Impact of mutant protein expression on clinical outcomes in patients with EGFR L858R-mutated non-small cell lung cancer

Introduction

Lung cancer is the leading cause of cancer-related death worldwide (1). Epidermal growth factor receptor (EGFR) mutations are major driver gene mutations in non-small cell lung cancer (NSCLC) (2). EGFR tyrosine kinase inhibitors (TKIs) improve the prognosis of patients with advanced EGFR-mutated NSCLC (3-6). Notably, monotherapy with osimertinib, a third-generation EGFR-TKI, elicits a high overall response rate (ORR), thereby improving the overall survival (OS) of patients with advanced EGFR-mutated NSCLC when compared to monotherapy with first-generation EGFR-TKIs (3,7,8). Based on these findings from pivotal trials, osimertinib monotherapy is strongly recommended for patients with advanced EGFR-mutated NSCLC (9-11). However, subgroup analyses suggested that the efficacy of osimertinib monotherapy may be inferior in patients with exon 21 codon Leu858Arg (L858R) point mutations when compared with that in patients with exon 19 deletions (3,8). Therefore, there is a need to develop predictors for the efficacy of osimertinib monotherapy in patients with L858R point mutations to optimize the therapeutic strategy among this patient population.

In patients treated with the first- or second-generation EGFR-TKIs, the expression level of mutant EGFR proteins, evaluated by performing immunohistochemical (IHC) staining, has been shown to predict the efficacy of EGFR-TKI treatment. Patients with high expression of mutant EGFR proteins reportedly experience longer progression-free survival (PFS) following treatment with first- or second-generation EGFR-TKIs than those with low expression (12-14). However, the association between the expression level of mutant EGFR proteins and the efficacy of osimertinib has not been investigated.

In the current retrospective study, we aimed to determine whether the expression of EGFR L858R-mutant protein (EGFR^L858R), as evaluated by IHC staining, could predict the efficacy and clinical outcomes of osimertinib monotherapy in patients with postoperative recurrent EGFR L858R-mutated NSCLC, where the therapeutic effect of osimertinib may be limited. Additionally, we assessed its potential utility as a predictive biomarker. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-126/rc).

Methods

Inclusion and exclusion criteria

We assessed consecutive patients with postoperative recurrent EGFR L858R-mutated NSCLC who received osimertinib monotherapy as first-line EGFR-TKI treatment at five hospitals in Japan (University Hospital Kyoto Prefectural University of Medicine, Japanese Red Cross Kyoto Daini Hospital, Kanazawa Medical University Hospital, Japanese Red Cross Kyoto Daiichi Hospital, and Otsu City Hospital) between September 2018 and February 2022. The primary endpoint of this study is to evaluate the association between the expression level of EGFR^L858R and the efficacy of osimertinib monotherapy. The secondary endpoint was to evaluate the association between the expression level of EGFR^L858R and the postoperative prognosis.

Inclusion criteria were as follows: (I) patient age ≥18 years; (II) Eastern Cooperative Oncology Group performance status (ECOG-PS) ≤2 at the start of osimertinib monotherapy; (III) histologically confirmed NSCLC; (IV) confirmed EGFR L858R-activating mutation; and (V) receipt of osimertinib monotherapy after postoperative recurrence as the first-line EGFR-TKI treatment. Patients were excluded if their residual specimens after pathological diagnosis could not be evaluated. Data pertaining to the following parameters at the start of osimertinib monotherapy were obtained from the medical records: age, sex, histological type, EGFR gene mutation status, ECOG-PS, smoking history, tumor PD-L1 expression stained with the 22C3 antibody (Agilent Technologies, Santa Clara, CA, USA), PFS after osimertinib monotherapy, OS after osimertinib monotherapy, disease-free survival (DFS), postoperative OS, and ORR. The eighth edition of the American Joint Commission on Cancer staging system was used to assess staging. Tumor response was determined based on RECIST (version 1.1).

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Ethical approval was obtained from the Ethics Committee of Kyoto Prefectural University of Medicine (No. ERB-C-2301-5), which reviewed and approved the study protocols on behalf of the five participating hospitals. Because this was a retrospective study, informed consent was waived and an official website was used as an opt-out method.

