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Background: RSPO fusions that lead to WNT pathway activation are potential therapeutic targets in colorectal cancer (CRC), but their clinicopathological significance remains unclear., Methods: We screened 1019 CRCs for RSPO fusions using multiplex reverse transcription-PCR. The RSPO fusion-positive tumours were subjected to whole-exome sequencing (WES)., Results: Our analysis identified 29 CRCs with RSPO fusions (2.8%), consisting of five with an EIF3E-RSPO2 fusion and 24 with PTPRK-RSPO3 fusions. The patients were 17 women and 12 men. Thirteen tumours (45%) were right-sided. Histologically, approximately half of the tumours (13/29, 45%) had a focal or extensive mucinous component that was significantly more frequent than the RSPO fusion-negative tumours (13%; P = 8.1 × 10 -7 ). Four tumours (14%) were mismatch repair-deficient. WES identified KRAS, BRAF, and NRAS mutations in a total of 27 tumours (93%). In contrast, pathogenic mutations in major WNT pathway genes, such as APC, CTNNB1 and RNF43, were absent. RSPO fusion status did not have a statistically significant influence on the overall or recurrence-free survival. These clinicopathological and genetic features were also confirmed in a pooled analysis of previous studies., Conclusion: RSPO fusion-positive CRCs constitute a rare subgroup of CRCs with several characteristic clinicopathological and genetic features., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins ( Adenomatous polyposis coli , β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis ( RNF43 , RSPO -fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours., Design: We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups., Results: Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes ( AXIN2 , NKD1 ) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93)., Conclusions: Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors., Competing Interests: Competing interests: SJL has received grant income from UCB Pharma. VHK is participant of a patent application co-owned by the Netherlands Cancer Institute (NKI-AVL) and the University of Basel on the assessment of cancer immunotherapy biomarkers by digital pathology and has served as an invited speaker on behalf of Indica Labs. ADB has received grant funding from InCyte for molecular pathology research; speaker fees and travel costs from Illumina UK for immuno-oncology research and consultancy fees from Bristol-Myers-Squibb for immuno-oncology research. All other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)