Marte A. J. Becker

Background and Aims: So far, there are no diagnostic tools to identify Crohn's disease (CD) patients at risk of failure or recurrence after perianal fistula closure surgery. This study aims to analyse if fistula scrapings calprotectin levels in CD patients undergoing surgical fistula closure can be predictive of postoperative outcomes. Methods: In this prospective study, fistula scrapings were collected from 47 TOpCLASS 2a patients who underwent a surgical closure procedure with or without mesenchymal stem cell (MST) between November 2019 and February 2024. Scraping calprotectin was determined by ELISA. The primary outcome parameter was the correlation between scraping calprotectin levels and clinical fistula closure and radiological remission (MAGNIFI score ≤ 6). Results: Eighteen patients underwent primary surgical closure and 29 patients underwent surgical closure with MST. Scraping calprotectin levels were significantly lower in the primary closure group when compared to MST (822 μg/g (IQR 134–3285) vs. 2698 μg/g (IQR 1042–6000), p = 0.041). After a median follow‐up of 18 months (IQR 14–51), overall, 68% clinical closure with 40% radiological remission was seen. In the primary surgical closure group, scraping calprotectin levels were significantly lower in patients with clinical fistula closure (586 μg/g (IQR 84–1454) vs. 5855 μg/g (IQR 3291–6000), p = 0.003). In contrast, in the MST group no significant differences were found for clinical closure, whereas higher scraping calprotectin levels were found in patients who reached radiological remission (4827 μg/g(IQR 2698–6000) vs. 1967 μg/g (IQR 428–3937), p = 0.033). Conclusions: These results suggest that scraping calprotectin can be used to identify patients amenable for primary surgical closure and select patients most likely to need additional treatment like MST.

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 behavior and therapy response. In this study, we aimed to identify biological factors involved in the clinical behavior of perianal fistulas, allowing better stratification. Forty-nine patients were included (Crohn's n = 36, cryptoglandular n = 13). Gene expression was analyzed in internal fistula openings and tracts by RNA sequencing. Data were verified using spatial transcriptomics and functionally in human adult organoid cultures. A gene-module score for keratinization activity was calculated and correlated to the Crohn's TOpClass classification as well as to long-term fistula outcomes. Perianal fistulas were associated with the development of stratified squamous epithelium, expressing specific keratins (KRT5, KRT7, and KRT13). In human organoid models, exposing intestinal mucosa to a pro-inflammatory cytokine mixture partially induced similar changes, supporting rectal mucosal redifferentiation in response to perianal wounding. A gene-module score based on the keratinization-associated markers showed a decreasing gradient from cryptoglandular to mild Crohn-related to severe Crohn-related fistula (0.448 vs. -0.125 vs. -0.448, P = .018). Interestingly, the score was also predictive of long-term outcomes: Patients with low keratinization often showed progressive disease (14/20 patients), whereas this was not seen in the high keratinization group (0/16, P < .001). We identify an epithelial redifferentiation process characterized by a keratinization-associated gene module as part of the healing process in perianal fistula. The extent of the keratinization not only distinguished Crohn-related from cryptoglandular fistulas but also aligned with disease severity and healing potential.

Wound healing is critical to homeostasis in particular in tissues exposed to environmental insults such as the skin and the intestine. At the intersection of these tissues a particular form of wounding occurs in the form of perianal fistula, i.e. abnormal tracts connecting the intestine with the skin. Importantly, a natural disparity is found in healing efficiency in perianal fistula depending on the underlying medical condition, which is currently not well understood. Using this disparity, we modelled appropriate and defective wound healing by comparing Crohn’s disease related fistula (poor healing) and non-IBD/idiopathic fistula (well healing). We show that during wound healing, cytokines TNFα and IL6 in conjunction with TGF-β induce differentiation of columnar intestinal mucosa into squamous epithelium with high expression of keratins 5 and 13 and WFDC2 . Specifically in poorly healing wounds, cytokines including IL17, IL22 and IFNγ induced expression of S100A8/9 in this intestinal derived tissue. Surprisingly, S100A8/9 was not released but rather functioned as a transcriptional (co)regulator, affecting downstream inflammatory mediators such as C3 and CXCL17. These data identify separate immune pathways contributing to healing in general and deficient healing, allowing for more targeted intervention approaches.

