The Inflammatory Bowel Disease Yearbook (State of the Art)

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Contents Blood tests in IBD: which are necessary? • Pediatric IBD: novel investigative approaches for diagnosis and follow-up • Progress in IBD genetics • Therapy: purine analogs and methotrexate • Biological therapies • Crohn’s perianal fistulas: current treatment paradigms Praise for The Inflammatory Bowel Disease Yearbook 2004 “This book is an outstanding review of selected topics in IBD. The clear style and the evidence-based approach make this edition an excellent quick reference for the practicing gastroenterologist.” Razvan Arsenescu, MD University of Kentucky College of Medicine

The Inflammatory Bowel Disease Yearbook VOLUME 3

This third volume in the ‘The Inflammatory Bowel Disease Yearbook’ series is a perfect reference guide to the hot topics in IBD. Investigators and critical thinkers from Europe, North America, and Israel unite to clearly communicate the latest research in continually evolving subject areas. Of interest to anyone seeking an update on diagnostic testing, perianal fistulas, pediatric IBD, genetics, and the latest treatment options, this Inflammatory Bowel Disease Yearbook is an essential addition to the gastroenterologist’s bookshelf.

Charles N Bernstein

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State of the Art

The Inflammatory Bowel Disease Yearbook VOLUME 3 Charles N Bernstein, Editor

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The Inflammatory Bowel Disease Yearbook VOLUME 3

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Remedica State of the Art series ISSN 1472-4626 Also available The Handbook of Diabetes Mellitus and Cardiovascular Disease The Inflammatory Bowel Disease Yearbook 2003 The Inflammatory Bowel Disease Yearbook 2004 Kidney Transplantation Management of Atherosclerotic Carotid Disease Multiple Myeloma Osteoporosis Rheumatoid Arthritis Viral Co-infections in HIV: Impact and Management Published by Remedica Commonwealth House, 1 New Oxford Street, London, WC1A 1NU, UK Civic Opera Building, 20 North Wacker Drive, Suite 1642, Chicago, IL 60606, USA Email: [email protected] www.remedicabooks.com Tel: +44 (0) 20 7759 2999 Fax: +44 (0) 20 7759 2951 Publisher: Andrew Ward In-house editors: Carolyn Dunn and Cath Harris Design: AS&K Skylight Creative Services While every effort is made by the publisher to see that no inaccurate or misleading data, opinions, or statements appear in this book, they wish to make it clear that the material contained in the publication represents a summary of the independent evaluations and opinions of the editor and contributors. As a consequence, the editor, authors, publisher, and any sponsoring company accept no responsibility for the consequences of any inaccurate or misleading data or statements. Neither do they endorse the content of the publication or the use of any drug or device in a way that lies outside its current licensed application in any territory. © 2006 Remedica All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. Remedica is a member of the AS&K Media Partnership. ISBN-13: 978 1 901346 88 6 ISBN-10: 1 901346 88 9 British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

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The Inflammatory Bowel Disease Yearbook VOLUME 3 Charles N Bernstein, MD, Editor Professor of Medicine Head, Section of Gastroenterology Director, Inflammatory Bowel Disease Clinical and Research Centre University of Manitoba Winnipeg, Manitoba, Canada

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Authors Devendra K Amre, MD, PhD Research Centre, Sainte-Justine Hospital Division of Gastroenterology and Nutrition Department of Pediatrics University of Montreal Montreal, Quebec, Canada Steven R Brant, MD Associate Professor of Medicine Meyerhoff Inflammatory Bowel Disease Center Gastroenterology Division, Department of Medicine Johns Hopkins University School of Medicine Baltimore, MD, USA Amir Karban, MD Department of Gastroenterology Rambam Medical Center Bruce Rappaport Faculty of Medicine Technion, Haifa, Israel Paul Rutgeerts, MD, PhD, FRCP Department of Medicine Division of Gastroenterology University Hospital Gasthuisberg Leuven, Belgium

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William J Sandborn, MD Professor of Medicine, Mayo College of Medicine Head, IBD Interest Group and Director IBD Clinical Research Unit Mayo Clinic, Rochester, MN, USA Jürgen Schölmerich, MD Department of Internal Medicine I University Medical Center Regensburg Regensburg, Germany David A Schwartz, MD Division of Gastroenterology Vanderbilt University Medical Center Nashville, TN, USA Ernest G Seidman, MD Bruce Kaufman Chair of IBD Professor of Medicine and of Pediatrics Research Institute of the McGill University Health Centre Faculty of Medicine, McGill University Montreal, Quebec, Canada

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A Hillary Steinhart, MD, MSc, FRCP(C) Associate Professor of Medicine Division of Gastroenterology Mount Sinai Hospital/University Health Network University of Toronto Toronto, Canada Gert Van Assche, MD, PhD Department of Medicine Division of Gastroenterology University Hospital Gasthuisberg Leuven, Belgium Severine Vermeire, MD, PhD Department of Medicine Division of Gastroenterology University Hospital Gasthuisberg Leuven, Belgium

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Preface In this third volume of the ‘Inflammatory Bowel Disease Yearbook’ series we have assembled another stellar roster of investigators and critical thinkers in IBD. Once again we have tackled hot topics that are important to both clinicians and researchers. The first two chapters review diagnostic testing in adults and children. Hillary Steinhart of the University of Toronto has reviewed the continually evolving area of blood testing and fecal markers of inflammation. Clinicians are keen to have markers that will help them diagnose patients noninvasively or to help them subgroup patients in a meaningful way. Ernest Seidman of McGill University and Devendra Amre of the University of Montreal have reviewed the evolving epidemiology and both blood- and imaging-related diagnostic testing in pediatric IBD. Through studying evolving trends in pediatric disease epidemiology and disease presentation, etiologic clues will emerge; perhaps to a greater extent in studying newly diagnosed children than adults. In Chapter 3, Steven Brant of Johns Hopkins University and Amir Karban of Technion University have comprehensively reviewed the state-of-the-art of IBD genetics. This chapter updates the reader on genes identified to be significantly associated with IBD but also on the potential function of these genes and how mutations in these genes might plausibly have a role in IBD. In Chapter 4, Severine Vermeire, Gert Van Assche, and Paul Rutgeerts of the University of Leuven have updated the use of azathioprine, 6-mercaptopurine, and methotrexate in both

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Crohn’s disease and ulcerative colitis. They have reviewed novel concepts regarding mechanisms of action of these agents and best strategies for drug administration and monitoring. In Chapter 5, Juergen Schölmerich of the University of Regensburg has provided a concise but comprehensive review of the current state-of-the-art regarding biological therapies in IBD. The excitement for these agents has been somewhat tempered by the number of agents that have not been proven to have clinically relevant effects. Prof. Schölmerich also reviews the concept of using CRP (a blood marker also reviewed by Dr. Steinhart) as a guide for clinical trial inclusion and possibly even therapy in the clinic today. In Chapter 6, David Schwartz of Vanderbilt University and William Sandborn of the Mayo Clinic School of Medicine comprehensively review perianal fistulas in Crohn’s disease including anatomical issues related to perineal fistulas and the diagnostic, medical, and surgical management of this difficult problem. On behalf of the authors, I hope you enjoy this Yearbook and find it to be an important updating resource in the ever-changing world of IBD. Charles N Bernstein, MD University of Manitoba Inflammatory Bowel Disease Clinical and Research Centre Winnipeg, Manitoba, Canada

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Contents Chapter 1: Blood tests in IBD: which are necessary? A Hillary Steinhart

1

Chapter 2: Pediatric IBD: novel investigative approaches for diagnosis and follow-up Ernest G Seidman and Devendra K Amre

25

Chapter 3: Progress in IBD genetics Steven R Brant and Amir Karban

41

Chapter 4: Therapy: purine analogs and methotrexate Severine Vermeire, Gert Van Assche, and Paul Rutgeerts

91

Chapter 5: Biological therapies Jürgen Schölmerich

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Chapter 6: Crohn’s perianal fistulas: current treatment paradigms David A Schwartz and William J Sandborn

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Abbreviations

195

Index

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1 Blood tests in IBD: which are necessary? A Hillary Steinhart

Introduction Patients with inflammatory bowel disease (IBD) typically undergo a wide range and large number of investigations throughout the course of their disease. These investigations can range from the simple, noninvasive, and inexpensive to the more complex, invasive, or expensive. The need for repeated investigations and their associated discomfort, inconvenience, and embarrassment can weigh heavily on the minds of patients and have a negative impact upon their quality of life. The most frequently performed investigations are blood tests. Although venipuncture is not a terribly invasive procedure and most analyses are not overly expensive, it is still important to consider which blood tests have value in the evaluation of IBD patients or patients suspected of having IBD and which do not. In general, blood tests that are performed in patients with IBD or suspected IBD can be divided into: •

those that are used to screen for or diagnose disease

•

those that monitor disease activity

•

those that diagnose or monitor disease complications, or predict natural history

•

those that predict or monitor drug response, adverse effects, or toxicity

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Diagnostic blood tests Traditionally, the diagnosis of IBD (either Crohn’s disease [CD] or ulcerative colitis [UC]) has been based upon clinical presentation combined with radiologic, endoscopic, histologic, and, occasionally, surgical evaluation. As a result of the invasive nature of some of these investigations or their associated radiation exposure, there has been a move to develop simple, noninvasive blood tests to either screen for the presence of IBD in patients, or differentiate between CD and UC in patients known to have IBD, but in whom the exact phenotype is not known. The first situation in which blood tests can be useful involves the investigation of patients who present with undiagnosed symptoms that could be compatible with IBD. This approach may be particularly useful in pediatric patients where invasive diagnostic testing might be deferred, and where, as a result, the diagnosis of IBD might be delayed by months from the onset of symptoms. Diagnostic delays can be problematic from the perspectives of nutrition and growth, in particular. The blood tests most commonly used when screening undiagnosed symptomatic individuals are the: •

complete blood count (CBC)

•

serum albumin (SA) level

•

erythrocyte sedimentation rate (ESR)

•

C-reactive protein (CRP) level

These are all easily available and relatively inexpensive. Additional tests, such as for the perinuclear anti-neutrophil cytoplasmic antibody (pANCA) and the anti-Saccharomyces cerevisiae antibody (ASCA), might be less readily available and

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Blood tests in IBD

are certainly more expensive [1,2]. They are discussed in more detail later. Other specialized blood tests have been studied, but are still considered experimental for this purpose [3–5]. Testing for the most common mutations of the NOD2/CARD15 gene (which have been found to be associated with an increased risk of developing IBD, see Chapter 3) might be available in some centers, but is still considered investigational for use as a diagnostic screening test. The clinical decision of whether to pursue further confirmatory diagnostic testing following an abnormality in one or more of hemoglobin, white blood cell count (WBCC), platelet count (PC), SA, ESR, or CRP generally depends on the pre-test likelihood of IBD based on history and physical examination, and the sensitivity and specificity of the test in question. The operating characteristics of the CBC elements, ESR, and SA are not completely known, but some more recent studies of CRP have attempted to define its sensitivity and specificity [6,7]. Little is known about the use of various tests in combination, or the effect this has on sensitivity and specificity. Complete blood count Anemia, elevated WBCC, and elevated PC can all be markers of a chronic inflammatory disorder such as IBD. However, taken individually any one of these indicators is unlikely to be sufficiently sensitive to serve as an appropriate screening tool for IBD. There are numerous situations unrelated to IBD in which these values may be abnormal (ie, low specificity) and their utility as a confirmatory diagnostic tool is, therefore, likely to be poor. The same arguments apply to ESR and SA, although a study of previously undiagnosed patients being assessed for abdominal symptoms at St Mark’s Hospital, UK, found that an elevated ESR level was present in 100% of patients with CD and 50% of patients with UC, which could be used to successfully diagnose IBD in conjunction with rectal biopsy and CRP level [8].

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A study from the Birmingham Children’s Hospital, UK, found that a combination of hemoglobin and PC had very high sensitivity (90.8%) and positive predictive value (94.4%) in a population of 153 children referred to a pediatric gastroenterology practice for the evaluation of possible IBD [6]. The investigators considered an abnormality in at least one out of two of the laboratory tests to be a positive test. Of the 153 children, 103 were found to have IBD following diagnostic work-up (60 CD, 37 UC, 6 indeterminate colitis). This high proportion suggests that IBD was highly likely or known to exist based upon the suspicions of the referring physician (eg, severe or typical symptoms) or based upon investigations performed prior to referral. As such, the sensitivity of the assay is likely to be an overestimate of the true value when used in the general pediatric population. C-reactive protein CRP is an acute-phase reactant, produced in increased amounts in the liver in response to a variety of inflammatory and neoplastic conditions. There are some data on the utility of CRP as a screening tool in individuals with gastrointestinal symptoms that could be suggestive of IBD [6–9]. In the study from St Mark’s Hospital described earlier, investigators assessed the performance characteristics of several laboratory indicators in 82 patients being assessed for undiagnosed abdominal symptoms [8]. All 19 patients who were found to have CD and 11 of the 22 patients with UC had elevated ESR and CRP. None of the patients with functional bowel disorders were found to have elevated ESR and CRP. In another study, 91 children presenting with ≥3 months of symptoms suggestive of IBD were evaluated [9]. Following complete work-up in all children, which included blood tests, colonoscopy, and small bowel follow-through, 42 were diagnosed with IBD (26 CD, 13 UC, 3 indeterminate colitis) and 37 had normal investigations. The remaining 12 had a variety of other diagnoses, including eight with polyps, two with tuberculous colitis, and two with nodular lymphoid hyperplasia. An elevated

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CRP level was present in all of the patients who were found to have CD, in eight out of 13 patients who were found to have UC, and in none of those who were found to have polyps or normal ileocolonoscopy and small bowel follow-through. In a similar study in adults, 203 new out-patients referred to a gastroenterology clinic for suspected lower bowel disorder were assessed [7]. Investigators performed a highly sensitive enzyme-linked immunoabsorbent assay test for CRP, and all patients underwent rigid sigmoidoscopy and rectal biopsy. Final diagnosis was determined by follow-up at 6 months. As a result, not all patients underwent complete investigation of the large and small intestine. By 6 months, a total of 20 patients were found to have IBD (13 CD, 7 UC). CRP worked reasonably well as an indicator of IBD, with a sensitivity of 70% when a cut-off of 5 mg/L was used. When a cut-off of 2.3 mg/L was used, CRP had a sensitivity of 100% and a specificity of 67% in differentiating IBD from functional bowel disorders. ASCA and pANCA Screening for IBD Since IBD is often considered an autoimmune disorder there has been great interest in finding associated autoantibodies. These can not only provide insight into disease pathogenesis and genetic heterogeneity, but can also be utilized for noninvasive diagnostic tests. Several of these autoantibodies, such as antilactoferrin, anti-cathepsin G, and anti-epithelial cell antibodies have not been found to be useful in the diagnosis of IBD. pANCA was first described in 1990, when initial reports found it to be present in 59–84% of patients with UC and 10–20% of CD patients [10,11]. Around the time that pANCA was first described in association with UC, the presence of ASCA was found in the serum of 63% of CD patients [12,13]. The use of these serologic tests was subsequently extended to the screening of symptomatic but undiagnosed individuals for the presence of IBD. Using a lower cut-off level than is

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generally used for differentiating CD from UC, a combination of the pANCA and ASCA tests (IBD First Step®, Prometheus Laboratories, San Diego, CA, USA) was reported to have a sensitivity of 81% and a specificity of 72% for IBD in a cohort of pediatric patients presenting with symptoms or signs of possible IBD [1]. The authors recommended that patients with a positive test undergo repeat testing using a higher and more specific cut-off level (IBD Diagnostic System, Prometheus Laboratories), and those who have a positive result using the higher cut-off undergo further diagnostic testing. In that study, the prevalence of IBD was relatively high in the study cohort (40%), suggesting the possibility of referral bias. A lower prevalence of disease in other populations would result in a higher negative predictive value for ruling out disease, but at the cost of a lower positive predictive value. It is also interesting to note that 91% of patients who were found to have IBD had an abnormality of one or more of hemoglobin, PC, ESR, or SA. This would suggest that there may be little to gain by adding ASCA and pANCA to these other simple and inexpensive blood tests. Differentiating between CD and UC The use of pANCA and ASCA to differentiate CD from UC might be more effective than their use as a screening panel for IBD. One of the earliest studies of ASCA reported an immunoglobulin (Ig)G subtype prevalence of 63% in CD patients, 15% in UC patients, and 8% in healthy controls [12]. The same study reported an IgA subtype prevalence of 43% in CD patients but 0% in UC patients. There are a number of different pANCA and ASCA assays that are available. These have various reported sensitivity and specificity, in part related to different cut-off levels used by the different assay systems. Although agreement among the assays is relatively good, direct comparison of absolute antibody levels is problematic and might not be possible [14]. For example,

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wide variation in the sensitivity of pANCA, from 0% to 63%, has been reported from five different laboratories using the same serum samples [15]. Given their apparent complementary pattern of positivity it has been suggested that the combination of pANCA and ASCA tests would provide a precise diagnostic tool with which to differentiate CD from UC. A combination of a positive pANCA and negative ASCA test has generally been associated with a diagnosis of UC, and a negative pANCA and positive ASCA test has been associated with a diagnosis of CD [2,15,16]. However, the ranges quoted in the literature for sensitivity, specificity, and predictive values have frequently been based upon studies in patients with known CD or UC. The patient populations in whom these tests would be particularly useful are those who have only colonic inflammation and in whom the clinical, radiologic, endoscopic, and histologic features are not diagnostic or are indeterminate in nature. In a study by Quinton et al., 61% of patients with CD were positive for ASCA. That proportion fell to only 46% in those with colonic disease only, suggesting that the presence of ASCA might not be a good discriminant tool in patients with colonic disease only [16]. Other studies have confirmed the association of ASCA primarily with ileal CD [12,17–21]. In one study, all of the patients who were ASCA-positive had ileal disease [20]. If this is borne out by other studies it would mean that ASCA is not useful in differentiating CD from UC, since the diagnosis in ASCA-positive patients will always be possible based on clinical grounds through the detection of small intestinal disease. Unfortunately, most studies have not reported results specifically in the group of patients with colonic inflammation only but, rather, have included many patients with confirmed CD with small intestinal involvement.

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A study by Joossens et al. examined the operating characteristics of ASCA and pANCA in a cohort of 97 patients with indeterminate colitis and no small intestinal involvement [22]. Blood was obtained for ASCA and pANCA determination at study inception, and investigators at three participating European centers followed the patients for a minimum of 1 year. Overall, 31/97 (32%) patients developed clinical, endoscopic, or histologic features diagnostic of either CD or UC, although definite UC was only diagnosed on surgical specimens. Of the 26 patients with a pANCA-negative and ASCA-positive serologic pattern, 10 received a definite diagnosis, of which eight had CD and two had UC. Of the 20 patients who were pANCA-positive and ASCA-negative, 11 developed a definite diagnosis, of which seven had UC and four had CD. Of note, almost half of the patients with indeterminate colitis had negative serologic markers, and over 80% of these did not develop a definitive diagnosis of either CD or UC over the study duration. Although the serologic patterns, when positive, demonstrated a tendency to predict a specific diagnosis, they were not sufficiently sensitive to provide much guidance in the management of patients with indeterminate colitis. Most studies have confirmed the relatively high specificity and low sensitivity for the serologic patterns using this combination of tests, suggesting that they might be more suitable for confirming a diagnosis rather than for screening. It is likely that serologic patterns within the IBD population will help to further refine phenotypic subtypes, and could possibly provide insights into pathogenic mechanisms.

Monitoring disease activity Endoscopy For most clinicians, the primary means of monitoring disease activity and response to therapy is to monitor changes in patient symptoms and physical signs. Endoscopy can be used to monitor

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response to therapy in UC patients, but although a well-described endoscopic scoring system has been developed for CD, endoscopic monitoring of disease activity is not commonly used in practice or in clinical trials. Blood tests Blood tests have tended to play an adjuvant role in evaluating disease activity and response to therapy, and have not been the main criterion used in making management decisions. However, due to the subjective nature of clinical assessments there has been a desire to develop simple, noninvasive, and objective measures of disease activity, both to monitor response to therapy and to predict clinical relapse in asymptomatic patients in remission. Several of the blood tests that are used to screen for IBD in undiagnosed patients with gastrointestinal symptoms appear to also have some utility in monitoring response to therapy. The tests that appear to be most responsive to change in clinical status or predictive of relapse in patients with clinical quiescent disease are CRP level in serum, WBCC, and PC [23–25]. The correlation between clinical disease activity and serum markers such as CRP is not perfect. A recent study from the Mayo Clinic, USA, found that serum CRP did not correlate well with clinical disease activity, but did seem to correlate with endoscopic and histologic appearance in CD, and with histologic appearance in UC [26]. A previous study found that approximately one-third of patients with quiescent clinical disease activity had elevated CRP, while one-third of those with active clinical disease had normal CRP [25]. Part of this lack of correlation might be due to the fact that clinical symptoms do not always correlate terribly well with underlying intestinal inflammation, which is better reflected by serum markers such as CRP. In addition, symptoms in CD patients can also be due to intestinal scarring, stenosis, or short bowel rather than active

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intestinal inflammation, and this can be responsible for the normal levels of serum markers in individuals with apparent clinical disease activity. The lack of correlation might also be a reflection of genetic heterogeneity in the determination of CRP response to inflammation. In one study, polymorphisms in the promoter region of the tumor necrosis factor (TNF)-α gene predicted whether CD patients had a correlation between disease activity and CRP levels [27]. Other blood tests that have been studied, such as hemoglobin and SA, might reflect underlying inflammatory disease or malnutrition, but might not be as specific nor as responsive to acute changes in clinical status or inflammatory activity. Although simple, inexpensive blood tests such as CRP, ESR, PC, and WBCC are commonly used by clinicians treating patients with IBD, it is unlikely that these tests will replace clinical assessment of disease activity as the ‘gold standard’ in making management decisions. However, these, and other newer blood tests, may provide additional evidence with respect to the effect of interventions on intestinal inflammation, particularly in the setting of clinical trials. Simple, inexpensive blood tests might become more important for clinicians if it can be demonstrated that continuation or escalation of treatment to normalization of blood tests (and not simply improvement in clinical symptoms) results in more sustained remission or improvement in long-term outcomes. Until such time, these investigations are likely to continue to be an adjunct to clinical decision making. Serum transglutaminase A study performed in 249 UC patients found that serum transglutaminase (factor XIIIa) levels correlated (as measured by the modified Rachmilewitz activity index) with clinical disease activity (r=–0.63, P<0.01). Factor XIIIa levels also correlated with endoscopic (r=–0.71, P<0.001) and histologic (r=–0.79, P<0.001)

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measures of inflammation, suggesting that this could be used as a noninvasive means of monitoring disease activity [28]. α Tumor necrosis factor-α TNF-α is an important proinflammatory cytokine whose key role in CD has been clinically demonstrated by the response of CD symptoms to treatment with anti-TNF-α antibodies [29]. TNF-α is thought to have some role in promoting intestinal inflammation in UC since recent studies have suggested similar outcomes at 1 year in UC as for Crohn’s disease [30]. At least one study has shown that the degree of disease activity in UC correlates with serum levels of TNF-α (P<0.05) [31]. Tenascin C Investigators in Germany have examined the relationship between serum levels of tenascin C, a matrix protein that is induced by inflammation and neoplasia and found to be correlated with tissue injury and remodeling in colitis, and clinical disease activity [32]. They found that the mean serum level of tenascin C was significantly higher in UC patients just prior to proctocolectomy than in familial adenomatous polyposis controls. They also found that levels in patients with active, nonsurgically treated UC and CD correlated with disease activity. This suggests that tenascin C is not specific to UC or CD, but is rather a general indicator of mucosal inflammation.

Predicting natural history and monitoring for complications Simple blood tests A Dutch study of patients with severe acute UC found that simple laboratory blood tests such as CRP, ESR, PC, and WBCC were poor predictors of clinical course and disease outcome, although disease activity did correlate with these measures [33]. A study of patients with severe UC from Oxford suggested that a CRP level >45 mg/L might help predict the need for colectomy [34]. However, the investigators also found that

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simple clinical measures such as more than eight bowel motions per 24 hours predicted the need for surgery. Antibody markers In CD, the presence of the IgA ASCA has been found to be strongly associated with the need for early surgery in both ileal and ileocolonic disease (P=0.0013) [35]. Mow et al. examined the predictive value of ASCA, as well as the serum markers pANCA, anti-ompC (Escherichia coli outer-membrane porin protein C), anti-I2 (Crohn’s disease related sequence from Pseudomonas fluorescens), and the three most common mutations of the NOD2/CARD15 gene [36]. They found that the presence of anti-I2 was associated with fibrostenosing disease and a need for surgery (P<0.001), whereas the presence of anti-ompC was associated with internal perforations (P<0.001). Overall, 72% of patients with the presence of at least one of ASCA, anti-ompC, or anti-I2 required surgery compared with 23% of those who were negative for all three markers (P<0.001). Higher titers of antibodies were also associated with a greater need for surgery (P<0.001). The potential identification of independent risk factors for early surgery might be very useful in patient management by allowing the selection of patients for aggressive medical management from the onset of disease. Nutritional deficiencies IBD patients are at risk for developing several disease-related complications; these can be monitored by means of periodic blood tests. Nutritional deficiencies such as those for iron, vitamin B12, calcium, vitamin D, and zinc are among the most common deficiencies seen in IBD patients, particularly those with CD. Monitoring by means of blood tests should be considered in all patients, but well-defined evidence-based guidelines for the types and frequency of testing do not exist. In the absence of such evidence it seems reasonable to individualize monitoring according to the patient’s disease type, extent, severity, and treatment.

