本文關(guān)鍵詞： 非小細(xì)胞肺癌 間變淋巴瘤激酶 表皮生長因子受體 靶向治療 耐藥　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：表皮生長因子受體酪氨酸激酶抑制劑(epidermal growth factor receptor tyrosine kinase inhibition, EGFR-TKI)和間變淋巴瘤激酶(anaplastic lymphoma kinase, ALK)抑制劑的臨床應(yīng)用顯著改善了非小細(xì)胞肺癌(non-small cell lung cancer, NSCLC)患者的生存。但最終不可避免的發(fā)生耐藥。因此,本研究將在晚期NSCLC患者中檢測ALK融合基因并分析其臨床預(yù)后意義,為NSCLC靶向治療的臨床實(shí)踐提供數(shù)據(jù)參考。另外,建立EGFR-TKI耐藥細(xì)胞系,分析其生物學(xué)性狀,開展組蛋白去乙�；敢种苿�(histone deacetylase inhibitor, HDACi)或聯(lián)合EGFR-TKI對NSCLC的抗腫瘤作用研究,為NSCLC治療新策略的開展提供思路。本論文研究內(nèi)容分為兩個(gè)部分：第一部分：晚期非小細(xì)胞肺癌中ALK融合基因的檢測和臨床意義分析第一章：晚期非小細(xì)胞肺癌中ALK融合基因的檢測。采用FISH、IHC和qRT-PCR技術(shù)分別檢測139例晚期非鱗NSCLC患者的ALK狀態(tài),以FISH檢測結(jié)果為參考,比較IHC和qRT-PCR檢測ALK的敏感度和特異度。結(jié)果顯示,IHC和FISH檢測結(jié)果的一致性為96.9%,qRT-PCR和FISH檢測結(jié)果的一致性為95.7%。IHC檢測的敏感性和特異度分別為97.7%和96.6%,qRT-PCR檢測的敏感性和特異度分別為89.2%和98.7%。結(jié)果提示,在臨床實(shí)踐中IHC技術(shù)可作為篩選ALK的有效方法,qRT-PCR技術(shù)可作為區(qū)分ALK具體變異類型的一種補(bǔ)充診斷手段。第二章：晚期非小細(xì)胞肺癌中ALK融合基因臨床意義分析。采用FISH.IHC和qRT-PCR技術(shù)分別檢測173例選擇性晚期非鱗NSCLC患者的ALK狀態(tài)。分析患者的臨床病理學(xué)特征,基因變異類型和臨床預(yù)后。結(jié)果顯示FISH、IHC和qRT-PCR檢測ALK的陽性率分別為35.5%(59/166)、35.7%(61/171)和27.9%(34/122)。ALK融合基因陽性更傾向發(fā)生于不吸煙或輕度吸煙、年輕、伴有印戒細(xì)胞特征、分化差的腺癌患者。ALK FISH陽性服用克唑替尼患者的中位無進(jìn)展生存時(shí)間(progression free survival, PFS)為7.6個(gè)月,總生存時(shí)間長于未接受克唑替尼治療和基因突變野生型患者,但與EGFR基因突變接受靶向治療患者的生存時(shí)間無顯著差異。與IHC 1+患者相比, IHC 3+/2+患者具有較長的生存時(shí)間(P=0.026)。結(jié)果提示根據(jù)臨床病理特征選擇性富集NSCLC患者能夠提高ALK融合基因陽性檢出率,并能高效篩選合適的患者進(jìn)行ALK抑制劑治療。ALK蛋白的表達(dá)強(qiáng)度對患者治療方案的選擇具有一定的參考意義。第二部分：非小細(xì)胞肺癌靶向治療耐藥相關(guān)研究第一章：非小細(xì)胞肺癌EGFR-TKI耐藥細(xì)胞系的建立及其生物學(xué)性狀分析和耐藥后治療新策略探索研究。采用�？