Lentivirus-mediated silencing of CNTN1 enhances gefitinib sensitivity by reversing epithelial-mesenchymal transition in lung adenocarcinoma A549 cells

Contactin-1 (CNTN1), a neuronal cell adhesion molecular, functions in nervous system development and has been associated with carcinogenesis and tumor progression. To investigate the role of CNTN1 in gefitinib resistance in lung adenocarcinoma, lentivirus-mediated short hairpin (sh)RNA was used to silence CNTN1 and its physiological function was analyzed in the A549 cell line. A cell cytotoxicity assay revealed that CNTN1 knockdown enhanced gefitinib sensitivity in the A549 cells. In addition, CNTN1 knockdown, together with gefitinib treatment, resulted in a significant inhibition of colony formation and migration, and promotion of apoptosis. Furthermore, CNTN1 knockdown also reversed the epithelial-mesenchymal transition (EMT) phenotype by increasing E-cadherin protein expression level, and decreasing N-cadherin and vimentin protein expression levels. The PI3K/Akt signaling pathway was also association with the effects of CNTN1 on EMT progression and gefitinib resistance in the A549 cells. Collectively, knockdown of CNTN1 reversed the EMT phenotype and enhanced gefitinib sensitivity in the A549 cells by inhibiting the activation of the PI3K/Akt signaling pathway. These results suggested that CNTN1 may represent a potential therapeutic target for reserving EGFR-tyrosine kinase inhibitor resistance in non-small cell lung cancer.     

Profile of the therascreen® EGFR RGQ PCR kit as a companion diagnostic for gefitinib in non-small cell lung cancer

Introduction: Gefitinib is recently approved by the US Food and Drug Administration as a first-line treatment for non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. The therascreen® EGFR RGQ PCR Kit is approved as a companion diagnostic to select patients with EGFR exon 19 deletions and L858R mutation for treatment with gefitinib.

Areas covered: This article reviews the methods for detecting EGFR mutations, the technology and indication of the therascreen® kit, and the clinical utility of the assay in phase 3 and phase 4 clinical trials. Studies that compared the performance of the therascreen® kit with other assays and assessed the kit’s application in non-tissue samples are also discussed.

Expert commentary: The therascreen® kit is a highly sensitive real-time polymerase chain reaction test that provides standardised testing and automated interpretation of EGFR mutation status in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Although not indicated for these applications, the test has also shown utility in detecting uncommon sensitizing EGFR mutations as well as mutations in non-tissue samples.     

The influence of autologous cytokine-induced killer cell treatment on the objective efficacy and safety of gefitinib in advanced non-small cell lung cancer

Objective The aim of the study was to observe the influence of autologous cytokine-induced killer cell(CIK) treatment on the objective efficacy and safety of gefitinib in advanced non-small cell lung cancer(NSCLC).Methods Sixty-six patients with NSCLC received gefitinib as second-line treatment. They were randomly divided into 2 groups, and informed consent forms were signed before grouping. Gefitinib was administrated to the control group, and autologous CIK treatment was added to the observation group. The objective treatment and adverse reactions were evaluated in both groups. Results The objective response rate(ORR) and the disease control rate(DCR) of the observation group were slightly higher than those of the control group, although no statistical differences were found between the 2 groups(P > 0.05). The incidences of diarrhea, fatigue, anorexia, oral ulcers, and myelosuppression in the observation group were much lower than those in the control group(P < 0.05). However, there were no statistical differences between the incidences of skin rash, and liver and kidney toxicities(P > 0.05). Conclusion Autologous CIK in combination with gefitinib is effective as second-line treatment for advanced NSCLC, and can significantly reduce adverse reactions and improve the objective efficacy.     

