Analysis of Clinical Features and Treatment Strategies in Advanced Lung Adenocarcinoma Patients With Acquired ALK Fusion After Resistance of EGFR-TKIs

Analysis of Clinical Features and Treatment Strategies in Advanced Lung Adenocarcinoma Patients With Acquired ALK Fusion After Resistance of EGFR-TKIs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of Clinical Features and Treatment Strategies in Advanced Lung Adenocarcinoma Patients With Acquired ALK Fusion After Resistance of EGFR-TKIs Yuan Yang, Baohua Lu, Liang Shi, Nana Zhang, Zhaoxin Chen, Jing Yu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4407880/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The mechanism of secondary drug resistance in advanced Non Small Cell Lung Cancer(NSCLC) patients with Epidermal Growth Factor Receptor (EGFR) gene sensitive mutation after EGFR-Tyrosine Kinase Inhibitors (TKIs) is complex. Acquired Anaplastic Lymphoma Kinase (ALK) fusion mutation is a rare type, and there are few reports on the clinical characteristics and treatment options for this group of patients. Methods Cases of 820 locally advanced or metastatic EGFR-sensitive mutations NSCLC patients whose gene status were detected by Next Generation Sequencing(NGS)after EGFR-TKIs resistance were retrospectively collected. Acquired ALK fusion gene mutation occurred in 4 of them. The clinical information, pathological types, gene mutation status, treatment plans, efficacies and prognoses of these 4 cases were analyzed. Results All 4 patients had lung adenocarcinoma. Three had EML4-ALK fusion and 1 had STRN-ALK fusion. EGFR gene mutation was detected negative in 2 cases after drug resistance, and the abundance of EGFR gene mutation decreased in 2 cases. The Progression Free Survival (PFS) of EGFR-TKIs ranged from 6 to 21 months, and after acquired ALK mutation objective response was all achieved using ALK-TKIs alone or the combination of ALK-TKIs and EGFR-TKIs, with PFS all exceeding 6 months. One patient developed small cell lung cancer transformation after ALK-TKIs resistance. Conclusion Acquired ALK fusion as a resistant mechanism of EGFR-TKIs is present and rare. EGFR is undetectable or abundance decreased when ALK fusion emerges. ALK-TKIs alone and ALK-TKIs combined with EGFR-TKIs are alternative treatment choices. lung cancer acquired resistance EGFR tyrosine kinase inhibitors ALK tyrosine kinase inhibitors Figures Figure 1 Introduction With a high incidence and high malignancy, lung cancer is currently the leading cause of death due to cancer worldwide, posing a serious threat to human health[ 1 ] [ 2 ]. Epidermal Growth Factor Receptor (EGFR) sensitive mutations population accounts for 10–50% in Non Small Cell Lung Cancer (NSCLC) patients[ 3 ] [ 4 ]. The conventional sensitive mutations are EGFR-19del and EGFR-L858R mutations. The application of EGFR-Tyrosine Kinase Inhibitors (TKIs) opens a new epoch in lung cancer treatment, significantly improves the survival of patients. Osimertinib, a third-generation EGFR-TKI, has a 18.9 months in median Progression Free Survival (PFS) and 38.6 months in median Overall Survival (OS)[ 5 ] [ 6 ]. Osimertinib has been recommended as the first-line treatment. However, NSCLC patients with EGFR-sensitive mutations will inevitably develop resistance after EGFR-TKIs treatment. The drug resistance mechanism of EGFR-TKIs is complicated, including on-target and off-target, epithelial mesenchymal transformation, small cell transformation and other mechanisms. Secondary fusion mutations are rare. Another key mutation Anaplastic Lymphoma Kinase (ALK) fusion has a low proportion, accounting for about 3–7% of newly diagnosed advanced NSCLC patients[ 7 ], while the use of second/third generation ALK-TKIs has resulted in a median PFS of 24–60 months. The application of ALK-TKIs makes NSCLC patients with ALK fusion gene mutation a truly chronic disease. Previous studies have suggested that EGFR and ALK fusion gene mutations are independent and mutually exclusive molecular events[ 8 ] [ 9 ] [ 10 ]. But with the development and application of Next Generation Sequencing (NGS), the coexistence of EGFR-sensitive mutations and fusion mutations has been gradually confirmed by a number of case reports and clinical studies[ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ]. Data show that the proportion of primary EGFR mutation and ALK fusion double mutation at initial diagnosis is about 0.3–1.6%[ 16 ] [ 17 ] [ 18 ] [ 19 ], while secondary ALK fusion mutation after EGFR-TKIs application is rarely reported. The clinicopathological features, treatment regimen, efficacies and side effects of these special patients remain unclear. To this end, we retrospectively analyzed this part of cases in Beijing Chest Hospital, Capital Medical University to provide real-world diagnosis and treatment basis for such patients. Methods Objects: A retrospective study was conducted on 820 cases with EGFR-TKIs drug-resistant NSCLC patients who were admitted to Beijing Chest Hospital, Capital Medical University from June 2019 to June 2023 with complete medical data. All patients underwent tumor tissue or plasma NGS gene detection after drug resistance. The 4 cases of ALK fusion after drug resistance were collected and analyzed. This study was approved by the Ethics Committee of Beijing Chest Hospital, Capital Medical University. the Number of Approval is LW-2024-001.Ethics Committee of Beijing Chest Hospital, Capital Medical University waived the need for informed consent. Inclusion criteria: 1. NSCLC was confirmed by histopathology; 2. At initial diagnosis, The deletion of exon 19 (19del) or the mutation of exon 21 (21L858R) of EGFR were confirmed by the tissue cell Fluorescence Quantitative Deoxyribonucleic Acid Amplification Refractory Mutation System Polymerase Chain Reaction (ARMSPCR) or NGS;3. Before EGFR-TKIs treatment, ALK fusion mutation was negative (VANTANA immunohistochemical or NGS). 4.According to the Union for International Cancer Control(UICC) lung cancer staging criteria (version 8),in stage IIIC or Ⅳ; 5. All patients had used EGFR-TKIs (including first-generation, second-generation or third-generation drugs) and developed resistance to EGFR-TKIs, and the definition of drug resistance followed the definition of acquired drug resistance by David Jackman[20]. 