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Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as gefitinib and osimertinib have primarily been used as first-line treatments for patients with EGFR-activating mutations in non-small cell lung cancer (NSCLC). Novel biomarkers are required to distinguish patients with lung cancer who are resistant to EGFR-TKIs. The aim of the study is to investigate the expression and functional role of YES1, one of the Src-family kinases, in EGFR-TKI-resistant NSCLC. YES1 expression was elevated in gefitinib-resistant HCC827 (HCC827/GR) cells, harboring EGFR mutations. Moreover, HCC827/GR cells exhibited increased reactive oxygen species (ROS) levels compared to those of the parent cells, resulting in the phosphorylation/activation of YES1 due to oxidation of the cysteine residue. HCC827/GR cells showed elevated expression levels of YES1-associated protein 1 (YAP1), NF-E2-related factor 2 (Nrf2), cancer stemness-related markers, and antioxidant proteins compared to those of the parent cells. Knockdown of YES1 in HCC827/GR cells suppressed YAP1 phosphorylation, leading to the inhibition of Bcl-2, Bcl-xL, and Cyclin D1 expression. Silencing YES1 markedly attenuated the proliferation, migration, and tumorigenicity of HCC827/GR cells. Dasatinib inhibited the proliferation of HCC827/GR cells by targeting YES1-mediated signaling pathways. Furthermore, the combination of gefitinib and dasatinib demonstrated a synergistic effect in suppressing the proliferation of HCC827/GR cells. Notably, YES1- and Nrf2-regulated genes showed a positive regulatory relationship in patients with lung cancer and in TKI-resistant NSCLC cell lines. Taken together, these findings suggest that modulation of YES1 expression and activity may be an attractive therapeutic strategy for the treatment of drug-resistant NSCLC. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma is the most common form of lung cancer, accounting for 60% of non-small cell lung cancer (NSCLC) cases in Asian patients. Importantly, nearly half of these patients have epithelial growth factor receptor (EGFR) mutations. Though EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are recommended as the first-line therapy for NSCLC patients, the development of resistance reduces their efficiency and limits their application. As the complicated and heterogeneous mechanism of acquired resistance among individuals, the efficiency of anti-angiogenesis therapy, immune checkpoint inhibitors, or chemo-radiotherapies is rather less promising. In this research, we investigated the role of the tumor stem cell marker DCLK1 in EGFR-TKI resistance of lung adenocarcinoma. We discovered that DCLK1 was critical in maintaining the stemness of tumor cells through the Wnt/β-Catenin pathway, which was conducive to the development of EGFR-TKI resistance. Inhibiting DCLK1 activity restored the sensitivity of TKI-resistant tumor cells and organoids. Moreover, our study showed that DCLK1 inhibitor had a synergistic effect in controlling tumor growth when combined with EGFR-TKIs. Overall, our study provides new insights into EGFR-TKI resistant lung adenocarcinoma through inhibition of DCLK1 expression. [ABSTRACT FROM AUTHOR]

