Analysis of Acquired Fusion Mutations in EGFR-TKIs-Resistant Patients with Advanced Lung Cancer

By Yuan Yang¹, Nana Zhang², Liang Shi¹, Zhaoxin Chen¹, Baohua Lu¹, Zhe Liu¹

Affiliations

1. Department of Oncology, Beijing Chest Hospital, Capital Medical University, and Beijing Tuberculosis and Thoracic Tumour Research Institute, Beijing, China
2. Department of Pathology, Beijing Chest Hospital, Capital Medical University, and Beijing Tuberculosis and Thoracic Tumour Research Institute, Beijing, China

doi: 10.29271/jcpsp.2026.03.402

ABSTRACT
To determine the characteristics of non-small cell lung cancer (NSCLC) patients with fusion genes emerging after resistance to EGFR-TKIs. This study retrospectively collected 2800 cases of NSCLC with EGFR-sensitive mutations who underwent tissue-based next- generation sequencing (NGS) testing at least once. Patients with acquired fusion mutations, including ALK, ROS1, RET, etc., were included in the study, and clinical data, gene mutation status, treatment strategies, and follow-up were collected. Given the small sample size and exploratory design of this case series (n = 9), the findings were reported descriptively. There were six cases of ALK fusion, two cases of RET fusion, and one case of ROS1 fusion. EGFR abundance decreased in five patients, while EGFR was detected as negative in the other four patients. When fusion mutations emerged, the use of ALK/RET/ROS1-TKIs or EGFR-TKIs combined with fusion-TKIs demonstrated efficacy. The progression-free survival ranged from 5 months to ≥39 months. One patient with acquired fusion gene mutations developed small-cell lung cancer transformation after ALK-TKIs resistance.

Key Words: Lung cancer, ALK fusion, RET fusion, ROS1 fusion, EGFR-TKIs-resistance, Targeted therapy.

INTRODUCTION

Due to the significant improvements in survival and quality of life brought by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), they have been prioritised and widely used in EGFR-sensitive non-small cell lung cancer (NSCLC) patients. However, acquiring resistance after EGFR-TKIs is inevitable. The currently known resistance mechanisms include EGFR-dependent resistance, bypass or downstream activation, organisational or phenotypic transformation. The proportion of off-target resistance is higher than that of on- target  resistance.¹

Acquired fusion gene mutations are a rare pathway activation mechanism that leads to resistance to EGFR-TKIs. Previous reports have shown that proto-oncogene tyrosine-protein kinase ROS1 (ROS1), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), neurotrophic  tyrosine  receptor  kinase 1 (NTRK1), fibroblast growth factor receptor 3 (FGFR3), and BRAF fusions  mediate  resistance  to  EGFR-TKIs.^1-3

This study retrospectively analysed cases in Beijing Chest Hospital to provide real-world data for such patients. This study aimed to determine the clinical-pathological features, drug selections, therapy responses, and prognoses of these unique patients.

METHODOLOGY

A retrospective study was conducted on 2,800 cases of NSCLC patients with EGFR-sensitive mutations who were admitted to the Department of Oncology, Beijing Chest Hospital, Capital Medical University, and Beijing Tuberculosis and Thoracic Tumour Research Institute, Beijing, China, from June 2019 to June 2024, with complete medical records. All patients received next-generation sequencing (NGS) testing either before EGFR-TKIs application or after resistance to EGFR-TKIs, with some undergoing both tests. This research specifically focused on patients with acquired fusion mutations, including ALK, ROS1, RET, BRAF, FGFR3, etc. The study period ranged from September 2024 to December 2024, during which data were gathered, and this investigation was conducted. The Ethical  Committee  of  Beijing  Chest Hospital approved  this study (LW-2024-001).

According to fusion mutation cases selection criteria, the diagnosis of NSCLC was established through histopathological examination. At the time of first diagnosis, the presence of either exon 19 deletion (19del) or exon 21 mutation (21L858R) in  the  EGFR  gene  was  confirmed  using  tissue-based  NGS.

Before EGFR-TKIs treatment, ALK/ROS1/RET/BRAF/FGFR3/ NTRK1 fusion mutations were negative (NGS and immuno-histochemistry). Stage IV disease was defined based on the UICC lung cancer staging criteria (version 8). Treatment with EGFR-TKIs was followed by the development of acquired resistance as per David Jackman's criteria. NGS was repeated in the re-biopsy tumour lesions after resistance, and the fusion gene mutation was detected. Exclusion criteria included: patients with other active malignancies requiring concurrent treatment; patients with severe comorbidities such as cardiovascular diseases, autoimmune disorders, vasculitis, interstitial pneumonia, or HIV positivity; and patients who received chemotherapy before or concurrently with EGFR-TKIs therapy after the detection of EGFR-sensitive mutations.

