Rebiopsy for patients with non‐small‐cell lung cancer after epidermal growth factor receptor‐tyrosine kinase inhibitor failure

Although third‐generation epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI) can overcome T790M‐mediated resistance in non‐small‐cell lung cancer (NSCLC), rebiopsy to confirm T790M status is occasionally difficult. We aimed to investigate the current tendency and the limitations of rebiopsy in clinical practice. This study included 139 consecutive NSCLC patients with EGFR mutations, who had experienced progressive disease (PD) after EGFR‐TKI treatment. We retrospectively reviewed patient characteristics, tumor progression sites and rebiopsy procedures. Of 120 patients (out of the original 139) who were eligible for clinical trials, 75 (63%) underwent rebiopsy for 30 pleural effusions, 32 thoracic lesions, four bone, two liver, and seven at other sites. Rebiopsy procedures included 30 thoracocentesis, 24 transbronchial biopsies, 13 computed tomography (CT)‐guided needle biopsies and 8 other procedures. Of the 75 rebiopsied patients, 71 (95%) were pathologically diagnosed with malignancy; and 34 (45%) had available tissue samples for EGFR analyses. Of the 75 biopsied patients, 61 (81%) were analyzed for EGFR mutation, using tissue or cytology samples; T790M mutations were identified in 20 (33%) of the 61 patients. Of the 120 patients, 45 (38%) did not undergo rebiopsy, because of inaccessible tumor sites (n = 19), patient refusal (n = 6) or decision of physician (n = 10). In conclusion, among patients with EGFR mutations who had PD after EGFR‐TKI treatment, 63% underwent rebiopsy. Most rebiopsy samples were diagnosed with malignancy. However, tissue samples were less available and T790M mutations were identified less frequently than in previous studies. Skill and experience with rebiopsy and noninvasive alternative methods will be increasingly important.     

Krüppel‐like factor 4 promotes c‐Met amplification‐mediated gefitinib resistance in non‐small‐cell lung cancer

Gefitinib has been widely used in the first‐line treatment of advanced EGFR‐mutated non‐small‐cell lung cancer (NSCLC). However, many NSCLC patients will acquire resistance to gefitinib after 9‐14 months of treatment. This study revealed that Krüppel‐like factor 4 (KLF4) contributes to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells. We observed that KLF4 was overexpressed in c‐Met‐overexpressing NSCLC cells and tissues. Knockdown of KLF4 increased tumorigenic properties in gefitinib‐resistant NSCLC cell lines without c‐Met overexpression, but it reduced tumorigenic properties and increased gefitinib sensitivity in gefitinib‐resistant NSCLC cells with c‐Met overexpression, whereas overexpression of KLF4 reduced gefitinib sensitivity in gefitinib‐sensitive NSCLC cells. Furthermore, Western blot analysis revealed that KLF4 contributed to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells by inhibiting the expression of apoptosis‐related proteins under gefitinib treatment and activating the c‐Met/Akt signaling pathway by decreasing the inhibition of β‐catenin on phosphorylation of c‐Met to prevent blockade by gefitinib. In summary, this study's results suggest that KLF4 is a promising candidate molecular target for both prevention and therapy of NSCLC with c‐Met overexpression.     

The insulin‐like growth factor 1 receptor causes acquired resistance to erlotinib in lung cancer cells with the wild‐type epidermal growth factor receptor

Epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) therapy often provides a dramatic response in lung cancer patients with EGFR mutations. In addition, moderate clinical efficacy of the EGFR‐TKI, erlotinib, has been shown in lung cancer patients with the wild‐type EGFR. Numerous molecular mechanisms that cause acquired resistance to EGFR‐TKIs have been identified in lung cancers with the EGFR mutations; however, few have been reported in lung cancers with the wild‐type EGFR. We used H358 lung adenocarcinoma cells lacking EGFR mutations that showed modest sensitivity to erlotinib. The H358 cells acquired resistance to erlotinib via chronic exposure to the drug. The H358 erlotinib‐resistant (ER) cells do not have a secondary EGFR mutation, neither MET gene amplification nor PTEN downregulation; these have been identified in lung cancers with the EGFR mutations. From comprehensive screening of receptor tyrosine kinase phosphorylation, we observed increased phosphorylation of insulin‐like growth factor 1 receptor (IGF1R) in H358ER cells compared with parental H358 cells. H358ER cells responded to combined therapy with erlotinib and NVP‐AEW541, an IGF1R‐TKI. Our results indicate that IGF1R activation is a molecular mechanism that confers acquired resistance to erlotinib in lung cancers with the wild‐type EGFR.     

Detection of epidermal growth factor receptor mutations in plasma by mutant‐enriched PCR assay for prediction of the response to gefitinib in patients with non‐small‐cell lung cancer

The high frequency of epidermal growth factor receptor (EGFR) mutations in tyrosine kinase inhibitor‐responsive non‐small‐cell lung cancer (NSCLC) cases is now well established, highlighting the predictive value of activating EGFR mutations in guiding the clinical use of EGFR‐targeted therapies. However, specimen source and methods for EGFR mutation analysis are limited by tissue availability and technical feasibility in clinical application. Therefore, the current study is designed to establish a blood‐based approach for the assessment of EGFR mutations in NSCLC patients, in particular the advanced stage, and to test its clinical application. Plasma samples were obtained from the enrolled 134 NSCLC patients. The detection rate of the EGFR exon19 deletions and exon21 L858R was 49.3% (66/134) by the blood‐based, mutant‐enriched polymerase chain reaction. In the paired tumor and plasma samples, the detected mutant types of each pair respectively by direct sequencing and mutant‐enriched polymerase chain reaction were concordant in 17 of 18 (94.4%). In the patients treated with gefitinib as a second‐line therapy, those with plasma EGFR mutation have a prolonged median progression‐free survival compared with those with EGFR wild type (7.609 vs. 2.877 months, p = 0.002). On comparing the efficacy of gefitinib with that of docetaxel, it was found that the median progression‐free survival was significantly longer for patients treated with gefitinib than those with docetaxel in those harboring plasma EGFR mutation (7.609 vs. 3.192 months, p = 0.006). These results suggest that the blood‐based EGFR mutations test has the ability to provide a reliable guidance for clinical decision making for the treatment of the advanced NSCLC patients. © 2009 UICC     

Role of interleukin‐17 in lymphangiogenesis in non‐small‐cell lung cancer: Enhanced production of vascular endothelial growth factor C in non‐small‐cell lung carcinoma cells

Interleukin‐17 (IL‐17), a potent pro‐inflammatory cytokine, plays an active role in inflammation and cancer. Recently, we found that increased IL‐17‐producing cells correlate with poor survival and increased lymphangiogenesis in non‐small‐cell lung cancer (NSCLC), but the mechanism is unknown. Here, we show that IL‐17 promotes lymphangiogenesis via inducing vascular endothelial growth factor‐C (VEGF‐C) production by lung cancer cells. We found that IL‐17 receptor (IL‐17R) is expressed on the surface of Lewis lung carcinoma (LLC) cells but not on lymphatic endothelial cells (LEC). Moreover, LEC chemotaxis and tube formation (measures of net lymphangiogenic potential) were increased by conditioned medium from recombinant mouse IL‐17 (rmIL‐17)‐stimulated LLC but not by rmIL‐17. Interleukin‐17 increased production of VEGF‐C in lung cancer cell lines. The enhanced chemotaxis and endothelial cord formation in the presence of LLC/rmIL‐17 was inhibited by addition of recombinant mouse VEGF R3/Fc chimera. Treatment of the A549 cells with rIL‐17 significantly increased VEGF‐C expression, which was extracellular signal‐regulated protein kinase 1/2 (ERK 1/2) dependent. Importantly, we found significant correlations between IL‐17 expression, VEGF‐C expression and lymphatic vascular density (LVD) in NSCLC. We conclude that IL‐17 is involved in lymphangiogenesis in NSCLC by enhancing production of VEGF‐C, and IL‐17 may be an important target for the treatment of NSCLC. (Cancer Sci 2010; 101: 2384–2390)     

