Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) accounting for 80–85% of all cases (1). Somatic activating mutations in the EGFR gene are the most frequent oncogenic driver mutations in Asian patients with NSCLC, with a prevalence of ~47% % (2). Mutations in EGFR exons 18–21 notably predict responses to targeted therapies therapies (3).

Among these mutations, the L858R substitution in exon 21 is one of the most common, alongside exon 19 deletions, together representing ~85–90% of all EGFR mutations in NSCLC. These mutations are typically sensitive to first–generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib erlotinib (4). By contrast, mutations in exon 18, including G719X, E709X and exon 18 deletions, are found in 3–4% of all EGFR mutations in NSCLC NSCLC (5). The most frequent point mutations in exon 18 are G719X and E709X, which together account for 84% of mutations in this exon. DelE709_T710insX, the most frequent exon 18 deletion, accounts for ~2.4%. For patients with these rare mutations, afatinib is considered a first–line treatment option option (6), which demonstrates moderate efficacy even against mutations that confer resistance to osimertinib.

E709X mutations, although rare, are notable for their sensitivity to targeted therapies. This mutation represents ~1.5% of all EGFR mutations and includes several variants, such as E709K, E709A, E709G, E709V, E709H, E709D and E709Q. Among these, E709K, E709A and E709G are the most common common (7,8). However, due to the low incidence of E709X mutations and their occurrence in combination with other EGFR mutations, systematic studies on the efficacy of TKIs for E709X mutations remain limited. The co-occurrence of E709K and L858R mutations, in particular, has garnered notable attention due to their impact on disease progression and treatment response.

The present study reported a patient with both E709K and L858R mutations, treated sequentially with osimertinib, afatinib and ametinib. A literature review was also conducted on the rare E709X mutation in exon 18 and its implications for treatment strategies.

A 64-year-old Chinese woman, a non-smoker with no family history of cancer, was admitted to The People's Hospital of Yingcheng City (Yincheng, China) in March 2022. The patient experienced swelling, numbness and limited movement in the right hip and knee for >10 days. Pelvic MRI indicated multiple abnormal lesions in the right iliac crest, pubic bone, ischium, upper right femur, bilateral femoral heads, L5 vertebra and bilateral sacrum. CT scans of the neck revealed enlarged lymph nodes in the left neck and left supraclavicular region. Chest and upper abdomen CT revealed a tumor in the left lower lobe (3.1×1.7 cm) with enlarged left hilar and mediastinal lymph nodes, bilateral lung nodules and some irregular ground-glass opacities. Cranial MRI indicated multiple brain metastases in both the frontal and temporal lobes.

A percutaneous lung biopsy was performed under CT guidance, which revealed invasive adenocarcinoma (solid + acinar type) (Fig. 1A and B), Hematoxylin and eosin staining was performed according to standard histopathological protocols routinely used in our pathology department. Programmed cell death-ligand 1 (PD-L1) expression was positive with 30% tumor cell interpretation, the test was performed on a biopsy specimen using Ventana SP263 reagent and the Ventana BenchMark Ultra platform (Fig. 1C). Immunohistochemistry revealed that tumor cells were positive for thyroid transcription factor 1 [TTF-1 (+)] (Fig. 1D), Napsin A (+) (Fig. 1E), cytokeratin (CK)7 (+) (Fig. 1F), Ki-67 (LI 10%) (Fig. 1G) and negative for P40 (Fig. 1H). Marker selection followed the International Association for the Study of Lung Cancer 2021 consensus guidelines (9). Formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples were prepared using 10% neutral-buffered formalin at room temperature for 24 h. Tissue sections were cut to a thickness of 4 µm using a microtome. For antigen retrieval and intracellular epitope exposure, slides were permeabilized with 0.2% Triton X-100 (Merck KGaA; Sigma-Aldrich) in PBS for 10 min at room temperature. To reduce nonspecific binding, tissue sections were blocked with 5% normal goat serum (Abcam) in PBS for 30 min at room temperature. Immunohistochemical staining was then performed using the following primary antibodies: Anti-TTF-1 (1:200 dilution; cat. no. M3575; Dako; Agilent Technologies, Inc.), anti-Napsin A (1:100 dilution; cat. no. 760-4867; Roche Diagnostics), anti-CK7 (1:300 dilution; cat. no. M7018; Dako; Agilent Technologies, Inc.), anti-Ki-67 (1:100 dilution; cat. no. M7240; Dako; Agilent Technologies, Inc.) and anti-P40 (1:200 dilution; cat. no. 760-4863; Roche Diagnostics). Primary antibodies were incubated at 4°C overnight. Secondary antibody incubation was performed using the EnVision+ System-HRP Labeled Polymer Anti-Mouse/Rabbit (cat. no. K4007; Dako; Agilent Technologies, Inc.) at room temperature for 30 min. Diaminobenzidine was used as the chromogen, followed by counterstaining with hematoxylin. All slides were examined using a Nikon Eclipse E200 light microscope at ×200 and ×400 magnifications.

