Explore the Primary Resistance Mechanisms of Anaplastic Lymphoma Kinase Inhibitors
Conditions: ALK-positive Advanced NSCLC Patients, Primary Resistance, NGS
Sponsor: National Taiwan University Hospital

                                                                        
 
Version 1.2/ 28 May, 2025 
1 
Clinical Trial Protocol 
Title：Explore the primary resistance mechanisms of anaplastic lymphoma 
kinase inhibitor. 
Principal Investigator and Co-Investigators Names 
• 
Principal Investigator: Jin-Shing Chen,  Title: Director,  
Department of Surgery, National Taiwan University Hospital 
• 
Co-Investigator: Hsao-Hsun Hsu,        Title: Director,  
Department of Oncology, National Taiwan University Cancer Center 
• 
Co-Investigator: Pei-Ming Huang,       Title: Director,  
Department of Thoracic Surgery, National Taiwan University Hospital 
• 
Co-Investigator: Shang-Gin Wu,         Title: Attending Physician,  
Department of Chest Medicine, National Taiwan University Hospital 
• 
Co-Investigator: Chih-Hsin Yang,        Title: Superintendent,  
National Taiwan University Cancer Center 
• 
Co-Investigator: Jin-Yuan Shih,          Title: Director,  
Department of Chest Medicine, National Taiwan University Hospital 
• 
Co-Investigator: Chao-Chi Ho,           Title: Associate Director,  
Department of Internal Medicine, National Taiwan University Hospital 
• 
Co-Investigator: Tung-Ming Tsai,         Title: Attending Physician,  
Department of Surgical Oncology, National Taiwan University Cancer Center 
• 
Co-Investigator: Pei-Hsing Chen,         Title: Attending Physician,  
Department of Thoracic Surgery, National Taiwan University Hospital 
• 
Co-Investigator: Yen-Ting Lin,           Title: Attending Physician,  
Department of Chest Medicine, National Taiwan University Hospital 
• 
Co-Investigator: Pin-Chih Liao,          Title: Attending Physician,  
Department of Oncology, National Taiwan University Hospital 
• 
Co-Investigator: Yen-Lin Huang,         Title: Attending Physician,  
Department of Pathology, National Taiwan University Hospital 
• 
Co-Investigator: Chia-Lang Hsu,         Title: Associate Research Fellow,  
Department of Medical Research, National Taiwan University Hospital 
  

                                                                        
 
Version 1.2/ 28 May, 2025 
2 
(1) Project Abstract: 
Anaplastic lymphoma kinase (ALK) gene rearrangement is a known oncogenic driver in 
non-small cell lung cancer (NSCLC). ALK tyrosine kinase inhibitors (TKIs) have been clearly 
shown to produce excellent therapeutic effects and prolong survival in patients with this 
gene mutation. According to current treatment guidelines, ALK inhibitors are the first-line 
treatment of choice for ALK-positive advanced NSCLC patients. However, although ALK TKIs 
are very effective, there is still a small group of patients who do not achieve good treatment 
outcomes, developing resistance and tumor progression within 3 to 6 months of initial ALK 
TKI use. This is called primary resistance. Intrinsic resistance to ALK inhibition occurs when 
the best clinical response after first-generation and second/third-generation TKI treatment is 
disease progression. Approximately 5-7% of cases after crizotinib treatment, 9% after 
ceritinib treatment, and 25% after lorlatinib treatment show no response to treatment, and 
no specific ALK mutation has been found to explain the occurrence of primary resistance. 
Currently, many different resistance mechanisms are known, some of which are still 
ALK-related, while others are ALK-independent alternative survival pathways. However, most 
research focuses on acquired resistance, with very few studies on primary resistance, only a 
few case reports. Therefore, this study aims to explore the primary ALK TKI resistance 
mechanisms. 
We plan to explore the incidence and mechanisms of primary ALK TKI resistance in 
ALK-positive advanced NSCLC patients who develop primary resistance or rapid progression 
(within 3-6 months) during ALK inhibitor treatment by re-obtaining tumor samples for 
genetic analysis. 
Keywords: Lung cancer, Anaplastic lymphoma kinase (ALK) gene rearrangement, Gene 
mutation, Primary resistance, Next-generation sequencing, Targeted therapy.

