Perianal fistulas in Crohn’s disease: new insights on pathogenesis, diagnosis and treatment.

Introduction Perianal fistulizing Crohn’s disease (pCD) develops in 20% and recurs in 30% of Crohn’s disease (CD) patients and typically follows a relapsing and remitting course. (1) The natural course of the disease is highly unpredictable. Most data are derived from outdated population-based studies and historical series. (2-5) After a mean duration of 10 years, 51% of patients with pCD are in stable remission, 9% have persistent drainage or recurrent abscesses and 40% have undergone a proctocolectomy. Predictive factors of disease severity and outcome are mainly related to the presence of proctitis, anorectal stricture and complexity of the fistula. (4, 5) Historically, perianal fistulas have been classified according to the anatomical model of Parks. This refers to the course of the fistula and its relationship to the anal sphincters (7) The American Gastroenterological Association (AGA) proposed a classification system, specifically designed for pCD, dividing fistulas into two categories: simple or complex. Fistula properties according to this classification has prognostic relevance as complex fistulas are less likely to heal than simple fistulas. (8, 9) Pelvic magnetic resonance image (MRI) represents the gold-standard imaging modality for the assessment of fistula characteristics and detection of abscesses, particularly in the setting of complex fistulas. (10) Failure in accurate assessment of the secondary extensions during surgery may be responsible for the high rate of recurrence in the setting of pCD. (11) To date few studies were designed to evaluate the additional clinical value of MRI imaging when dealing with complex CD fistulas. (12) Recently, there is growing interest in a better understanding of the fistula anatomy. Three dimensions fistula models with reconstruction of all anatomical components might serve to further improve surgeon understanding and might have the potential to alter surgical practice and to reduce the rate of recurrences. (13) Pathogenesis of perianal fistulas remains an unexplored area with paucity of recent literature on this topic. On histomorphological level, CD fistulas reveal a central fissure that penetrates in the deeper layers of the of the underlaying gut wall (deep ulcer). Uncontrolled inflammatory response has probably a major role in determining fistula persistence. Epithelial-to-mesenchymal transition (EMT) and matrix remodeling enzymes seem to be the main mechanisms at the basis of such an aggressive disease course. (14) However, other factors such as microbiological environment and epithelialization of the fistula track are possible additional pathogenetic drivers of the onset and perpetuation of CD fistulas. (15) Anti-TNF therapy whether or not in combination with immunomodulators (particularly thiopurines) is to date the cornerstone of medical therapy for patients with complex perianal fistulas, particularly in the presence of proctitis. (16, 17) Infliximab is the only anti-TNF drug with documented label use for pCD. It recently was established that higher doses are needed to obtain fistula regression and healing (unpublished data AMC, Amsterdam). However, efficacy of the medical treatment in pCD is moderate and with healing rates of roughly 50% remain largely unsatisfactory. Modern surgery for fistula tries to address several of the above mentioned pathogenetic aspects by for instance interrupting the connection between the rectal lumen and the fistula complex by closing the internal fistula opening [rectal advancement flap (RAF)], or by disrupting the fistula tract [(Ligation of the intersphincteric fistula tract (LIFT), laser fistula treatment, video assisted ablation of the fistula tract (VAAFT)]. However, probably because of the persistent active perianal inflammation, surgery is prone to fail in a consistent percentage of patients making fistulas in CD a perplexing condition for the colorectal surgeon. (18) Starting from these premises, this thesis will embrace several aspects of the epidemiology, pathophysiology, diagnostics and treatment of pCD. Epidemiologic, translational and clinical research methods will be used. The ultimate objective is to provide a synthesis of the current knowledge and understanding of factors impacting on success/failure of treatment strategies. Projects Project 1 Surgical options for complex Crohn’s disease fistulas: systematic review and meta-analysis. Once active proctitis is treated and after setons placement and withdrawal in absence of active disease in the fistula tract, a definitive surgical repair for complex fistula can be attempted. Due to the complexity of the disease and its recurrent nature, reiterative surgeries can lead to fecal incontinence, and sphincter-sparing techniques