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For example for a patient with only colonic disease, whether Crohn’s colitis or UC, vitamin B12 deficiency would not be expected because of the lack of involvement of the ileum; the area of absorption of vitamin B12. However, for a patient with previous ileal resections or known ileal CD, periodic measurement of serum vitamin B12 level would be indicated. Liver enzymes Hepatic complications of IBD, such as sclerosing cholangitis, can be detected before the onset of symptoms by monitoring liver enzymes. However, there is no evidence that earlier detection of abnormal liver enzymes in asymptomatic individuals, or making an earlier definitive diagnosis and intervention, changes the course of the disease. The knowledge that an individual has sclerosing cholangitis does permit counseling and heightens awareness of the potential complications of this liver disease.

Predicting response to therapy Response to therapy varies widely among IBD patients. Although clinical trials can provide reasonable estimates regarding the likelihood of response to a given treatment in a given patient population, it would be of great benefit if the likelihood of response could be determined for individuals. Response to therapy can be assisted by demographic or phenotypic characteristics such as the disease severity, location, and the presence of complications such as strictures or abscesses. These items are readily available. In addition, there has been considerable interest in developing blood tests to help predict treatment response (Table 1). Infliximab A study by Esters et al. examined the relationship between the presence of pANCA or ASCA and response to infliximab in a cohort of 279 patients, who received the drug for either inflammatory intestinal or fistulizing disease [37]. Esters et al. found no overall relationship between the presence or absence

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Diagnostic

Disease activity

Complete blood count

X

X

CRP

X

X

pANCA/ASCA

X

Natural Response/ history/ intolerance prognosis/ to therapy complications

Blood tests

Serum transglutaminase

X

TNF-α

X

X X

X

X

X

Anti-ompC

X

Anti-I2

X

NOD-2/CARD-15 mutations

X

Liver enzymes

X

IL-1 receptor antagonist allele 2

X

TPMT enzyme activity

X

TPMT genotype

X

6-TGN

X

Fecal tests Lactoferrin

X

X

X

Calprotectin

X

X

X

Table 1. Diagnostic tests in IBD. 6-TGN: 6-thioguanine nucleotide; ASCA: anti-Saccharomyces cerevisiae anibody; CARD-15: caspase recruitment domain-15; CRP: C-reactive protein; I2: Crohn's disease related sequence from Pseudomonas fluorescens; IL: interleukin; NOD-2: nucleotide-binding oligomerization domain; ompC: outer membrane porin protein C; pANCA: perinuclear anti-neutrophil cytoplasmic antibody; TNF-α: tumor necrosis factor-α; TPMT: thiopurine methyl transferase.

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of antibody in serum and response to therapy, but subgroup analysis demonstrated a trend towards a lower response rate in patients with refractory intestinal disease who were positive for pANCA and negative for ASCA. Out of 10 patients with that serologic profile, five (50%) responded. This compared with 61/75 (81.3%) of those who were pANCA-negative and ASCApositive, and 74/92 (80.4%) of those who were negative for both markers (P=0.067). It is possible that the lower response in the pANCA+/ASCA– group might simply be a reflection of the fact that many of those patients had UC, which might not respond as well to infliximab as CD [38]. Pierik et al. found no relationship between specific alleles of the TNF receptor 1 or TNF receptor 2 genes and response to infliximab therapy in 166 CD patients [39]. Blood tests might also help to predict the potential for complications or adverse effects of therapeutic interventions. Carter et al. prospectively followed 82 patients undergoing ileal pouch anal anastomosis surgery for UC. They found an association between allele 2 of the interleukin-1 receptor antagonist gene and the occurrence of pouchitis, with a calculated adjusted odds ratio (OR) of 6.5 [40]. The presence of allele 2 also seemed to be associated with chronic refractory colitis rather than acute severe colitis prior to surgery, although the OR of 1.9 was not statistically significantly different than 1. The finding of an association between this particular allele and pouchitis has not yet been replicated by others. Azathioprine and 6-mercaptopurine Azathioprine (AZA) and 6-mercaptopurine (6-MP) are analogs of the purine nucleotides that are critical to the process of DNA synthesis. These analogs interfere with DNA synthesis and, as a result, interfere with the replication of various cell lines such as white blood cells. The development of assays for the enzyme responsible for the metabolism of 6-MP and AZA, and genotyping for variations in the gene that determines enzyme

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activity, have led to the commercialization of these tests for the purposes of predicting which patients are at risk of drug toxicity. Therefore, these tests can allow avoidance of the drug, or encourage the use of lower doses in certain individuals. Low or absent levels of the thiopurine methyl transferase (TPMT) enzyme are associated with an increased risk of bone marrow suppression and leucopenia. This is because when TPMT activity is reduced, 6-MP is no longer converted to 6-methyl mercaptopurine (6-MMP) and a competing enzyme, hypoxanthine guanine phosphoribosyltransferase, increases production of the active metabolite 6-thioguanine nucleotide (6-TGN). Low TPMT enzyme activity generally occurs in individuals with one or two mutant, or variant, alleles of the TPMT gene and correlates with higher 6-TGN levels [41]. In a study of 30 IBD patients given 6-MP, the TPMT genotype was found to correlate with 6-TGN levels and to predict the development of leucopenia (relative risk 12.0, 95% confidence interval [CI] 1.7–92.3) [42]. Unfortunately, the presence of normal levels of TPMT or the presence of the wild-type alleles of the TPMT gene do not guarantee that intolerance to AZA or 6-MP will not occur [43,44]. In a study from New Zealand, only 6/50 (12%) IBD patients who demonstrated intolerance to AZA had one or more of the two common TPMT mutant alleles, TPMT*1 and TPMT*3 [43]. In that same study, a group of 50 AZA-tolerant patients were examined and variant alleles were found in three patients (6%). Colombel et al. examined the TPMT genotypes of 41 patients who developed myelosuppression during AZA or 6-MP therapy [45]. The study found that 11/41 patients (73%) did have one of the common variant alleles. On the other hand, it appears that low levels of the enzyme (as seen in heterozygotes for the mutant allele) do not necessarily predict the occurrence of leucopenia, but that the presence of two variant alleles is predictive of some increased risk of serious leucopenia and pancytopenia.

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In the study from New Zealand, one patient was homozygous for a mutant allele (TPMT*3/TPMT*3 genotype) – that particular individual developed pancytopenia requiring hospitalization. It appears that intermediate levels of TPMT activity, as predicted by the presence of a single mutant allele, might be associated with a higher likelihood of intolerance to AZA (50%) than normal or high levels of TPMT activity, as seen in patients who are homozygous for the wild-type alleles (16% intolerance) [41]. Measuring low TPMT enzyme activity Cuffari et al. followed 142 consecutive IBD patients treated with AZA or 6-MP and measured TPMT enzyme activity prior to initiation of therapy in 41 patients with active disease [46]. They found that patients with low TPMT activity, as defined by a level <12 U/mL blood, had higher mean 6-TGN levels and a higher rate of response as compared with those with TPMT activity >12 U/mL blood (69% vs 29%, P<0.001). Although it might predict a higher response rate to acute therapy, the presence of low or intermediate levels of TPMT activity appears, in several studies, to be associated with a higher risk of early myelosuppression. It would, therefore, seem reasonable to suggest that if it is known that a patient has intermediate TPMT activity prior to initiation of AZA therapy, a low dose (1–1.5 mg/kg/day) should be used until tolerance is demonstrated [47]. In those with absent or low TPMT activity the use of AZA or 6-MP is best avoided, although a case has been reported where a TPMT-deficient patient was safely treated with AZA [48]. The need for routine TPMT activity or genotype testing prior to initiation of therapy continues to be controversial. There appears to be little question that the presence of two mutant alleles and/or absent or very low enzyme activity puts an individual at significantly increased risk for leucopenia or pancytopenia.

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The prevalence of absent TPMT activity is estimated to be between 1:220 and 1:330 of the population [49–51]. Given the potential seriousness of the bone marrow toxicity, however, routine genotype-based TPMT testing might be reasonable despite this relatively rare occurrence. This is based in part upon the fact that cost-effectiveness modeling in a population of patients with rheumatoid disorders has shown TPMT testing to be more effective and less costly than a routine dosing strategy [52]. However, it should be recognized that normal or high TPMT levels do not guarantee that important side effects will not occur with long-term therapy, so ongoing monitoring of blood counts is still necessary [45,53]. Measuring 6-thioguanine nucleotide level The measurement of the active metabolite of 6-MP and AZA (6-TGN) might help with the appropriate dosing of these medications. In a study of 92 pediatric patients receiving 6-MP, response to therapy was more likely to occur if the 6-TGN level was >235 pmol/8×108 erythrocytes [54]. In that study, leucopenia was also found to be associated with higher 6-TGN levels. In another study, Cuffari et al. suggested that the cut-off for response should be 292 pmol/8×108 erythroctyes, with a positive predictive value for response of 85.7% with this cut-off level [46]. Measurement of purine analog metabolite levels may help to determine the reason why some patients have a suboptimal response to AZA. In a large survey of thioguanine metabolite panels from 9,187 patients from a single laboratory in the US, 3% of patients were found to be noncompliant, and 46% were under-dosed [49]. Dubinsky et al. have suggested that 6-TGN levels can be used to guide dose escalation in nonresponders [55]. Only 14/51 patients who had not responded to 6-MP because of low 6-TGN levels achieved remission when the dose was escalated and there was a small but significant increase in 6-TGN levels (P=0.046). However, dose escalation resulted in a significant increase in the metabolite 6-MMP. This increase was associated with hepatotoxicity in 12 patients (24%) [55].

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Another study from the University of Manitoba evaluated the utility of measuring purine metabolites in patients on AZA and 6-MP therapy [56]. They found that 6-TGN levels did not correlate well with either remission or WBCC. However, low levels of both 6-TGN and 6-MMP indicated patient noncompliance. Fecal lactoferrin and calprotectin There has been considerable interest in the development of stool tests to assess inflammatory disease activity in patients with IBD because of their noninvasive nature and because markers in stool might be more direct indicators of mucosal inflammation than serum markers. Stool tests could be a useful way to follow response to therapy or to predict clinical relapse. They might also be useful in differentiating patients with IBD from those with functional bowel disorders. Lactoferrin and calprotectin are the two stool tests that have been most studied. Lactoferrin Lactoferrin is a glycoprotein that is released by activated neutrophils. It is stable in stool, and fecal concentrations are higher in patients with active IBD than in normal control subjects [57]. A study of 184 IBD patients, 31 irritable bowel syndrome patients, and 56 normal controls from the University of Chicago, found an elevated fecal lactoferrin level to be 100% specific in detecting an absence of irritable bowel syndrome and 90% sensitive for detecting inflammation in patients with active IBD [58]. Kayazawa et al. compared the levels of lactoferrin to those of other neutrophil-derived proteins (polymorphonuclear leucocyte elastase, myeloperoxidase, and lysozyme) in whole gut lavage fluid and found lactoferrin to have the best operating characteristics [59]. In a further small series of pediatric patients, fecal lactoferrin was shown to correlate with response to infliximab therapy [60]. In another study from the Cleveland

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Clinic, USA, fecal lactoferrin levels could be used to distinguish pouchitis from irritable pouch syndrome in patients with ileal pouch anal anastomosis [61]. Calprotectin Calprotectin is another neutrophil-derived protein for which fecal testing is commercially available. Fecal calprotectin has been shown to correlate with fecal neutrophil excretion as measured by Indium111-labeled neutrophil excretion in stool [62]. It has also been shown to correlate well with the degree of intestinal inflammation in studies of patients with CD and UC [63,64]. A calprotectin level <60 μg/g of stool has a negative predictive value of 100% for eliminating active small intestinal CD, as determined by abnormal small bowel barium studies [65]. Fecal calprotectin has also been studied in children and suggested as a possible means of differentiating children with abdominal pain due to IBD from those with functional abdominal pain [66]. In a study of 281 children referred for evaluation of gastrointestinal symptoms and 76 healthy pediatric controls, fecal calprotectin was found to be normal in all of the control individuals [67]. In that study, elevated calprotectin levels were found in children with diseases characterized by mucosal inflammation, but not in other noninflammatory gastrointestinal disorders. Fecal calprotectin was found to be highly sensitive and specific for detecting or eliminating IBD in a group of adults and children investigated for chronic diarrhea [68]. Similar to lactoferrin, fecal calprotectin concentrations have been shown to fall with response to therapy, in this case granulocyte apheresis [69]. It appears that fecal testing for neutrophil-derived proteins is a useful, noninvasive way to eliminate IBD in patients presenting with gastrointestinal symptoms. Although fecal levels are frequently elevated in inflammatory disorders, they do not differentiate between CD and UC, and do not differentiate IBD from other inflammatory disorders or even intestinal neoplasia.

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Conclusion The use of blood tests in IBD is often not based on traditional, evidence-based guidelines. Ordering such tests should be individualized for the particular patient circumstances. The potential impact of test results on patient management should be considered before the tests are ordered. The use of blood tests in patients presenting with undiagnosed gastrointestinal symptoms, as a means of determining who to subject to more invasive testing, is an area of potential utility, particularly in pediatric patients. Some blood tests, such as the CRP level, may be helpful in following disease activity and other blood tests are useful in monitoring for and detecting complications of IBD. Yet other blood tests, such as for TPMT activity or 6-TGN levels, may help to prevent drug toxicity and maximize patient response to therapy.

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Bloomfeld RS, Onken JE. Mercaptopurine metabolite results in clinical gastroenterology practice. Aliment Pharmacol Ther 2003;17:69–73. Holme SA, Duley JA, Sanderson J, et al. Erythrocyte thiopurine methyl transferase assessment prior to azathioprine use in the UK. QJM 2002;95:439–44. Sandborn WJ. Pharmacogenomics and IBD: TPMT and thiopurines. Inflamm Bowel Dis 2004;10:S35–S37. Marra CA, Esdaile JM, Anis AH. Practical pharmacogenetics: the cost effectiveness of screening for thiopurine s-methyltransferase polymorphisms in patients with rheumatological conditions treated with azathioprine. J Rheumatol 2002;29:2507–12. Lennard L. TPMT in the treatment of Crohn’s disease with azathioprine. Gut 2002;51:143–6. Dubinsky MC, Lamothe S, Yang HY, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology 2000;118:705–13. Dubinsky MC, Yang H, Hassard PV, et al. 6-MP metabolite profiles provide a biochemical explanation for 6-MP resistance in patients with inflammatory bowel disease. Gastroenterology 2002;122:904–15. Goldenberg BA, Rawsthorne P, Bernstein CN. The utility of 6-thioguanine metabolite levels in managing patients with inflammatory bowel disease. Am J Gastroenterol 2004;99:1744–8. Uchida K, Matsuse R, Tomita S, et al. Immunochemical detection of human lactoferrin in feces as a new marker for inflammatory gastrointestinal disorders and colon cancer. Clin Biochem 1994;27:259–64. Kane SV, Sandborn WJ, Rufo PA, et al. Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol 2003;98:1309–14. Kayazawa M, Saitoh O, Kojima K, et al. Lactoferrin in whole gut lavage fluid as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived proteins. Am J Gastroenterol 2002;97:360–9. Buderus S, Boone J, Lyerly D, et al. Fecal lactoferrin: a new parameter to monitor infliximab therapy. Dig Dis Sci 2004;49:1036–9. Parsi MA, Shen B, Achkar JP, et al. Fecal lactoferrin for diagnosis of symptomatic patients with ileal pouch-anal anastomosis. Gastroenterology 2004;126:1280–6. Roseth AG, Schmidt PN, Fagerhol MK. Correlation between fecal excretion of indium-111-labelled granulocytes and calprotectin, a granulocyte marker protein, in patients with inflammatory bowel disease. Scand J Gastroenterol 1999;34:50–4. Costa F, Mumolo MG, Bellini M, et al. Role of fecal calprotectin as non-invasive marker of intestinal inflammation. Dig Liver Dis 2003;35:642–7. Summerton CB, Longlands MG, Wiener K, et al. Fecal calprotectin: a marker of inflammation throughout the intestinal tract. Eur J Gastroenterol Hepatol 2002;14:841–5. Dolwani S, Metzner M, Wassell JJ, et al. Diagnostic accuracy of fecal calprotectin estimation in prediction of abnormal small bowel radiology. Aliment Pharmacol Ther 2004;20:615–21. Olafsdottir E, Aksnes L, Fluge G, et al. Fecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children. Acta Paediatr 2002;91:45–50. Canani RB, Rapacciuolo L, Romano MT, et al. Diagnostic value of fecal calprotectin in paediatric gastroenterology clinical practice. Dig Liver Dis 2004;36:467–70. Carroccio A, Iacono G, Cottone M, et al. Diagnostic accuracy of fecal calprotectin assay in distinguishing organic causes of chronic diarrhea from irritable bowel syndrome: a prospective study in adults and children. Clin Chem 2003;49:861–7. Hanai H, Takeuchi K, Iida T, et al. Relationship between fecal calprotectin, intestinal inflammation, and peripheral blood neutrophils in patients with active ulcerative colitis. Dig Dis Sci 2004;49:1438–43.

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2 Pediatric IBD: novel investigative approaches for diagnosis and follow-up Ernest G Seidman and Devendra K Amre Introduction The diagnosis and follow-up of inflammatory bowel disease (IBD) is particularly challenging in the pediatric age group. Children and adolescents can have low grade and nonspecific symptoms, leading to delays in diagnosis. Furthermore, young patients are reluctant to undergo endoscopic procedures, especially repeatedly in order to ascertain disease recurrence, or complications over time. In this chapter, we appraise the most important recent clinical advances in the following areas: • epidemiology • novel imaging methods to diagnose and follow-up IBD • innovative uses of serologic markers to screen for IBD and distinguish between types of colitis, as well as to predict outcomes

Epidemiology Several studies have reported an increased incidence of pediatric Crohn’s disease (CD) in developed nations over the past few decades. Hildebrand et al. recently studied the incidence and characteristics of IBD in Sweden [1]. In a population-based catchment area of 180,000 individuals in Stockholm, the records of children up to 15 years of age who were suspected of having IBD between 1990 and 2001 were examined using defined

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diagnostic criteria. A total of 152 children were diagnosed with IBD, corresponding to an overall incidence of 7.4 cases per 100,000. The incidence of CD was 4.9, ulcerative colitis (UC) 2.2, and indeterminate colitis 0.2. There was a marked increase in CD incidence over the decade of study, whereas UC incidence was essentially unchanged. Of pediatric patients with CD and UC, respectively, 14% and 11% had a first- or second-degree relative with IBD, and 18% and 10% had a concomitant autoimmune disease. These results concur with the trend towards a shift in presentation and diagnosis from UC towards CD, as well as a net increase in IBD overall, as seen in North America. Recent studies also point to an increasing incidence of IBD in developing countries. Appleyard et al. examined the incidence of IBD in Puerto Rico [2]. The total incidence of IBD (pediatric and adult cases) increased significantly between 1996 and 2000 (3.07/100,000 to 7.74/100,000; P<0.001). This increase was significantly higher for CD (4-fold increase, P<0.01) and unspecified IBD (4-fold increase, P<0.005) than for UC (1.7-fold increase). The prevalence of CD was higher in males, with an earlier age of onset (P<0.05). Similarly, a study in Saudi Arabia noted an increased incidence of CD over the past decade [3]. Multivariate analysis of environmental risk factors If the genetic pool is stable, what explains the global trend towards an increased incidence of CD? A report from France attempted to examine this issue by studying environmental risk factors prior to the development of IBD in a pediatric population-based case-control study [4]. A total of 222 incident cases of CD and 60 incident cases of UC occurring before 17 years of age between 1988 and 1997 were matched with control subjects by sex, age, and geographic location. The investigators looked at 140 study variables in a questionnaire that covered: family history of IBD, events during the perinatal period, diet during infancy and childhood, vaccinations and childhood diseases, household amenities, and the family’s socioeconomic status.

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In a multivariate model, significant risk factors for CD included family history of IBD (odds ratio [OR] 4.3, 95% confidence interval [CI] 2.3–8), breast feeding (OR 2.1, 95% CI 1.3–3.4), bacille Calmette–Guerin vaccination (OR 3.6, 95% CI 1.1–11.9), and history of eczema (OR 2.1, 95% CI 1–4.5). Interestingly, regular drinking of tap water was a protective factor (OR 0.56, 95% CI 0.3–1). Risk factors for UC included family history of IBD (OR 12.5, 95% CI 2.2–71.4), disease during pregnancy (OR 8.9, 95% CI 1.5–52), and bedroom sharing (OR 7.1, 95% CI 1.9–27.4), whereas appendicectomy was protective (OR 0.06, 95% CI 0.01–0.36). The results of this study might provide new etiologic clues for IBD arising in the pediatric age group. Diet and risk of IBD A very recent study examined dietary factors potentially associated with onset of IBD in Japan [5]. IBD patients aged 15–34 years (111 UC patients, 128 CD patients) were studied within 3 years of diagnosis in 13 centers. One control subject was recruited for each case of IBD, matched for sex, age, and hospital. A semi-quantitative food frequency questionnaire was used to estimate pre-illness intakes of food groups and nutrients. All the available control subjects (n=219) were pooled and unconditional logistic models were applied to calculate risk. A higher consumption of sweets was positively associated with UC risk (OR for the highest versus lowest quartile 2.86, 95% CI 1.24–6.57). The consumption of sugars and sweeteners (OR 2.12, 95% CI 1.08–4.17), sweets (OR 2.83, 95% CI 1.38–5.83), fats and oils (OR 2.64, 95% CI 1.29–5.39), and fish and shellfish (OR 2.41, 95% CI 1.18–4.89) were positively associated with CD risk. With respect to consumption of nutrients, vitamin C intake (OR 0.45, 95% CI 0.21–0.99) was negatively related to UC risk, while the intake of total fat (OR 2.86, 95% CI 1.39–5.90), monounsaturated fatty acids (OR 2.49, 95% CI 1.23–5.03), polyunsaturated fatty acids (OR 2.31, 95% CI 1.12–4.79), vitamin E (OR 3.23, 95% CI 1.45–7.17), n-3 fatty acids (OR 3.24, 95% CI 1.52–6.88),

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and n-6 fatty acids (OR 2.57, 95% CI 1.24–5.32) were positively associated with CD risk. Although this study suffers from the shortcoming of recall bias, inherent in most retrospective studies, the findings support the importance of dietary factors for IBD risk and thus, potentially, its prevention.

Diagnostic imaging methods The traditional set of imaging methods used to diagnose IBD and to document its extent in the pediatric age group is, as in adults, based upon colonoscopy with ileal and colonic biopsies, and small bowel contrast radiography (SBCR) [6]. Other tests have been proposed that are either less invasive (scintigraphy and ultrasonography) or that also permit visualization of bowel segments that are inaccessible to standard endoscopes (capsule endoscopy). Scintigraphy The most common scintigraphy agent used is 99m Tc-hexamethyl-propylene-amine-oxime (HMPAO). However, this agent has limitations. The 99m Tc-HMPAO leukocyte scan was used in 95 children undergoing investigation for IBD in a tertiary center [7]. Diagnosis was based on conventional investigations, including SBCR, upper gastrointestinal endoscopy (UGIE), colonoscopy, and endoscopic biopsy (the ‘gold standards’). IBD was confirmed in 73 children (57 CD, 10 UC, 6 indeterminate colitis) and excluded in 22 (controls). Scintigraphy was evaluated as a screening test, compared with individual conventional tests, and assessed for each gut segment (Table 1). Comparison with biopsies paralleled that with endoscopy (UGIE and colonoscopy). False negative results were especially common (negative predictive value [NPV] ≤0.2) in cases with IBD involving the proximal gut. The authors concluded that 99m Tc-HMPAO leukocyte scintigraphy should not be relied

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Test

Sensitivity (95% CI)

Specificity (95% CI)

PPV

NPV

Scintigraphy

0.75 (0.63–0.85)

0.82 (0.59–0.94)

0.93

0.5

SBCR

0.87 (0.72–0.96)

0.57 (0.39–0.73)

0.69

0.2

UGIE Colonoscopy

NA

0.9 (0.79–0.96)

NA

0.13

0.57 (0.41–0.73)

0.71 (0.54–0.85)

0.71

0.42

Table 1. Evaluation of scintigraphy and conventional tests for the diagnosis of IBD in children. CI: confidence interval; NA: not available; NPV: negative predictive value; PPV: positive predictive value; SBCR: small bowel contrast radiography; UGIE: upper gastrointestinal endoscopy. Adapted from Grahnquist et al [28]. Reprinted with permission from Elsevier Science, Inc.

on as a screening test for IBD, because false negative results are too common. In a retrospective study, Peacock et al. assessed the use of 99m Tc-stannous colloid leukocyte scintigraphy for the initial evaluation of children with suspected IBD [8]. A total of 64 patients (35 male and 29 female; mean age 12.5 years; age range 2–19 years) had scintigraphy performed, with IBD subsequently diagnosed in 34 cases. 99m Tc-stannous colloid leukocyte scintigraphy had 88% sensitivity, 90% specificity, and an 8.8 likelihood ratio for initial investigation of IBD. However, agreement was poor for topographic localization of disease. Small bowel series had 75% sensitivity, 50% specificity, and a 1.5 likelihood ratio for detecting endoscopic disease of the terminal ileum and proximal colon. The results suggest that leukocyte scintigraphy with this reagent is a useful imaging technique for the initial evaluation of patients with suspected IBD. 99m Tc-stannous colloid had results at least comparable to those of other scintigraphy agents. The authors concluded that, in children, 99m Tc-stannous colloid leukocyte scintigraphy should be preferred to 99m Tc-HMPAO due to lower cost, shorter preparation time, and the smaller blood volumes required.

