EGFR Tyrosine Kinase Inhibitors In Non-small Cell Lung Cancer Research Articles

Challenges of EGFR-TKIs in NSCLC and the potential role of herbs and active compounds: From mechanism to clinical practice.

Epidermal growth factor receptor (EGFR) mutations are the most common oncogenic driver in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are widely used in the treatment of lung cancer, especially in the first-line treatment of advanced NSCLC, and EGFR-TKIs monotherapy has achieved better efficacy and tolerability compared with standard chemotherapy. However, acquired resistance to EGFR-TKIs and associated adverse events pose a significant obstacle to targeted lung cancer therapy. Therefore, there is an urgent need to seek effective interventions to overcome these limitations. Natural medicines have shown potential therapeutic advantages in reversing acquired resistance to EGFR-TKIs and reducing adverse events, bringing new options and directions for EGFR-TKIs combination therapy. In this paper, we systematically demonstrated the resistance mechanism of EGFR-TKIs, the clinical strategy of each generation of EGFR-TKIs in the synergistic treatment of NSCLC, the treatment-related adverse events of EGFR-TKIs, and the potential role of traditional Chinese medicine in overcoming the resistance and adverse reactions of EGFR-TKIs. Herbs and active compounds have the potential to act synergistically through multiple pathways and multiple mechanisms of overall regulation, combined with targeted therapy, and are expected to be an innovative model for NSCLC treatment.

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway.

For patients exhibiting non-small-cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are a first-line treatment. However, most patients who initially responded to EGFR-TKIs eventually developed acquired resistance, limiting the effectiveness of therapy. It has long been known that epithelial-mesenchymal transition (EMT) leads to acquired resistance to EGFR-TKIs in NSCLC. However, the mechanisms underlying the resistance dependent on EMT are unknown. This research aimed to reveal the effects of LMNA in the regulation of acquired resistance to erlotinib by EMT in NSCLC. The acquired erlotinib-resistant cells (HCC827/ER) were induced by gradual increase of concentrations of erlotinib in erlotinib-sensitive HCC827 cells. RNA sequencing and bioinformatics analysis were performed to uncover the involvement of LMNA in the EMT process that induced acquired resistance to erlotinib. The effect of LMNA on cell proliferation and migration was measured by clone-formation, wound-healing, and transwell assays, respectively. The EMT-related protein, nuclear shape and volume, and cytoskeleton changes were examined by immunofluorescence. Western blot was used to identify the underlying molecular mechanism of LMNA regulation of EMT. HCC827/ER cells with acquired resistance to erlotinib underwent EMT and exhibited lower LMNA expression compared to parental sensitive cells. LMNA negatively regulated the expression of EMT markers; HCC827/ER cells showed a significant up-regulation of mesenchymal markers, such as CDH2, SNAI2, VIM, ZEB1, and TWIST1. The overexpression of LMNA in HCC827/ER cells significantly inhibited EMT and cell proliferation, and this inhibitory effect of LMNA was enhanced in the presence of 2.5 μM erlotinib. Furthermore, a decrease in LMNA expression resulted in a higher nuclear deformability and cytoskeletal changes. In HCC827/ER cells, AKT, FGFR, ERK1/2, and c-fos phosphorylation levels were higher than those in HCC827 cells; Furthermore, overexpression of LMNA in HCC827/ER cells reduced the phosphorylation of AKT, ERK1/2, c-fos, and FGFR. In conclusion, our findings first demonstrated that downregulation of LMNA promotes acquired EGFR-TKI resistance in NSCLC with EGFR mutations by EMT. LMNA inhibits cell proliferation and migration of erlotinib-resistant cells via inhibition of the FGFR/MAPK/c-fos signaling pathway. These findings indicated LMNA as a driver of acquired resistance to erlotinib and provided important information about the development of resistance to erlotinib treatment in NSCLC patients with EGFR mutations.

Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation

Lung cancer is the leading cause of cancer-related death worldwide. The development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and immune checkpoint inhibitors (ICIs) has brought favorable survival benefits to patients with non–small-cell lung cancer (NSCLC); unfortunately, acquired drug resistance remains a major barrier to the treatment of NSCLC. Recent studies have demonstrated that the transcriptional co-activator with a PDZ-binding motif (TAZ, also called WWTR1) induces tumor immune evasion by directly modulating the expression of programmed death ligand 1 (PD-L1), a key therapeutic target for checkpoint immunotherapy. Moreover, aberrant activation of TAZ is also a major mechanism of acquired resistance to EGFR-TKIs in NSCLC. Therefore, TAZ signaling blockade might be an effective strategy to overcome resistance to ICIs and EGFR-TKIs in NSCLC. In this study, we showed for the first time that artesunate effectively reduced TAZ and PD-L1 expression in NSCLC. We further demonstrated that artesunate suppressed TAZ/PD-L1–induced T-cell growth inhibition in vitro and enhanced anti-tumor immunity by recruiting infiltrating CD8 + T-cells in syngeneic mouse models. Artesunate also inhibited the stem cell–like properties of NSCLC cells and suppressed tumor growth in xenografts bearing gefitinib-resistant tumors. In addition, our results of molecular docking and cellular thermal shift assay analysis suggested that artesunate might directly target the TAZ–TEAD complex and induce proteasome-dependent TAZ degradation in NSCLC cells. These results suggest that artesunate enhanced anti-tumor immunity and overcame EGFR-TKI resistance in NSCLC at least in part by suppressing TAZ/PD-L1 signaling.

Efficacy and Safety of EGFR Tyrosine Kinase Inhibitors Combined with Cranial Radiotherapy for Brain Metastases from Non-Small-Cell Lung Cancer: A Protocol for a Systematic Review and Meta-Analysis.

Introduction. Brain metastases (BMs) are common in non-small-cell lung cancer (NSCLC), which leads to a poor prognosis. As the two most effective strategies available, the use of combination of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and radiotherapy (RT) is still controversial. This protocol proposes a methodology for carrying out a systematic review and meta-analysis that is aimed at (1) focusing on the efficacy and safety role of EGFR-TKIs combined with RT for BMs from NSCLC and (2) displaying the difference in efficacy of EGFR-TKIs owing to the sites and number of BMs, different types of RT, EGFR mutation status, and the subtypes of EGFR mutations by subgroup analysis. Methods and Analysis. Electronic databases including PubMed, Embase, CENTRAL, Web of Science, CBM, CNKI, Wanfang database, and VIP database will be searched from their inception until May 2022. Only randomized controlled trials evaluating the clinical efficacy and safety of EGFR-TKIs combined with RT on BMs of NSCLC will be included. Two reviewers will select the articles, assess the risk of bias, and extract data independently and in duplicate. The RoB 2 tool will be used to assess the quality of included studies. The meta-analysis of data synthesis will be performed with Stata 16. Publication bias will be assessed with the funnel plot method and the Egger test. Quality of the evidence will be evaluated by the GRADE system. Discussion. The approval of an ethical committee is not required. All the included trials will comply with the current ethical standards and the Declaration of Helsinki. Given the ongoing controversies regarding the optimal sequencing of the available and expanding treatment options for EGFR-TKIs in NSCLC with BMs, a synthesis of available, high-quality clinical research evidence is essential to advance our understanding in the treatment of this complex and common disease. This systematic review will evaluate available evidence, will try to provide optimized advice in the applications of EGFR-TKIs, and will be published in a high-quality journal. This study is registered with PROSPERO registration number CRD42021291509.

Integrin β3 Promotes Resistance to EGFR-TKI in Non-Small-Cell Lung Cancer by Upregulating AXL through the YAP Pathway.

Integrin β3 plays a key role in the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), but the development of integrin β3 inhibitors has been stalled due to the failure of phase III clinical trials for cancer treatment. Therefore, it is imperative to find a potentially effective solution to the problem of acquired resistance to EGFR-TKI for patients with integrin-β3 positive non-small-cell lung cancer (NSCLC) by exploring novel downstream targets and action mechanisms of integrin β3. In the present study, we observed that the expression of integrin β3 and AXL was significantly upregulated in erlotinib-resistant NSCLC cell lines, which was further confirmed clinically in tumor specimens from patients with NSCLC who developed acquired resistance to erlotinib. Through ectopic expression or knockdown, we found that AXL expression was positively regulated by integrin β3. In addition, integrin β3 promoted erlotinib resistance in NSCLC cells by upregulating AXL expression. Furthermore, the YAP pathway, rather than pathways associated with ERK or AKT, was involved in the regulation of AXL by integrin β3. To investigate the clinical significance of this finding, the current well-known AXL inhibitor R428 was tested, demonstrating that R428 significantly inhibited resistance to erlotinib, colony formation, epithelial–mesenchymal transformation and cell migration induced by integrin β3. In conclusion, integrin β3 could promote resistance to EGFR-TKI in NSCLC by upregulating the expression of AXL through the YAP pathway. Patients with advanced NSCLC, who are positive for integrin β3, might benefit from a combination of AXL inhibitors and EGFR-TKI therapy.