                                                                        
 
Version 1.2/ 28 May, 2025 
3 
(2)  Background and Objectives of the Research Project: 
Lung cancer is the most common cause of cancer death in Taiwan and worldwide [1]. 
The traditional chemotherapy, platinum-doublet chemotherapy, provided limited survival 
benefit. In the recent decades, the major evolution in lung cancer is the molecular subtyping 
of NSCLC, especially lung adenocarcinoma, which has resulted in a paradigm shift in the 
management of patients in advanced disease. The target therapy provided better treatment 
response and longer overall survival. For example, patients with NSCLC harboring Epidermal 
growth factor receptor (EGFR) mutations can get remarkable benefit and longer survival 
from EGFR tyrosine kinase inhibitors (TKIs) treatment comparing with the traditional 
platinum-base chemotherapy [2].  
Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular 
subset of NSCLC, typically occurring in younger, non-smoking individuals with 
adenocarcinoma histology [3]. The discovery of ALK fusions, particularly EML4-ALK, 
revolutionized the treatment of these patients with the development of ALK TKIs. 
First-generation ALK inhibitors, such as crizotinib, demonstrated remarkable initial responses, 
significantly improving progression-free survival (PFS) compared to standard chemotherapy 
[4]. However, the emergence of resistance, both primary and acquired, remains a major 
clinical challenge.    
Primary resistance, defined as the lack of initial response or early progression within the 
first few months of ALK TKI therapy, occurs in a subset of patients and represents a 
significant obstacle to optimal treatment outcomes [5]. Primary resistance to ALK inhibition 
occurs, even with advanced TKI therapies, when the best clinical outcome is disease 
progression. Notably, approximately 5-7% of patients following crizotinib, 9% following 
ceritinib, and 25% following lorlatinib demonstrated a lack of response, and in these cases, 
no identifiable ALK mutations were detected [6]. Unlike acquired resistance, which typically 
arises from secondary mutations within the ALK kinase domain or activation of bypass 
signaling pathways, the mechanisms underlying primary resistance are less well 
understood.    
Several potential factors contribute to primary resistance. One key aspect involves 
inherent tumor heterogeneity. NSCLC tumors are complex ecosystems with diverse 
subclones, some of which may harbor pre-existing genetic alterations that confer resistance 
to ALK inhibition [7]. These alterations might include co-occurring mutations in genes 

                                                                        
 
Version 1.2/ 28 May, 2025 
4 
involved in cell signaling pathways, such as KRAS, EGFR, or TP53, which can bypass ALK 
dependence [6]. Furthermore, variations in ALK fusion isoforms or copy number alterations 
may also influence initial drug sensitivity. For example, some less common ALK fusion 
variants might possess distinct structural properties that reduce their affinity for ALK TKIs.    
Another potential mechanism involves the tumor microenvironment (TME). The TME, 
composed of stromal cells, immune cells, and extracellular matrix, plays a critical role in 
tumor growth and drug response [8]. Components of the TME, such as cancer-associated 
fibroblasts (CAFs) and immunosuppressive cells, can secrete growth factors and cytokines 
that activate alternative signaling pathways, thereby promoting resistance to ALK inhibition. 
Additionally, the TME can create a physical barrier that limits drug penetration and 
efficacy.    
Furthermore, the complexity of ALK signaling networks and the potential for activation 
of bypass pathways contribute to primary resistance. For example, activation of the EGFR or 
MET signaling pathways can provide alternative growth signals, bypassing the need for ALK 
activation [9]. Understanding these bypass pathways is crucial for developing combination 
therapies that can overcome primary resistance.    
Investigating primary resistance requires a comprehensive approach that integrates genomic, 
transcriptomic, and proteomic analyses. Next-generation sequencing (NGS) can identify 
pre-existing genetic alterations that confer resistance. Single-cell RNA sequencing can dissect 
tumor heterogeneity and identify resistant subclones. Proteomic profiling can reveal 
activation of bypass signaling pathways. Clinical studies analyzing pre-treatment tumor 
samples and correlating genomic and clinical data are essential for identifying predictive 
biomarkers of primary resistance.    
In summary, primary resistance to ALK TKIs in NSCLC is a complex phenomenon driven 
by multiple factors, including tumor heterogeneity, TME interactions, pharmacokinetic 
variations, and activation of bypass signaling pathways. Further research is needed to 
elucidate the precise mechanisms underlying primary resistance and to develop strategies to 
overcome this clinical challenge, ultimately improving outcomes for patients with 
ALK-positive NSCLC. 
 
