Visceral crisis in a patient with non-small cell lung cancer and ROS1::SDC4 fusion: intrinsic resistance to entrectinib via L2026M mutation—a case report
Highlight box
Key findings
• The L2026M resistance mutation in the ROS1 kinase domain was identified in a patient with ROS1::SDC4 fusion-positive non-small cell lung cancer (NSCLC) who exhibited early disease progression with entrectinib.
• The L2026M mutation confers resistance by reducing drug-binding affinity, promoting tumor cell survival.
What is known and what is new?
• ROS1 inhibitors, such as entrectinib, are effective in ROS1-rearranged NSCLC, but resistance mechanisms remain incompletely characterized.
• This case reports an early resistance case due to the L2026M mutation, highlighting the need for alternative therapeutic strategies.
What is the implication, and what should change now?
• Early detection of resistance mutations through advanced molecular profiling is crucial for guiding therapeutic decisions in ROS1-positive NSCLC.
• New ROS1 inhibitors and combination therapies are required to overcome L2026M-mediated resistance.
Introduction
Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC) by targeting specific oncogenic driver mutations, such as EGFR, ALK, and ROS1 (1-3). The proto-oncogene ROS1 encodes a receptor tyrosine kinase that undergoes somatic chromosomal fusions to produce chimeric oncoproteins, leading to the development of various types of cancers, including NSCLC (1). Genetic mutation testing is crucial in the current approach to treating NSCLC patients, and targeting the ROS1 fusion protein with TKIs has significantly improved treatment outcomes with considerable efficacy for this subset of patients (3). Among these, ROS1 rearrangements are found in approximately 1–2% of NSCLC cases, predominantly in younger, non-smoking patients with adenocarcinoma histology (1).
Crizotinib, the first TKI approved for ROS1-positive NSCLC, demonstrated significant efficacy in the PROFILE 1001 trial, with an objective response rate (ORR) of approximately 72% and a median progression-free survival (mPFS) of 19.2 months. However, its efficacy against central nervous system (CNS) metastases was not evaluated (4). Entrectinib, a second-generation TKI, has also shown strong clinical effectiveness, particularly for patients with ROS1 fusion-positive NSCLC, including those with CNS metastasis. The STARK 1 and 2 trials reported an ORR of 68% and an mPFS of 15.7 months (5).
In general, patients with NSCLC who receive targeted therapy for driver mutations eventually develop resistance to TKIs (2,3). Similarly to EGFR and ALK, ROS1 fusion-positive NSCLC frequently develops resistance, with mechanisms well-documented for crizotinib. Resistance to ROS1 TKIs can be classified as intrinsic (on-target), involving mutations within the ROS1 kinase domain, or extrinsic (off-target), involving bypass pathways such as EGFR, KRAS, or MET activation (6). While resistance mechanisms are well-documented for crizotinib, data on intrinsic and extrinsic resistance to second-generation TKIs like entrectinib remain limited (6,7). Among intrinsic mechanisms, gatekeeper mutations like L2026M, which disrupt TKI binding by altering the hydrophobic pocket in the kinase domain, are particularly challenging as they confer resistance to multiple ROS1 inhibitors, including entrectinib (7). Other known resistance mutations, such as G2032R and D2033N, similarly impair drug efficacy, necessitating the development of next-generation therapies (6).
In this report, we present the case of a patient with ROS1::SDC4 fusion NSCLC who exhibited early progression during entrectinib therapy, driven by the L2026M gatekeeper mutation, a leucine-to-methionine substitution at position 2026 in the ROS1 kinase domain. This case highlights the clinical and molecular challenges posed by intrinsic resistance mechanisms in ROS1-positive NSCLC and underscores the critical need for next-generation therapies targeting such mutations. We present this article in accordance with the CARE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1149/rc).