Emission CT scan demonstrated clear body bone imaging with strong distribution of radioactivity in the parietal, right shoulder, left humerus, left clavicle, multiple ribs, T2-4 vertebrae, sacrum, right sacroiliac joint, right iliac, bilateral hips and right femur, with physiological distribution elsewhere (Fig. 1I). Next-generation sequencing (NGS) of the transthoracic lung biopsy tissue identified EGFR exon 21 p.L858R mutation (15.59%) and EGFR exon 18 p.E709K mutation (21.2%) (Table I; Fig. 1J and K). Genomic DNA was extracted from FFPE lung biopsy tissue using a commercial DNA extraction kit (Auto-Pure 96; Hangzhou Allsheng Instruments Co., Ltd). The quality and integrity of the extracted DNA were assessed using standardized procedures, and the sample passed internal quality control metrics (average sequencing depth: 1191.02×; Q30: 94.25%). NGS was conducted using a hybrid capture-based 26-gene panel targeting lung cancer-related genes (WuHan Kingmed Center for Clinical Laboratory Co., Ltd). Paired-end sequencing was performed using the Illumina NovaSeq platform, with a read length of 150 bp (2×150 bp). Library preparation and enrichment were performed according to the manufacturer's protocols (KM Miniseq-DX; Guangzhou Jinqi Rui Biotechnology Co., Ltd). The final library was quantified using a Qubit fluorometer and normalized to a loading concentration of 2.0 nM, calculated using standard molar concentration formulas. Variant calling and visualization were performed using Integrative Genomics Viewer software [analytical software 1: bcl2fastq (v2.19.0.316); analytical software 2: fastp (v0.23.2); analytical software 3: BWA (v0.7.17-r1188); analytical software 4: Mutect 2 (v4.2.3.0); Analytical software 5: annovar (:v2020-06-08); Analytical software 6: msisensor-pro (v1.0.2); Analytical software 7: manta (v1.6.0); Analytical software 8: Delly (v0.9.1); and Analytical software 9: cnvkit (v0.9.10)]. For bioinformatic analysis, the laboratory used proprietary pipelines and quality standards validated under ISO15189 guidelines. The serum tumor biomarker CEA level was 17.82 ng/ml, above the normal range (<5.0 ng/ml) (Fig. 1L). Based on these findings, the patient was diagnosed with stage IV left lower lung adenocarcinoma with lymphatic, intracranial and extensive bone metastasis (TNM staging, cT2N3M1; according to the guidelines from the International Association for the Study of Lung Cancer, 9th edition) (10). Osimertinib targeted therapy was recommended in accordance with the National Comprehensive Cancer Network guidelines (11).

In March 2022, the patient initially reported improvement in right hip pain, the chest CT and head MRI of the patient at that time are shown in Fig. S1A. Oral osimertinib therapy was initiated in March 2022 (80 mg, once daily). After 10 days, the patient experienced notable relief from lower back pain. A follow-up in April 2022, 1 month after starting osimertinib, indicated partial response (PR) (Fig. S1B). A 3-month follow-up in July 2022 also indicated PR (Fig. S1C).

However, by November 2022, after 8 months of osimertinib treatment, progression of disease (PD) was observed (Fig. S1D). Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) performed at Tongji Hospital (Wuhan, China) confirmed non-small cell carcinoma with pathology consistent with lung adenocarcinoma (Fig. 1M and N). NGS of the EBUS-TBNA biopsy tissue revealed EGFR exon 21 p.L858R (12.4%) and exon 18 p.E709K (12.5%) mutations and PD-L1 expression was again 30% (Fig. 1C).

In December 2022, the patient tested positive for COVID-19 and was admitted to the First People's Hospital of Tianmen City (Tianmen, China). The patient received 50 Gy/25 fractions of radiotherapy to the lower left lung and 1 month of oral afatinib therapy during the radiation (40 mg, once daily). However, afatinib was discontinued due to poor tolerance and osimertinib was resumed. In February 2023, following radiotherapy, afatinib was reintroduced for 1 month and the patient was again reviewed, which indicated PR (Fig. S1E).

