循环肿瘤DNA的突变分析用于监测靶向EGFR单抗治疗晚期结直肠癌继发耐药的价值

Anti-EGFR monoclonal antibodies, such as commonly used cetuximab, are commonly used in treatment of metastatic colorectal cancer patients with RAS wild type. However, those patients who do respond, acquired resistance to antibodies would eventually emerge in 6 months. More than 50% of acquired resistance cannot be explained by using RAS mutation. Whether the EGFR and its downstream pathway of other key genes play a role in the secondary cetuximab resistance? So far no clear answer. The main reason is difficult to obtain each patient’s tumor tissue after failure to treatment. Some studies have used Next Generation Sequecing (NGS) to dynamically monitor specific mutations in the circulating tumor DNA (ctDNA) from longitudinal plasma samples instead of tumor tissues. But the challenge is how to screen effective mutations associated with acquired cetuximab resistance from a large number of low frequency mutations. By using the ultra deep amplicon sequencing platform and quartile analysis we established, we are going to monitor all key genes of EGFR pathway mutations whose variant frequencies are significantly higher in plasma samples collected after disease progression than before cetuximab treatment. Through clinical data comparison and in vitro experiments to identify candidate mutations and verify the function of effective mutations. Lastly, we will evaluate the value of this innovative approach used in effective mutation selection of ctDNA of NGS to understand acquired cetuximab resistance.

靶向表皮生长因子受体 (EGFR) 的西妥昔单抗是治疗RAS基因野生型晚期结直肠癌的重要药物。但是治疗有效的患者仍会在6个月左右产生继发耐药,其中超过50%无法用RAS的突变解释。EGFR通路的其他关键基因是否在其中发挥了作用，至今还没有明确的答案。主要原因是很难获取耐药后的肿瘤组织进行突变分析。二代测序技术通过对循环肿瘤DNA(ctDNA)中的基因突变进行检测，可以替代肿瘤组织实现对继发耐药的动态监测。但如何从众多低频突变基因中筛选出与继发耐药相关的有效突变，是目前面临的重要课题。本课题组拟通过前期建立的针对ctDNA低频突变的深度测序平台和四分位数据分析法，动态监测西妥昔单抗治疗患者的ctDNA中EGFR通路中所有关键基因的突变，寻找与EGFR单抗继发耐药相关的基因突变，并通过临床数据比对和体外实验验证突变基因的功能。最终确认这种新的突变筛选策略对继发耐药研究的价值。

结直肠癌（colorectal cancers; CRC）是全球发病率居第三位的恶性肿瘤。西妥昔单抗单药应用或与化疗联合可以显著改善转移性结直肠癌(mCRC）患者的疗效和生存期。然而，大部分患者在治疗过程中仍会发生继发耐药。因此，寻找可以动态监测继发耐药的方法，全面认识EGFR单抗的继发耐药的机制，对于改善晚期结直肠癌患者的疗效和预后至关重要。本课题组通过自己创建的针对ctDNA新发突变和四分位数据分析法，监测68例西妥昔单抗治疗患者的ctDNA中EGFR通路中10个关键基因的动态变异。通过临床数据比对和体外实验验证，发现并证明PIK3CA第19外显子多个位点的突变对西妥昔单抗获得性耐药的贡献，同时评价了基于二代测序技术的ctDNA突变筛选策略对发现西妥昔单抗继发耐药机制的价值。另外，本课题组发现癌细胞分泌的丝氨酸蛋白酶（PRSS）表达水平与西妥昔单抗的耐药性有关，并通过细胞实验、裸鼠荷瘤实验以及临床标本等研究发现并证实PRSS可通过在Val 115和Thr 116位点之间降解西妥昔单抗，使其疗效减弱进而导致耐药。通过动态监测结直肠癌患者治疗前、中、后血清外泌体DNA中的目的基因片段突变频率的变化，本课题组发现外泌体DNA检测可以提示西妥昔单抗治疗的继发耐药突变。研究结果为结直肠癌患者更好地实现个体化治疗提供了理论依据。