Next Article in Journal
TREM2 in Neurodegenerative Diseases: Mechanisms and Therapeutic Potential
Previous Article in Journal
Machine Learning for Multi-Omics Characterization of Blood Cancers: A Systematic Review
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cells
Volume 14
Issue 17
10.3390/cells14171386
cells-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Efficacy of Conventional and Novel Tyrosine Kinase Inhibitors for Uncommon EGFR Mutations—An In Vitro Study

by
Hana Oiki
1,
Kenichi Suda
1,2,*,
Akira Hamada
1,
Toshio Fujino
1,3,4,
Keiko Obata
1,
Yoshihisa Kobayashi
5,
Kazuko Sakai
6,
Shota Fukuda
1,
Shuta Ohara
1,
Masaoki Ito
1,
Junichi Soh
1,7,
Kazuto Nishio
6,
Tetsuya Mitsudomi
1,2 and
Yasuhiro Tsutani
1
1
Division of Thoracic Surgery, Department of Surgery, Kindai University Faculty of Medicine, Osaka-Sayama 589-8511, Japan
2
Division of Thoracic Surgery, Izumi City General Hospital, Izumi 594-0073, Japan
3
Krantz Family Center for Cancer Research, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA
4
Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
5
Division of Molecular Pathology, National Cancer Center Research Institute, Tokyo 104-0045, Japan
6
Department of Genome Biology, Kindai University Faculty of Medicine, Osaka-Sayama 589-8511, Japan
7
Department of Thoracic Surgery, Graduate School of Medicine, Osaka Metropolitan University, Osaka 558-8585, Japan
*
Author to whom correspondence should be addressed.
Cells 2025, 14(17), 1386; https://doi.org/10.3390/cells14171386
Submission received: 30 July 2025 / Revised: 2 September 2025 / Accepted: 3 September 2025 / Published: 4 September 2025
(This article belongs to the Section Cellular Pathology)
Browse Figures
Versions Notes

Abstract

Afatinib and osimertinib are current treatment options for non-small cell lung cancer (NSCLC) patients with uncommon epidermal growth factor receptor (EGFR) mutations, although their efficacy is limited. To explore potentially effective drugs for these patients, we evaluated the efficacy of conventional EGFR tyrosine kinase inhibitors (TKIs) and novel third-generation (3G) TKIs using in vitro models. Ba/F3 cells transformed with each of the five most frequent uncommon EGFR mutations, Del18 (delE709_T710insD), E709K, G719A, S768I, and L861Q, were used. The growth inhibitory effects of five novel 3G-TKIs, almonertinib, lazertinib, furmonertinib, rezivertinib, and befotertinib, in addition to currently available TKIs, were evaluated. We also explored for secondary resistant mutations to afatinib or osimertinib and TKIs that can overcome these resistances. Afatinib was active against all uncommon EGFR mutations tested. The 3G-TKIs were all active against the L861Q mutation and were inactive against the S768I mutation. Furmonertinib and befotertinib showed efficacy against exon 18 mutations (Del18, E709K, and G719A). In the acquired resistance models to afatinib or osimertinib, we found T790M or a novel T725M secondary mutation, respectively, both of which could be overcome by lazertinib. However, some afatinib-resistant cells acquired V769L/M secondary mutations that were refractory to all EGFR-TKIs tested. In conclusion, afatinib exhibited broad activity and some 3G-TKIs showed promising efficacy in the front-line setting. Lazertinib is a potential second-line option after acquisition of resistance to afatinib or osimertinib.

1. Introduction

Mutations in the epidermal growth factor receptor (EGFR) gene are the most frequent driver mutations in non-small cell lung cancer (NSCLC), especially in patients with East-Asian ethnicity and no history of smoking [1]. Numerous subtypes of EGFR mutations have been reported to date [2], and these mutations are usually classified as common mutations (L858R point mutation or exon 19 in-frame deletions) and uncommon mutations (all other mutations). Uncommon mutations are usually detected in about 10% of patients with EGFR mutations, irrespective of disease stage [3]. The common versus uncommon classification is useful when considering treatment strategies in advanced-stage settings, because uncommon mutations are usually less sensitive to some of the currently available EGFR tyrosine kinase inhibitors (TKIs) [4]. This observation has been validated in structure-based analysis; many of the uncommon EGFR mutations are classified into the P-loop alphaC-helix compressing subtype that is usually insensitive to first- (1G) and third-generation (3G) TKIs, while some (such as L861Q/R) are classified as classical-like EGFR mutations [5].
In the Lux-Lung clinical trials [6], afatinib monotherapy demonstrated a progression-free survival (PFS) of 10.7 months (95% confidence interval 5.6–14.7) in patients with NSCLC harboring uncommon EGFR mutations (excluding exon 20 insertion- and T790M-positive groups) in the front-line setting. This finding has been validated in the phase III ACHILLES study, which reported superior PFS with afatinib (10.6 months) over platinum plus pemetrexed (5.7 months) in NSCLC patients with uncommon EGFR mutations [7]. Osimertinib has also shown some activity against NSCLC in these patients, with phase II studies reporting a PFS of 9.4 (3.7–15.2) months in Japanese patients [8] and 8.2 months (5.9–10.5) in Korean patients [9]. As a result, afatinib or osimertinib are often used in daily clinical practice to treat advanced NSCLC with uncommon EGFR mutations. However, the PFS reported for these agents is shorter than that reported for patients harboring common EGFR mutations; for example, a PFS of 18.9 months has been reported in patients with common mutations receiving osimertinib [10]. Although the recent CHRYSALIS-2 study (cohort C) demonstrated promising efficacy for amivantamab plus lazertinib in patients with NSCLC harboring uncommon EGFR mutations [11] research into single-agent TKI regimens is still warranted because of the high toxicity associated with the amivantamab plus lazertinib combination.
Several novel 3G-TKIs are currently under clinical development, some of which may have activity against uncommon EGFR mutations. In addition, some of these new drugs may overcome the acquired resistance that can occur during treatment with currently available TKIs, including afatinib and osimertinib. Here, we used Ba/F3 cell models of NSCLC driven by uncommon EGFR mutations to evaluate the efficacy of conventional TKIs and novel 3G-TKIs in the first-line setting and the second line after afatinib or osimertinib treatment failure.

2. Materials and Methods

2.1. Data Collection from the cBioPortal Database

Data on EGFR mutation subtypes and their frequencies in NSCLC were extracted from the cBioPortal database (https://www.cbioportal.org) as of April 2024. We counted the total number of each mutation in exons 18–21 of EGFR, excluding L858R point mutation, exon 19 in-frame deletions, and exon 20 in-frame insertions.

