Assessing and Evaluating the Scope and Constraints of Idylla Molecular Assays by Using Different Source Materials in Routine Diagnostic Settings

For cancer treatment, diagnostics concerning tumor type and determination of molecular markers in short TAT is critical. The fully automated, real-time PCR-based molecular diagnostic Idylla assays are well established in many laboratories for qualitative detection, short TAT and routine screening of clinically relevant oncogenic mutations. According to the manufacturer, all IVD assays are recommended for use only with FFPE tissue samples of 5–10 µM dissections with at least 10% tumor content. In this study, we tested the performance and accuracy of the IVD assays along with the gene fusion assay (RUO) with different tissue/source materials like isolated DNA/RNA, cryomaterial, etc. The study also included testing archival FFPE tissue sections dating back from 20 years and a performance check for different pan-cancer samples individually. All the assays tested with FFPE sections and gDNA/RNA input showed above 96% accuracy and sensitivity, individually with 100% specificity. The Idylla assays also performed exceptionally well on the archival FFPE tissues, and the use of assays for other solid tumors was also remarkable. The performance test and accuracy of Idylla assays showed high efficiency with certain limitations. For the use of Idylla assays, both qualitative and quantitative applicability of different tumor source materials could produce efficient results in different diagnostic settings within a short TAT.     

Novel Emerging Molecular Targets in Non-Small Cell Lung Cancer

In the scenario of systemic treatment for advanced non-small cell lung cancer (NSCLC) patients, one of the most relevant breakthroughs is represented by targeted therapies. Throughout the last years, inhibitors of the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-Ros oncogene 1 (ROS1), and V-raf murine sarcoma viral oncogene homolog B (BRAF) have been approved and are currently used in clinical practice. However, other promising molecular drivers are rapidly emerging as therapeutic targets. This review aims to cover the molecular alterations with a potential clinical impact in NSCLC, including amplifications or mutations of the mesenchymal–epithelial transition factor (MET), fusions of rearranged during transfection (RET), rearrangements of the neurotrophic tyrosine kinase (NTRK) genes, mutations of the Kirsten rat sarcoma viral oncogene (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), as well as amplifications or mutations of human epidermal growth factor receptor 2 (HER2). Additionally, we summarized the current status of targeted agents under investigation for such alterations. This revision of the current literature on emerging molecular targets is needed as the evolving knowledge on novel actionable oncogenic drivers and targeted agents is expected to increase the proportion of patients who will benefit from tailored therapeutic approaches.     

EGFR T751_I759delinsN Mutation in Exon19 Detected by NGS but Not by Real-Time PCR in a Heavily-Treated Patient with NSCLC

The detection of driver gene mutations can determine appropriate treatment strategies for non-small cell lung cancer (NSCLC) by identifying the presence of an effective druggable target. Mutations in the gene encoding the epidermal growth factor receptor (EGFR) are common driver mutations in NSCLC that can be effectively targeted by the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, without the detection of driver mutations, appropriate therapeutic decisions cannot be made. The most commonly applied methods for detecting driver gene mutations are assays based on polymerase chain reaction (PCR). However, the underlying mechanism of PCR-based assays limits the detection of rare mutations. Therefore, patients harboring rare mutations may not receive optimal treatment. We report a heavily-treated patient with NSCLC who harbored a T751_I759delinsN mutation in exon 19 of EGFR that was not detected by real-time PCR but was successfully detected by next-generation sequencing (NGS). The detection of a driver mutation using NGS resulted in the administration of targeted therapy, leading to favorable progression-free survival for the patient. Our report highlights the importance and potential of routine NGS testing among NSCLC patients for whom traditional assays fail to detect driver mutations when determining treatment options.     

EGFR-Mutated Non-Small Cell Lung Cancer and Resistance to Immunotherapy: Role of the Tumor Microenvironment

Lung cancer is a leading cause of cancer-related deaths worldwide. About 10–30% of patients with non-small cell lung cancer (NSCLC) harbor mutations of the EGFR gene. The Tumor Microenvironment (TME) of patients with NSCLC harboring EGFR mutations displays peculiar characteristics and may modulate the antitumor immune response. EGFR activation increases PD-L1 expression in tumor cells, inducing T cell apoptosis and immune escape. EGFR-Tyrosine Kinase Inhibitors (TKIs) strengthen MHC class I and II antigen presentation in response to IFN-γ, boost CD8+ T-cells levels and DCs, eliminate FOXP3+ Tregs, inhibit macrophage polarization into the M2 phenotype, and decrease PD-L1 expression in cancer cells. Thus, targeted therapy blocks specific signaling pathways, whereas immunotherapy stimulates the immune system to attack tumor cells evading immune surveillance. A combination of TKIs and immunotherapy may have suboptimal synergistic effects. However, data are controversial because activated EGFR signaling allows NSCLC cells to use multiple strategies to create an immunosuppressive TME, including recruitment of Tumor-Associated Macrophages and Tregs and the production of inhibitory cytokines and metabolites. Therefore, these mechanisms should be characterized and targeted by a combined pharmacological approach that also concerns disease stage, cancer-related inflammation with related systemic symptoms, and the general status of the patients to overcome the single-drug resistance development.     

