For a study, researchers compared the molecular features of mutated (mut) RSPOfp/RNF43 colorectal cancers to wild-type (WT) colorectal cancers to gain insight into potential therapeutic strategies. RSPOfp/RNF43 status was determined in a discovery cohort using DNA/RNA sequencing and IHC. The findings were validated using an independent cohort. There were 7,245 colorectal cancer samples in the discovery cohort. RSPOfp and RNF43 mutations were found in 1.3% (n=94) and 6.1% (n=443) of cases. Investigators discovered 5 previously unknown RSPO fusion events (e.g., IFNGR1–RSPO3). RNF43-must tumors were linked to primary tumors on the right side. RNF43 mutations were not found in any RSPOfp tumors. RSPOfp colorectal cancers had a higher frequency of BRAF, BMPR1A, and SMAD4 mutations than WT colorectal cancers. APC mutations were found in a minority of RSPOfp-positive cases compared to WT cases (4.4%vs.81.4%). In comparison to WT samples, RNF43 mutations had a higher rate of KMT2D and BRAF mutations. Although RNF43 mutations were linked to microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%) and a tumor mutation burden of more than equal to 10 mt/Mb (65.8%), RSPOfp was not. Study group genetic findings were replicated in the validation cohort. It was the largest reported series of RSPOfp/RNF43-mut colorectal cancers. Comprehensive molecular analyses confirmed the RSPO/RNF43 molecular landscape and proposed potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.