TROY Modulates Cancer Stem-Like Cell Properties and Gefitinib Resistance Through EMT Signaling in Non-Small Cell Lung Cancer.

Targeted therapy has made breakthrough progress in the treatment of advanced non–small cell lung cancer (NSCLC) in the last 20 years. Despite that, acquired resistance of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is an urgent clinical problem. Our study established an acquired gefitinib-resistant cell line, which exhibited epithelial–mesenchymal transition (EMT) and stem cell–like properties. Transcriptional sequencing and bioinformatics analysis revealed that TROY was significantly increased in gefitinib-resistant cells. Gene set enrichment analysis (GSEA) showed EMT was the core enriched hallmark in the resistant cells. TROY siRNA interference could overcome the gefitinib resistance with the downregulated expression of EMT and CSC markers. In addition, immunohistochemistry indicated that TROY was overexpressed in tumor samples from patients who acquired resistance to first-generation EGFR-TKI without T790M mutation and the expression of TROY was associated with poor prognosis in LUAD. Here, we provided the potential role of TROY in the resistance of targeted therapy and a new strategy to overcome the acquired resistance to EGFR-TKI in NSCLC.

P76.76 EGFR Tyrosine Kinase Inhibitors in Patients with Non-Small Cell Lung Cancer and the Role of Diabetes Mellitus and Metformin

The EGFR tyrosine kinase inhibitors (TKI) had increased survival in non-small cell lung cancer (NSCLC) patients in the presence of response predictive EGFR mutations. Previously, these drugs were used in wild type patients as a 2nd line or beyond. Some laboratory studies and phase I/II trials had demonstrated a possible synergistic effect between EGFR TKI and metformin. The aim of this study is to analyze patients treated with EGFR TKI and assessing the impact of diabetes mellitus (DM) and metformin on the outcomes. A retrospective study including 75 patients with stage IV NSCLC submitted to EGFR TKI between January 2013 and December 2019. Clinicopathological characteristics were described. The progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier with Log Rank test and Cox regression, with 5% statistical significance. Thirty-one patients (n=31, 41.3%) had EGFR mutations and were treated with EGFR TKI as 1st line treatment (group 1) and 44 (58.7%) were wild type, treated with EGFR TKI in 2nd line or beyond (group 2). Twenty patients had DM and 12 patients were on metformin. The median follow-up time was 54.73 months. In group 1, the median PFS and OS were 10.37 and 18.70 months, respectively. There were no statistically significant differences in PFS and OS between patients with or without DM (p=0.28 and p=0.53), and patients treated or not with metformin (p=0.20 and p=0.50). In group 2, the median PFS and OS were 2.73 and 9.87 months, respectively. Inversely from the group 1, the median PFS was superior in patients with DM: 5.77 vs 2.63 months, HR 0.44 (95%CI 0.21-0.91), p=0.023; and in patients on metformin: 5.77 vs 2.53 months, HR 0.43 (95%CI 0.20-0.95), p=0.031. The median OS was also superior in patients with DM: 14.90 vs 7.10 months, HR 0.44 (95%CI 0.22-0.89), p=0.019. Patients that received metformin had a median OS of 12.57 months vs 7.73 months in the remaining, HR 0.47 (95%CI 0.21-1.02), p=0.051. In group 1, there were no differences in median survival among patients with DM and on metformin. However, the number of patients with DM and on metformin in this group was very small. In group 2, the median PFS and OS were superior in patients with DM, and the median PFS was superior in patients on metformin. This study has some limitations: it is retrospective, with a limited sample and it includes patients that nowadays wouldn’t be treated with EGFR TKI following the current international guidelines. Nevertheless, it will be important to study the potential association between metformin and EGFR TKI in NSCLC, regarding the fact that this drug is easily accessed in daily practice.