2.2. Cell Lines and Reagents

The Ba/F3 cell line was purchased from Riken Bio Resource Center (Tsukuba, Japan). Ba/F3 cell lines driven by uncommon EGFR mutations, E709K, G719A, exon 18 deletion (delE709_T710insD), and S768I, as well as wild-type human EGFR were established in our previous studies [12,13]. In this study, Ba/F3 cells driven by EGFR L861Q mutation were established as reported previously [13], and the cells were cultured as previously described elsewhere [12,13]. Human recombinant EGF was purchased from Thermo Fisher Scientific (Waltham, MA, USA). First-generation (1G) EGFR-TKIs (gefitinib and erlotinib), second-generation (2G) EGFR-TKI (afatinib), and 3G EGFR-TKIs (osimertinib, furmonertinib, lazertinib, almonertinib, rezivertinib, and befotertinib) were purchased from Selleck Chemicals (Houston, TX, USA).

2.3. Establishment of Ba/F3 Cells Harboring EGFR L861Q Mutation

Ba/F3 cells driven by the EGFR L861Q mutation were established as described previously [13]. Briefly, the pBABE retroviral vector with a full-length cDNA fragment of human EGFR with the L861Q point mutation was purchased from Addgene (Cambridge, MA, USA). The pBABE construct was co-transfected into gpIRES-293 cells with the pVSV-G vector (Clontech, Mountain View, CA, USA) using FuGENE6 transfection reagent (Promega, Madison, MI, USA). Viral envelopes were generated to produce viral particles. After 48 h of transfection, the culture medium was collected and centrifuged at 1500× g for 45 min at 4 °C to concentrate the virus particles. Viral pellets were resuspended in DMEM (Sigma-Aldrich) and stored at −80 °C.

2.4. Growth Inhibition Assay

Ba/F3 cells were seeded in 96-well plates at a density of 2500 cells/well. After 24 h incubation, cells were exposed to each TKI at the concentrations determined based on the ranges of clinically achievable drug concentration. After 72 h, 10 µL of the tetrazolium salt WST-8 was added to each well (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) and the plates were incubated for an additional 1.5–3 h. The absorbance was read at 450 nm using a multiplate reader (Tecan, Mannedorf, Switzerland), and the growth inhibitory effect was calculated by comparing with DMSO-treated control cells. Growth inhibitory curves were generated and, at the same time, the half-maximal inhibitory concentration (IC50) values were automatically calculated using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). After calculating the IC50 values, the sensitivity index (SI), defined as the IC50 value divided by the trough concentration of each drug at the recommended dose (IC50/Ctrough × 100), was calculated. SIs are more appropriate parameters than IC50 values when comparing the efficacy of drugs because clinically achievable blood concentrations vary greatly. SIs have been shown to correlate with the clinical efficacy, such as response rates, of various molecular targeted drugs with one potential cut-off value of 5 [14]. We also evaluated the selectivity index, which was defined as the SI divided by the SI of Ba/F3 cells with wild-type EGFR. A smaller value of selectivity index indicates a stronger inhibitory effect on mutant EGFR expressed on tumor cells compared to wild-type EGFR expressed on non-cancerous cells.

2.5. Establishment of Resistant Clones to Afatinib and Osimertinib

The N-ethyl-N-nitrosourea (ENU, Sigma-Aldrich, St. Louis, MO, USA) mutagenesis technique was used to accelerate the establishment of afatinib- and osimertinib-resistant cells, as previously described [15]. Ba/F3 parental cells were initially treated with 100 mg/mL ENU for 24 h. After washing twice with cell culture medium, cells were cultured for 24 h and then plated in 96-well plates (10,000 cells/well) with 10 nM afatinib or 100 nM osimertinib. These drug concentrations were selected to be between the IC50 values of uncommon mutations and that of wild-type EGFR. We cultured the cells for 14–28 days, with a change in medium every 3 to 5 days. After establishing resistant cells, DNA was extracted using a DNeasy Blood & Tissue Kit (250) (QIAGEN, Venlo, The Netherlands), and secondary EGFR mutations were detected by direct sequencing as previously described [16]. We examined all wells with regrowth or randomly selected 12 wells if there were 13 or more wells with confluent cells. If no cells grew in the plate, the drug concentration was reduced by orders of magnitude (5 nM and 2.5 nM for afatinib, and 50 nM and 25 nM for osimertinib), and the same experiments were repeated.

3. Results

3.1. Frequency of Uncommon EGFR Mutations in cBioPortal Database

By evaluating data obtained from cBioPortal, we found that G719X, L861X, S768I, Del18 (delE709_T710insD), and E709X were the five most frequent uncommon EGFR mutations in patients with NSCLC (Figure 1). Therefore, we decided to evaluate the efficacies of the conventional and novel EGFR-TKIs using Ba/F3 cells harboring these five mutations.

3.2. Efficacy of Novel TKIs Against Uncommon EGFR Mutations

We evaluated the inhibitory effects of gefitinib and erlotinib (1G-), afatinib (2G-), and osimertinib, almonertinib, lazertinib, furmonertinib, rezivertinib, and befotertinib (3G-TKIs) against Ba/F3 cells harboring one of uncommon EGFR mutations to identify TKIs with activity against these mutations. In addition, the efficacies of these drugs were evaluated in Ba/F3 cells harboring wild-type EGFR supplemented with 20 ng/mL of human recombinant EGF to assess the side effects of the drugs on non-cancerous cells. Growth inhibitory curves are shown in Figure 2a,b, and in Supplementary Figure S1. IC50 values and SIs, which indicate efficacy of drug adjusted with clinically achievable drug concentration, are summarized in Figure 2c and Figure 2d, respectively.
As shown in Figure 2c,d, afatinib showed the greatest inhibitory effect against all tested Ba/F3 cell models with uncommon EGFR mutations. Erlotinib was active against L861Q and G719A mutations, and osimertinib was active against the L861Q mutation only when we defined being active by an SI of <5. However, we observed that the ratios of SIs between uncommon EGFR mutations and wild-type, hereafter defined as the selectivity index, were smallest in osimertinib (≤15), followed by afatinib (≤17), gefitinib (≤85), and erlotinib (≤200), suggesting that osimertinib may also work clinically, while preserving the phosphorylation of wild-type EGFR in non-cancerous cells (Supplementary Figure S2). Among novel 3G-TKIs, we found that all were active against Ba/F3 cells with the L861Q mutation, and all had low efficacy (SIs over 10) against Ba/F3 cells with the S768I mutation. Comparing among the 3G-TKIs, befotertinib was active against all uncommon EGFR mutations other than S768I, whereas furmonertinib had the smallest selectivity index (6.9 or smaller) versus osimertinib and afatinib (Supplementary Figure S2).
These results suggest that befotertinib or furmonertinib (especially for higher dosing) are potentially useful 3G-TKIs against NSCLC in patients harboring uncommon EGFR mutations. Afatinib and osimertinib are also reasonable treatment options. The potential utility of other drugs (gefitinib, erlotinib, lazertinib, almonertinib, and rezivertinib) could be considered for each mutation variant in accordance with the results summarized in Figure 2d.

