肺腺癌中不同形态成分间演化关系及关键事件的研究

Lung cancer is the leading cause of cancer-related deaths in China. Studies about clonal evolution of early-stage lung cancer would greatly improve our understanding about lung cancer scientifically and clinically. Yet, after solving difficulties of library from tiny amount of nucleic acids and affordable deep sequencing, many types of cancers have made major progress in evolution study, but not lung cancer. Based on our previous work and preliminary results from other groups, and by learning from recent progress in the study of cancer involution in other types of cancers, we hypothesize that lung adenocarcinoma evolves in a non-linear fashion, i.e. the pre-cancerous and adenocarcinoma in situ regions and different subtypes of invasive adenocarcinoma regions progress independently from a common genetic precursor which fits the parallel evolution model. This proposed study will be a collaborated project by Peking University (PKU) People's Hospital who will provide samples and PKU Biodynamic Optical Imaging Center (BIOPIC) who will construct library from micro-dissected tissues using their unique Multiple Annealing and Looping Based Amplification Cycles (MALBAC) methods. Then whole exome and transcriptome sequencing with Illumina Hiseq x Ten sequencer will be performed. Bioinformatic analysis of single nucleotide polymorphism (SNP), chromatin rearrangement and copy number variation (CNV) by BIOPIC will result in phylogenetic trees indicative of evolution relationships and key events of lung adenocarcinoma evolution.

肺癌是国人第一位肿瘤杀手。阐明早期肺癌的演化过程，具有重大科学和临床价值。随着近年微量核酸建库及测序等技术发展，多个肿瘤的演化研究取得突破，但肺癌研究仍滞后。根据新近国际发现和我们前期工作，参考其它肿瘤研究结果，我们提出假说：早期肺腺癌从浸润前病变到浸润癌的演化，不是传统理论的渐进线性发展，而是类似其它肿瘤的间断平衡进化。本课题计划克服现有肺癌研究中形态成分取材粗糙、测序分析简单等不足，并增加转录组测序，由北京大学人民医院胸外科进行病例和标本采集，经病理医师选取典型区域行显微切割后，由北京大学生物动态光学成像中心采用多重退火和环化循环的扩增技术(MALBAC)进行核酸扩增建库，采用最新最准的Illumina HiSeq X Ten平台进行全外显子组和转录组测序；随后完成单核苷酸多态性、染色体重组、基因拷贝数变化和转录组差异分析，描绘进化树，明确肺腺癌不同形态成分间的演化关系，以及关键事件。

本研究的目的在于揭示早期肺腺癌从浸润前病变到浸润癌的演化过程，以期发现其进展过程中的关键基因事件。通过全外显子测序技术明确了极早期肺腺癌中磨玻璃（GGO）（非浸润）成分和实性（浸润）成分之间的差异。测序结果显示混合密度GGO这一早期肺癌中即已经存在明显的异质性和分支进化现象（branched evolution）。此外，我们还发现同一病灶中，实性成分和GGO成分中肿瘤突变负荷(TMB)类似，而且实性成分和GGO成分之间没有各自显著富集的突变基因，即GGO向浸润癌的转化不是通过基因组水平突变产生。通过GGO病灶单细胞转录组测序，并与正常肺组织、浸润肺腺癌单细胞转录组数据对比，发现GGO较正常肺组织的T细胞浸润增多，髓系细胞减少。此外GGO中的成纤维细胞表达谱与正常组织无明显差别，但血管内皮细胞与浸润腺癌更相似。这说明，早期肺腺癌演化不仅仅是肿瘤细胞本身。