are indicated. There are several alternatives that can be used in this situation, and the proper choice depends on the surgeon’s experience and fistula anatomy. RAF, LIFT and, recently, fistula anal plugs, fibrin glue injections, VAAFT and laser fistula treatment are the most popular surgical options to approach complex fistulas in CD. A temporary diverting stoma may be required in case of severe penetrating disease. However, rates of successful establishment of recontinuity are disappointing. Proctectomy can be needed in 8-40% of patients. (2) To date, data describing surgical intervention for Crohn’s anal fistula are heterogeneous and with a high degree of bias. Published papers reporting on results (success rate, treatment failure) of modern surgery for pCD are scarce and underpowered. Comparative studies between the different surgical techniques are lacking. (19) A systematic review aiming at clarifying the outcomes of modern sphincter sparing surgery for Crohn’s disease patients with complex perianal fistula will be performed. LIFT, laser fistula treatment and VAAFT only will be considered in the analysis. The focus will be laid on: - Indications - Association of medical therapy (anti-TNF agents) - Outcomes (response, recurrence, maintenance of healing) Project 2 Exploring the role of stem cells for the treatment of perianal Crohn’s disease. Stem cell-based therapy has emerged as a promising alternative to conventional medical and surgical treatment for tissue regeneration and repair. Mesenchymal stem cells (MSC) are an adherent fibroblast-like population with the capacity to differentiate into various mesodermal cell lines. In general, MSCs display the property to migrate to the site of injury and inflammation suggesting a possible role in tissue repair and regeneration. (20) It has been proposed that the effect of MSC relies on their immunomodulatory and anti-inflammatory properties, through cell-to-cell contact and paracrine secretion of soluble factors, inducing cytoprotective, pro-vascular, anti-inflammatory, and anti-fibrotic effect and finally stimulating endogenous tissue regeneration. (21, 22) The mechanisms underlying these specific effects remain largely unexplained.  MSC were initially isolated from bone marrow, but similar populations have then been found in adipose tissue, placenta, amniotic fluid, and umbilical cord blood. Cx601-darvadstrocel (Alofisel®, Takeda) is a suspension of expanded human allogenic mesenchymal adult stem cells extracted from adipose tissue (expanded adipose stem cells) of a single healthy donor. To date, MSC can only be used under two regulatory statuses in Europe: approved clinical trials or compassionate use programs regulated by the European Medicines Agency. Aim of this project is double: 1. to perform a detailed review on the role of MSC (both autologous and allogenic) for the treatment of pCD 2. to investigate the results of the administration of Cx601-darvadstrocel (compassionate use) at our department. Adult patients (≥18 years) with treatment-refractory CD complex perianal fistulas will be included. - Primary endpoint: combined remission at week 24 and 52 (defined as closure of all treated external openings and absence of collections >2 cm of the treated perianal fistulas confirmed by MRI). - Secondary endpoints: clinical remission (defined as closure of all treated external openings) and clinical response (defined as closure of at least 50% of all treated external opening), relapse and time to relapse at 24 and 52 weeks - Safety endpoints: adverse events, including treatment emergent adverse event (TEAEs), serious TEAEs and deaths Project 3 Exploring the role of inflammation, epithelialization and microbiological factor in onset and persistence of perianal Crohn’s disease. An uncontrolled inflammatory response has probably a major role in determining fistula persistence in perianal Crohn’s disease. Epithelial-to-mesenchymal transition (EMT) and matrix remodeling enzymes seem to be the main drivers for this aggressive basis of such an aggressive disease course. (23) EMT consists of trans-differentiation of epithelial cells into motile mesenchymal cells which migrate and penetrate in different tissues. In CD, some aspects of the EMT seem to be inappropriately activated in response to microenvironmental alterations and aberrant stimuli such as bacterial antigens as consequence of fecal impaction in the fistula tract. (24-26) Other factors such as microbiological environment and epithelialization of the fistula track are possible additional pathogenetic drivers of the onset and perpetuation of CD fistulas. One recent study from St. Mark’s hospital has failed to demonstrate the presence of luminal bacteria in the fistula tract. (27) However, the isolation of peptidoglycan and endotoxin, major components of bacteria