6. NGS detection was performed again in rebiopsy tumor tissues or plasma of all patients after acquired resistance and the ALK gene fusion were positive. Methods: The clinical and pathological data of these 4 patients have been collected, including age, gender, pathological type, EGFR gene mutation type, gene detection method, Computed Tomography(CT)scan results, medication regimen for EGFR-TKIs therapy, PFS for first-line EGFR-TKIs therapy, fusion partner of ALK fusion gene, abundance of ALK fusion gene, treatment plan and PFS after acquired resistance and OS etc. Assessment of curative effect: Evaluation of curative effects is characterized by Complete Response (CR), Partial Response (PR), Stable Disease (SD), and Progressive Disease (PD)based on Response evaluation Criteria In Solid tumors (RECIST1.1)[21] . CR+PR is objective response, CR+PR+SD is disease control. PFS of a treatment was defined as the time from the initiation of this treatment regime until PD or death from any cause. OS was defined as from the time of diagnosis of advanced lung cancer to death from any cause. The last follow-up period of this study was January 1, 2024. Treatment strategies in this study: EGFR-TKIs：Icotinib (first generation):125mg three times a day; Gefitinib (first generation):250mg once a day; Afatinib (second generation):40mg once a day; Dacomitinib (second generation):45mg once a day; Almonertinib (third generation) :110mg once a day. ALK-TKIs：Alectinib(second generation):600mg twice a day; Ensartinib (second generation):225mg once a day. Genetic analysis: Tumor tissue NGS: Tumor tissue NGS test was performed by the DA8600 NGS system of Novogene Co., Ltd. and at least 25 genes were detected. Plasma NGS: Plasma high-throughput NGS was performed by the sequencing Illumina Novaseq platform of Genetron Co., Ltd. using PairEnd150 sequencing strategy for sequencing samples. The depth of normal white plasma cell control is 100X, and plasma sequencing is 10000X Fluorescence Quantitative Deoxyribonucleic Acid ARMSPCR: Scorpion shaped probe amplification and blocking mutation system (ARMS PCR) was used to detect gene variations such as EGFR, ALK, Rearranged during Transfection(RET), proto-oncogene tyrosine-protein kinase ROS1(ROS1), and Neurotrophic Tyrosine Receptor Kinase (NTRK) in tumor tissue wax blocks. The reagents were provided by Xiamen Aide Biopharmaceutical Technology Co., Ltd. The experimental instrument Cobas z480 real-time fluorescence quantitative PCR instrument was provided by Roche Diagnostic Products Shanghai Co., Ltd. Mutation abundance: The concept of mutation abundance is the ratio of the number of mutated reads at a specific site to the total number of reads at that site. Expressed as a percentage, it represents the proportion of clones with mutations in the sample. Results All the 4 patients had lung adenocarcinoma, of which 3 had Echinoderm Microtubule-associated protein-Like 4 gene (EML4)-ALK fusion (Case1,3,4) and one had striatin(STRN)-ALK fusion (Case2). EGFR gene mutation was detected negative in two patients after drug resistance (Case1, 4), and EGFR gene abundance decreased in two patients (Case2, 3). After acquired ALK mutation objective response was all achieved using ALK-TKIs alone or the combination of ALK-TKIs and EGFR-TKIs, The patients' chest CT images before EGFR-TKIs are shown in Fig. 1 , and the patients' clinical features, pathological characteristics, PFS of each treatment and OS are shown in Table 1 . Table 1 Genetic mutation status and treatments notes of four patients: Sex/ Age Genomic Alterations before EGFR TKIs (NGS and abundance) Regime of EGFR-TKIs(PFS1) Genomic Alterations after acquired resistance / (NGS and abundance) Treatment after ALK fusion detection (PFS2) OS Case1 M/60 years Tissue NGS: EGFR L858R / (3.43%) EGFR G719A/(8.65%) Icotinib (10 months) Tissue NGS: EML4-ALK V3/ (4.99%) Alectinib (12months: ceased due to hemolytic anemia) Followed by Ensartinib (> 8months) Not reached Case2 F/63years Tissue NGS: EGFR exon19 p.L747-T751del/ (56.98%) TP53 PIK3CA Icotinib (10 months); Followed by Almonertinib after PD (11 months) Tissue NGS: STRN-ALK/ (12.58%) EGFR exon19 p.L747-T751del/ (48.34%) TP53 CDK6 CTNNB1 Alectinib+ Almonertinib (> 8month) Not reached Case3 F/62years Tissue NGS: EGFR exon 19 p.E746-A750del (25.10%) MDM2 Dacomitinib: (10 months); Followed by Gefitinib after PD (2months) Plasma NGS: ALK EML4-ALK V1 fusion(2.98%) EGFR exon 19 p.E746-A750del (18.28%) Ensartinib +Almonertinib (2months, ceased due to edema); Followed by Ensartinib + Osimertinib (> 4months) Not reached Case4 F/42years Tissue NGS: EGFR exon 20 L858R (19.35%) Afatinib (6 months) Tissue NGS: ALK EML4-ALK V1fusion(2.31% STK11 Alectinib (21months)Followed by chemotherapy due to Small cell lung cancer transformation 52 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;PD: Progressive Disease;EML4: Echinoderm Microtubule-associated protein-Like 4 gene. STRN:striatin.TP53:Tumor Protein 53. PIK3CA: Phosphatidylinositol-3-kinase catalytic subunit α. CDK6: cyclin dependent kinase 6.CTNNB1: catenin (cadherin-associated protein), beta 1.MDM2: murine double minute 2. STK11: Serine/ Threonine kinase 11. PFS1: PFS time of EGFR-TKIs treatment; PFS2: PFS time of ALK-TKIs or ALK-TKIs + EGFR-TKIs after EGFR-TKIs Sex: M: Male; F: Female Discussion Basic characteristics of secondary ALK fusion mutation after EGFR-TKIs resistance The probability of ALK mutation after EGFR-TKIs resistance is very low. According to previous reports, the calculated probability of occurrence is about 0.01%-0.2%[22] [23] [24]. In our analysis, 4 of 820 patients with acquired drug resistance after EGFR-TKIs developed ALK fusion mutations, with an incidence of 0.49%, which is close with previous reports. Haiyuan Xu et al. reviewed the NGS genome map data of 3873 patients with EGFR (exon 18-21) mutation and found that 5 new ALK fusion mutations were acquired during EGFR-TKIs treatment[24].Fusion partners of ALK identified in the 5 patients included EML4 (3 cases), STRN(1 case) and CCAAT-enhancer binding protein zeta(CEBPZ) (1 case). All three EML4–ALK fusions emerging after EGFR-TKI resistance were the rare variant E2:A20 (V5)[24]. Other literature reported one case of secondary STRN-ALK fusions in 62 patients with EGFR-mutated metastatic NSCLC with tissue or plasma biopsies at EGFR-TKIs progression[25].Alexa B Schrock et al. Identified a case of Pleckstrin homology domain containing A7 (PLEKHA7)-ALK fusion mutation after application of Erlotinib followed by Osimertinib[26]. A patient with EGFR L858R lung adenocarcinoma developed disease progression after 72.7 months of gefitinib therapy, emerging a novel ALK-R3HDM1 and EML4-ALK dual fusion that might be a delicate mechanism for the acquired resistance of EGFR- TKIs [27]. In our study, 3 EML4-ALK fusion mutations emerged, of which 2 cases were V2 subtype and 1 case was V3 subtype.1 STRN-ALK fusion mutation was detected. Our another study showed that classical EML4-ALK fusion accounted for 94.9% of patients with primary ALK fusion[28]. From this research and other previous literature, non-classical ALK fusions may be more common in acquired EGFR-TKIs resistance. In this study, EGFR gene mutation was detected negative in 2 of the 4 patients, and the abundance of EGFR-19DEL mutation decreased from 56.98% to 48.34% in 1 case, and from 25.10% to 18.28% in the other. In only four patients, the abundance of EGFR mutation was either reduced or undetectable at the time of resistance. Analysis