Targeting the IL-1β/EHD1/TUBB3 axis overcomes resistance to EGFR-TKI in NSCLC

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) significantly prolong the survival time of non-small-cell lung cancer (NSCLC) patients with EGFR-activating mutations, but resistance develops universally. Activation of the phosphatidyl inositol-3 kinase (PI3K)/AKT signaling pathway and phenotypic alterations in epithelial-mesenchymal transition (EMT) are both mechanisms of acquired resistance to EGFR-TKIs. However, the mechanisms underlying this resistance remain unclear. In this study, EHD1 depletion significantly increased NSCLC cell sensitivity to EGFR-TKI, which was accompanied by EMT reversal. Microarray analysis showed that the PTEN/PI3K/AKT signaling pathway is a crucial pathway regulated by EHD1. Moreover, a PTEN inhibitor abolished EHD1 shRNA regulation of EGFR-TKI sensitivity, EMT, and cancer progression. Mass spectrometry showed that TUBB3 is a novel EHD1-interacting protein. EHD1 modulated microtubule stability by interacting with TUBB3. Furthermore, TUBB3 depletion significantly attenuated EHD1-induced EGFR-TKI resistance and EMT. Bioinformatics analysis revealed that EHD1 is significantly associated with the gene set, "Cellular Response to Interleukin-1β (IL-1β)". As expected, treatment with IL-1β led to increased expression of EHD1, activation of PTEN/PI3K/AKT signaling, and induction of EMT in NSCLC cells. In patient specimens, EHD1 was highly expressed in EGFR-TKI-refractory specimens. EHD1 was positively associated with TUBB3 and IL-1R1 but negatively associated with PTEN. In addition, targeting the IL-1β/EHD1/TUBB3 axis mitigated cancer progression by inhibiting cell proliferation and metastasis and promoting apoptosis. Our study demonstrates the involvement of the IL-1β/EHD1/TUBB3 axis in EGFR-TKI resistance and provides a potential therapeutic approach for treating patients with NSCLC that has acquired EGFR-TKI resistance.

Traditional Chinese Medicine Prolongs Progression-Free Survival and Enhances Therapeutic Effects in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR-TKI)Treated Non-Small-Cell Lung Cancer (NSCLC) Patients Harboring EGFR Mutations.

BackgroundLung cancer is the most common cause of cancer-associated deaths worldwide. This study aimed to investigate the efficacy and safety of Traditional Chinese Medicine combining EGFR-TKIs in treatment of NSCLC patients harboring EGFR mutations.Material/MethodsThis study involved 153 advanced-stage NSCLC patients harboring EGFR mutations. Patients were divided into a Control group (administered EGFR-TKI, n=61) and an Experimental group (administered Traditional Chinese Medicine combining EGFR and TKI, n=92). Progression-free survival (PFS) was evaluated for exon 19 deletion and/or 21 deletion patients. Disease control rate (DCR) was assessed to observe therapeutic effects. Adverse effects, including rashes, diarrhea, ALT/AST increase, dental ulcers, and onychia lateralis, were also evaluated.ResultsTCM combining EGFR-TKI (90.11%) demonstrated no DCR improvement compared to single EGFR-TKI (83.33%) (p>0.05). Median PFS (mPFS) of TCM combining EGFR-TKI (13 months) was significantly longer compared to that in the single EGFR-TKI group (8.8 months) (p=0.001). For 19DEL mutant NSCLC, the mPFS (11 months) in TCM combining EGFR-TKI was significantly longer compared to single EGFR-TKI (8.5 months) (p=0.007). The mPFS of L858 mutant NSCLC patients in EGFR-TKI combining CTM (14 months) was significantly longer compared to single EGFR-TKI (9.5 months) (p=0.015). TCM combining EGFR-TKI was more inclined to prolong mPFS of NSCLC with exon 21 deletion. TCM combining EGFR-TKI illustrated no additional adverse effects in NSCLC patients (p=0.956).ConclusionsApplication of Traditional Chinese Medicine prolonged progression-free survival and enhanced therapeutic effect in NSCLC patients harboring EGFR mutations receiving EGFR-TKI treatment. Meanwhile, adjunctive Chinese medicine combining EGFR-TKI in NSCLC with EGFR mutations caused no adverse effects.

P3.01-016 Factors Associated with Symptoms Improvement and HRQoL for First-Line EGFR-TKIs in NSCLC: A Multicenter Prospective SMILE Study

First-line epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) offer an advantage compared to doublet chemotherapy in progression free survival, tolerability, and quality of life (QOL) in EGFR-mutated advanced non-small cell lung cancer (NSCLC) patients. In Taiwan, gefitinib, erlotinib and afatinib are all reimbursed as first-line therapy. It provides a rare opportunity to investigate factors associated with the extent of symptoms and QOL improvement in real-world patient population.

Identification of microRNAs involved in gefitinib resistance of non-small-cell lung cancer through the insulin-like growth factor receptor 1 signaling pathway.