cell wall, seems to suggest an effective host response to bacterial inflammation in which bacteria are destroyed but a paroxysmal inflammatory response is driven by these bacteria remnants. Furthermore, symptomatic improvement of pCD after faecal diversion furnish indirect evidence of the possible role of the microbioma in triggering and perpetuating inflammation (28) Antibiotics (ciprofloxacin and/or metronidazole) are effective in the control of acute local septic manifestations, reducing fistula discharge and abscesses. However, they do not result in fistula healing and relapse rates are large upon discontinuation. Epithelialization is thought to be one of the most important determinant in fistula persistence. Variable rates and various degrees of epithelialization along the fistula tract have been reported in literature. (29) However, studies addressing this specific topic are scarce and probably affected by consistent sampling bias. Samples obtained from full fistulectomies in both Crohn’s and idiopathic patients will be used to perform a detailed histopathological examination (fixation, haematoxylin and eosin staining -H&E-, laser microdissection on fresh biopsies). Moreover, a microbiological analysis on samples taken from the fistula tract and from the rectum (luminal biopsies) with either traditional (microbiological culture using selective or differential medium, microscopy, Gram-staining and biochemical tests) and immunological methods (fluorescence in situ hybridization (FISH)) will be performed. A complete characterization of the fistula tract using metagenomics and metatranscriptomics will lead to: a. Assess the presence of bacteria (in particular luminal bacteria) in the fistula tract b. Assess any difference in the bacteria population between Crohn’s and idiopathic (cryptoglandular) fistulas c. Suggest a possible therapeutic action of specific antibiotics Project 4 Incidence, classification and clinical course of symptomatic perianal Crohn’s disease. Analysis of a patient cohort from a tertiary referral center. Penetrating lesions develop in 20% of CD patients and recur in 30% of the cases. (1, 2) The cumulative incidence increases with disease duration, being 12% after 1 year and up to 26% after 20 years from the diagnosis. (3) The probability to develop perianal fistulizing lesions is related to disease location; the likehood is higher, the more distal the intestinal disease is. Perianal fistulas are present in 42% of patients with colonic disease and in 92% of cases with rectal involvement. (2, 4) After a mean duration of 10 years, 51 % of patients are in stable remission, 9% have persistent drainage or abscesses and 40% have undergone a proctocolectomy. (2) Further data suggest that the risk of proctectomy in pCD patients is as high as one in three and that those lesions which do heal take a median of 4 years and 6 interventions to do so. (7) Predictive factors of disease severity and outcome are mainly related to presence of proctitis, anorectal stricture and complexity of the fistula. Active proctitis is recognized as independent factor affecting healing and increasing recurrence rate. (6) Rectal involvement doubles up the risk of proctectomy compared to rectal sparing. (7, 8) An epidemiologic analysis will be performed by examining the clinical course of those patients who developed fistulizing perianal disease in a previously identified Crohn’s disease inception cohort (Leuven, Universitary Hospitals, IBD clinic). Data will be retrieved making a query in KWS (klinisch werkstation) database in the period 2000-2018 (after the introduction of anti-TNF agents). The aim is to determine: 3. Incidence 4. Classification (according to AGA) (31) 5. Time course (with particular focus on the timing between the onset of perianal disease and CD diagnosis) 6. Relationship between perianal and luminal disease (incidence/location) 7. Prognosis of fistulizing pCD. This in order to better delineate the burden of perianal Crohn’s fistulas and the course of the disease in the era of biologics. Project 5 Additional value of three-dimensional reconstruction models for the treatment of complex perianal fistulas. Pelvic MRI should be considered in any primary fistula deemed after clinical assessment to be complex. It should be considered in patients with recurrent fistula. MRI is recognized as ‘gold standard’ for the assessment of anal sepsis. The importance of MRI in this context lies in its ability to demonstrate hidden areas of sepsis and secondary extensions, both of which contribute to the high rate of recurrence after surgery (3). Furthermore, MR imaging can be used to define the anatomic relationships of the fistula to predict the likelihood of postoperative fecal incontinence To date, the most appropriate protocol used for evaluation of perianal fistulas consists