Case presentation
A 50-year-old man presented with a persistent cough, fatigue, and moderate somatic pain localized in the anterior left thoracic region. He was a lifelong non-smoker without significant medical history or known family history of cancer. Positron emission tomography-computed tomography (PET-CT) revealed a left parahilar pulmonary mass measuring 65 mm × 82 mm with necrotic features and a standardized uptake value (SUV) max of 21. Widespread metastatic involvement was observed, including lymph nodes (supraclavicular, retroclavicular, mediastinal, left hilar, para-aortic, retro-pancreatic, and peri-gastric), different organs (left adrenal gland, pancreas tail, body, and head, and transverse colon wall), skeletal muscles (left thigh, right arm, left rectus abdominis), and other sites (subcutaneous tissue in the right cheek and posterior cervical region). Magnetic resonance imaging (MRI) of the brain showed a solitary intra-axial lesion in the superior right frontal lobe, with no evidence of other metastases (Figure 1). Histopathological evaluation of a tissue biopsy confirmed lung adenocarcinoma with acinar and solid patterns. Immunohistochemistry demonstrated positive staining for thyroid transcription factor 1 (TTF-1) and napsin A and negative staining for p40 and CK5/6. Molecular testing using Oncomine DxTT identified ROS1 rearrangement with an ROS1::SDC4 fusion. Based on these findings, the patient was diagnosed with advanced-stage (cT4N3M1c, stage IVB) lung adenocarcinoma with ROS1::SDC4 fusion and PD-L1 expression of 70%.

Treatment course
The disease involved nodal, adrenal, pancreatic, subcutaneous, and cerebral regions. Treatment began with stereotactic radiosurgery (SRS) for a metastatic brain lesion. During this period, the patient developed venous thrombosis, requiring anticoagulation with apixaban, later switched to low-molecular-weight heparin after recurrent pulmonary embolism. Entrectinib was initiated as first-line systemic therapy and was well-tolerated initially. However, one month into entrectinib treatment, the patient developed biliary obstruction secondary to pancreatic metastases, necessitating biliary stent placement. Subsequently, he experienced leptomeningeal carcinomatosis, which was managed with intensity-modulated radiation therapy (IMRT). After 6 weeks of treatment, his condition deteriorated with worsening dyspnea, hemoptysis, dysphagia, and somnolence. Recurrent pulmonary embolism was suspected, and thoracic vessel imaging revealed disease progression with extrinsic compression of the bronchus, left pulmonary artery, and vein. Despite interventions, the disease advanced to respiratory failure, and the patient succumbed to his illness 8 weeks after initiating first-line therapy. At this moment, marked interstitial lung involvement, increased lymph node disease, and meningeal dissemination were found. During exposure to the TKI, the dose used was 600 mg of entrectinib once a day until biliary tract obstruction was found, when it was reduced to 400 mg every day for 2 weeks. The patient did not receive chemotherapy, including pemetrexed or an alternative TKI such as lorlatinib, due to the complexity of the clinical complications and rapid functional decline.
The residual tumor tissue was analyzed using real-time polymerase chain reaction (PCR), which identified the L2026M mutation, a leucine-to-methionine substitution in the ROS1 kinase domain. In addition, confirmatory Sanger sequencing was performed (Figure S1). This mutation is associated with intrinsic resistance to TKIs. This case highlights the importance of early molecular profiling in patients with atypical progression during TKI therapy. It highlights the urgent need for next-generation inhibitors to overcome resistance mechanisms like the L2026M mutation. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committees, and with the Helsinki Declaration and its subsequent amendments. Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.
Discussion
This case underscores a rare presentation of rapid disease progression in a ROS1::SDC4 fusion-positive NSCLC patient treated with entrectinib; despite a dose adjustment, it is important to highlight an intrinsic resistance mechanism driven by the subclonal presentation of the L2026M mutation. To our knowledge, this is the first reported case linking ROS1::SDC4 fusion with primary resistance due to the L2026M gatekeeper mutation, which results in a leucine-to-methionine substitution at a critical kinase domain residue. Furthermore, the patient exhibited early systemic progression, highlighting the clinical challenges posed by intrinsic resistance mechanisms to ROS1 TKIs. Notably, the initial next-generation sequencing (NGS) test did not detect the L2026M mutation, probably due to a lack of coverage or because it was present in a smaller proportion of cells that made rapid selection during treatment. However, the retrospective review confirmed the presence of the resistance mutation by two complementary techniques. Likewise, it is impossible to rule out the presence of additional commutations not covered by the NGS panel or due to overexpression of genes such as AXL (8).