颂婺岬蜐舛瘸掷m(xù)加量法誘導(dǎo),建立EGFR-TKI耐藥NSCLC細(xì)胞系。對其生物學(xué)性狀進(jìn)行分析,探索其耐藥機(jī)制及治療新策略。結(jié)果發(fā)現(xiàn)成功構(gòu)建的耐藥細(xì)胞系其個(gè)別生物學(xué)性狀不同于親代細(xì)胞,對另外兩種EGFR-TKI類藥物吉非替尼和厄洛替尼呈高度交叉耐藥性,MET基因擴(kuò)增是引起耐藥的重要原因。耐藥前后兩株細(xì)胞系對西達(dá)本胺均敏感,耐藥后細(xì)胞對克唑替尼敏感�？诉蛱婺峄蚵�(lián)合�？颂婺崮軌蛎黠@抑制耐藥細(xì)胞的增殖,減弱耐藥細(xì)胞中MET及其信號通路中相關(guān)調(diào)配分子的活化程度,阻滯細(xì)胞在G2/M期并誘導(dǎo)細(xì)胞凋亡,同時(shí)能明顯抑制HCC827IR細(xì)胞裸鼠移植瘤的生長。研究結(jié)果為臨床EGFR-TKI類藥物的應(yīng)用及耐藥后治療方案的選擇提供了實(shí)驗(yàn)參考,也為EGFR-TKI耐藥機(jī)制研究及耐藥逆轉(zhuǎn)藥物篩選等提供了必要的實(shí)驗(yàn)?zāi)Ｐ�。第二章：組蛋白去乙�；敢种苿┗蚵�(lián)合表皮生長因子受體酪氨酸激酶抑制劑對非小細(xì)胞肺癌的抗腫瘤作用研究。體內(nèi)外實(shí)驗(yàn)研究西達(dá)苯胺或聯(lián)合埃克替尼對攜有不同遺傳背景NSCLC的抗腫瘤作用及其相關(guān)分子機(jī)制。結(jié)果發(fā)現(xiàn)西達(dá)本胺單藥對A549、HCC827、HCC827IR具有明顯的增殖抑制作用,聯(lián)合�？颂婺釋1975細(xì)胞具有明顯的協(xié)同抑制作用。西達(dá)本胺單藥或聯(lián)合埃克替尼能抑制細(xì)胞中RAS/MAPK、PI3K/AKT和／或JAK/STAT信號通路的活化,阻滯細(xì)胞周期,激活caspase家族凋亡相關(guān)蛋白表達(dá),誘導(dǎo)細(xì)胞凋亡。西達(dá)苯胺單藥或聯(lián)合埃克替尼能通過降低HCC827、HCC827IR和H1975細(xì)胞中P-catenin的表達(dá)而發(fā)揮抗腫瘤作用。西達(dá)苯胺能夠上調(diào)H1975細(xì)胞中E-cadherin的表達(dá)而增強(qiáng)其對�？颂婺岬拿舾行浴Ａ硗�,西達(dá)本胺能夠明顯抑制HCC827IR細(xì)胞裸鼠移植瘤的生長,聯(lián)合�？颂婺崮軌蛎黠@抑制H1975細(xì)胞裸鼠移植瘤的生長,且未發(fā)生體重減輕及其他毒副反應(yīng)。本研究結(jié)果將為HDACi在NSCLC中的臨床應(yīng)用提供實(shí)驗(yàn)數(shù)據(jù)參考。以上兩部分研究明確了不同檢測技術(shù)檢測ALK融合基因的臨床適用性,揭示了ALK融合基因在中國NSCLC患者中的臨床病理學(xué)特征及預(yù)后意義：同時(shí),建立了NSCLC EGFR-TKI耐藥細(xì)胞模型,分析了其生物學(xué)性狀及耐藥分子機(jī)制,并對耐藥后治療新策略進(jìn)行了初步探索研究；另外,通過體內(nèi)外實(shí)驗(yàn)揭示了西達(dá)苯胺或聯(lián)合埃克替尼對攜有不同遺傳背景NSCLC的抗腫瘤作用及其相關(guān)分子機(jī)制。本研究結(jié)果能為NSCLC靶向治療的臨床實(shí)踐和治療新策略的開展提供思路。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R734.2
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本文編號：1518469