Assessment of efficacy

PFS was defined as the time from the first administration of osimertinib to disease progression or death due to any cause. OS was defined as the time from surgery and the first administration of osimertinib to death due to any cause. DFS was defined as the time from surgery to disease recurrence. PFS and OS were censored upon final survival confirmation in patients whose disease did not progress and in those who survived, respectively.

Tumor genetic analysis

EGFR L858R mutations were identified using either the PCR-invader method (LSI Medience, Tokyo, Japan), therascreen EGFR RGQ PCR Kit (Qiagen, Hilden, Germany), Cobas EGFR Mutation Test (Roche, Basel, Switzerland), or Oncomine^TM Dx Target Test (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the respective kit instructions.

IHC staining and evaluation of EGFR^L858R and Ki-67

Formalin-fixed, paraffin-embedded tissue blocks, which were residual specimens at the time of operation, were cut into serial 4–5 µm-thick sections. The sections were deparaffinized and immunostained with the following antibodies: anti-EGFR (L858R mutation-specific) rabbit monoclonal primary antibodies (Cell Signaling Technology, Danvers, MA, USA) and anti-Ki-67 mouse monoclonal primary antibodies (Agilent Technologies, Danvers, CA, USA). For the EGFR^L858R expression, in accordance with a previous report, the percentage of positive cells was evaluated at five levels (0, none; 1, 1–10%; 2, 11–30%; 3, 31–50%; 4, 51–70%; 5, 71–100%) and the intensity of staining was evaluated at four levels (0, none; 1, weak; 2, moderate; 3, strong) and the values were added (range of total values, 0–8). Because the median value of the EGFR^L858R expression was 4.5, we set the cutoff value at 4 (11). For the Ki-67 expression, the percentage of Ki-67-positive cells in tumor cells (Ki-67 index) was determined. Two pathologists (A.M.H. and N.T.) independently evaluated the expression of EGFR^L858R and Ki-67.

Statistical analysis

Categorical variables were analyzed using Fisher’s exact test, while continuous variables were analyzed using the Mann-Whitney U test. The Kaplan-Meier method was used to analyze PFS and OS, and differences were compared using the log-rank test. Cox proportional hazard models were performed to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Linear regression analysis was performed to evaluate the correlation between EGFR^L858R expression levels and DFS. Statistical significance was set at P<0.05. Data analyses were performed using the EZR software (version 1.54) (15). The graphs for the violin plot and linear regression analysis were created using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).

Results

Patient characteristics and evaluation of EGFR^L858R expression

A total of 23 patients [median age, 72 (range, 63–84) years, 10 (43.5%) males] from five institutions across Japan were included. Ten (43.5%) patients had a history of smoking, and 8 (34.8%) had pathological stage III. Tumors with expression scores of 0 to 3 were categorized into the low EGFR^L858R group (43.5%), and those with expression scores of 4 to 8 were categorized into the high EGFR^L858R group (56.5%). There were no significant differences in background between patients in the low and high EGFR^L858R groups (Table 1). Representative images of IHC staining with anti-EGFR (L858R mutation-specific) antibodies are shown in Figure 1.

Figure 1 Representative images of immunohistochemical staining showing EGFR^L858R expression in tumors. EGFR, epidermal growth factor receptor; L858R, exon 21 codon Leu858Arg.

Efficacy of osimertinib therapy in patients with EGFR L858R mutation according to EGFR^L858R expression

The median follow-up time of censored cases for PFS and OS after the first osimertinib administration was 43.3 (range, 28.6–61.3) months and 57.1 (range, 28.6–68.1) months, respectively. The ORR of patients with recurrent EGFR L858R-mutated NSCLC who received osimertinib therapy was 65.2% (Table 2). The median PFS with osimertinib therapy was 17.1 months (95% CI: 14.1–45.9 months) (Figure 2A). The median OS with osimertinib therapy was not reached (NR) (95% CI: 20.7 months–NR) (Figure 2B). There were no significant differences in ORR, PFS, or OS between the low and high EGFR^L858R groups (70.0% versus 61.5%, P>0.99; 25.5 versus 16.9 months, log-rank test P=0.69; and NR versus NR, log-rank test P=0.83, respectively) (Table 2, Figure 2C,2D).