3.3. Secondary Resistance Mutations to Osimertinib and Strategies to Overcome Resistance

To explore useful TKIs in the second-line setting, we examined potential secondary mutations that may confer acquired resistance to osimertinib using Ba/F3 cells with the three most frequent uncommon mutations (G719A, S768I, and L861Q). After exposing the Ba/F3 cells to ENU, we treated them with osimertinib (starting at 100 nM) for a few weeks.
We identified the T725M secondary EGFR mutation (Figure 3 and Figure 4a) in Ba/F3 cells with the G719A mutation in all viable wells tested. In contrast, we detected no secondary mutations in the kinase domain of the EGFR gene in Ba/F3 cells with S768I or L861Q mutations after treatment with osimertinib (Figure 3).
In the growth inhibitory analysis (Figure 4b), Ba/F3 cells with G719A/T725M mutations showed an IC50 value of 165.6 nM for osimertinib that was 4.1-fold higher than parental Ba/F3 cells with only the G719A mutation. We observed that Ba/F3 cells with G719A/T725M were insensitive to all novel 3G-TKIs except for lazertinib. In addition, we found that afatinib was active against Ba/F3 cells with G719A/T725M mutations (Figure 4c,d). The selectivity index was also summarized in Supplementary Figure S3.

3.4. Secondary Resistance Mutations to Afatinib and Strategies to Overcome Resistance

To explore useful TKIs in the second-line setting after afatinib treatment, we examined potential secondary mutations that may confer acquired resistance to afatinib. After ENU exposure, we treated these Ba/F3 cells with afatinib (starting at 10 nM) for a few weeks.
In contrast to osimertinib-resistant cells, we detected several different secondary mutations depending on the activating EGFR mutation subtype (Figure 5). In the G719A model, secondary T790M mutation emerged in all six established wells. Ba/F3 cells with G719A/T790M had a 7.1-fold higher IC50 value compared with G719A parental cells (Figure 4c). T790M secondary mutation also emerged in cells with S768I mutation at the lowest drug concentration (2.5 nM), and Ba/F3 cells with S768I/T790M had a 146-fold higher IC50 value compared with the parental cells. Furthermore, several different substitutions involving V769 were found in Ba/F3 cells with S768I or L861Q mutations.
To explore EGFR-TKIs that may overcome these secondary mutations induced by afatinib, we also evaluated the efficacy of osimertinib and other novel 3G-TKIs. We observed that most of the 3G-TKIs tested were active against Ba/F3 cells with G719A/T790M and S861Q/V769L; however, only lazertinib was effective against Ba/F3 cells with S768I/T790M mutations (Figure 4d). In contrast, Ba/F3 cells with S768I/V769L or S768I/V769M mutations were refractory to all EGFR-TKIs tested. The selectivity index was also summarized in Supplementary Figure S3.

4. Discussion

Various studies have reported that afatinib and osimertinib are effective against NSCLCs harboring uncommon EGFR mutations; therefore, these drugs are usually the treatment of choice in clinical practice [6,8,9,31,32,33]. However, treatment outcomes with afatinib or osimertinib in NSCLC patients with uncommon EGFR mutations are not satisfactory compared with osimertinib treatment of NSCLC in patients with common EGFR mutations. Therefore, as a first step in this study, we screened novel 3G-TKIs to identify the most effective agent using in vitro models with uncommon EGFR mutations. Because Ba/F3 cells transformed by mutated EGFR are solely depend their proliferation and survival on signaling from the mutated (and phosphorylated) EGFR and we have observed correlation between growth inhibitory effects and decreased phosphorylation of driver mutation in some of our previous studies [12], we did not perform Western blotting in this study. As the first result, we found that afatinib was active (SI < 5) against all uncommon EGFR mutations, but that osimertinib was less active against many of the uncommon mutations tested. This result is in agreement with a recent pooled analysis comparing the efficacy of afatinib versus osimertinib using propensity score-matching in patients with NSCLC harboring uncommon EGFR mutations [34]. However, in our analysis of novel 3G-TKIs, we observed that befotertinib and furmonertinib could be promising TKIs for patients with many of the uncommon EGFR mutations, such as L861Q, G719A, or E709K, when considering IC50 values, clinically achievable drug concentrations, and the inhibitory effect of wild-type EGFR (selectivity index as defined in the Methods section). Although no clear cut-off value was defined for the selectivity index in this study, it would be possible to increase the dosage of TKIs with small selectivity index, such as furmonertinib (Supplementary Figure S2) to enhance the efficacy similar to a previous study of high-dose TKI treatment [35]. Based on these results, we suggest that NSCLC harboring uncommon EGFR mutations should not be treated as a single disease; rather, we should determine an appropriate TKI (and appropriate drug concentrations as reported recently [36]) for each mutation subtype.
As the next step, we explored TKIs that can overcome acquired resistance to front-line osimertinib or afatinib. To evaluate potential secondary mutations associated with acquired resistance to osimertinib or afatinib, we used ENU mutagenesis to establish cells with acquired resistance. Although this experimental model has been widely used to detect secondary resistant mutations that may occur in patients who experience acquisition of resistance to TKI(s) [14], as a limitation of this model, we could not rule out the possibility that ENU-induced mutations in genes other than EGFR affected the growth inhibition results. However, because Ba/F3 cells with secondary EGFR mutations, which were established in this study, showed relatively low IC50 values to afatinib and/or lazertinib, we consider that these cells were still dependent on signaling from mutated EGFR. As one of secondary mutations, we detected T725M in Ba/F3 cells with G719A that acquired resistance to osimertinib. Although a machine-learning approach in a previous study has suggested the transforming ability of the EGFR T725M mutation [37], this mutation is very rare in clinical practice. By exploring the COSMIC database, we observed that five cases of EGFR T725M mutation have been reported (Supplementary Table S1). None had a concurrent G719X mutation, although some patients had concurrent L858R mutation. In addition, through a literature search, we found two studies that reported detecting T725M mutations in tumors after osimertinib treatment [38,39]. Therefore, it is reasonable that the T725M secondary mutation emerged after osimertinib exposure in our in vitro model. Our results also suggest that afatinib or lazertinib could overcome the T725M secondary mutation in EGFR G719A-positive patients.
Furthermore, we observed that T790M or V769M/L secondary mutations emerged after afatinib exposure in Ba/F3 cell models with G719A, S768I, or L861Q mutations. We had expected the emergence of the T790M secondary mutation because it has been reported as an acquired resistance mechanism to afatinib in NSCLC among patients with common EGFR mutations. Furthermore, it is reasonable that the T790M secondary mutation could be overcome by 3G-TKIs, which were designed to overcome the T790M mutation. Our results showed that lazertinib was the most effective 3G-TKI in this setting.
Among secondary resistant mutations to afatinib, we also observed V769M and V769L mutations in models with S768I and L861Q EGFR-activating mutations. Previous studies have reported that the EGFR V769M mutation is a frequent EGFR germ-line mutation [40,41]; however, it has also been reported that V769M/L mutations have emerged as somatic mutations, usually together with another EGFR-activating mutation, as summarized in Supplementary Table S2 [42,43]. Interestingly, V769L often co-exists with the EGFR S768I mutation, although the molecular mechanisms responsible for this co-existence are unclear. Two of the affected patients had efficacy data for TKI treatment; neither showed a response to erlotinib or afatinib (Supplementary Table S2). In addition to COSMIC data, additional case studies have reported TKI efficacy against NSCLCs with S768I plus V769L compound mutation; only one patient responded to full-dose afatinib [44], whereas the other two patients showed inherent resistance to gefitinib [45] or lower-dose afatinib [46]. Currently, few case studies have reported the emergence of V769X as a secondary mutation [47]. Therefore, future studies are needed to evaluate the frequency of this mutation after acquiring afatinib resistance in patients with NSCLC harboring uncommon EGFR mutations. It should be noted that one patient with the G719A plus V769M compound mutation responded to upfront osimertinib [48].