Treatment of Brain Metastases of Non-Small Cell Lung Carcinoma

Lung cancer is one of the most common malignant neoplasms. As a result of the disease’s progression, patients may develop metastases to the central nervous system. The prognosis in this location is unfavorable; untreated metastatic lesions may lead to death within one to two months. Existing therapies—neurosurgery and radiation therapy—do not improve the prognosis for every patient. The discovery of Epidermal Growth Factor Receptor (EGFR)—activating mutations and Anaplastic Lymphoma Kinase (ALK) rearrangements in patients with non-small cell lung adenocarcinoma has allowed for the introduction of small-molecule tyrosine kinase inhibitors to the treatment of advanced-stage patients. The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein with tyrosine kinase-dependent activity. EGFR is present in membranes of all epithelial cells. In physiological conditions, it plays an important role in the process of cell growth and proliferation. Binding the ligand to the EGFR causes its dimerization and the activation of the intracellular signaling cascade. Signal transduction involves the activation of MAPK, AKT, and JNK, resulting in DNA synthesis and cell proliferation. In cancer cells, binding the ligand to the EGFR also leads to its dimerization and transduction of the signal to the cell interior. It has been demonstrated that activating mutations in the gene for EGFR-exon19 (deletion), L858R point mutation in exon 21, and mutation in exon 20 results in cancer cell proliferation. Continuous stimulation of the receptor inhibits apoptosis, stimulates invasion, intensifies angiogenesis, and facilitates the formation of distant metastases. As a consequence, the cancer progresses. These activating gene mutations for the EGFR are present in 10–20% of lung adenocarcinomas. Approximately 3–7% of patients with lung adenocarcinoma have the echinoderm microtubule-associated protein-like 4 (EML4)/ALK fusion gene. The fusion of the two genes EML4 and ALK results in a fusion gene that activates the intracellular signaling pathway, stimulates the proliferation of tumor cells, and inhibits apoptosis. A new group of drugs—small-molecule tyrosine kinase inhibitors—has been developed; the first generation includes gefitinib and erlotinib and the ALK inhibitor crizotinib. These drugs reversibly block the EGFR by stopping the signal transmission to the cell. The second-generation tyrosine kinase inhibitor (TKI) afatinib or ALK inhibitor alectinib block the receptor irreversibly. Clinical trials with TKI in patients with non-small cell lung adenocarcinoma with central nervous system (CNS) metastases have shown prolonged, progression-free survival, a high percentage of objective responses, and improved quality of life. Resistance to treatment with this group of drugs emerging during TKI therapy is the basis for the detection of resistance mutations. The T790M mutation, present in exon 20 of the EGFR gene, is detected in patients treated with first- and second-generation TKI and is overcome by Osimertinib, a third-generation TKI. The I117N resistance mutation in patients with the ALK mutation treated with alectinib is overcome by ceritinib. In this way, sequential therapy ensures the continuity of treatment. In patients with CNS metastases, attempts are made to simultaneously administer radiation therapy and tyrosine kinase inhibitors. Patients with lung adenocarcinoma with CNS metastases, without activating EGFR mutation and without ALK rearrangement, benefit from immunotherapy. This therapeutic option blocks the PD-1 receptor on the surface of T or B lymphocytes or PD-L1 located on cancer cells with an applicable antibody. Based on clinical trials, pembrolizumab and all antibodies are included in the treatment of non-small cell lung carcinoma with CNS metastases.     