of treatment strategies and curative effects of patients: Previous literature has focused on fusion gene mutations that may be acquired after EGFR-TKIs resistance. Drug resistance mechanisms include B-Raf proto-oncogene(BRAF),ALK,RET,NTRK,fibroblast growth factor receptor 3 (FGFR3) and other mutation types[24] [25] [26]. Few reports have analyzed the therapeutic strategy and efficacy of secondary ALK fusion mutation therapy and there are no relevant recommendations in current guidelines and expert consensus. Previous reports mostly focused on patients with co-existing of primary EGFR-sensitive mutation and ALK fusion mutation, and the medication strategy for patients with primary double mutations was also controversial. A literature review of 100 patients with primary double mutation found that the Disease control rate(DCR) and Objective response rate(ORR)of first-line use of ALK-TKIs (including Crizotinib and Ceritinib ) were 73.1% and 42.3%, respectively, that were higher than first-line use of EGFR-TKIs (including Gefitinib and Erlotinib) with DCR and ORR of 61.5% and 23.1%, respectively. Therefore, ALK-TKIs may be the first choice for patients with primary double mutation because NSCLC with ALK gene rearrangement tends to be more aggressive. ALK-TKIs seem to be more effective than EGFR-TKIs[29]. ALK inhibitors can be preferentially used in double-positive patients, especially in those with low EGFR mutation abundance[30]. However, there are different views that EGFR-TKIs may be more effective than ALK-TKIs in this group of patients[31] [32], and in patients with ALK/EGFR double mutation, the effectiveness of ALK-TKIs and EGFR-TKIs are worse than those in patients with a single mutation. Treatment options for ALK fusion after secondary resistance have only been reported in few cases. This study analyzed the diagnoses and treatments of 4 patients with acquired ALK fusion mutation after EGFR-TKIs, and found that the ORR and PFS of ALK-TKIs were similar to those reported in previous literature. In two patients with ALK/EGFR double mutations after resistance, one treated with EGFR-TKI plus ALK-TKI and obtained a long PFS; the other was treated with Ensartinib plus Almonertinib for 2 months, which caused severe systemic edema. The treatment plan was adjusted to Ensartinib every other day plus Osimertinib and showed good tolerance and effect. Previous literature has also reported that the combination therapy of EGFR-TKIs and ALK-TKIs has a good effect[33] [34]. The other two patients in our study used ALK-TKIs alone, Case 1 changed Alectinib to Ensartinib due to a side effect of hemolytic anemia and obtained disease control over 20 months after EGFR-TKI resistance and is still being treated. Case 4 used Alectinib monotherapy and reached PFS of 21 months. The above analysis suggests that AlK-TKIs alone or in combination with EGFR-TKIs are both alternative. It is worth noting that Case 4, after PD of Alectinib, this patient showed Small Cell Lung Cancer (SCLC)transformation in rebiopsy. Chemotherapy achieved certain effects. To our knowledge, this is the first case of ALK fusion followed by acquired SCLC transformation after EGFR-TKIs resistance, which reveals the complexity of resistance and the need for rebiopsy after disease progression. However, due to the small number of patients in this study, more sample size analysis is needed to guide the treatment of patients with acquired ALK fusion mutations. Tumor origin and tumor heterogeneity of EGFR/ALK co-mutation There are many studies on the tumor origin of primary EGFR/ALK co-mutation. The basic theory is that EGFR mutation and ALK fusion, both genetic changes, exist from the initial proliferation of tumors[18]. One view is that the changes of these two drivers can develop in the same tumor cell clone and coexist in the process of cancer development through cell line studies[35]. Immunohistochemical analysis of lung cancer tissues from patients with primary EGFR and ALK co-mutation found that EGFR mutation and ALK fusion were co-localized and co-expressed in the same cell group, and the two proteins may play synergistic driving roles in the same cancer cells[18]. Another view is that ALK/EGFR co-mutated lung adenocarcinoma is composed of two tumor cell subpopulations carrying EGFR mutations and ALK fusion mutations, reflecting genetic heterogeneity within the tumor[36] [37]. Cai et al. [38]used laser capture microdissection (LCM) to capture tumor cells with similar or different growth patterns from patients with ALK/EGFR co-mutations, and deduced that the gene changes were divided into four subclones: coexisting with EGFR mutation and ALK rearrangement, single EGFR mutation, single ALK rearrangement, and no gene changes. Our study found that 2 out of 4 patients had co-existing EGFR and ALK fusion mutations at the time of EGFR-TKIs resistance (case2 had NGS result of metastatic lymph node rebiopsy and case3 had the NGS result of plasma). At present, for secondary ALK fusion gene mutations, it is possible that there was a small number of ALK fusion gene mutations initially. Due to the pressure of EGFR-TKIs therapy and cloning selection, the subsequent ALK fusion gene mutations then could be detected. In the case of primary double mutation, if one of TKIs is selected for first treatment, the remaining uninhibited mutant cell lines of ALK or EGFR will continue to grow[11]. But in the case of secondary EGFR-TKIs resistance, the need for combined application of EGFR-TKIs and ALK-TKIs may be related to the phosphorylation level of downstream proteins activated by the signaling pathway[35]. From our study, it may also be related to the abundance of ALK and EGFR after drug resistance. Therefore, detecting the abundance of EGFR mutation and ALK fusion, as well as the phosphorylation level of downstream proteins, may be beneficial to guide the selection of TKIs in clinical practice. This study analyzed the clinical characteristics and diagnoses and treatments of four very rare patients with acquired ALK fusion mutation after EGFR-TKIs treatment and only individual cases were reported in the previous literature. Our patients have dynamic results of NGS method detection, indicating the dynamic gene mutation status of these patients. Our study discussed the specific treatment plans of this kind of patients and analyzed the curative effect and benefit of them. Conclusion ALK fusion gene mutation may occur in EGFR-TKIs resistant patients with EGFR sensitive mutation and the incidence is very low. EGFR abundance may be reduced or undetectable in the presence of ALK fusion. Patients may benefit from ALK-TKIs alone or ALK-TKIs combined with EGFR-TKIs. In patients with secondary ALK fusion mutation after EGFR-TKIs resistance, SCLC transformation can also occur after ALK-TKIs resistance, which shows rebiopsy is an important step in developing treatment plans. Declarations Ethics approval