5. Conclusions

Our in vitro study demonstrated that afatinib showed universal activity against various uncommon EGFR mutations, while 3G-TKIs, especially furmonertinib and befotertinib, also showed high efficacy against all these mutations, except S768I. Therefore, we suggest that NSCLC with uncommon EGFR mutations should not be treated as a single disease but should be treated based on mutation subtype. Furthermore, we detected several on-target resistance mutations of EGFR, such as T725M, T790M, and V769M/L, after exposure to osimertinib or afatinib, and our results suggest that lazertinib may overcome some of these secondary mutations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells14171386/s1, Figure S1: Growth inhibitory curves of EGFR-TKIs tested in this study; Figure S2: Summaries of the growth inhibitory effects of EGFR-TKIs tested against Ba/F3 cells harboring an uncommon EGFR mutation based on the selectivity index; Figure S3: Summaries of the growth inhibitory effects of EGFR-TKIs tested against Ba/F3 cells with secondary resistant mutation based on the selectivity index; Table S1: T725M mutation reported in lung cancers in COSMIC database (accessed at 31 January 2025); Table S2: V769L/M mutations reported in lung cancers in COSMIC database (accessed at 31 January 2025).

Author Contributions

Conceptualization, K.S. (Kenichi Suda) and A.H.; methodology, H.O., K.S. (Kenichi Suda), T.F., Y.K. and T.M.; validation, K.S. (Kenichi Suda), K.O., M.I., J.S. and Y.T.; formal analysis, H.O. and K.S. (Kenichi Suda); investigation, H.O., T.F., K.O., S.F., S.O. and M.I.; resources, K.S. (Kenichi Suda), T.F., Y.K., K.S. (Kazuko Sakai), K.N. and T.M.; writing—original draft preparation, H.O. and K.S. (Kenichi Suda); writing—review and editing, all authors; visualization, H.O., K.S. (Kenichi Suda) and T.F.; supervision, K.S. (Kenichi Suda), K.N., T.M. and Y.T.; project administration, K.S. (Kenichi Suda), T.M. and Y.T.; funding acquisition, K.S. (Kenichi Suda), A.H. and J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (grants 22K07291 to Kenichi Suda, 23K15569 to A.H., and 22K08986 to J.S.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The findings of this study are available from the corresponding author (K.S.) upon reasonable request.

Acknowledgments

The authors thank Yoshihiro Mine (Center for Instrumental Analyses, Central Research Facilities, Kindai University Faculty of Medicine) for his technical assistance.

Conflicts of Interest

Dr. Suda received research funding from AstraZeneca K.K.; honoraria from Chugai Pharmaceutical Co., Ltd., Boehringer Ingelheim and AstraZeneca K.K.; and has previously undertaken an advisory role for AstraZeneca K.K. and Chugai Pharmaceutical Co., Ltd. Dr. Hamada has received research funding from AstraZeneca and Chugai Pharmaceutical Co., Ltd.; honoraria from AstraZeneca K.K. and Chugai Pharmaceutical Co., Ltd.; and has undertaken an advisory board for AstraZeneca and Chugai Pharmaceutical Co., Ltd. Dr. Kobayashi has received honoraria from AstraZeneca K.K. and Chugai Pharmaceutical Co., Ltd. Dr. Soh has received research funding from Chugai Pharmaceutical Co., Ltd. and honoraria from Chugai Pharmaceutical Co., Ltd. Dr. Nishio has received research funding from Nippon Boehringer Ingelheim Co., Ltd. and honoraria from Chugai Pharmaceutical Co Ltd., Janssen Pharmaceutical K.K., and AstraZeneca K.K. Dr. Mitsudomi has received honoraria from AstraZeneca K.K., Chugai Pharmaceutical Co, Janssen Pharmaceutical K.K. and has a patent for AstraZeneca K.K. pending. Dr. Tsutani has undertaken an advisory role for AstraZeneca K.K. and Chugai Pharmaceutical Co, Ltd.; received honoraria from AstraZeneca K.K., Chugai Pharmaceutical Co, Ltd.; and has received research funding from Nippon Boehringer Ingelheim Co, Ltd. and Chugai Pharmaceutical Co, Ltd. The remaining authors declare no conflicts of interest related to this work.

Abbreviations

The following abbreviations are used in this manuscript:
EGFREpidermal growth factor receptor
ENUN-ethyl-N-nitrosourea
IC50Half maximal inhibitory concentration
NSCLCNon-small cell lung cancer
PFSProgression-free survival
SISensitivity index
TKITyrosine kinase inhibitor
1GFirst-generation
2GSecond-generation
3GThird-generation