BACKGROUND: EGFR mutations widely exists in NSCLC patients, which are involved in cancer development. OBJECTIVE: The function of EGFR mutations in the resistance to TKI treatments of NSCLC was evaluated to provide theoretical support for the clinical management of NSCLC patients. METHODS: A total of 150 NSCLC patients including 118 patients with EGFR mutation and 32 without, were included in this study. The EGFR mutation status and subtypes were analyzed in recruited patients. The distribution of EGFR mutation subtypes and their association with clinicopathological features were also assessed. The prognostic value of EGFR mutation was evaluated by the overall survival of recruited patients. The function of EGFR mutation was estimated, in vitro, in the TKI resistant NSCLC cells with different subtypes of EGFR mutation. RESULTS: The exon 19 deletion was the most common subtype of EGFR mutation in the enrolled patients followed by the exon 21 L858R point mutation. The EGFR mutations were closely associated with the differentiation degree and the histological types of NSCLC cases. EGFR mutation was an independent prognostic factor of NSCLC with a close relationship with the overall survival of patients. The exon 20 T790M mutation results in the erlotinib resistance through the PI3K/Akt signaling pathway. CONCLUSIONS: The EGFR mutation is a critical factor in the prognosis and for the resistance to TKI treatment in NSCLC. The exon 20 T790M mutation was involved in the erlotinib resistance through PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Non-Small Cell Lung Cancer (NSCLC) represents ∼85% of all lung cancers and ∼15–20% of them are characterized by mutations affecting the Epidermal Growth Factor Receptor (EGFR). For several years now, a class of tyrosine kinase inhibitors was developed, targeting sensitive mutations affecting the EGFR (EGFR-TKIs). To date, the main burden of the TKIs employment is due to the onset of resistance mutations. This scoping review aims to resume the current situation about the cell line models employed for the in vitro evaluation of resistance mechanisms induced by EGFR-TKIs in oncogene-addicted NSCLC. Adenocarcinoma results the most studied NSCLC histotype with the H1650, H1975, HCC827 and PC9 mutated cell lines, while Gefitinib and Osimertinib the most investigated inhibitors. Overall, data collected frame the current advancement of this topic, showing a plethora of approaches pursued to overcome the TKIs resistance, from RNA-mediated strategies to the innovative combination therapies. [Display omitted] • 228 original articles have been collected from 1999 to March 2023. • H1650, H1975, HCC827 and PC9 are the most employed NSCLC mutated cell lines. • Afatinib, Erlotinib, Gefitinib and Osimertinib are the most investigated EGFR-TKIs. • Knockdown and combined approaches were pursued to overcome the TKIs resistance. [ABSTRACT FROM AUTHOR]

Abstract Background Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification. Methods The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model. Results T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance. Conclusions Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC.

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification. Methods: The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibodydependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model. Results: T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinibresistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance. Conclusions: Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC. [ABSTRACT FROM AUTHOR]

Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcinoma. Nonetheless tumour consists of heterogeneous cell subpopulations with diverse phenotypes and genotypes, and cancer cells can actively release extracellular vesicles (EVs) to modulate the phenotype of other cells in the tumour microenvironment. We hypothesized that EVs derived from a drug-resistant subpopulation of cells could induce drug resistance in recipient cells. We have established ALK-translocated lung adenocarcinoma cell lines and subclones. The subclones have been characterized and the expression of EV-RNAs determined by quantitative polymerase chain reaction. The effects of EV transfer on drug resistance were examined in vitro. Serum EV-RNA was assayed serially in two patients prescribed ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. We demonstrated that the EVs from an ALK-TKI-resistant subclone could induce drug resistance in the originally sensitive subclone. EV-RNA profiling revealed that miRNAs miR-21-5p and miR-486-3p, and lncRNAs MEG3 and XIST were differentially expressed in the EVs secreted by the resistant subclones. These circulating EV-RNA levels have been found to correlate with disease progression of EML4-ALK-translocated lung adenocarcinoma in patients prescribed ALK-TKI treatment. The results from this study suggest that EVs released by a drug-resistant subpopulation can induce drug resistance in other subpopulations and may sustain intratumoural heterogeneity.

The induction of resistance mechanisms represents an important problem for the targeted therapy of patients with non-small-cell lung cancer (NSCLC). The best-known resistance mechanism induced during treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is EGFR T790M mutation for which specific drugs are have been developed. However, other molecular alterations have also been reported as induced resistance mechanisms to EGFR-TKIs. Similarly, there is growing evidence of acquired resistance mechanisms to anaplastic lymphoma kinase (ALK)-TKI treatment. A better understanding of these acquired resistance mechanisms is essential in clinical practice as patients could be treated with specific drugs that are active against the induced alterations. The use of free circulating tumor nucleic acids or circulating tumor cells (CTCs) enables resistance mechanisms to be characterized in a non-invasive manner and reduces the need for tumor re-biopsy. This review discusses the main resistance mechanisms to TKIs and provides a comprehensive overview of innovative strategies to evaluate known resistance mechanisms in free circulating nucleic acids or CTCs and potential future orientations for these non-invasive approaches. [ABSTRACT FROM AUTHOR]

