Abstract Background Perianal fistulas remain a major complication in Crohn’s disease, profoundly impacting quality of life. Although often therapy refractory, strong heterogeneity exists between patients, both in clinical behaviour and therapy response. In this study we aimed to identify biological factors involved in the clinical behavior of perianal fistulas, allowing better stratification. Methods Forty-nine fistula tracts patients were included (Crohn’s n = 36, cryptoglandular n = 13). Gene expression was analyzed by whole transcriptome RNA sequencing. An integrated gene-module score for keratinization activity was calculated and correlated to the Crohn’s disease TOpClass fistula classification as well as to long-term fistula outcomes. Results Perianal fistulas overall were associated with development of stratified squamous epithelium, expressing specific keratins (e.g. KRT5, KRT7, KRT13). A gene-module score was developed based on the keratinization-associated markers and calculated for each individual sample. This score clearly separated Crohn’s related fistulas from cryptoglandular fistulas (median score 0.448 vs -0.321, p = 0.0018). Interestingly, within the Crohn’s-related fistulas keratinization was significantly lower in the more severe phenotypes (TOpClass 2c/3) when compared to those more amenable to surgical intervention(TOpClass 2a/b, median score -0.448 vs - 0.125, p 0.05). Interestingly, the score was also predictive of long-term outcomes: After a follow-up receiving routine care for a median of 63 months, the group with low keratinization scores showed severe disease in a large proportion of patients (14/20 patients), versus none of the patients with a high keratinization score (0/16, p 0.001). Conclusion We show here that fistula pathology is marked by epithelial changes, including altered keratin expression. The extent of this process not only distinguishes Crohn-related from cryptoglandular fistulas, but also aligns with disease severity and healing potential. These findings provide a mechanistic explanation for the heterogeneity observed in Crohn’s disease associated perianal fistulas and establish a foundation for biomarker-driven stratification and better targeted therapeutic strategies. Conflict of interest: Becker, Marte: No conflict of interest Koelink, Pim: No conflict of interest Nabozny, Gerald: Is an employee of Boehringer Ingelheim Pharmaceuticals Li, Frank: Is an employee of Boehringer-Ingelheim Pharmaceuticals Bemelman, Willem: Other: Speakersfees from Applied 4.9% shares Semiflex bv D’Haens, Geert: Grant: Pfizer, BMS, Johnson and Johnson, Abbvie, Alimentiv BV, Eli Lilly, Takeda, Prometheus Laboratories Personal Fees: Abbvie, Abivax, Agomab, Alimentiv, Anaptys Bio, AstraZeneca, Bristol Meiers Squibb, Boehringer Ingelheim, Celltrion, Eli Lilly, Exeliom Biosciences, Galapagos, Glaxo Smith Kline, Dr Falk Pharma, Pfizer, Johnson and Johnson, Merck, Mirador, Polpharma, Procise Diagnostics, Prometheus Biosciences, Sorriso Pharma, Spyre, Takeda, Ventyx Buskens, Christianne J.: Grant: C. Buskens has received an unrestricted grant from Boehringer Ingelheim and Roche Personal Fees: C. Buskens has received consultancy fees and/or speaker’s honoraria from Tillotts, Takeda, MSD and Janssen Dr. Wildenberg, Manon: Received research grant support from Hoffman-La Roche, Boehringer-Ingelheim
Becker et al. (Thu,) studied this question.