of the following sequences: oblique axial T1-weighted FSE, oblique axial T2-weighted FSE, and oblique axial and oblique coronal fat-suppressed T1-weighted FSE with gadolinium-based contrast material, oriented perpendicular or parallel (in the case of the latter) to the long axis of the anal canal. Classically fat-suppressed T2-weighted sequences such as short inversion time inversion-recovery (STIR) or frequency-selective fat-satured T2-weighted FSE have been used to increase the conspicuity of fluid-containing tracks or abscesses. Recently, great interest has raised over the fact that a three-dimensional (3-D) model representation of the fistula tract will improve the surgeon understanding of the fistula anatomy with regard to the relationship with the sphincter complex. (13) From a technical point of view, the 3D imaging technique has several advantages over two-dimensional (2-D) imaging: there is no operator dependence in acquiring images in any obliquity, a larger volume can be covered, thinner sections without intersection gaps can be obtained, a higher signal-to-noise ratio can be achieved, and imaging time can be reduced. The availability of new sequences offers opportunities to improve efficiency and diagnostic capability. Three-dimensional T2-weighted turbo spin-echo (TSE) sequences will provide source data for postprocessing reformation of images into any desired plane. Therefore, a single 3-D T2-weighted sequence with postprocessing reformation of images in the axial, coronal, and sagittal planes will replace 2-D sequences in those three planes, decreasing the number of sequences performed from three to one. Fixed parameters to be reported in the conventional 2-D MRI radiological report will be: a. Position of the internal opening b. Anatomical description of the fistula tract and, whether present, of secondary tracts c. Percentage of the length of the external anal sphincter distal to the fistula tract d. Presence of undrained collections On the basis of this 2-D MRI reports and on the clinical findings a first surgical strategy will be defined after discussion of the case in a multidisciplinary team (radiologist, gastroenterologist and surgeon). Subsequently 3-D reconstructions of the fistula tract will be reviewed independently by two colorectal surgeons. When necessary the surgical strategy will be redefined. Finally, intraoperative findings will be compared to both preoperative MRI 2-D and 3-D findings. Aim of this project will be to evaluate the additional clinical value of pelvic MRI for complex CD fistulas with regard to: a. Spatial orientation for the surgeon b. Identification of all tracts and collections c. Modification of original surgical strategy d. Outcomes (recurrence) Project 6 Role of video assisted ablation of the fistula tract (VAAFT) as symptom control strategy in complex refractory fistulas of both cryptoglandular and Crohn’s origin. Perianal Crohn’s disease is associated with debilitating symptoms and poor quality of life. Video assisted ablation of the fistula tract (VAAFT) is a sphincter-sparing minimally invasive technique comprising diagnostic and operative phases. The diagnostic phase involves visualization from inside of the main and secondary fistula tracts through a rigid fistuloscope. The operative phase employs cautery for fistula tract ablation with various options (suture or stapled closure of the internal opening as well as rectal advancement flap. (32) The role of VAAFT for the treatment of complex fistulas of cryptoglandular origin remains debated with great variations in success rate described in several series. (33-35) Data on Crohn’s disease are scarce. (36) To date, the aim of VAAFT is achieving fistula closure. Recently, the role of VAAFT as a means of symptoms improvement has been investigate in a small cohort of CD patients with therapy refractory perianal penetrating disease. (37) The objective of this project will be to further investigate the role of VAAFT in complex fistula with focus on: - confirmation of the course of the primary/secondary tracts of the fistula obtained in a preoperative phase using 3-D pelvic MRI (project 5) - healing of the fistula (fistula closure) in both cryptoglandular and Crohn’s disease - symptoms amelioration and improvement of quality of life. Recently a core outcome set for fistulizing pCD has been published on behalf of the ENiGMA group from St. Mark’s hospital. (38) Pre and postoperative assessment of quality of life (QoL) will be mainly based on lifestyle restrictions (in general + related to toilet need), depression, inability to attend school/work, restriction of sexual activity and avoidance of intimacy and global assessment of incontinence. Reoperation rate at 30-day together with rate of abscesses formation will be strictly monitored to assess the safety of the procedure.