The induction of resistance mechanisms represents an important problem for the targeted therapy of patients with non-small-cell lung cancer (NSCLC). The best-known resistance mechanism induced during treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is EGFR T790M mutation for which specific drugs are have been developed. However, other molecular alterations have also been reported as induced resistance mechanisms to EGFR-TKIs. Similarly, there is growing evidence of acquired resistance mechanisms to anaplastic lymphoma kinase (ALK)-TKI treatment. A better understanding of these acquired resistance mechanisms is essential in clinical practice as patients could be treated with specific drugs that are active against the induced alterations. The use of free circulating tumor nucleic acids or circulating tumor cells (CTCs) enables resistance mechanisms to be characterized in a non-invasive manner and reduces the need for tumor re-biopsy. This review discusses the main resistance mechanisms to TKIs and provides a comprehensive overview of innovative strategies to evaluate known resistance mechanisms in free circulating nucleic acids or CTCs and potential future orientations for these non-invasive approaches.

Abstract: Aim: Non-small cell lung cancer (NSCLC)-patients with an epidermal growth factor receptor (EGFR)-mutation have median progression-free survival (PFS) of 12 months on tyrosine kinase inhibitors (TKIs). Resistance is mediated by the EGFR T790M-mutation in the majority of patients. Longitudinal follow-up data are lacking. We retrospectively evaluated EGFR-mutated NSCLC-patients who were rebiopsied after TKI-treatment. A subgroup was sequentially rebiopsied along the course of the disease. Patients and methods: Advanced EGFR-mutated NSCLC-patients who had both a pre-TKI biopsy and post-TKI biopsy available were included. Information on treatments and (re)biopsies was collected chronologically. Primary endpoint was the incidence of the T790M-mutation. Results: Sixty-six patients fulfilled the inclusion criteria. In first post-TKI biopsies, T790M-mutation was detected in 34 patients (52%) of patients. Twenty-seven patients had subsequent post-TKI rebiopsies with mutation analysis available; in 10 patients (37%) the T790M-status in subsequent post-TKI rebiopsies was not consistent with the T790M-status of the first post-TKI biopsy. Progression free survival (PFS) on TKI-treatment was 12.0 months. Objective response rate on TKI-treatment was 81%. Patients developing T790M-mutation at post-TKI biopsy had longer median PFS compared to T790M-negative patients (14.2 versus 11.1 months respectively (P =0.034)) and longer overall survival (45.9 months versus 29.8 months respectively (P =0.213)). Transformation to SCLC was detected in 1 patient (2%). Conclusion: Incidence of T790M-mutation at first post-TKI biopsy in this cohort of EGFR-mutated NSCLC-patients was 52%. Detection of T790M-mutation was not consistent over time; some patients who were T790M-positive at first post-TKI biopsy became T790M-negative in later post-TKI rebiopsies and vice versa. T790M-positive patients showed longer PFS than T790M-negative patients. Whether the low incidence of transformation to SCLC is justifying post-TKI rebiopsy in EGFR-mutated NSCLC-patients with acquired TKI-resistance in regular clinical practice is debatable. [Copyright &y& Elsevier]

Overall benefits of EGFR-TKIs are limited because these treatments are largely only for adenocarcinoma (ADC) with EGFR activating mutation. The treatments also usually lead to development of resistances. We have established a panel of patient-derived xenografts (PDXs) from treatment naïve Asian NSCLC patients, including those containing 'classic' EGFR activating mutations. Some of these EGFR-mutated PDXs do not respond to erlotinib: LU1868 containing L858R/T790M mutations, and LU0858 having L858R mutation as well as c-MET gene amplification, both squamous cell carcinoma (SCC). Treatment of LU0858 with crizotinib, a small molecule inhibitor for ALK and c-MET, inhibited tumor growth and c-MET activity. Combination of erlotinib and crizotinib caused complete response, indicating the activation of both EGFR and c-MET promote its growth/survival. LU2503 and LU1901, both with wild-type EGFR and c-MET gene amplification, showed complete response to crizotinib alone, suggesting that c-MET gene amplification, not EGFR signaling, is the main oncogenic driver. Interestingly, LU1868 with the EGFR L858R/T790M, but without c-met amplification, had a complete response to cetuximab. Our data offer novel practical approaches to overcome the two most common resistances to EGFR-TKIs seen in the clinic using marketed target therapies. [ABSTRACT FROM AUTHOR]

