Clinical and Experimental Studies in Idiopathic and Crohn's-Related Anal Fistula

Clinical and experimental studies in idiopathic and Crohn's-related anal fistula By Philip James Tozer MBBS MRCS Eng MCEM Thesis submitted for the degree of MD(Res) Department of Surgery and Cancer Imperial College London 2011 From The Fistula Research Unit, St Mark’s Hospital Watford Road, Harrow, HA1 3UJ Declaration of originality The following thesis and all studies embodied therein represent my own work including data collection and analysis, specimen retrieval, transport and processing of tissue, walk out, flow cytometry and other immunological techniques, histology, immunohistochemistry and fluorescent in situ hybridisation. Surgical procedures and colonoscopies were undertaken by the clinical staff at St Mark’s Hospital. Electron microscopy was kindly performed by the Centre for Ultrastructural Imaging at Kings College, London, and assistance in multiplex analysis was provided by the Antigen Presenting Research Group, Imperial College London. A proportion of the 3 year MRI data presented (up to 18 months follow up in around three quarters of the patients) were collected before I took over the running of the study. All other work reported in this thesis is appropriately referenced. 2 Abstract The factors leading to the creation and persistence of anal fistula in Crohn’s disease are poorly understood. As with luminal Crohn’s disease genetic, microbiological and immunological factors are implicated but the immunological and microbiological composition of Crohn’s and idiopathic anal fistulae have been obscure. My data demonstrate a lack of clinically relevant organisms within fistula tracts, a luminally driven immune response and subtle differences in this response between Crohn’s and idiopathic fistulae which may provide the basis for diagnostic tests, interventions and further research. Surgical treatment of anal fistula is characterised by a compromise between risk of recurrence and impairment of continence. In complex, recurrent and multiply operated anal fistulae, fistulotomy can still provide a high success rate with low additional risk of impairment of continence. Rectovaginal fistulae are also difficult to manage both surgically and medically. In the infliximab era, successful healing of Crohn’s RVF remains disappointingly rare. Surgery for RVF requires a variety of approaches but remains a valuable tool in the treatment of both Crohn’s and non-Crohn’s tracts. Medical treatment of Crohn’s anal fistulae with combination thiopurine and anti-TNFα agents has demonstrated good short term results. Clinical and radiological data to 3 years follow up demonstrate that around a third of patients maintain healing on infliximab, radiological healing lags behind clinical remission by around a year, and cessation may lead to recurrence in spite of a healed tract on MRI. A treatment for anal fistula with high success and low risk of impairment of continence in complex anal fistulae eludes colorectal surgeons and gastroenterologists. A treatment combining the best aspects of current fistula management with novel elements prompted by improved aetiological understanding must be the goal for fistula surgeons and is the inspiration behind this thesis. 