EGFR扩增亚型诱导三代EGFR酪氨酸激酶抑制剂耐药的机制及逆转耐药研究

Although the 1st, 2nd and 3rd generation drugs targeted for epidermal growth factor receptor (EGFR) have excellent efficacy in lung cancer, they will eventually become resistant. The resistance mechanism of 1st and 2nd generation drugs is mainly T790M mutation, which of the 3rd generation is diverse, including C797S mutation, c-met amplification, T790M deletion, EGFR amplification and BRAF mutation. Thus, explaining resistance mechanism of the 3rd generation drug and formulating corresponding strategies to overcome drug resistance are unanswered questions. Our pilot study found that EGFR amplification not only plays an important role in 3rd generation targeted drug resistance, but also that there are different subtypes of EGFR amplification, and different subtypes play different roles in drug resistance. This project will construct three different Lentivirus vectors for EGFR amplification (wild-type amplification, sensitive mutation amplification, and sensitive accompanied with resistance mutant amplification), as well as establish and screen for cell lines overexpressing different EGFR subtypes. Not only verify the role of each EGFR amplification subtype in inducing drug resistance of 3rd generation EGFR-TKI respectively, but also further explored the feasibility of three different reversal resistance strategies through in vitro models and animal models. By exploring the pattern and timing of overcoming resistance, we would be able to explain the mechanism of EGFR amplification subtypes in inducing 3rd generation EGFR-TKI resistance and strategies of overcoming drug resistance. This work will provide us a theoretical basis for formulating strategies to overcome drug resistance in clinical practice, and promote the concept of accurately target therapy known as “treatment based on type” to accurately overcoming drug resistance, which we called “treatment based on subtype”, and eventually achieving full-scale management of precise targeted therapies.

针对表皮生长因子受体（EGFR）的一代、二代和三代靶向治疗药物在肺癌中均疗效卓越但终将耐药，一代和二代药物的耐药机制主要集中于T790M突变，而三代药物的耐药机制则呈现高度异质性，阐明其耐药机制并制订逆转耐药策略是亟待解决的问题。本课题组前期采用二代测序技术勾画出三代靶向药物艾维替尼的耐药变异图谱，EGFR扩增占整体图谱中的27%，并发现，EGFR扩增存在三种不同的亚型，不同亚型诱导耐药的机制及逆转耐药策略各有差异。本课题将构建三种EGFR扩增亚型的慢病毒载体，建立并筛选出不同亚型稳定过表达的细胞株，分别检测三种EGFR扩增亚型诱导耐药的作用，并进一步通过体外模型和动物模型探索三种逆转耐药策略的可行性，确定逆转耐药的方式和时机，从而阐明EGFR扩增亚型诱导三代药物耐药的机制及逆转耐药策略，为临床制订克服耐药策略提供分子依据，将“分型而治”的精准医学理念延伸到“分亚型而治“的领域中。

第三代EGFR-TKIs有效地克服了既往EGFR-TKIs耐药后产生的T790M突变，但终将耐药。本研究前期研究勾画出三代EGFR-TKIs的耐药变异图谱，发现EGFR扩增诱导三代EGFR-TKIs产生耐药且EGFR扩增存在不同的亚型。本课题探讨介导EGFR-TKIs耐药后的EGFR扩展亚型、对疗效的影响程度以及克服耐药策略，为临床制订克服耐药策略提供分子依据，使精准靶向治疗的全程化管理得到进一步的延伸。.1、第三代EGFR-TKIs在晚期EGFR T790M突变患者中独特的耐药基因谱：前期研究通过二代测序技术勾画出三代靶向药物耐药变异图谱以及EGFR扩增的独特特征、不同亚型和治疗价值。.2、EGFR扩增介导第三代EGFR-TKIs耐药的体外功能学验证及克服耐药策略探索：通过慢病毒转染构建不同的细胞亚型，发现野生型EGFR过表达的细胞株对三代EGFR-TKI IC50升高，且EGFR，AKT及ERK磷酸化无法被抑制。部分研究结果已参加国际会议交流。.3、罕见突变患者使用EGFR-TKIs的疗效和耐药机制，以及EGFR扩增的介导作用：总结了非小细胞肺癌EGFR-TKIs耐药机制和克服EGFR-TKIs耐药性的最新治疗策略，探索EGFR扩增合并EGFR 20号外显子插入突变患者使用EGFR-TKIs的疗效分析，发现伴EGFR扩增是不良疗效预测因子，以及罕见突变ERBB2基因在NSCLC中的突变谱及突变特征。.4、EGFR-TKIs耐药后的治疗选择：通过分析不同化疗方案对EGFR常见突变肺癌患者疗效，发现含铂化疗方案在EGFR常见突变肺腺癌患者中疗效并无差异。EGFR-TKIs耐药后获得性RET融合病例和ICIs在具有驱动基因改变的癌症中，RET融合阳性患者，不能从免疫治疗中获益。.5、探讨导致耐药的相关因素以及逆转耐药的策略研究：探究了非小细胞肺癌血浆外泌体 PD-L1的可行性，揭示血浆外泌体miRNA的特征谱及与免疫治疗疗效相关。分析晚期NSCLC患者化疗后生存获益和影响化疗疗效分子特征，总结化疗后突变图谱。.6、本课题研究结果已以论文形式公开发表，共15篇（SCI收录论文9篇，中文核心期刊6篇），其中第一标注5篇，第二标注9篇，第六标注1篇。培养硕士研究生6名，博士研究生2名，技术员2名。项目负责人在项目执行期间获奖2项。