Management of acquired resistance to epidermal growth factor receptor kinase inhibitors in patients with advanced non‐small cell lung cancer

The widespread adoption of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors for the first‐line treatment of patients with advanced EGFR‐mutated non‐small cell lung cancer has resulted in acquired tyrosine kinase inhibitor resistance becoming a ubiquitous clinical problem. The identification of specific mechanisms of acquired resistance has allowed a better understanding of the biology and natural history of resistant disease, but is only now starting to impact treatment decisions. Strategies for managing acquired resistance in patients with advanced non‐small cell lung cancer are complex and must be adapted to the individual characteristics of each patient's cancer. Although combination chemotherapy is the presumed standard of care for most patients, prospective trial data are lacking, highlighting the importance of offering patients participation in clinical trials in this setting. Emerging data from trials of third‐generation mutant‐specific EGFR kinase inhibitors suggests particular promise with this class of agents. Cancer 2014;120:2289–2298. © 2014 American Cancer Society.     

下调miR-23a对EGFR突变非小细胞肺癌细胞吉非替尼耐药的影响研究

目的 探讨抑制miR-23a对EGFR T790M突变非小细胞肺癌H1975细胞吉非替尼耐药性的影响及其可能机制。方法 选取吉非替尼耐药的对数生长期H1975细胞为研究样本,合成miR-23a抑制物（miR-23a inhibitor）,应用脂质体转染H1975细胞以抑制其miR-23a表达（抑制组）,同时设未经转染处理的非转染组和转染无关序列的阴性对照组,实时荧光定量PCR（qRT-PCR）验证转染效果;采用CCK-8法检测转染24、48、72及96 h后各组的增殖情况,同时于转染48 h后用吉非替尼（0.03～300 μmol／L）处理各组细胞以评价对吉非替尼的药物敏感性变化情况;分别用流式细胞术检测各组的凋亡率（FITC-Annexin V／PI双染）和细胞周期（PI单染）情况,免疫印迹法检测各组多药耐药相关蛋白1（MRP1）、肺耐药相关蛋白（LRP）及谷胱甘肽转移酶π（GST-π）的表达变化。结果 抑制组在转染24～96 h出现miR-23a水平的持续下降,其miR-23a水平均低于其余两组,转染 96 h后的miR-23a水平分别为非转染组的（32.06±4.68）％和阴性对照组的（31.77±3.18）％,且吉非替尼对抑制组的半数抑制浓度（IC₅₀）为（2.82±0.46）μmol／L,均低于其余两组,以上差异均有统计学意义（P＜0.05）;与其余两组相比,抑制组转染后增殖抑制率、凋亡率及G0／G1期比例均升高,S期、G2／M期比例均下降且耐药蛋白MRP1、LRP及GST-π蛋白水平亦下调,以上差异均有统计学意义（P＜0.05）。结论 下调miR-23a可逆转H1975细胞的吉非替尼耐药性,同时可抑制细胞增殖、诱导凋亡及细胞周期阻滞,可能与降低耐药蛋白表达有关。     

吉非替尼治疗表皮生长因子受体突变型非小细胞肺癌患者的效果及影响因素分析

目的 观察吉非替尼治疗表皮生长因子受体(EGFR)突变型非小细胞肺癌(NSCLC)患者的疗效并探讨影响因素.方法 选取150例EGFR突变型NSCLC患者的临床资料,所有患者均给予吉非替尼口服治疗,连续治疗6个疗程,评估其临床疗效,并对可能影响EGFR突变型NSCLC患者临床疗效的相关因素进行单因素和多因素分析.结果 150例患者中,总缓解患者48例(32.00％)设为缓解组,剩余102例患者设为对照组.缓解组和对照组患者性别、肿瘤分期、病理类型和肿瘤直径比较,差异均有统计学意义(P﹤0.05).Logistic回归分析结果显示,性别、肿瘤分期、病理类型和肿瘤直径均为EGFR突变型NSCLC患者临床疗效的影响因素(P﹤0.05).治疗期间,150例患者药物不良反应总发生率为28.67％.结论 吉非替尼治疗EGFR突变型NSCLC患者有一定的临床疗效,性别、肿瘤分期、病理类型和肿瘤直径均为EGFR突变型NSCLC患者临床疗效的影响因素.     

MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib

Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib. Acquired resistance to EGFR-TKIs develops after prolonged treatments. The study was prompt to explore effective strategies against resistance to EGFR-TKIs. We established gefitinib resistant PC-9 cells which harbor EGFR exon 19 deletion. Known mechanisms for intrinsic or acquired EGFR-TKI resistance, including KRAS mutation, HER2 mutation, EGFR T790M mutation and MET gene amplification, were studied, and we did not observe any known mechanisms for intrinsic or acquired resistance to EGFR-TKIs in the resistant cells. In the parental PC-9 cells, labeled as PC-9/wt, gefitinib completely inhibited EGF-induced phosphorylation of EGFR, AKT and ERK. Gefitinib inhibited EGFR phosphorylation, but was unable to block EGF-induced phosphorylation of ERK in resistant cells, labeled as PC-9/gef cells, including PC-9/gefB4, PC-9/gefE3, and PC-9/gefE7 subclones. We detected NRAS Q61K mutation in the PC-9/gef cells but not the PC-9/wt cells. MEK inhibitors, either AZD6244 or CI1040, inhibited ERK phosphorylation and sensitized gefitinib-induced cytotoxicity in PC-9/gef cells. Whereas MEK inhibitors or gefitinib alone did not activate caspases in PC-9/gef cells, combination of gefitinib and AZD6244 or CI1040 induced apoptosis. Our in vivo studies showed that gefitinib inhibited growth of PC-9/wt xenografts but not PC-9/gef xenografts. Furthermore, combination of a MEK inhibitor and gefitinib inhibited growth of both PC-9/wt xenografts and PC-9/gefB4 xenografts. To conclude, persistent activation of ERK pathway contributes to the acquired gefitinib-resistance. Combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistance lung adenocarcinoma cells harboring EGFR mutations.     

Enhanced gefitinib‐induced repression of the epidermal growth factor receptor pathway by ataxia telangiectasia‐mutated kinase inhibition in non‐small‐cell lung cancer cells

The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR‐TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non‐small‐cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR‐TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR‐TKIs, we examined the cross‐talk of the EGFR pathways with ataxia telangiectasia‐mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR‐TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib‐dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib‐dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.     

Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.

PURPOSE: Non-small cell lung cancers carrying activating mutations in the gene for the epidermal growth factor receptor (EGFR) are highly sensitive to EGFR-specific tyrosine kinase inhibitors. However, most patients who initially respond subsequently experience disease progression while still on treatment. Part of this "acquired resistance" is attributable to a secondary mutation resulting in threonine to methionine at codon 790 (T790M) of EGFR. EXPERIMENTAL DESIGN: We sequenced exons 18 to 21 of the EGFR gene to look for secondary mutations in tumors with acquired resistance to gefitinib in 14 patients with adenocarcinomas. Subcloning or cycleave PCR was used in addition to normal sequencing to increase the sensitivity of the assay. We also looked for T790M in pretreatment samples from 52 patients who were treated with gefitinib. We also looked for secondary KRAS gene mutations because tumors with KRAS mutations are generally resistant to tyrosine kinase inhibitors. RESULTS: Seven of 14 tumors had a secondary T790M mutation. There were no other novel secondary mutations. We detected no T790M mutations in pretreatment specimens from available five tumors among these seven tumors. Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration. None of the tumors had an acquired mutation in the KRAS gene. CONCLUSIONS: A secondary T790M mutation of EGFR accounted for half the tumors with acquired resistance to gefitinib in Japanese patients. Other drug-resistant secondary mutations are uncommon in the EGFR gene.     