The clinical and pathological characteristics of the selected patient cohort were comprehensively analysed. The assess-ment of curative effect was based on response evaluation criteria in solid tumours (RECIST1.1). The last follow-up period of this study was December 1, 2024. This study also incorporated analysis of mutation abundance, quantitatively defined as the proportion of mutated reads at a specific genomic location relative to the total number of reads at that same site.

Table I: Characteristics of nine patients.

Clinical features		Frequencies	Percentages
Age	≤60 years	5	56%
-	>60 years	4	44%
Gender	Male	3	33%
-	Female	6	67%
Histological subtype	Adenocarcinoma	9	100%
Smoking history	Current	2	22%
-	Never	7	78%
PDL-1	0%	2	22%
-	1-49%	5	56%
-	>50%	2	22%
EGFR type	19 DEL	5	56%
-	L858R	4	44%
PDL-1: Programmed cell death ligand 1; EGFR: Epidermal growth factor receptor.

This small-scale case series (n = 9) was exploratory in nature; thus, all results are presented descriptively. Categorical variables are shown as absolute numbers and percentages. Due to the limited sample size, no hypothesis-testing statistical methods were employed. Statistical software SPSS version 26.0 (Armonk, NY: IBM Corp) was used for data analysis.

RESULTS

There were six cases of ALK fusion, two cases of RET fusion, and one case of ROS1 fusion. There were no secondary mutations in the fusion genes of NTRK1, BRAF, and FGFR3.

The abundance of the EGFR gene decreased in five patients, and four patients tested negative for EGFR. All nine patients had lung adenocarcinoma. Five cases exhibited echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion, while one displayed striatin (STRN)-ALK fusion. Following the acquisition of ALK mutations, all cases achieved objective responses through treatment with ALK-TKIs or in combination with EGFR-TKIs. Patients with acquired RET and ROS1 fusion mutations also achieved a partial response after undergoing fusion-TKIs. Patients' clinical features and pathological characteristics are shown in Table I. The patients' gene status, treatment plans, efficacy, and prognoses are shown in Table II. Regarding adverse events associated with second-line therapy, eight patients experienced Grade 1 events or higher. The most common adverse events included rash (5 cases, 55.6%), diarrhoea (4 cases, 44.4%), nausea (4 cases, 44.4%), and fatigue (2 cases, 22.2%), all of which were Grade 1 or 2 in severity. No Grade 3 or higher adverse events were reported.

Table II: Changes in NGS and treatment plans after resistance to EGFR-TKIs.

Case	New fusion genes (NGS abundance)	EGFR alterations after acquired fusion gene (NGS abundance)	Treatment after fusion detected (second-line therapy)	PFS of fusion-TKIs ±EGFR-TKIs	OS
Case1   Case2	EML4-ALK V1 (2.98%)   EML4-ALK V1 (2.30%)	EGFR 19 del (25.10%→18.28%)	Ensartinib+Osimertinib	>17 months	>39 months
		EGFR exon 20 L858R (19.35%→0)	Alectinib followed by chemotherapy due to SCLC transformation	=21 months	=33 months
Case3	EML4-ALK V2 (3.9%)	EGFR 19 del (22.4%→3.7%)	Ensartinib	>22 months	>42 months
Case4   Case5	EML4-ALK V3 (7.94%) EML4-ALK V3 (0.29%)	EGFR L858R (3.4%→0) EGFR G719A (8.7%→0)	Alectinib	>32 months	>42 months
		EGFR 19 del (12.4%→0)	Ensartinib	>18 months	>39 months
Case6	STRN-ALK (12.6%)	EGFR 19 del (56.98%→48.34%)	Alectinib+ Almonertinib	>20 months	>46 months
Case7   Case8	CCDC6-RET (1.86%) CCDC6-RET (0.32%)	EGFR 19 del (61.18%→57.03%)	Pralsetinib+ Almonertinib	=5 months	=43 months
		EGFR 19 del insP (41.25%→0)	Pralsetinib	=6 months	=51 months
Case9	CD74-ROS1 (4.25%)	EGFR L858R (12.56%→1.94%)	Iruplinalib	>39 months	>41 months
EGFR: Epidermal growth factor receptor; NGS: Next generation sequencing; ALK: Anaplastic lymphoma kinase; TKIs: Tyrosine kinase inhibitors; PFS: Progression-free survival; OS: Overall survival; EML4: Echinoderm Microtubule-associated protein-Like 4 gene; STRN: Striatin; RET: Rearranged during transfection; ROS1: Proto-oncogene tyrosine-protein kinase ROS1; CCDC6: Coiled coil domain containing 6 gene; CD74: Cluster of differentiation 74.

