肺腺癌肿瘤异质性在EGFR-TKI耐药中的作用和机制研究

Mutational activation of epidermal growth factor receptor(EGFR) is the most prevalent genetic alteration in non-small cell lung cancer (NSCLC) of East Asian population. Moreover, the marked tumor regression and improved survival of advanced EGFR-mutated lung cancer patients in response to treatment with EGFR tyrosine kinase inhibitors(EGFR TKI) demonstrated the essential role of EGFR in lung cancers. However,resistance to EGFR TKI treatment invariably occurs, and there is no effective therapy for patients who develop such resistance..Recent studies have identified extensive heterogeneity in tumors using large-scale sequencing and single cell analyses. Intratumor heterogeneity arises through the evolution of genetically diverse subclones varying in proliferation, persistence, and drug tolerance. Thereby chemotherapeutic agents or targeted drugs, such as EGFR-TKI, could destroy the sensitive clones but promote the dominance of previously insensitive lineages. We previously analyzed adenosquamous cell lung cancer tissues by microdissection technique, and found that discrepancy of driver mutations between the adeno and squamous component frequently occurred. We also identified the major mechanisms associated with EGFR-TKI resistance by large scale library screening. This finding further indicated our current hypothesis that therapy sensitivity is associated with subclonal heterogeneity..In this study, we further investigate the mechanism of development of EGFR-TKI sesitive/resistant subclones. Then we will analyze whether those genetic alterations related to EGFR TKI resistance exist in multiple spatially surgically resected samples obtained from prmary lung tumors by direct sequencing, chromosome aberration analysis and immunohistochemical analysis. In addition, we will also construct a heterogeneous mixture of EGFR-TKIs resistant and sensitizing lung cancer cell model. With this evolutionary model, we will predict alternative therapeutic strategies for drug-resistant lung cancers. We will also explore the genetic changes associated with resistance to EGFR TKIs at a single-cell nucleotide level. Our study may contribute to overcoming the resistance of EGFR TKIs to achieve curative therapy for EGFR mutant lung cancer patients， and improve our management of lung adenocarcinoma.

表皮生长因子受体（EGFR）突变是东亚肺腺癌最常见的驱动基因突变之一，以其为靶点的酪氨酸激酶抑制剂（TKI）在临床获得了巨大成功，但绝大多数患者最终将发生耐药并导致治疗失败。目前研究显示肿瘤具有亚克隆异质性，EGFR-TKI敏感亚克隆在药物作用下被大量杀灭，但耐药亚克隆则持续发展引起耐药和肿瘤的进展。我们通过对显微切割样本的分析，初步对药物敏感性与亚克隆异质性的关联进行了阐述，通过大规模文库筛选确定了与EGFR-TKI治疗抵抗的主要相关基因。在此基础上，本项目将进一步研究肺腺癌EGFR-TKI治疗敏感/耐受异质性亚克隆产生的内在机制，研究这些耐药基因异常在肿瘤组织中的分布，同时通过耐药细胞株，建立模拟肿瘤内异质性的细胞模型，预测EGFR-TKI疗效。这将有望革新我们对肿瘤异质性在耐药中作用的认知，推动针对异质性耐药的临床试验开展，以期提高肺癌的整体诊治水平。

表皮生长因子受体（EGFR）突变是东亚人群肺腺癌最常见的驱动基因突变之一，以其为靶点的酪氨酸激酶抑制剂（TKI）在临床获得了巨大成功，延长了许多进展期肺腺癌的预期存活时间，但绝大多数患者最终将发生耐药并导致治疗失败。目前研究显示肿瘤具有亚克隆异质性，EGFR-TKI敏感亚克隆在药物作用下被大量杀灭，但耐药亚克隆则持续发展引起耐药和肿瘤的进展。本研究通过全外显子组测序、RNA测序、CRISPR/Cas9等技术探究肿瘤异质性在EGFR-TKI耐药中的作用和机制。.首先，我们对肺腺癌致癌基因突变异质性/形态异质性与EGFR-TKI治疗进行了探索。我们发现了FGFR融合基因阳性患者的临床病理特征，这一发现为肺癌靶向治疗增添了一个新靶点。通过系统综述，我们发现EGFR-TKI作为辅助治疗可显著改善生存，这一结果为EGFR-TKI作为复发高危患者辅助治疗的替代方案提高了证据。通过研究发现，“血统基因”与腺癌亚型和EGFR突变相关，本研究有助于鉴别肿瘤间异质性。同时，我们也发现EGFR 18号外显子突变肺腺癌复发时表现出对EGFR-TKI治疗的良好反应。通过分析肿瘤标本中的NF1突变和B7-CD28家族基因表达水平，我们了解到EGFR/NF1共突变患者对EGFR-TKI疗效较差，NF1突变和B7-CD28表达水平可以区分预后，这阐明了基因突变异质性对肺腺癌预后的影响。.其次，我们对肺腺癌形态异质性与个体化手术治疗、术后随访进行了进一步研究。我们发现术中冰冻病理诊断的原位腺癌/微浸润腺癌是行亚肺叶切除的精确指征，该成果为早期非小细胞肺癌外科精准治疗重要的一步。而且，肺腺癌亚型的异质性是重要的复发预测因子，我们建立了网页版预测模型，能够帮助临床医生制定个体化的肺癌患者术后随访策略。我们还基于影像学形态异质性定义了亚实性结节是肺腺癌中一个独特的亚型。.最后，通过对第三代EGFR-TKI奥西替尼耐药和未经治疗的小鼠模型研究，初步鉴定了CCL23基因可以介导奥希替尼的耐药，我们也通过实验证实了SETD2基因突变可以介导EGFR-TKI耐药。同时，我们也通过全外显子组测序和RNA测序阐明了肺腺癌形态异质性背后的遗传背景异质性。