Comprehensive Analysis of R-Spondin Fusions and <i>RNF43</i> Mutations Implicate Novel Therapeutic Options in Colorectal Cancer

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Purpose:</jats:title> <jats:p>Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies.</jats:p> </jats:sec> <jats:sec> <jats:title>Experimental Design:</jats:title> <jats:p>A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings.</jats:p> </jats:sec> <jats:sec> <jats:title>Results:</jats:title> <jats:p>The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1–RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions:</jats:title> <jats:p>This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.</jats:p> </jats:sec>