A novel acquired EGFR-SEPT14 fusion confers differential drug resistance to EGFR inhibitors in lung adenocarcinoma

Around 10%e30% of non-small cell lung cancer (NSCLC) patients harbored epidermal growth factor receptor (EGFR) mutations, with L858R and exon-19 deletions (19-Del) accounting for 90% of cases. EGFR tyrosine kinase inhibitors (TKIs) showed significant efficacy against common EGFR mutations. However, the therapeutic relevance of uncommon EGFR mutations remained insufficiently investigated. EGFR fusions are extremely rare (0.05%e0.13%) in NSCLC, and Konduri group reported only 5 EGFR fusions from 10,097 patients. Additional EGFR fusions were reported in NSCLC, all of which were oncogenic drivers and sensitive to EGFR TKIs. Herein, we reported an NSCLC patient with leptomeningeal metastasis (LM) who acquired a novel EGFR-SEPT14 fusion during TKI resistance and showed promising responses to certain EGFR TKIs and intrathecal pemetrexed (IP). A 51-year-old male was diagnosed with lung adenocarcinoma (T1cN0M0, IA3) in May 2015 (Fig. S1A, B). After thoracoscopic resection, EGFR 19-Del was detected using an amplification refractory mutation system. No adjuvant therapy was performed. In July 2016, the patient presented symptoms of progressive disease (PD) such as a progressive headache, while no abnormality was shown on magnetic resonance imaging (MRI) (Fig. S1C), so anxiolytic treatment was performed according to symptoms. Four months later, he presented clinical symptoms of intracranial hypertension, such as blurred vision, obvious dizziness, and syncope. Kamofsky performance status (KPS) score was 70. A lumbar puncture was performed to reduce intracranial pressure, and cerebrospinal fluid (CSF) was sent for pathological diagnosis confirming LM. From December 2016 to September 2017, he received erlotinib and AZD3759, and his symptoms were controlled. Meanwhile, next-generation sequencing (NGS)