EGFR Fragmentation for Topological Transformation Nanobarcoding

As the market for personalized lung cancer medicine expands, the demand for molecular diagnostic tools in general, and methods of detecting multiple genes with qualitative, quantitative, and high specificity in particular, have grown. Here, we propose a system for the effective detection of lung cancer-specific, long-length epidermal growth factor receptor (EGFR) gene mutations by using a topological transformation nano-barcoding technique (TNT). In former TNT studies, EGFR was successfully detected in cell environments and at test stages in the presence of a reference gene. However, because typical EGFR target concentrations are significantly lower at the clinical stage and the probe-binding ability of long-length targets is lower that of short targets, our system employs polymerase chain reaction (PCR) amplification, restriction, and filtering (PRF) for EGFR fragmentation to maximize performance. In a PRF system, the target is amplified by PCR, cut to a suitable size by a restriction enzyme, and filtered by a magnetic bead. With detection limits of 0.3555 % and 1.500 % for EGFR Del 19 and L858R mutations, respectively, the proposed TNT with PRF can effectively distinguish mutant cell lines and efficiently detect various lengths of genetic variations in clinical trials.     

Landscape of Genomic Alterations and PD-L1 Expression in Early-Stage Non-Small-Cell Lung Cancer (NSCLC)—A Single Center,Retrospective Observational Study

Precision oncology and immunotherapy have revolutionized the treatment of advanced non-small-cell lung cancer (NSCLC). Emerging studies show that targeted therapies are also beneficial for patients with driver alterations such as epidermal growth factor receptor (EGFR) mutations in early-stage NSCLC (stages I–IIIA). Furthermore, patients with elevated programmed death-ligand 1 (PD-L1) expression appear to respond favorably to adjuvant immunotherapy. To determine the frequency of genomic alterations and PD-L1 status in early-stage NSCLC, we retrospectively analyzed data from 2066 unselected, single-center patients with NSCLC diagnosed using next-generation sequencing and immunohistochemistry. Nine-hundred and sixty-two patients (46.9%) presented with early-stage NSCLC. Of these, 37.0% had genomic alterations for which targeted therapies have already been approved for advanced NSCLC. The frequencies of driver mutations in the early stages were equivalent to those in advanced stages, i.e., the rates of EGFR mutations in adenocarcinomas were 12.7% (72/567) and 12.0% (78/650) in early and advanced NSCLC, respectively (p = 0778). In addition, 46.3% of early-stage NSCLC cases were PD-L1-positive, with a tumor proportion score (TPS) of ≥1%. With comparable frequencies of driver mutations in early and advanced NSCLC and PD-L1 overexpression in nearly half of patients with early-stage NSCLC, a broad spectrum of biomarkers for adjuvant and neoadjuvant therapies is available, and several are currently being investigated in clinical trials.     

Linear Ubiquitination Mediates EGFR-Induced NF-κB Pathway and Tumor Development

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.     

Challenges of Systemic Therapy Investigations for Bone Sarcomas

Bone sarcoma is a rare component of malignant solid tumors that accounts for only ~0.2% of malignancies. Bone sarcomas present various histological types, and genomic mutations differ markedly by the histological types. Although there are vast mutations in various bone sarcomas, most of them are non-actionable, and even potential targetable mutations that are actionable targets in other malignancies have not shown the appropriate responses in clinical trials for bone sarcomas. Investigations of new systemic therapy, including molecular targeted therapies for bone sarcomas, have thus not progressed like those for other solid tumors. Another problem is that high rates of pediatric/adolescent and young adult patients have bone sarcomas such as osteosarcoma, and patient recruitment for clinical trials (especially randomized trials) is challenging. For pediatric patients, evaluations of tolerability and appropriate dose modifications of new drugs are needed, as their findings could provide the threshold for investigating new drugs for bone sarcomas. To solve these problems, improvements in registry systems, real world data, and pediatric extrapolation have been attempted. We review the issues regarding targeted drug investigations for bone sarcomas, focusing on the current clinical evidence and efforts to resolve these issues.     

