Objective Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. Design We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. Results We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. Conclusions These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

中文翻译：

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早发性结直肠癌的类器官培养物揭示了独特而罕见的遗传特征

目的散发性早发性结直肠癌（EOCRC）预后不良，但细胞系模型的代表性较差。我们检查了富含 EOCRC 的类器官生物库中的关键突变和转录组改变。设计 我们从 20 名富含微卫星稳定 EOCRC 的患者中建立了配对癌症 (n=32) 和正常类器官 (n=18)。进行了外显子组和转录组分析。结果我们观察到分子表型的惊人多样性，包括 PTPRK-RSPO3 融合。在转录方面，RSPO 融合类器官类似于正常结肠类器官，与 APC 突变类器官不同，具有高 BMP2 和低 PTK7 表达。单细胞转录组分析证实了 RSPO 融合类器官和正常类器官之间的相似性，具有在 Wnt 退出时成熟的倾向，而 APC 突变类器官则被锁定在祖细胞阶段。正常人结肠类器官中 APC 的 CRISPR/Cas9 工程突变导致 PTK7 蛋白的上调和 BMP2 的抑制，但对于工程 RNF43 突变则较少。在 TCGA 数据库搜索中证实了 RSPO 融合与 SMAD4 或 BMPR1A 突变的频繁同时发生。在白血病幸存者的类器官中发现了 RNF43 突变，具有新的突变特征；具有 POLE 校对突变的类器官显示出超突变。癌症类器官基因组在长期培养期间是稳定的，而正常的人类结肠类器官随着时间的推移往往会受到克隆优势的影响。