3 1 Table of Contents 2 1 Table of Contents 4 3 1 Introduction 18 3.1 1.1 A background to Crohn’s-related perianal fistulae 19 3.1.1 1.1.1 Historical context 19 3.1.2 1.1.2 The impact of perianal disease on Crohn’s 21 3.1.3 1.1.3 Factors associated with perianal Crohn’s disease 22 3.1.4 1.1.4 Hidradenitis Suppurativa 22 3.2 1.2 Aetiological factors 23 3.2.1 1.2.1 Genetic factors 25 3.2.2 1.2.2 Microbiological factors 26 3.2.3 1.2.3 Immunological factors 28 3.2.4 1.2.4 Aetiological implications of immunosuppressant therapy 31 3.2.5 1.2.5 Comparison with idiopathic fistulae 33 3.3 1.3 Assessing perianal fistulae in Crohn’s disease 34 3.3.1 1.3.1 Fistula classification 34 3.3.2 1.3.2 Assessment of perianal Crohn’s disease 34 3.4 1.4 Medical treatment of perianal Crohn’s disease 36 3.4.1 1.4.1 Corticosteroids 37 3.4.2 1.4.2 Metronidazole and ciprofloxacin 37 3.4.3 1.4.3 Immunomodulators 38 3.4.4 1.4.4 Infliximab 39 3.4.5 1.4.5 Local injection of infliximab 41 3.4.6 1.4.6 Adalimumab 41 3.4.7 1.4.7 Other medical options 43 3.4.8 1.4.8 Monitoring response to therapy 46 3.5 1.5 Surgery for perianal Crohn’s disease 48 3.5.1 1.5.1 Surgery and infliximab 49 4 3.5.2 1.5.2 Lay open 50 3.5.3 1.5.3 Advancement flaps 50 3.5.4 1.5.4 Glues and plugs 51 3.5.5 1.5.5 Modified infill materials 53 3.5.6 1.5.6 Defunctioning and proctectomy 54 3.6 1.6 Fistula-associated carcinoma in Crohn’s disease 55 3.7 1.7 Summarising the current management of Crohn’s anal fistulae 55 3.8 1.8 Idiopathic anal fistulae 56 3.8.1 1.8.1 Background 56 3.8.2 1.8.2 Aetiology 57 3.8.3 1.8.3 Classification 57 3.8.4 1.8.4 Clinical assessment 59 3.8.5 1.8.5 Investigations 60 3.8.6 1.8.6 Surgical treatment 60 3.8.7 1.8.7 Optimal treatment of high anal fistulae 71 3.9 1.9 Rectovaginal fistulae 72 3.9.1 1.9.1 Background 72 3.9.2 1.9.2 Clinical assessment 72 3.9.3 1.9.3 Imaging 73 3.9.4 1.9.4 Treatment 75 3.9.5 1.9.5 Medical treatment of Crohn’s rectovaginal fistulae 75 3.9.6 1.9.6 Surgery 77 3.10 1.10 Summary 84 4 2 The aetiology of anal fistulae 86 4.1 2.1 Abstract 86 4.2 2.2 Introduction 87 4.2.1 2.2.1 Microbiology 87 4.2.2 2.2.2 Immunology 88 5 4.3 2.3 Hypothesis and Aims 89 4.3.1 2.3.1 Hypothesis 89 4.3.2 2.3.2 Aims 89 4.4 2.4 Methods 90 4.4.1 2.4.1 Patient selection 90 4.4.2 2.4.2 Sample collection 90 4.4.3 2.4.3 Surgery 90 4.4.4 2.4.4 Colonoscopy 91 4.4.5 2.4.5 Proctitis 91 4.5 2.5 Microbiology methods - Quantification of Fistula Tract and Anal/Rectal Mucosa-Associated Microbiota using Fluorescent In Situ Hybridisation 92 4.5.1 2.5.1 Patients 92 4.5.2 2.5.2 Anal/rectal and fistula tract sample processing 92 4.6 2.6 Histology and Immunohistology methods 101 4.6.1 2.6.1 Patients 101 4.6.2 2.6.2 Histology sample processing 101 4.6.3 2.6.3 Histological staining 102 4.6.4 2.6.4 Histological assessment 102 4.6.5 2.6.5 Immunohistochemistry sample processing 103 4.6.6 2.6.6 Two dimensional stereology 104 4.7 2.7 Immunology methods 107 4.7.1 2.7.1 Patients 107 4.7.2 2.7.2 FACS sample processing 107 4.7.3 2.7.3 Multiplex sample processing 110 4.7.4 2.7.4 Statistical methods 111 4.8 2.8 Results 112 4.8.1 2.8.1 Patient demographics 112 4.8.2 2.8.2 Reduced bacteria in anal fistula tracts 114 6 4.8.3 2.8.3 Reduced