新辅助放化疗驱动的食管鳞癌克隆进化及基因突变对食管鳞癌放化疗敏感性影响的研究

Neoadjuvant chemoradiotherapy (neo-CRT) is an important strategy for advanced esophageal squamous cell carcinoma (ESCC) treatment. Clinical practice found that neo-CRT response is heterogeneous, and the percent of tumor cells after neo-CRT determines ESCC patients’ prognoses. According to the theory of tumor clonal evolution and tumor heterogeneity, CRT could promote tumor clonal evolution, and the genome characteristics of pre- and post-CRT tumor tissues could be related to primary or secondary therapy resistance. In this study, we are going to compare differences of genomic characteristics between pre- and post-neo-CRT ESCC samples with whole exome sequencing, so as to illuminate the ESCC clonal evolution driven by neo-CRT. Furthermore, potential gene mutations related to ESCC CRT response are intended to be identified by analyzing the current exome and previous transcriptome data of pre- and post-neo-CRT ESCC samples. By generating ESCC cells containing site-specific mutation via CRISPR-Cas9, we will verify the role of specific gene mutation on ESCC chemo- and (or) radio-sensitivity by in vitro and in vivo studies, and further explore the underlying molecular mechanisms, which will be further validated by analyzing the relationship between specific gene mutation and ESCC CRT response in clinical samples. This study would lay foundation for identifying useful predictive markers for ESCC neo-CRT response.

新辅助放化疗联合手术是治疗局部晚期食管鳞癌的重要模式。临床发现，新辅助放化疗疗效在食管鳞癌患者存在异质性；放化疗后残余肿瘤细胞比例与患者预后密切相关。根据肿瘤克隆进化及肿瘤异质性理论，放化疗可促使肿瘤细胞发生克隆进化；放化疗前后肿瘤基因组学特征可能与原发或继发治疗耐受相关。本研究中，我们拟通过全外显子组测序，比较新辅助放化疗前后食管鳞癌组织基因组特征的差异，阐明新辅助放化疗驱动的食管鳞癌克隆进化；分析放化疗前后食管鳞癌基因突变与放化疗敏感性的关系，联合前期新辅助放化疗转录组学数据，寻找潜在的食管鳞癌放化疗敏感性相关基因突变，通过CRIPSR-Cas9技术构建特异基因定点突变的食管鳞癌细胞系，在细胞-动物-分子三个层次阐明该基因突变影响食管鳞癌放和（或）敏感性的作用与机制，在食管鳞癌组织验证该突变与患者放化疗敏感性及预后的相关性，为寻找新的食管鳞癌放化疗敏感性分子标志物奠定基础。

食管癌是世界常见恶性肿瘤之一，在我国，95%的食管癌为食管鳞癌，与西方国家更为普遍的食管腺癌在病因学、治疗方法及预后等方面均有所差异。目前，对于占绝大部分临床病例的可手术局部晚期食管鳞癌患者来说，新辅助放化疗并手术的治疗模式较单纯手术治疗显著改善患者预后，已经作为标准治疗模式写入治疗指南。肿瘤由多个遗传学上不同的细胞亚克隆组成；在肿瘤发生发展及治疗过程中，多种不断变化的环境压力作用于肿瘤细胞群体，并选择具有最适生存能力以适应环境变化的肿瘤细胞克隆。根据肿瘤克隆进化及肿瘤异质性理论，放化疗可促使肿瘤细胞发生克隆进化；放化疗前后肿瘤基因组学特征可能与原发或继发治疗耐受相关。本研究中，我们对来自于29例局部晚期食管鳞癌患者的29个新辅助放化疗前和27个新辅助放化疗后肿瘤组织进行全外显子测序，结果发现：治疗前食管鳞癌组织中NEF2L2和RYR3突变在无反应组较有反应组突变升高；Nrf2通路突变在无反应组富集；3p11.2缺失的比例在有反应组高于无反应组。单碱基替换突变C>G突变在有反应组新辅助治疗后显著降低，而C>T和C>A突变在无反应组新辅助治疗后显著降低。肿瘤突变负荷在有反应组新辅助治疗后显著降低；并且，新辅助治疗后的实际突变负荷显著低于期望突变负荷。此外，我们构建了基于8个基因的肿瘤突变细胞比例的预后预测模型，并且发现根据基因突变进行食管鳞癌基因分型，可有效预测食管鳞癌患者预后。通过本研究，我们了解了新辅助.放化疗对食管鳞癌细胞克隆进化的影响，为进一步实现食管鳞癌个体化治疗奠定基础。

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