and consent to participate The study is approved by Ethics Committee of Beijing Chest Hospital, Capital Medical University and the Number of Approval is LW-2024-001. Ethics Committee of Beijing Chest Hospital, Capital Medical University waived the need for informed consent. All methods and experiments of this study were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests None Funding None Authors contributions: Conceptualization(Yuan Yang, Jing Yu, Zhe Liu); Data curation (Yuan Yang, Baohua Lu, Liang Shi, Nana Zhang, Zhaoxin Chen);Formal analysis(Yuan Yang, Baohua Lu, Jing Yu,Zhe Liu);Investigation(Yuan Yang, Jing Yu, Zhe Liu); Methodology(Yuan Yang, Baohua Lu, Jing Yu,Zhe Liu); Project administration(Yuan Yang,Baohua Lu); Resources(Yuan Yang, Baohua Lu, Liang Shi, Nana Zhang, Zhaoxin Chen); Software(Yuan Yang,Baohua Lu); Supervision(Jing Yu, Zhe Liu); Roles/Writing - original draft(Yuan Yang); Writing - review &(Yuan Yang,Baohua Lu) Acknowledgements None References Romaszko AM, Doboszyńska A. Multiple primary lung cancer: A literature review. Adv Clin Exp Med. 2018;27:725–30. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209–49. 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Acquired EML4-ALK fusion and EGFR C797S in cis mutation as resistance mechanisms to osimertinib in a non-small cell lung cancer patient with EGFR L858R/T790M. Anticancer Drugs. 2023;34:1146–50. Hou H, Sun D, Zhang C, Liu D, Zhang X. ALK rearrangements as mechanisms of acquired resistance to osimertinib in EGFR mutant non-small cell lung cancer. Thorac Cancer. 2021;12:962–9. Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res. 2008;14:4275–83. Zhao D, Fan J, Peng L, Huang B, Zhu Y, Shi H, et al. Two different patterns of lung adenocarcinoma with concomitant EGFR mutation and ALK rearrangement. Tumori. 2022;108:12–8. Kim T-J, Park CK, Yeo CD, Park K, Rhee CK, Kim J, et al. Simultaneous diagnostic platform of genotyping EGFR, KRAS, and ALK in 510 Korean patients with non-small-cell lung cancer highlights significantly higher ALK rearrangement rate in advanced stage. J Surg Oncol. 2014;110:245–51. Cai W, Lin D, Wu C, Li X, Zhao C, Zheng L, et al. Intratumoral Heterogeneity of ALK-Rearranged and ALK/EGFR Coaltered Lung Adenocarcinoma. J Clin Oncol. 2015;33:3701–9. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4407880","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":306912258,"identity":"2aed41d5-0fda-4539-b181-145e167d4e04","order_by":0,"name":"Yuan Yang","email":"","orcid":"","institution":"Oncology Department of Beijing Chest Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yang","suffix":""},{"id":306912259,"identity":"74de9f35-8369-4879-8723-e8239a36e02c","order_by":1,"name":"Baohua Lu","email":"","orcid":"","institution":"Oncology Department of Beijing Chest Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baohua","middleName":"","lastName":"Lu","suffix":""},{"id":306912260,"identity":"633350d6-27b7-4081-9aaa-8f606394e3dd","order_by":2,"name":"Liang Shi","email":"","orcid":"","institution":"Oncology Department of Beijing Chest Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Shi","suffix":""},{"id":306912261,"identity":"f2b7fea9-5724-477e-b34b-4571d2cd059a","order_by":3,"name":"Nana Zhang","email":"","orcid":"","institution":"Pathology Department of Beijing Chest Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Zhang","suffix":""},{"id":306912262,"identity":"ec46c3c1-0dd0-4214-9065-234b363ec54c","order_by":4,"name":"Zhaoxin Chen","email":"","orcid":"","institution":"Oncology Department of Beijing Chest Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhaoxin","middleName":"","lastName":"Chen","suffix":""},{"id":306912263,"identity":"283066d4-070c-4d77-b20b-d519ed20c0f7","order_by":5,"name":"Jing Yu","email":"","orcid":"","institution":"Oncology Department of Beijing Friendship Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yu","suffix":""},{"id":306912264,"identity":"f8264b0c-d586-4e3b-8c01-53912d4cb9a8","order_by":6,"name":"Zhe Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYDCCA2DSgoeBAYg+GNjYEaOFsYGBQQKshXFGQVoy0VoYQFqYeT4cAvHwA77jzc8ffNwjIWNw/uzBxzYGB5gZ2A8f3YBPi+SZY4aNM55J8BgcOJdsnGNwh4+BJy3tBj4tBjdyGJt5DgC1HOwxk84xeMYM9JcZkVoO85j/tjA4zNhAvJZjPGbMDMRoAfll5gygFskzPMaSPQZpyWyE/AIMsQcfPhywsec7f8bww48/Nnb87IeP4dWCCdhIUz4KRsEoGAWjABsAAGl5SpqG03PyAAAAAElFTkSuQmCC","orcid":"","institution":"Oncology Department of Beijing Chest Hospital, Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-05-12 09:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4407880/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4407880/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57518461,"identity":"e7d54922-20da-4cf7-99fe-8800b8299074","added_by":"auto","created_at":"2024-05-31 20:35:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":368952,"visible":true,"origin":"","legend":"\u003cp\u003eThe patients' chest CT images before Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR-TKIs)\u003c/p\u003e\n\u003cp\u003eA:case1, B:case 2,C:case 3,D:case 4.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4407880/v1/f55fb77f8d9d975d555953bf.png"},{"id":60688343,"identity":"c09f6e91-3a06-458a-bccc-2341836324c1","added_by":"auto","created_at":"2024-07-19 14:26:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":954427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4407880/v1/0c5e8267-b335-46da-8d01-b63924d62481.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of Clinical Features and Treatment Strategies in Advanced Lung Adenocarcinoma Patients With Acquired ALK Fusion After Resistance of EGFR-TKIs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith a high incidence and high malignancy, lung cancer is currently the leading cause of death due to cancer worldwide, posing a serious threat to human health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Epidermal Growth Factor Receptor (EGFR) sensitive mutations population accounts for 10\u0026ndash;50% in Non Small Cell Lung Cancer (NSCLC) patients[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The conventional sensitive mutations are EGFR-19del and EGFR-L858R mutations. The application of EGFR-Tyrosine Kinase Inhibitors (TKIs) opens a new epoch in lung cancer treatment, significantly improves the survival of patients. Osimertinib, a third-generation EGFR-TKI, has a 18.9 months in median Progression Free Survival (PFS) and 38.6 months in median Overall Survival (OS)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Osimertinib has been recommended as the first-line treatment. However, NSCLC patients with EGFR-sensitive mutations will inevitably develop resistance after EGFR-TKIs treatment. The drug resistance mechanism of EGFR-TKIs is complicated, including on-target and off-target, epithelial mesenchymal transformation, small cell transformation and other mechanisms. Secondary fusion mutations are rare.