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Ultrasonography Many studies, including our own, have established that bowel wall thickening and mucosal blood flow imaged using transabdominal ultrasound (US) with Doppler correlates well with CD activity [9,10]. A recent study by Bremner et al. compared US and SBCR in the assessment of CD in 24 pediatric patients [11]. All 13 cases of terminal ileum disease had abnormal US and an abnormality with SBCR. However, US did not detect five cases of an affected terminal ileum and two cases of isolated proximal small bowel CD. The authors concluded that SBCR is a more sensitive indicator of small bowel CD than standard US (without Doppler). In another recent report, Baud et al. suggested that color Doppler US can be useful in children in order to distinguish between an inflammatory colitis (eg, active CD or neutropenic colitis) and an ischemic colitis (eg, hemolytic uremic syndrome) [12]. Capsule endoscopy The small bowel has been termed the ‘last frontier’ in terms of endoscopic visualization, as the vast majority of it is out of the range of current endoscopes and enteroscopes. In adults, push enteroscopy can theoretically visualize approximately 150 cm distal to the pylorus, but the use of an overtube measuring 15 mm limits its applicability for children. The ingenious Israeli invention of a wireless capsule endoscope enables the unique opportunity to visualize the entire small bowel in a noninvasive manner [13]. A number of potential indications for capsule endoscopy have been proposed for children and adolescents [14]. Capsule endoscopy appears to have great promise for the diagnosis and perhaps follow-up of small bowel CD. It might ultimately also assist in differentiating between UC and CD in cases considered ‘indeterminate’ [14]. Capsule endoscopy is also likely to be very suitable for objectively documenting, in a noninvasive manner, mucosal healing of small bowel CD after therapy [14].

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In the last 2 years, two pediatric studies have examined the utility of capsule endoscopy to diagnose or rule out CD that affects the small bowel only [15,16]. In both studies, CD was suspected, but not confirmed, by traditional imaging (normal colonoscopy with ileal and colonic biopsies, as well as SBCR). In these reports, the diagnostic yield for small bowel inflammatory disorders missed by the traditional methods (‘obscure’ CD or eosinophilic enteropathies) was close to 60% [15,16]. Patients with dysphagia, or those too young to swallow the capsule, require that the capsule be deposited into the proximal duodenum endoscopically under anesthesia, as we recently described [14]. Upper endoscopy In about 10% of cases, discrimination between UC and CD can be difficult using ileocolonoscopy and SBCR because of a lack of definitive lesions. Our group has suggested that UGIE might be helpful in confirming the diagnosis in such cases of colitis [17]. A recent study from the UK showed that UGIE helped to confirm a diagnosis of CD in a further 20% of pediatric IBD patients after clinical, radiologic, and histologic analysis [18]. The authors noted that the absence of specific upper gastrointestinal symptoms did not preclude the presence of upper gastrointestinal inflammation. They recommended that UGIE be part of the first-line investigations in all new suspected pediatric IBD cases [18].

Serologic markers In mild cases, the nonspecific symptoms of IBD (abdominal pain, diarrhea, etc.) can be easily mistaken for a functional bowel disorder, thus delaying diagnosis. This is particularly true for pediatric CD, where growth failure, arthralgias, or fever can be the presenting complaint, at times in the absence of any gastrointestinal symptoms. School-aged CD patients might complain of insidious abdominal pain, without other more

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alarming symptoms or signs. In contrast, in most cases of UC, hematochezia generally leads to rapid consultation and subsequently, to a diagnostic colonoscopy. The diagnosis of CD in the pediatric age group is more often delayed than for UC, not uncommonly for over a year. Therefore, there is a clinical need for accurate, noninvasive screening tests for IBD, similar to what has been achieved for celiac disease using anti-tissue transglutaminase and antiendomysial antibodies (arguably amongst the most highly performing screening tests in medicine). The usefulness of novel serologic assays to screen for IBD, as well as their ability to discriminate between types of IBD, has been the subject of considerable clinical research of late. Perinuclear anti-neutrophil cytoplasmic autoantibodies (pANCA) have been established as an autoimmune marker most characteristic of UC in the pediatric age group [19]. Alternatively, anti-Saccharomyces cerevisiae antibodies (ASCA) have been shown to be a reliable marker of CD [19]. The potential sensitivity of these markers as screening tests for IBD is maximized when the two assays are combined. Discriminating between types of IBD (CD vs UC) CD is typically diagnosed utilizing routine clinical, endoscopic, radiologic, and histologic criteria. Discrimination of CD from UC is straightforward when involvement of the colon is segmental, or if the disease extends into the small bowel. However, diffuse or homogeneous colitis, while typical of UC, might also be seen in a subgroup of CD patients. Furthermore, some cases of treated UC can have a more patchy distribution of inflammation. Although the treatment of UC and CD is quite similar, the precise classification of these entities becomes highly relevant when surgery is contemplated. For clinicians, one of the major benefits of the pANCA and ASCA tests is their potential ability

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to distinguish between UC and CD when the extent of IBD is restricted to the colon. As in adults, the former is highly specific for UC in the pediatric age group, whereas the latter is most suggestive of CD [19,20]. It must be noted, however, that CD patients with a ‘UC-like’ presentation might also be pANCApositive [19]. Therefore, positivity for ASCA is a much more specific result (88–95% specificity for IgA or IgG, 100% for both). In a recent study, the results of ASCA and pANCA testing were concordant with clinical diagnosis in 76 of 107 (71%) pediatric patients [21]. In view of the important differences in outcome after colectomy in UC versus CD, the available data suggest that ASCA and pANCA testing should be carried out prior to elective ‘curative’ colectomy in the pediatric age group. More recently, antibody testing to Escherichia coli outer membrane porin (anti-ompC) was added to the serologic armamentarium for IBD. A recent study examined these three serologic tests in parallel in 81 children with CD, 54 children with UC, and 63 controls [22]: • ASCA antibodies (IgA or IgG) were observed in 44% of CD patients, 0% of UC patients, and only 1.5% of controls. • Deoxyribonuclease-sensitive pANCA antibodies were found in 70% of UC patients, 18% of CD patients (predominantly Crohn’s colitis, as noted above), and 3% of controls. • Anti-ompC as an isolated assay had low sensitivity for both CD (24%) and UC (11%), and displayed a 5% false positive result rate. However, anti-ompC did identify a small number of IBD patients who were not detected by any other assay. If one or more of the three antibody assays was positive, the panel’s overall sensitivity was 65% for CD and 76% for UC, with a specificity of 94% [22]. As seen in other studies, patients who were ASCA-positive were more likely to have disease of the

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ileum or the ileum and right colon than patients who were ASCA-negative (58% vs 18% respectively, P<0.001). Screening for IBD As noted previously, the diagnosis of CD is delayed in some pediatric patients due to nonspecific presenting complaints. Reliable screening tests would potentially be helpful in these circumstances, resulting in both earlier diagnosis and treatment. Furthermore, the high NPV of the serologic assays in excluding IBD could potentially avoid unnecessary, invasive, and costly testing, such as colonoscopy and radiologic studies of the upper gastrointestinal tract and small bowel (ie, SBCR). Our report, described below, is the only prospective study to date that has analyzed the clinical utility of pANCA and ASCA testing in pediatric patients in order to distinguish between IBD and irritable bowel syndrome (IBS) [23]. In series with routine laboratory test results (hemoglobin level, platelet count, erythrocyte sedimentation rate, serum albumin and iron levels), enzyme-linked immunoabsorbent assay-based pANCA and ASCA testing was found to be useful to ‘ rule out’ IBD in children and adolescents with functional bowel disorders, without resorting to more invasive testing. The positive predictive value was 90%, although the false negative result rate was 20% [23]. The incorporation of such sequential noninvasive testing into a diagnostic strategy (Figure 1) might avoid unnecessary, costly evaluations, and facilitate clinical decision making when the diagnosis of IBD in children is initially uncertain. This approach has been found to be an effective and economically sound approach to such IBS-like presentations [24]. However, studies have not yet addressed this issue in adult patients. Patients with a high degree of suspicion for IBD should undergo the full set of investigations, regardless of serologic results (Figure 2).

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Pediatric IBD

Symptoms: IBS or IBD?

Alarm symptoms

YES

Follow algorithm in Figure 2

NO

IBD serology: positive

IBD serology: negative

Follow algorithm in Figure 2

Follow-up visit

Figure 1. Diagnostic algorithm for pediatric patients suspected of having IBD, but without the presence of physical signs suggestive of the diagnosis (such as right lower quadrant mass, malnutrition, perianal disease, etc.) or ‘alarm’ symptoms (hematochezia, fever, weight loss, arthritis, etc.). All patients are first screened using ASCA and pANCA enzyme-linked immunoabsorbent assay (ELISA)-based assays. Coincidentally, blood is drawn for other routine parameters to screen for IBD, including hemoglobin level, platelet count, and C-reactive protein, as well as serum iron and albumin levels. According to this algorithm, patients who have negative serology are observed in follow-up, without further work-up at that time. Positive ELISA-based assay results should be confirmed with the traditional ASCA/pANCA assays. Those patients with positive ELISA-based and traditional assays would then undergo a complete diagnostic investigation, including colonoscopy with biopsies, as well as barium upper gastrointestinal series and small bowel contrast radiography, as shown in Figure 2.

Predicting disease activity and outcome Other studies have aimed to determine whether serologic tests are useful to predict or monitor disease activity in pediatric IBD. Limited data in pediatrics concur with studies in adults showing that pANCA activity persists even after colectomy, reflecting its status as a true marker of autoimmunity in UC [19].

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Symptoms: IBS or IBD?

Alarm symptoms

YES

NO

Colonoscopy + biopsies + SBCR

NORMAL

DIAGNOSTIC OF IBD

Obstructive symptoms

No obstructive symptoms

CT enteroclysis

Capsule endoscopy

Treat IBD

Figure 2. Diagnostic algorithm for pediatric patients with a high degree of suspicion for IBD, either because of physical signs highly suggestive of IBD (right lower quadrant mass, malnutrition, perianal disease, etc.) and/or ‘alarm’ symptoms (hematochezia, fever, weight loss, arthritis, etc.). All such patients should undergo standard investigations, including colonoscopy with biopsies, as well as small bowel contrast radiography (SBCR). If negative, either computed tomography (CT) enteroclysis or capsule endoscopy should be pursued.

More recently, we conducted a study aimed to analyze the correlation between clinical prognosis of selected outcomes in relation to baseline (at time of diagnosis), as well as serial antibody measurements in pediatric CD patients [25]. Serum ASCA and pANCA antibodies were measured at baseline (n=154) and again during follow-up (n=61) using standard techniques in a cohort of CD patients identified at Sainte-Justine Hospital, Montreal, Canada, between 1996 and 1998. Clinical information was abstracted from medical charts and antibody patterns were examined using mixed modeling techniques.

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The prognostic ability of antibodies for selected outcomes was evaluated using logistic regression. We found that relatively few patients with serial antibody measurements changed their ASCA-IgA, ASCA-IgG, and pANCA status (24.5%, 29.5%, and 18%, respectively). No distinct patterns in the evolution of antibody titers were noted. However, patients who were positive for baseline ASCA-IgA were more than twice as likely to experience a relapse during their disease course compared with those who were negative (OR 2.9, 95% CI 1.33–6.35, P<0.05). Serial antibody measurements did not add further to the ability to predict the occurrence of clinical outcomes [25]. In another pediatric follow-up study in Italy, ASCA titers significantly correlated with disease activity, and children with severe active disease showed higher ASCA values compared to those in remission [26]. Moreover, a significant reduction of ASCA was observed during the follow-up of CD children when clinical remission was achieved [26]. As in adult series, pediatric ASCA-positive patients were found to be more likely to require ileocecal resection than ASCA-negative patients (36% vs 13% respectively, P<0.05) [22]. In the Italian study, significantly lower ASCA titers were observed in CD children after intestinal resection compared to those who did not undergo surgical resection (P<0.05) [26]. A recent long-term follow-up study showed that CD activity does not ‘burn out’ with time [27]. Approximately one-quarter of the patients followed had active disease 20 years after diagnosis. Taken together, the data from recent serologic studies in pediatric CD suggest that ASCA positivity at time of diagnosis, particularly if of IgA isotype or high titer, predicts a poor outcome (higher frequency of relapse and requirement for surgery). These data would support the early use of immunosuppressive therapy in pediatric CD cases in order to improve outcomes. However, studies to determine whether this “preventive approach” is valid in young ASCA-positive patients have not yet been carried out.

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Conclusion This chapter highlights some of the recent advances in the epidemiology, diagnosis, and follow-up of IBD in the pediatric age group. The incidence of CD, but not UC, is on the increase in many countries around the world. Although the gene–environmental interactions underlying this increase are poorly understood, dietary factors might play a key role. Estimation of intestinal wall vessel density by color Doppler abdominal sonography appears to provide information about bowel inflammation in a noninvasive manner. The use of leukocyte scintigraphy has yielded inconsistent results. On the other hand, capsule endoscopy has greatly improved our ability to detect ‘obscure’ small bowel CD, especially when traditional endoscopic and imaging techniques have not confirmed or excluded the diagnosis (see Figure 2). Serologic assays facilitate the distinction between UC and CD when the latter is restricted to the colon. Furthermore, these tests can assist clinicians in screening for IBD in pediatric cases where the symptoms are nonspecific and suggestive physical findings are absent. Finally, serologic testing can assist in predicting an aggressive course in CD, and might thus serve to identify patients who warrant more aggressive immunomodulatory drugs at an early stage in their disease course.

References 1. 2. 3. 4. 5.

6.

38

Hildebrand H, Finkel Y, Grahnquist L, et al. Changing pattern of pediatric inflammatory bowel disease in northern Stockholm 1990–2001. Gut 2003;52:1432–4. Appleyard CB, Hernandez G, Rios-Bedoya CF. Basic epidemiology of inflammatory bowel disease in Puerto Rico. Inflamm Bowel Dis 2004;10:106–11. Al-Ghamdi AS, Al-Mofleh IA, Al-Rashed RS, et al. Epidemiology and outcome of Crohn’s disease in a teaching hospital in Riyadh. World J Gastroenterol 2004;10:1341–4. Baron S, Turck D, Leplat C, et al. Environmental risk factors in pediatric inflammatory bowel diseases: a population based case control study. Gut 2005;54:357–63. Sakamoto N, Kono S, Wakai K, et al. Epidemiology Group of the Research Committee on Inflammatory Bowel Disease in Japan. Dietary risk factors for inflammatory bowel disease: a multicenter case-control study in Japan. Inflamm Bowel Dis 2005;11:154–63. Seidman EG. Role of endoscopy in pediatric inflammatory bowel disease. Gastrointest Endosc Clin N Am 2001;11:641–57.

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7.

8. 9.

10.

11.

12. 13. 14.

15. 16.

17. 18.

19. 20. 21.

22.

23. 24.

25. 26.

Grahnquist L, Chapman SC, Hvidsten S, et al. Evaluation of 99mTc-HMPAO leukocyte scintigraphy in the investigation of pediatric inflammatory bowel disease. J Pediatr 2003;143:48–53. Peacock K, Porn U, Howman-Giles R, et al. 99mTc-stannous colloid white cell scintigraphy in childhood inflammatory bowel disease. J Nucl Med 2004;45:261–5. Spalinger J, Patriquin H, Miron M-C, et al. Doppler US in patients with Crohn’s disease: vessel density in the diseased bowel reflects disease activity. Radiology 2000;217:787–91. Scholbach T, Herrero I, Scholbach J. Dynamic color Doppler sonography of intestinal wall in patients with Crohn’s disease compared with healthy subjects. J Pediatr Gastroenterol Nutr 2004;39:524–8. Bremner AR, Pridgeon J, Fairhurst J, et al. Ultrasound scanning may reduce the need for barium radiology in the assessment of small-bowel Crohn’s disease. Acta Paediatr 2004;93:479–81. Baud C, Saguintaah M, Veyrac C, et al. Sonographic diagnosis of colitis in children. Eur Radiol 2004;14:2105–19. Iddan G, Meron G, Glukhovsky A, et al. Wireless capsule endoscopy. Nature 2000;405:417. Seidman EG, Sant’Anna AM, Dirks MH. Potential applications of wireless capsule endoscopy in the pediatric age group. Gastrointest Endosc Clin N Am 2004;14:207–17. Arguelles-Arias F, Caunedo A, Romero J, et al. The value of capsule endoscopy in pediatric patients with a suspicion of Crohn’s disease. Endoscopy 2004;36:869–73. Guihan de Araujo Sant’Anna AM, Dubois J, Miron MC, et al. Wireless capsule endoscopy for obscure small bowel disorders: final results of the first pediatric controlled trial. Clin Gastroenterol Hepatol 2005;3:264–70. Lenaerts C, Roy CC, Vaillancourt M, et al. High incidence of upper gastrointestinal tract involvement in children with Crohn’s disease. Pediatrics 1989;83:777–81. Castellaneta SP, Afzal NA, Greenberg M, et al. Diagnostic role of upper gastrointestinal endoscopy in pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2004;39:257–61. Ruemmele FM, Targan SR, Levy G, et al. Diagnostic accuracy of serologic assays in pediatric inflammatory bowel disease. Gastroenterology 1998;115:822–9. Hoffenberg EJ, Fidanza S, Sauaia A. Serologic testing for inflammatory bowel disease. J Pediatr 1999;134:447–52. Gupta SK, Fitzgerald JF, Croffie JM, et al. Comparison of serologic markers of inflammatory bowel disease with clinical diagnosis in children. Inflamm Bowel Dis 2004;10:240–4. Zholudev A, Zurakowski D, Young W, et al. Serologic testing with ANCA, ASCA, and anti-OmpC in children and young adults with Crohn’s disease and ulcerative colitis: diagnostic value and correlation with disease phenotype. Am J Gastroenterol 2004;99:2235–41. Dubinsky MC, Ofman JJ, Urman M, et al. Clinical utility of serodiagnostic testing in suspected pediatric inflammatory bowel disease. Am J Gastroenterol 2001;96:758–65. Dubinsky MC, Johanson JF, Seidman EG, et al. Suspected inflammatory bowel disease – the clinical and economic impacts of competing diagnostic strategies. Am J Gastroenterol 2002;97:2333–42. Desir B, Amre DK, Lu SE, et al. Utility of serum antibodies in determining clinical course in pediatric Crohn’s disease. Clin Gastroenterol Hepatol 2004;2:139–46. Canani RB, Romano MT, Greco L, et al. Effects of disease activity on antiSaccharomyces cerevisiae antibodies: implications for diagnosis and follow-up of children with Crohn’s disease. Inflamm Bowel Dis 2004;10:234–9.

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27. 28.

40

Etienney I, Bouhnik Y, Gendre JP, et al. Crohn’s disease over 20 years after diagnosis in a referral population. Gastroenterol Clin Biol 2004;28:1233–9. Grahnquist L, Chapman SC, Hvidsten S, et al. Evaluation of 99mTc-HMPAO leukocyte scintigraphy in the investigation of pediatric inflammatory bowel disease. J Pediatr 2003;143:48–53.

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3 Progress in IBD genetics Steven R Brant and Amir Karban Introduction Important progress has been made in inflammatory bowel disease (IBD) genetics over the past 2 years. Genetic epidemiology research has further solidified the importance of NOD2 (nucleotide-binding oligomerization domain 2, also known as CARD15 [caspase recruitment domain 15]), the major Crohn’s disease (CD) susceptibility gene in Caucasian populations. Cell biology studies have begun to unravel how mutant NOD2 can lead to CD. The IBD5 haplotype is now the second most established genetic risk factor for CD. The leading candidate genes for the IBD5 haplotype are the organic cation transporter (OCTN) genes, OCTN1 and OCTN2. Several novel IBD candidate genes have recently been identified, some with provocative functional polymorphisms genetically associated with IBD. We will review progress over the past 2 years, firstly by focusing on progress in the two genes/regions that can be considered definitive IBD genetic risk factors: NOD2 and the IBD5 haplotype. We will then report on the present status of linkage studies that are defining the chromosomal regions likely to contain IBD susceptibility genes. Finally, we will review progress in the identification of additional IBD susceptibility genes.

NOD2 Genetic epidemiology of NOD2 Since Hugot et al. in France and Ogura et al. in the United States simultaneously identified three CD-predisposing mutations in the

41

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NOD2 gene in 2001, more than 20 published studies in Caucasian populations have confirmed that these mutations are more frequent in persons with CD compared with healthy controls [1,2]. Therefore, the three major genetically independent NOD2 mutations (R702W, G908R, and Leu1007fs) are established CD risk factors for people with European ancestry. Ethnicity The frequencies of the three major NOD2 mutations differ by ethnicity (Table 1) [3]. NOD2 mutations are not present in Pacific Asian populations (eg, Japanese, Chinese, and Koreans), and seem to be unimportant in populations of African ancestry (eg, African Americans with CD) [4–8]. It remains to be determined whether the absence of these major mutations accounts for the lower prevalence of CD in Pacific Asian and African populations. However, preliminary studies suggest that there might be some ancestral African NOD2 risk variants [8]. NOD2 mutation allele frequency is lower in Israeli Sephardic Jews (immediate ancestry from Middle East/north Africa/ southern Mediterranean countries) compared with Israeli Ashkenazi Jews (central and eastern European), perhaps accounting for the lower CD incidence in Sephardic relative to Israeli Ashkenazi Jews [9]. The G908R variant is the most common risk allele for non-Jewish Caucasians, whereas the R702W variant is the most common risk allele in all Jewish populations (see Table 1). Regional heterogeneity Arnott et al. recently identified regional heterogeneity in NOD2 risk within European populations [10]. NOD2 risk in northern Europeans, specifically Scottish and Finnish populations, is significantly lower compared with European populations at lower latitudes, ie, English and French cohorts (P<0.00001) (see Table 1) [10]. However, the overall control allele frequency in the Scottish control population is similar to that reported for French and English control populations. It is noteworthy that more northern populations have a higher incidence of CD,

42

144.1 NA 144.8 NA 147 198.5

US – NJ

US – Jewish

England

Finland

Scotland

Canada

14.6

11.6

9

8.3

0/0

0

2.6

8.7/3.5

3.3 NA 12.9

28.4/5.6

7.2

21.3/7.6

13.6/7.7

12.5

18.6/9.6 25.2/8.2

4.5

10.7

23.4/9.4 17.9/10.2

8.4

6.7

15.7/7.8 21.9/8.4

13.7

11.5

5.2

5.9

1.5

11.2

36.2/10.7

24.2/10.5

29.0/6.3

16.7/3.7

2.3/1.6

20.3/6.2

5.2

NA

1.8

0.6

3.3

8.7

7.6

4.3

8.4

4.5

8.3

3.7

15.0

9.8

0.0

2.4

0

G908R AF

10.3

NA

4.6

4.8

9.4

7.3

5.8

8.4

5.1

4.5

14.2

9

8.8

1.0

0.8

6.7

0

1007fs AF

45

35.5

22.8

15.5

38.5

NA

33

NA

30.1

27.9

32.8

38

47.4

27.5

4.6

NA

0

NOD2 carrier (%)

229/71

216/336

252/189

271/300

244/349

172/62

112/79

303/288

166/143

165/165

204/104

205/95

51/54

97/105

110/127

267/409

350/292

Patients/ controls

123,125

123,124

115,122

120,121

118,119

14

117

14

3,116,117

113,115

113,114

111,112

9

9

8,109

107,108

4,106

Ref.

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NA

6.9

6.08

4.9

NA

NA

2.7

2.1

0.5

R702W AF

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Table 1. NOD2 in Crohn’s disease (CD) in different populations. AA: African American; AF: allele frequency; Israel Ashk: Israel Ashkenazi Jewish; Israel Seph: Israel Sephardic Jewish; NA: not available; NJ: non-Jewish; Ref.: reference number.

NA

116.47

Israel Ashk.

Spain

30–80

Israel Seph.

