Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 80% -85% of the cases (1). In recent years, significant progress has been made in the molecular genetics of lung cancer, and the treatment of NSCLC has entered the era of targeted therapy (2). The most common driver gene mutation in NSCLC is the epidermal growth factor receptor (EGFR), which is found in 45% of Asian patients with adenocarcinoma histology and in 20% of Caucasian patients (3). The most common gene mutations include EGFR 19 del and EGFR 21 L858R. In patients with advanced NSCLC harboring sensitizing EGFR mutations, EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment was significantly better effective than conventional chemotherapy. Globally, neither treatment guidelines nor clinical practice considers EGFR-TKIs as the first-line treatment option for EGFR-mutated metastatic NSCLC patients (4).

However, the vast majority of patients inevitably develop acquired drug resistance, which seriously affects the survival prognosis of patients (5). The resistance mechanisms can be generally classified into two categories: on-target and off-target resistance. We report two cases of rare resistance mechanisms involving ALK and BRAF pathway that occurred under EGFR-TKI treatment. To some extent, our cases contribute to previously reported new treatment approaches.

A 60-year-old male smoker presented with complaints of cough and fatigue. A chest CT scan revealed multiple nodules in both lungs, with the largest measuring approximately 1.1x0.8cm, suggesting the presence of lung cancer with multiple pulmonary metastases. Additionally, multiple liver metastases were observed, with the largest measuring about 2.1x1.3cm. The patient was diagnosed with stage cT1bN3M1c IVB based on biopsy from the tumor in the right lung. Real-Time PCR indicated the presence of EGFR 19 E746_A750del(1) deletion mutation. As the first-line treatment, the patient received furmonertinib (80 mg once daily) for 27 months. A follow-up chest CT scan revealed an enlargement of the right lung nodule to approximately 2.6x2.1cm. Subsequently, the patient underwent 4 cycles of chemotherapy with pemetrexed combined with cisplatin. One month later, the chest CT scan showed the development of massive pleural effusion on the left side and enlarged liver lesions, measuring about 5.5x4.6cm. PCR genetic testing of tumor liver tissue revealed a mutation in V600E in exon 15 of the BRAF gene. The patient was then treated with dabrafenib (150 mg twice daily) and trametinib (2 mg once daily), and a follow-up CT scan after eight weeks showed improvement in the left pleural effusion and liver metastasis ( Figure 1 ). So far, the patient responses well to targeted drugs and remains healthy with no signs of recurrence.

A 77-year-old female non-smoking patient presented with a cough. Imaging examination revealed the presence of nodules (1.7x1.5 cm in size) in the left lower lobe, along with multiple brain metastases. Genetic testing revealed a mutation in EGFR18-G719X. The patient was initially treated with gefitinib (250 mg once daily) with good tolerance and treatment for 20 months. NGS analysis of peripheral blood showed no mutations. Subsequently, the treatment was changed to icotinib, but a re-evaluation after 4 months demonstrated progression of the lung and brain metastases. Then the patient was treated with 6 cycles of Pemetrexed and Bevacizumab. The following MRI of the brain revealed enlarging lesions with extensive adjacent edema. Rebiopsy from progression lung tumor investigated by illumina NGS showed the presence of ALK-fusion, leading to the initiation of treatment with Alectinib. After eight weeks, reexamination showed a reduction in the size of lung lesions and disappearance of brain lesions ( Figure 2 ). So far, the patient remains stable with no signs of recurrence.

We present two cases of advanced lung adenocarcinoma with two EGFR -mutations at initial diagnosis. In both cases progression on EGFR-TKIs was observed, leading to the identification of acquired resistance mechanisms in form of ALK(EML4 exon20) fusion and BRAF mutation. Interestingly, both patients achieved long-term survival with the use of ALK and BRAF inhibitors. These findings offer valuable insights for selecting therapeutic drugs in patients with acquired ALK fusion and BRAF mutation.