References

  1. Shigematsu, H.; Lin, L.; Takahashi, T.; Nomura, M.; Suzuki, M.; Wistuba, I.I.; Fong, F.M.; Lee, H.; Toyooka, S.; Shimizu, N.; et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J. Natl. Cancer Inst. 2005, 97, 339–346. [Google Scholar] [CrossRef] [PubMed]
  2. Du, Z.; Sun, J.; Zhang, Y.; Hesilaiti, N.; Xia, Q.; Cui, H.; Fan, N.; Xu, X. Structure-Guided Strategies of Targeted Therapies for Patients with EGFR-Mutant Non-Small Cell Lung Cancer. Biomolecules 2023, 13, 210. [Google Scholar] [CrossRef] [PubMed]
  3. Suda, K.; Mitsudomi, T.; Shintani, Y.; Okami, J.; Ito, H.; Ohtsuka, T.; Toyooka, S.; Mori, T.; Watanabe, S.I.; Asamura, H.; et al. Clinical Impacts of EGFR Mutation Status: Analysis of 5780 Surgically Resected Lung Cancer Cases. Ann. Thorac. Surg. 2021, 111, 269–276. [Google Scholar] [CrossRef]
  4. Gomez-Randulfe, I.; Scanlon, L.A.; Carter, M.; Moliner, L.; Cil, E.; Califano, R.; Summers, Y.; Blackhall, F.; Lindsay, C.R.; Lewis, J.; et al. First-line osimertinib compared to earlier generation TKIs in advanced EGFR-mutant NSCLC: A real-world survival analysis. Lung Cancer 2025, 200, 108084. [Google Scholar] [CrossRef]
  5. Robichaux, J.P.; Le, X.; Vijayan, R.S.K.; Hicks, J.K.; Heeke, S.; Elamin, Y.Y.; Lin, H.Y.; Udagawa, H.; Skoulidis, F.; Tran, H.; et al. Structure-based classification predicts drug response in EGFR-mutant NSCLC. Nature 2021, 597, 732–737. [Google Scholar] [CrossRef]
  6. Yang, J.C.; Sequist, L.V.; Geater, S.L.; Tsai, C.M.; Mok, T.S.; Schuler, M.; Yamamoto, N.; Yu, C.J.; Ou, S.H.; Zhou, C.; et al. 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. 2015, 16, 830–838. [Google Scholar] [CrossRef]
  7. Miura, H.T.S.; Misumi, T. LBA66 Afatinib versus chemotherapy for treatment-naïve non-small cell lung cancer with a sensitizing uncommon epidermal growth factor receptor mutation: A phase III study (ACHILLES/TORG1834). Ann. Oncol. 2023, 34, S1310–S1311. [Google Scholar] [CrossRef]
  8. Okuma, Y.; Kubota, K.; Shimokawa, M.; Hashimoto, K.; Kawashima, Y.; Sakamoto, T.; Wakui, H.; Murakami, S.; Okishio, K.; Hayashihara, K.; et al. First-Line Osimertinib for Previously Untreated Patients with NSCLC and Uncommon EGFR Mutations: The UNICORN Phase 2 Nonrandomized Clinical Trial. JAMA Oncol. 2024, 10, 43–51. [Google Scholar] [CrossRef]
  9. Cho, J.H.; Lim, S.H.; An, H.J.; Kim, K.H.; Park, K.U.; Kang, E.J.; Choi, Y.H.; Ahn, M.S.; Lee, M.H.; Sun, J.M.; et al. Osimertinib for Patients with Non-Small-Cell Lung Cancer Harboring Uncommon EGFR Mutations: A Multicenter, Open-Label, Phase II Trial (KCSG-LU15–09). J. Clin. Oncol. 2020, 38, 488–495. [Google Scholar] [CrossRef] [PubMed]
  10. 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. 2018, 378, 113–125. [Google Scholar] [CrossRef]
  11. Catherine, A.; Shu, K.G.; Cho, B.C.; Griesinger, F.; Yang, J.C.-H.; Felip, E.; Xie, J.; Chen, J.; Mahoney, J.; Thayu, M.; et al. CHRYSALIS-2: A phase 1/1b study of lazertinib as monotherapy and in combination with amivantamab in patients with EGFR-mutant NSCLC. J. Clin. Oncol. 2021, 39, TPS9132. [Google Scholar] [CrossRef]
  12. Banno, E.; Togashi, Y.; Nakamura, Y.; Chiba, M.; Kobayashi, Y.; Hayashi, H.; Terashima, M.; de Velasco, M.A.; Sakai, K.; Fujita, Y.; et al. Sensitivities to various epidermal growth factor receptor-tyrosine kinase inhibitors of uncommon epidermal growth factor receptor mutations L861Q and S768I: What is the optimal epidermal growth factor receptor-tyrosine kinase inhibitor? Cancer Sci. 2016, 107, 1134–1140. [Google Scholar] [CrossRef]
  13. Kobayashi, Y.; Togashi, Y.; Yatabe, Y.; Mizuuchi, H.; Jangchul, P.; Kondo, C.; Shimoji, M.; Sato, K.; Suda, K.; Tomizawa, K.; et al. 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. 2015, 21, 5305–5313. [Google Scholar] [CrossRef]
  14. Koga, T.; Suda, K.; Mitsudomi, T. Utility of the Ba/F3 cell system for exploring on-target mechanisms of resistance to targeted therapies for lung cancer. Cancer Sci. 2022, 113, 815–827. [Google Scholar] [CrossRef]
  15. Bradeen, H.A.; Eide, C.A.; O’Hare, T.; Johnson, K.J.; Willis, S.G.; Lee, F.Y.; Druker, B.J.; Deininger, M.W. Comparison of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: High efficacy of drug combinations. Blood 2006, 108, 2332–2338. [Google Scholar] [CrossRef]
  16. Suda, K.; Murakami, I.; Katayama, T.; Tomizawa, K.; Osada, H.; Sekido, Y.; Maehara, Y.; Yatabe, Y.; Mitsudomi, T. Reciprocal and complementary role of MET amplification and EGFR T790M mutation in acquired resistance to kinase inhibitors in lung cancer. Clin. Cancer Res. 2010, 16, 5489–5498. [Google Scholar] [CrossRef]
  17. Nakagawa, K.; Tamura, T.; Negoro, S.; Kudoh, S.; Yamamoto, N.; Yamamoto, N.; Takeda, K.; Swaisland, H.; Nakanishi, I.; Hirose, M.; et al. Phase I pharmacokinetic trial of the selective oral epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (‘Iressa’, ZD1839) in Japanese patients with solid malignant tumors. Ann. Oncol. 2003, 14, 922–930. [Google Scholar] [CrossRef] [PubMed]
  18. Hidalgo, M.; Siu, L.L.; Nemunaitis, J.; Rizzo, J.; Hammond, L.A.; Takimoto, C.; Eckhardt, S.G.; Tolcher, A.; Britten, C.D.; Denis, L.; et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J. Clin. Oncol. 2001, 19, 3267–3279. [Google Scholar] [CrossRef] [PubMed]
  19. Yap, T.A.; Vidal, L.; Adam, J.; Stephens, P.; Spicer, J.; Shaw, H.; Ang, J.; Temple, G.; Bell, S.; Shahidi, M.; et al. Phase I trial of the irreversible EGFR and HER2 kinase inhibitor BIBW 2992 in patients with advanced solid tumors. J. Clin. Oncol. 2010, 28, 3965–3972. [Google Scholar] [CrossRef] [PubMed]
  20. Tsubata, Y.; Watanabe, K.; Saito, R.; Nakamura, A.; Yoshioka, H.; Morita, M.; Honda, R.; Kanaji, N.; Ohizumi, S.; Jingu, D.; et al. Osimertinib in poor performance status patients with T790M-positive advanced non-small-cell lung cancer after progression of first- and second-generation EGFR-TKI treatments (NEJ032B). Int. J. Clin. Oncol. 2022, 27, 112–120. [Google Scholar] [CrossRef] [PubMed]
  21. Jänne, P.A.; Yang, J.C.; Kim, D.W.; Planchard, D.; Ohe, Y.; Ramalingam, S.S.; Ahn, M.J.; Kim, S.W.; Su, W.C.; Horn, L.; et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. N. Engl. J. Med. 2015, 372, 1689–1699. [Google Scholar] [CrossRef] [PubMed]
  22. Kruithof, P.D.; Brouns, A.; Degens, J.; Hendriks, L.E.L.; Wong, D.R.; van Geel, R.; Croes, S. Osimertinib Dose Optimization Guided by Therapeutic Drug Monitoring in Patients with EGFR-Mutated Advanced Non-Small Cell Lung Cancer: A Case series. Clin. Lung Cancer 2025, in press. [Google Scholar] [CrossRef]
  23. Wu, Y.L.; Xue, Y.R.; Guo, Z.T.; Chen, Z.D.; Ge, X.Y.; Zhong, D.F.; Diao, X.X. Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes. Acta Pharmacol. Sin. 2022, 43, 747–756. [Google Scholar] [CrossRef]
  24. Ahn, M.J.; Han, J.Y.; Lee, K.H.; Kim, S.W.; Kim, D.W.; Lee, Y.G.; Cho, E.K.; Kim, J.H.; Lee, G.W.; Lee, J.S.; et al. Lazertinib in patients with EGFR mutation-positive advanced non-small-cell lung cancer: Results from the dose escalation and dose expansion parts of a first-in-human, open-label, multicentre, phase 1–2 study. Lancet Oncol. 2019, 20, 1681–1690. [Google Scholar] [CrossRef] [PubMed]