State-of-the-Art Molecular Oncology of Lung Cancer in Taiwan

Lung cancers are life-threatening malignancies that cause great healthcare burdens in Taiwan and worldwide. The 5-year survival rate for Taiwanese patients with lung cancer is approximately 29%, an unsatisfactorily low number that remains to be improved. We first reviewed the molecular epidemiology derived from a deep proteogenomic resource in Taiwan. The nuclear factor erythroid 2-related factor 2 (NRF2)antioxidant mechanism was discovered to mediate the oncogenesis and tumor progression of lung adenocarcinoma. Additionally, DNA replication, glycolysis and stress response are positively associated with tumor stages, while cell-to-cell communication, signaling, integrin, G protein coupled receptors, ion channels and adaptive immunity are negatively associated with tumor stages. Three patient subgroups were discovered based on the clustering analysis of protein abundance in tumors. The first subgroup is associated with more advanced cancer stages and visceral pleural invasion, as well as higher mutation burdens. The second subgroup is associated with EGFR L858R mutations. The third subgroup is associated with PI3K/AKT pathways and cell cycles. Both EGFR and PI3K/AKT signaling pathways have been shown to induce NRF2 activation and tumor cell proliferation. We also reviewed the clinical evidence of patient outcomes in Taiwan given various approved targeted therapies, such as EGFR-tyrosine kinase inhibitors and anaplastic lymphoma kinase (ALK)inhibitors, in accordance with the patients’ characteristics. Somatic mutations occurred in EGFR, KRAS, HER2 and BRAF genes, and these mutations have been detected in 55.7%, 5.2%, 2.0% and 0.7% patients, respectively. The EGFR mutation is the most prevalent targetable mutation in Taiwan. EML4-ALK translocations have been found in 9.8% of patients with wild-type EGFR. The molecular profiling of advanced NSCLC is critical to optimal therapeutic decision-making. The patient characteristics, such as mutation profiles, protein expression profiles, drug-resistance profiles, molecular oncogenic mechanisms and patient subgroup systems together offer new strategies for personalized treatments and patient care.     

Role of NEU3 Overexpression in the Prediction of Efficacy of EGFR-Targeted Therapies in Colon Cancer Cell Lines

The epidermal growth factor receptor (EGFR), through the MAP kinase and PI3K-Akt-mTOR axis, plays a pivotal role in colorectal cancer (CRC) pathogenesis. The membrane-associated NEU3 sialidase interacts with and desialylates EGFR by promoting its dimerization and downstream effectors’ activation. Among the targeted therapies against EGFR, the monoclonal antibody cetuximab is active only in a subgroup of patients not carrying mutations in the MAP kinase pathway. In order to better understand the EGFR-NEU3 interplay and the mechanisms of pharmacological resistance, we investigated the role of NEU3 deregulation in cetuximab-treated CRC cell lines transiently transfected with NEU3 using Western blot analysis. Our results indicate that NEU3 overexpression can enhance EGFR activation only if EGFR is overexpressed, indicating the existence of a threshold for NEU3-mediated EGFR activation. This enhancement mainly leads to the constitutive activation of the MAP kinase pathway. Consequently, we suggest that the evaluation of NEU3 expression cannot entirely substitute the evaluation of EGFR because EGFR-negative cases cannot be stimulated by NEU3. Furthermore, NEU3-mediated hyperactivation of EGFR is counterbalanced by the administration of cetuximab, hypothesizing that a combined treatment of NEU3- and EGFR-targeted therapies may represent a valid option for CRC patients, which must be investigated in the future.     

Personalized Targeted Therapy for Lung Cancer

Lung cancer has long been recognized as an extremely heterogeneous disease, since its development is unique in every patient in terms of clinical characterizations, prognosis, response and tolerance to treatment. Personalized medicine refers to the use of markers to predict which patient will most likely benefit from a treatment. In lung cancer, the well-developed epidermal growth factor receptor (EGFR) and the newly emerging EML4-anaplastic lymphoma kinase (ALK) are important therapeutic targets. This review covers the basic mechanism of EGFR and EML4-ALK activation, the predictive biomarkers, the mechanism of resistance, and the current targeted tyrosine kinase inhibitors. The efficacy of EGFR and ALK targeted therapies will be discussed in this review by summarizing the prospective clinical trials, which were performed in biomarker-based selected patients. In addition, the revolutionary sequencing and systems strategies will also be included in this review since these technologies will provide a comprehensive understanding in the molecular characterization of cancer, allow better stratification of patients for the most appropriate targeted therapies, eventually resulting in a more promising personalized treatment. The relatively low incidence of EGFR and ALK in non-Asian patients and the lack of response in mutant patients limit the application of the therapies targeting EGFR or ALK. Nevertheless, it is foreseeable that the sequencing and systems strategies may offer a solution for those patients.     

Pharmacological and In Silico Analysis of Oat Avenanthramides as EGFR Inhibitors: Effects on EGF-Induced Lung Cancer Cell Growth and Migration

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.     