France

29.8

12–55

USA – AA

5.8 NA

Australia

Prevalence Incidence NOD2 AF CD per 105 CD per 105 CD/controls

Japan

Country/ ethnicity

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and NOD2 mutation has a lower population attributable risk for CD in Scottish versus English populations (11% vs 27%) [10]. One potential interpretation of these findings is that environmental risk factors in these populations might account for the greater CD incidence. Genetic factors other than NOD2 or unique environmental factors might account for the very high CD incidence in North American Jews. NOD2 allele frequencies in North American Jewish controls are higher than North American non-Jewish Caucasians, but, overall, NOD2 mutations in North American Jews with CD are slightly less frequent than in non-Jewish Caucasians (see Table 1). Variants and mutations A recent meta-analysis of 42 studies (37 in Caucasians) by Economou et al. determined that the overall relative risk (RR) for a person carrying one mutation (simple heterozygotes) was 2.4, while the RR for those carrying two mutations (homozygotes and compound heterozygotes) was 17.1 [3]. However, in Jews, the risk of carrying at least one NOD2 mutation was significantly lower than in non-Jews (odds ratio [OR] 1.55, 95% confidence interval [CI] 0.99–2.42 vs OR 3.15, 95% CI 2.76–3.59, respectively). Another possible explanation for the high Ashkenazim CD prevalence could be a unique Ashkenazi NOD2 risk variant. Sugimura et al. detected the presence of an Ashkenazi-specific risk haplotype, independent of the three common NOD2 mutations [11]. The 268S-JW1 haplotype is specified by two alleles: a single nucleotide polymorphism (SNP) in NOD2 intron 8 (IVS8+158C/T), and the non-physiologically active P268S variant. The 268S-JW1 haplotype was significantly more common in CD North American Ashkenazim versus ethnically matched controls (20.5% vs 6.3%, OR 5.75, P=0.0005) and had a population attributable risk of 15.1%. This result potentially accounts for a large fraction of the increased CD risk in Ashkenazim compared with non-Jewish Caucasians.

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Demonstration of a functional alteration of NOD2-containing cells from people homozygous for the 268S-JWI haplotype is anxiously awaited. Our own study did not find evidence for an increase in this haplotype in Israeli Ashkenazim CD patients, nor was there replication evidence in a small combined US/Israeli study [12,13]. However, significantly larger studies are necessary for ultimate validation of this potentially important risk allele. Do each of the three common NOD2 mutations cause equal risk for developing CD? The Leu1007fs mutation (which results in a truncation of the 33 C-terminal amino acids and an L1007P missense alteration) was suspected as being associated with greater risk compared with the G908R and R702W NOD2 variants. This is because the Leu1007fs variant shows a far greater decrease in normal response to muramyl dipeptide (MDP) stimulation relative to the other variants in transfection experiments, and shows a greater reduction in vivo in human defensin secretion [14,15]. The NOD2 meta-analysis of Economou et al. confirmed suspicions that Leu1007fs was a more significant risk allele: in non-Jewish Caucasians, the CD ORs for the R702W and G908R alleles were 2.20 (95% CI 1.84–2.62) and 2.99 (95% CI 2.38–3.74), respectively, whereas the Leu1007fs OR was 4.09 (95% CI 3.23–5.18) [3]. The trend was identical in Jews (although the values were slightly smaller). A recent prospective pediatric study similarly found a disproportionate risk for the Leu1007fs variant [16]. NOD2 and CD phenotype In The Inflammatory Bowel Disease Yearbook 2003, we reported on seven studies that looked at all three NOD2 variants and CD phenotype [17]. These studies showed a strong association for NOD2 mutants with greater risk for ileal disease involvement and intestinal structuring, with one study also showing greater risk for internal (non-perianal) fistulizing complications.

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Multiple studies have since yielded further support for each of these findings; most consistently, greater risk for ileal involvement as opposed to colonic-only disease and youthful disease onset. Likewise, meta-analysis found NOD2 mutations to be most consistently associated with small bowel disease (OR 2.53), with a significant risk for stenosing complications (OR 1.94) [3]. Studies have not found a disproportionate risk with NOD2 mutations for perianal disease, perhaps because perianal disease is most commonly associated with the colonic site. Nonetheless, it would be incorrect to state that NOD2 mutations are a risk factor for ileal CD only. As seen in several studies and confirmed by the Economou et al. meta-analysis, NOD2 mutations are associated with colonic-only CD compared with healthy controls, but the risk is relatively modest (meta-analysis OR 1.49, 95% CI 1.18–1.87) [3]. In contrast to studies that show NOD2 mutations associate with stricturing and intestinal fistulizing complications independent of ileal disease, a French study of 163 patients observed that early stricturing or fistulizing complications (ie, within 5 years of diagnosis) were associated with disease site, number of flares, and tobacco use, but not NOD2 mutations [18]. A potential explanation for this observation comes from a longitudinal Scottish study, which showed that rapid progression of complications was associated with anti-Saccharomyces cerevisiae antibodies (ASCA), whereas delayed progression was associated with NOD2 mutations [19]. Regarding other disease phenotypes, two studies have found no evidence to suggest that NOD2 is associated with granulomas in CD patients, even though distinct NOD2 mutations (eg, L469F, R334Q, R334W) have been identified as responsible for the autosomal dominant granulomatous disorder, Blau syndrome [20–22].

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How does NOD2 cause CD risk? Studies in human cells and tissues Two major functions of NOD2 have been elucidated: •

NOD2 functions as an intracellular receptor of the innate immune system’s response to bacteria.

•

NOD2 appears to interact with and regulate the activity of other molecules involved in innate immunity.

These seemingly disparate functions of NOD2 appear to be both defective or altered in cells with a NOD2 CD-predisposing mutation. How these defective functions lead to the development of CD is uncertain, and recent studies have been conflicting. Mutant NOD2 has a defective response to bacteria This was the first defect discovered from NOD2 CD-associated mutations [2,14]. The specific bacterial element that activates NOD2 is MDP, the minimal NOD2 activating component found in peptidoglycans from the cell walls of gram-positive and negative bacteria [23]. MDP is believed to bind to the NOD2 leucine-rich repeat (LRR) region. This results in NOD2 binding to receptor interacting protein 2 (RIP2), which in turn leads to activation of the major inflammatory transcription factor, nuclear factor (NF)-κB, and hence transcription of inflammatory cytokines (Figure 1, left). LRR region Exactly why mutant NOD2 does not respond normally to MDP is unclear. The G908R and Leu1007fs variants are both found in the LRR region, the structural domain important for binding MDP, and thus it has been postulated that these mutations might affect MDP binding. In a recent analysis of >500 NOD2 mutations, Tanabe et al. observed that the C-terminal LRR amino acids formed the β-strand/β-turn structure critical for response to MDP [24].

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M

D P

Steven R Brant and Amir Karban

PGN

PGN

N2 wt

N2 Ci RIP2

NF-κB

RIP2

NF-κB

Figure 1. Diagrams of wild-type and mutant nod2 (nucleotide-binding oligomerization domain 2) observed in different studies. Left, wild-type nod2 (N2 wt) is activated by muramyl dipeptide (MDP, derived from peptidoglycan [PGN]) to stimulate receptor interacting protein 2 (RIP2). RIP2 causes (via IκB kinase) phosphorylation of IκB protein (inhibitor of κB, represented by the hatched circle). This triggers IκB degradation and releases free active nuclear factor (NF)-κB to enter the nucleus and activate proinflammatory transcription. In cells with either mutant Leu1007fs N2 (N2 Ci) (right) or from nod2 ‘knockout’ mice (Kobayashi et al. model [32]) there is minimal activation of RIP2.

Albrecht et al. used computer modeling and predicted that the G908R and Leu1007fs regions of NOD2 mutations were important for interaction of the LRR domain with the NOD2 nucleotide-binding (NACHT) domain, which is responsible for NOD2 activation of RIP2 [25]. However, the R702W mutation is not located in the LRR, and modeling could not predict how this variant might effect the LRR–NACHT interaction. Abbott et al. recently proposed that mutant NOD2 has a decreased ability to bind RIP2, which could lead to reduced RIP2 activation [26].

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Transfection experiments A defective response of mutant NOD2 to MDP (as determined by NF-ΚB activation studies in stably transfected NOD2 wild-type and mutant expression constructs in human embryonic kidney [HEK]-293 cell lines as illustrated in Figure 1) correlates with experimental evidence that shows that mutant NOD2 results in impaired immunity to bacteria. Salmonella typhi grows and replicates intracellularly in colonic epithelial cells and in human colonic epithelial cell cultures that normally express only minimal NOD2. Hisamatsu et al. showed that S. typhi grew very poorly in cells that were transfected to highly express wild-type NOD2, whereas S. typhi grew very well in cells transfected to highly express NOD2 Leu1007fs mutant protein (similar to untransfected cells) [27]. This indicates that a normal NOD2 antibacterial effect is defective for the mutant Leu1007fs protein. Complementing these studies, Opitz et al. found that Pneumococcus activation of NF-κB in HEK-293 transfection models depends on normal NOD2 protein. Opitz et al. also found that NOD2 is upregulated in lung tissues from mice experimentally infected with Pneumococcus [28]. MDP stimulation Studies of NOD2 transfection, using cells that normally express minimal or no NOD2 protein, have received criticism as being artificial. How then do cells that highly express NOD2 protein, particularly monocytes and macrophages, differ in function according to NOD2 genotype? Li et al. exposed monocytederived macrophages cultured from the peripheral blood of wild-type controls and wild-type CD patients, as well as from Leu1007fs homozygote CD patients, to MDP [29]. This resulted in significantly altered expression of >2,000 genes for wild-type macrophages, but <200 genes for Leu1007fs macrophages.

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For wild-type but not Leu1007fs homozygotes, MDP stimulation resulted in a marked increase in the mRNA of a number of inflammatory chemokines and cytokines, including: interleukin (IL)-8; the ‘regulated upon activation, normal T-cell expressed and secreted’ gene (RANTES); small inducible cytokine (SCYA)3; SCYA4; IL-1β; tumor necrosis factor (TNF)-α and IL-6. More modest increases were seen in IL-18, and no increase was seen in IL-12. MDP stimulation of primary macrophage cultures from all three NOD2 mutant homozygotes failed to activate NF-κB p50/p65 complex. MDP strongly induced IL-8 protein secretion in cell cultures from CD wild-type and CD heterozygous patients compared with cultures from healthy controls. However, the R702W and G908R homozygotes showed no induction of IL-8 secretion with low-dose MDP (10 ng/mL), and cells from Leu1007fs homozygotes did not induce IL-8 secretion even at high-dose MDP (1,000 ng/mL). Treatment with MDP and TNF-α together also induced IL-1β secretion in macrophages from wild-type, but not Leu1007fs, homozygous individuals. These results showed that macrophages from CD patients with NOD2 mutations have a parallel lack of NF-κB activation, as observed in the HEK-293 transfectants. Activation of NF-κB observed in macrophages from heterozygotes correlates with their marked decreased risk for CD relative to homozygotes. Cells from a few individuals who were simple NOD2 heterozygotes were found to have defects similar to cells from double-mutant patients (Li and Cho, personal communication, 2005). Sequencing NOD2 coding and promoter regions in these few heterozygote patients did not reveal rare second mutations; the reason that their macrophages responded like double-mutant carriers is therefore unclear. NOD2 expression is involved in innate immunity NOD2 expression Studies of NOD2 expression in the ileum have yielded surprising results, perhaps explaining the disproportionate ileal involvement associated with NOD2 mutation carriers. Lala et al. used

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in situ hybridization and immunochemistry to assess NOD2 in ileal biopsy tissues [30]. Surprisingly, significant NOD2 mRNA and protein expression was present in ileal crypt epithelial cells. NOD2 specifically localized to Paneth cells. These findings were extended using monoclonal anti-NOD2 antibodies that located NOD2 to the cytosol of Paneth cells, in close proximity to the granules that contain antimicrobial peptides [31]. In CD patients, staining for NOD2 was also present in colonic metaplastic Paneth cells, in addition to newly recruited monocytes/macrophages in the colonic lamina propria. Defensins Paneth cells seem to be the only cells in the body that secrete the antimicrobial peptides known as ‘defensins’ and defensin secretion increases in response to the presence of bacterial products. Wehkamp et al. showed that biopsies of the inflamed ileum of CD patients expressed less human defensin (HD)-5 than biopsies of noninflamed ileal tissue. They also demonstrated that inflamed ileum from patients carrying NOD2 mutations (both heterozygotes as well as homozygotes) had significantly lower HD5 expression than biopsies from wild-type patients [15]. Biopsies from the colon of NOD2 mutation patients also showed less HD5 expression. Leu1007fs patients showed the greatest reduction in colonic Paneth cell HD5 expression and also showed a significant reduction in HD6 expression. The number of Paneth cells in the ileum was independent of NOD2 mutation status. NOD2 studies in animal models NOD2 knockout (nod2–/–) mice Kobayashi et al. recently reported studies in a nod2 ‘knockout’ mouse model (nod2–/– mice) that gave support to the concept developed from observations in human cells: that NOD2 dysfunction results in a defect in innate immunity [32]. Complementing the findings of Wehkamp et al. [15], the Kobayashi nod2–/– mice showed reduced ileal expression of the murine homologs to the human defensin proteins (cryptidins)

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defensin-related cryptidin (DEFCR)-4 and DEFCR-10 [15,32]. In addition, nod2–/– mice had a paradoxical decrease in cryptidin expression and secretion with IG infection from Listeria monocytogenes. Furthermore, nod2–/– mice showed a specific defect in innate immunity of the gut through their inability to handle bacterial infection: there was a relative failure to inhibit growth of L. monocytogenes given IG, but a normal response to IV or IP administration. The nod2–/– mice did show several features suggesting that NOD2 is required for MDP activation of macrophages. These features included inability of bone marrow-derived macrophages (BMDM) to respond properly to MDP, lack of MDP sensitization to endotoxin shock, lack of MDP-enhanced activation of serum immunoglobulin G1 in response to foreign antigen stimulation, and defective secretion of IL-6 and TNF-α from immature bone marrow-derived dendritic cells (BMDC). Watanabe et al. created a nod2–/– mouse model and, similarly to the Kobayashi model, these splenocytes failed to respond to MDP [33]. However, in response to peptidoglycan exposure (which yields MDP to activate NOD2, as well as other lipoproteins or associated lipoteichoic acids that stimulate TLR [toll-like receptor]2, an extracellular receptor that also activates NF-κB), the nod2–/– mice showed a paradoxical enhanced production of IL-12, IL-18, and interferon (IFN)-γ, with an overall increase in NF-κB activity. Similar results were observed with MDP and Pam3Cys (a synthetic TLR2 agonist) co-stimulation. In wild-type nod2 mice, MDP was observed to down-regulate Pam3Cys-driven TLR2 activation of IL-12. This inhibition was not present in the nod2 –/– mice, and thus an apparent negative feedback of nod2 to a TLR2-related pathway was uncovered.

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PGN

N2 wt

TLR2

M

TLR2

M

D P

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PGN

N2 Ci RIP2

NF-κB

RIP2

NF-κB

Figure 2. Diagrams of wild-type and mutant nod2 (nucleotide-binding oligomerization domain 2) observed in different studies. Left, Watanabe et al. observed that MDP activation of N2 wt results in inhibition of PGN-mediated activation of NF-κB C-rel isoform via toll like receptor 2 (TLR2) [33]. In the Watanabe et al. models of nod2 ‘knockout’ mice, N2 Ci mice, or human N2 Ci/TLR2 doubly transfected HT29 cells, nod2 fails to properly down-regulate PGN-mediated TLR2 activation, resulting in hyperstimulation of NF-κB C-Rel isoform (and thus IL-12).

C-insertion double-mutant mice and human cell lines Watanabe et al. also created a C-insertion double-mutant mouse model (a C-insertion frameshift mutation of mouse nod2, which creates a homologous defect to human Leu1007fs) and observed splenocytes to behave similarly to nod2–/– cells. Furthermore, HT-29 cells (a human colonic epithelial cell line that does not contain NOD2 or TLR2) were cotransfected with TLR2 and either wild-type or Leu1007fs human NOD2. The doubly cotransfected Leu1007fs cells showed no stimulation with MDP alone, but showed enhanced stimulation of NF-κB in response to peptidoglycan (as compared with the HT-29 cells cotransfected with TLR2 and wild-type NOD2, see Figure 2).

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The results of the Watanabe et al. study suggest that the extracellular response of macrophage and splenocyte populations to peptidoglycan (via TLR2) is down-regulated by intracellularprocessed peptidoglycan via wild-type NOD2. The form of NF-κB most strongly induced by peptidoglycan was cRel, the form required for IL-12 production. Netea et al. observed that human monocytes from Leu1007fs homozygotes had defective release of IL-10 after stimulation with TLR2 and NOD2 ligands, peptidoglycan, and the TLR2 agonist, Pam3Cys-KKKK [34]. This result could also imply a similar proinflammatory imbalance to that observed in the Watanabe studies. Kobayashi et al. could not replicate these results in their nod2–/– mice BMDM and BMDC, using co-stimulation with both MDP and Pam3Cys [32]. There is no ready explanation as to why the Kobayashi et al. mice behaved differently. Factors might include the difference in background strain and the source of cells studied (bone marrow-derived vs splenocyte-derived). Recently, Maeda et al. also developed a C-nucleotide frameshift mutation at the corresponding position in mouse nod2, similar to the Watanabe et al. C-nucleotide mutant mice [35]. Surprisingly, these mice showed a relative increase in NF-κB activity with MDP exposure alone (ie, without TLR2 stimulation), as well as an increase in transcription of il6, cox2 (cytochrome oxidase subunit 2), a20 (mouse tumor necrosis factor α), mip2 (chemokine ligand 2), il1a, and il1b mRNA, and an increase in IL-1β secretion. The increases were significantly greater for MDP than for peptidoglycan. Also, unlike the nod2–/– mice, the nod22397cins/2397cins mice were markedly sensitive to dextran sodium sulfate, producing large areas of colonic ulceration, and greater mortality. Further experiments directly comparing the different models in the same laboratory are needed in order to resolve the various findings of these different mouse nod2–/– and C-insertion mutation models. We presently favor the Kobayashi et al. and the

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Watanabe et al. findings as they have demonstrated parallels in NOD2 studies in human cells and/or transfected proteins. NOD2 and other diseases NOD2 nuclear-binding mutations (eg, R334Q) are well-established, known causes of the autosomal dominant granulomatous disorder, Blau syndrome. Tanabe et al. demonstrated that Blau mutations result in constitutive hyperactivity of NOD2 induction of NF-κB [24]. The NOD2 CD-predisposing mutations have recently been implicated in other diseases: preliminary evidence suggests that NOD2 is one of several genes associated with a worse prognosis following sepsis [36]. NOD2 mutations might also be responsible for increased instance of graft versus host disease, particularly when both donor and recipient carry NOD2 mutations (mortality rates: 20% when both donor and recipient were wild-type, 49% when only the recipient carried a mutation, 59% when only the donor carried a mutation, and 83% when both the donor and recipient carried NOD2 mutations) [37].

The IBD5 haplotype and the OCTN genes IBD5 In The Inflammatory Bowel Disease Yearbook 2003, we reported the discovery of a 600,000 base-pair haplotype at the IBD5 chromosome 5q31 cytokine locus, which is highly associated with CD in 605 parent/CD-child ‘trio’ pedigrees (P<0.0000002) [17,38]. The IBD5 haplotype (H2) could be specified by any of 11 highly correlated, anonymous SNP alleles that span the region. H2 is now established as a CD risk factor, and this has been confirmed in four different replication study populations: one using the case-control method of association; two using the more rigorous within-family, transmission disequilibrium test (TDT); and one using both these methods (Table 2) [39–42]. A Spanish replication study showed a nonsignificant increase in H2 alleles in CD patients versus controls (P=0.085) [43].

55

56 2.1 (1.6–2.8) non-Jewish NS Jewish 1.4 (1.1–1.8) 1.2 (1.1,1.4)

1.3 (1.1–1.6) 1.4 (1.2–1.7)

NS

Canadian (OCTN-TC) [48]

Oxford, UK [39]

British [40]

German [41]

Oxford, UK [42]

Spanish [43]

0.48/0.44

0.46/0.40

274/511 (211)

368 CD trios 187 UC trios 330/870 (242)

282 CD trios 252 UC trios 684/701 (388)

CD cases/ controls (UC cases) 605 total CD trios 507/352 (216) (70% non-Jewish)

No

No

P=0.002

No

No

No

NA

UC association

Significant phenotype Other features association (P<0.05) NA Original discovery of H2 Ileal site TC/TC: Overlap with OR 4.5 discovery cohort Surgery P<0.05 No NOD2 epistasis None for onset or family history None for onset No NOD2 or TNFα epistasis Early onset TDT association (P<0.05) with CD in 511 None for site trios P=0.016 None (site, NOD2 and H2 behavior, onset) epistasis for UC Perianal (Pc=0.0005) No NOD2 epistasis H2H2 RR=1.7 None for behavior, onset, EIM Ileocolon; any H2/H2 greater fistulizing compared in infliximab to controls nonresponders vs responders

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0.56 carrier

0.48/0.43

NA

0.49/0.35 non-Jewish 0.51/0.44 Jewish

Haplotype (gene) frequency (CD/controls) NA

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Table 2. IBD5 haplotype (H2) association studies; CD: Crohn’s disease; CI: confidence interval; EIM: extraintestinal manifestations; NA: not available; NOD2: nucleotide-binding oligomerization domain 2 gene; NS: not significant; OCTN-TC: organic cation transporter gene C1672T/G-207C haplotype; OR: odds ratio; Pc: corrected P-value; Ref.: reference; TC/TC: TC homozygote; TDT: transmission disequilibrium test; TNFα: TNF-α gene; UC: ulcerative colitis.

Canadian [38]

Relative risk of CD (95% CI) 2.1 (1.5–3.2)

Population [Ref.]

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The RR of CD conferred by the four positive replication studies is substantially weaker (average RR 1.30) than the original discovery population (RR 2.13) [44]. Only one of the four replication studies found significant association evidence for UC, and this evidence was greater in NOD2 carriers [41]. However, in the largest UC case sample, UC allele frequency (0.40) was less than that of the control allele frequency (0.43) (see Table 2) [40]. Most studies did not find evidence for NOD2 epistasis. In general, NOD2 and H2 risks were additive. All studies observed a greater association with CD in homozygote carriers compared with heterozygote carriers. Phenotype associations for CD were inconsistent, and only a perianal disease association observed in the Oxford cohort was significant after correction (see Table 2) [42]. Interestingly, in the Spanish study the H2/H2 homozygote genotype was significantly increased in CD patients lacking response to infliximab (RR 3.88, 95% CI 1.2–12.0) [43]. Candidate genes The IBD5 haplotype involves multiple candidate genes, including IL3, IL4, IL5, IL13, and IFN regulatory factor 1 (IRF1), with the monocyte differentiation antigen gene – CD14 (see later section on candidate genes) – just outside the cytokine cluster. Initial sequencing in the Rioux et al. study failed to identify functional variants of the haplotype [38]. Functional variants of IL4 and CD14 (not known to be on the 5q haplotype) were evaluated for association with CD with inconsistent results [17]. OCTN1 and OCTN2 Evaluating the Canadian discovery cohort (also a cohort with strong linkage to the 5q31 region), Peltekova et al. reported that two functional polymorphisms in the OCTN1 and OCTN2 genes (both located near the midpoint of H2) were in strong linkage disequilibrium with H2. In addition, two variants in these genes form a haplotype (TC) more highly associated with CD susceptibility than H2 [38,45,46]. These two variants are a

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C1672T/ Leu503Phe missense substitution in the OCTN1 gene and a G –207C OCTN2 promoter polymorphism. OCTN transporters The OCTN transporters are highly expressed in the apical epithelial membrane of the kidney as well as in other tissues, including the trachea, liver, bone marrow, and cardiac and skeletal muscle (OCTN2), and also in intestinal epithelium [46]. The proteins are responsible for carnitine transport, and, in particular, OCTN2 mutations are responsible for primary systemic carnitine deficiency (PSCD). Both OCTN1 and OCTN2 are responsible for epithelial transport of multiple drugs and potentially toxic xenobiotics. The OCTN1 503Phe variant had a 2.7-fold reduced transport activity for carnitine, but greater transport of tetraethyl ammonium and greater binding affinity for other xenobiotics. The OCTN2 –207C variant disrupted a heat-shock element of the OCTN2 promoter, causing the –207C variant to have nearly 2-fold promoter activation (by exposure to heat-shock factors) in luciferase reporter assays. OCTN animal knockout models have not been reported; however, no intestinal inflammatory manifestations have been reported in PSCD. Nonetheless, OCTN disruption could theoretically result in a greater risk of inflammation in patients who are unable to properly clear xenobiotics, parallel to multidrug resistant gene 1 (MDR1) knockout mice, where loss of xenobiotic clearance in the presence of intestinal bacteria is believed to cause chronic colitis [47]. Demography and phenotype A more detailed phenotypic/demographic analysis was recently reported (see Table 2) by Newman et al. [48]. The C1672T/G207C haplotype (TC) was strongly associated (P<0.0001) with CD in the non-Jewish, but not the Jewish, subgroup of patients. Risk (OR) was 3.73 (95% CI 2.13–6.54) for TC/TC homozygosity and 2.49 (95% CI 1.61–3.85) for heterozygosity. There was also a modest increase in risk for ileal versus nonileal disease site in

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TC/TC patients (OR 2.42), which was increased further in those carrying a common NOD2 risk allele (OR 4.5). Further investigation is required to discover whether the OCTN TC haplotype is responsible for overall IBD5 (H2) CD risk. This will involve finding evidence that TC alleles are, when found present on chromosomes that do not also contain the H2 risk haplotype (particularly those H2 alleles closest in proximity to the OCTN genes), significantly associated with CD.

