Molecular Sub‐Classification Suggests IBD Unclassified as a Distinct Subtype of Inflammatory Bowel Disease: Insights From a 3‐Gene Mucosal Signature

ABSTRACT

Differentiating Crohn's disease (CD) from ulcerative colitis (UC) is challenging, often resulting as Inflammatory bowel disease unclassified (IBDU). Based on targeted RNA sequencing, we recently reported a 3‐gene signature algorithm that distinguished CD from UC. This study evaluated its ability to classify IBDU cases and explore disease biology. We analyzed 197 mucosal FFPE biopsies (inflamed and non‐inflamed) from 113 individuals at diagnosis: CD ( n  = 42), UC ( n  = 41) and IBDU ( n  = 102; follow‐up: 0–23 years (IQR)). Expression levels of ANXA1, PI3, and VDR mRNAs were measured by RT‐qPCR, and assessed using logistic regression, and ROC curve analysis. The algorithm discriminated CD from UC with AUC values > 0.70 in 6 out of 8 patient subsets, peaking in inflamed adult biopsies (0.78, 95% CI: 0.64–0.92). At 80% sensitivity, specificity ranged from 46% to 58%, replicating previous cohort findings and confirming reproducibility. Application of the algorithm to the IBDU cases showed low classification accuracy, with frequent misclassification of cases that were later received definitive diagnoses. Incorporation of additional IBDU‐specific features did not improve performance. While the 3‐gene signature algorithm demonstrates moderate ability to distinguish CD from UC, its limited performance in IBDU cases highlights fundamental constraints of binary classification approaches in IBD.