Circulating miRNAs is a potential marker for gefitinib sensitivity and correlation with EGFR mutational status in human lung cancers

miRNA expression is deregulated in non-small cell lung cancer (NSCLC), and some miRNAs are associated with gefitinib sensitivity. Here, we investigated if circulating miRNAs could be a useful biomarker for the prediction of EGFR mutation and the patient’s prognosis. The differential miRNAs related to gefitinib sensitivity were screened and identified by microRNA array. Using Taqman-based real-time RT-PCR, we analyzed the expression of selected miRNAs in tumor tissues and plasma of 150 NSCLC patients. Kaplan-Meier survival analysis and Cox proportional hazards regression were used to determine the association between miRNAs expression and survival. Receiver operating characteristic curve analysis was also performed. Compared with PC9 cell line, 41 microRNAs detected by microarray were significantly differentially expressed in A549 and H1299 cells. The 5 selected hsa-miRNAs were all found differently expressed between wild and mutant EGFR carriers (all P<0.01). Down-regulation of 5 selected miRNAs were independently associated with lymphatic invasion (all P<0.01) and clinical stage (all P<0.01), respectively. Both down-regulation of has-miR-195 (P=0.012) and has-miR-21 (P=0.004) were associated with poor differentiation. All up-regulation of 5 has-miRNAs were associated with smoking (All P<0.05). 5 hsa-miRNAs were up-regulated both in plasma and tissue samples. A model including 4 hsa-miRNAs may predict EGFR mutational status and gefitinib-sensitivity (both AUC: 0.869). Plasma levels of has-miR-125b expression were associated with disease-free survival (P=0.033) and overall survival in the patients (P=0.028). In a word, Circulating 5 selected miRNAs may especially be useful in predicting EGFR mutation, and circulating hsa-miR-125b may have prognostic values in NSCLC patients.     

参苓白术颗粒联合吉非替尼对晚期非小细胞肺癌的治疗作用

目的观察参苓白术颗粒联合吉非替尼对晚期非小细胞肺癌(NSCLC)的治疗效果。方法选取经病理组织学或细胞学确诊为NSCLC患者40例,随机分为治疗组及对照组。治疗组患者给予参苓白术颗粒口服,6 g/次,3次/d,吉非替尼250 mg/d口服;对照组仅给予吉非替尼250 mg/d口服。2组均连续治疗21 d为1个化疗周期,至少治疗3个周期,对比2组患者近期疗效、生活质量、中医症状改善及T细胞亚群变化。结果 2组近期疗效比较无统计学意义(P0.05);治疗组治疗后生活质量KPS评分改善,中医证候临床疗效显著高于对照组(P0.05),治疗组治疗后CD4较治疗前升高(P0.05)。结论参苓白术颗粒联合吉非替尼可显著改善晚期NSCLC患者生活质量,调节免疫功能。     

Gefitinib in advanced non-small cell lung cancer

BACKGROUND: Gefitinib is an oral, selective epidermal growth factor receptor (EGFR) inhibitor that has activity in non-small cell lung cancer (NSCLC). AIM: To evaluate the tolerability, safety-profile and response of single agent gefitinib in patients with advanced stage NSCLC. METHODS: Twenty-seven patients of good performance status with stage IIIB or IV NSCLC were entered on the study at the Sydney Cancer Centre. Gefitinib was prescribed at an oral dose of 250 mg daily, as a continuous dose. Radiological evaluation of indicator lesions occurred at baseline and were repeated every 2-3 months until disease progression. Toxicity was graded using standard measures at baseline and at every month. RESULTS: The response rate was 17% in the patients eligible for evaluation. Symptom improvement was observed in 75% of patients. No patients withdrew because of adverse events. Toxicity was observed in 15 patients and consisted mainly of rash (59%), which was usually mild in severity. CONCLUSION: Gefitinib is active in NSCLC. It is well tolerated with minimal side-effects. Symptomatic improvement was found in the majority of patients treated with gefitinib. There may be a role for gefitinib in the palliation of symptoms in patients with advanced NSCLC.     