Background and Aims: Previous literature suggests that faecal calprotectin (FC) discriminates Crohn's disease perianal fistulas from cryptoglandular fistulas, irrespective of luminal disease. This study aims to prospectively validate this and analyse if increased local fistula calprotectin levels are associated with fistula characteristics.Methods: In this prospective study, all consecutive patients with an active perianal fistula undergoing examination under anaesthesia were included. Faecal and fistula tract scraping calprotectin levels were determined. The primary objective was to analyse whether FC levels could be used to differentiate between Crohn's disease and cryptoglandular perianal fistulas. Secondary outcome parameters were the levels of local calprotectin in fistula scrapings and their correlation with fistula characteristics.Results: Sixty‐three patients were included in this study (perianal Crohn's disease; 45, cryptoglandular; 18). Faecal calprotectin levels were significantly higher in Crohn's disease patients compared with cryptoglandular fistula (354.3 [58.8–1076.3] vs. 47.3 [14.6–233.6] μg/g, p = 0.003). Faecal calprotectin could accurately discriminate Crohn's disease patients with active luminal disease from patients without luminal disease (median [interquartile range]) (1167.0 [557.0–2806.3] vs. 93.0 [47.5–571.6] μg/g, p = 0.001). Faecal calprotectin was not related to calprotectin levels in fistula scrapings. No fistula characteristic was found to be correlated to scraping calprotectin, but a correlation was found with the TOpCLASS classification system, which stratifies fistulas according to disease severity and outcome: class 2a (amenable for repair), class 2b (symptom control) and class 2c (gradually debilitating disease): 140[31.0–149.0]) μg/g versus 706[198.5–1936] μg/g versus 4000[1337–5894] μg/g, p < 0.001). Scraping calprotectin was also related to pronounced hyperintensity of the fistula tract on MRI in Crohn's disease patients: (69.0[30.0–821.0] vs. 1284.0[204.3–4185.5]; p = 0.01)) and cryptoglandular patients: (30.0[13.5–80.5] vs. 3012.0 [923.8–5021.0]; p = 0.002).Conclusion: Crohn's disease and cryptoglandular perianal fistulas differ in FC levels. Local fistula calprotectin production did not explain this difference, implying FC reflects the luminal condition. A correlation exists between scraping calprotectin levels and Crohn's disease fistula severity, which could be clinically relevant for prognostic cohorts and tailored treatment.