  25. Cho, B.C.; Han, J.Y.; Kim, S.W.; Lee, K.H.; Cho, E.K.; Lee, Y.G.; Kim, D.W.; Kim, J.H.; Lee, G.W.; Lee, J.S.; et al. A Phase 1/2 Study of Lazertinib 240 mg in Patients with Advanced EGFR T790M-Positive NSCLC After Previous EGFR Tyrosine Kinase Inhibitors. J. Thorac. Oncol. 2022, 17, 558–567. [Google Scholar] [CrossRef]
  26. Yang, J.C.; Camidge, D.R.; Yang, C.T.; Zhou, J.; Guo, R.; Chiu, C.H.; Chang, G.C.; Shiah, H.S.; Chen, Y.; Wang, C.C.; et al. Safety, Efficacy, and Pharmacokinetics of Almonertinib (HS-10296) in Pretreated Patients with EGFR-Mutated Advanced NSCLC: A Multicenter, Open-label, Phase 1 Trial. J. Thorac. Oncol. 2020, 15, 1907–1918. [Google Scholar] [CrossRef]
  27. Shi, Y.; Zhao, Y.; Yang, S.; Zhou, J.; Zhang, L.; Chen, G.; Fang, J.; Zhu, B.; Li, X.; Shu, Y.; et al. Safety, Efficacy, and Pharmacokinetics of Rezivertinib (BPI-7711) in Patients with Advanced NSCLC with EGFR T790M Mutation: A Phase 1 Dose-Escalation and Dose-Expansion Study. J. Thorac. Oncol. 2022, 17, 708–717. [Google Scholar] [CrossRef]
  28. Shi, Y.; Wu, S.; Wang, K.; Cang, S.; Yao, W.; Fan, Y.; Wu, L.; Huang, M.; Li, X.; Pan, Y.; et al. Efficacy and Safety of Rezivertinib (BPI-7711) in Patients with Locally Advanced or Metastatic/Recurrent EGFR T790M-Mutated NSCLC: A Phase 2b Study. J. Thorac. Oncol. 2022, 17, 1306–1317. [Google Scholar] [CrossRef]
  29. Lu, S.; Zhang, Y.; Zhang, G.; Zhou, J.; Cang, S.; Cheng, Y.; Wu, G.; Cao, P.; Lv, D.; Jian, H.; et al. Efficacy and Safety of Befotertinib (D-0316) in Patients with EGFR T790M-Mutated NSCLC That Had Progressed After Prior EGFR Tyrosine Kinase Inhibitor Therapy: A Phase 2, Multicenter, Single-Arm, Open-Label Study. J. Thorac. Oncol. 2022, 17, 1192–1204. [Google Scholar] [CrossRef]
  30. Lu, S.; Zhou, J.; Jian, H.; Wu, L.; Cheng, Y.; Fan, Y.; Fang, J.; Chen, G.; Zhang, Z.; Lv, D.; et al. Befotertinib (D-0316) versus icotinib as first-line therapy for patients with EGFR-mutated locally advanced or metastatic non-small-cell lung cancer: A multicentre, open-label, randomised phase 3 study. Lancet Respir. Med. 2023, 11, 905–915. [Google Scholar] [CrossRef] [PubMed]
  31. Chang, L.C.; Lim, C.K.; Chang, L.Y.; Chen, K.Y.; Shih, J.Y.; Yu, C.J. Non-small cell lung cancer harbouring non-resistant uncommon EGFR mutations: Mutation patterns, effectiveness of epidermal growth factor receptor-tyrosine kinase inhibitors and prognostic factors. Eur. J. Cancer 2019, 119, 77–86. [Google Scholar] [CrossRef]
  32. Tanaka, I.; Morise, M.; Kodama, Y.; Matsui, A.; Ozawa, N.; Ozone, S.; Goto, D.; Miyazawa, A.; Hase, T.; Hashimoto, N.; et al. Potential for afatinib as an optimal treatment for advanced non-small cell lung carcinoma in patients with uncommon EGFR mutations. Lung Cancer 2019, 127, 169–171. [Google Scholar] [CrossRef]
  33. Yang, J.C.; Schuler, M.; Popat, S.; Miura, S.; Heeke, S.; Park, K.; Märten, A.; Kim, E.S. Afatinib for the Treatment of NSCLC Harboring Uncommon EGFR Mutations: A Database of 693 Cases. J. Thorac. Oncol. 2020, 15, 803–815. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, C.; Zhao, K.; Hu, S.; Dong, W.; Gong, Y.; Xie, C. Clinical Outcomes of Afatinib Versus Osimertinib in Patients with Non-Small Cell Lung Cancer with Uncommon EGFR Mutations: A Pooled Analysis. Oncologist 2023, 28, e397–e405. [Google Scholar] [CrossRef] [PubMed]
  35. Fang, W.F.; Bu, Q.; Wang, Q.; Zhao, W.; Wang, L.; Dong, X.; Chen, P.; Wen, Z.; Jia, J.; Jiang, G.; et al. 57P: Safety and efficacy of aumolertinib treatment in patients with advanced NSCLC harboring 20ins and uncommon EGFR mutations (AIM). J. Thorac. Oncol. 2025, 20 (Suppl. 1), S45–S46. [Google Scholar] [CrossRef]
  36. Planchard, D.; Le, X.; Yu, Y.; Zhao, Y. FURTHER (FURMO-002): A Global, Randomized Study of Firmonertinib at Two Dose Levels in TKI-Naïve, Advanced NSCLC with EGFR PACC Mutations. In Proceedings of the 2024 World Conference on Lung Cancer, San Diego, CA, USA, 7 October 2024. [Google Scholar]
  37. ManChon, U.; Talevich, E.; Katiyar, S.; Rasheed, K.; Kannan, N. Prediction and prioritization of rare oncogenic mutations in the cancer Kinome using novel features and multiple classifiers. PLoS Comput. Biol. 2014, 10, e1003545. [Google Scholar]
  38. Lin, Q.L.; Ran, C.; Ou, Q.X.; Ma, Y.T.; Bao, H.; Wu, X.; Shao, Y.; Wang, Z.X. Acquired rare recurrent EGFR mutations as mechanisms of resistance to Osimertinib in lung cancer and in silico structural modelling. Am. J. Cancer Res. 2020, 10, 4005. [Google Scholar]
  39. Janning, M.; Süptitz, J.; Albers-Leischner, C.; Delpy, P.; Tufman, A.; Velthaus-Rusik, J.L.; Reck, M.; Jung, A.; Kauffmann-Guerrero, D.; Bonzheim, I.; et al. Treatment outcome of atypical EGFR mutations in the German National Network Genomic Medicine Lung Cancer (nNGM). Ann. Oncol. 2022, 33, 602–615. [Google Scholar] [CrossRef]
  40. Hellmann, M.D.; Hayashi, T.; Reva, B.; Yu, H.A.; Riely, G.J.; Adusumilli, P.S.; Travis, W.D.; Wilkins, O.; Bramletta, N.; Chandramohan, R.; et al. Identification and Functional Characterization of EGFR V769M, a Novel Germline Variant Associated with Multiple Lung Adenocarcinomas. JCO Precis. Oncol. 2017, 1, 1–10. [Google Scholar] [CrossRef] [PubMed]
  41. Lin, X.; Peng, M.; Chen, Q.; Yuan, M.; Chen, R.; Deng, H.; Deng, J.; Liu, O.; Weng, Y.; Chen, M.; et al. Identification of the Unique Clinical and Genetic Features of Chinese Lung Cancer Patients with EGFR Germline Mutations in a Large-Scale Retrospective Study. Front. Oncol. 2021, 11, 774156. [Google Scholar] [CrossRef]
  42. Huang, S.F.; Liu, H.P.; Li, L.H.; Ku, Y.C.; Fu, Y.N.; Tsai, H.Y.; Chen, Y.T.; Lin, Y.F.; Chang, W.C.; Kuo, H.P.; et al. High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin. Cancer Res. 2004, 10, 8195–8203. [Google Scholar] [CrossRef]
  43. Wu, J.Y.; Yu, C.J.; Chang, Y.C.; Yang, C.H.; Shih, J.Y.; Yang, P.C. Effectiveness of tyrosine kinase inhibitors on “uncommon” epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clin. Cancer Res. 2011, 17, 3812–3821. [Google Scholar] [CrossRef] [PubMed]
  44. Zhang, H.; Shao, Y.W.; Xia, Y. Responsiveness to Full-Dose Afatinib in a Patient with Lung Adenocarcinoma Harboring EGFR S768I and V769L Mutations. J. Thorac. Oncol. 2019, 14, e25–e27. [Google Scholar] [CrossRef] [PubMed]
  45. Asahina, H.; Yamazaki, K.; Kinoshita, I.; Yokouchi, H.; Dosaka-Akita, H.; Nishimura, M. Non-responsiveness to gefitinib in a patient with lung adenocarcinoma having rare EGFR mutations S768I and V769L. Lung Cancer 2006, 54, 419–422. [Google Scholar] [CrossRef] [PubMed]
  46. Niogret, J.; Coudert, B.; Boidot, R. Primary Resistance to Afatinib in a Patient with Lung Adenocarcinoma Harboring Uncommon EGFR Mutations: S768I and V769L. J. Thorac. Oncol. 2018, 13, e113. [Google Scholar] [CrossRef]
  47. Deng, Q.; Xie, B.; Wu, L.; Ji, X.; Li, C.; Feng, L.; Fang, Q.; Bao, Y.; Li, J.; Jin, S.; et al. Competitive evolution of NSCLC tumor clones and the drug resistance mechanism of first-generation EGFR-TKIs in Chinese NSCLC patients. Heliyon 2018, 4, e01031. [Google Scholar] [CrossRef]
  48. Simionato, F.; Calvetti, L.; Cosci, M.; Scarparo, S.; Aprile, G. Case Report: A Metabolic Complete Response to Upfront Osimertinib in a Smoker Non-Small Cell Lung Cancer Patient Harbouring EGFR G719A/V769M Complex Mutation. Onco Targets Ther. 2020, 13, 12027–12031. [Google Scholar] [CrossRef]
