非小细胞肺癌靶向治疗和耐药机制研究

发布时间：2018-02-20 01:45

  本文关键词： 非小细胞肺癌 间变淋巴瘤激酶 表皮生长因子受体 靶向治疗 耐药　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibition, EGFR-TKI)和间变淋巴瘤激酶(anaplastic lymphoma kinase, ALK)抑制剂的临床应用显著改善了非小细胞肺癌(non-small cell lung cancer, NSCLC)患者的生存。但最终不可避免的发生耐药。因此,本研究将在晚期NSCLC患者中检测ALK融合基因并分析其临床预后意义,为NSCLC靶向治疗的临床实践提供数据参考。另外,建立EGFR-TKI耐药细胞系,分析其生物学性状,开展组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor, HDACi)或联合EGFR-TKI对NSCLC的抗肿瘤作用研究,为NSCLC治疗新策略的开展提供思路。本论文研究内容分为两个部分：第一部分：晚期非小细胞肺癌中ALK融合基因的检测和临床意义分析第一章：晚期非小细胞肺癌中ALK融合基因的检测。采用FISH、IHC和qRT-PCR技术分别检测139例晚期非鳞NSCLC患者的ALK状态,以FISH检测结果为参考,比较IHC和qRT-PCR检测ALK的敏感度和特异度。结果显示,IHC和FISH检测结果的一致性为96.9%,qRT-PCR和FISH检测结果的一致性为95.7%。IHC检测的敏感性和特异度分别为97.7%和96.6%,qRT-PCR检测的敏感性和特异度分别为89.2%和98.7%。结果提示,在临床实践中IHC技术可作为筛选ALK的有效方法,qRT-PCR技术可作为区分ALK具体变异类型的一种补充诊断手段。第二章：晚期非小细胞肺癌中ALK融合基因临床意义分析。采用FISH.IHC和qRT-PCR技术分别检测173例选择性晚期非鳞NSCLC患者的ALK状态。分析患者的临床病理学特征,基因变异类型和临床预后。结果显示FISH、IHC和qRT-PCR检测ALK的阳性率分别为35.5%(59/166)、35.7%(61/171)和27.9%(34/122)。ALK融合基因阳性更倾向发生于不吸烟或轻度吸烟、年轻、伴有印戒细胞特征、分化差的腺癌患者。ALK FISH阳性服用克唑替尼患者的中位无进展生存时间(progression free survival, PFS)为7.6个月,总生存时间长于未接受克唑替尼治疗和基因突变野生型患者,但与EGFR基因突变接受靶向治疗患者的生存时间无显著差异。与IHC 1+患者相比, IHC 3+/2+患者具有较长的生存时间(P=0.026)。结果提示根据临床病理特征选择性富集NSCLC患者能够提高ALK融合基因阳性检出率,并能高效筛选合适的患者进行ALK抑制剂治疗。ALK蛋白的表达强度对患者治疗方案的选择具有一定的参考意义。第二部分：非小细胞肺癌靶向治疗耐药相关研究第一章：非小细胞肺癌EGFR-TKI耐药细胞系的建立及其生物学性状分析和耐药后治疗新策略探索研究。采用埃克替尼低浓度持续加量法诱导,建立EGFR-TKI耐药NSCLC细胞系。对其生物学性状进行分析,探索其耐药机制及治疗新策略。结果发现成功构建的耐药细胞系其个别生物学性状不同于亲代细胞,对另外两种EGFR-TKI类药物吉非替尼和厄洛替尼呈高度交叉耐药性,MET基因扩增是引起耐药的重要原因。耐药前后两株细胞系对西达本胺均敏感,耐药后细胞对克唑替尼敏感。克唑替尼或联合埃克替尼能够明显抑制耐药细胞的增殖,减弱耐药细胞中MET及其信号通路中相关调配分子的活化程度,阻滞细胞在G2/M期并诱导细胞凋亡,同时能明显抑制HCC827IR细胞裸鼠移植瘤的生长。研究结果为临床EGFR-TKI类药物的应用及耐药后治疗方案的选择提供了实验参考,也为EGFR-TKI耐药机制研究及耐药逆转药物筛选等提供了必要的实验模型。第二章：组蛋白去乙酰化酶抑制剂或联合表皮生长因子受体酪氨酸激酶抑制剂对非小细胞肺癌的抗肿瘤作用研究。体内外实验研究西达苯胺或联合埃克替尼对携有不同遗传背景NSCLC的抗肿瘤作用及其相关分子机制。结果发现西达本胺单药对A549、HCC827、HCC827IR具有明显的增殖抑制作用,联合埃克替尼对H1975细胞具有明显的协同抑制作用。西达本胺单药或联合埃克替尼能抑制细胞中RAS/MAPK、PI3K/AKT和／或JAK/STAT信号通路的活化,阻滞细胞周期,激活caspase家族凋亡相关蛋白表达,诱导细胞凋亡。西达苯胺单药或联合埃克替尼能通过降低HCC827、HCC827IR和H1975细胞中P-catenin的表达而发挥抗肿瘤作用。西达苯胺能够上调H1975细胞中E-cadherin的表达而增强其对埃克替尼的敏感性。另外,西达本胺能够明显抑制HCC827IR细胞裸鼠移植瘤的生长,联合埃克替尼能够明显抑制H1975细胞裸鼠移植瘤的生长,且未发生体重减轻及其他毒副反应。本研究结果将为HDACi在NSCLC中的临床应用提供实验数据参考。以上两部分研究明确了不同检测技术检测ALK融合基因的临床适用性,揭示了ALK融合基因在中国NSCLC患者中的临床病理学特征及预后意义：同时,建立了NSCLC EGFR-TKI耐药细胞模型,分析了其生物学性状及耐药分子机制,并对耐药后治疗新策略进行了初步探索研究；另外,通过体内外实验揭示了西达苯胺或联合埃克替尼对携有不同遗传背景NSCLC的抗肿瘤作用及其相关分子机制。本研究结果能为NSCLC靶向治疗的临床实践和治疗新策略的开展提供思路。
[Abstract]:Epidermal growth factor receptor tyrosine kinase inhibitors (epidermal growth factor receptor tyrosine kinase inhibition, EGFR-TKI) and anaplastic lymphoma kinase (anaplastic lymphoma, kinase, ALK) inhibitors can significantly improve the clinical application of non-small cell lung cancer (non-small cell lung cancer, NSCLC) the survival of patients. But the resistance occurred inevitably. Therefore, this study in patients with advanced NSCLC detection of ALK fusion gene and analyze its clinical prognostic significance for NSCLC targeted therapy in clinical practice to provide reference data. In addition, to establish EGFR-TKI resistant cell line, and analyze its biological characters, development of histone deacetylase inhibitors (histone deacetylase, inhibitor, HDACi) study