Advances in linkage studies There are now 12 published genome-wide screens, five of which have been published since our last review [45,49–59]. A total of 1,303 families, multiply affected by IBD, were studied in the 12 screens: 67% of relative pairs with CD and 18% with UC. These screens analyzed alleles on chromosomal markers for evidence of increased sharing of alleles in affected relatives (greater than that expected by their familial relationship), thus providing evidence that the chromosomal region specified by the markers is ‘linked’ to the IBD phenotype [60]. All screens genotyped pedigrees with microsatellite markers (primarily dinucleotide, trinucleotide, or tetranucleotide repeat polymorphisms), with an average marker density of 10 cM. Seven screens included the X chromosome. The six North American screens had an average of 30% of pedigrees of Jewish ethnicity. All screens were essentially limited to Caucasian pedigrees. The five recent genome-wide screens included three novel populations, a 63-pedigree Nova Scotia IBD screen, a 92-pedigree Finnish screen, and an 89-pedigree Flemish screen [55,56,59]. The remaining two screens were of new pedigrees assembled from the Oxford (UK) IBD center (112 pedigrees) and from three North American centers (139 pedigrees – University of Pittsburgh, Johns Hopkins IBD Center, and University of Chicago) [57,58].

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The Finnish screen was the only IBD screen where the majority of affected relative pairs were either both UC (55% of pairs) or mixed (ie, UC and CD pairs, 27%) [56]. This screen provided suggestive evidence of linkage (P<0.001) for a novel UC locus on chromosome 2p11 and further established the chromosome 12 IBD2 locus, with nominal UC evidence at 12p13 and suggestive CD evidence at 12q23. The Nova Scotia screen provided additional evidence for the IBD3 HLA region locus and showed suggestive linkage evidence for a potential novel locus at chromosome 11p [55]. The recent Oxford screen found evidence to replicate a locus on chromosome 3q25–28, first seen in the first North American genome-wide screen, and a locus on chromosome Xp22, first identified in a German genome-wide screen and perhaps the first X-linked IBD locus [51,52,58]. By stratifying linkage evidence according to NOD2 and IBD5 haplotype status, the Oxford screen also found evidence to support IBD1 (chromosome 16cen) and particularly IBD6 (chromosome 19), with evidence more on the q-arm of chromosome 19 than in the original IBD6 discovery report [45,58]. The recent North American screen found suggestive UC linkage evidence at chromosome 2q23–35, a region first identified with nominal evidence for IBD overall in the first Oxford screen [50,57]. A recent meta-analysis suggested that the locus 2q24–34 has the strongest overall linkage evidence for a UC-specific locus [61]. The North American screen also provided suggestive evidence of linkage for a novel IBD locus on chromosome 6q [57]. The recent Flemish genome screen, primarily of CD pedigrees (85% CD, 15% mixed), found evidence to support this 6q linkage evidence. It also found replication evidence for IBD4 (14q11–12, P=0.008), IBD7 (1p36–32), and a region on chromosome 10p12 that was seen in a prior German genome screen [52,59].

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Table 3 provides a list of loci that demonstrated either significant (genome-wide) linkage evidence in a genome screen, or suggestive evidence and replication evidence in more than one study. These loci that had linkage evidence (P<0.10) in a meta-analysis by van Heel et al. that analyzed the autosomal chromosome linkage results of 11 genome screens are also noted (all but the Flemish study, which was published subsequently) [61]. In the meta-analysis the IBD3/HLA locus was the most significant locus, and IBD3 was the only locus with significant linkage evidence when corrections were made for multiple testing (ie, genome-wide linkage evidence). In descending order, IBD1, IBD3, and IBD6/chromosome 19 were the strongest CD loci. The 2q, IBD2, and 19q (region just telomeric to IBD6) loci were the strongest UC loci. The IBD4 chromosome 14q11–12 locus did not demonstrate significance in the meta-analysis, perhaps because of the exclusion of the Flemish study and because IBD4 is located more centromeric than markers used in several genome screens. The International IBD Genetics Consortium genotyped the IBD4 candidate region in 733 IBD sibling pair pedigrees (432 both with CD) and found evidence confirming linkage for CD (P≤0.01), with nominal evidence (P<0.04) in pedigrees not included in the initial screens that first identified IBD4 [62]. Particularly noteworthy was the identification of tobacco as a possible interactive factor with IBD4 (mean allele sharing in smokers vs nonsmokers, 59% vs 52%, respectively), the first environmental/molecular genetic IBD association that has been reported. In the next generation of planned genome screens, difficulty with marker coverage of potential linkage regions will likely be eliminated. This is because these screens use closely spaced SNP markers, giving nearly complete chromosomal coverage.

61

62 IBD (CD) IBD/CD IBD IBD/CD/UC

6q22–q27

7p13–q21

11p15.5–11p12

12p13.2–q24 (IBD2)

NA

0.007 (CD)

0.003

–

0.02 (UC)

0.06

0.02 (IBD)

–

0.0001

–

0.04 (UC)

0.004 (IBD)

0.09

0.01

45,52,58

45,51,54,56,58

49,51,52,55,58

51,53,54,59

50,52,53,56–58

51,55,56

45,50,51,56,58

53,57,59

45,52,53,55,57

45,53

45,51,52

45,51,58

45,50,56,58

45,50,54,57

51,54,59

Genome screen references

135

62

135–139

130–134

38

127–129

126,127

Replication study references

Table 3. Significant loci identified in genome screens. Loci with either genome-wide linkage evidence (P<0.00002), or suggestive evidence of linkage (P<0.001) in one genome screen with replication evidence in at least two studies. CD: Crohn’s disease; IBD: inflammatory bowel disease; NA: not analyzed; UC: ulcerative colitis.

IBD/CD

IBD

6p21–p23 (IBD3)

CD/IBD

CD

5q31–q35 (IBD5)

19p13–q13 (IBD6)

IBD/UC

4q22–q31

Xp22–p21

IBD/CD

3q25–q28

CD

IBD

3p25–p14

CD

UC

2q23.2–q35

–

P value, meta-analysis

12:44

16p11–q21 (IBD1)

IBD (UC)

1p36.2–p34 (IBD7)

20/4/06

14q11–q12 (IBD4)

IBD phenotype

IBD loci

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Genetic studies of other IBD candidate genes since 2003 Table 4 lists candidate gene association studies that have been reported since our last detailed IBD Genetics chapter in The Inflammatory Bowel Disease Yearbook 2003 [17]. Noteworthy recent positive candidate gene reports, with the exception of OCTN (reviewed previously), are discussed in the following paragraphs. HLA associations were reviewed extensively in the 2003 yearbook. Very consistent associations for UC (particularly DRB1*0103 and DRB1*1502) were noted, with less consistent associations for CD, and novel, strong associations for IBD with TNF-α promoter allele –857C. Our current review identified only two recent TNF-α reports (both negative, see Table 4) and two HLA reports: a Canadian study that extends CD evidence for DRB1*0103 (reported in a prior study of the same population), and an Italian report of novel HLA phenotype influences (see Table 4) [63–66]. MDR1 (multidrug resistance gene 1) The multidrug resistance gene, MDR1, encodes the membrane transport protein, P-glycoprotein 1. It has been proposed as a candidate gene for IBD on the basis of: •

Its location within the region of linkage detected on chromosome 7q22.

•

Its possible function as a barrier to xenobiotics in intestinal epithelial cells.

•

The development of colitis in mdr1a knockout mice [47,50,61].

Schwab et al. genotyped the C3435T synonymous exon 26 polymorphism, associated in some studies with lower MDR1 gene expression and activity. There was a borderline association with UC in 149 patients compared with 126 controls (P=0.49) [67].

63

64 2q14

IL-1β, MMP-1, MMP-3, iNOS

IL-1RN (VNTR polymorphism)

P450 1A1 (CYP1A1), GSTM1, GSTT1, GSTP1, EPXH

MCP-1 (G/A SNP in distal regulatory region)

1q32

1q42 (EPXH gene)

Gene/ polymorphism

Chromosome

140

Locus

96 UC, 106 controls

229 UC, 239 controls

151 CD, 149 controls

179 CD, 189 controls

Samples tested

No UC associations

Allele 2 carriage rate is associated with lower age of diagnosis (P=0.002) and pancolitis (P=0.025)

Tyr113 allele in exon 3 of the EPXH gene increased in CD (P<0.0001)

GA and GG alleles are decreased in CD patients with later onset (P<0.05) and fistulizing disease (P<0.06)

Findings

Japanese

Japanese

Dutch

German

Ethnicity

12:44

2q14.2

+

Association

20/4/06

105

103

102

Ref.

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2q33

3p21

+

+

+

142

143

80

78

4q24

NFKB1 (–94ins/ delATTG)

The rare variants of two intronic polymorphisms are more common in controls than CD (P<0.00001)

–94delATTG is associated with UC by TDT (P=0.047–0.052) and case-control (P=0.021)

235 IBD pedigrees 350 UC, 802 controls

–1237C allele association with CD (P<0.01)

SAMP1/YitFc mice and 134 CD, 125 controls

174 CD, 138 UC, 265 controls

TLR9 –1237 (T/C) and 2848 (G/A)

No IBD associations

No IBD associations

Non-Jewish Caucasian

Caucasian

German

Dutch/ Chinese

German

12:44

PPARG

163 CD, 139+35 UC, 174+62 controls

166 CD, 63 UC, 187 controls

CTLA4 (C-318T, A+49G)

LCT

20/4/06

3p25

2q21

141

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65

66 IBD3

IBD3

64

65

Locus

IBD3

+

Association

63

87 (see also 84)

Ref.

HLA-DRB1

507 unrelated CD

128 CD, 103 controls

TNF-α (–1031T/C, –863C/A, –857C/T, –308 G/A, –238 G/A)

HLA-DRB1*0103 allele is associated with CD in non-Jewish, familial cases (P=0.0002)

No significant IBD associations

No significant IBD associations

T allele and TT genotype increased in CD with NOD2 variants (P=0.02 and 0.0002, respectively)

Findings

Canadian

Canadian

German

German

Ethnicity

12:44

6p21.3

95 CD, 93 UC, 119 controls

253 CD, 650 controls

Samples tested

TNF-α (–308 G/A), EGFR (codon 497), VDR (TaqI polymorphism)

CD14 (–159C/T)

Gene/ polymorphism

20/4/06

6p21

6p21 (TNF-α), 7p12 (EGFR), 12q12 (VDR)

5q31.1

Chromosome

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6p25

7q21

7q21

145

146

67

+

6p21–23

IBD3

144

66

MDR1 3435 C/T

Type-1 PAI-1 (4G/4G polymorphism)

3435T association with UC in smaller and larger control sets (P<0.05, P<0.01, respectively)

4G/4G genotype is associated with penetrating CD (P<0.0001)

157 CD, 350 controls

275 IBD, 275 controls

No IBD associations

No IBD associations

DR13 allele increased in UC with pancolitis and extraintestinal manifestations (P<0.0001) DR3 allele increased in CD with colonic extent (P<0.0001) and extraintestinal manifestations (P=0.0003)

German

Spanish

Italian

Canadian

Italian

12:44

62 CD, 90 UC, 130 controls

126 CD, 105 controls

102 CD, 114 UC, 264 controls

20/4/06

Factor XIII (Val34Leu)

SEEK1 (3 SNPs: +39604, +39709, +26680)

HLA-DRB1

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67

68

+

147

MDR1 (1236C/A, A893S, 3435C/T)

MDR1 (Asn21Asp, Ala893Ser/Thr, C3435T)

+

71

163 CD, 144 UC, 355 controls

558 IBD (409 CD, 119 UC)

CD 120, 85 UC, 100 controls

Caucasian (US)

Slovenian

Haplotypes of the SNPs associated with IBD

Greek

No associations

The common Ala893 allele is associated with IBD by case-control analysis (P=0.002) and the PDT (P<0.0004)

German

German/ British

Ethnicity

Association only of T carriage and UC (P=0.03)

No associations

Findings

12:44

MDR1 3435 C/T

CD 135, 123 UC, 265 controls

562 CD, 307 UC, 538 controls, 553 IBD trios

Samples tested

20/4/06

70

MDR1 3435 C/T

+

Chromosome Gene/ polymorphism

69

Locus

MDR1 3435 C/T

Association

68

Ref.

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+

85

348 CD, 163 UC, 140 controls, 318 IBD trios

120 CD, 85 UC, 100 controls

234 CD, 246 UC, 189 HC

102 CD, 98 UC, 145 controls

268 CD, 335 UC, 370 controls

299Gly association with CD (P=0.004), UC (P=0.03) in case-control and in IBD trios (P=0.01)

299Gly increased in CD vs controls (P=0.03); CD14 –159T increased in CD vs controls (P<0.01)

No association

Thr399Ile is associated with UC (P=0.014)

3435TT genotype (P=0.04), T allele (P=0.02) and Ala893–G2677 haplotype (P=0.03) associated with UC

Belgian

Greek

Scottish

German

Scottish

12:44

TLR-4 (Asp299Gly)

TLR-4 (Asp299Gly), CD14 (–159C/T)

+

TLR-4 (Asp299Gly, Thr399Ile), CD14 (–159C/T)

86

9q32 (TLR-4) 5q31 (CD14)

TLR-4 (Asp299Gly), CD14 (–159C/T)

+

84

MDR1 (Ala893Ser, C3435T)

20/4/06

10

+

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69

70 IBD2

IBD4

148

149

183 CD, 141 UC, 173 controls 151 CD, 111 UC, 119 controls

IL-25 (c424C/A)

14q11.2

228 CD, 151 UC, 495 controls

482 CD and 339 controls

302 CD trios and 155 UC trios

Samples tested

STAT6 (G2964A)

VDR (BsmI polymorphism)

DLG5

DLG5 (113G/A)

Gene/ polymorphism

12q13.3

12q12–14

10q23

Chromosome

No IBD associations

No IBD associations

BB genotype increased in Ashkenazi UC patients (P=0.042)

DLG5 INV15-137(C/T), CC genotype association with CD (P=0.02)

Two distinct haplotypes are associated with IBD (P=0.000023 and P=0.004 for association with IBD, P=0.00012 and P=0.04 for association with CD). 113G/A is associated with IBD (P=0.001)

Findings

German

Dutch

Israeli

Japanese

German

Ethnicity

12:44

IBD2

+

75

Locus

20/4/06

100 (see also 63)

+

Association

74

Ref.

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75 CD, 90 UC, 187 controls

103 CD, 100 UC, 120 controls

61 CD, 61 controls

259 CD, 145 UC, 441 controls

EE genotype increased in IBD (P=0.000230). E469 allele increased in ileocolonic (P=0.00072) and penetrating CD (P<0.00001)

Italian

German

German

Association with severe CD

T allele and TT genotype increased in CD (P<0.01)

German

The AA genotype in the 3'-UTR increased in CD patients (P<0.04), especially in patients without the NOD2predisposing allele (P<0.003)

12:44

ICAM-1 (K469E)

IL-16 (–295 T/C)

HSP70-2 (PstIpolymorphism)

NFKB1A (SNP at 3'-UTR and at position –420 in the promoter)

20/4/06

19p13

+

93

IBD6

15q26

+

98

14q13

14q24

+

151

150

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71

72

Association Locus

22q11.2

Chromosome

Samples tested

221 UC, 438 controls

Gene/ polymorphism MIF (–173 G/C)

CC genotype is associated with pancolitis (P=0.0074)

Findings

Japanese

Ethnicity

Table 4. Update in IBD candidate gene reports. CD: Crohn’s disease; CTLA4: cytotoxic T lymphocyte-associated 4; DLG5: Drosophila discs, large homolog 5; DR: down-regulator of transcription; EGFR: epidermal growth factor receptor; EPXH: microsomal epoxide hydrolase; GSTM1: glutathione S-transferase μ-1; GSTP1: glutathione S-transferase π-1; GSTT1: glutathione S-transferase θ-1; HLA-DRB1: human leukocyte antigen-DRβ1; IBD: inflammatory bowel disease; ICAM-1: intercellular adhesion molecule 1; IL: interleukin; iNOS: inducible nitric oxide synthase; LCT: lactase phlorizin hydrolase; MCP: membrane cofactor protein; MDR1: multidrug resistance protein 1; MIF: macrophage migration inhibitory factor; MMP: matrix metalloproteinase; NFκB1: nuclear factor κB 1; PAI-1: plasminogen activator inhibitor 1; PDT: pedigree disequilibrium test; PPARG: peroxisome proliferator-activated receptor γ; SEEK1: psoriasis susceptibility 1 protein; SNP: single nucleotide polymorphism; STAT6: signal transducer and activator of transcription 6; TDT: transmission disequilibrium test; TLR: toll-like receptor; TNF: tumor necrosis factor; UC: ulcerative colitis; VDR: vitamin D receptor; VNTR: variable number tandem repeat.

92

Ref.

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These findings were not supported in a German/British study of 307 UC cases, 538 controls, and >200 UC trios, nor in a small Greek study, but equivocal results were found in a second German population [68–70]. Brant et al. sequenced exonic regions of the gene and tested two missense polymorphisms, asn21asp (exon 2) and the tri-allelic ala893ser/thr (G2677T/A, exon 21), as well as the C3435T variant in a TDT analysis of North American IBD trio pedigrees and a case-control analysis [71]. The ala893 allele was associated with reduced MDR1 activity in transfection experiments [72]. Significant association of the ala893 (but not of the asn21 to asp or C3435T polymorphisms) allele was found with IBD by both case-control analysis (P=0.002) and the pedigree disequilibrium test (P=0.00020–0.00030). Similar trends were observed for CD and UC association with ala893, providing a significant CD association (P=0.001). In this cohort, there was also no support for the 3435T allele with UC, as it was under-transmitted to UC offspring. A study of haplotypes defined by 10 MDR1 polymorphisms including exon 21 (a rare A allele not genotyped) and 26 polymorphisms in a Slovenian population failed to identify associations with specific alleles, although there was a weak UC association with a 1236T–2677T–3435T haplotype (P=0.03). Ho et al. performed a case-control analysis of MDR1 C3435T and ala893ser polymorphisms in a Scottish IBD cohort (the 2677ser allele was genotyped in a subset of affected individuals) [73]. The MDR1 3435 TT genotype and T-allelic frequencies were significantly higher in patients with UC (P=0.04 and P=0.02, respectively) compared with controls. The strongest UC association was observed with an ala893–3435T haplotype (OR 1.44, P=0.03). The confusing array of MDR1 studies suggest: • A genetic risk of MDR1, if present, is likely to be small, and much larger studies are needed with genotyping done on all the same polymorphisms.

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• There might be genetic heterogeneity, perhaps involving interactions with other genes. • The ala893 and 3435T alleles have most consistently demonstrated functional associations with decreased MDR1 protein activity and are leading contenders for MDR1/IBD risk alleles. DLG5 (Drosophila discs large homolog 5) Hampe et al. identified a locus in the pericentromeric region of chromosome 10 that was associated with susceptibility to IBD in a genome-wide linkage scan involving 282 families of European descent [52]. Stoll et al. refined this linkage region on 10q23 and used positional cloning to identify genetic variants in Drosophila discs large homolog 5 (DLG5) associated with IBD [74]. DLG5 encodes a scaffolding protein involved in the maintenance of epithelial integrity. The study identified two distinct haplotypes with a replicable distortion of transmission in trios. One of the risk-associated DLG5 haplotypes was distinguished from the common haplotype by a nonsynonymous SNP, 113G–A, resulting in the amino acid substitution arg30 to gln (R30Q) in the DUF622 domain of DLG5. The mutation was predicted to impede scaffolding of DLG5. Stoll et al. stratified the study sample according to the presence of risk-associated variants of NOD2 to study potential gene–gene interactions. They found a significant difference in association of the 113A variant of DLG5 with CD in affected individuals carrying the risk-associated NOD2 alleles versus those carrying nonrisk-associated NOD2 alleles. This suggested a complex pattern of gene–gene interaction between DLG5 and NOD2. In a Japanese study, out of eight DLG5 SNPs tested, a weak association of DLG5 with CD was found with one intron 15 variant [75]. κB p50 isoform) NFKB1 (nuclear factor-κ NFKB1 is the gene for the NF-κB p50 isoform, one of the two major NF-κB peptides. The major susceptibility locus for

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two different mouse colitis models maps tightly to mouse nfkb1 [76,77]. Karban et al. identified a novel 4 base-pair NFKB1 insertion/deletion promoter polymorphism, –94ins/delATTG [78]. The deletion allele was associated with reduced luciferase reporter activity of the NFKB1 promoter and disrupted promoter–transcription factor binding. Our study found the deletion allele over-transmitted to UC offspring in 131 UC trios (P=0.05), and observed replication evidence for association in a separate set of non-Jewish UC cases and ethnically matched controls (P=0.02). Overall risk for UC was modest (OR 1.51, 95% CI 1.1–2.2). Preliminary evidence for replication was recently reported in a Dutch cohort [77]. PPARG (peroxisome proliferator-activated receptor γ) Peroxisome proliferator-activated receptor γ (PPAR-γ) is a ligand-activated transcription factor that plays a central role in adipocyte differentiation and insulin sensitivity, but is also central to signal transduction pathways involved in controlling inflammatory responses. A recent study has also shown that it is expressed at decreased levels in colonic tissue from UC patients [79]. The SAMP1/YitFc (SAMP1/Fc) mouse strain expresses many features of CD in humans, including chronic inflammation of the ileum and discontinuous, transmural inflammatory lesions. Sugawara et al. performed a quantitative trait linkage analysis for ileitis in offspring of nonileitis phenotype AKR x SAMP1/Fc mice backcrossed to SAMP1/Fc. They identified a major recessive ileitis locus on mouse chromosome 6, a region with synteny (ie, genes that occur in the same order) to the human chromosome 3p25 candidate region (see Table 3) [80]. Sugawara et al. identified a 7 base-pair PPARG promoter insert in SAMP mice that corresponded with reduced SAMP PPARG epithelial mRNA. A treatment trial of SAMP1/B6 mice with the PPAR-γ agonist rosiglitazone ameliorated colitis as effectively as dexamethasone.

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A case-control study was conducted, where three noncoding SNPs spanning the coding region of PPARG were genotyped in 134 CD patients and 125 healthy controls. Allele frequencies in the CD cohort differed significantly from those in the controls (P<0.0001 for SNP1). Replication studies, particularly in cohorts with 3p25 linkage evidence, will be helpful to determine whether PPARG accounts for the 3p25 locus. Toll-like receptors and CD14 Toll-like receptors (TLRs) are activated by the molecular structures of microbes. Activation of these receptors initiates an inflammatory cascade that attempts to clear the offending pathogen. Inappropriate TLR signaling can contribute to common diseases such as severe sepsis, atherosclerosis, IBD, and autoimmune syndromes [81]. Alterations in the expression of TLRs might initiate or perpetuate intestinal inflammation. Among the TLRs, TLR4 was found to be strongly upregulated in both UC and CD [82]. TLR4 binds to lipopolysaccharides together with CD14, and through internalization prevents inappropriate NF-κB activation [83]. Torok et al. conducted a study in 102 patients with CD, 98 patients with UC, and 145 healthy controls. The investigators evaluated two polymorphisms of the TLR4 gene (Asp299Gly and Thr399Ile) that affect lipopolysaccharide signaling; the –159C/T promoter polymorphism of the CD14 gene, and polymorphisms of the lipopolysaccharide-binding protein gene [84]. The allele and carrier frequencies for the Thr399Ile mutation in the TLR4 gene were significantly increased in UC when compared with controls (P=0.014 and P=0.018, respectively). None of the other five polymorphisms were associated with IBD. Similarly, Arnott et al. found no differences in the TLR4 Asp299Gly or –159C/T polymorphisms among Scottish CD and UC patients and healthy controls [10].

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In contrast, Franchimont et al., analyzing only the Asp299Gly variant, observed an association in both CD and UC cohorts, as well as significantly increased transmission of the 299Gly variant in 318 IBD trios (P=0.01) [85]. Similarly, Gazouli et al. reported that the TLR4 299Gly and CD14 –159T allele frequencies were significantly higher among Greek CD patients compared with healthy controls (P=0.026 and P=0.0048, respectively) [86]. Additionally, 51.7% of CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one NOD2 risk allele, compared with 27% of the UC patients. The results suggest that coexistence of a risk allele in the TLR4 or CD14 gene and a NOD2 allele is associated with an increased susceptibility to developing CD compared with UC. Klein et al. also reported evidence for potential –159T allele/NOD2 epistasis, finding a significant increase in T allele and TT genotype in CD patients with at least one variation in the NOD2 gene, compared with controls (P=0.02 and P=0.0002, respectively) [87]. However, no CD14 –159T/C association was present independent of NOD2. A previously reviewed Japanese study found higher T allele frequencies in UC patients compared with CD patients, with UC allele frequencies significantly greater than in controls (0.57 vs 0.45, P=0.007) [17,88]. A recent letter by Torok et al. evaluated the TLR9*1237 T/C allele in their German IBD population and found association of the minor C allele with CD, compared with controls [88]. TLR9 maps to the 3p21 locus. It functions as a receptor for unmethylated CpG DNA motifs common in bacteria and appears to be the innate immune receptor by which the effect of probiotic bacteria is mediated [89]. Further research in larger and more diverse populations is needed to elucidate the importance of TLRs, CD14, and their polymorphisms in the pathogenesis of IBD.