Around 10%e30% of non-small cell lung cancer (NSCLC) patients harbored epidermal growth factor receptor (EGFR) mutations, with L858R and exon-19 deletions (19-Del) accounting for 90% of cases. EGFR tyrosine kinase inhibitors (TKIs) showed significant efficacy against common EGFR mutations. However, the therapeutic relevance of uncommon EGFR mutations remained insufficiently investigated. EGFR fusions are extremely rare (0.05%e0.13%) in NSCLC, and Konduri group reported only 5 EGFR fusions from 10,097 patients. 1,2 Additional EGFR fusions were reported in NSCLC, 3,4 all of which were oncogenic drivers and sensitive to EGFR TKIs. Herein, we reported an NSCLC patient with leptomeningeal metastasis (LM) who acquired a novel EGFR-SEPT14 fusion during TKI resistance and showed promising responses to certain EGFR TKIs and intrathecal pemetrexed (IP).
A 51-year-old male was diagnosed with lung adenocarcinoma (T1cN0M0, IA3) in May 2015 (Fig. S1A, B). After thoracoscopic resection, EGFR 19-Del was detected using an amplification refractory mutation system. No adjuvant therapy was performed.
In July 2016, the patient presented symptoms of progressive disease (PD) such as a progressive headache, while no abnormality was shown on magnetic resonance imaging (MRI) (Fig. S1C), so anxiolytic treatment was performed according to symptoms. Four months later, he presented clinical symptoms of intracranial hypertension, such as blurred vision, obvious dizziness, and syncope. Kamofsky performance status (KPS) score was 70. A lumbar puncture was performed to reduce intracranial pressure, and cerebrospinal fluid (CSF) was sent for pathological diagnosis confirming LM. From December 2016 to September 2017, he received erlotinib and AZD3759, and his symptoms were controlled. Meanwhile, next-generation sequencing (NGS) analysis of CSF confirmed EGFR 19-Del. Osimertinib treatment was then included, and the patient underwent a second CSF NGS, with only EGFR 19-Del detected. Until April 2019, the disease was stable with a continual decrease in carcinoembryonic antigen (CEA) levels and the relief of symptoms (KPS: 90). No progression was observed according to the image scan (Fig. S1D).
In May 2019, the patient suffered from PD and was treated with bevacizumab, nab-paclitaxel, and carboplatin. As carcinoembryonic antigen (CEA) levels increased sharply (Fig. 1A), he received osimertinib plus cabozantinib in December 2019; however, no symptomatic remission was observed. By a third CSF NGS, EGFR-SEPT14 fusions were newly detected (Fig. 1B).
We treated the patient with osimertinib plus erlotinib, but his disease conditions became worsened. From February to June 2020, he received dacomitinib plus osimertinib, resulting in noted improvement in his symptoms (KPS: 90) with a reduction in CEA level (Fig. 1A). However, in June 2020, the patient presented with headache and vomiting again, and the result of the increasing CEA suggested PD. Therefore, he then underwent 4 cycles of chemotherapy with bevacizumab, nab-paclitaxel, and carboplatin and maintained a double dose of osimertinib until PD in November 2020. By administering dacomitinib plus osimertinib, he experienced a short period of disease control. However, the patient suffered hearing dysesthesia and blurred vision (KPS: 40) soon, which displayed evidence of PD.
In January 2021, the fourth CSF NGS showed increased mutant allele frequency (MAF) of EGFR-SEPT14 (from 7.2% to 57.0%), together with EGFR amplification (Fig. 1B). The patient was subjected to IP to treat LM and osimertinib for maintenance therapy. After 3 months, his CEA level decreased from 38.42 ng/mL to 12.36 ng/mL, accompanied by a relief of symptoms. No recurrence or abnormality was found according to the patient's last CT and MRI scan Peer review under responsibility of Chongqing Medical University.
Available online at www.sciencedirect.com ScienceDirect journal homepage: www.kea ipublishing.com/en/ journals/gene s-disea se s (Fig. S1E, F). Based on in vitro findings (results below), the patient received afatinib plus osimertinib for maintenance treatment in June 2021, and his CEA levels further dropped to 8.24 ng/mL (Fig. 1A). The fifth CSF NGS (July 2021) showed that the MAF of EGFR-SEPT14 had decreased from 57.0% to 44.8% and EGFR copy number was decreased from 6.9 to 4.2 (Fig. 1B).
After initial TKI resistance, the patient acquired two forms of EGFR-SEPT14 fusions. The first one fused EGFR exon-24 with SEPT14 exon-10 (E24:S10), which was reported in glioblastomas 5 and was unlikely to confer drug resistance due to its sensitivity to erlotinib 5 and low MAF (Fig. 1B). The second one linked EGFR exon-25 to SEPT14 exon-7 (E25:S7) (Fig. 1C, D), whose MAFs were well-corresponded to disease conditions (Fig. 1A, B), implying to be a novel TKI resistant mechanism. Simulated structural analyses revealed that EGFR-SEPT14 (E25:S7) and wild-type EGFR had similar secondary but distinct tertiary structures (Fig. S2A, B), suggesting that the fusion protein might have different functions and/or drug-binding capacities.
To further simulate the pathological scenario, we cotransfected EGFR 19-Del and EGFR-SEPT14 (E25:S7) into Ba/ F3 cells, which could proliferate in the absence of IL3 and/ or EGF (Fig. 1E). Notably, the co-transfected cells also maintained high sensitivity to some second-and thirdgeneration TKIs but gained drug resistance to first-generation TKIs and osimertinib (Fig. 1H, I and Table S3).
Our results suggested that EGFR-SEPT14 (E25:S7) was not oncogenic, but it can function together with EGFR 19-Del. Previous studies demonstrated that EGFR could form heterodimers to stimulate downstream signaling. Therefore, we suspected that EGFR-SEPT14 (E25:S7) could couple with EGFR 19-Del to form heterodimers, conferring oncogenicity and hindering the access of certain TKIs. Overall, we identified a novel EGFR-SEPT14 (E25:S7) fusion as a resistant mechanism to EGFR TKIs. Despite resistance to first-generation TKIs and osimertinib, EGFR-SEPT14 (E25:S7) responded well to second-generation and some third-generation TKIs.

Ethics declaration
The patient included in this research signed an informed consent according to the Research Ethics Committee of The First Affiliated Hospital, School of Medicine, Zhejiang University.

Data availability
The human sequence data generated in this study are not publicly available due to patient privacy requirements but are available upon reasonable request from the corresponding author. Other data generated in this study are available within the article and its supplementary data files.