A comprehensive clinical trajectory of A763_Y764insFQEA-positive non-small cell lung cancer treated with fifth-line osimertinib following circulating tumor DNA-based detection: a case report.

Introduction
Epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations account for approximately 5–12% of all EGFR mutations in non-small cell lung cancer (NSCLC), and are generally associated with resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) (1,2). Among them, the A763_Y764insFQEA variant is a rare but distinct subtype that has been shown in preclinical studies by Yasuda et al. to be sensitive to multiple generations of EGFR-TKIs (3,4). Early clinical reports have also demonstrated a favorable response to first-generation EGFR-TKIs such as gefitinib in this variant (5). Structurally, insertion of the FQEA motif disrupts hydrophobic interactions that stabilize the inactive conformation of EGFR, thereby promoting an active conformation and enhancing TKI binding affinity (3,6).
In recent years, advances in next-generation sequencing (NGS) using circulating tumor DNA (ctDNA) have enabled the detection of rare EGFR mutations in later-line settings, even in patients who are not eligible for tissue rebiopsy (7,8). In particular, polymerase chain reaction (PCR)-based diagnostic platforms such as the cobas®
EGFR Mutation Test v2 often fail to detect the A763_Y764insFQEA variant, whereas hybrid-capture-based assays such as FoundationOne® CDx and FoundationOne® Liquid (F1L) have demonstrated greater sensitivity (8-10).
This report describes a Japanese patient in whom the A763_Y764insFQEA variant was identified via ctDNA-based testing using F1L. The administration of osimertinib as fifth-line treatment provides important clinical insight into managing this rare EGFR ex20ins mutation. We present this case in accordance with the CARE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-869/rc).

Case presentation
A 63-year-old Japanese woman with no history of smoking presented to a local clinic in December 2020 with right-sided chest pain. Chest imaging revealed a mass in the right S2 segment, mediastinal and supraclavicular lymphadenopathy, and bone metastases. She was referred to our pulmonary department for further evaluation.
Bronchoscopic biopsy confirmed lung adenocarcinoma with positive thyroid transcription factor-1 (TTF-1) immunostaining. The clinical stage was cT1cN3M1c (bone and lymph node metastases), corresponding to stage IVB. PCR-based molecular testing on the initial tissue specimen was negative for EGFR, ALK, ROS1, and BRAF mutations. The PD-L1 tumor proportion score (TPS) was 70%.
First-line therapy, initiated in January 2021, consisted of four cycles of pembrolizumab plus carboplatin and pemetrexed, followed by maintenance pemetrexed monotherapy. Disease control was achieved for approximately 10 months.
Subsequently, the patient developed neurological symptoms due to spinal metastases and received multiple courses of palliative radiotherapy. She underwent second-line treatment with docetaxel plus ramucirumab, third-line treatment with carboplatin plus S-1, and fourth-line treatment with vinorelbine; all regimens failed to achieve satisfactory disease control.
Rebiopsy was attempted, but insufficient tissue prevented NGS testing via FoundationOne® CDx. In August 2022, comprehensive genomic profiling was performed using plasma ctDNA with F1L, which identified the EGFR A763_Y764insFQEA mutation.
Osimertinib (80 mg daily) was initiated as fifth-line therapy in October 2022. A full clinical timeline is shown in Figure 1, and a partial response was confirmed by December 2022 and sustained at least through March 2023 (Figure 2). Treatment was continued despite progression, following a beyond PD strategy (i.e., treatment continuation beyond RECIST-defined progression). She also received palliative radiotherapy to the spine, skull, and brain during osimertinib therapy. The patient died on August 18, 2023, ten months after starting osimertinib. Molecular confirmation is shown in Figure S1.
All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The patient had passed away at the time of manuscript preparation; therefore, written informed consent for publication could not be obtained. All possible efforts were made to protect patient anonymity.