Figure 2 Efficacy of osimertinib therapy in patients with EGFR L858R mutation according to EGFR^L858R expression. PFS (A) and OS (B) of osimertinib monotherapy in all patients (n=23). PFS (C) and OS (D) of osimertinib monotherapy according to EGFR^L858R expression. CI, confidence interval; EGFR, epidermal growth factor receptor; L858R, exon 21 codon Leu858Arg; NR, not reported; OS, overall survival; PFS, progression-free survival.

Impact of EGFR^L858R expression on DFS and OS after surgery

Next, we examined the relationship between EGFR^L858R expression and DFS or OS post-surgery. The median follow-up time of censored cases for OS post-surgery was 76.6 (range, 35.0–113.7) months. The median DFS and OS post-surgery were 17.9 months (95% CI: 12.1–26.4 months) and 78.4 months (95% CI: 48.6 months–NR), respectively (Figure 3A,3B). The median DFS was significantly shorter in patients with high EGFR^L858R expression than in those with low EGFR^L858R expression (13.8 versus 32.0 months, log-rank test P=0.008; HR: 3.62, 95% CI: 1.32–9.94, P=0.01) (Figure 3C). However, EGFR^L858R expression did not significantly impact the median OS post-surgery (the low EGFR^L858R group versus the high EGFR^L858R group, NR versus 68.1 months, log-rank test P=0.31) (Figure 3D). A moderate correlation was observed between EGFR^L858R expression levels and DFS (R²=0.26, P=0.01) (Figure 4A).

Figure 3 Impact of EGFR^L858R expression on DFS and OS after surgery. DFS (A) and OS (B) after surgery in all patients (n=23). DFS (C) and OS (D) after surgery according to EGFR^L858R expression. CI, confidence interval; DFS, disease-free survival; EGFR, epidermal growth factor receptor; L858R, exon 21 codon Leu858Arg; NR, not reported; OS, overall survival.

Figure 4 Association between EGFR^L858R expression and Ki-67 index in tumors. (A) Correlation between EGFR^L858R and DFS. (B) Ki-67 index according to EGFR^L858R expression. DFS, disease-free survival; EGFR, epidermal growth factor receptor; L858R, exon 21 codon Leu858Arg.

Association between EGFR^L858R expression and Ki-67 index in tumors

To further investigate the association between recurrence risk and EGFR^L858R expression levels, we measured the Ki-67 index, a marker of tumor proliferation. As expected, the Ki-67 index was significantly higher in the high EGFR ^L858R group than in the low EGFR ^L858R group (P=0.03) (Figure 4B).

Discussion

In the current study, the expression level of EGFR L858R-mutant protein within the tumor, assessed using surgical specimens, did not significantly impact the efficacy of osimertinib monotherapy after recurrence. Reportedly, the expression of EGFR L858R-mutant protein can predict the efficacy of the first- or second-generation EGFR-TKIs (12-14). Similar to the current study, Azuma et al. (12) conducted a study examining surgical specimens, and found that in the low EGFR^L858R group, the median PFS with the first-generation EGFR-TKI gefitinib was only 3.4 months, suggesting that several tumors with low EGFR^L858R expression may have primary resistance to first-generation EGFR-TKIs. In our study, osimertinib monotherapy was effective even in patients with low EGFR^L858R-expressing tumors. The discrepancy between our findings and those of the previous reports remains unclear; that is, whether it is due to the ability of third-generation EGFR-TKIs to overcome resistance mechanisms associated with first-generation EGFR-TKIs or the influence of the small sample size. Further large-scale studies are needed to clarify this issue.

In recent years, postoperative adjuvant osimertinib monotherapy was shown to substantially prolong DFS when compared with placebo in patients with resected EGFR-mutated NSCLC (16). Furthermore, osimertinib monotherapy could markedly prolong PFS when compared with placebo therapy in patients with unresectable stage III EGFR-mutated NSCLC following chemoradiotherapy (17). Based on these trials, osimertinib monotherapy is anticipated to be used more frequently in patients with early-stage EGFR-mutated NSCLC. Identifying risk factors for early recurrence is crucial to ensure that the timing of osimertinib administration is not missed. Although the utility of plasma biomarkers, such as circulating tumor DNA levels, as predictive factors has been reported (18-22), there are challenges in implementing these methods in routine clinical practice from the perspective of their general applicability.