Clinical impact of amphiregulin expression in patients with epidermal growth factor receptor (EGFR) wild‐type nonsmall cell lung cancer treated with EGFR‐tyrosine kinase inhibitors

BACKGROUND:

In patients with nonsmall cell lung cancer (NSCLC), several studies have demonstrated a positive correlation between somatic mutation in the epidermal growth factor receptor (EGFR) tyrosine kinase domain and clinical outcomes with the use of EGFR tyrosine kinase inhibitors (TKIs). However, some patients with wild‐type (WT) EGFR also responded to EGFR TKIs and remained stable. Recently, amphiregulin (AR) has been suggested as a predictive marker for EGFR TKIs in patients with WT EGFR‐positive NSCLC. The objective of the current study was to evaluate the association between AR expression and the efficacy of using EGFR TKIs in the treatment of patients with WT EGFR‐positive NSCLC.

METHODS:

Seventy‐three patients with WT EGFR‐positive NSCLC received treatment with gefitinib or erlotinib between May 2005 and December 2008. AR expression was assessed by immunohistochemistry.

RESULTS:

The clinical response to EGFR TKIs was reassessed for all patients as follows: 16 of 73 patients had a partial response (21.9%), 12 patients had stable disease (16.5%), and 45 patients had progressive disease (61.6%). AR expression was positive in 24 of 40 patients (60%). The ability to achieve disease control did not differ significantly between AR‐positive patients and AR‐negative patients (P = .188). At a median follow‐up of 25.4 months (range, 10.5‐53.3 months), progression‐free survival was 8.1 weeks in AR‐positive patients and 4 weeks in AR‐negative patients (P = .025), and overall survival was significantly longer in AR‐positive patients than in AR‐negative patients (12.2 months vs 4.1 months; P = .001).

CONCLUSIONS:

The current results suggested that patients with WT EGFR‐positive NSCLC who have AR‐positive tumors may benefit clinically from treatment with EGFR TKIs, indicating that AR expression may be a potential marker for the selection of EGFR‐TKI treatment for patients with WT EGFR‐positive NSCLC. Cancer 2011. © 2010 American Cancer Society.     

Impact of tumor microenvironment on the efficacy of epidermal growth factor receptor‐tyrosine kinase inhibitors in patients with EGFR‐mutant non‐small cell lung cancer

We retrospectively investigated the impact of the tumor microenvironment (TME) on the efficacy of epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs) as first‐line treatment in 70 patients with advanced EGFR‐mutant non‐small cell lung cancer and who were seen at Osaka City University Hospital (Osaka, Japan) between August 2013 and December 2017. Using immunohistochemical staining with 28‐8 and D7U8C Abs, the tumor proportion score was assessed for programmed cell death‐1 ligand‐1 (PD‐L1), as high (50% or more) or low (less than 50%), and ligand‐2 (PD‐L2) expression, respectively. The extent of CD8⁺ tumor‐infiltrating lymphocytes was evaluated on a scale of 0‐3, with 0‐1 as low and 2‐3 as high. The TME of the 52 evaluable pretreatment specimens was categorized into 4 subtypes, according to the respective PD‐L1 tumor proportion and CD8⁺ scores, as follows: (a) high/high (13.5%, n = 7); (b) low/low (42.3%, n = 22); (c) high/low (17.3%, n = 9); and (d) low/high (26.9%, n = 14). Expression of PD‐L2 was significantly the highest in type 1 (57.1% vs 4.5% vs 11.1% vs 7.1%, respectively; P = .0090). Response rate was significantly the lowest in type 1 (14.3% vs 81.8% vs 66.7% vs 78.6%, respectively; P = .0085). Progression‐free survival was the shortest in type 1 and the longest in type 4 (median, 2.4 vs 11.3 vs 8.4 vs 17.5 months, respectively; P = .00000077). The efficacy of EGFR‐TKIs differed according to the TME, and the phenotype with high PD‐L1 and CD8⁺ expression might be the subset that would poorly benefit from such treatment.     