Discussion
The EGFR A763_Y764insFQEA mutation is one of the few EGFR ex20ins variants known to be sensitive to EGFR-TKIs (2). Early clinical reports demonstrated a favorable response to first-generation TKIs such as gefitinib (3). Preclinical studies have shown that the insertion of four amino acids (FQEA) disrupts hydrophobic interactions involving residue I759, leading to constitutive kinase activation and increased binding affinity for EGFR-TKIs (2). This variant also exhibits a lower binding free energy for TKIs compared to other ex20ins mutations, providing a structural basis for its drug sensitivity (2,6).
Clinically, a cohort study involving 16 patients with the A763_Y764insFQEA mutation reported an objective response rate (ORR) of 62.5%, a median progression-free survival (PFS) of 5.5 months, and a median overall survival (OS) of 22.0 months following EGFR-TKI therapy (4). In contrast, the overall efficacy of first- and second-generation EGFR-TKIs against ex20ins mutations has been limited (1), underscoring the need for mutation-specific treatment strategies based on drug sensitivity.
Recent developments in agents such as amivantamab have expanded our understanding of subtype-specific drug reactivity, further emphasizing the importance of accurately identifying highly drug-sensitive variants like A763_Y764insFQEA (5).
Several large-scale analyses have recently characterized the clinical and molecular spectrum of EGFR ex20ins mutations across diverse populations (11-13). These studies provide valuable insights into the heterogeneity of ex20ins and their overall clinical outcomes. Within this heterogeneous group, however, the A763_Y764insFQEA variant represents a structurally distinct and relatively TKI-sensitive subtype, underscoring the need for continued detailed reporting and mechanistic investigation to refine treatment strategies for affected patients.
Several case reports describing clinical responses to EGFR-TKIs in patients with the A763_Y764insFQEA mutation, including those summarized in Table 1, have been published. Although most prior reports originated from Western countries, recent years have seen an increase in case reports and registry analyses from Asia. In mainland China and Taiwan, the clinical spectrum of ex20ins mutations, including A763_Y764insFQEA, has been described (9,16). In the United States, Coleman et al. reported a case in which the A763_Y764insFQEA mutation was identified by tissue-based genomic profiling (FoundationOne® CDx) and responded to afatinib (14). In Japan, there is a notable report of a relapsed case where this mutation was detected using a tissue-based panel and osimertinib successfully reversed carcinomatous meningitis (15). In that case, Kunimasa et al. identified the variant via the OncoGuideTM NCC Oncopanel after standard treatments, and a favorable response to EGFR-TKI was observed.
In the present case, EGFR-TKI was not introduced until the fifth line of therapy. If the mutation had been identified earlier, molecular targeted therapy could have been initiated at an earlier treatment stage.
The mutation was initially missed using the cobas®
EGFR Mutation Test but was later detected via F1L, underscoring the diagnostic importance of selecting appropriate molecular tests for rare actionable variants. This case reinforces the clinical value of hybrid-capture-based ctDNA analysis in precision oncology.
This case represents a rare fifth-line administration of osimertinib in a Japanese NSCLC patient and contributes valuable real-world data. The full clinical trajectory—from diagnosis through multiple lines of therapy to death—is documented, making this case particularly educational.
Furthermore, the initial treatment choice of immune checkpoint inhibitor (ICI)–based chemoimmunotherapy was appropriate, considering the PD-L1 TPS of 70% in accordance with current clinical guidelines. Other reported cases have shown partial responses or disease stabilization with EGFR-TKIs, and the present case aligns with those outcomes. This case also illustrates that continuation of EGFR-TKIs beyond radiological progression may be justified when clinical benefit persists, consistent with findings from meta-analyses evaluating subtype-specific efficacy of EGFR-TKIs (17).
A763_Y764insFQEA is an extremely rare EGFR mutation that differs structurally and pharmacologically from other ex20ins variants. Despite this, such variants are often grouped under the umbrella term “ex20ins” in clinical practice, potentially leading to inappropriate treatment decisions. The poor sensitivity of Cobas® for detecting this mutation suggests that undiagnosed cases likely exist in Japan.
While both preclinical and clinical data support the efficacy of EGFR-TKIs against this mutation, additional evidence is needed to solidify its place in treatment guidelines and ensure broader access to appropriate care for patients with this rare alteration. Accumulating further cases and elucidating optimal timing and prognostic markers for treatment intervention are critical next steps.

Conclusions
This report describes a Japanese patient in whom the EGFR A763_Y764insFQEA mutation was identified using plasma-based ctDNA NGS, leading to the initiation of osimertinib as fifth-line therapy. Our findings demonstrate that even rare EGFR variants can be appropriately diagnosed and treated when suitable testing methods are selected. This report may contribute to the establishment of future clinical guidelines and the optimization of therapeutic interventions for patients harboring rare EGFR mutations.

Supplementary
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