In March 2023, the patient's condition progressed again and the patient developed intermittent cough, dizziness, nausea and vomiting. A CT scan indicated pericardial effusion and cancerous lymphangitis (Fig. S1F), with PD observed. In January 2023, 75 ml of bloody pericardial effusion was drained and pathological examination confirmed metastatic lung adenocarcinoma. Immunohistochemistry results were positive for PCK, CK7, TTF-1 and Napsin A, and negative for P40, CK5/6 and P40, with Ki-67 expression at ~40% (Anti-PCK: 1:150 dilution; cat. no. M3515; Dako; Agilent Technologies, Inc. Other antibody information is the same as stated above). NGS of the pericardial effusion revealed the same EGFR mutations (L858R and E709K). Given disease progression and suspected resistance to targeted therapy, cisplatin chemotherapy (cavum pericardii: 40 mg; vein: 60 mg, once) was administered in March 2023. Systemic chemotherapy with the paclitaxel-platinum (PP) (cisplatin: 100 mg; pemetrexed: 750 mg, once a month) regimen was continued, which led to PR after one cycle (Fig. S1G). After one cycle, bevacizumab (400 mg, once a month) was added to the PP regimen, which resulted in another PR (Fig. S1H).

In August 2023, after four cycles of the PP regimen, the treatment was switched to a combination of pemetrexed (750 mg, once a month) and bevacizumab (400 mg, once a month). After one cycle, a SD was achieved (Fig. S1I). However, after three cycles in November 2023, PD was observed and compliance with chemotherapy in the patient was suboptimal. Because the patient's self-perceived tolerance was poor, treatment intervals were extended to 45 days (Fig. S1J).

In February 2024, after six cycles of pemetrexed and bevacizumab, brain MRI revealed sporadic nodular enhancement and CT scans demonstrated worsening left lower lobe tumor and metastasis (Fig. S1K). The patient reported occasional dizziness and headaches and PD was confirmed. Whole-brain radiotherapy (WBRT) was initiated. NGS of peripheral blood indicated EGFR exon 21 p.L858R (0.92%) and exon 18 p.E709K (1.18%), with Bcl-2-like protein 11 interacting mediator of cell death intron 2 deletion (Table I). In May 2024, treatment with ameritinib (110 mg, once daily) and bevacizumab (400 mg, once a month) was started and after 1 month of WBRT, the patient demonstrated PR (Fig. S1L). The reexamination in August 2024 showed SD (Fig. S1M).

By November 2024, after 6 months of combined treatment with ameritinib and bevacizumab, the patient developed proteinuria (3+) and marked blood pressure fluctuations (≤170/120 mmHg), suspected to be side effects of bevacizumab. Consequently, bevacizumab was discontinued and intravenous pemetrexed chemotherapy (750 mg, once a month) was started. The patient subsequently developed symptoms of dizziness, headache, vomiting, somnolence, decreased vision, hearing loss, reduced appetite, lower limb weakness and unsteady gait. Brain MRI revealed slight enlargement of ventricles and cisterns, mild widening of sulci and possible leptomeningeal metastasis (Fig. S1N).

Following the confirmation of leptomeningeal metastasis, an Ommaya reservoir implantation (Fig. 2A) was performed and cerebrospinal fluid examination confirmed the presence of tumor cells (Fig. 2B). Intrathecal administration of pemetrexed (30 mg, once a month) via the Ommaya reservoir, in combination with oral ameritinib (220 mg, once daily), led to notable improvement in the symptoms of the patient. The treatment regimen is ongoing and the patient receives an intrathecal injection once a month. The timeline of the diagnosis and treatment of the patient is displayed on the left side of Fig. S1.

The treatment of EGFR mutations has undergone notable advancements, with targeted therapies serving a key role in the improvement of patient outcomes. Among the various mutations in the EGFR gene, the E709X mutation, including the E709K mutation, has gained attention due to its implications for treatment response to different generations of EGFR TKIs. A comprehensive summary of previous studies on the E709X mutation is provided in Table II.