\u003c/p\u003e \u003cp\u003eAnother key mutation Anaplastic Lymphoma Kinase (ALK) fusion has a low proportion, accounting for about 3\u0026ndash;7% of newly diagnosed advanced NSCLC patients[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], while the use of second/third generation ALK-TKIs has resulted in a median PFS of 24\u0026ndash;60 months. The application of ALK-TKIs makes NSCLC patients with ALK fusion gene mutation a truly chronic disease.\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that EGFR and ALK fusion gene mutations are independent and mutually exclusive molecular events[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. But with the development and application of Next Generation Sequencing (NGS), the coexistence of EGFR-sensitive mutations and fusion mutations has been gradually confirmed by a number of case reports and clinical studies[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Data show that the proportion of primary EGFR mutation and ALK fusion double mutation at initial diagnosis is about 0.3\u0026ndash;1.6%[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], while secondary ALK fusion mutation after EGFR-TKIs application is rarely reported. The clinicopathological features, treatment regimen, efficacies and side effects of these special patients remain unclear. To this end, we retrospectively analyzed this part of cases in Beijing Chest Hospital, Capital Medical University to provide real-world diagnosis and treatment basis for such patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eObjects:\u003c/strong\u003e A retrospective study was conducted on 820 cases with EGFR-TKIs drug-resistant NSCLC patients who were admitted to Beijing Chest Hospital, Capital Medical University from June 2019 to June 2023 with complete medical data. All patients underwent tumor tissue or plasma NGS gene detection after drug resistance. The 4 cases of ALK fusion after drug resistance were collected and analyzed. This study was approved by the Ethics Committee of Beijing Chest Hospital, Capital Medical University. the Number of Approval is LW-2024-001.Ethics Committee of Beijing Chest Hospital, Capital Medical University waived the need for informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion criteria:\u003c/strong\u003e 1. NSCLC was confirmed by histopathology; 2. At initial diagnosis, The deletion of exon 19 (19del) or the mutation of exon 21 (21L858R) of EGFR were confirmed by the tissue cell Fluorescence Quantitative Deoxyribonucleic Acid Amplification Refractory Mutation System Polymerase Chain Reaction (ARMSPCR) or NGS;3. Before EGFR-TKIs treatment, ALK fusion mutation was negative (VANTANA immunohistochemical or NGS). 4.According to the Union for International Cancer Control(UICC) lung cancer staging criteria (version 8),in stage IIIC or Ⅳ; 5. All patients had used EGFR-TKIs (including first-generation, second-generation or third-generation drugs) and developed resistance to EGFR-TKIs, and the definition of drug resistance followed the definition of acquired drug resistance by David Jackman[20]. 6. NGS detection was performed again in rebiopsy tumor tissues or plasma of all patients after acquired resistance and the ALK gene fusion were positive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The clinical and pathological data of these 4 patients have been collected, including age, gender, pathological type, EGFR gene mutation type, gene detection method, Computed Tomography(CT)scan results, medication regimen for EGFR-TKIs therapy, PFS for first-line EGFR-TKIs therapy, fusion partner of ALK fusion gene, abundance of ALK fusion gene, treatment plan and PFS after acquired resistance and OS etc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of curative effect:\u0026nbsp;\u003c/strong\u003eEvaluation of curative effects is characterized by Complete Response (CR), Partial Response (PR), Stable Disease (SD), and Progressive Disease (PD)based on Response evaluation Criteria In Solid tumors (RECIST1.1)[21]\u0026nbsp;. CR+PR is objective response, CR+PR+SD is disease control.\u0026nbsp;PFS of a treatment was defined as the time from the initiation of this treatment regime until PD or death from any cause. OS was defined as from the time of diagnosis of advanced lung cancer to death from any cause.\u0026nbsp;The last follow-up period of this study was January 1, 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment strategies in this study:\u0026nbsp;\u003c/strong\u003eEGFR-TKIs：Icotinib (first generation):125mg three times a day; Gefitinib (first generation):250mg once a day; Afatinib (second generation):40mg once a day; Dacomitinib (second generation):45mg once a day; Almonertinib (third generation) :110mg once a day. ALK-TKIs：Alectinib(second generation):600mg twice a day; Ensartinib (second generation):225mg once a day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor tissue NGS:\u003c/strong\u003e Tumor tissue NGS test was performed by the\u0026nbsp;DA8600 NGS system of Novogene Co., Ltd.\u0026nbsp;and at least 25 genes were detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasma\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNGS:\u0026nbsp;\u003c/strong\u003ePlasma\u0026nbsp;high-throughput\u0026nbsp;NGS was\u0026nbsp;performed by the sequencing Illumina Novaseq platform of Genetron\u0026nbsp;Co., Ltd.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eusing PairEnd150 sequencing strategy for sequencing samples. The depth of normal white plasma cell control is 100X, and plasma sequencing is 10000X\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence Quantitative Deoxyribonucleic Acid ARMSPCR:\u003c/strong\u003e Scorpion shaped probe amplification and blocking mutation system (ARMS PCR) \u0026nbsp;was used to detect gene variations such as EGFR, ALK,\u0026nbsp;Rearranged during Transfection(RET), proto-oncogene tyrosine-protein kinase ROS1(ROS1), and Neurotrophic Tyrosine Receptor Kinase (NTRK)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ein tumor tissue wax blocks.\u0026nbsp;The reagents were provided by Xiamen Aide Biopharmaceutical Technology Co., Ltd. The experimental instrument Cobas z480 real-time fluorescence quantitative PCR instrument was provided by Roche Diagnostic Products Shanghai Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMutation abundance:\u0026nbsp;\u003c/strong\u003eThe concept of mutation abundance is the ratio of the number of mutated reads at a specific site to the total number of reads at that site. Expressed as a percentage, it represents the proportion of clones with mutations in the sample.