on the anti tumor effect of NSCLC or joint EGFR-TKI, new treatment strategies for NSCLC provide ideas. This thesis is divided into two parts: the first Part one: detection and clinical significance of ALK fusion gene in small cell lung cancer: analysis of the first chapter of advanced detection of ALK fusion gene non small cell lung cancer in advanced non. Using FISH, ALK were detected in 139 patients with advanced non squamous NSCLC IHC and qRT-PCR technology, to take the FISH results as reference, comparison of IHC and qRT-PCR detection of the sensitivity and specificity of ALK. The results showed that the consistency of IHC and FISH test results for 96.9%, consistency of qRT-PCR and FISH detection results for the sensitivity of 95.7%.IHC was 97.7% and specificity was 96.6%, the sensitivity of qRT-PCR was 89.2% and specificity was 98.7%. results suggest that in clinical practice IHC technology can be used as an effective method for screening ALK, qRT-PCR technology can be used as a supplementary diagnostic tool to distinguish ALK specific variation types. The second chapter: the clinical significance of ALK fusion gene in advanced non small cell lung cancer Semantic analysis. Using FISH.IHC and qRT-PCR techniques were used to detect 173 cases of selective NSCLC in patients with advanced non squamous ALK. Clinicopathologic features of patients with the gene mutation types and clinical prognosis. The results showed that FISH, IHC and qRT-PCR to detect the positive rate of ALK were 35.5% (59/166), 35.7% (61/171) and 27.9% (34/122).ALK fusion gene positive tend to occur in no smoking or smoking, young, with signet ring cell characteristics, poorly differentiated adenocarcinoma in patients with.ALK FISH positive crizotinib patients with median progression free survival time (progression free, survival, PFS) for 7.6 months, the total survival time was longer than that of untreated g with imatinib therapy and gene mutation of wild type patients, but associated with mutations in the EGFR gene targeted therapy the survival time of patients with no significant difference. Compared with IHC 1+ patients, IHC patients with 3+/2+ had longer survival time (P=0.026). According to clinical and pathological features of patients with NSCLC can improve the selective enrichment of ALK fusion gene positive rate, has certain reference significance to choose expression intensity and efficient screening of appropriate patients for treatment with a ALK inhibitor of.ALK protein in cancer treatment. The second part: the target therapy of non-small cell lung cancer drug resistance related research first chapter: new strategy establishment and biological characteristics analysis and treatment of non-small cell lung cancer EGFR-TKI resistant cell line after the study. The icotinib with continuous low concentration by induction, the establishment of EGFR-TKI resistant NSCLC cell line. The biological character of analysis, to explore the mechanism of drug resistance and therapeutic strategies. The results show that the properties of drug resistant cell line was successfully constructed the individual is different from the parent