboring EGFR exon 20 insertion mutations, originally established by the authors.

    “To our knowledge BID007 is the only cell line harboring EGFR exon 20 insertion mutations, which we originally established [20].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Sequist, 2013, Structural, Biochemical, and Clinical Characterization of Epidermal Growth Factor Receptor (EGFR) Exon 20 Insertion Mutations in Lung Cancer, Sci Transl Med

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the phase I study, patients were assigned to once-daily dosing from 20 mg to 240 mg.

    “The patients were assigned to once-daily dosing from 20 mg to 240 mg.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Brown, 2015, AZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer, New Engl J Med source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In human clinical trials, osimertinib and rociletinib showed promising safety and efficacy, even though some hyperglycemia was observed in patients treated with rociletinib.

    “In human clinical trials, osimertinib and rociletinib showed promising safety and efficacy, even though some hyperglycemia was observed in patients treated with rociletinib [38, 39].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [38] Brown, 2015, AZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer, New Engl J Med source

      Not checked in the free run.

    • cites [39] Logan, 2015, Rociletinib in EGFR-Mutated Non-Small-Cell Lung Cancer, New Engl J Med source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    EGFR-TKI in vivo concentrations may vary among EGFR-TKIs.

    “Hence, EGFR-TKI in vivo concentrations may vary among EGFR-TKIs.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The pharmacokinetics and pharmacodynamics may vary among EGFR-TKIs.

  • May combine two claims.

    Interpretationown data

    Osimertinib may present a wide therapeutic window and is effective for several exon 20 insertion mutations.

    “These data indicate that osimertinib may present a wide therapeutic window and is effective for several exon 20 insertion mutations.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cell authentication for H1975 and H3255 was performed in June 2015.

    “Cell authentication for H1975 and H3255 was performed in June 2015.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    EGFR-TKIs highly selective to mutated EGFR but not wild-type EGFR can efficiently inhibit mutated EGFR in lung cancer cells without affecting wild type EGFR expressed in epithelial cells.

    “Hence, EGFR-TKIs highly selective to mutated EGFR, but not to wild-type EGFR, can efficiently inhibit mutated EGFR in lung cancer cells without affecting the wild type EGFR expressed in epithelial cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Side effects of EGFR-TKIs are most likely due to inhibition of wild type EGFR expressed in epithelial cells of skin, lungs, and gastrointestinal tract.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All experiments were performed at least three times.

    “All experiments were performed at least three times.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Actin was used as a loading control in immunoblotting.

    “Actin was used as a loading control.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The study biologically confirmed the efficacy and therapeutic window of osimertinib for EGFR exon 20 insertion mutations.

    “In summary, we biologically confirmed the efficacy and therapeutic window of osimertinib for EGFR exon 20 insertion mutations.”
    • Also stated in: § DISCUSSION · plain
      “Furthermore, we identified the therapeutic window of osimertinib for 1st generation EGFR-TKIs resistant exon 20 insertion mutations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Osimertinib and rociletinib are effective and safe EGFR-TKIs for EGFR T790M positive mutations.

    “These data indicate that osimertinib and rociletinib are effective and safe EGFR-TKIs for EGFR T790M positive mutations.”
    Quote in the paperSubstantial claim
47 of 47

  • The paper relies on this without support.

    There is little information regarding mutation-specific potency of EGFR-TKIs against various types of EGFR mutations.

    “However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations.”

  • The paper relies on this without support.

    There is no clear guideline regarding which EGFR-TKIs should be used for which mutation.

    “However, there is no clear guideline regarding which EGFR-TKIs should be used for which mutation.”

  • The paper relies on this without support.

    For 1st generation EGFR-TKI resistant exon 20 insertion mutations, no potent inhibitor has been reported.

    “For these 1st generation EGFR-TKIs resistant exon 20 insertion mutations, no potent inhibitor has been reported.”

  • The paper relies on this without support.

    Until now, there is no EGFR-TKI reported to be effective against exon 20 insertion mutations.

    “Until now, there is no EGFR-TKI reported to be effective against exon 20 insertion mutations.”

  • The authors state this as an assumption.

    The ratio of IC50 values of wild-type to mutated EGFR (selectivity index) estimates the therapeutic window of EGFR-TKIs.

    “To provide an estimation of the therapeutic window of EGFR-TKIs, we created an in vitro model by calculating the ratio of IC50 values of the wild type EGFR and mutated EGFR, referred to as the selectivity index (SI) (Figure 3B). In this model, the selectivity of each EGFR-TKI is high if the SI value is low.”