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAll the 4 patients had lung adenocarcinoma, of which 3 had Echinoderm Microtubule-associated protein-Like 4 gene (EML4)-ALK fusion (Case1,3,4) and one had striatin(STRN)-ALK fusion (Case2). EGFR gene mutation was detected negative in two patients after drug resistance (Case1, 4), and EGFR gene abundance decreased in two patients (Case2, 3). After acquired ALK mutation objective response was all achieved using ALK-TKIs alone or the combination of ALK-TKIs and EGFR-TKIs, The patients\u0026apos; chest CT images before EGFR-TKIs are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, and the patients\u0026apos; clinical features, pathological characteristics, PFS of each treatment and OS are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eGenetic mutation status and treatments notes of four patients:\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex/ Age\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenomic Alterations before EGFR TKIs\u003c/p\u003e\n \u003cp\u003e(NGS and abundance)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRegime of EGFR-TKIs(PFS1)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenomic Alterations after acquired resistance /\u0026nbsp;(NGS and abundance)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment after ALK fusion detection (PFS2)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOS\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCase1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM/60 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS:\u003c/p\u003e\n \u003cp\u003eEGFR L858R / (3.43%)\u003c/p\u003e\n \u003cp\u003eEGFR G719A/(8.65%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIcotinib\u003c/p\u003e\n \u003cp\u003e(10 months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS: EML4-ALK V3/\u003c/p\u003e\n \u003cp\u003e(4.99%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlectinib\u003c/p\u003e\n \u003cp\u003e(12months: ceased due to hemolytic anemia)\u003c/p\u003e\n \u003cp\u003eFollowed by Ensartinib\u003c/p\u003e\n \u003cp\u003e(\u0026gt;\u0026thinsp;8months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot reached\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCase2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF/63years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS: EGFR exon19\u003c/p\u003e\n \u003cp\u003ep.L747-T751del/\u003c/p\u003e\n \u003cp\u003e(56.98%)\u003c/p\u003e\n \u003cp\u003eTP53\u003c/p\u003e\n \u003cp\u003ePIK3CA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIcotinib\u003c/p\u003e\n \u003cp\u003e(10 months);\u003c/p\u003e\n \u003cp\u003eFollowed by Almonertinib\u0026nbsp;after PD\u003c/p\u003e\n \u003cp\u003e(11 months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS: STRN-ALK/\u003c/p\u003e\n \u003cp\u003e(12.58%)\u003c/p\u003e\n \u003cp\u003eEGFR exon19 p.L747-T751del/\u003c/p\u003e\n \u003cp\u003e(48.34%)\u003c/p\u003e\n \u003cp\u003eTP53\u003c/p\u003e\n \u003cp\u003eCDK6\u003c/p\u003e\n \u003cp\u003eCTNNB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlectinib+\u003c/p\u003e\n \u003cp\u003eAlmonertinib\u003c/p\u003e\n \u003cp\u003e(\u0026gt;\u0026thinsp;8month)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot reached\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCase3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF/62years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS: EGFR exon 19 p.E746-A750del\u003c/p\u003e\n \u003cp\u003e(25.10%)\u003c/p\u003e\n \u003cp\u003eMDM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDacomitinib:\u003c/p\u003e\n \u003cp\u003e(10 months);\u003c/p\u003e\n \u003cp\u003eFollowed by\u003c/p\u003e\n \u003cp\u003eGefitinib after PD\u003c/p\u003e\n \u003cp\u003e(2months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlasma NGS: ALK EML4-ALK V1 fusion(2.98%)\u003c/p\u003e\n \u003cp\u003eEGFR exon 19 p.E746-A750del\u003c/p\u003e\n \u003cp\u003e(18.28%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEnsartinib\u003c/p\u003e\n \u003cp\u003e+Almonertinib\u003c/p\u003e\n \u003cp\u003e(2months, ceased due to edema);\u003c/p\u003e\n \u003cp\u003eFollowed by Ensartinib\u0026thinsp;+\u0026thinsp;Osimertinib\u003c/p\u003e\n \u003cp\u003e(\u0026gt;\u0026thinsp;4months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot reached\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCase4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF/42years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS: EGFR exon 20 L858R\u003c/p\u003e\n \u003cp\u003e(19.35%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAfatinib\u003c/p\u003e\n \u003cp\u003e(6 months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTissue NGS: ALK EML4-ALK V1fusion(2.31%\u003c/p\u003e\n \u003cp\u003eSTK11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlectinib\u003c/p\u003e\n \u003cp\u003e(21months)Followed by chemotherapy due to\u003c/p\u003e\n \u003cp\u003eSmall cell lung cancer transformation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003cp\u003emonths\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eEGFR: Epidermal Growth Factor Receptor; NGS:Next Generation Sequencing; ALK: Anaplastic Lymphoma Kinase; TKIs: Tyrosine Kinase Inhibitors; PFS: Progression Free Survival; OS: Overall Survival;PD: Progressive Disease;EML4: Echinoderm Microtubule-associated protein-Like 4 gene. STRN:striatin.TP53:Tumor Protein 53. PIK3CA: Phosphatidylinositol-3-kinase catalytic subunit \u0026alpha;. CDK6: cyclin dependent kinase 6.CTNNB1: catenin (cadherin-associated protein), beta 1.MDM2: murine double minute 2. STK11: Serine/ Threonine kinase 11.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003ePFS1: PFS time of EGFR-TKIs treatment;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003ePFS2: PFS time of ALK-TKIs or ALK-TKIs\u0026thinsp;+\u0026thinsp;EGFR-TKIs after EGFR-TKIs Sex: M: Male; F: Female\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eBasic characteristics of secondary ALK fusion mutation after EGFR-TKIs resistance\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe probability of ALK mutation after EGFR-TKIs resistance is very low. According to previous reports, the calculated probability of occurrence is about 0.01%-0.2%[22]\u0026nbsp;[23]\u0026nbsp;[24]. In our analysis, 4 of 820 patients with acquired drug resistance after EGFR-TKIs developed ALK fusion mutations, with an incidence of 0.49%, which is close with previous reports.\u003c/p\u003e\n\u003cp\u003eHaiyuan Xu et al. reviewed the NGS genome map data of 3873 patients with EGFR (exon 18-21) mutation and found that 5 new ALK fusion mutations were acquired during EGFR-TKIs treatment[24].Fusion partners of ALK identified in the 5 \u0026nbsp;patients included EML4 (3 cases), STRN(1 case) and CCAAT-enhancer binding protein zeta(CEBPZ) (1 case). All three EML4\u0026ndash;ALK fusions emerging after EGFR-TKI resistance were the rare variant E2:A20 (V5)[24]. Other literature reported one case of secondary STRN-ALK fusions in 62 patients with EGFR-mutated metastatic NSCLC with tissue or plasma biopsies at EGFR-TKIs progression[25].Alexa B Schrock \u0026nbsp;et al. Identified a case of Pleckstrin homology domain containing A7 (PLEKHA7)-ALK fusion mutation after application of Erlotinib followed by Osimertinib[26]. A patient with EGFR L858R lung adenocarcinoma developed disease progression after 72.7 months of gefitinib therapy, emerging a novel ALK-R3HDM1 and EML4-ALK dual fusion that might be a delicate mechanism for the acquired resistance of EGFR- TKIs [27].