Potential mechanism of primary resistance to icotinib in patients with advanced non–small cell lung cancer harboring uncommon mutant epidermal growth factor receptor: A multi‐center study

The incidence of epidermal growth factor receptor uncommon mutation (EGFRum) is relatively low and patients harboring EGFRum are resistant to the first‐generation tyrosine kinase inhibitors (TKI). However, the mechanism of primary resistance remains unclear. Medical records of 98 patients who had never been treated by TKI and who accepted icotinib treatment were collected and followed. The circulating tumor DNA (ctDNA) were detected and analyzed using the next‐generation sequencing (NGS) platform after progression on icotinib. The potential primary resistance mechanism of icotinib was explored. A total of 21 (21.4%) and 48 (49%) patients developed primary and acquired resistance to icotinib, respectively. The median progression‐free survival (PFS) of primary resistance patients was 1.8 months (0.5‐2.3, 95% CI = 1.50‐2.10). Before treatment, 52.4% (11/21) of patients carried S768I, 23.8% (5/21) L861Q, 14.3% (3/21) G719X and 14.3% (3/21) exon 20‐ins mutations. Approximately 23.8% (5/21) of patients harbored the combined pattern mutations and 76.2% (16/21) of patients harbored the single pattern mutations. The combined pattern with EGFR classical mutation (EGFRcm) had worse PFS than the combined with EGFRum and single pattern (P < .05). There were 6 (28.57%) patients with acquired EGFR extracellular domain mutation, 5 (23.81%) with BCL2L11 loss (BIM deletion polymorphism), 3 (14.29%) with MET amplification, 1 (4.76%) with ERBB2 amplification, 1 (4.76%) with MYC amplification, 1 (4.76%) with PTEN mutation, 1 (4.76%) with PIK3CA mutation and 3 (14.29%) with unknown status. EGFR extracellular domain mutation, BCL2L11 loss, PI3K‐AKT‐mTOR signaling pathway (PTEN and PIK3CA mutations), MET amplification, ERBB2 amplification or MYC amplification might contribute to molecular mechanisms of primary resistance to icotinib in patients with advanced non‐small cell lung cancer harboring uncommon mutant epidermal growth factor receptor. Combined targeted therapy or chemotherapy should be considered in this population.     

Enhanced anti‐angiogenic effect of E7820 in combination with erlotinib in epidermal growth factor receptor–tyrosine kinase inhibitor‐resistant non‐small‐cell lung cancer xenograft models

Most non‐small‐cell lung cancers (NSCLCs) harboring activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR‐TKIs); however, they invariably develop resistance to these drugs. E7820 is an angiogenesis inhibitor that decreases integrin‐α2 expression and is currently undergoing clinical trials. We investigated whether E7820 in combination with erlotinib, an EGFR‐TKI, could overcome EGFR‐TKI‐resistance in the NSCLC cell lines A549 (KRAS; G12S), H1975 (EGFR; L858R/T790M), and H1650 (PTEN; loss, EGFR; exon 19 deletion), which are resistant to erlotinib. Immunohistochemical analysis was carried out in xenografted tumors to investigate anti‐angiogenesis activity and endothelial cell apoptosis levels by endothelial cell marker CD31 and TUNEL staining, respectively. Treatment with E7820 (50 mg/kg) with erlotinib (60 mg/kg) showed a synergistic antitumor effect in three xenograft models. Immunohistochemical analysis indicated that combined treatment with E7820 and erlotinib significantly decreased microvessel density and increased apoptosis of tumor‐associated endothelial cells compared with use of only one of the agents. This combination increased apoptosis in HUVECs through activation of both intrinsic and extrinsic apoptosis pathways in vitro. The combination of E7820 with erlotinib is an alternative strategy to overcome erlotinib resistance in NSCLC by enhancement of the anti‐angiogenic activity of E7820.