A new class of 2-amino-4-(1,2,4-triazol)pyridine derivatives were designed and synthesized as potent epidermal growth factor receptor inhibitors. In particular, compound 10c exhibited significant inhibitory against EGFRL858R/T790M, and also displayed potent anti-proliferative activity against non-small cell lung cancer cell line H1975. Besides, compound 10j showed potent inhibitory activity against glioblastoma cell line U87-EGFRvⅢ, which was at least 3-fold more potent than Osimertinib and 25-fold superior to Lazertinib. Moreover, molecular modeling and molecular dynamics simulations disclosed the binding model of the most active compound to the domain of EGFR. This contribution provides 2-amino-4-(1,2,4-triazol)pyridines as a new scaffold for EGFRT790M and/or EGFRvⅢ inhibitor. Image 1 • Sixteen 2-amino-4-(1,2,4-triazol)pyridine derivatives were designed and synthesized. • Compound 10c exhibited strong inhibitory activities against EGFRL858R/T790M enzyme. • Compound 10j showed potent inhibitory activity against U87-EGFRvIII cell. • Molecular docking and molecular dynamics simulation gave a probable explanation for the activity of 10c. [ABSTRACT FROM AUTHOR]

Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcinoma. Nonetheless tumour consists of heterogeneous cell subpopulations with diverse phenotypes and genotypes, and cancer cells can actively release extracellular vesicles (EVs) to modulate the phenotype of other cells in the tumour microenvironment. We hypothesized that EVs derived from a drug-resistant subpopulation of cells could induce drug resistance in recipient cells. We have established ALK-translocated lung adenocarcinoma cell lines and subclones. The subclones have been characterized and the expression of EV-RNAs determined by quantitative polymerase chain reaction. The effects of EV transfer on drug resistance were examined in vitro. Serum EV-RNA was assayed serially in two patients prescribed ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. We demonstrated that the EVs from an ALK-TKI-resistant subclone could induce drug resistance in the originally sensitive subclone. EV-RNA profiling revealed that miRNAs miR-21-5p and miR-486-3p, and lncRNAs MEG3 and XIST were differentially expressed in the EVs secreted by the resistant subclones. These circulating EV-RNA levels have been found to correlate with disease progression of EML4-ALK-translocated lung adenocarcinoma in patients prescribed ALK-TKI treatment. The results from this study suggest that EVs released by a drug-resistant subpopulation can induce drug resistance in other subpopulations and may sustain intratumoural heterogeneity. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP1) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP1 in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. In this study, we detected a correlation between YAP1 level and EGFR mutation status in lung adenocarcinoma tissues. Using lung adenocarcinoma cell lines, enhanced YAP1 expression and activity mediated by EGFR signaling was detected through enhanced protein stability. A SRC family protein, YES, was involved in EGFR-regulated YAP1 expression and this pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP1 levels by mechanisms bypassing EGFR signaling were effective in reducing viability in EGFR-dependent cells including those with EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP1 in EGFR mutant lung adenocarcinomas and identified YAP1 as a promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflict of interest.