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MIF (macrophage migration inhibitory factor) Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been shown to be involved in the development of chronic murine colitis [90]. In patients with UC, the level of MIF and the functions of dendritic cells are upregulated in the peripheral blood, with increased numbers of MIF-expressing cells and mature dendritic cells at the colonic mucosa [91]. In the +173 G/C polymorphism of the MIF gene, the presence of the C allele creates the binding motif of activator protein 4. Nohara et al. genotyped the polymorphism in a Japanese UC cohort and controls [92]. No significant difference in genotype distribution was found between UC patients and healthy controls. However, when the relationship of the C/C genotype to clinical parameters in UC patients was evaluated, the frequency of the C/C genotype was found to be higher in pancolitis-type UC patients than in those with types restricted to the distal or left-sided colon. ICAM1 (intercellular adhesion molecule 1) The ICAM1 (intercellular adhesion molecule 1) gene maps to the IBD6 region of chromosome 19p13. The protein is expressed on vascular endothelium and plays a key role in transendothelial migration of neutrophils. Two recent European studies have provided conflicting results suggesting that ICAM1 probably has no real IBD association. An Italian study reported an association of IBD with 469E homozygotes, while allele 469E was associated with a subgroup of CD patients with more extensive location of disease and penetrating behavior [93]. This IBD EE association provides replication evidence for an earlier German study that found significant association with both CD and UC [17,94]. However, a study from the Oxford group found the opposite results, with 469E homozygosity increased in CD patients (40% vs 29%) [95]. Consistent with the Italian study, the 469E allele was increased in patients with fistulating disease. A Japanese

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study had previously reported significant 469E association with both UC and CD [96]. IL16 (interleukin-16) IL-16 is a T lymphocyte-derived cytokine that uses CD4 as its receptor and hence selectively recruits CD4-bearing cells. Infiltrating CD4+ T cells are a feature of CD. Colonic mucosal IL-16 levels are elevated in CD, suggesting a role for IL-16 in the pathophysiology of IBD [97]. Recently, a T to C polymorphism at position –295 in the promoter region of the IL16 gene was reported. Glas et al. found that the frequencies of the T allele and the TT genotype were significantly increased (P<0.01) in patients with CD compared to controls, regardless of the disease phenotype or the site of intestinal involvement [98]. An association with UC was not observed. VDR (vitamin D receptor) The VDR (vitamin D receptor) gene maps to the chromosome 12, IBD2 locus. It is the cellular receptor for 1,25(OH)2D3 (calcitriol), which has a wide range of different regulatory effects on the immune system. Simmons et al. reported significantly more homozygotes for the TaqI polymorphism at codon 352 of exon 8 (genotype ‘tt’) among patients with CD than in patients with UC or controls (OR 1.99, P=0.017) [99]. A smaller German study that examined the TaqI polymorphism failed to identify a VDR–IBD association [63]. An Israeli case-control study determined the association between the BsmI VDR gene polymorphism and IBD [100]. The frequency of the BB genotype was higher in Ashkenazi patients with UC compared with Ashkenazi controls (OR 2.27, P=0.042). MCP1 (monocyte chemoattractant protein 1) The monocyte chemoattractant protein (MCP)-1 is thought to be important for the recruitment of mononuclear cells and the maintenance of inflammation in IBD. In both CD and UC, there is a marked increase in tissue levels of MCP-1, in addition

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to a whole array of other chemokines [101]. Herfarth et al. studied whether a functional SNP (G or A) located in the distal regulatory region of the MCP1 gene is associated with CD and/or its phenotype [102]. The gene frequency of the different MCP1 alleles did not differ from healthy controls. However, the Gcontaining genotypes were significantly decreased in patients with later onset of the disease and also less frequent in patients with fistulizing disease behavior. Therefore, the alleles might have an influence on disease presentation and behavior. EPHX1 (epoxide hydrolase) Mucosal biotransformation enzymes can modify toxic compounds in the gut. As chemical or oxidative stress might be involved in the etiology of CD, genes encoding enzymes involved in the prevention of such stress could be candidates for genetic susceptibility to CD. To assess the association of CD with genetic polymorphisms in cytochrome P450 1A1, glutathione S-transferases (μ-1, π-1, and θ-1), and epoxide hydrolase, de Jong et al. studied a CD cohort of 151 patients and 149 controls [103]. A polymorphism in exon 3 of the microsomal epoxide hydrolase gene, EPHX1, was distributed significantly differently in CD patients when compared with controls (P<0.0001). All other polymorphisms tested were equally distributed between patients and controls. de Jong et al. concluded that microsomal EPHX1, located on chromosome 1q close to a region previously linked to CD, might play a role in the pathophysiology of CD. IL1RN (IL-1 receptor antagonist) The role of the IL1RN (IL-1 receptor antagonist) gene in predisposing an individual to IBD is controversial. The variable number tandem repeat polymorphism in intron 2 of IL1RN has been shown to associate with UC in some populations. Carter et al. performed a meta-analysis of seven previously reported studies in northern Europeans, in addition to a new set of 320 UC patients and 827 controls from the UK [104]. For the

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total meta-analysis set, there was a minor but significant IL1RN*2 UC association (OR 1.23, P=0.01). Recently, Nohara et al. assessed the relationship between the IL1RN polymorphism and UC in a Japanese population [105]. No significant difference of genotype distribution was found between UC patients and controls. However, when the relationship of the carriage rate of allele 2 with clinical parameters was evaluated, it was found that the allele 2 carriage rate was higher in patients with lower ages at diagnosis (P=0.002). In addition, it tended to be higher in patients with pancolitis-type UC (P=0.025). Variability in results might arise from different sample sizes or patients with different ethnic backgrounds. More rigorous within-family control studies with TDT analysis might help to clarify the variations in IL1RN*2 UC association results.

Conclusion Determination that variants of an IBD candidate gene are truly responsible for IBD genetic susceptibility is a difficult task. The relatively straightforward establishment of NOD2 variants as CD genetic risk factors will prove to be the exception: NOD2 is located in the middle of the most successfully replicated CD genetic locus, IBD1; NOD2 has a polymorphism, Leu1007fs, that results in protein truncation and obliteration of normal function in vitro; and the relative risk of NOD2 mutant homozygotes/compound heterozygotes was ten-fold greater than that of all other present IBD candidate genes. Establishment of the other IBD susceptibility genes will likely be similar for the HLA and IBD5 regions: variants will have a modest effect on function; there will be replications – considerably weaker than the initial studies – as well as multiple nonreplications; variants of candidate genes found on the same risk haplotype will need to be excluded as responsible for the observed associations; and extensive testing will be required to determine the potential relevant biological function of these variants.

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The power to ultimately determine which gene variants are truly responsible for IBD genetic susceptibility will require: testing DNA samples from very large numbers (ie, several thousands) of IBD cases and ethnically matched controls; and examining associations in more phenotypically homogenous subsets, such as CD patients with ileal disease or disease complications, to maximize power. IBD genetic consortiums such as the International IBD Genetics Consortium and more recently the National Institutes of Diabetes, Digestion, and Kidney Diseases IBD Genetics Consortium have been established to develop these essential collaborative studies. Linkage disequilibrium genome screens using >100,000 SNP markers that cover nearly all human genes, are underway for several complex genetic diseases and are being planned for IBD. This will likely result in even more candidate genes that will require validation in large collaborative studies. These future studies will bring us ever closer to realizing the promise of IBD genetics, already partially visualized in the progress seen with the NOD2 discovery. This includes the ability to define the etio-pathogenesis of IBD, determine the causes of variations in IBD phenotype expression, predict who will develop IBD, and define targets for future preventive and therapeutic strategies. Recently, McGovern et al. further elucidated the promise of identification of multiple IBD susceptibility genes [152]. While presence of one disease gene polymorphism (eg, NOD2) is associated with low likelihood ratios and has no use by itself in prediction of CD, the presence of three different risk genes (ie, NOD2, IBD5 and a risk polymorphism on the candidate gene Tucan) gave 98% specificity for prediction of CD. Therefore, as multiple susceptibility gene polymorphisms are established, we will likely see the development of diagnostic panels with high specificity and greater sensitivity for predicting CD. Such panels will be useful in directing strategies to prevent IBD, the ultimate goal of IBD genetics research.

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103. de Jong DJ, van der Logt EM, van Schaik A, et al. Genetic polymorphisms in biotransformation enzymes in Crohn’s disease: association with microsomal epoxide hydrolase. Gut 2003;52:547–51. 104. Carter MJ, di Giovine FS, Jones S, et al. Association of the interleukin 1 receptor antagonist gene with ulcerative colitis in Northern European Caucasians. Gut 2001;48:461–7. 105. Nohara H, Inoue N, Hibi T, et al. Association between the interleukin-1 receptor antagonist polymorphism and ulcerative colitis with younger age at diagnosis. Immunol Lett 2003;90:53–7. 106. Morita N, Toki S, Hirohashi T, et al. Incidence and prevalence of IBD in Japan: nationwide epidemiological survey during the year 1991. J Gastroenterol 1995;30(Suppl 8):1–4. 107. Anseline PF. Crohn’s disease in the Hunter Valley region of Australia. Aust NZ J Surg 1995;65:564–9. 108. Cavanaugh JA, Adams KE, Quak EJ, et al. CARD15/NOD2 risk alleles in the development of Crohn’s disease in the Australian population. Ann Hum Genet 2003;67:35–41. 109. Kurata JH, Kantor-Fish S, Frankl H, et al. Crohn’s disease among ethnic groups in a large health maintenance organization. Gastroenterology 1992;102:1940–8. 110. Calkins BM, Lilienfeld AM, Garland CF, et al. Trends in incidence rates of ulcerative colitis and Crohn’s disease. Dig Dis Sci 1984;29:913–20. 111. Heresbach D, Gicquel-Douabin V, Birebent B, et al. NOD2/CARD15 gene polymorphisms in Crohn’s disease: a genotype-phenotype analysis. Eur J Gastroenterol Hepatol 2004;16:55–62. 112. Gower-Rousseau C, Salomez JL, Dupas JL, et al. Incidence of IBD in northern France (1988–1990). Gut 1994;35:1433–8. 113. Saro Gismera C, Lacort Fernandez M, Arguelles Fernandez G, et al. Incidence and prevalence of IBD in Gijon, Asturias, Spain. Gastroenterol Hepatol 2000;23:322–7. 114. Mendoza JL, Murillo LS, Fernandez L, et al. Prevalence of mutations of the NOD2/CARD15 gene and relation to phenotype in Spanish patients with Crohn disease. Scand J Gastroenterol 2003;38:1235–40. 115. Nunez C, Barreiro M, Dominguez-Munoz JE, et al. CARD15 mutations in patients with Crohn’s disease in a homogeneous Spanish population. Am J Gastroenterol 2004;99:450–6. 116. Loftus EV Jr, Silverstein MD, Sandborn WJ, et al. Crohn’s disease in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and survival. Gastroenterology 1998;114:1161–8. 117. Sugimura K, Taylor KD, Lin YC, et al. A novel NOD2/CARD15 haplotype conferring risk for Crohn disease in Ashkenazi Jews. Am J Hum Genet 2003;72:509–18. 118. Rubin GP, Hungin AP, Kelly PJ, et al. IBD: epidemiology and management in an English general practice population. Aliment Pharmacol Ther 2000;14:1553–9. 119. Ahmad T, Armuzzi A, Bunce M, et al. The molecular classification of the clinical manifestations of Crohn’s disease. Gastroenterology 2002;122:854–66. 120. Helio T, Halme L, Lappalainen M, et al. CARD15/NOD2 gene variants are associated with familially occurring and complicated forms of Crohn’s disease. Gut 2003;52:558–62. 121. Halme L, von Smitten K, Husa A. The incidence of Crohn’s disease in the Helsinki metropolitan area during 1975–1985. Ann Chir Gynaecol 1989;78:115–19. 122. Kyle J. Crohn’s disease in the northeastern and northern Isles of Scotland: an epidemiological review. Gastroenterology 1992;103:392–9. 123. Bernstein CN, Blanchard JF, Rawsthorne P, et al. Epidemiology of Crohn’s disease and ulcerative colitis in a central Canadian province: a population-based study. Am J Epidemiol 1999;149:916–24.

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124. Wang MH, Shugart YY, Rawsthorne P, et al. A population based assessment of NOD2/CARD15 and risk of developing Crohn’s disease. Gastroenterology 2004:126;1533–49. 125. Vermeire S, Wild G, Kocher K, et al. CARD15 genetic variation in a Quebec population: prevalence, genotype-phenotype relationship, and haplotype structure. Am J Hum Genet 2002;71:74–83. 126. Cho JH, Nicolae DL, Ramos R, et al. Linkage and linkage disequilibrium in chromosome band 1p36 in American Chaldeans with IBD. Hum Mol Genet 2000;9:1425–32. 127. Paavola P, Helio T, Kiuru M, et al. Genetic analysis in Finnish families with IBD supports linkage to chromosome 3p21. Eur J Hum Genet 2001;9:328–34. 128. Hampe J, Lynch NJ, Daniels S, et al. Fine mapping of the chromosome 3p susceptibility locus in IBD. Gut 2001;48:191–7. 129. Duerr RH, Barmada MM, Zhang L, et al. Evidence for an IBD locus on chromosome 3p26: linkage, transmission/disequilibrium and partitioning of linkage. Hum Mol Genet 2002;11:2599–606. 130. Yang H, Plevy SE, Taylor K, et al. Linkage of Crohn’s disease to the major histocompatibility complex region is detected by multiple non-parametric analyses. Gut 1999;44:519–26. 131. Satsangi J, Welsh KI, Bunce M, et al. Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotype in IBD. Lancet 1996;347:1212–17. 132. Hampe J, Shaw SH, Saiz R, et al. Linkage of IBD to human chromosome 6p. Am J Hum Genet 1999;65:1647–55. 133. Dechairo B, Dimon C, van Heel D, et al. Replication and extension studies of IBD susceptibility regions confirm linkage to chromosome 6p (IBD3). Eur J Hum Genet 2001;9:627–33. 134. Fisher SA, Hampe J, MacPherson AJ, et al. Sex stratification of an IBD genome search shows male-specific linkage to the HLA region of chromosome 6. Eur J Hum Genet 2002;10:259–65. 135. Cavanaugh J. International collaboration provides convincing linkage replication in complex disease through analysis of a large pooled data set: Crohn disease and chromosome 16. Am J Hum Genet 2001;68:1165–71. 136. Parkes M, Barmada MM, Satsangi J, et al. The IBD2 locus shows linkage heterogeneity between ulcerative colitis and Crohn disease. Am J Hum Genet 2000;67:1605–10. 137. Duerr RH, Barmada MM, Zhang L, et al. Linkage and association between IBD and a locus on chromosome 12. Am J Hum Genet 1998;63:95–100. 138. Curran ME, Lau KF, Hampe J, et al. Genetic analysis of IBD in a large European cohort supports linkage to chromosomes 12 and 16. Gastroenterology 1998;115:1066–71. 139. Yang H, Ohmen JD, Ma Y, et al. Additional evidence of linkage between Crohn’s disease and a putative locus on chromosome 12. Genet Med 1999;1:194–8. 140. Nohara H, Saito Y, Higaki S, et al. Polymorphisms of the IL-1beta and IL-1betainducible genes in ulcerative colitis. J Gastroenterol 2002;37(Suppl 14):107–10. 141. Buning C, Ockenga J, Kruger S, et al. The C/C(-13910) and G/G(-22018) genotypes for adult-type hypolactasia are not associated with IBD. Scand J Gastroenterol 2003;38:538–42. 142. Xia B, Crusius JB, Wu J, et al. CTLA4 gene polymorphisms in Dutch and Chinese patients with IBD. Scand J Gastroenterol 2002;37:1296–300. 143. Torok HP, Glas J, Tonenchi L, et al. Crohn’s disease is associated with a toll-like receptor-9 polymorphism. Gastroenterology 2004;127:365–6. 144. Peddle L, Zipperlen K, Melay B, et al. Association of SEEK1 polymorphisms in Crohn’s disease. Hum Immunol 2004;65:706–9.

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145. Saibeni S, Vecchi M, Faioni EM, et al. Val34Leu factor XIII polymorphism in Italian patients with IBD. Dig Liver Dis 2003;35:32–6. 146. Sans M, Tassies D, Pellise M, et al. The 4G/4G genotype of the 4G/5G polymorphism of the type-1 plasminogen activator inhibitor (PAI-1) gene is a determinant of penetrating behaviour in patients with Crohn’s disease. Aliment Pharmacol Ther 2003;17:1039–47. 147. Potocnik U, Ferkolj I, Glavac D, et al. Polymorphisms in multidrug resistance 1 (MDR1) gene are associated with refractory Crohn disease and ulcerative colitis. Genes Immun 2004;5:530–9. 148. Xia B, Crusius JB, Wu J, et al. Signal transducer and activator of transcription 6 gene G2964A polymorphism and IBD. Clin Exp Immunol 2003;131:446–50. 149. Buning C, Genschel J, Weltrich R, et al. The interleukin-25 gene located in the inflammatory bowel disease (IBD) 4 region: no association with inflammatory bowel disease. Eur J Immunogenet 2003;30:329–33. 150. Klein W, Tromm A, Folwaczny C, et al. A polymorphism of the NFKBIA gene is associated with Crohn’s disease patients lacking a predisposing allele of the CARD15 gene. Int J Colorectal Dis 2004;19:153–6. 151. Debler J, Schiemann U, Seybold U, et al. Heat-shock protein HSP70-2 genotypes in patients with Crohn’s disease: a more severe clinical course with intestinal complications in presence of the PstI-polymorphism. Eur J Med Res 2003;8:120–4. 152. McGovern DPB, Ahmad T, Van Heel DA, et al. From the benchside to bedside? A genetic panel strongly predicts Crohn’s disease. Gut 2004;53:A68.

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4 Therapy: purine analogs and methotrexate Severine Vermeire, Gert Van Assche, and Paul Rutgeerts Introduction Patients with inflammatory bowel disease (IBD) often require extensive treatment as a result of the chronic relapsing nature of their disease. Medical therapy can be divided into drugs effective in inducing remission and drugs capable of maintaining remission. Patients who relapse within 6–12 months after discontinuation of induction therapy should be started on long-term maintenance therapy. The most frequently used drugs are the purine analogs azathioprine (AZA) and 6-mercaptopurine (6-MP), and randomized controlled trials have clearly shown their benefit in maintaining remission. Less evidence and experience is available for methotrexate (MTX), although this drug is effective in inducing and maintaining remission in patients with Crohn’s disease (CD). Both the purine analogs and MTX have been proven to modify the course of the disease, to be steroid sparing, and to reduce the need for surgery.

Azathioprine and 6-mercaptopurine Metabolism and mechanism of action The pathway of AZA metabolism is illustrated in Figure 1. In a first nonenzymatic step, AZA is rapidly converted into 6-MP, which is subsequently metabolized by three competing catabolic and anabolic enzymatic pathways. One pathway includes metabolization by xanthine oxidase into the inactive 6-thiouric acid. Alternatively, 6-MP may be methylated to 6-methyl MP (6-MMP) by the enzyme thiopurine methyl

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Intracellular: 6-MP taken up by erythrocytes and organ tissues

Circulation

6-MMP inactive

6-MMP ribonucleotides

TPMT AZA

6-MP HGPRT XO

Purine synthesis

TPMT 6-TGN

6-TIMP IMPDH

DNA/RNA Cytotoxicity and immunosuppression

6-TU inactive

Figure 1. Pathway of azathioprine (AZA) metabolism. 6-MP: 6-mercaptopurine; 6-MMP: 6-methyl mercaptopurine; 6-TGN: 6-thioguanine nucleotide; 6-TIMP: 6-thioinosine monophosphate; 6-TU: 6-thiouric acid; HGPRT: hypoxanthine guanine phosphoribosyltransferase; IMPDH: inosine monophosphate dehydrogenase; TPMT: thiopurine methyl transferase; XO: xanthine oxidase.

transferase (TPMT). Lastly, metabolization by hypoxanthine phosphoribosyltransferase leads to the formation of the active metabolites, 6-thioguanine nucleotides (6-TGNs). Until recently, the mechanism of action of AZA was unknown. This changed with the study of Tiede et al., who showed that a metabolite of AZA induces apoptosis of T cells when co-stimulated with CD28 [1]. The AZA-generated metabolite, 6-thioguanine (6-TG) triphosphate, will bind to a small guanosine triphosphate (GTP) enzyme called Rac1, instead of binding to GTP, leading to inhibition of Rac1. By doing so, the activation of Rac1 target genes such as mitogen-activated protein kinase, nuclear factor-κB, and anti-apoptosis regulatory protein is suppressed, leading to a mitochondrial pathway of apoptosis.

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Study

N

Drug

Induction of remission Present et al., 1980 [2]

83

6-MP

1.35

Ewe et al., 1993 [4]

42

AZA

2.5

Candy et al., 1995 [3]

63

AZA

2.5

Markowitz et al., 2000 [5]

55

6-MP

1.5

63

AZA

2.5

55

AZA

1.5

51

AZA

2

Maintenance of remission Candy et al., 1995 [3]

Markowitz et al., 2000 [5] O’Donoghue et al., 1978 [6]

Dose Outcome (mg/kg/day) vs placebo 67% vs 8% remission 76% vs 38% remission 76% vs 67% remission 89% remission in both groups 42% vs 7% remission at 15 months 9% vs 47% relapse at 18 months 5% vs 41% relapse at 1 year

Table 1. Placebo-controlled randomized trials with azathioprine (AZA) or 6-mercaptopurine (6-MP) in Crohn’s disease.

AZA and 6-MP in the treatment of CD Induction of remission In 1980, a pivotal, placebo-controlled trial by Present et al. randomized 83 CD patients to treatment with 6-MP or placebo for a total period of 2 years [2]. Cross-over data showed improvement in 26/39 courses of 6-MP (67%) compared with 3/39 courses of placebo (8%) (P<0.001). 6-MP was also more effective than placebo in closing fistulas (31% vs 6%) and in discontinuation or reduction of steroid intake (75% vs 36%) (P<0.001). However, the onset of action of the drug required an average of 3–4 months. Maintenance of remission Subsequent studies confirmed the superiority of the purine analogs over placebo in inducing, but particularly in maintaining, remission (Table 1) [2–6]. In the Candy–Wright study, 63 patients

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with active CD treated with prednisolone were randomized to either AZA (2.5 mg/kg/day) or placebo [3]. At 15 months, 42% of patients receiving AZA versus 7% of patients receiving placebo were in remission (P=0.001). A prospective, placebo-controlled, multicenter trial in 55 children, who were randomized to treatment with 6-MP (1.5 mg/kg/day) or placebo showed similar results [5]. Both treatment arms also received prednisone (40 mg/day). The observed rate of induction of remission was the same in both groups (89%). The duration of steroid use was shorter, however, in the group treated with 6-MP (P<0.001) and the cumulative steroid dose was lower at 6, 12, and 18 months in this group (P<0.01). Furthermore, only 9% of the remitters in the 6-MP group relapsed compared with 47% of controls (P=0.007). These studies clearly show that the addition of AZA or 6-MP to a regimen of corticosteroids significantly decreases the need for prednisone and improves maintenance of remission. A Cochrane systematic review showed that the pooled odds ratio (OR) for response to AZA or 6-MP compared with placebo in active CD is 2.36 (95% confidence interval [CI] 1.57–3.53), with a number needed to treat (NNT) of 5 [7]. The OR for steroid sparing is 3.86 (95% CI 2.14–6.96) in favor of AZA and 6-MP, with an NNT of 3. The same analyses for maintenance of remission in quiescent disease give a pooled OR of 2.16 (95% CI 1.35–3.47), with an NNT of 7 [8]. The maintenance studies also showed a steroid-sparing effect for AZA, with an OR of 5.22 (95% CI 1.06–25.68) and an NNT of 3. AZA and 6-MP in the treatment of UC In contrast to CD, there are few controlled studies on the use of AZA in ulcerative colitis (UC) (Table 2). Kirk and Lennard-Jones conducted a double-blind controlled trial of AZA with a dose of 2–2.5 mg/kg/day in 44 patients with UC [9]. Induction of remission and tapering of prednisolone was greater in the AZA group than in the placebo group (P<0.001). Hawthorne et al. randomized

94

Case series

Fernandez-Banares et al., 1996 [13]

13

56

79

44 346

N

AZA

AZA

AZA

AZA AZA

Drug

2–2.5

Median dose of 100 mg/day 2

2–2.5 –

Dose (mg/kg/day)

36% vs 59% (placebo) relapse (P<0.05) 64–69% remission from years 1–3 75% relapse reduction over 3 years 10% relapse rate after mean 16.3 months

(P<0.001) 58% remission (P=0.0001)

Outcome

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Table 2. Studies using azathioprine (AZA) in ulcerative colitis. RCT: randomized controlled trial.