Cells 14 01386 g001
Figure 1. Frequencies of uncommon EGFR mutations, excluding exon 20 insertion mutations, reported to the cBioPortal database in patients with NSCLC.
Figure 1. Frequencies of uncommon EGFR mutations, excluding exon 20 insertion mutations, reported to the cBioPortal database in patients with NSCLC.
Cells 14 01386 g001
Cells 14 01386 g002
Figure 2. Efficacy of 1G-, 2G-, and 3G EGFR-TKIs against Ba/F3 cells harboring one of uncommon EGFR mutations (Del18, E709K, G719A, S768I, or L861Q). (a,b) Growth inhibitory curves of osimertinib (a) and furmonertinib (b) against Ba/F3 cells transformed by various common (Del19 or L858R) or uncommon (G719A, E709K, Del18, L861Q, and S768I) EGFR mutations. Curves for the other TKIs are shown in Supplementary Figure S1. Data are presented as the mean values from three individual experiments. Error bars indicate the standard deviation. (c,d) Summaries of the growth inhibitory effects of EGFR-TKIs tested in this study. IC50 values (nM) are summarized for each TKI in (c). The measured IC50 values are color-coded as follows: green (≤10 nM); yellow (10–100 nM); and red (≥100 nM). The SI of each TKI, which was defined as the IC50 value/Ctrough × 100, is summarized in (d). The calculated SIs are color-coded as follows: green (≤5); yellow (5–10); and red (≥10). The estimated Ctrough value for each TKI and the supporting reference(s) are as follows; gefitinib, 591 nM [17]; erlotinib, 2969 nM [18]; afatinib, 69 nM [19]; osimertinib, 400 nM [20,21,22]; furmonertinib, 53 nM [23]; lazertinib, 281 nM [24,25]; almonertinib, 380 nM [26]; rezivertinib, 493 nM [27,28]; and befotertinib, 1000 nM [29,30]. EGFR, epidermal growth factor receptor; IC50, half maximal (50%) inhibitory concentration; SI, sensitivity index; TKI, tyrosine kinase inhibitor.
Figure 2. Efficacy of 1G-, 2G-, and 3G EGFR-TKIs against Ba/F3 cells harboring one of uncommon EGFR mutations (Del18, E709K, G719A, S768I, or L861Q). (a,b) Growth inhibitory curves of osimertinib (a) and furmonertinib (b) against Ba/F3 cells transformed by various common (Del19 or L858R) or uncommon (G719A, E709K, Del18, L861Q, and S768I) EGFR mutations. Curves for the other TKIs are shown in Supplementary Figure S1. Data are presented as the mean values from three individual experiments. Error bars indicate the standard deviation. (c,d) Summaries of the growth inhibitory effects of EGFR-TKIs tested in this study. IC50 values (nM) are summarized for each TKI in (c). The measured IC50 values are color-coded as follows: green (≤10 nM); yellow (10–100 nM); and red (≥100 nM). The SI of each TKI, which was defined as the IC50 value/Ctrough × 100, is summarized in (d). The calculated SIs are color-coded as follows: green (≤5); yellow (5–10); and red (≥10). The estimated Ctrough value for each TKI and the supporting reference(s) are as follows; gefitinib, 591 nM [17]; erlotinib, 2969 nM [18]; afatinib, 69 nM [19]; osimertinib, 400 nM [20,21,22]; furmonertinib, 53 nM [23]; lazertinib, 281 nM [24,25]; almonertinib, 380 nM [26]; rezivertinib, 493 nM [27,28]; and befotertinib, 1000 nM [29,30]. EGFR, epidermal growth factor receptor; IC50, half maximal (50%) inhibitory concentration; SI, sensitivity index; TKI, tyrosine kinase inhibitor.
Cells 14 01386 g002
Cells 14 01386 g003
Figure 3. Secondary EGFR mutations found in osimertinib-resistant clones established through N-ethyl-N-nitrosourea (ENU) mutagenesis. Numbers in pie charts indicate established or analyzed clones at each drug concentration (maximum 12 clones).
Figure 3. Secondary EGFR mutations found in osimertinib-resistant clones established through N-ethyl-N-nitrosourea (ENU) mutagenesis. Numbers in pie charts indicate established or analyzed clones at each drug concentration (maximum 12 clones).
Cells 14 01386 g003
Cells 14 01386 g004
Figure 4. Exploration of TKIs that can overcome T725M and other secondary mutations found in this study. (a) Identification of secondary mutation T725M. Sequencing results for parental cells (G719A only) and resistant cells (G719 plus T725M) are shown. (b) Growth inhibition curves of each TKI against Ba/F3 cells with G719A plus T725M. Data are presented as the mean values of three individual experiments. Error bars indicate the standard deviation. (c,d) Inhibitory activities of each TKI used to treat cells with various uncommon EGFR mutations were compared using the IC50 values (nM, c) and SI (IC50/Ctrough of each drug × 100, d). The measured SI values are color-coded as follows: green (≤5); yellow (5–10); and red (≥10). The estimated Ctrough value for each TKI and the supporting reference(s) are as follows: afatinib, 69 nM [19]; osimertinib, 400 nM [20,21,22]; furmonertinib, 53 nM [23]; lazertinib, 281 nM [24,25]; almonertinib, 380 nM [26]; rezivertinib, 493 nM [27,28]; and befotertinib, 1000 nM [29,30]. EGFR, epidermal growth factor receptor; IC50, half maximal (50%) inhibitory concentration; SI, sensitivity index; TKI, tyrosine kinase inhibitor.
Figure 4. Exploration of TKIs that can overcome T725M and other secondary mutations found in this study. (a) Identification of secondary mutation T725M. Sequencing results for parental cells (G719A only) and resistant cells (G719 plus T725M) are shown. (b) Growth inhibition curves of each TKI against Ba/F3 cells with G719A plus T725M. Data are presented as the mean values of three individual experiments. Error bars indicate the standard deviation. (c,d) Inhibitory activities of each TKI used to treat cells with various uncommon EGFR mutations were compared using the IC50 values (nM, c) and SI (IC50/Ctrough of each drug × 100, d). The measured SI values are color-coded as follows: green (≤5); yellow (5–10); and red (≥10). The estimated Ctrough value for each TKI and the supporting reference(s) are as follows: afatinib, 69 nM [19]; osimertinib, 400 nM [20,21,22]; furmonertinib, 53 nM [23]; lazertinib, 281 nM [24,25]; almonertinib, 380 nM [26]; rezivertinib, 493 nM [27,28]; and befotertinib, 1000 nM [29,30]. EGFR, epidermal growth factor receptor; IC50, half maximal (50%) inhibitory concentration; SI, sensitivity index; TKI, tyrosine kinase inhibitor.
Cells 14 01386 g004
Cells 14 01386 g005
Figure 5. Secondary EGFR mutations found in afatinib-resistant clones established through N-ethyl-N-nitrosourea (ENU) mutagenesis. Numbers in pie charts indicate established or analyzed clones at each drug concentration (maximum 12 clones).
Figure 5. Secondary EGFR mutations found in afatinib-resistant clones established through N-ethyl-N-nitrosourea (ENU) mutagenesis. Numbers in pie charts indicate established or analyzed clones at each drug concentration (maximum 12 clones).
Cells 14 01386 g005
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Oiki, H.; Suda, K.; Hamada, A.; Fujino, T.; Obata, K.; Kobayashi, Y.; Sakai, K.; Fukuda, S.; Ohara, S.; Ito, M.; et al. Efficacy of Conventional and Novel Tyrosine Kinase Inhibitors for Uncommon EGFR Mutations—An In Vitro Study. Cells 2025, 14, 1386. https://doi.org/10.3390/cells14171386