DISCUSSION

From the basic clinical information of this research, it was found that female patients, non-smokers, and patients with high programmed cell death ligand 1(PDL-1) expression had a higher proportion of secondary fusion gene mutations.

The incidence of primary fusion gene mutations is very low in NSCLC patients. In newly diagnosed advanced NSCLC, the proportion of ALK fusion is about 5%. RET fusions occur in 1–2% of NSCLC patients,⁴ and approximately 2.59% of patients carry the ROS1 fusion gene in China.⁵

EGFR-TKIs treatment induces activation of bypass and/or downstream signalling pathways, promoting cell survival and proliferation, which is a form of EGFR-TKIs resistance.⁶ Previous studies have shown that ALK/RET/ROS1 fusion mutations are acquired during EGFR-TKIs treatment.^2,7-9 Secondary fusion mutations after EGFR-TKIs are even rarer. The overall changes in the status of secondary fusion gene mutations after resistance to EGFR-TKIs and the treatment plans remain unclear.

In this study, after acquired resistance to EGFR-TKIs, there were ALK, RET, ROS1 mutations rather than NTRK1, BRAF, and FGFR3. There were six cases of ALK fusion mutations, including five EML4-ALK and one STRN-ALK fusion mutations. This research also screened two cases of CCDC6-RET fusion mutations and one case of cluster of differentiation 74 (CD74)-ROS1 fusion gene mutation. Fusion gene partners of ALK/RET/ROS1 were consistent with previous research, and no new rare fusion partners were found.

This research found that the abundance of the EGFR mutation was significantly reduced in five patients. The EGFR gene mutation in the other four patients were completely undetectable. This finding indicates that when acquired resistance occurs, the abundance of EGFR mutations is significantly reduced or even undetectable.

There are a few reports analysing the treatment approach and the effectiveness of therapy for acquired fusion mutations. The combination of ALK-TKIs and EGFR-TKIs has achieved therapeutic effects in previous case reports.¹⁰ In this study, the PFS of two patients treated with combined TKIs (case 1 and case 6) exceeded 17 and 20 months, respectively. At follow-up, they remained in a state of disease control. In vitro experiments have found that EGFR-TKIs activated ALK in solid tumours, which can induce EGFR-TKIs resistance. Gefitinib combined with ALK-TKIs had a more effective inhibitory effect on tumour cell activity.¹¹ In this study, four patients who received ALK-TKIs alone also achieved drug response.

In a multicentre retrospective real-world study, it was found that among acquired RET fusion patients who developed resistance after EGFR/ALK-TKIs therapy, immediate use of a pralsetinib-containing regimen after resistance was superior to delayed or non-immediate use of a pralsetinib-based regimen.¹² In this study, RET patients were treated with pralsetinib, and partial response was achieved; however, the PFS was five and six months, respectively. This outcome may be related to the poor performance status of patients before subsequent treatment.

It is worth noting that one case of acquired EML4-ALK fusion occurred after treatment failure of alectinib. On re-biopsy, this patient presented with small-cell lung cancer transformation. Chemotherapy showed a degree of effectiveness. To the best of the authors’ knowledge, this represents the first documented instance of ALK rearrange-ments followed by SCLC transformation as a source of resistance after EGFR-TKIs.

CONCLUSION

In the presence of fusion mutations, EGFR abundance may decrease or become undetectable. The use of fusion TKIs or EGFR-TKIs in combination with fusion TKIs in an optional treatment strategy. ALK-TKIs resistance can lead to small-cell lung cancer transformation in patients with secondary ALK fusion, indicating the importance of re-biopsy.

ETHICAL APPROVAL:
Ethical approval was obtained from the Ethical Committee of Beijing Chest Hospital, Capital Medical University, Beijing, China (Approval No. LW-2024-001).

PATIENTS’ CONSENT:
Informed consent was obtained from all participants.

COMPETING INTEREST:
The authors declared no conflict of interest.

AUTHORS’ CONTRIBUTION:
YY, BL, ZL: Conceptualisation, data curation, formal analysis, investigation, methodology, project administration, resources, software, roles/writing of the original draft, and review.
NZ, LS, ZC: Data curation and resources.
All authors approved the final version of the manuscript to be published.

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