The ROS1 receptor, a receptor tyrosine kinase (RTK) structurally similar to ALK, lacks a known ligand and is involved in cellular proliferation, survival, and migration. Genetic rearrangements of ROS1 lead to fusion proteins with constitutive kinase activity, activating oncogenic signaling pathways such as MAPK, PI3K/AKT, JAK/STAT, and RHOA/VAV3, thereby driving tumor progression (9-12). ROS1 fusions, including CD74::ROS1 (44%), EZR::ROS1 (16%), ROS1::SDC4 (14%), and others, are associated with distinct biological behaviors (Figure 2). ROS1::SDC4 fusion proteins, localized to endosomal compartments, enhance MAPK pathway activation, likely contributing to the aggressive phenotype and early progression observed in this case (12).

ROS1 inhibitors are classified into first-generation (e.g., crizotinib), second-generation (e.g., entrectinib, ceritinib, cabozantinib), and next-generation TKIs (e.g., lorlatinib, taletrectinib, repotrectinib), designed to overcome resistance (13). Entrectinib, a type I TKI, demonstrates high CNS penetration and superior ORR (68%) in treatment-naïve patients (5,14). However, resistance mutations limit its efficacy. The L2026M gatekeeper mutation, observed in this case, alters the kinase domain conformation, reducing TKI binding affinity, similar to resistance patterns seen with G2032R and D2033N mutations (15).
Additionally, resistance mechanisms involve solvent-front mutations (G2032R, D2033N, S1986F, L1951R) and gatekeeper mutations (L2026M) (Table 1) (19). The L2026M mutation induces conformational changes in the ROS1 ATP-binding pocket, impairing TKI interaction. Preclinical studies demonstrated that L2026M confers high-level resistance to entrectinib, with half maximal inhibitory concentration (IC50) values exceeding adequate therapeutic levels (19,20). Furthermore, the L2026M mutation enhances autophagy via the MEK/ERK pathway, promoting tumor invasion, metastasis, and clonal heterogeneity, complicating second-line treatment strategies (21-23). Given entrectinib’s lack of activity against L2026M, next-generation TKIs, such as lorlatinib, taletrectinib, repotrectinib, unecritinib, and zidesamtinib, remain the most viable alternatives. These inhibitors effectively overcome solvent-front and gatekeeper mutations, demonstrating superior CNS penetration and ORR (79–91%) (16-18,24,25). Additionally, chemotherapy (e.g., pemetrexed-based regimens, ORR 60%) remains a viable salvage option, given ROS1 fusion-positive tumors’ intrinsic sensitivity to thymidylate synthase inhibition (26,27).
Table 1
Medication | Resistance mutations |
---|---|
Crizotinib | E1935G, L1947R, L1951R, G1971E, L1982F, S1986F/Y, L2026M, G2032R, D2033N, C2060G, V2098I, L2155S |
Ceritinib | E1990G, F1994L |
Entrectinib | F2004C/I, G2032R, L2026M (preclinical) |
Lorlatinib | S1986F, G2032K/R, S1986F, L2086F |
Type II TKIs | E1974K, F2004V/C, E2020K, V2089M, D2113N/G, M2134I, F2075V/C (16-18) |
NSCLC, non-small cell lung cancer; TKI, tyrosine kinase inhibitor.
Conclusions
In conclusion, this case highlights the aggressive nature of adenocarcinoma with ROS1::SDC4 fusion and the L2026M gatekeeper mutation. The subcellular localization of the ROS1::SDC4 fusion protein and the presence of the L2026M mutation significantly enhance MAPK pathway activation, contributing to a more aggressive cancer phenotype. This is consistent with the patient’s clinical course, characterized by a transient response to entrectinib followed by early systemic progression. It is crucial to assess the efficacy of TKIs in patients who exhibit an initial unfavorable response or early progression, as this may indicate the presence of resistance mutations. Both intrinsic mechanisms, such as kinase domain mutations, and extrinsic mechanisms, such as associated commutations, should be systematically investigated in these scenarios.