rectal bifidobacteria and Bacteroides in Crohn’s patients 117 4.8.4 2.8.4 Histopathological features of anal fistula tracts 118 4.8.5 2.8.5 Similar levels of acute and chronic inflammation in Crohn’s and idiopathic fistulae 118 4.8.6 2.8.6 Inflammation is greatest near the luminal surface in Crohn’s and idiopathic fistula tracts 119 4.8.7 2.8.7 Higher T lymphocytes in Crohn’s than idiopathic anal fistula tracts 121 4.8.8 2.8.8 Dendritic cells lack homing markers in Crohn’s anal fistula tracts 123 4.8.9 2.8.9 Crohn’s and idiopathic anal fistula contain similar levels of TNF but IL-17a is reduced in Crohn’s compared to idiopathic anal fistulae 123 4.8.10 2.8.10 Associations between bacteria and immunity 123 4.9 2.9 Discussion 124 4.10 2.10 Summary 128 4.11 2.11 Limitations 128 5 3 An audit of the surgical management of anal fistulae: Fistulotomy in the tertiary setting can achieve high rates of fistula cure with an acceptable risk of deterioration in continence 130 5.1 3.1 Abstract 130 5.2 3.2 Introduction 131 5.3 3.3 Hypothesis and Aims 132 5.3.1 3.3.1 Hypotheses 132 5.3.2 3.3.2 Aims 132 5.4 3.4 Methods 132 5.4.1 3.4.1 Patient identification 132 5.4.2 3.4.2 Review of records 132 5.4.3 3.4.3 Database and statistics 133 5.4.4 3.4.4 Surgical procedures 133 5.5 3.5 Results 134 5.5.1 3.5.1 Demographic and baseline disease data 134 7 5.5.2 3.5.2 Fistula anatomy 135 5.5.3 3.5.3 Impairment of continence 136 5.5.4 3.5.4 Recurrence 137 5.5.5 3.5.5 Complications 137 5.6 3.6 Tertiary referral patients 138 5.7 3.7 Complex fistulae 138 5.8 3.8 Inflammatory bowel disease 138 5.9 3.9 Setons 139 5.10 3.10 Conclusions 139 5.11 3.11 Summary 141 5.12 3.12 Future work: a prospective database 141 5.13 3.13 Limitations 142 6 4 An audit of the medical and surgical management of Crohn’s and non-Crohn’s rectovaginal fistulae 143 6.1 4.1 Surgical management of rectovaginal fistulae in a tertiary referral centre: many techniques are needed 143 6.1.1 4.1.1 Abstract 143 6.1.2 4.1.2 Introduction 144 6.1.3 4.1.3 Hypothesis and Aims 148 6.1.4 4.1.4 Methods 148 6.1.5 4.1.5 Results 151 6.1.6 4.1.6 Discussion 157 6.2 4.2 The clinical course of rectovaginal fistula in Crohn’s disease in the infliximab era 159 6.2.1 4.2.1 Abstract 159 6.2.2 4.2.2 Introduction 159 6.2.3 4.2.3 Aims 161 6.2.4 4.2.4 Methods 161 6.2.5 4.2.5 Results 161 6.2.6 4.2.6 Discussion 163 8 6.3 4.3 Limitations 164 7 5 Long-term MRI-guided combined anti-TNF and thiopurine therapy for Crohn’s perianal fistulae 166 7.1 5.1 Abstract 166 7.2 5.2 Introduction 167 7.3 5.3 Hypothesis and Aims 169 7.3.1 5.3.1 Hypotheses 169 7.3.2 5.3.2 Aims 169 7.4 5.4 Methods 169 7.4.1 5.4.1 Patients 169 7.4.2 5.4.2 Study design 170 7.4.3 5.4.3 Radiology 171 7.4.4 5.4.4 Fistula complexity 171 7.4.5 5.4.5 Treatment 171 7.4.6 5.4.6 Clinical follow-up 172 7.4.7 5.4.7 Radiological follow-up 172 7.4.8 5.4.8 Statistical Analysis 173 7.4.9 5.4.9 Ethical considerations 174 7.5 5.5 Results 174 7.5.1 5.5.1 Baseline data 174 7.5.2 5.5.2 Clinical and radiological outcome up to three years 175 7.5.3 5.5.3 Delay between clinical remission and radiological healing 176 7.5.4 5.5.4 Factors influencing clinical and radiological outcome 177 7.5.5

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