BACKGROUND: Endorectal advancement flap repair is often performed for the treatment of cryptoglandular transsphincteric fistulas. However, this procedure fails in approximately 1 of 4 patients. Based on its supposed healing properties, platelet-rich plasma might enhance the outcome of this procedure. OBJECTIVE: To evaluate and compare the short-term and long-term outcomes after endorectal advancement flap repair with and without platelet-rich plasma injection in patients with a cryptoglandular transsphincteric fistula. DESIGN: Retrospective cohort study. SETTING: Tertiary referral hospital for proctology in the Netherlands. PATIENTS: Consecutive patients with a cryptoglandular transsphincteric fistula. Inverse propensity score–weighted comparison was used to adjust for confounding and selection bias. INTERVENTIONS: Endorectal advancement flap repair with and without platelet-rich plasma injection. MAIN OUTCOME MEASURES: Clinical fistula closure within 1 year without need for a reintervention (primary healing), clinical fistula closure within 1 year corrected for reinterventions (secondary healing), overall fistula healing within 1 year, and long-term outcomes assessed by a questionnaire. RESULTS: In total, 219 patients underwent an endorectal advancement flap repair. In 88 patients (40.2%), platelet-rich plasma was injected. No significant difference was observed in primary healing (67.0% vs 69.5%, p = 0.71), secondary healing (37.5% vs 43.5%, p = 0.60), or overall healing (73.9% vs 77.1%, p = 0.58) between patients treated with and without platelet-rich plasma injection. Long-term follow-up was available in 67.1% of the patients with a mean follow-up of 6.8 years (SD: 3.7 years). Among all patients who reached fistula healing, whether primary or secondary, within 1 year and had available long-term follow-up data, recurrence rates were also not significantly different (6.3% vs 2.9%, p = 0.37). Propensity score–weighted analysis showed that patients treated with a platelet-rich plasma injection were not more likely to achieve primary healing (OR 1.0; 95% CI, 0.5–1.9), secondary healing (OR 1.1; 95% CI, 0.2–3.2), overall healing (OR 0.9; 95% CI, 0.5–1.7), or recurrence at long-term follow-up (OR 1.1; 95% CI, 0.4–18.8) compared with patients without platelet-rich plasma injection. LIMITATIONS: Retrospective design, lack of postoperative imaging, and assessment of long-term follow-up using a questionnaire. CONCLUSION: Addition of platelet-rich plasma injection does not improve the short-term and long-term outcomes of endorectal advancement flap repair in patients with a cryptoglandular transsphincteric fistula treated in a tertiary referral center. See Video Abstract. ADICIÓN DE PLASMA RICO EN PLAQUETAS A LA REPARACIÓN DEL COLGAJO DE AVANCE ENDORRECTAL NO MEJORA LA CURACIÓN DE LAS FÍSTULAS TRANSESFINTERIANAS CRIPTOGLANDULARES: ANTECEDENTES: La reparación con colgajo de avance endorrectal a menudo se realiza para el tratamiento de fístulas transesfinterianas criptoglandulares. Sin embargo, este procedimiento falla en aproximadamente uno de cada cuatro pacientes. Basándose en sus supuestas propiedades curativas, el plasma rico en plaquetas (PRP) podría mejorar el resultado de este procedimiento. OBJETIVO: Evaluar y comparar los resultados a corto y largo plazo después de la reparación con colgajo de avance endorrectal con y sin inyección de PRP en pacientes con una fístula transesfintérica criptoglandular. DISEÑO: Estudio de cohorte retrospectivo. ÁMBITO: Hospital terciario de referencia para proctología en los Países Bajos. PACIENTES: Pacientes consecutivos con fístula transesfintérica criptoglandular. Se utilizó una comparación ponderada por puntuación de propensión inversa para ajustar los factores de confusión y el sesgo de selección. INTERVENCIONES: Reparación del colgajo de avance endorrectal con y sin inyección de PRP. PRINCIPALES MEDIDAS DE VALORACIÓN: Cierre clínico de la fístula dentro de un año sin necesidad de reintervención (cicatrización primaria), cierre clínico de la fístula dentro de un año corregido por reintervenciones (cicatrización secundaria), curación general de la fístula dentro de un año y resultados a largo plazo evaluados mediante un cuestionario. RESULTADOS: En total, 219 pacientes se sometieron a una reparación con colgajo de avance endorrectal. En 88 pacientes (40,2%) se inyectó PRP. No se observaron diferencias significativas en la curación primaria (67,0% frente a 69,5%, p = 0,71), curación secundaria (37,5% frente a 43,5%, p = 0,60) y curación general (73,9% frente a 77,1%, p = 0,58).) entre pacientes con y sin inyección de PRP, respectivamente. El seguimiento a largo plazo estuvo disponible en el 67,1% de los pacientes con un seguimiento medio de 6,8 años (desviación estándar: 3,7 años). Dentro de todos los pacientes que alcanzaron la curación de la fístula, tanto primaria como secundaria, dentro de un año y tenían datos de seguimiento a largo plazo disponibles, las tasas de recurrencia tampoco fueron significativamente diferentes (6,3% vs. 2,9%, p = 0,37). El análisis ponderado por puntuación de propensión mostró que los pacientes tratados con una inyección de PRP no tenían más probabilidades de lograr la curación primaria (odds ratio [OR] 1,0; intervalo de confianza [IC] del 95 %: 0,5 – 1,9), curación secundaria (OR 1,1; IC del 95 % 0,2 – 3,2), curación general (OR 0,9; IC 95 % 0,5 – 1,7) o recurrencia en el seguimiento a largo plazo (OR 1,1; IC 95 % 0,4 – 18,8) en comparación con pacientes sin inyección de PRP. LIMITACIONES: Diseño retrospectivo, falta de imágenes postoperatorias y evaluación del seguimiento a largo plazo mediante un cuestionario. CONCLUSIÓN: La adición de la inyección de PRP no mejora el resultado a corto y largo plazo de la reparación con colgajo de avance endorrectal en pacientes con una fístula transesfintérica criptoglandular tratados en un centro de referencia terciario. (Traducción— Dr. Ingrid Melo)

Background: Myeloid cells are critical determinants of the sustained inflammation in Crohn’s Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. Methods: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14⁺ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. Results: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1β, IL6, and TNFα secretion from healthy donor CD14⁺ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14⁺ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. Conclusions: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.

