大肠癌Cetuximab治疗中获得性KRAS突变动态变化及克服耐药的机制研究

The exact mechanism of inevitable resistance to cetuximab during targeted therapy in colorectal cancer (CRC) patients is still unknown, let alone the theoratical instruction for the subsequent medication for patients in this category. Based on our established patient-derived tumor tissue KRAS wild-type (wt) xenograft models of CRC, we demonstrated that xenograft may develop cetuximab resistance accompanied by more cells displaying KRAS mutations. Therefore, we hypothesized that resistance to cetuximab in CRC may be induced by acquired KRAS mutations.Aiming to study the mechanism of acquired KRAS mutation, here we will investigate the dynamic change of KRAS status and the related EGFR key pathway in KRAS wt CRC cell lines and xenograft models from initiation of cetuximab treatment to the development of resistance. Then we will explore the best strategy and its mechnanism to overcome the resistance to cetuximab by the "EGFR key pathway analysis - resistance or mutation triggered intervention medication" model. Furthermore, we will apply digital PCR to monitor KRAS mutations and related pathway alterations in circulating tumor DNA from the blood samples of CRC patients in order to make early detection of resistance to cetuximab and the prospective modification for treatment strategy. This study may provide the theoratical evidence for the relationship between acquired KRAS mutation and cetuximab resistance in CRC patients. And also the mechanism of the means to overcome the drug resistance.All these expected results would render the important information for clincical medication.

大肠癌经Cetuximab治疗后产生继发耐药，其机制尚不明确，临床更缺乏对后续用药的理论指导。项目组前期构建了患者新鲜大肠癌组织来源的KRAS野生型移植瘤模型，发现经Cetuximab作用后移植瘤出现耐药并检测到了KRAS突变型细胞，提示获得性KRAS突变可能是产生耐药的关键机制。本研究拟通过KRAS野生型细胞系和移植瘤，分析经Cetuximab治疗直至耐药过程中KRAS突变及伴随的EGFR关键信号通路的动态变化，探讨获得性KRAS突变的产生机制，并根据"EGFR关键信号通路分析- - 耐药或突变触发式给药"的模式探索克服耐药的最适途径。在此指导下，通过数字PCR技术检测患者外周血循环DNA中KRAS突变的动态变化来早期监测耐药并前瞻性指导治疗策略的改变。本研究将为阐明获得性KRAS突变与大肠癌Cetuximab耐药产生及克服的相关机制提供理论依据，并为临床用药提供重要信息。

结直肠癌是常见的消化道肿瘤，全身化疗联合靶向治疗是晚期结直肠癌的标准治疗，然而，西妥昔单抗治疗后耐药的情况十分普遍，除了已知的KRAS,NRAS和BRAF基因突变等原发耐药外，对EGFR单抗继发耐药还有待于进一步的研究。我们的研究发现，通过动态检测患者外周血ctDNA可以早于影像学检查发现耐药基因的出现，便于监测病情变化，目标基因如TP53的VAF变化也与治疗疗效明确相关，检测方法上NGS的比ddPCR更为全面和准确,可以发现更多ddPCR无法检测到的基因变异。本研究还开发了一种更为经济有效的点突变及SNP检测方法F-CAUM PCR，此方法与既往点突变检测方法相比具有良好的检测敏感性和特异性。通过流式细胞学检测外周血免疫细胞的方法，我们发现西妥昔单抗治疗后外周血CD8+T细胞以及NK细胞明显增多，提示西妥昔单抗可以增强细胞免疫。另外，我们通过对KRAS野生型移植瘤裸鼠进行西妥昔单抗治疗，6次传代后成功获得耐药模型并进行了全基因组，LncRNA以及microRNA测序分析，进一步结合信号通路探索了耐药机制。在细胞试验上发现wnt通路抑制剂与西妥昔单抗在KRAS突变的肠癌细胞系上有协同疗效作用，而在野生型细胞系上没有发现协同作用，提示wnt通路抑制剂联合西妥昔单抗治疗KRAS突变肠癌患者的可能性。

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