Mutations such as E709K and L858R have demonstrated varying responses to different generations of EGFR TKIs: i) First-generation TKIs: Both L858R and E709K mutations exhibit sensitivity to first-generation TKIs, although E709K generally exhibits a slightly lower response compared with L858R. Previous studies indicated that while first-generation TKIs are initially effective, resistance typically develops over time (7,12,13); ii) second-generation TKIs: Afatinib and dacomitinib, as second-generation EGFR TKIs, irreversibly bind to the EGFR receptor. Clinical data suggested that afatinib is particularly effective in treating E709K mutations, offering a notable progression-free survival (PFS) advantage. Afatinib is also highly efficacious for L858R mutations and is often preferred due to its potency and broader mutation coverage (5); and iii) third-generation TKIs: Osimertinib, a third-generation TKI, is typically reserved for patients with acquired T790M resistance mutations but has also demonstrated efficacy in L858R mutation cases after resistance to earlier-generation TKIs. However, the effectiveness of osimertinib and other third-generation TKIs in the treatment of E709K mutations remains elusive and warrants further investigation (12).

Previous studies reported three lung cancer cases with dual EGFR mutations (L858R and E709K) treated using afatinib: 1 patient achieved PR and 2 patients had stable disease (SD) (14). The E709 amino acid alteration in the EGFR gene is relatively rare in NSCLC and is often associated with other sensitive EGFR mutations (e.g., G719X and L858R) (15,16). A study suggested that the E709K mutation may be sensitive to afatinib-targeted therapy but demonstrates reduced sensitivity to gefitinib and erlotinib (6), a finding confirmed by a retrospective case-control study conducted by Kuiper et al (17). Furthermore, patients with uncommon EGFR mutations (e.g. S768I and exon 20 insertion mutation) (18,19) tend to have shorter PFS and overall survival (OS) on EGFR-TKI treatment compared with those with classic EGFR mutations (e.g. Del19 and L858R point mutation) (20), although there is considerable variability depending on the specific mutation.

A previous study by Kobayashi et al (5) compared the inhibitory effects of various EGFR TKIs on Ba/F3 cells expressing the E709K/E709_T710delinsD mutation. The results indicated that second-generation TKIs, such as afatinib, were more potent in inhibiting the E709K/E709_T710delinsD Ba/F3 cell line compared with first-generation TKIs. This suggested that second-generation TKIs may be more effective in the treatment of patients with NSCLC with the E709_T710delinsD mutation. However, clinical evidence confirming the efficacy of second-generation TKIs in patients with E709_T710delinsX mutations remains sparse.

The findings of the present study align with those of previous studies (5,8,18), which demonstrated moderate to good responsiveness of dual EGFR mutations (E709K and L858R) to targeted EGFR-TKIs, notably afatinib and osimertinib. Similar to Wu et al (24), the present study observed initial sensitivity followed by the development of resistance. By contrast, while Kobayashi et al (5) and Wei et al (12) reported a notable clinical benefit with afatinib for exon 18 mutations, the present patient exhibited limited tolerance to afatinib, which necessitates a treatment switch to third-generation TKI amitinib.

The novel insight provided by the present study is the clinical efficacy and tolerability of sequential treatment involving osimertinib, afatinib and amitinib, including comprehensive radiotherapy and chemotherapy management strategies. These findings suggest individualized therapeutic strategies and highlight the importance of real-time genomic monitoring to promptly manage resistance mechanisms. However, notable gaps remain, particularly regarding standardized treatment protocols for rare EGFR mutations, mechanisms underlying acquired resistance and the optimal sequencing and combination of TKIs in clinical practice. Future large-scale studies are key to filling these knowledge gaps.

A notable case study reported a patient with lung adenocarcinoma with the rare E709_T710delinsD mutation who responded well to afatinib, which achieved a PFS of 23 months (12). The present case highlighted the potential of second-generation TKIs, such as afatinib, in the management of mutations involving exon 18. Additionally, broader analyses suggested that patients with E709K and other exon 18 mutations exhibited an improved response to afatinib or neratinib compared with first-generation TKIs (5,25,26).

Functional studies using transfected cell models (Ba/F3 and NIH/3T3) have demonstrated that the E709K mutation, similar to G719X, induces oncogenic transformations. These mutations exhibit greater sensitivity to second-generation TKIs, such as afatinib and neratinib, compared with first-generation TKIs. These findings underscore the importance of precise molecular diagnostics in guiding optimal treatment strategies for patients with rare EGFR mutations (6).

