EGFR-TKI治疗肺腺癌的疗效与血清肿瘤标志物的相关性

目的探讨表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinaseinhibitor,EGFR-TKI)对肺腺癌的疗效和血清肿瘤标志物间的关系。方法 回顾分析48例应用EGFR-TKI治疗的晚期肺腺癌患者的临床特征、生存时间和治疗前血清肿瘤标志物水平的相关性。结果 EGFR-TKI治疗后有效率为58.3%,控制率为65.6%;中位生存时间为13.2月。统计学分析显示:吸烟史、血清CEA和CA19-9水平与EGFR-TKI的疗效相关(P<0.05);治疗前血清CEA,CA19-9水平高者有着更高的治疗有效率、控制率和更长的生存期(P<0.05)。结论 EGFR-TKI治疗前血清CA19-9和CEA的水平对预测EGFR-TRI对肺腺癌患者的疗效有参考价值。     

Exon 16–Skipping HER2 as a Novel Mechanism of Osimertinib Resistance in EGFR L858R/T790M–Positive Non–Small Cell Lung Cancer

IntroductionOsimertinib is the current recommended treatment for EGFR T790M–positive NSCLC after EGFR tyrosine kinase inhibitor therapy. However, resistance to osimertinib therapy is inevitably acquired after a period of effective treatment. We had a patient with EGFR L858R/T790M–positive NSCLC who initially responded to osimertinib therapy but eventually experienced development of resistance. Plasma cell–free DNA analysis revealed the occurrence of exon 16–skipping HER2, which may have resulted in the erb-b2 receptor tyrosine kinase 2 gene (HER2) splice variant HER2D16. HER2D16 has never been reported in lung cancer, and HER2D16-driven signaling is known to be regulated by Src kinase in breast cancer. We investigated the role of HER2D16 as an osimertinib-resistant mechanism.MethodsWe constructed and established H1975 cells stably expressing HER2D16. The dimeric formation of HER2D16 was tested by using nonreducing polyacrylamide gel electrophoresis. The effects of the study drugs on signaling transduction were examined by using Western blot. Synergistic effect was assessed by using the Chou-Talalay method.ResultsWe found that HER2D16 can form a homodimer in NSCLC cells. HER2D16-expressing H1975 cells were resistant to osimertinib treatment. We also found that mutant EGFR and HER2D16 cooperated to activate downstream signaling for osimertinib resistance. In addition, cotreatment with osimertinib and an Src kinase inhibitor failed to reverse resistance, indicating that HER2D16-driven signaling in NSCLC did not occur through a canonical pathway. Finally, we revealed that the combination of osimertinib with the pan-HER small-molecule inhibitor afatinib could synergistically repress cell growth and signaling in H1975-HER2D16 cells.ConclusionHER2D16 can contribute to osimertinib resistance through an Src-independent pathway. HER2D16 should be included in the molecular diagnosis panel for lung cancer.     

EGFR and ERBB2 Germline Mutations in Chinese Lung Cancer Patients and Their Roles in Genetic Susceptibility to Cancer

Introduction

Inherited genetic determinants of lung cancer risk remain relatively elusive. Germline mutations in EGFR and erb-b2 receptor tyrosine kinase 2 (ERBB2) have been previously reported in lung cancers that may be associated with genetic susceptibility to lung cancer.

Epithelial phenotype as a predictive marker for response to EGFR‐TKIs in non‐small cell lung cancer patients with wild‐type EGFR

Epithelial‐to‐mesenchymal transition (EMT) has profound impacts on cancer progression and also on drug resistance, including epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs). Nowadays, there is still no predictive biomarker identified for the use of EGFR‐TKIs in non‐small cell lung cancer (NSCLC) patients with wild‐type EGFR. To clarify the role of EMT phenotype as a predictive marker for EGFR‐TKI, we performed a retrospective study in 202 stage IV or recurrent NSCLC patients receiving gefitinib or erlotinib therapy from June 2008 to September 2012 in our institute. Clinical data and EGFR mutational status were collected, while epithelial, epithelial to mesenchymal, not specified or mesenchymal phenotype were classified according to EMT markers such as E‐cadherin, fibronectin, N‐cadherin and vimentin by immunohistochemistry. Epithelial phenotype was more frequently found in patients with EGFR mutation (p = 0.044). Epithelial phenotype was associated with a significantly higher objective response rate (23.5 vs. 11.1 vs. 0.0 vs. 2.4%, p = 0.011), longer progression‐free survival (4.4 vs. 1.9 vs. 1.7 vs. 1.0 months, p < 0.001) and longer overall survival (11.5 vs. 8.9 vs. 4.5 vs. 4.9 months, p < 0.001) compared to epithelial to mesenchymal, not specified and mesenchymal phenotype in the wild‐type EGFR subgroup. In the subgroup with EGFR mutation, the trend remained but without a statistically significant difference. In conclusion, epithelial phenotype was more likely expressed in patients with EGFR mutation and was associated with a better outcome in advanced NSCLC patients with wild‐type EGFR, which indicates that the EMT phenotype might be a potential marker to guide EGFR‐TKI therapy in this population.     