Multiple clinical and experimental studies have suggested that epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) may be effective at treating advanced non-small cell lung cancer (NSCLC), however, the molecular basis of primary resistance to EGFR-TKIs in NSCLC remains unclear. In the current study, the insulin-like growth factor 1 receptor (IGF-1R) gene in the gefitinib-resistant human lung adenocarcinoma epithelial cell line A549 (A549/GR) was silenced using small interfering RNA (siRNA) in order to determine the role of microRNA (miRNA) in the development of resistance against epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The relative gefitinib-resistant capacity in A549 and A549/GR cells was determined using a cell counting kit 8. A549/GR cells were transfected with chemically synthesized siRNA to silence the IGF-1R gene. A total of 48 h after siRNA transfection, IGF-1R expression in A549/GR cells was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. miRNA expression in A549/GR cells and A549/GR cells with silenced IGF-1R was analyzed using a miRNA microarray. The microarray results of 10 miRNAs were then compared with the results of RT-qPCR. The results demonstrated that the gefitinib-resistance capacity of A549/GR cells was six times higher than that of A549 cells. Additionally, RT-qPCR and western blotting demonstrated that the IGF-1R gene in A549/GR cells was successfully silenced by siRNA. The highest silencing rate (72%) of the IGF-1R gene was obtained using siRNA-2. The microarray identified 72 miRNAs with significantly different expression in A549/GR cells with silenced IGF-1R compared with A549/GR cells. Of the 72 differentially expressed miRNAs, 13 miRNAs (including miR-497-3p and miR-1273c) were up-regulated and 59 miRNAs (including miR-361-3p and miR-345-3p) were down-regulated in A549/GR cells with silenced IGF-1R compared with A549/GR cells. The changes in the expression of 10 different miRNAs were confirmed by RT-qPCR. Thus, the present study successfully established an A549/GR cell line with silenced IGF-1R. The results suggest that a number of miRNAs associated with the IGF-1R signaling pathway, including miR-497-3p and miR-144-5p, were involved in the development of resistance against EGFR-TKIs in A549 cells. These miRNAs may provide novel targets to treat lung adenocarcinoma exhibiting resistance against EGFR-TKIs.

Mechanism and clinical efficacy of third-generation epidermal growth factor receptor tyrosine kinase inhibitor in non-small cell lung cancer

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are the standard treatment for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, most of patients will develop resistance to TKIs treatment due to the emergence of the T790M mutation. The third-generation EGFR-TKI is highly selective and efficient for activating mutants (EGFR sensitive mutations) and resistance mutant (T790M+ ). This review summarizes the mechanism and clinical efficacy of the third-generation EGFR-TKI in NSCLC patients.

Iron Oxide Nanoparticles Synergize with Erlotinib to Suppress Refractory Non-Small Cell Lung Cancer Cell Proliferation Through the Inhibition of ErbB/PI3K/AKT and PTEN Activation.

Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases. EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib and gefitinib, are currently approved for the management of NSCLC. However, primary and acquired resistances to EGFR-TKIs are the major obstacles in the treatment of NSCLC. These resistances have been associated with the development of secondary mutations in EGFR or continued oncogenic signaling despite TKI treatment. In this study, NSCLC cells with wild-type EGFR, A549, H460, H358, H157 which do not respond to EGFR-TKIs, were used. We investigated whether a combination therapy of erlotinib plus iron oxide nanoparticles (IONPs) could sensitize NSCLC cells to erlotinib-induced cancer inhibition. In the 4 NSCLC cells investigated, erlotinib and IONPs combination therapy obviously inhibited NSCLC proliferation in vitro and in vivo, compared with erlotinib treatment alone. This effect was not dependent on erlotinib dose. Activation of ErbB3 was observed in these refractory NSCLC cells. Combined with IONPs, erlotinib could block ErbB3 activity and induce the expression of PTEN, which in turn inhibited the downstream PI3KAKT signaling pathway. These data demonstrate the therapeutic potential of biocompatible IONPs in combination with EGFR-TKIs in NSCLC, thus expanding and repurposing the current therapy for NSCLC.

