微卫星不稳定高（MSI-H）结肠癌（CRC）的肿瘤内异质性（ITH）研究较少。我们旨在阐明MSI-H CRC的ITH如何在癌症进化中生成，以及免疫选择性压力如何影响ITH。我们重新分析了246个MSI-H CRC的公共全外显子测序数据。此外，我们还对6个MSI-H CRC进行了多区域分析。为了验证亚克隆免疫逃逸积累的过程，我们开发了一个新的癌症进化计算模型，考虑了免疫压力的影响。我们的分析显示了抗原呈递机制（APM）中功能性基因组变化的富集。通过新抗原的相关分析，显示出通过HLA变异产生免疫逃逸机制。多区域分析揭示了驱动突变的克隆获取和APM缺陷的亚克隆积累在MSI-H CRC中。突变等位基因频率的检查表明，亚克隆突变往往受到选择扫荡。肿瘤进展与免疫细胞相互作用的计算模拟成功验证了免疫逃逸突变的亚克隆积累，并建议早期启动免疫检查点抑制剂（ICI）为基础的治疗的功效。我们的结果表明了MSI-H CRC中免疫逃逸机制的异质性获取是达尔文选择的结果，为ICI治疗策略提供了新的见解。© 2023. 作者，独家许可给Springer Nature Limited出版。

Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH.We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed.Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment.Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.© 2023. The Author(s), under exclusive licence to Springer Nature Limited.