 

                                                                        
 
Version 1.2/ 28 May, 2025 
5 
(3) Research Methods and Procedures: 
Number of Subjects, Selection and Exclusion Criteria: 
n 
Trial Period and Progress: From July 1, 2026, to July 1, 2028. Approximately 20 
participants are expected to be enrolled for testing. 
n 
Enrollment Locations: Outpatient clinics and wards of Thoracic Surgery, Chest 
Medicine, and Oncology Departments at National Taiwan University Hospital and 
National Taiwan University Cancer Center. 
Lung cancer patients who initially use ALK TKI as first-line treatment will be invited 
to enroll and sign the consent form. Only those who meet the criteria for primary 
resistance or rapid progression will proceed with tumor sample collection for genetic 
analysis. Alternatively, patients who develop primary resistance or rapid progression 
after using ALK TKI and are about to undergo tumor re-biopsy can also sign the subject 
consent form to be included in this study. 
Expected number of enrolled cases is approximately 20 patients with primary resistance 
to ALK TKI. 
 
<Inclusion Criteria> 
Patient must meet the following criteria for study entry 
1. Age ≥ 18 years old. 
2. Histologically or cytologically diagnosed NSCLC  
3. ALK gene fusion detected at initial diagnosis of NSCLC 
4. Patients exhibiting primary resistance to ALK-TKI therapy, defined as the absence 
of initial response or progression within 3 to 6 months of treatment initiation, 
were included.  
5. Able and willing to provide written informed consent prior to performing any study 
related procedures and to comply with the study protocol. 
<Exclusion Criteria> 
Patient who meet any of the following criteria will be excluded from study entry 
1. Unable to provide written inform consent 
2. The patients had received other systemic treatments except ALK TKI. 
3. Unable to undergo tumor biopsy or venipuncture  

                                                                        
 
Version 1.2/ 28 May, 2025 
6 
This study is planned to be conducted at National Taiwan University Hospital, National 
Taiwan University Cancer Center, Tri-Service General Hospital, Linkou Chang Gung Memorial 
Hospital, and Taipei Veterans General Hospital, adopting a competitive enrollment 
approach. 
3.1  Overall study design: 
With appropriate written inform consent, we will collect all of the patients with advanced 
NSCLC harboring ALK rearrangement who received 2nd-or 3rd- generation ALK TKI as first-line 
treatment. A subtype of ALK TKI-treated patients suffered from primary resistance or rapid 
progression to ALK TKI treatment. Re-biopsy of the enlarged tumor will be arranged for NGS 
analysis. If tumor re-biopsy is not feasible, 20 ml peripheral blood samples form subjects 
who fulfill the inclusion/exclusion criteria of this study will be collected for NGS study, too. If 
clinically feasible, archival tissues at initial diagnosis of lung cancer will also be collected if 
the specimens were obtained when patients developed tumor progression on ALK inhibitor 
treatment. Both the blood and tissue samples will be sent for NGS analysis. Patients’ clinical 
characteristics, tumors’ pathological features, and the ALK inhibitor treatment response, will 
be reviewed. A correlation analysis between the clinicopathological characteristics and the 
genetic alterations in ALK and other associated genes will be performed.  
3.2  Sample preparation 
The collected tissue samples, including at initial diagnosis of lung cancer and re-biopsy after 
disease progression, will be collected. The formalin-fixed, paraffin-embedded tissue, if 
available, will also used for mutation testing by next generation analysis. One of the 
consecutive sections will be stained with hematoxylin and eosin and reviewed by 
pathologists to select tumor region(s) for genomic DNA extraction. The selected tumor 
region(s) will be marked on deparaffined tissue sections by the experienced pathologists. 
If the patient will not have adequate tumor for re-biopsy, peripheral blood samples 
after disease progression will be collected into Cell-Free DNA BCT tubes (Streck, La Viintsta, 

                                                                        
 
