Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles.

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.

A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening.

Gastric cancer displays marked molecular heterogeneity with aggressive behavior and treatment resistance. Therefore, good in vitro models that encompass unique subtypes are urgently needed for precision medicine development. Here, we have established a primary gastric cancer organoid (GCO) biobank that comprises normal, dysplastic, cancer, and lymph node metastases (n = 63) from 34 patients, including detailed whole-exome and transcriptome analysis. The cohort encompasses most known molecular subtypes (including EBV, MSI, intestinal/CIN, and diffuse/GS, with CLDN18-ARHGAP6 or CTNND1-ARHGAP26 fusions or RHOA mutations), capturing regional heterogeneity and subclonal architecture, while their morphology, transcriptome, and genomic profiles remain closely similar to in vivo tumors, even after long-term culture. Large-scale drug screening revealed sensitivity to unexpected drugs that were recently approved or in clinical trials, including Napabucasin, Abemaciclib, and the ATR inhibitor VE-822. Overall, this new GCO biobank, with linked genomic data, provides a useful resource for studying both cancer cell biology and precision cancer therapy.

RNF43 germline and somatic mutation in serrated neoplasia pathway and its association with BRAF mutation.

OBJECTIVE: Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1me+). We investigate genetic alterations in the serrated polyposis pathway. DESIGN: We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies. RESULTS: In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1me+ (85%) versus MLH1me- (33.3%) group (p<0.001). These findings were validated in the TCGA MSI CRCs (p=0.005), which further delineated a significant differential involvement of three Wnt pathway genes between these two groups (RNF43 in MLH1me+; APC and CTNNB1 in MLH1me-); and identified significant co-occurrence of BRAF and RNF43 mutations in the MSI (p<0.001), microsatellite stable (MSS) (p=0.002) and MLH1me+ MSI CRCs (p=0.042). Functionally, organoid culture of serrated adenoma or mouse colon with CRISPR-induced RNF43 mutations had reduced dependency on R-spondin1. CONCLUSIONS: These results illustrate the importance of RNF43, along with BRAF mutation in the serrated neoplasia pathway (both the sporadic and familial forms), inform genetic diagnosis protocol and raise therapeutic opportunities through Wnt inhibition in different stages of evolution of serrated polyps.