The present case included a patient with EGFR mutations, specifically E709K and L858R, who was treated with multiple targeted therapies, including osimertinib (8 months), afatinib (1 month) and amitinib (ongoing). Due to the complexity of the treatment regimen, which combined targeted therapies with radiation for the primary lesions, it was difficult to assess the efficacy of the treatment. However, both osimertinib (PFS, 8 months) and amitinib (ongoing therapy) demonstrated favorable responses, which resulted in remission. The present case highlighted the potential effectiveness of third-generation EGFR TKIs in the treatment of the rare E709K mutation. Although resistance ultimately developed, these treatments markedly alleviated the condition of the patient and produced tolerable side effects, rendering them a promising therapeutic option for similar cases in the future.

Resistance mechanisms to third-generation EGFR TKIs, such as osimertinib, include secondary mutations such as C797S, MET amplification and histological transformations (e.g., small cell transformation). The patient of the present case study eventually demonstrated progression on osimertinib, which suggested potential activation of such resistance pathways. Future treatment strategies should integrate comprehensive genomic monitoring to promptly identify and target these resistance mechanisms.

Additionally, the patient began chemotherapy in April 2023, which included a combination of bevacizumab, the PP regimen, pemetrexed chemotherapy, whole-brain radiotherapy and continued targeted therapy with amitinib and intrathecal injections. Xu et al (27) also suggested that continuing EGFR-TKI therapy in combination with bevacizumab is a rational strategy for patients with NSCLC who experience gradual progression after initial EGFR-TKI treatment. Clinical studies have reported that the addition of bevacizumab to standard chemotherapy (carboplatin/paclitaxel) notably increases survival in patients with NSCLC. In the pivotal Phase III study (ECOG 4599), chemotherapy plus bevacizumab resulted in improved OS (12.3 vs. 10.3 months) and longer PFS (6.2 vs. 4.5 months) (28).

The present case underscores the value of incorporating targeted therapies and bevacizumab into treatment regimens for advanced NSCLC, particularly for patients with rare mutations such as E709K, where conventional therapies may offer limited efficacy. Further studies are warranted to optimize these combination therapies and enhance the understanding of their long.term impact on patient survival and quality of life.

The present case study demonstrated the complex treatment process for a patient with advanced lung adenocarcinoma. Through multidisciplinary collaboration, the patient received effective symptomatic treatment and an improvement in quality of life. The present case underscores the importance of individualized treatment plans in oncology.

The treatment of patients with EGFR mutations, particularly the E709X and E709_T710delinsX mutations, poses notable challenges due to their unique properties and varying responses to different EGFR TKIs. While first-generation TKIs exhibit certain efficacy, second-generation TKIs, particularly afatinib, appear to offer a more favorable therapeutic option for patients with these mutations, the literature on E709X and E709_T710delinsX mutations was searched and their views seem to support this conclusion (29–36). However, further clinical studies with larger sample sizes are required to validate these findings and optimize treatment strategies for these patients. Furthermore, the role of third-generation TKIs, such as osimertinib, in the treatment of these mutations remains to be elucidated in future research.

The E709K and L858R mutations in EGFR are key factors in the pathogenesis and treatment response of NSCLC. While the L858R mutation is well-characterized and commonly targeted with established treatment protocols, the E709K mutation, although less frequent, demonstrates promising responses to second-generation EGFR TKIs, particularly afatinib. Ongoing research and clinical trials are necessary to refine therapeutic strategies and improve patient outcomes for those with these mutations. For clinicians, understanding the specific EGFR mutation profile is essential for the selection of the most effective targeted therapies, which ultimately enhances both survival and quality of life for patients.

The limited number of cases including rare or complex mutations poses challenges for the design of large-scale prospective trials. Therefore, translational studies and the establishment of national and international biobanks will be key to addressing several of the unresolved questions in this field.

In conclusion, the management of NSCLC with rare EGFR mutations such as E709X and E709_T710delinsX remains challenging due to their heterogeneity and limited clinical data. This case highlights the clinical value of individualized treatment strategies combining sequential EGFR TKIs, including afatinib and osimertinib, and adjunct therapies such as bevacizumab and radiotherapy. Second-generation TKIs, particularly afatinib, appear to offer improved efficacy for exon 18 mutations compared to first-generation TKIs, while third-generation TKIs may serve as effective alternatives upon resistance. The integration of genomic monitoring and multidisciplinary approaches is essential for optimizing treatment in these complex cases. Further prospective studies and real-world evidence are needed to establish standardized protocols and improve outcomes for patients harboring uncommon EGFR mutations.