A regenerative stem cell phenotype marked by ANXA1 defines aggressive colorectal cancer biology

Background: LGR5 and ANXA1 are markers for specific intestinal stem cell states. LGR5+/ANXA1- crypt base columnar cells (CBCs) maintain homeostasis, whereas ANXA1+/LGR5- regenerative stem cells (RSCs) are present in fetal and repairing epithelium. Both states have been described in colorectal cancer (CRC), yet their clinical significance remains unclear. We aimed to determine how ANXA1 and LGR5 expression relates to molecular subtype, immune microenvironment and outcome in CRC. Methods: Dual-colour in situ hybridisation for ANXA1 and LGR5 was performed on tissue microarrays from 2163 patients in the randomised SCOT trial ( ISRCTN59757862 ). AI-based digital pathology quantified marker-positive area by compartment and image based consensus molecular subtype (imCMS). Markers were related to clinicopathological, molecular and immune data, and to survival using Kaplan-Meier and multivariable Cox analyses. Results: ANXA1 and LGR5 showed inverse enrichment among a diverse group of parameters. ANXA1 was highest in CMS1 and LGR5 in CMS2, with CMS3 and CMS4 being intermediate. ANXA1 dominance was associated with right-sidedness, mismatch repair deficiency, BRAF-V600E mutation, high stromal content, T4 stage, low stromal FoxP3 and high epithelial CD68 density. Combining both markers improved prognostic discrimination compared with either alone (cancer-specific survival p<0.001; recurrence-free interval p=0.004), with ANXA1 dominance remaining an independent predictor of recurrence after adjustment (HR 1.43, 95% CI 1.08-1.90, p=0.014). Conclusion: Relative ANXA1 enrichment defines an aggressive, stroma-rich, macrophage-enriched CRC subgroup with poor prognosis.