Mechanisms of EGFR-TKI resistance include EGFR mutations such as C797S, T790M, G796D, G724S, and L718Q, with T790M being the most common (50-60%). Additionally, resistance can arise from MET amplification (20%), HER2 amplification (13%), activation of bypass pathways through mutation or amplification of tyrosine kinase receptor genes like c-MET, FGFR, and HER2, mutations in downstream signaling genes like KRAS, BRAF, PIK3CA, and RAS/RAF/MEK/ERK, and transformation into small cell lung cancer (10%) (6). ALK fusion is also involved in mediating EGFR-TKI resistance and may occur as intrinsic or acquired resistance mechanism (7). Primary fusion occurs in patients with dual alteration of EGFR mutation and ALK fusion genes, with a prevalence ranging from 0.9% to 6% in NSCLC (8). For patients with double alteration, the effectiveness of EGFR-TKI or ALK-TKI may depend on the mutation level of the involved gene (9, 10). Secondary fusion refers to ALK fusion may occur as a rare acquired resistance mechanism under treatment with EGFR-TKI ( 11). However, cases of secondary ALK fusion following acquired resistance are rarely reported, and the optimal choice of targeted therapy remains inconclusive. In 2016, a case study reported the first patient with EGFR exon 19 deletion and ALK wild-type NSCLC. The study demonstrated the coexistence of primary EGFR mutation and acquired EML4-ALK gene fusion in plasma under EGFR-TKI treatment. The patient was treated with the EGFR inhibitor Osimertinib along with crizotinib, which resulted in sustained efficacy in the liver metastatic lesions (Extrahepatic lesions were stable) (12). In a study involving 3505 patients treated with EGFR-TKI, new ALK-fusions were detected in 7 patients. These fusion partners included EML4 (n=4), STRN (n=1), TGF (n=1), and PLEKHA7 (n=1). The patient with acquired STRN-ALK fusion did not experience the effect of Crizotinib. Additionally, in the patient with acquired PLEKHA7-ALK fusion, partial remission in six months was observed under the treatment with Alectinib and Osimertinib (13). Based on the hitherto published reports and the current case, combining of EGFR- and ALK-TKI may be an effective treatment in EGFR-mutated NSCLC with ALK-fusion acquired on the EGFR-TKI therapy. It is important to note that further research with a larger sample size is necessary to validate these results.

As one of the rare driver genes in NSCLC, BRAF mutations play a crucial role in the development and occurrence of tumors. Among these mutations, BRAF V600 is the most commonly observed. With significant advancements in precision therapy, targeted therapy has emerged as a primary treatment approach for BRAF mutations. Dual-target combination therapy, in particular, has gained widespread recognition for its clinical value. This therapy involves the use of dabrafenib, a BRAF inhibitor, and trametinib, a MAPK-inhibitor. The combination of these two drugs allows for precise targeting of dual pathways and complete inhibition of the upstream and downstream pathways of MAPK (14). The results of the BRF113928 trial demonstrated an overall response rate (ORR) of 63.9% and a 5-year overall survival (OS) rate of 22% in first-line treatment with dabrafenib + Trametinib (D+T). Among the 36 subjects treated with D + T as after line, the ORR reached 68.4%, and the duration of remission (DOR) was 9.8 (95% CI 6.9-18.3) months. Furthermore, the trial confirmed the safety of this treatment regimen (15). The Chinese Lung Cancer Registration Clinical Study also provided evidence of the favorable antitumor activity and safety of dabrafenib + trametinib in Chinese NSCLC patients. Consequently, in 2023, dabrafenib and trametinib were recommended as grade I treatments in the Clinical Diagnosis and Treatment Guidelines of the Chinese Society of Clinical Oncology (CSCO).

BRAF mutation is also considered one of the mechanisms of EGFR-TKI resistance. However, the occurrence of BRAF mutation in EGFR-TKI resistance is rare, with an incidence of only about 1%. On the other hand, the rate of BRAF mutation after osimertinib resistance is higher, ranging from 3% to 10% (16). There was a study (17) showing that a combination treatment of dabrafenib, trametinib, and osimertinib resulted in clinical remission with an overall response rate (ORR) of 80% in five EGFR+ patients who had received osimertinib as a primary treatment. This suggests that the combination treatment may have a better therapeutic effect for patients with BRAF mutation after EGFR resistance. However, it is important to note that this study had a small sample size and there were significant differences in patient baseline data. Therefore, further studies with larger sample sizes are needed to validate these findings.

While TKIs are widely used in NSCLC treatment, the occurrence of drug resistance is inevitable. The resistance mechanisms of 3rd generation EGFR-TKIs are complex and lack specific treatment. ALK-fusion and BRAF-mutations, although rare, cannot be ignored as they may emerge as mechanisms of EGFR-TKIs acquired resistance in a small number of NSCLC-patients. To develop more accurate personalized treatment plans, it is crucial to continuously explore potential drug resistance mechanisms and for finding therapeutic options under EGFR-TKIs resistance. This will further optimize the application of targeted therapy in NSCLC and provide precise guidance for individualized treatment of patients.