The prognostic role of EGFR-TKIs for patients with advanced non-small cell lung cancer

Clinical trials have shown that epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) did not improve the survival of patients with EGFR-mutated non-small cell lung cancer (NSCLC) because of the high crossover of treatments. Realistically, the role of EGFR-TKIs in NSCLC with mutated EGFR is not well known. We retrospectively analysed data from patients with recurrent or metastatic NSCLC. Clinical prognostic factors were identified by Cox proportional hazards modelling. Among 503 patients, the median overall survival (OS) for all of patients was 11.7 months. Cox analysis showed that PS 0–1, recurrent disease, EGFR mutations, or EGFR-TKI treatment were associated with improved OS. In patients with EGFR-activating mutations, Cox analysis showed that patients with adenocarcinoma, recurrent disease, or EGFR-TKI treatment had significantly longer survival. Patients with EGFR-activating mutations who received EGFR-TKI therapy had a median OS of 24.3 months, which was significantly longer than those who had not received EGFR-TKI therapy (10.8 months). Patients with wild-type EGFR had a median OS of 9.7 months and Cox analysis showed that PS score and disease type were independent predictors. EGFR-TKI therapy is an independently prognostic factor for NSCLC with mutated EGFR. A more effective therapy is needed for patients with wild-type EGFR.

Relationship between ID1 and EGFR-TKI Resistance in Non-small Cell Lung Cancer

背景与目的非小细胞肺癌(non-small cell lung cancer, NSCLC)是当今世界上发病率和死亡率最高的恶性肿瘤之一，而表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitors, EGFR-TKI)整体有效率为30%-40%，无进展生存期(progression-free survival, PFS)为12个月。但EGFR-TKI在临床中的耐药现象也很普遍，严重影响了其抑瘤作用。因此，克服耐药、寻找一种新的与肺癌耐药相关的预后因子势在必行。本研究旨在通过体内外实验探讨DNA结合抑制因子1(differentiation inhibitory factor 1, ID1)与NSCLCEGFR-TKI耐药之间的关系，看其是否有统计学意义，并初步探讨其机制。方法免疫组化(immunohistochemistry, IHC)检测手术标本(肺癌组织和癌旁组织)1D1的表达；qRT-PCR、Western-blot检测并比较肺癌细胞敏感株与耐药株中ID1的表达变化；MTT检测吉非替尼对ID1慢病毒载体处理肺癌细胞的细胞增殖情况，将肺癌细胞接种至裸鼠腋下，待肿瘤生长至一定体积使用吉非替尼治疗，估算肿瘤体积。结果ID1在肺癌组织中的表达明显高于正常组织(P < 0.05)；ID1的表达与NSCLC EGFR-TKI耐药呈正相关(P < 0.05)。结论ID1在NSCLC中高表达，并且参与了NSCLC EGFR-TKI的耐药，其机制可能与STAT3磷酸化程度增加有关。

H34 - KRAS has a role in acquired resistance to EGFR-TKIs in NSCLC: an analysis on circulating tumor DNA

Background: Activating mutations of KRAS oncogene drive resistance to EGFR inhibition by providing an alternative signal transduction pathway [1]. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations that are mutually exclusive with KRAS mutations. However, pharmacological inhibition of EGFR signaling has the potential to select cells whose growth may depend, at least in part, on alternative proliferation pathways or continued EGFR signaling due to the c.2369C > T (p.T790M) gatekeeper mutation within the ATP-binding pocket of EGFR. NSCLC heterogeneity can drive the therapeutic decisions; therefore, tissue availability is increasingly recognized as a crucial issue [2]. Unfortunately, the location of the tumor and the risk of complications are serious limitations to re-biopsies in NSCLC. Alternatively, the detection of somatic mutations in cell-free tumor DNA (cftDNA) released in plasma could be instrumental for a better understanding of the genetic modifications driven by the selective pressure of drug treatments on NSCLC [3]. Material and methods: This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 KRAS and p.T790M EGFR mutations in 33 advanced NSCLC patients that progressed after an EGFR-TKI. Six ml of blood samples were drawn from patients at disease progression and cftDNA was extracted by Circulating Nucleic Acid extraction kit (Qiagen) and analysed by digital droplet PCR (BioRad). Results:KRAS mutation at codon 12 alone or in combination with p.T790M was demonstrated in 3 (9.1%) and 13 patients (39.4%), respectively. p.T790M was detected in 11 subjects (33.3%) alone and in 13 patients (39.4%) with mutant KRAS. Six patients (18.2%) were negative for both KRAS and p.T790M. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62,5% for p.T790M and 37,5% for KRAS. Conclusions: In conclusion, mutation of KRAS could be an additional mechanism of escape to EGFR-TKI and cftDNA is a feasible approach to monitor the molecular development of drug resistance. Therefore, the clinical relevance of this finding, expecially for what concerns mutKRAS, needs to be evaluated prospectively. References 1Han SW, et al Clin Cancer Res 2006;12:2538–44. 2Bosc C, et al. Target Oncol 2014 [DOI 10.1007/s11523-014-0332-y]. 3Del Re M, et al. Ex Rev Mol Diagn 2014;14:453–68