In this study, the EGFR^L858R expression significantly correlated with DFS in enrolled patients with EGFR L858R-mutated NSCLC. To the best of our knowledge, this is the first study to demonstrate that the expression of EGFR-mutant protein may predict DFS in patients with EGFR-mutated NSCLC. Interestingly, the EGFR^L858R expression was significantly associated with the percentage of Ki-67-positive cells. Ki-67 is a nuclear protein that is expressed in proliferating cells but not resting cells (23). Tumors with high EGFR^L858R expression may exhibit stronger proliferative signaling from mutant EGFR proteins and a faster rate of tumor growth than those with low EGFR^L858R expression, resulting in a shorter time for tumor detection post-surgery.

In patients with early-stage NSCLC who have undergone surgery, disease recurrence is associated with worsening of quality of life (QOL) (24); therefore, extending DFS means extending the period during which the QOL is maintained. Osimertinib prolongs DFS while maintaining the same level of QOL as placebo (25). However, there are concerns regarding the administration of osimertinib from the perspectives of toxicity and cost, and the development of biomarkers related to its selectivity is needed. Based on the results of this study, EGFR^L858R expression level in surgical specimens may serve as a potential biomarker for selecting patients eligible for postoperative adjuvant therapy with osimertinib. Additionally, identifying patients at a high risk of recurrence could help optimize postoperative follow-up strategies and is expected to contribute to improved survival outcomes.

In the future, it will be necessary to investigate whether a similar approach using IHC analysis is also applicable to postoperative NSCLC with other EGFR mutation subtypes or other driver gene alterations. However, to validate the benefits of this approach, large-scale studies with sufficient statistical power are required.

This study has several limitations that need to be addressed. First, because this study included only patients with postoperative recurrence, it is unclear whether EGFR^L858R expression is a risk factor for postoperative recurrence in all patients with EGFR-mutated NSCLC who underwent surgery. However, we considered it meaningful that EGFR^L858R expression could predict postoperative recurrence in patients with EGFR-mutated NSCLC. The relevance of this IHC staining would be further clarified by analysis including recurrence-free patients. Second, the sample size of this study was small, and multivariate analysis could not be performed. We cannot rule out the possibility that confounding factors may influence the results. Finally, as the cohort size was small, it is not possible to conclude that there was no clinically significant difference even for results that did not show a significant difference in this study. In addition, as this study was conducted only in hospitals in Japan, the generalizability of the findings, including their applicability to other races, needs to be considered with caution. Further studies with larger and more diverse cohorts are warranted.

Conclusions

This observational study revealed that the expression of EGFR L858R-mutant protein, evaluated using IHC staining, may predict the time to postoperative recurrence in patients with EGFR-mutated NSCLC; however, there was no significant difference in other aspects (survival and treatment efficacy). Given the need for early introduction of osimertinib therapy in patients with EGFR-mutated NSCLC, further prospective large-cohort studies are necessary to validate these findings.

Acknowledgments

We thank Editage (www.editage.jp) for help with English-language editing.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-126/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-126/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-126/prf

Funding: This work was supported by the Takeda Science Foundation and the Princess Takamatsu Cancer Research Fund.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-126/coif). T.Y. serves as an unpaid editorial board member of Translational Lung Cancer Research from October 2023 to September 2025. T.Y. received commercial research grants from Ono Pharmaceutical, Janssen Pharmaceutical K.K., AstraZeneca, and Takeda Pharmaceutical Company Limited and speaking honoraria from Eli Lilly and Chugai-Roche. K.T. received research grants from Chugai-Roche and Ono Pharmaceutical and personal fees from AstraZeneca, Chugai-Roche, MSD-Merck, Eli Lilly, Boehringer-Ingelheim, and Daiichi-Sankyo. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Ethical approval was obtained from the Ethics Committee of Kyoto Prefectural University of Medicine (No. ERB-C-2301-5), which reviewed and approved the study protocols on behalf of the five participating hospitals. Because this was a retrospective study, informed consent was waived and an official website was used as an opt-out method.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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