Association of wnt signalling with metachronous colonic polyp risk.
Authors: Edwards Joanne , Al-Badran Sara SF , Hillson Lily , Ammar Aula , Hayman Hannah Louise , Johnstone Mark Scott , Hay Jennifer , Jawny Jakub , Maka Noori , Andersen Ditte , Dunne Philip D , Roxburgh Campbell SD , Lynch Gerard , McSorley Stephen Thomas ,   INtegrated teChnologies for Improved polyp SurveillancE (INCISE)
Published: January 2023

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Background: Following colorectal polypectomy, 20-50% of patients develop metachronous polyps, and a proportion have increased colorectal cancer (CRC) risk. However, there are currently no molecular biomarkers for predicting metachronous polyp risk. Constitutive activation of the Wnt signalling cascade is a hallmark of polyp development and carcinogenesis, therefore biomarkers from this pathway were investigated for association with detection of metachronous polyps. Methods: A retrospective study was performed on a tissue microarray (TMA) of left sided colonic polyps from 279 patients undergoing screening polypectomy (May’09-Dec’16) followed by surveillance colonoscopy (up to 6 years). Mutational analysis was performed using the GPOL cancer plus panel, protein expression by Immunohistochemistry and RNA-sequencing by TempO-Seq (BioClavis). Log rank statistics determined associations with time to detection of metachronous polyp. Index polyp number, histology and size were compared to metachronous lesion outcome using χ2/ANOVA and multivariate polynomial regression identified independent predictors of advanced future lesions. Results: APC (91%), KRAS (30%) and SOX9 (23%) were the most frequently mutated genes and APC and SOX9 are both implicated in Wnt signalling. Mutations in APC and co-mutations in SOX9 with ARID1 were associated with a shorter time to detection of metachronous polyps. Neither variant classification nor location of mutations were associated with metachronous polyp detection. Luminal epithelial cells with high expression of E-Cadherin or SOX9 were significantly associated with earlier detection of metachronous polyps or CRC (HR 2.3, 95%CI 1.6-3.3: p<0.001 and: HR 2.0, 95%CI 1.3-3.0; p=0.001 respectively). By χ2/ANOVA analysis, E-Cadherin and SOX9 expression was associated with detection of metachronous polyp (p>0.001, p=0.037 respectively). On multivariate regression, number of polyps (HR 1.2, 95%CI 1.1-1.3; p<0.001), and E-Cadherin expression (HR 2.1, 95%CI 1.5-3; p<0.001), independently predicted metachronous lesions. Gene set enrichment analysis (GSEA) was used to explore differential biology between high and low E-Cadherin or SOX9 expression. For E-Cadherin, all seven immune-annotated MSigDB hallmarks had negative enrichment, however Wnt--catenin signaling was positively enriched (NES=1.50, p-adj<0.1). The top signature enriched for SOX9 was ‘NFB signaling via TNF (NES=1.70, p-adj<0.001). The LGR5 stemness signature was enriched in both high E-Cadherin (NES=1.97 p-adj<0.0001) and high SOX9 (NES=1.48, p-adj<0.0001) groups. Conclusions: Wnt signalling-associated protein expression within index polyp tissue is valuable for predicting metachronous polyp risk. In addition, expression of E-Cadherin and SOX9 in colonic polyps are linked to immune and stemness signatures, suggesting immune and stemness biology may be an underlying driver of metachronous polyp development.