Vorinostat synergizes with EGFR inhibitors in NSCLC cells by increasing ROS via up-regulation of the major mitochondrial porin VDAC1 and modulation of the c-Myc-NRF2-KEAP1 pathway

In non-small-cell lung cancer (NSCLC) patients, the activation of alternative pathways contributes to the limited efficacy of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The present study examines a panel of EGFR wild-type, K-Ras mutated, NSCLC lines, which were all intrinsically resistant to EGFR-TKIs, and demonstrates that the histone deacetylase inhibitor vorinostat can improve the therapeutic efficacy of gefitinib or erlotinib, inducing strong synergistic antiproliferative and pro-apoptotic effects that are paralleled by reactive oxygen species accumulation and by increased DNA damage. By knockdown experiments, we suggested that the up-regulation of voltage-dependent anion-selective channel protein 1 (VDAC1), the major mitochondrial porin of the outer mitochondrial membrane, which was induced by vorinostat and further increased by the combination, could be functionally involved in oxidative stress-dependent apoptosis. Significantly, we also observed the attenuation of the expression of both the enzyme hexokinase1, a negative VDAC1 regulator, and the anti-apoptotic porin VDAC2, only in the combination setting, suggesting convergent mechanisms that enhanced mitochondria-dependent apoptosis by targeting VDAC protein functions. Furthermore, the prosurvival capacities of the cells were also inhibited by the combination treatments, as shown by complete pAKT deactivation, increased GSK3β expression, and c-Myc down-regulation. Finally, we observed that the combination treatment of vorinostat and either of the EGFR-TKIs induced the down-regulation of the c-Myc-regulated nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor and the up-regulation of the NRF2 repressor Kelch-like ECH-associated protein 1 regulator (KEAP1). These two genes are crucial for the redox stress response, often dysfunctional in NSCLC, and involved in EGFR-TKI resistance. Taken together, these results are the first to demonstrate that altering redox homeostasis is a new mechanism underlying the observed synergism between vorinostat and EGFR TKIs in NSCLC.

TGFb expression as predictor of outcome in EGFR wild type (WT) metastatic non-small cell lung cancer (NSCLC) patients (PTS) treated with EGFR tyrosine kinase inhibitors (TKIs).

e18104 Background: Gefitinib and erlotinib have shown to be highly effective in pts harboring EGFR mutations, while the majority of EGFR wt pts does not respond to treatment.Preclinical data suggested that TGFb and HGF could affect response to TKIs. Aim of the present study is to investigate molecular predictors for primary resistance to TKIs in metastatic NSCLC pts. Methods: This retrospective study included 120 metastatic NSCLC patients treated with gefitinib (68/56.7%) or erlotinib (52/43.3%) in first line (20/16.7%) or after prior chemotherapy (100/83.4%) with at least one measurable lesion and availability of paraffin-embedded tumour tissue from primary cancer. Analyses included presence of mutations in EGFR and KRAS genes, assessment of ALK rearrangement by fluorescence in situ hybridization (FISH) and protein expression of HGF and TGFb by immunohistochemistry (IHC). Pts were grouped in IHC+ and IHC- according to staining intensity. Results: In the whole study cohort response rate (RR) was 9.2%, median progression free survival (PFS) 2.5 months, and overall survival (OS) 7.8 months. EGFR mutations were observed in 9.2% of pts and were significantly associated with better pts outcome. KRAS was mutated in 23.3% of pts without any correlation with clinical end points. ALK translocations were observed in 5 pts (4.2%), and none responded to treatment. IHC was successfully performed for HGF and TGFb in 95 and 75 pts respectively. No difference was seen in HGF+ (9/9.5%) vs HGF- pts in terms of RR, PFS and OS. TGFb+ pts (41/54.7%) had a significantly shorter OS than TGFb- pts (6.7 vs 10.3 months, p=0.020) although no difference in terms of RR and PFS was observed. When restricting survival analysis to EGFR wt/KRAS wt pts (n=80) TGFb+ subjects had a significantly worse PFS (1.9 vs 2.9 months, HR 2.16, CI 95% 1.18;3.95, p=0.013) and OS (5.3 vs 8.8 months, HR 2.07, CI 95% 1.10;3.91, p=0.025) when compared with the TGFb- group. Conclusions: Overexpression of TGFb is associated with a poor outcome in EGFR and KRAS wt pts treated with gefitinb or erlotinib. Prospective validation of the role of TGFb as a mediator of intrinsic resistance to EGFR TKIs in NSCLC is warranted.

