我们通过二代测序发现，ALK阳性非小细胞肺癌患者中有18.7%（28/150）存在ALK双融合；且多重免疫荧光证实，与ALK单融合患者相比，ALK双融合患者PD-L1表达显著升高，且和克唑替尼治疗预后差相关，进一步通过RNA-seq发现，ALK双融合患者中BAG-1高表达，我们推测PD-L1诱发BAG-1高表达介导了克唑替尼耐药，然而其机制不清。本课题拟通过复合免疫组化及RNA-seq进一步验证ALK阳性非小细胞肺癌单、双融合患者的关键差异功能基因，进一步通过生物信息学分析、Western blot及PDC实验明确PD-L1上调BAG-1的分子生物学机制，最后通过体外共培养及PDX模型验证PD-1单抗联合克唑替尼可以逆转ALK双融合细胞耐药。本研究结果不仅有助于揭示ALK双融合肺癌患者克唑替尼耐药机制，还能为PD-1单抗联合治疗逆转其对克唑替尼耐药提供理论基础及实验依据。

Through next-generation sequencing, we found that 18.7% (28/150) of ALK-positive non-small cell lung cancer patients harboring ALK double fusion. Compared with ALK single fusion patients, multiple immunofluorescences confirmed that PD-L1 expression was significantly increased, which was related to the poor prognosis of crizotinib. RNA-seq revealed that BAG-1 was highly expressed in patients with ALK double fusion. However, the resistance mechanism is unclear. Our study aims to identify key functional genes in single and double fusion ALK-positive non-small cell lung cancer patients through composite immunohistochemistry and RNA-sequence; next step, we will further identify the mechanism of PD-L1 up-regulates BAG-1 through bioinformatic analysis, western blotting and PDC experiments. Finally, we verify the resistance of ALK double fusion cells by combining the PD-1 monoclonal antibody with crizotinib through the in vitro co-culture and PDX model. The results of this study will not only help to reveal the mechanism of resistance to crizotinib in patients with ALK double fusion lung cancer, but also provide a theoretical basis and experiment for PD-1 monoclonal antibody combination therapy to reverse the resistance of crizotinib.