Retrospective

RCT

Maintenance of remission Hawthorne et al., 1992 [10]

Ardizzone et al., 1997 [12]

RCT 30-year review

Type of study

Induction of remission Kirk et al., 1982 [9] Fraser et al., 2002 [11]

Study

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79 patients with UC who had been on AZA for 6 months or more to either continuation of the drug or withdrawal [10]. A total of 67 patients who had been in remission for 2 months or more were randomized to AZA (n=33) or placebo (n=34). The observed relapse rate after 1 year was 36% in patients continuing AZA compared to 59% for those on placebo (P<0.05). Twelve patients had chronic low-grade or corticosteroid-dependent disease (five were randomized to AZA and seven to placebo). In this group, no benefit was found from continued AZA therapy. The authors concluded that continuing AZA therapy is beneficial in UC if patients have achieved remission while taking the drug. Some caution with this conclusion is necessary given the small sample size of the study and larger studies are needed before firm recommendations can be given. In a 30-year review of the efficacy of AZA in patients attending the Oxford IBD clinic (272 CD and 346 UC patients), the overall remission rate was 52% and the remission rate for UC was 58% [11]. There was no difference in relapse rates between CD and UC. An Italian retrospective study evaluated the outcome of 56 patients who received AZA for steroid-resistant (n=10) or steroid-dependent (n=46) UC [12]. Remission with complete withdrawal of corticosteroids was achieved in 64%, 66%, and 69% of patients in the first, second, and third year of AZA therapy. Compared with the 2 years before AZA treatment, a significant 75% decrease in steroid intake and the number of clinical relapses was observed. Finally, AZA has been shown to be safe and effective in maintaining remission induced by cyclosporine in steroid-refractory severe UC [13]. Efficacy in the postoperative setting Although AZA and 6-MP have often been used to prevent postoperative relapse in IBD patients, until recently there were no published randomized controlled studies to support this. In 2004, a prospective, open-label study randomized 142 CD patients to AZA (2 mg/kg/day) or mesalamine (3 g/day) [14]. At 24 months, the risk of clinical relapse was not significantly

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different in the AZA (17.4%) and mesalamine (28.2%) groups, on an intention-to-treat analysis (OR 2.04, 95% CI 0.89–4.67). In the per-protocol analysis, again no statistical difference in clinical relapse was seen, although the relapse rate reported in the AZA group (34%) was lower than in the mesalamine group (45.9%) (OR 1.79, 95% CI 0.80–3.97). No difference was observed with respect to surgical relapse at 24 months between the two groups. In a subgroup analysis of patients with previous intestinal resections, however, AZA was more effective than mesalamine (12.8% vs 35.9% relapse, respectively; OR 4.83, 95% CI 1.47–15.8, P=0.03). In the multicenter randomized study by Hanauer et al., 131 patients were randomized to receive 6-MP (50 mg/day), mesalamine (3 g/day), or placebo in a double-blind, doubledummy fashion [15]. Clinical recurrence rates (intentionto-treat) at 24 months were 50% (95% CI 34%–68%), 58% (95% CI 41%–75%), and 77% (95% CI 61%–91%) in patients receiving 6-MP, mesalamine, and placebo, respectively. Endoscopic recurrence rates were 43% (95% CI 28%–63%), 63% (95% CI 47%–79%), and 64% (95% CI 46%–81%), respectively. The authors concluded that 6-MP is more effective than placebo (P<0.05) at preventing clinical and endoscopic recurrence over 2 years. This study should also be interpreted with caution. Firstly, the clinical recurrence rate (77%) was higher than the endoscopic recurrence rate (64%) in the placebo group – something that has never been demonstrated before. Secondly, 20% of patients were not assessed for clinical or endoscopic recurrence, and this might have introduced bias. From a practical point, we recommend that CD patients carrying a high risk for recurrence (eg, active smokers, second or third surgery, patients with penetrating disease) should be given AZA 2–2.5 mg/kg/day postoperatively. Patients at low risk for recurrence may be given mesalamine, bearing in mind that this drug is probably only 10% more effective than placebo in preventing relapse and that the NNT is 10.

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Side effects AZA and 6-MP are associated with side-effects in 15–30% of patients. The most reported adverse events include dose-related bone marrow toxicity, pancreatitis and hepatotoxicity, skin rash, fever, gastrointestinal intolerance, myalgias, arthralgias, and risk of infection [16]. TPMT mutations Some adverse events can be explained by the metabolism of AZA by TPMT. The activity of TPMT is genetically determined and various polymorphisms have been reported in the gene [17]. The most common variants in the Caucasian population are the TPMT*3A and TPMT*3C alleles [18,19]. Approximately 11% of the population is heterozygous for the common TPMT polymorphisms, and approximately 0.3% is homozygous mutant. Heterozygous patients have a lower TPMT enzyme activity and homozygous patients have almost absent TPMT activity. Bone marrow toxicity A lower TPMT enzyme activity results in less 6-MP metabolism through the TPMT pathway, and therefore more metabolism via the pathway leading to increased 6-TGN levels. This increases the risk of bone marrow suppression and neutropenia. It must be said, however, that there is a degree of variability in TPMT activity within both the wild-type and the heterozygous genotypes. Some patients with a heterozygous genotype still exhibit high TPMT enzyme activity, whereas some wild-type patients have a more intermediate activity. These discrepancies arise because mutations are not the only factor regulating the activity of the enzyme. The TPMT genotype accounts for approximately two thirds of the total level of enzyme activity. Other factors could also play a role, including promoter polymorphisms, drug interactions, and environmental factors [20]. This was clearly demonstrated by Colombel et al., who genotyped 41 patients with signs of bone marrow toxicity

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(leucopenia or thrombocytopenia) upon AZA treatment [21]. Only 11/41 (26.8%) of patients had TPMT mutations: four (10%) were homozygous and seven (17%) heterozygous. However, the time to onset of bone marrow suppression was shorter in the homozygous patients (<1.5 months) compared with the wild-type patients (0.5–87 months). The heterozygous patients had a duration of cytopenia onset between that of wild-type and of homozygous patients (1–18 months). The authors concluded that factors other than genetic polymorphisms in the TPMT gene are responsible for bone marrow suppression. More recent data suggest that polymorphisms in other genes, such as the ITPA (inosine triphosphate pyrophosphatase) gene, might be associated with side effects to AZA [22]. In a cohort of 62 IBD patients suffering adverse drug reactions to AZA, the ITPA 94C>A polymorphism was associated with flu-like symptoms (OR 4.7, 95% CI 1.2–18.1, P=0.0308) and with rash (OR 10.3, 95% CI 4.7–62.9, P=0.0213). Hepatotoxicity Besides bone marrow toxicity, hepatotoxicity is a second adverse event of the purine analogs that is possibly related to TPMT enzyme activity. Although hepatotoxicity is a relatively rare event, certainly compared to leucopenia, one should always be aware of it and check liver tests on a routine basis. Dubinsky et al. showed that hepatotoxicity might be related to the metabolite 6-matrix metalloproteinase (6-MMP) [23]. In her study, 16/92 CD patients (17%) showed hepatotoxicity, and 6-MMP levels in this group were significantly higher (median 6-MMP 5463 pmol/8×108 red blood cells) than in patients without those symptoms (median 6-MMP 2213 pmol/8×108 red blood cells; P<0.05). A cut-off of 6-MMP >5700 pmol/8×108 red blood cells was associated with a greater risk for hepatotoxicity. Remarkably, none of the patients with hepatotoxicity carried genetic variants in the TPMT gene. Therefore, it appears that certain patients might preferentially metabolize through TPMT,

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resulting not only in higher 6-MMP levels and possibly more risk of hepatotoxicity, but also in lower 6-TG levels. Type 1 hypersensitivity reactions Other side effects of AZA, including pancreatitis, fever, myalgias, and arthralgias, are type I hypersensitivity reactions. In the unlikely event of such an adverse side effect, AZA should be stopped and re-challenge is not recommended. Given that these drugs suppress immunity, opportunistic infections should be considered where applicable. Rare cases of cytomegalovirus pneumonitis and other opportunistic infections have been reported [24]. Carcinomas There is agreement that AZA is probably not associated with an increased risk of malignancy, including the occurrence of nonHodgkin’s lymphoma, except maybe an increase in the frequency of colorectal carcinomas after 5 years of use, as demonstrated in the study by Connell et al. (OR 6.7, P=0.00001) [25]. However, these tumours are recognized complications of chronic IBD. Birth defects No increased prevalence of birth defects has been reported, and therefore it is likely that the drug can be safely continued during pregnancy. Since small amounts of the drug are traceable in breast milk, breast feeding is not recommended. 6-TG as therapy for IBD? To overcome the problems of TPMT polymorphism activity, the administration of 6-TG, a closely related thiopurine, has been proposed. In a pilot study of 21 IBD patients resistant to 6-MP/AZA, 6-TG was both efficacious and well tolerated [26]. However, the occurrence of nodular regenerative hyperplasia after 6-TG treatment in these patients raised serious concerns [27] and, therefore, 6-TG should not be considered as therapy for patients with IBD.

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Which dose to use? There are no scientific data to show that AZA is more efficacious than 6-MP or vice versa. However, 6-MP might be better tolerated than AZA in some cases. A dose of 2–2.5 mg/kg/day is usually recommended for AZA. For 6-MP, the comparable dose is 1–1.25 mg/kg/day. An IV loading dose does not decrease the time to response [28]. Some authors advocate dosing of AZA and 6-MP based on the patient’s genotype. In patients with the wild-type genotype, the standard dose (as stated above) would be safe. Heterozygous patients should be started at 50%–60% of the standard dose. Patients with the homozygous mutant genotype are known to develop early pancytopenia after administration of the drug, and AZA is therefore not recommended in these patients. However, more prospective studies are probably necessary before making general recommendations on TPMT genotyping and/or measurement of TPMT enzyme activity in clinical practice. Efficacy monitoring When a patient is started on AZA or 6-MP, monitoring of white blood cell count, hemoglobin, platelets, and liver and pancreas enzymes should be performed after 1 week, after 4 weeks, and then 8-weekly in the first year and 12-weekly during the following years, assuming that the dose of the drug is stable. Assessing blood counts 1 week after initiation will identify TPMT homozygous mutant patients, since they will develop early pancytopenia. It is also important that blood counts be checked earlier than 8 weeks every time a dose adjustment is made. There is much discussion regarding the measurement of 6-TGN metabolites in patients who do not respond to AZA or 6-MP. A relationship has been reported between 6-TGN levels and clinical response [23,29]. In the study by Dubinsky et al., 6-TGN <235 pmol/8×108 was associated with a lower response compared with 6-TGN >235 pmol/8×108 [23]. However, another study was not able to find a good correlation between metabolite

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testing and clinical response [30]. It therefore remains debatable whether measuring metabolites is helpful for the clinician. These metabolites could be clinically useful when noncompliance is suspected, since this most often results in extremely low 6-TG levels [30,31]. Duration of therapy with purine analogs O’Donoghue et al. randomized 51 CD patients on AZA (2 mg/kg/day) for at least 6 months to continuation of AZA or to placebo [6]. The cumulative probability of relapse at 1 year was 5% in the AZA group compared with 41% in the control group (P<0.01). A larger French study from the GETAID (Group d’Etude Thérapeutique des Affections Inflammatoires du tube Digestif) group in patients on AZA or 6-MP (2 mg/kg/day) who were in prolonged clinical remission (>6 months without steroids) showed that, of 157 patients who continued to take the therapy, cumulative probabilities of relapse at 1 and 5 years were 11% and 32%, respectively [32]. In 42 patients who stopped therapy, probabilities of relapse at 1 and 5 years were 38% and 75%, respectively. After 4 years of remission on these drugs, the risk of relapse appeared to be similar, whether the therapy was maintained or stopped. The authors speculated that therapy could be stopped after 4 years. However, follow-up data from the same group later suggested that the risk for relapse remains even beyond 3.5 years [33]. Therefore, at present, it is unclear how long therapy with purine analogs should be continued. However, taking into account the most recent data from the GETAID group, even after a long period of AZA therapy, stopping the drug is associated with a 10% increased relapse rate per year [33]. While this should be discussed with the patient, some patients will probably be able to discontinue the drug. Furthermore, it must be said that the efficacy of AZA in a given patient is not lost after withdrawal. Therefore, in the case of a relapse, AZA should be reintroduced at the same dose as before and is likely to be as effective as before.

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Study

N

Dose Mode of Outcome vs (mg/week) administration placebo

Induction of remission Feagan et al., 141 1995 [38] Oren et al., 84 1997 [36]

Arora et al., 1999 [37]

33

Maintenance of remission Feagan et al., 76 2000 [39]

25

Intramuscular

12.5

Oral

15

Oral

15

Intramuscular

39.4% vs 19.1% remission (P=0.025) 38% vs 46% remission (P=NS) 54% vs 20% remission (P=NS) P<0.1

65% vs 39% remission (P=0.04)

Table 3. Placebo-controlled randomized trials with methotrexate (MTX) in Crohn’s disease. NS: not significant.

Methotrexate Mechanisms of action MTX is a folate antagonist. It is used as an immunomodulator not only in CD, but also in other inflammatory conditions such as rheumatoid arthritis and psoriasis. The mechanism of action of MTX in IBD is incompletely understood. It is believed that MTX has anti-inflammatory actions by decreasing chemotaxis and cytokine production, inhibiting production of purines, and increasing adenosine levels (although the latter was not a uniform finding in all studies [34]). MTX has a short half-life of 5–6 hours. It is taken up by tissues and is converted into MTX polyglutamates, which have a much longer duration of activity. The main action of MTX polyglutamates is the inhibition of dihydrofolate reductase. The drug is mainly excreted through the kidneys. MTX in the treatment of IBD MTX has been studied in IBD both for induction of remission and maintenance of remission. It has become the main alternative to AZA or 6-MP therapy.

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Induction of remission In a nonrandomized, open-label preliminary trial, MTX was given as a 25 mg IM injection weekly for 12 weeks in 21 patients with refractory IBD (14 CD and 7 UC) [35]. A total of 16 patients (76%) responded, 11 with CD and five with UC. The dose of prednisone was able to be decreased significantly, and >80% of patients showed endoscopic and/or histologic healing. These findings led to the further development of controlled trials. There have been three placebo-controlled randomized clinical trials evaluating the efficacy of MTX for induction of remission (Table 3) [36–38]. Two studies that used low doses of orally administered MTX showed no significant difference in treatment with MTX versus placebo [36,37]. In the study by Arora et al., fewer MTX-treated patients (46%) had flares of CD when compared with placebo-treated patients (80%), but this did not achieve statistical significance, probably due to the small sample size [37]. From these studies, it seems that orally administered MTX is not effective – this might be because absorption of MTX is highly variable. Compared with oral dosing, IM administration gives more consistent blood levels. A subsequently performed large, double-blind, placebocontrolled multicenter study of IM administered MTX, showed that MTX at a dose of 25 mg/week was more effective than placebo in improving symptoms and in reducing the need for prednisone [38]. The NNT in this study was 5. However, MTX was only better than placebo in patients who received a daily dose of >20 mg of prednisone (remission in 39% vs 10%, respectively; P=0.003). There was no significant difference in the low-dose prednisone stratum (<20 mg prednisone) between MTX (40%) and placebo (35.3%). Maintenance of remission A follow-up study of the induction of remission trial by Feagan et al. investigated the efficacy of maintenance therapy with MTX [39]. In this study, patients with chronically active CD

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who had entered remission after 4–6 months of treatment with 25 mg/week of MTX were randomized to either MTX IM 15 mg/week or placebo. At week 40, 65% of MTX patients were in remission, compared with 39% of placebo-treated patients (P=0.04). The absolute reduction in the risk of relapse was 26.1% (95% CI 4.4%–47.8%). In the MTX group, fewer patients required prednisone for relapse (28%) than in the placebo group (58%) (P=0.01). Retrospective evaluation of remission Besides the limited data from randomized placebo-controlled trials, there are more data available through retrospective evaluation of the use of MTX in IBD. Fraser et al. reviewed 70 patients (48 CD, 22 UC) who had received treatment with MTX [40]. In their cohort, 34/55 (62%) of patients who completed more than 3 months of treatment entered remission. If treatment was continued, the chances of remaining in remission at 12, 24, and 36 months were 90%, 73%, and 51%, respectively. If treatment was stopped, the chances of remaining in remisson at 6, 12, and 18 months were 42%, 21%, and 16%, respectively. A French study treated 49 CD patients (most of them intolerant or failures to AZA) with MTX for a minimum of 6 months; 41 patients (83%) achieved complete clinical remission [41]. Our own center analyzed data from 20 patients treated between January 1995 and June 1997 with MTX because of corticosteroid-dependent or corticosteroid-refractory CD [42]. All patients were AZA resistant or intolerant. At 12 weeks, a clinical response was obtained in 14/20 patients (70%). These response rates decreased to 10/20 patients at 6 months, 8/17 patients at 9 months, and 4/14 evaluable patients at 12 months. In initial responders (n=14), maintenance of remission was observed in 9/14, 6/11, and 3/9 patients at 6, 9, and 12 months, respectively. MTX allowed corticosteroid tapering in 85% of patients and discontinuation in 60% of patients at 6 months.

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Few studies have addressed the efficacy of MTX for the treatment of UC, and results have not always shown agreement. The initial study by Kozarek et al. in 1989 showed that MTX induced remission in 5/7 patients with UC [35]. In a small study by Baron et al., eight patients with UC were given MTX orally 15 mg/week [43]. The daily prednisone dose dropped from 26.3±3.2 mg/day to 12.7±2.0 mg/day (P<0.001). Oren et al. randomized 67 patients with active UC to MTX orally 12.5 mg/week (30 patients) or placebo (37 patients) for 9 months [44]. The proportion of patients entering remission (MTX 46.7% vs placebo 48.6%), the time to reach remission (MTX 4.1±1.9 months vs placebo 3.4±1.7 months), and the proportions of patients having a relapse after first remission (MTX 64.3% vs placebo 44.4%) were not significantly different between the two groups. In a Spanish study, 72 steroid-dependent IBD patients (34 with UC and 38 with CD) were randomized to 6-MP 1.5 mg/kg/day (group A), MTX 15 mg/week (group B), or 5-amino salicylic acid 3 g/day (group C) [45]. A significantly higher remission rate was seen for UC patients in group A (78.6%) than in group C (25%) (P<0.05), with no statistical differences in group B (58.3%) versus group C. For CD patients, remission rates were significantly higher in groups A (93.7%) and B (80%) versus C (14%) (P<0.001 and P<0.01, respectively). In addition, there was a significant difference in maintenance of remission between group B and group C in CD patients (P<0.001), but not for UC patients. Finally, a UK review of 22 patients with UC found similar remission rates to those observed in CD patients treated with similar doses of MTX [40]. Which dose to use? Although in the North American Crohn’s Study Group Investigators multicenter study MTX was administered at a dose of 25 mg/week to induce remission, later studies showed that a dose of 15 mg/week might be as effective as 25 mg/week in inducing remission [46]. Indeed, in this study of 32 patients,

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39% of patients randomized to 15 mg/week and 33% of patients randomized to 25 mg/week improved, and 17% of patients in each group achieved remission. However, some patients who did not respond to 15 mg/week improved after dose escalation to 25 mg/week. Therefore, for induction of remission, initial doses of 15 (SC or IM) and 25 (IM) mg/week seem equally efficacious, although 25 mg/week has been tested in a much more rigorous study. For maintenance of remission, a dose of 15 mg/week (either IM or SC) is recommended. Side effects and duration of therapy Side effects associated with MTX therapy are usually mild. They include gastrointestinal symptoms of nausea and diarrhea, leucopenia, rises in transaminase levels, and, rarely, cases of interstitial pneumonitis. A French study including 49 CD patients treated with MTX for a minimum of 6 months recorded adverse reactions in 24 patients, requiring discontinuation of MTX in five [41]. In this study, a liver biopsy was performed in 11 patients: of note, a mild steatosis was found in five patients, a slight dilation of the sinusoids in one patient, a granulomatous hepatitis with a mild portal fibrosis in one patient, and a slight periportal fibrosis in one patient. Whereas it was previously recommended to perform a liver biopsy after a cumulative administered dose of 1.5 g MTX, this is no longer supported and the drug can be given in addition to this dose with 8-weekly laboratory controls for as long as the patient is receiving MTX. In animals, an increased risk for embryotoxicity and malformations has been observed, and MTX should therefore not be used in women who want to become pregnant [47].

Conclusion IBD patients who require corticosteroids for longer than 3 months or who relapse within 6–12 months after discontinuation of induction therapy should be started on long-term maintenance therapy. The drug of choice is AZA 2–2.5 mg/kg/day or 6-MP 1–1.25 mg.kg/day. Biochemistry (full blood count, liver tests)

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should be checked regularly (at week 1 and at week 4 following start of treatment and every 2–3 months thereafter, given that the dose is kept stable) as long as the patient is on AZA. If patients fail to respond, IM or SC MTX should be given. We feel this strategy may be used for both CD and UC, even though there are few studies of either agent in UC. Finally, novel biologicals such as infliximab should always be given with concomitant AZA or MTX to avoid immunogenicity (see Chapter 5 for further details).

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Hanauer SB, Korelitz BI, Rutgeerts P, et al. Postoperative maintenance of Crohn’s disease remission with 6-mercaptopurine, mesalamine, or placebo: a 2-year trial. Gastroenterology 2004;127:723–9. Lamers CB, Griffioen G, van Hogezand RA, et al. Azathioprine: an update on clinical efficacy and safety in inflammatory bowel disease. Scand J Gastroenterol Suppl 1999;230:111–15. Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet 1980;32:651–62. McLeod HL, Siva C. The thiopurine S-methyltransferase gene locus – implications for clinical pharmacogenomics. Pharmacogenomics 2002;3:89–98. Yates CR, Krynetski EY, Loennechen T, et al. Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med 1997;126:608–14. Vuchetich JP, Weinshilboum RM, Price RA. Segregation analysis of human red blood cell thiopurine methyltransferase activity. Genet Epidemiol 1995;12:1–11. Colombel JF, Ferrari N, Debuysere H, et al. Genotypic analysis of thiopurine S-methyltransferase in patients with Crohn’s disease and severe myelosuppression during azathioprine therapy. Gastroenterology 2000;118:1025–30. Marinaki AM, Ansari A, Duley JA, et al. Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase). Pharmacogenetics 2004;14:181–7. Dubinsky MC, Lamothe S, Yang HY, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology 2000;118:705–13. Hookey LC, Depew W, Boag A, et al. 6-mercaptopurine and inflammatory bowel disease: hidden ground for the cytomegalovirus. Can J Gastroenterol 2003;17:319–22. Connell WR, Kamm MA, Dickson M, et al. Long-term neoplasia risk after azathioprine treatment in inflammatory bowel disease. Lancet 1994;343:1249–52. Dubinsky MC, Feldman EJ, Abreu MT, et al. Thioguanine: a potential alternate thiopurine for IBD patients allergic to 6-mercaptopurine or azathioprine. Am J Gastroenterol 2003;98:1058–63. Dubinsky MC, Vasiliauskas EA, Singh H, et al. 6-thioguanine can cause serious liver injury in inflammatory bowel disease patients. Gastroenterology 2003;125:298–303. Sandborn WJ, Tremaine WJ, Wolf DC, et al. Lack of effect of intravenous administration on time to respond to azathioprine for steroid-treated Crohn’s disease. North American Azathioprine Study Group. Gastroenterology 1999;117:527–35. Cuffari C, Hunt S, Bayless T. Utilisation of erythrocyte 6-thioguanine metabolite levels to optimise azathioprine therapy in patients with inflammatory bowel disease. Gut 2001;48:642–6. Goldenberg BA, Rawsthorne P, Bernstein CN. The utility of 6-thioguanine metabolite levels in managing patients with inflammatory bowel disease. Am J Gastroenterol 2004;99:1744–8. Dubinsky MC, Yang H, Hassard PV, et al. 6-MP metabolite profiles provide a biochemical explanation for 6-MP resistance in patients with inflammatory bowel disease. Gastroenterology 2002;122:904–15. Bouhnik Y, Lemann M, Mary JY, et al. Long-term follow-up of patients with Crohn’s disease treated with azathioprine or 6-mercaptopurine. Lancet 1996;347:215–19. Lemann M, Jean-Yves M, Colombel JF, et al. A randomized double-blind controlled withdrawal trial in Crohn’s disease patients in long-term remission on azathioprine. Gastroenterology 2005;128:1812–18.