EGFR-Targeted Pentacyclic Triterpene Analogues for Glioma Therapy

Glioma, particularly its most malignant form, glioblastoma multiforme (GBM), is the most common and aggressive malignant central nervous system tumor. The drawbacks of the current chemotherapy for GBM have aroused curiosity in the search for targeted therapies. Aberrantly overexpressed epidermal growth factor receptor (EGFR) in GBM results in poor prognosis, low survival rates, poor responses to therapy and recurrence, and therefore EGFR-targeted therapy stands out as a promising approach for the treatment of gliomas. In this context, a series of pentacyclic triterpene analogues were subjected to in vitro and in silico assays, which were conducted to assess their potency as EGFR-targeted anti-glioma agents. In particular, compound 10 was the most potent anti-glioma agent with an IC₅₀ value of 5.82 µM towards U251 human glioblastoma cells. Taking into account its low cytotoxicity to peripheral blood mononuclear cells (PBMCs), compound 10 exerts selective antitumor action towards Jurkat human leukemic T-cells. This compound also induced apoptosis and inhibited EGFR with an IC₅₀ value of 9.43 µM compared to erlotinib (IC₅₀ = 0.06 µM). Based on in vitro and in silico data, compound 10 stands out as a potential orally bioavailable EGFR-targeted anti-glioma agent endowed with the ability to cross the blood–brain barrier (BBB).     

Machine Learning-Based Radiomics Signatures for EGFR and KRAS Mutations Prediction in Non-Small-Cell Lung Cancer

Early identification of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations is crucial for selecting a therapeutic strategy for patients with non-small-cell lung cancer (NSCLC). We proposed a machine learning-based model for feature selection and prediction of EGFR and KRAS mutations in patients with NSCLC by including the least number of the most semantic radiomics features. We included a cohort of 161 patients from 211 patients with NSCLC from The Cancer Imaging Archive (TCIA) and analyzed 161 low-dose computed tomography (LDCT) images for detecting EGFR and KRAS mutations. A total of 851 radiomics features, which were classified into 9 categories, were obtained through manual segmentation and radiomics feature extraction from LDCT. We evaluated our models using a validation set consisting of 18 patients derived from the same TCIA dataset. The results showed that the genetic algorithm plus XGBoost classifier exhibited the most favorable performance, with an accuracy of 0.836 and 0.86 for detecting EGFR and KRAS mutations, respectively. We demonstrated that a noninvasive machine learning-based model including the least number of the most semantic radiomics signatures could robustly predict EGFR and KRAS mutations in patients with NSCLC.     

EGFR Mutations in Head and Neck Squamous Cell Carcinoma

EGFR is a prototypical receptor tyrosine kinase that is overexpressed in multiple cancers including head and neck squamous cell carcinoma (HNSCC). The standard of care for HNSCC remains largely unchanged despite decades of research. While EGFR blockade is an attractive target in HNSCC patients and anti-EGFR strategies including monoclonal antibodies and kinase inhibitors have shown some clinical benefit, efficacy is often due to the eventual development of resistance. In this review, we discuss how the acquisition of mutations in various domains of the EGFR gene not only alter drug binding dynamics giving rise to resistance, but also how mutations can impact radiation response and overall survival in HNSCC patients. A better understanding of the EGFR mutational landscape and its dynamic effects on treatment resistance hold the potential to better stratify patients for targeted therapies in order to maximize therapeutic benefits.     

Future Perspectives in Detecting EGFR and ALK Gene Alterations in Liquid Biopsies of Patients with NSCLC

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.     

Structure–Activity Relationship (SAR) Study of Spautin-1 to Entail the Discovery of Novel NEK4 Inhibitors

Lung cancer is one of the most frequently diagnosed cancers accounting for the highest number of cancer-related deaths in the world. Despite significant progress including targeted therapies and immunotherapy, the treatment of advanced lung cancer remains challenging. Targeted therapies are highly efficacious at prolonging life, but not curative. In prior work we have identified Ubiquitin Specific Protease 13 (USP13) as a potential target to significantly enhance the efficacy of mutant EGFR inhibition. The current study aimed to develop lead molecules for the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) by developing potent USP13 inhibitors initially starting from Spautin-1, the only available USP13 inhibitor. A SAR study was performed which revealed that increasing the chain length between the secondary amine and phenyl group and introducing a halogen capable of inducing a halogen bond at position 4’ of the phenyl group, dramatically increased the activity. However, we could not confirm the binding between Spautin-1 (or its analogues) and USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but do not exclude binding under physiological conditions. Nevertheless, we found that the anti-proliferative activity displayed by Spautin-1 towards EGFR-mutant NSCLC cells in vitro was at least partially associated with kinase inhibition. In this work, we present N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. These analogues are significantly more effective towards EGFR-mutant NSCLC cells than Spautin-1 and act as potent never in mitosis A related kinase 4 (NEK4) inhibitors (IC₅₀~1 µM) with moderate selectivity over other kinases.