Background: RNF43 is a tumor suppressor gene that represses the WNT/β-catenin pathway. Emerging data show that RNF43 alt are commonly present in MSI-H CRC with improved outcomes to immune checkpoint inhibitors (ICI). We evaluated whether genomic and transcriptomic analysis of MSI-H RNF43 alt CRC defines distinct subsets with potential therapeutic vulnerabilities. Methods: We analyzed CRC tumors (any stage/ subtype) sequenced with Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). RNF43 alt were defined as pathogenic/likely pathogenic per OncoKB, or copy number aberrations (CNA); deep deletions, or amplifications (>8x). RNF43 alt vs. wildtype (wt) were compared using Chi-squared/Fisher’s Exact tests or Wilcoxon rank-sum tests to evaluate differences in biomarkers. Gene Set Variation Analysis (GSVA) was applied to gene expression data to derive single-sample pathway enrichment scores. ICI-treated MSI-H CRC from the MSKCC cohort (n = 74), publicly available in AACR GENIE v13, was used to assess the overall survival (OS) using the Kaplan-Meier method. Results: RNF43 alt was identified in 5.2% (635 of 12,243) CRC tumors. CNA accounted for 6% of RNF43 alt CRC. RNF43 alt vs. RNF43 wt CRC was associated with older age (median 69 vs. 59 years, p < 0.001), more prevalent in females (56% vs 43%; p < 0.001). A detailed comparison of RNF43 alt vs. RNF43 wt CRC with p < 0.001 are shown (Table). Of MSI-H CRC (n = 714), 51% were RNF43 alt, with 80% being RNF43 p.GLY659fs. Among MSI-H RNF43 alt CRC, BRAF alt was co-mutated in 59%, with BRAF^V600E constituting 99%. Median TMB was higher for MSI-H RNF43 alt vs. MSI-H RNF43 wt (40 vs. 31 mut/MB, p < 0.001). Among MSI-H CRC, GSVA identified significant downregulation of Cetuximab_benefit pathway and upregulation of IGF1R and PI3K/AKT pathways in RNF43 alt vs RNF43 wt. Among ICI-treated MSI-H CRC (n = 74) from the MSKCC cohort, no significant OS difference (HR 0.81 [95%CI: 0.34 – 1.97]; p = 0.65) was noted for RNF43 alt (n = 42) vs. wt (n = 32). Conclusions: RNF43 alt are common in MSI-H CRC and associated with potentially actionable genomic and transcriptomic signatures. Functional characterization of interplay between the WNT signaling and therapeutically relevant pathways (PI3K/AKT, BRAF, IGF1R, Cetuximab benefit) to better define optimal ICI combination approaches in MSI-H RNF43 alt CRC is ongoing.

*Of tumors with known sidedness, ⁺of tumors with internal IHC. Bold denominators reflect non-missing data.