吉非替尼治疗老年晚期非小细胞肺癌57例临床分析

目的观察表皮生长因子受体酪氨酸激酶抑制剂吉非替尼单药治疗老年晚期非小细胞肺癌的疗效和毒副作用。方法2003年9月至2006年11月期间，57例65岁以上老年晚期非小细胞肺癌患者，单药口服吉非替尼250mg 1次／d。应用方差分析、t检验、Kaplan-Meier进行统计分析。结果男31例，女26例；年龄65～86岁，中位年龄71岁；Ⅲ期10例，Ⅳ期47例。近期疗效：完全缓解（CR）1例（1．8％），部分缓解（PR）14例（25．5％），有效率（CR＋PR）27．3％；疾病稳定（SD）24例（43．6％），疾病控制率（CR＋PR＋SD）70．9％；疾病进展16例（29．1％）；症状改善率63．6％，症状改善中位时间为8d（3-17d），症状改善持续中位时间为5．8个月；中位疾病进展时间5．3个月（27d至24个月），中位生存期7．8个月（18d至34个月），1年生存率33％。主要不良反应为皮疹42．1％、腹泻31．6％，大部分腹泻患者为轻度，经对症处理后可缓解，仅1例患者因腹泻停药。皮肤瘙痒4例（7．3％），皮肤干燥4例（7．3％），口腔疼痛伴溃疡1例（1．8％），还有1例（1．8％）患者因出现双足皮肤干燥、脱皮和趾甲皲裂而影响行走。结论吉非替尼单药治疗老年晚期非小细胞肺癌疗效肯定，毒副作用相对较小，患者耐受性好。     

Binding of gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase, to plasma proteins and blood cells: in vitro and in cancer patients

Summary Gefitinib exhibits wide inter-subject pharmacokinetic variability which may contribute to differences in treatment outcome. Unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug. Thus it is desirable to determine gefitinib binding in plasma and factors affecting this process. An equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (fu) gefitinib in human plasma. Gefitinib binding in plasma from four different species and isolated protein solutions as well as drug partitioning in human blood cells were investigated. Unbound gefitinib plasma concentrations were measured in 21 cancer patients receiving daily oral gefitinib 250 mg or 500 mg. It was found that gefitinib was extensively bound in human rat mouse and dog plasma with mean fu values of 3.4%, 3.8%, 5.1% and 6.0% respectively. In isolated protein solutions approximately 90% and 78% of gefitinib was bound to human serum albumin (HSA) (40 mg/dL) and alpha1-acid glycoprotein (AAG) (1.4 mg/dL) with binding constants of 1.85 × 10⁴ M⁻¹ and 1.13 × 10⁵ M⁻¹ respectively. In whole blood 2.8% of gefitinib existed as the free drug while 79.4% and 17.8% was bound to plasma proteins and blood cells respectively. In plasma from cancer patients fu at pre-treatment varied 2.4-fold (mean 3.4 ± 0.6%; range 2.2–5.4%) and fu was constant over the 28-days of treatment (P > 0.05). Pre-treatment AAG concentration was negatively correlated with pre-treatment fu (R² = 0.28, P = 0.01). In conclusion gefitinib is highly protein bound (∼ 97%) in human plasma. Variable AAG concentrations observed in cancer patients may affect gefitinib fu with implications for inter-subject variation in drug toxicity and response.     

Epidermal growth factor receptor dimerization status determines skin toxicity to HER-kinase targeted therapies

Skin toxicity, a common drug-related adverse event observed in cancer patients treated with epidermal growth factor receptor (EGFR)-directed therapies is rarely seen with therapies targeting HER2. This study reports the significance of the EGFR and HER2 dimerization status in skin with regard to these dermatologic side effects. We demonstrate the differential effect of HER-directed therapies on the ligand driven activation status of EGFR, HER2 and MAPK in normal human epidermal keratinocytes. EGFR-directed therapies, such as gefitinib and cetuximab, inhibited ligand-induced activation of EGFR and MAPK in human keratinocytes. Pertuzumab, an antibody interfering with functional HER2 heterodimerization, failed to block ligand-induced HER signaling in primary keratinocytes. Using a novel proximity-based dimerization assay (eTagtrade mark) we show that EGFR homodimers are the predominant HER dimer pair in normal primary kertinocytes and in normal skin tissue from 16 patients with solid malignancies. The presence of [p]EGFR and [p]MAPK, but the absence of [p]HER2, demonstrates productive signaling via EGFR but not HER2 in human skin. These data illustrate the importance of the EGFR dimerization partner in human skin and suggests that inhibition of EGFR homodimer signaling rather than EGFR/HER2 heterodimer signaling maybe the key molecular event determining dermatologic toxicity discrepancies observed between EGFR-targeted versus HER2-targeted therapies.