Background: The PISA-II trial showed that short-term anti-TNF therapy followed by surgical closure induces radiological healing of Crohn’s perianal fistulas more frequently than anti-TNF therapy after 18 months. This study aimed to compare long-term outcomes of both treatment arms. Methods: Follow-up data were collected from patients who participated in the PISA-II trial, an international patient preference RCT. Data were collected until May 2022. Primary outcome was radiological (MRI) healing. Findings: Data were collected from 91/94 patients (36/38 anti-TNF+surgical closure, 55/56 anti-TNF). Median follow-up was 6 years (IQR 5-7). Radiological healing occurred significantly more often after anti-TNF+surgical closure (42% versus 18%; P=0·014). Clinical closure was comparable (72% versus 62%; P=0·183). Results were after re-interventions in 15% versus 53%, including (redo-)surgical closure procedures. Recurrences occurred in 0/25 patients with radiological healing versus 27/76 patients with clinical closure only, sometime during follow-up. Anti-TNF serum concentrations were higher in patients with long-term clinical closure in both groups (P=0·01). In 6/11 patients in the anti-TNF group with available serum concentrations, recurrences were diagnosed within three months of a drop under 3.5ug/ml. 36 patients stopped anti-TNF, after which 0/14 patients with radiological healing developed a recurrence and 9/22 with clinical closure only. Continence was comparable between groups, and 79% of patients indicated comparable/improved continence after treatment. Decision-regret analysis showed that all anti-TNF+surgical closure patients (strongly) agreed that surgery was the right decision versus 78% in the anti-TNF arm. All surgical closure patients would go for the same treatment again, whereas this was 89% in the anti-TNF arm. Interpretation: This study confirms that surgical closure should be considered in amenable patients with Crohn’s perianal fistulas as long-term outcomes are favourable, and that radiological healing should be the aim of treatment as recurrences only occurred in patients without radiological healing. In patients with complete MRI closure, anti-TNF could be safely stopped. Funding Information: None. Declaration of Interests: EMMP, MAJB, KLR, KATGMW, JS, MGWD, and AS have nothing to declare. GRH has received grants from Bristol Meiers Squibb, Pfizer and Takeda, has served as a consultant and/or received speaker’s fees from AbbVie, Alimentiv, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Celltrion Healthcare, Cytoki Pharma, Eli Lilly, Ferring, Galapagos, Glaxo SmithKline, Gossamer Bio, Immunic, InDex Pharmaceuticals, Janssen, Johnson & Johnson, Kaleido, Pfizer, Progenity, Prometheus Biosciences, Prometheus Laboratories, Protagonist Therapeutics, Takeda, Tillotts, Ventyx Biosciences. He furthermore received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Pfizer, Boehringer Ingelheim, Takeda and Abbvie, and participated on a Data Safety Monitoring Board or Advisory Board of Astrazeneca, Abbvie and Seres Health. CYP has received grants from Perspectum and Gilead; consulting fees from Shire, and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Tillotts. KBG has received grants from Pfizer, Galapagos, Celltrion, consultancy fees from AbbVie, Arena Pharmaceuticals, Galapagos, Gilead, ImmunicTherapeutics, Janssen Pharmaceuticals, Novartis, Pfizer Inc., Samsung Bioepis and Takeda, and speaker’s honoraria from Celltrion, Ferring, Janssen Pharmaceuticals, Novartis, Pfizer Inc, Samsung Bioepis, Takeda and Tillotts. SD has received consulting fees from Abbvie, Alimentiv, Allergan, Amgen, AstraZaneca, Athos Therapeutics, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Dr Falk Pharma, Eli Lilly, Enthera, Ferring Pharmaceuticals Inc., Gilead, Hospira, Inotrem, Janssen, Johnson & Johnson, Morphic, MSD, Mundipharma, Mylan, Pfizer, Roche, Sandoz, Sublimity Therapeutics, Takeda, Teladoc Health, TiGenix, UCB Inc., Vial and Vifor. He furthermore received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Abbvie, Amgen, Ferring Pharmaceuticals Inc., Gilead, Janssen, Mylan, Pfizer and Takeda. WAB has received grants from Braun and Vifor, and has served as speaker for Takeda, Johnson and Johnson and Braun. CJB has an unrestricted grant from Boehringer Ingelheim and Roche. She has also received consultancy fees and/or speaker’s honoraria from AbbVie, MSD, Tillotts, Janssen and Takeda. Ethics Approval Statement: The PISA-II2 study received central approval from the Medical Ethical Committee at the Amsterdam University Medical Centers, location University of Amsterdam, and all other participating centres and was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. For the current study, formal approval and individual patient informed consent was waived by the Medical Ethics Review Committee of the Amsterdam University Medical Centers.