ObjectivePathological 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.DesignWe 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.ResultsWnt 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).ConclusionsEpigenetic 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.

Background & Aims MicroRNA (miRNA) is highly stable in biospecimens and provides tissue-specific profiles, making it a useful biomarker of carcinogenesis. We aimed to discover a set of miRNAs that could accurately discriminate Barrett’s esophagus (BE) from normal esophageal tissue and to test its diagnostic accuracy when applied to samples collected by a non-invasive esophageal cell sampling device. Methods We analyzed miRNA expression profiles of 2 independent sets of esophageal biopsy tissues collected during endoscopy from 38 patients with BE tissues and 90 patients with non-BE esophagus (controls) using Agilent microarray and Nanostring counter assays. Consistently upregulated miRNAs were quantified by real-time PCR in esophageal tissues collected by Cytosponge from patients with BE or without BE. miRNAs from plasmids and anti-sense oligonucleotides were expressed in NES normal esophageal squamous cells and effects on proliferation and gene expression patterns were analyzed. Results We identified 15 miRNAs that were significantly upregulated in BE vs control tissues. Of these, 11 (MIR215, MIR194, MIR 192, MIR196a, MIR199b, MIR10a, MIR145, MIR181a, MIR30a, MIR7, MIR199a) were validated in Cytosponge samples. The miRNAs with the greatest increases in BE tissues (7.9-fold increase inexpression or more, P<0.0001: MIR196a, MIR192, MIR194, and MIR215) each identified BE vs control tissues with area under the curve (AUC) values of 0.82 or more. We developed an optimized multivariable logistic regression model based on expression levels of 6 miRNAs (MIR7, MIR30a, MIR181a, MIR192, MIR196a, and MIR199a) that identified patients with BE with an AUC value of 0.89, 86.2% sensitivity, and 91.6% specificity. Expression level of MIR192, MIR196a, MIR199a, combined with Trefoil Factor 3 (TFF3), identified patients with BE with an AUC of 0.93, 93.1% sensitivity, and 93.7% specificity. Hypo-methylation was observed in the promoter region of the highly upregulated cluster MIR192-194. Overexpression of these miRNAs in NES cells increased their proliferation, via GRHL3 and PTEN signaling. Conclusions In analyses of miRNA expression patterns of BE vs non-BE tissues, we identified a profile that can identify Cytosponge samples from patients with BE with an AUC of 0.93. Expression of MIR194 is increased in BE samples via epigenetic mechanisms that might be involved in BE pathogenesis.