Tyrosine kinase inhibitors (TKI) have improved CML response rates, and some are effective against resistance-promoting point mutations in BCR-ABL1. However, in the absence of point mutations, resistance still occurs. Here, we identify a novel pathway mediating resistance which connects p47phox, the organizer subunit of NADPH oxidase-2 (NOX2), with early growth response-1 (Egr-1) and the Src family kinase Fyn. We found up-regulation of p47phox, Egr-1, and Fyn mRNA and protein using paired isogenic CML cell lines and mined data. Isolation of CD34+ cells and tissue microarray staining from blast crisis CML patients confirmed in vivo over-expression of components of this pathway. Knockdown studies revealed that p47phox modulated reactive oxygen species and Egr-1 expression, which, in turn, controlled Fyn expression. Interestingly, Fyn knockdown sensitized TKI-resistant cells to dasatinib, a dual BCR-ABL1/Src inhibitor. Egr-1 knockdown had similar effects, indicating the utility of targeting Fyn expression over activation. Pointedly, p47phox knockdown also restored TKI-sensitivity, indicating that targeting the NOX2 complex can overcome resistance. The NOX2/Egr-1/Fyn pathway was also conserved within TKI-resistant EGFRΔIII-expressing glioblastoma and patient-derived glioblastoma stem cells. Thus, our findings suggest that targeting the NOX2/Egr-1/Fyn pathway may have clinical implications within multiple cancer types; particularly where efficacy of TKI is compromised.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been used as a powerful targeting therapeutic agent for treatment of lung adenocarcinoma for years. Nevertheless, the efficacy of TKI was hampered by the appearance of acquired TKI-resistance. In the present study, we aimed to search, predict, and screen the agents that can overcome the acquired TKI-resistance of lung adenocarcinoma by using the expression profiles of differentially expressed genes (DEGs) and Connectivity map (CMAP). The profiles of DEGs were obtained by searching GEO microarray database, and then, they were submitted to CMAP for analysis in order to predict and screen the agent that might reverse the TKI-resistance of lung cancer cells. Next, the effects of the selected agent on TKI-resistant cancer cells were tested and the possible signaling pathways were also evaluated. As a result, valproic acid (VPA) was selected. Then, we used a low-concentration of VPA that has little effect on the cell growth for analysis. Interestingly, the results showed that treatment with a combination of VPA and Erlotinib significantly led to a decrease in cell viability and an increase in cell apoptosis for TKI-resistant HCC827-ER cells, relative to those treated with VPA or Erlotinib alone. Further experiments confirmed that inhibition of MAPK and AKT might be involved in this process. Analyzing the DEGs through the CMAP is a good strategy for exploitation of anti-tumor agents. VPA might markedly increase the sensitivity of TKI-resistant lung adenocarcinoma cells to Erlotinib, thus reversing the acquired TKI-resistance of cancer cells and raising VPA as a potential agent for TKI-resistant lung cancer therapy.

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown improved progression-free survival (PFS) and overall survival (OS) over chemotherapy in a molecularly defined subgroup of advanced non-small-cell lung cancer (NSCLC) patients (ie, patients with an activating mutation in the EGFR gene). Nevertheless, all EGFR-mutated NSCLC patients develop TKI resistance eventually and there is no registered treatment or therapeutic strategy available for these patients. Several retrospective or small cohort studies have described patients who re-responded to EGFR-TKI treatment after a TKI-free interval ('drug holiday'). To date, no large prospective evaluation of the clinical effects of EGFR-TKI rechallenge in EGFR-mutated NSCLC patients has been performed., Patients and Methods: The IRENE (Iressa RE-challenge in advanced, EGFR-mutated NSCLC patients who responded to an EGFR-TKI used as first-line or previous treatment) (Dutch association for pulmonologists [NVALT]-16) trial is a multicenter, open-label, single-arm, single-stage, phase II study to evaluate gefitinib rechallenge in EGFR-mutated NSCLC patients who were previously treated with a TKI followed by a subsequent line of treatment (excluding EGFR-TKIs). The primary objective is disease control rate according to Response Evaluation Criteria in Solid Tumors criteria. Secondary objectives are objective response rate, PFS, OS, mutation characterization of sequential biopsies, VeriStrat correlation to PFS and OS, analysis of tumor-derived RNA in blood platelets and analysis of cell-free DNA in blood plasma., Results: The IRENE (NVALT-16) trial will evaluate the safety, efficacy, and feasibility of readministration of gefitinib after an EGFR-TKI-free interval in EGFR-mutated NSCLC patients., Conclusion: The study will evaluate gefitinib re-challenge in EGFR-mutated NSCLC patients. The study will also provide more insight into the dynamic development of molecular characteristics of EGFR-mutated NSCLC along the course of the disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)