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, 3 EML4-ALK fusion mutations emerged, of which 2 cases were V2 subtype and 1 case was V3 subtype.1 STRN-ALK fusion mutation was detected. Our another study showed that classical EML4-ALK fusion accounted for 94.9% of patients with primary ALK fusion[28]. From this research and other previous literature, non-classical ALK fusions may be more common in acquired EGFR-TKIs resistance.\u003c/p\u003e\n\u003cp\u003eIn this study, EGFR gene mutation was detected negative in 2 of the 4 patients, and the abundance of EGFR-19DEL mutation decreased from 56.98% to 48.34% in 1 case, and from 25.10% to 18.28% in the other. In only four patients, the abundance of EGFR mutation was either reduced or undetectable at the time of resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of treatment strategies and curative effects of patients:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious literature has focused on fusion gene mutations that may be acquired after EGFR-TKIs resistance. Drug resistance mechanisms include B-Raf proto-oncogene(BRAF),ALK,RET,NTRK,fibroblast growth factor receptor 3 (FGFR3) and other mutation types[24]\u0026nbsp;[25]\u0026nbsp;[26]. Few reports have analyzed the therapeutic strategy and efficacy of secondary ALK fusion mutation therapy and there are no relevant recommendations in current guidelines and expert consensus.\u003c/p\u003e\n\u003cp\u003ePrevious reports mostly focused on patients with co-existing of primary EGFR-sensitive mutation and ALK fusion mutation, and the medication strategy for patients with primary double mutations was also controversial. A literature review of 100 patients with primary double mutation found that the Disease control rate(DCR) and Objective response rate(ORR)of first-line use of ALK-TKIs (including Crizotinib and Ceritinib\u0026nbsp;) were 73.1% and 42.3%, respectively, that were higher than first-line use of EGFR-TKIs (including Gefitinib and Erlotinib) with DCR and ORR of 61.5% and 23.1%, respectively. Therefore, ALK-TKIs may be the first choice for patients with primary double mutation because NSCLC with ALK gene rearrangement tends to be more aggressive. ALK-TKIs seem to be more effective than EGFR-TKIs[29]. ALK inhibitors can be preferentially used in double-positive patients, especially in those with low EGFR mutation abundance[30]. However, there are different views that EGFR-TKIs may be more effective than ALK-TKIs in this group of patients[31]\u0026nbsp;[32], and in patients with ALK/EGFR double mutation, the effectiveness of ALK-TKIs and EGFR-TKIs are worse than those in patients with a single mutation.\u003c/p\u003e\n\u003cp\u003eTreatment options for ALK fusion after secondary resistance have only been reported in few cases. This study analyzed the diagnoses and treatments of 4 patients with acquired ALK fusion mutation after EGFR-TKIs, and found that the ORR and PFS of ALK-TKIs were similar to those reported in previous literature. In two patients with ALK/EGFR double mutations after resistance, one treated with EGFR-TKI plus ALK-TKI and obtained a long PFS; the other \u0026nbsp;was treated with Ensartinib plus Almonertinib for 2 months, which caused severe systemic edema. The treatment plan was adjusted to Ensartinib every other day plus Osimertinib and showed good tolerance and effect. Previous literature has also reported that the combination therapy of EGFR-TKIs and ALK-TKIs has a good effect[33]\u0026nbsp;[34]. The other two patients in our study used ALK-TKIs alone, Case 1 changed Alectinib to Ensartinib due to a side effect of hemolytic anemia and obtained disease control over 20 months after EGFR-TKI resistance and is still being treated. Case 4 used Alectinib monotherapy and reached PFS of 21 months. The above analysis suggests that AlK-TKIs alone or in combination with EGFR-TKIs are both alternative. It is worth noting that Case 4, after PD of Alectinib, this patient showed Small Cell Lung Cancer (SCLC)transformation in rebiopsy. Chemotherapy achieved certain effects. To our knowledge, this is the first case of ALK fusion followed by acquired SCLC transformation after EGFR-TKIs resistance, which reveals the complexity of resistance and the need for rebiopsy after disease progression. However, due to the small number of patients in this study, more sample size analysis is needed to guide the treatment of patients with acquired ALK fusion mutations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor origin and tumor heterogeneity of EGFR/ALK co-mutation\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are many studies on the tumor origin of primary EGFR/ALK co-mutation. The basic theory is that EGFR mutation and ALK fusion, both genetic changes, exist from the initial proliferation of tumors[18]. One view is that the changes of these two drivers can develop in the same tumor cell clone and coexist in the process of cancer development through cell line studies[35]. Immunohistochemical analysis of lung cancer tissues from patients with primary EGFR and ALK co-mutation found that EGFR mutation and ALK fusion were co-localized and co-expressed in the same cell group, and the two proteins may play synergistic driving roles in the same cancer cells[18]. Another view is that ALK/EGFR co-mutated lung adenocarcinoma is composed of two tumor cell subpopulations carrying EGFR mutations and ALK fusion mutations, reflecting genetic heterogeneity within the tumor[36]\u0026nbsp;[37]. Cai et al.\u0026nbsp;[38]used laser capture microdissection (LCM) to capture tumor cells with similar or different growth patterns from patients with ALK/EGFR co-mutations, and deduced that the gene changes were divided into four subclones: coexisting with EGFR mutation and ALK rearrangement, single EGFR mutation, single ALK rearrangement, and no gene changes.\u003c/p\u003e\n\u003cp\u003eOur study found that 2 out of 4 patients had co-existing EGFR and ALK fusion mutations at the time of EGFR-TKIs resistance (case2 had NGS result of metastatic lymph node rebiopsy and case3 had the NGS result of plasma). At present, for secondary ALK fusion gene mutations, it is possible that there was a small number of ALK fusion gene mutations initially. Due to the pressure of EGFR-TKIs therapy and cloning selection, the subsequent ALK fusion gene mutations then could be detected. In the case of primary double mutation, if one of TKIs is selected for first treatment, the remaining uninhibited mutant cell lines of ALK or EGFR will continue to grow[11]. But in the case of secondary EGFR-TKIs resistance, the need for combined application of EGFR-TKIs and ALK-TKIs may be related to the phosphorylation level of downstream proteins activated by the signaling pathway[35]. From our study, it may also be related to the abundance of ALK and EGFR after drug resistance. Therefore, detecting the abundance of EGFR mutation and ALK fusion, as well as the phosphorylation level of downstream proteins, may be beneficial to guide the selection of TKIs in clinical practice.