Version 1.2/ 28 May, 2025 
7 
NE) and stored at room temperature (RT). Plasma will be separated from blood cells by 
centrifugation at 1,600 × g for 10 min at RT using a swing-out rotor. The supernatant will be 
transferred and centrifuged a second time at 6,000 × g for 10 min at RT. Plasma will then be 
gently mixed and aliquoted in cryogenic vials, and frozen at -80°C until DNA extraction.  
3.3  DNA and RNA extraction 
3.3.1  Genomic DNA Extraction 
Genomic DNA will be extracted from three to five unstained, 5×5mm2 with 5-µm thick FFPE 
sections using Cobas® DNA Sample Preparation Kit (Roche) according to the manufacturer's 
protocol. Cell-free DNA (cfDNA) will be isolated from peripheral blood using the QIAamp 
Circulating Nucleic Acid Kit (Qiagen, Venlo, Netherlands). Isolated DNA and cfDNA will be 
quantified using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). 
DNF-474 High Sensitivity NGS Fragment Analysis Kit (AATI, Ankeny, IA, USA) along with 
Fragment Analyzer Automated CE System (AATI) will be used for quality control, including 
concentration and the extent of degradation of extracted genomic DNA. 
3.3.2  Total RNA Extraction 
The total RNA of the FFPE sections will be isolated using RecoverAll™ Total Nucleic Acid 
Isolation Kit (Ambion, Austin, TX). Isolated RNA will be quantified using the Qubit RNA HS 
Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNF-474 High Sensitivity NGS 
Fragment Analysis Kit (AATI, Ankeny, IA, USA) along with Fragment Analyzer Automated CE 
System (AATI) will be used for quality control, including RNA concentration and the extent of 
degradation of extracted RNA. 
3.4  Next Generation Sequencing 
Then, the DNA libraries and RNA samples will be sequenced by next generation sequence of 
Guardant 360. The panel, Guardant360 TissueNextTM, will be used, and this panel can detect 
742 genes whose structures are related to tumor message transmission pathways, analyze 
741 single gene variation (SNV) or insertion/deletion fusion (InDel), 34 copy number 

                                                                        
 
Version 1.2/ 28 May, 2025 
8 
variation (CNV), 28 genes (fusion genes), and 47 promoter methylation genes, analyze the 
causes of tumor formation and characteristic fusion, and find suitable drugs.  
(4)  Expected Trial Results: 
    Different ALK fusion variants are also not created equal. In addition, in vitro data have 
suggested different secondary mutations at ALK kinase domain will result in different drug 
sensitivity to different kinds of ALK inhibitors [10]. In addition, the bypass survival pathway 
will contribute to treatment failure if it is not targeted appropriately. In this observational 
study, we will be able to comprehensively evaluate the primary resistance mechanisms of 
ALK TKIs. Meanwhile, we will also be able to evaluate the usefulness of liquid biopsy in 
ALK-positive NSCLC patients. We believe our study results will not only to provide the 
landscape of primary resistance mechanisms of ALK TKIs but also offer a potential solution to 
future clinical patients who are not able to undergo invasive tissue re-biopsy when their 
ALK-positive lung tumors become resistant to ALK inhibitors 
(5) Data Collection, Processing, Evaluation, and Statistical Analysis Methods:     
All categorical variables will be analyzed with Pearson’s χ2 tests, except where a small size 
required the use of Fisher's exact test. The time to tumor recurrence, progression-free 
survival curve and overall survival will be plotted by the Kaplan–Meier method and 
compared by a log-rank test. Two-sided p-values of less than 0.05 will be considered 
significant. All analyses will be performed using the SPSS software (version 29.0 for Windows; 
SPSS Inc.) 

                                                                        
 
Version 1.2/ 28 May, 2025 
9 
(6) Clinical Adverse Reactions and Management Methods: This study does not 
involve treatment. 
(7)Funding Source: This is an investigator-initiated study. The shipping and NGS testing of 
blood and tissue samples are sponsored by Taiwan Society of Thoracic Surgeons (TSTS). 
There is no consideration relationship. It has no impact nor responsibility on the prescription 
of clinical physicians.  
(8) Potential Psychological and Physical Risks and Benefits: 
There is no directly interested relationship between participating patients and sponsored 
company. Possible AEs related to study medication are mentioned above. Participating 
institutes and investigators will take the responsibility of patient follow-up and potential risk 
management of blood sampling.  
(9) Potential Financial Risks and Benefits: 
Although the NGS test is sponsored by Taiwan Society of Thoracic Surgeons (TSTS). 
There is no other conflict of interest related to sponsored TSTS. The research results will be 
published in academic literature, without any other intellectual property and substantial 
benefits related to TSTS. The National Taiwan University Hospital will be engaged in medical 
purposes such as disease diagnosis, prevention, treatment, and research. 
(10) Conflicts of Interest: 
Although the study is sponsored by Taiwan Society of Thoracic Surgeons (TSTS). There is 
no other conflict of interest related to sponsored TSTS. Investigators are responsible for 
study conception, design, protocol writing, IRB application/approval, operational execution, 
data handling, and data analysis, and interpretation, along with subsequent publication. 
Participating institutes and investigators will take the responsibility of patient follow-up. 
(11) Research Personnel: The principal investigator and co-investigators will apply 
to the Department of Pathology to send samples to Guardant Health testing laboratory 
(Taiwan general agent: Welgene Biomedical Co., Ltd.) for testing according to the 