Overall, the L2026M mutation exemplifies the complex and multifaceted nature of resistance mechanisms in ROS1-positive NSCLC. Addressing these challenges requires developing next-generation inhibitors and combination therapies to target primary and compensatory pathways effectively. Ongoing research and clinical trials are critical to refining these strategies and improving outcomes for patients with ROS1-rearranged NSCLC and resistance mutations. The main limitations of this study were the inability to perform cell-based assays with tissue culture to validate L2026M resistance due to technical challenges, high costs, and the availability of additional tissue to perform a more comprehensive analysis by Oncomine clinical next-generation sequencing. Additionally, another limitation of the case analysis is the impossibility of having access to the BAM file to review the presence of the L2026M mutation, which would allow validation of the importance of somatic variant refinement using these files (28) (Figure S1).
Acknowledgments
None.
Footnote
Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1149/rc
Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1149/prf
Funding: None.
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1149/coif). J.A.Z.L. reports the payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from AstraZeneca, Merck Sharp & Dome, Boehringer Ingelheim, Bristol-Myers Squibb; payment for expert testimony from AstraZeneca, Bristol-Myers Squibb, Pfizer; support for attending meetings and/or travel from Addium Pharma, AstraZeneca, Merck Sharp & Dome, Boehringer Ingelheim, Roche, Bristol-Myers Squibb, Pfizer, Novartis, Eli Lilly; and receipt of equipment, materials, drugs, medical writing, gifts or other services from AstraZeneca, Pfizer, Roche. S.I.M.J. reports the payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Astrazeneca, Medtronic, Johnson & Johnson, MSD. C.A.C.F. reports payment or honoraria as the Astrazeneca speaker. A.R.P. reports the payment for presentation honoraria from Astra Zeneca, Amgen, Bayer, Bristol Myers Squibb, Böhringer Ingelheim, Glaxo Smith Kline, Johnson and Johnson, Merck Sharpe and Dohme, Pfizer, and support for attending meetings and/or travel from Johnson and Johnson. L.C. reports the payment for presentations from Roche. A.F.C. reports the grants from Merck Sharp & Dohme, Boehringer Ingelheim, Roche, Bristol-Myers Squibb, Foundation Medicine, Roche Diagnostics, Termo Fisher, Broad Institute, Amgen, Flatiron Health, Teva Pharma, Rochem Biocare, Bayer, INQBox and The Foundation for Clinical and Applied Cancer Research – FICMAC; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from EISAI, Merck Serono, Jannsen Pharmaceutical, Merck Sharp & Dohme, Boehringer Ingelheim, Roche, Bristol-Myers Squibb, Pfizer, Novartis, Celldex Therapeutics, Foundation Medicine, Eli Lilly, Guardant Health, Illumina, and Foundation for Clinical and Applied Cancer Research-FICMAC; payment for expert testimony from Merck Sharp & Dohme, Boehringer Ingelheim, Roche, Bristol-Myers Squibb, Pfizer, Novartis, Foundation Medicine, Guardant Health, Illumina, and Foundation for Clinical and Applied Cancer Research-FICMAC; support for attending meetings and/or travel from Merck Serono, Merck Sharp & Dohme, Boehringer Ingelheim, Roche, Bristol-Myers Squibb, Pfizer, Novartis, Celldex Therapeutics, Foundation Medicine, Eli Lilly, and Foundation for Clinical and Applied Cancer Research-FICMAC; participation on a Data Safety Monitoring Board or Advisory Board of Roche, Merck Sharp & Dohme; and receipt of equipment, materials, drugs, medical writing, gifts or other services from Roche, Roche diagnostics, Rochem Biocare. 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. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committees, and with the Helsinki Declaration and its subsequent amendments. Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.
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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