  • The paper relies on this without support.

    Side effects of EGFR-TKIs are most likely due to inhibition of wild type EGFR expressed in epithelial cells of skin, lungs, and gastrointestinal tract.

    “These side effects most likely due to the inhibition of the wild type EGFR expressed in the epithelial cells present in the skin, lungs, and gastrointestinal tract by EGFR-TKIs.”

  • The paper relies on this without support.

    EGFR is ubiquitously expressed in epithelial cells, and EGFR-TKIs induce toxicity when they affect the wild type EGFR in epithelial cells.

    “Because EGFR is ubiquitously expressed in epithelial cells, EGFR-TKIs induce toxicity when EGFR-TKIs affect the wild type EGFR in epithelial cells.”

  • The paper relies on this without support.

    The pharmacokinetics and pharmacodynamics may vary among EGFR-TKIs.

    “The pharmacokinetics and pharmacodynamics may vary among EGFR-TKIs.”

  • The paper relies on this without support.

    PC-9ER cells become resistant to erlotinib after chronic exposure to erlotinib through acquisition of EGFR T790M second mutation.

    “PC-9ER cells become resistant to erlotinib after chronic exposure to erlotinib through acquisition of EGFR T790M second mutation.”

  • The paper relies on this without support.

    This is the first report to clearly demonstrate the difference in mutation spectrum of EGFR-TKIs against various types of EGFR mutations.

    “To our knowledge, this is the first report, which clearly demonstrates the difference in term of mutation spectrum of EGFR-TKIs against various types of EGFR mutations.”
10 of 10

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Selectivity index is defined as a ratio but reported as negative

    Minor

    A ratio of positive IC50 values cannot be negative. The reported SI values around −2 therefore contradict the stated definition of SI as a ratio; a logarithmic transformation may have been intended but is not specified.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Mitsudomi, 2004, Mutations of the epidermal growth factor receptor gene in lung cancer: Biological and clinical implications, Cancer Res, 64, 8919, 10.1158/0008-5472.CAN-04-2818 · DOI
  2. Bezjak, 2005, Erlotinib in previously treated non-small-cell lung cancer, N Engl J Med, 353, 123, 10.1056/NEJMoa050753 · DOI · Paper page

Cited by

Sorted by citations

Authors
Hirano, T., Yasuda, H., Tani, T., Hamamoto, J., Oashi, A., Ishioka, K., Arai, D., Nukaga, S., Miyawaki, M., Kawada, I., Naoki, K., Costa, D. B., Kobayashi, S. S., Betsuyaku, T., & Soejima, K.
Affiliations
  • Keio University (JP)
  • Beth Israel Deaconess Medical Center (US)
Corresponding author
  • Hirano, Toshiyuki
  • Tani, Tetsuo
ORCID
Venue
Oncotarget
Publisher
Impact Journals, LLC
Published
2015-10-15
Volume
6
Issue
36
Pages
38789-38803
ISSN
1949-2553 (ISSN-L)
DOI
10.18632/oncotarget.5887
PMC
PMC4770737
OpenAlex
W1840732701
Cited by
165
References
42
Citations by year
2026: 3 · 2025: 10 · 2024: 15 · 2023: 12 · 2022: 21 · 2021: 28 · 2020: 22 · 2019: 23 · 2018: 17 · 2017: 8 · 2016: 6
FWCI
8.17
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Oncotarget (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion)
Funders
  • Japan Society for the Promotion of Science (15K09229)
  • National Cancer Institute (CA090578, R01CA169259, P50CA090578, P20CA090578, R21CA178301, R21CA17830)
Topics
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Lung Cancer Treatments and Mutations
  • Health Sciences › Medicine › Oncology › HER2/EGFR in Cancer Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
Keywords
EGFR tyrosine kinase inhibitors, EGFR mutation, non-small cell lung cancer, exon 20 insertion mutations, Ba/F3 cells, erlotinib, afatinib, osimertinib, rociletinib, gefitinib, EGFR inhibitor resistance, therapeutic window, drug potency comparison, lung cancer cell lines
MeSH terms
Carcinoma, Non-Small-Cell Lung, Drug Screening Assays, Antitumor, Exons, Humans, Lung Neoplasms, Mutation, Phosphorylation, Substrate Specificity, Cell Line, Tumor, Protein Kinase Inhibitors, ErbB Receptors

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

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