                                                                        
 
Version 1.2/ 28 May, 2025 
10 
general sample outsourcing procedures. No laboratory tests will be conducted in the 
hospital, so no research personnel are required. 
(12) Research Funding Source: Taiwan Society of Thoracic Surgeons 
(13) Subject Information and Consent Form Format: Please refer to the Subject 
Consent Form 
(14) Required Drugs or Medical Devices and Quantity: None. 
(15) Patient Data Confidentiality and Privacy: 
Regarding patient data confidentiality, relevant clinical data records will be collected using a 
code instead of the patient's real medical record number and name. The relevant codes and 
corresponding patient list will be stored in Dr. Shang-Jun Wu's locked office. Relevant data 
will be stored electronically or filed for statistical analysis and will be kept in a dedicated 
computer with a password and appropriate anti-virus software (Dr. Shang-Jun Wu's 
in-hospital office). This data will be completely deleted after the publication of the thesis. In 
addition, it also includes: 
(1) Only use hospital (NTU or NTUH) email to transmit data. 
(2) Do not register for outpatient clinics in the name of patients without authorization. 
(3) Follow the regulations that data should not be taken out of the hospital, and patient 
personal data will not be taken out of the hospital without reason. 
(4) In principle, business will be conducted on public computers, and personal computers 
need to be encrypted when used. 
(5) Patient data will be marked with codes instead of identifiable data. 
Regarding patient privacy: To protect the patient's personal privacy, a trial number will be 
used instead of the name and related personal data to ensure that relevant retrospective data 
are fully confidential. At the same time, related protection methods are provided: 

                                                                        
 
Version 1.2/ 28 May, 2025 
11 
(1) Comply with the outpatient medical privacy maintenance regulations issued by the 
Department of Health. 
(2) Do not use official duties to inquire about unrelated patient test reports. 
(3) Implement the National Taiwan University Hospital's "Small Sealing Movement." 
(16) Potential Psychological and Physical Risks and Benefits: This study 
retrospectively collects patients' clinical data and drug treatment effects, and there is 
no special interventional treatment, which does not affect patients' rights and does not 
subject subjects to any coercion or interference. 
(17) Potential Financial Risks and Benefits: This study is not expected to generate 
commercial interests, nor will it generate patent rights or other commercial interests. 
(18) Ownership and Use of Research Results: The research results belong to 
National Taiwan University Hospital and the Taiwan Society of Thoracic Surgeons, 
and the results may be published in academic papers in the future. 
(19) References: 
1 
Siegel RL, Kratzer TB, Giaquinto AN, et al. Cancer statistics, 2025. CA Cancer J Clin 
2025;75:10-45. 
2 
Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor 
receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 
2004;350:2129-2139. 
3 
Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients 
with non-small-cell lung cancer who harbor eml4-alk. J Clin Oncol 2009;27:4247-4253. 
4 
Camidge DR, Bang YJ, Kwak EL, et al. Activity and safety of crizotinib in patients with 
alk-positive non-small-cell lung cancer: Updated results from a phase 1 study. Lancet Oncol 
2012;13:1011-1019. 

                                                                        
 
Version 1.2/ 28 May, 2025 
12 
5 
Katayama R, Shaw AT, Khan TM, et al. Mechanisms of acquired crizotinib resistance in 
alk-rearranged lung cancers. Sci Transl Med 2012;4:120ra117. 
6 
Gainor JF, Dardaei L, Yoda S, et al. Molecular mechanisms of resistance to first- and 
second-generation 
alk 
inhibitors 
in 
alk-rearranged 
lung 
cancer. 
Cancer 
Discov 
2016;6:1118-1133. 
7 
Jamal-Hanjani M, Wilson GA, McGranahan N, et al. Tracking the evolution of 
non-small-cell lung cancer. N Engl J Med 2017;376:2109-2121. 
8 
Hinshaw DC and Shevde LA. The tumor microenvironment innately modulates cancer 
progression. Cancer Res 2019;79:4557-4566. 
9 
Shaw AT, Kim DW, Mehra R, et al. Ceritinib in alk-rearranged non-small-cell lung cancer. 
N Engl J Med 2014;370:1189-1197. 
10 Yoda S, Lin JJ, Lawrence MS, et al. Sequential alk inhibitors can select for 
lorlatinib-resistant compound alk mutations in alk-positive lung cancer. Cancer Discov 
2018;8:714-729.