\u003c/p\u003e\n\u003cp\u003eThis study analyzed the clinical characteristics and diagnoses and treatments of four very rare patients with acquired ALK fusion mutation after EGFR-TKIs treatment and only individual cases were reported in the previous literature. Our patients have dynamic results of NGS method detection, indicating the dynamic gene mutation status of these patients. Our study discussed the specific treatment plans of this kind of patients and analyzed the curative effect and benefit of them.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eALK fusion gene mutation may occur in EGFR-TKIs resistant patients with EGFR sensitive mutation and the incidence is very low. EGFR abundance may be reduced or undetectable in the presence of ALK fusion. Patients may benefit from ALK-TKIs alone or ALK-TKIs combined with EGFR-TKIs. In patients with secondary ALK fusion mutation after EGFR-TKIs resistance, SCLC transformation can also occur after ALK-TKIs resistance, which shows rebiopsy is an important step in developing treatment plans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is approved by Ethics Committee of Beijing Chest Hospital, Capital Medical University and the Number of Approval is LW-2024-001.\u003c/p\u003e\n\u003cp\u003eEthics Committee of Beijing Chest Hospital, Capital Medical University waived the need for informed consent.\u003c/p\u003e\n\u003cp\u003eAll methods and experiments of this study were carried out in accordance with relevant guidelines and regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization(Yuan Yang, Jing Yu, Zhe Liu); Data curation (Yuan Yang, Baohua Lu, Liang Shi, Nana Zhang, Zhaoxin Chen);Formal analysis(Yuan Yang, Baohua Lu, Jing Yu,Zhe Liu);Investigation(Yuan Yang, Jing Yu, Zhe Liu); Methodology(Yuan Yang, Baohua Lu, Jing Yu,Zhe Liu); Project administration(Yuan Yang,Baohua Lu); Resources(Yuan Yang, Baohua Lu, Liang Shi, Nana Zhang, Zhaoxin Chen);\u003c/p\u003e\n\u003cp\u003eSoftware(Yuan Yang,Baohua Lu); Supervision(Jing Yu, Zhe Liu); Roles/Writing - original draft(Yuan Yang); Writing - review \u0026amp;(Yuan Yang,Baohua Lu)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRomaszko AM, Doboszyńska A. 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Characterization of acquired receptor tyrosine-kinase fusions as mechanisms of resistance to EGFR tyrosine-kinase inhibitors. Cancer Manag Res. 2019;11:6343\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnrico D, Lacroix L, Chen J, Rouleau E, Scoazec J-Y, Loriot Y, et al. Oncogenic Fusions May Be Frequently Present at Resistance of EGFR Tyrosine Kinase Inhibitors in Patients With NSCLC: A Brief Report. JTO Clin Res Rep. 2020;1:100023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrock AB, Zhu VW, Hsieh W-S, Madison R, Creelan B, Silberberg J, et al. Receptor Tyrosine Kinase Fusions and BRAF Kinase Fusions are Rare but Actionable Resistance Mechanisms to EGFR Tyrosine Kinase Inhibitors. J Thorac Oncol. 2018;13:1312\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng Z, Wang T, He J, Wang Y. 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Tumori. 2022;108:12\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim T-J, Park CK, Yeo CD, Park K, Rhee CK, Kim J, et al. Simultaneous diagnostic platform of genotyping EGFR, KRAS, and ALK in 510 Korean patients with non-small-cell lung cancer highlights significantly higher ALK rearrangement rate in advanced stage. J Surg Oncol. 2014;110:245\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai W, Lin D, Wu C, Li X, Zhao C, Zheng L, et al. Intratumoral Heterogeneity of ALK-Rearranged and ALK/EGFR Coaltered Lung Adenocarcinoma. J Clin Oncol. 2015;33:3701\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"lung cancer, acquired resistance, EGFR tyrosine kinase inhibitors, ALK tyrosine kinase inhibitors","lastPublishedDoi":"10.21203/rs.3.rs-4407880/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4407880/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe mechanism of secondary drug resistance in advanced Non Small Cell Lung Cancer(NSCLC) patients with Epidermal Growth Factor Receptor (EGFR) gene sensitive mutation after EGFR-Tyrosine Kinase Inhibitors (TKIs) is complex. Acquired Anaplastic Lymphoma Kinase (ALK) fusion mutation is a rare type, and there are few reports on the clinical characteristics and treatment options for this group of patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCases of 820 locally advanced or metastatic EGFR-sensitive mutations NSCLC patients whose gene status were detected by Next Generation Sequencing(NGS)after EGFR-TKIs resistance were retrospectively collected. Acquired ALK fusion gene mutation occurred in 4 of them. The clinical information, pathological types, gene mutation status, treatment plans, efficacies and prognoses of these 4 cases were analyzed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll 4 patients had lung adenocarcinoma. Three had EML4-ALK fusion and 1 had STRN-ALK fusion. EGFR gene mutation was detected negative in 2 cases after drug resistance, and the abundance of EGFR gene mutation decreased in 2 cases. The Progression Free Survival (PFS) of EGFR-TKIs ranged from 6 to 21 months, and after acquired ALK mutation objective response was all achieved using ALK-TKIs alone or the combination of ALK-TKIs and EGFR-TKIs, with PFS all exceeding 6 months. One patient developed small cell lung cancer transformation after ALK-TKIs resistance.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAcquired ALK fusion as a resistant mechanism of EGFR-TKIs is present and rare. EGFR is undetectable or abundance decreased when ALK fusion emerges. ALK-TKIs alone and ALK-TKIs combined with EGFR-TKIs are alternative treatment choices.\u003c/p\u003e","manuscriptTitle":"Analysis of Clinical Features and Treatment Strategies in Advanced Lung Adenocarcinoma Patients With Acquired ALK Fusion After Resistance of EGFR-TKIs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-31 20:35:13","doi":"10.21203/rs.3.rs-4407880/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a66e5b8-0c43-4f54-a555-6731be96025f","owner":[],"postedDate":"May 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-19T14:18:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-31 20:35:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4407880","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4407880","identity":"rs-4407880","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}