cell biology, on the other two kinds of EGFR-TKI drugs gefitinib and erlotinib are highly cross Cross resistance, MET gene amplification is an important cause of the resistance. The resistance before and after the two cell lines were sensitive to chidamide, resistance to crizotinib sensitive cells. Crizotinib or combined with icotinib could significantly inhibit the proliferation of resistant cells, reduced the level of activation in resistant cells MET and its signal pathway in the deployment of related molecules, cell block in G2/M phase and induce cell apoptosis, and can inhibit HCC827IR cell growth in nude mice. The results provide the experimental reference for clinical application and treatment of drug-resistant EGFR-TKI drugs after the selection, also provides an experimental model for the study of the necessary mechanism of EGFR-TKI resistance and the reversal of drug resistance drug screening. The second chapter: histone deacetylase inhibitors or a combination of epidermal growth factor receptor tyrosine kinase inhibitor on the antitumor effect of non-small cell lung cancer. And the related molecular mechanism of antitumor effects in vivo and in vitro. Combined with aniline or icotinib for carrying different genetic background of NSCLC. The results showed that HCC827 chidamide alone on A549, HCC827IR significantly inhibited the proliferation, combined with Ike could synergistically inhibit the proliferation of H1975 cells in imatinib chidamide. A drug or a combination of icotinib can inhibit RAS/MAPK cell activation, PI3K/AKT and / or JAK/STAT signaling pathway, cell cycle arrest, activate the expression of caspase family proteins related to apoptosis, induce cell apoptosis. Western aniline monotherapy or in combination with icotinib by reducing HCC827, HCC827IR and P-catenin expression in H1975 cells and the anti tumor. Polyaniline can upregulate the expression of E-cadherin. H1975 cells and enhance the sensitivity of icotinib. In addition, chidamide can inhibit HCC827IR Cell growth in nude mice, combined with icotinib can inhibit H1975 cell growth in nude mice, and did not produce weight loss and other side effects. The results of this study will provide experimental data for the clinical application of HDACi in NSCLC. The above two parts of clear clinical applicability to detect ALK detection the technology of fusion gene, reveals the clinical and pathological features of ALK fusion gene in China in patients with NSCLC characteristics and prognostic significance. At the same time, to establish EGFR-TKI resistant NSCLC cell model, analyzed its biological characteristics and molecular mechanisms of drug resistance, and conducted a preliminary exploration and Research on a new strategy for the treatment of drug resistance; in addition, the in vitro and in vivo. And reveal the molecular mechanism of the antitumor effect of icotinib. Aniline or combined with different genetic background of NSCLC. The results of this study for NSCLC targeted therapy Provide ideas for the development of new strategies for clinical practice and treatment.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
，

本文编号：1518469