Innovative Therapies for Children with Cancer pediatric phase I study of erlotinib in brainstem glioma and relapsing/refractory brain tumors

This multicenter phase I study aimed to establish the recommended dose (RD) of the epidermal growth factor receptor (EGFR) inhibitor erlotinib, given as monotherapy or with radiotherapy to children with malignant brain tumors. Group 1 included patients with refractory or relapsing brain tumors receiving erlotinib alone, and group 2 included newly diagnosed patients with brainstem gliomas receiving radiotherapy and erlotinib. A conventional 3 + 3 dose escalation and a continual reassessment method, respectively, were utilized in 4 dose levels: 75, 100, 125, and 150 mg/m2 per day. Fifty-one children were enrolled (30 and 21, respectively); 50 received treatment. The RD of erlotinib was 125 mg/m2 per day as monotherapy or in combination with radiotherapy. Overall, 230 adverse events in 44 patients were possibly treatment related (216, grades 1 and 2; 9, grade 3; 1, grade 4; 4, grade 5). Dermatologic and neurologic symptoms were common; intratumoral hemorrhage was confirmed in 3 patients. In group 1, 8 of 29 patients (28%) had stable disease with tumor regression approaching 50% in a malignant glioma and an anaplastic oligoastrocytoma. In group 2, overall survival was 12.0 months. EGFR overexpression by immunohistochemistry was found in 17 of 38 (45%) tumor samples analyzed, with a partial gain of 7p11.2 in 1 glioblastoma; phosphate and tensin homolog loss was frequent in brainstem glioma (15 of 19). Mean (95% CI) apparent clearance and volume of distribution for erlotinib were 4.0 L/h (3.4-4.5 L/h) and 98.6 L (69.8-127.0 L), respectively, and were independent of the dose level; mean half-life was 16.6 hours. Thus, erlotinib 125 mg/m2 per day has an acceptable tolerability profile in pediatric patients with brain tumors and can be combined with radiotherapy.     

Study of the Correlation of EGFR and K-RAS Gene Mutations with Its Protein Expression in Non-small Cell Lung Cancer

OBJECTIVE To investigate gene mutations of epidermal growth factor receptor (EGFR) and K-RAS (Kirsten rat sarcoma viral oncogene) in Chinese patients with non-small cell lung cancer (NSCLC), and study the correlation with its protein expression and its clinical significance on gefitinib. METHODS Detect the EGFR and K-RAS gene mutations status by gene sequencing and use the method of Immunohistochemistry to detect EGFR and K-RAS protein expression. RESULTS The frequency of EGFR mutations was 33%, mainly located in exon 19 and exon 21. The frequency of K-RAS mutations was 5.5%, mainly located in codon 12. There was no case which both had EGFR and K-RAS mutations, suggesting a mutually exclusive relationship between the two. EGFR mutations are more common in adenocarcinomas (particularly those with bronchioloalveolar features), nonsmokers and females. 16% were detected EGFR positive expression and had no correlation with EGFR mutation (P > 0.05), but had significant correlation with mutation in exon 19 (P < 0.05). The frequency of K-RAS positive expression was 52.5% and had no correlation with K-RAS mutation (P > 0.05). Twelve (8 cases were protein-negative) out of 15 gefitinib-treated NSCLC patients with disease control carry EGFR mutations. CONCLUSION EGFR protein expression has some correlation with exon 19 mutations. Combined detection of EGFR and K-RAS gene mutations can help clinicians to choose patients who may benefit from EGFR tyrosine kinase inhibitor (EGFR-TKI) and to predict the response and prognosis of gefitinib.     

大肠癌细胞FoxQ1与EGFR基因表达的相关性

目的 探讨大肠癌细胞中FoxQ1与EGFR基因间的相关性,为研究大肠癌中FoxQ1基因在EGFR通路中的作用机制奠定基础。 方法 应用荧光定量PCR以293-T细胞中FoxQ1及EGFR基因相对表达量为1作为参照,检测大肠癌细胞系DLD1、HT29、LOVO、HCT116中FoxQ1及EGFR基因mRNA相对表达量;荧光定量检测经shRNA-FoxQ1慢病毒干扰后的DLD1细胞（命名为DLD1-shRNAFoxQ1）中EGFR的相对表达量改变;DLD1-shRNA-FoxQ1经EGFR酪氨酸激酶抑制剂Erlotinib HCl和siRNA-EGFR处理后,荧光定量PCR分别检测FoxQ1和EGFR基因mRNA相对表达量。结果 （1）FoxQ1在DLD1、HT29、LOVO、HCT116细胞系中的相对表达量分别为83.09、59.58、0.06、0.03,EGFR的相对表达量分别为4.95、3.67、2.08、1.36;（2）经shRNA-FoxQ1干扰的DLD1 细胞EGFR表达量随FoxQ1表达量的降低而增高;（3）细胞DLD1-shRNA-FoxQ1、DLD1-shRNA-Control分别经 siRNA-EGFR处理抑制EGFR的表达后,FoxQ1表达量随EGFR表达量的降低而增高,经Erlotinib HCl阻断EGFR酪氨酸激酶后,FoxQ1表达量增高。结论 大肠癌细胞系中FoxQ1与EGFR基因的表达趋势基本一致;同时两者间可能相互存在负反馈调节机制,从而维持大肠癌细胞中FoxQ1与EGFR高表达的状态。     

Phase I first-in-human study of TAK-285, a novel investigational dual HER2/EGFR inhibitor, in cancer patients

Background:

This phase I first-in-human study was conducted in Japanese patients to investigate the safety, pharmacokinetics (PKs), and determine the maximum tolerated dose (MTD) of oral TAK-285, a novel dual erbB protein kinase inhibitor that specifically targets human epidermal growth factor receptor (EGFR) and HER2.