AMA Style

Oiki H, Suda K, Hamada A, Fujino T, Obata K, Kobayashi Y, Sakai K, Fukuda S, Ohara S, Ito M, et al. Efficacy of Conventional and Novel Tyrosine Kinase Inhibitors for Uncommon EGFR Mutations—An In Vitro Study. Cells. 2025; 14(17):1386. https://doi.org/10.3390/cells14171386

Chicago/Turabian Style

Oiki, Hana, Kenichi Suda, Akira Hamada, Toshio Fujino, Keiko Obata, Yoshihisa Kobayashi, Kazuko Sakai, Shota Fukuda, Shuta Ohara, Masaoki Ito, and et al. 2025. "Efficacy of Conventional and Novel Tyrosine Kinase Inhibitors for Uncommon EGFR Mutations—An In Vitro Study" Cells 14, no. 17: 1386. https://doi.org/10.3390/cells14171386

APA Style

Oiki, H., Suda, K., Hamada, A., Fujino, T., Obata, K., Kobayashi, Y., Sakai, K., Fukuda, S., Ohara, S., Ito, M., Soh, J., Nishio, K., Mitsudomi, T., & Tsutani, Y. (2025). Efficacy of Conventional and Novel Tyrosine Kinase Inhibitors for Uncommon EGFR Mutations—An In Vitro Study. Cells, 14(17), 1386. https://doi.org/10.3390/cells14171386

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop