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

Abstract

Purpose: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer (CRC). Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared to wildtype CRCs to gain insights into potential rationales for therapeutic strategies. Experimental Design: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and immunohistochemistry. An independent cohort was used to validate our findings. Results: The discovery cohort consisted of 7,245 CRC 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 to wildtype CRCs, 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 to wildtype cases (4.4 vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared to wildtype samples. While RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a TMB {greater than or equal to}10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings. Conclusions: This is the largest series of RSPOfp/RNF43-mut CRCs 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.

Exome sequencing revealed comparable frequencies of RNF43 and BRAF mutations in Middle Eastern colorectal cancer

“…Our analysis revealed that 5.9% of the Middle Eastern CRC patients had RNF43 mutations. This is similar to what has been reported in the largest existing RNF43 study of Seeber et al (6.1%) 32 . Similar frequency of RNF43 mutant cases has also been observed in TCGA cohort where RNF43 mutations were seen in about 5.7% (12/212) of CRC patients 33 .…”

“…Interestingly, RNF43 mutations were found to be significantly enriched in right sided CRCs, which has also previously been reported in other studies 16 , 17 , 32 , 41 , and suggests that RNF43 mutations are associated with distinct primary tumor locations within the colon, further supporting regional differences for Wnt pathway alterations.…”

R-spondin family biology and emerging linkages to cancer

“…Based on recent research reports, the authors identified five previously unreported RSPO fusion events (e.g. IFNGR1 - RSPO3 ) [ 23 ]. Seshagiri et al.…”

The emerging era of personalized medicine in advanced colorectal cancer

“…RNF43 mutations are frequently truncated frameshift mutations with tandem repeats of microsatellites 46 . Thus, these mutations often occur in sporadic MSI colorectal tumors, with MLH1 mismatch repair gene being the most frequently associated mutation, of right colonic distribution, and CMS1 tumor molecular subtype 47–49 . Fifty percent of sessile serrated lesions are thought to have RNF43 mutations.…”

“…46 Thus, these mutations often occur in sporadic MSI colorectal tumors, with MLH1 mismatch repair gene being the most frequently associated mutation, of right colonic distribution, and CMS1 tumor molecular subtype. [47][48][49] Fifty percent of sessile serrated lesions are thought to have RNF43 mutations. Ligand-dependent tumors are rich in mucins, 50 reflected by the fact that RNF43 mutations are seen in 34.5% of genetic mutations responsible for mucinous adenocarcinoma, which are also more commonly associated with MSI.…”