Methods:

The TAK-285 dose was escalated until MTD was determined. A second patient cohort received TAK-285 at the MTD for at least 4 weeks.

Results:

In all, 26 patients received TAK-285 at doses ranging from 50 to 400 mg once daily (q.d.) or twice daily (b.i.d.); 20 patients made up the dose escalation cohort and the remaining 6 patients were the repeated administration cohort. TAK-285 was well tolerated. Dose-limiting toxicities noted in two patients who received 400 mg b.i.d. were grade 3 increases in aminotransferases and grade 3 decreased appetite. Consequently, the MTD was determined to be 300 mg b.i.d. Absorption of TAK-285 was rapid after oral dosing, and plasma exposure at steady-state increased in a dose-proportional fashion for doses ranging from 50 to 300 mg b.i.d. A partial response was observed for one patient with parotid cancer who received 300 mg b.i.d.

Conclusion:

The toxicity profile and PK properties of oral TAK-285 warrant further evaluation.     

A phase I dose escalation study of BIBW 2992, an irreversible dual inhibitor of epidermal growth factor receptor 1 (EGFR) and 2 (HER2) tyrosine kinase in a 2-week on, 2-week off schedule in patients with advanced solid tumours

To assess tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and clinical activity of the dual epidermal growth factor receptor (EGFR) 1 and 2 (HER2) tyrosine kinase inhibitor BIBW 2992. An escalating schedule of once-daily (OD) BIBW 2992 for 14 days followed by 14 days off medication was explored. Thirty-eight patients were enrolled. Dose levels were 10, 20, 30, 45, 70, 85, and 100 mg. At 100 mg dose-limiting toxicity (DLT) (common toxicity criteria grade 3 skin rash and grade 3 diarrhoea despite treatment with loperamide) occurred in two patients. In the next-lower dose of 70 mg, DLT (grade 3 fatigue and ALAT elevation) occurred in one of six patients. An intermediate dose level of 85 mg was studied. Here DLT occurred in two patients (grade 3 diarrhoea despite treatment and grade 2 diarrhoea lasting more than 7 days despite treatment). An additional 12 patients were treated at 70 mg. BIBW 2992 PK after single and multiple doses revealed moderately fast absorption, and no deviation from dose proportionality. Pharmacodynamics analysis in skin biopsies did not show significant changes in EGFR-associated biomarkers. However, a significant inhibitory effect on the proliferation index of epidermal keratinocytes was observed. No partial or complete responses were observed, stable disease lasting more than four cycles was seen in seven patients. The recommended dose for studies with BIBW 2992 for 14 days followed by 14 days off medication is 70 mg OD.     

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.     

Induction of cell cycle arrest and apoptosis in human nasopharyngeal carcinoma cells by ZD6474, an inhibitor of VEGFR tyrosine kinase with additional activity against EGFR tyrosine kinase

ZD6474 is a vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. The present study was undertaken to investigate the direct antiproliferative effect of ZD6474 on human nasopharyngeal carcinoma (NPC) in vitro and the antitumor activity on NPC xenografts in vivo. Results indicated that ZD6474 treatment inhibited EGFR phosphorylation and led to a dose- and time-dependent decrease in NPC cell (CNE-1, CNE-2 and C666-1) proliferation. Further investigation demonstrated G0/G1 cell cycle arrest in all 3 cell lines, which was associated with an upregulation of p21 and/or p27, and downregulation of CDK4, CDK6 and CDK2. ZD6474 treatment also induced apoptosis in CNE-1 and CNE-2 cells. The apoptosis mechanisms involved reduction of Bcl-2 and/or Bcl-XL, induction of Bak and/or Bax, and activation of caspases-3, -9 and/or -8. The in vivo antitumor activity was evaluated in CNE-2 and C666-1 xenografted nude mice. Administration of ZD6474 (25-100 mg/kg/day, once-daily, p.o.) produced a dose-dependent inhibition of tumor growth and prolonged survival in both models. This study suggests that ZD6474 exerts direct antiproliferative effects on NPC cell lines in vitro by inducing G0/G1 arrest and apoptosis, and potent antitumor effects on NPC xenografts in vivo. It indicates that ZD6474 may offer a new and effective treatment for human NPC.     

Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK rearranged lung cancer

Introduction

The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK.

Methods

We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 μM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases.

Results

All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as “bypass” tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib + erlotinib (reversible EGFR TKI) and crizotinib + afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib.

Conclusions

We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.