Mechanisms of Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients with Advanced Non-Small-Cell Lung Cancer: Clinical and Molecular Considerations

Non-Small-Cell Lung Cancer (NSCLC) with somatic mutations of the epidermal growth factor receptor (EGFR) is anticipated to respond to small-molecule tyrosine kinase inhibitors (TKIs) of the EGFR tyrosine kinase. There are, however, patients with EGFR mutated tumors who do not demonstrate tumor response. The most widely accepted mechanism of 'de novo' (inherent) resistance to these TKIs involves mutations of the KRAS gene. KRAS is a downstream mediator of EGFR-induced cell signaling, such mutations appear to be mutually exclusive from EGFR mutations in lung cancer. The first molecular modifier of resistance identified in patients who developed resistance (termed 'acquired resistance') to TK inhibition was a new acquired somatic EGFR mutation (T790M). Today there is an ever-growing series of molecular events that have recently come to the forefront to explain other instances of TKI resistance not attributable to T790M or KRAS. These include a number of molecules that interact with EGFR or form part of its downstream signaling pathway such as HER-2, IGFR-1, MET and B-RAF. Considering that the majority of studies carried out to date with respect to the identification of resistant clones have not used highly sensitive techniques (e.g. allelic discrimination to identify somatic mutations), coupled with the relatively low number of studies examining multiple molecular markers and the accepted molecular heterogeneity of NSCLC raise question as to the existence of 'acquired' versus 'de-novo' resistance. By examining the current knowledge base with respect to mechanisms of resistance to EGFR TKIs in NSCLC, we explore whether 'acquired' resistance is 'de-novo' resistance in disguise, and discuss the promises and limitations of molecular stratification with respect to strategies incorporating TKIs in the treatment of NSCLC.

Activated c-Met[pY1003]: A potential marker of intrinsic resistance and therapy target to restore sensitivity to gefitinib

7624 Background: Epidermal growth factor receptor tyrosine-kinase inhibitors (EGFR-TKIs) show anti-tumor activity in only 10% of Caucasian non-small cell lung cancer (NSCLC) patients. Aim of this study was to evaluate several biological parameters potentially related to EGFR, including c-Met activation, as potential markers of intrinsic resistance to EGFR-TKIs in NSCLC. Methods: P-Akt, p-Erk, c-Src, E-Cadherin, c-Met[pY1003] and c-Met[pY1230/1234/1235] status was immunohistochemically determined on a tissue micro-array of tumor samples from 51 NSCLC patients treated with gefitinib. EGFR, k-ras, and c-Met mutation analysis was also carried out. A panel of NSCLC cell lines expressing c-Met[pY1003] were treated with gefitinib (0.01–100μ M) alone or in combination with hepatocyte growth factor (40 ng/ml) and the c-Met-agonistic antibody DN-30 (80 μg/ml) for 72 hours in 0.5% FCS medium. Drug interaction between gefitinib and DN-30 was assessed, at a non-constant concentration ratio, using the combination index (CI) method. Results: There was no association between p-Erk, c-Src, E-Cadherin, c-Met[pY1230/1234/1235], and k-ras status and response or survival. EGFR exon 19 deletion and p-Akt nuclear staining were significantly associated with response (P&lt;0.0001; Fisher's exact test) and longer time to progression (TTP) (P=0.007; log-rank test), respectively. High c-Met[pY1003] membrane staining was significantly associated with progressive disease (P=0.019; Fisher's exact test) and shorter TTP (P=0.0416; log-rank test), but not with survival. Multivariate analysis confirmed a significant relationship between c-Met[pY1003] and increased risk of disease progression (HR=2.464, 95% CI 1.293–4.696, P=0.006). No c-Met mutations were found. In vitro, the combination with DN- 30 synergistically (CI&lt;1) enhanced gefitinib-induced growth inhibition in all c-Met[pY1003]-expressing NSCLC cells studied (H460, SW1573, A549 and H292). Conclusions: Activation of c-MET may be a biological marker of intrinsic resistance to gefitinib in NSCLC patients, and combined inhibition of c-Met and EGFR may be a suitable therapeutic approach in patients with activated c-Met[pY1003] tumors. No significant financial relationships to disclose.