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Egan LJ, Sandborn WJ, Mays DC, et al. Plasma and rectal adenosine in inflammatory bowel disease: effect of methotrexate. Inflamm Bowel Dis 1999;5:167–73. Kozarek RA, Patterson DJ, Gelfand MD, et al. Methotrexate induces clinical and histologic remission in patients with refractory inflammatory bowel disease. Ann Intern Med 1989;110:353–6. Oren R, Moshkowitz M, Odes S, et al. Methotrexate in chronic active Crohn’s disease: a double-blind, randomized, Israeli multicenter trial. Am J Gastroenterol 1997;92:2203–9. Arora S, Katkov W, Cooley J, et al. Methotrexate in Crohn’s disease: results of a randomized, double-blind, placebo-controlled trial. Hepatogastroenterology 1999;46:1724–9. Feagan BG, Rochon J, Fedorak RN, et al. Methotrexate for the treatment of Crohn’s disease. The North American Crohn’s Study Group Investigators. N Engl J Med 1995;332:292–7. Feagan BG, Fedorak RN, Irvine EJ, et al. A comparison of methotrexate with placebo for the maintenance of remission in Crohn’s disease. North American Crohn’s Study Group Investigators. N Engl J Med 2000;342:1627–32. Fraser AG, Morton D, McGovern D, et al. The efficacy of methotrexate for maintaining remission in inflammatory bowel disease. Aliment Pharmacol Ther 2002;16:693–7. Lemann M, Zenjari T, Bouhnik Y, et al. Methotrexate in Crohn’s disease: long-term efficacy and toxicity. Am J Gastroenterol 2000;95:1730–4. Vandeputte L, D’Haens G, Baert F, et al. Methotrexate in refractory Crohn’s disease. Inflamm Bowel Dis 1999;5:11–15. Baron TH, Truss CD, Elson CO. Low-dose oral methotrexate in refractory inflammatory bowel disease. Dig Dis Sci 1993;38:1851–6. Oren R, Arber N, Odes S, et al. Methotrexate in chronic active ulcerative colitis: a double-blind, randomized, Israeli multicenter trial. Gastroenterology 1996;110:1416–21. Mate-Jimenez J, Hermida C, Cantero-Perona J, et al. 6-mercaptopurine or methotrexate added to prednisone induces and maintains remission in steroiddependent inflammatory bowel disease. Eur J Gastroenterol Hepatol 2000;12:1227–33. Egan LJ, Sandborn WJ, Tremaine WJ, et al. A randomized dose-response and pharmacokinetic study of methotrexate for refractory inflammatory Crohn’s disease and ulcerative colitis. Aliment Pharmacol Ther 1999;13:1597–604. Jordan RL, Wilson JG, Schumacher HJ. Embryotoxicity of the folate antagonist methotrexate in rats and rabbits. Teratology 1977;15:73–9.

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5 Biological therapies Jürgen Schölmerich

Introduction The last review on biological therapies in The Inflammatory Bowel Disease Yearbook 2003 ended with the conclusion “The breadth of targeted biological agents is only limited by our ability to delineate the finest of molecular details involved in the development and maintenance of the immune activation that characterizes [inflammatory bowel disease]. The future of targeted biological therapies will be inundated with as many questions as discoveries. Ultimately, strategies need to be developed to determine which new agents will be used, and how, with respect to currently available therapies” [1]. This topic has actually led to innumerable reviews, many of them enthusiastic, but others more conservative or even skeptical [2–7]. Currently available therapy has significantly increased survival in inflammatory bowel disease (IBD) patients, which is nowadays almost normal. Social integration is preserved in >90% of patients, as evidenced by their ability to go to work or school, and to participate in leisure activities [8]. When looking at population-based data it becomes evident that less than half of all patients with Crohn’s disease (CD) and even fewer of those with ulcerative colitis (UC) have received steroids [9]. However, there is still a group of patients who cannot be successfully treated with conventional therapy. Even in patients who can be treated, quality of life is still less than optimal. Side effects of steroids and other drugs must be considered and treatments have not led to proven mucosal healing, at least not in CD.

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Therefore, for CD in particular, the relapse rate is high and there is no effective maintenance treatment available. The picture becomes even more complicated since genetic as well as clinical analyses have demonstrated that CD and probably UC are syndromes rather than diseases. They therefore include different subgroups, which have different etiologies and probably different treatment needs [10]. Although effective and safe treatments for chronic IBD are available, many new therapeutic principles are being studied worldwide. Most of them result from more recent pathophysiologic knowledge and belong to the group of ‘biological therapies’. For some therapies a biological effect has been proven in patients. In many cases, however, the effects are unfortunately still limited and long-term results and risks are unknown. Many approaches have been futile. The concentration on mostly expensive treatment principles has led to a situation where alternative concepts and approaches are overlooked due to lack of interest from the pharmaceutical industry. During the last few decades, many pathophysiologic mechanisms of IBD have been demonstrated using animal models and human cells. However, the causes of IBD have not been definitively clarified, although a first susceptibility gene has been proven and two more suggested [11–15]. Probably due to a defective interaction between the mucosal immune system and the enteric flora, an increased immune response develops where cytokines such as interferon (IFN)-γ, interleukin (IL)-2, IL-12, IL-18, and others are expressed; this leads to the production of proinflammatory mediators such as tumor necrosis factor (TNF)-α and IL-1. For a number of years, intensive attempts have been made to modulate the mucosal response by interacting with these cytokines or the cells that produce them. This has been named ‘biological therapy’.

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This rather trendy name, which attempts to indicate that no chemical substances are used, comprises a large number of very different therapeutic principles. For IBD, mostly recombinant peptides or proteins (ie, IL-10, IFN-β, growth factors), partially or almost completely humanized antibodies (ie, anti-TNF, anti-ILs), antisense constructs (ie, anti-intercellular adhesion molecule [ICAM] 1, anti-nuclear factor [NF]-κB p65), or cellular growth factors (ie, granulocyte-macrophage colony-stimulating factor [GM-CSF]) have been tested. The majority of these substances need to be applied IV or SC. More recently, small molecules have been found by screening molecular libraries and developed to inhibit given steps in signal transduction pathways (ie, mitogen-activated protein [MAP] kinase inhibitors). Table 1 gives an overview of biological therapies studied in IBD. In the following section, only data published after the 2003 review will be presented according to the categories of biological therapies given in Table 1 [1].

Inhibitors of proinflammatory cytokines Interleukin-1 and TNF inhibition There have been no recent attempts to inhibit IL-1, which has been successful in rheumatoid arthritis, although to a lesser extent than TNF inhibition [16]. There have, however, been many attempts to target TNF. The first attempt, using the chimerized antibody infliximab, was biologically and clinically successful. This obviously sparked further interest in harnessing TNF. While the effects of infliximab were well studied 2 years ago and published in numerous papers, in particular the ACCENT (A Crohn’s disease Clinical trial Evaluating infliximab in a New long term Treatment regimen) I and ACCENT II studies [17,18], a number of papers regarding its mechanism of action and side-effect profile have recently appeared. Furthermore, data on infliximab use in UC have emerged.

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Inhibitors of proinflammatory cytokines Interleukin-1 antibodies TNF antibodies Infliximab CDP571 CDP870 Adalimumab Soluble TNF receptors Etanercept (p75) Onercept (p55) TNF inhibitors MAP kinase inhibitors Thalidomide Transcription factor inhibitors NF-κB p65 antisense Inhibitors of Th1 polarization and T-cell proliferation Interleukin-12 antibodies Interleukin-18 antibodies Interferon-γ antibodies Interleukin-6 receptor antibodies Interleukin-2 receptor antibodies Daclizumab Basiliximab CD4 antibodies CD3 antibodies CD40L antibodies Cytokines Interleukin-10 Inhibitors of leukocyte trafficking α4 integrin antibodies Natalizumab α4‚7 integrin antibodies (LDP02) ICAM antisense oligonucleotide Alicaforsen

Table 1. Biological therapies studied in IBD. G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte macrophage colony-stimulating factor; ICAM: intercellular adhesion molecule; MAP: mitogen-activated protein; NF-κB: nuclear factor-κB; Th1: T helper cell type 1; TNF: tumor necrosis factor.

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Growth factors and hormones Keratinocyte growth factor Repifermin Epidermal growth factor Human growth hormone Immunostimulating factors G-CSF Filgrastim GM-CSF Sargramostim Immunomodulators Interferon-α Interferon-β1a Interleukin-11 (also barrier repair)

Table 1. cont’d.

Infliximab While the effects of infliximab in CD are undisputed (although it is not of benefit to all patients), data in UC thus far have been rather disappointing. Some small studies found effects as good as with steroids, while others found a sustained response rate of only 25% after 6 months [19–22]. One small, randomized placebo-controlled trial did not find any difference in remission rates [23]. The initial results of two large studies (ACT [Active Ulcerative Colitis] I and ACT II) did show efficacy but “remissions off steroids” at week 30 was disappointing [24]. Nonetheless, the results for remission and improvement at week 48 are similar to that which has been seen in CD. Furthermore, it can be effective in severe steroid-refractory UC [25]. Hence infliximab will become an option for subjects with steroid intolerance or steroid-refractory UC. With regard to the mechanism of action, more data have indicated that infliximab appears to act by inducing apoptosis in activated lymphocytes and perhaps monocytes (Figure 1) [26,27]. Using nuclear medicine technology, this has been shown to occur

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TN

F

TNF-AB

Apoptosis

TN

F

TNF-Trimer

TN

F

cAMP IL-10 Thalidomide

Metalloproteinase inhibitor

TNF-AB

Figure 1. Effects of anti-tumor necrosis factor (TNF) strategies. AB: antibody; cAMP: cyclic adenosine monophosphate; IL-10: interleukin 10.

in vivo [28]. Apoptosis might be due to binding of membranebound TNF on activated cells, and also seems to involve downregulation of TNF-α-induced growth factor expression [29,30]. Recent studies have indicated that a considerable number of patients develop antibodies to infliximab due to the chimeric nature of this protein: the presence of high concentrations of antibodies (>8 μg/mL) before an infusion predicted a shorter duration of response and a higher risk for infusion reactions [31]. Concomitant immunosuppressive therapy can reduce antibodies to infliximab, hence treatment with azathioprine (AZA) or methotrexate (MTX) is indicated when using infliximab. Furthermore, continued maintenance treatment might be preferable to episodic treatment. However, the best strategy will probably be to develop more humanized compounds to avoid antibody formation as much as possible. It is unclear, however, to what extent this will be possible.

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Since infliximab has been licensed for use a significant number of women directly exposed to infliximab have become pregnant. A series of 146 identified pregnancies revealed that live births occurred in 67%, miscarriages in 15%, and therapeutic terminations in 19% of exposed women. These results are similar to those expected for the general US population of pregnant women or pregnant women with CD who have not been exposed to infliximab. Of 10 pregnancies indirectly exposed through the male partner, nine resulted in live births and one in a miscarriage [32]. Two large series studied the safety profile of infliximab in daily clinical practice. The series from the Mayo Clinic, USA, including 500 patients, revealed that 43 (8.6%) patients experienced a serious adverse event, 30 (6%) of which were related to infliximab. In addition, 3.8% of patients experienced acute infusion reactions, 2.8% of patients had serum sicknesslike disease, and 0.6% of patients had drug-induced lupus [33]. One patient had a demyelination disorder. The number of patients who had infectious events related to infliximab was 41 (8.2%), 20 (4%) of whom had serious infections: two patients had lethal sepsis, eight patients had pneumonias (two lethal), six patients had viral infections, two patients had abdominal abscesses requiring surgery, one patient had arm cellulitis, one patient had histoplasmosis, and none had tuberculosis. Malignant disorders were present in nine patients, three of which were possibly related to infliximab. Death was possibly related to the drug in 1% of all patients. The second series from Stockholm County, Sweden included 217 patients. In this cohort, 42 severe adverse events occurred in 41 patients. Of the 41 patients, three developed lymphoma (two fatal), two had opportunistic infections (one fatal), and two died due to sepsis. One additional patient died from pulmonary embolism [34]. Thus, an overall death rate of 1% due to the treatment seems to be a reasonable estimate, and needs to be considered when using this approach [35].

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125 consecutive patients with CD

71/125 (56.8%) ANA+ after 24 months (75% after<3 infusions)

15/71 seronegative after median 12 months

14/43 DS DNA AB+ 17/43 SS DNA AB+ 9/43 antihistone AB+

2/71 SLE 1/71 AIHA

Figure 2. ‘Autoimmunity’ under treatment with infliximab [36]. AB: antibody; AIHA: autoimmune hemolytic anemia; ANA: antinuclear antibodies; DS: double-stranded; SLE: systemic lupus erythematosus; SS: single-stranded.

Treatment with infliximab seems to induce the formation of antibodies (antinuclear, double-stranded DNA, and others) in >50% of patients, but frank lupus or autoimmune hepatitis is uncommon (Figure 2) [36]. Finally, a recent report indicated that treatment with infliximab induced the occurrence of anticardiolipin antibodies in patients treated for rheumatoid arthritis, which might explain associated thromboembolic complications [37]. As already mentioned, due to the immunogenicity of infliximab, other, more humanized concepts have emerged. These include antibodies such as CDP571, CDP870, and the fully humanized antibody adalimumab, as well as the soluble receptor fusion proteins etanercept (p75) and onercept (p55). CDP571 The humanized monoclonal antibody CDP571 was studied in a number of small trials; however, a large Phase III randomized, double-blind, placebo-controlled trial in 396 patients with moderate to severe active CD, given either 10 mg/kg drug or

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Outcome

Placebo

CDP870 (mg) 100

200

400

Clinical response (CDAI ↓ >100) week 12 (%)

35.6

36.5

36.1

44.4

Clinical remission when initial CRP ≥10 mg/L (%)

10.7

35.5

32.1

53.1*

Table 2. Effect of CDP870, an anti-tumor necrosis factor antibody fragment in ‘active’ Crohn’s disease (n=292) [39]. Dosing weeks 0, 4, and 8. *Significant vs placebo. CDAI: Crohn’s disease activity index; CRP: C-reactive protein.

placebo, demonstrated that a clinical response was significantly more often achieved with CDP571 (34.2%) versus placebo (21.2%). The effect was not maintained through week 28, although the treatment was only given until week 24 [38]. Patients with an activated baseline C-reactive protein (CRP) ≥10 mg/dL responded better and more often remained in clinical remission at week 28. This is not really surprising since an anti-inflammatory treatment should only work in patients who show signs of inflammation. The development of CDP571, however, has been abandoned due to these results. CDP870 A humanized anti-TNF antibody fragment, CDP870 (linked to polyethylene glycol to increase its biological half-life), was more recently developed. In a Phase II study, 292 patients were treated with placebo or 100, 200, or 400 mg CDP870 SC at weeks 0, 4, and 8. Clinical efficacy was seen early, but the effect was not sustained to week 12 (the primary endpoint). However, in the subgroup of patients with elevated CRP, a clear dose–effect relationship was found, and there was a significant difference versus placebo (Table 2) [39]. Phase III studies are currently underway. Adalimumab Adalimumab is a fully humanized monoclonal antibody to TNF. It binds soluble membrane-bound TNF, fixes complement,

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Outcome

Placebo

Adalimumab (mg) 40/20

80/40

160/80

CDAI ↓ >100 (%)

25

34

40

50*

Remission (%)

12

18

24

36*

Table 3. Effect of subcutaneous adalimumab in active Crohn’s disease (n=299) [43]. Dosing weeks 0 and 2. *P<0.05 vs placebo. CDAI: Crohn’s disease activity index.

and induces T-cell apoptosis [40]. Pilot studies have been successful [41]. A study in 299 patients comparing two injections of 40 and 20 mg, 80 and 40 mg, and 160 and 80 mg of adalimumab with placebo at weeks 0 and 2 showed 36% remission at the highest dose of adalimumab versus placebo (12%), and a Crohn’s disease activity index (CDAI) decrease of >100 points in 50% versus 25% of patients. The lower doses were associated with lower response rates (Table 3) [42,43]. An open-label trial in 16 patients with prior loss of response or intolerance to infliximab showed clinical response in 46% of patients and remission in 8% of patients treated with adalimumab at week 4, suggesting that adalimumab might be helpful in this situation [44]. Further work through Phase III studies is currently being undertaken. Soluble receptors While the negative outcome of studies using etanercept had already been described 2 years ago, at that time only a pilot trial of onercept (the fusion protein using the p55 receptor of TNF) had been reported with a positive outcome [45]. However, a large placebo-controlled trial of four different doses of onercept, SC three times weekly for 8 weeks in 207 patients, did not show any difference regarding remission, response, or side effects [46]. The negative outcomes from these trials could be due to the treatments not inducing cell apoptosis, although this effect has only been studied in etanercept [27,40].

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Other TNF-inhibiting strategies Thalidomide and MAP kinase inhibitors

Thalidomide was shown in one series to have a rather low efficacy and a high frequency of side effects [47]. MAP kinase inhibitors inhibit the phosphorylation of protein kinases, which are important in the signal transduction cascade leading to TNF-α production by macrophages. Based on results in experimental animal models and pilot studies, a larger trial was initiated of MAP kinase inhibitors, but was halted when liver toxicity was reported in parallel studies in rheumatoid arthritis [48,49]. RDP58 RDP58 blocks TNF production at a posttranscriptional level and also inhibits the production of IFN-γ, IL-2, and IL-12. It is not absorbed systemically and lacks toxicity when given orally. After testing in animal models and a Phase I study in healthy volunteers, RDP58 was given to 127 patients with mild to moderate active UC in a Phase II study [50]. Patients were randomized to either placebo or to 100, 200, or 300 mg RPD58 daily for 28 days. Clinical remission was achieved in 72% of patients in the 300-mg group, 70% of patients in the 200-mg group, and 29% of patients in the 100-mg group versus 40% of patients in the placebo group. Histologic scores improved in both higher-dose groups compared with placebo. Studies in CD have been started and results are awaited. κB Inhibition of nuclear factor-κ The transcription factor NF-κB controls expression and release of proinflammatory cytokines, in particular IL-1 and TNF-α. A number of currently used drugs (such as 5-AZA and steroids) interact with the signal transduction that leads to the nuclear translocation of this transcription factor. The first human trial treated 11 IBD patients with an oligonucleotide antisense molecule against NF-κB p65 – these patients showed an improvement at 1 week. In two steroid-refractory patients a long-lasting steroid remission was obtained [51]. The oligonucleotides were applied topically since systemic blockade

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is probably difficult in daily life, as has been demonstrated in animal experiments [52].

Inhibitors of T helper cell type 1 polarization and T-cell proliferation Antibodies against interleukin-12 IL-12 is a key cytokine driving the T helper cell type 1 (Th1) inflammatory response, which is associated with inflammation in CD. Antibodies against IL-12 and other inhibitors of this cytokine have been studied in animals, and a trial of human anti-IL-12 (anti-p40) monoclonal antibody in 79 patients with active CD has also been performed [53,54]. Patients were randomly assigned to receive seven weekly SC injections of 1 or 3 mg/kg of antibody or placebo, with either a 4-week interval between the first and the second injection (cohort 1) or no interruption (cohort 2). In cohort 2, patients receiving 3 mg/kg gained a striking benefit from treatment: compared with placebo, rates of clinical response (reduction ≥100 CDAI points) were 75% versus 25% at the end of treatment, and 69% versus 13% after 12 weeks of follow-up. Remission rates were 38% versus 0% at the end of treatment, and 50% versus 0% after 12 weeks of follow-up (Table 4). The beneficial effects were durable through 18 weeks. In cohort 1, the rates of response and remission with the higher dose were consistently higher than with placebo, but not significant. Antibodies against interleukin-18 Antibodies against this Th1 cytokine were studied in animals with some success, but have not been tested in humans. Antibodies against interferon-γγ IFN-γ is an important cytokine in the proinflammatory setting of the Th1 response. A genetically engineered humanized monoclonal antibody against IFN-γ did not show a positive effect

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Week

Remission (%) Placebo

3 mg/kg anti-IL-12

3

0

31

7

0

38

13

0

44

19

0

50

25

0

38

Table 4. Effect of anti-interleukin (IL)-12 p40 antibodies in active Crohn’s disease [54]: seven weekly subcutaneous injections. Week

Remission (%) Placebo

4 mg/kg fontolizumab

10 mg/kg fontolizumab

2

0

23

7

4

0

46

20

6

0

54*

57*

Table 5. Effect of fontolizumab (anti-interferon γ) in active Crohn’s disease. Subanalysis of patients (n=35) with C-reactive protein >10 mg/L, two infusions on days 0 and 28, two doses. In the total population of 133 there was a similar trend, but no significance [55]. *Significant vs placebo.

in an initial pilot study. However, since unusually high responses were noticed in the placebo group, a larger Phase II study was performed. This revealed a significant effect in those patients with an increased CRP level after two infusions given at days 0 and 28. There was no significant difference between 4 mg/kg and 10 mg/kg doses in this rather small group of 35 patients. In the total population of 133 there was a trend towards benefit with this treatment, but it was not significant (Table 5) [55]. Antibodies against interleukin-6 receptor IL-6 is a pluripotent cytokine with a central role in immune regulation and inflammation, and is required for the development of Th1 in UC [56,57]. A humanized monoclonal

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antibody against the IL-6 receptor was studied in 36 active-CD patients (8 mg/kg) for 12 weeks in a placebo-controlled trial. Of the patients given the antibody every other week, 80% had clinical response compared with 31% of placebo-treated patients. Of patients treated with anti-IL-6, 20% showed remission versus 0% of placebo-treated patients. In a treatment group dosed every 4 weeks, the response rate was 42%. The incidence of adverse events was similar between placebo and both treatment regimens [58]. Anti-CD4 antibodies Pursuant to the observation that a patient with HIV infection achieved complete remission of CD when his CD4 cells decreased, a number of CD4 antibodies eliminating CD4+ T-cells have been developed [59]. Investigation proceeded in four studies, but this concept has not been actively pursued during the last 2 years due to concerns of persistent lymphopenia [2]. Anti-CD3 antibodies The CD3 antibody visilizumab is postulated to cause apoptosis of activated, but not resting, T-cells and to do this much faster than AZA [60]. It also induces apoptosis in dexamethasoneinsensitive T-cells. A Phase I dose-finding study in patients with severe UC who did not respond to 5 days of IV corticosteroids showed positive results for eight patients treated with 15 μg/kg visilizumab and 18 patients treated with 10 μg/kg visilizumab on days 1 and 2. Patients with Epstein–Barr virus DNA levels were excluded. Of these patients, 16 maintained clinical improvement for up to 16 months. Some decreases in peripheral blood T-cell counts were observed, but T-cells were recovered in all patients by day 60. Mild to moderate cytokine-release symptoms occurred in two thirds of patients [61]. Anti-CD40 ligand antibodies CD40 ligand (CD40L) is expressed on T-lymphocytes after activation, and is an important costimulatory molecule.

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A humanized monoclonal antibody to CD40L was developed and a study in CD was underway, until it was stopped prematurely due to concerns regarding thromboembolism [2]. Antibodies against interleukin-2 receptor Th1 lymphocytes produce IL-2 as an effector molecule, which is necessary for the proliferation of activated T-cells. Two antibodies have been studied: the humanized monoclonal antibody daclizumab, and the chimeric antibody basiliximab. In a pilot study, 10 patients with UC were treated with a single bolus of 40 mg basiliximab IV plus steroids for 24 weeks [62]. After 8 weeks, nine patients achieved clinical remission; at 24 weeks, seven patients were still in remission. Out of nine responders eight relapsed, but remission was achieved by giving AZA in addition to the corticosteroids. A follow-up to this study included 30 patients: 20 with moderately active disease and 10 with severe disease. In this study, 80% of patients improved their disease activity score; in addition, 70% of moderate patients and 50% of severe patients achieved remission. Adverse events included herpes zoster in two patients. Overall, improvement was seen in 80% of patients and remission was seen in 63% of patients [63]. Our own experience in five patients with UC and CD also demonstrated positive effects [64]. Daclizumab was used in 10 patients, nine of whom completed the study receiving 1 mg/kg IV twice with a 4-week interval [65]. The primary endpoint was clinical improvement at week 8. There was a decrease in the disease activity score (P<0.005) and in the endoscopic score (P<0.01). Furthermore, mucosal biopsies showed a significant decrease in IL-2 receptor positive cells. It is possible that the anti-IL-2 receptor antibodies might have a steroid-sensitizing property in addition to their immunosuppressive actions. Larger trials are underway.

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Inhibition of cell migration and adhesion Since the elimination of activated immune cells by infliximab or similar antibodies has a positive effect, it is reasonable to consider that inhibiting adhesion molecules and thereby the migration of inflammatory cells would also be beneficial. This approach has been followed using several antibodies and antisense oligonucleotides. Antibodies against α4 integrin The recombinant humanized monoclonal immunoglobulin (Ig)G4 antibody natalizumab is directed against the human α4 integrin. The antibody blocks the function of both α4β1 and α4β7 integrins, and therefore could be useful in the treatment of a broad range of inflammatory diseases. A more selective inhibitor of intestinal inflammatory cell migration, such as an inhibitor of the α4β7–MAdCAM (mucosal addressin cell adhesion molecule) interaction, could have less systemic effects. Also, selective inhibition of α4β1–vascular adhesion molecule (VCAM)-1 binding could be useful due to fewer potential adverse effects, as constitutive immune cell migration to the intestine would not be affected. The approach that involves blocking the α4 integrin subunit has been used in multiple animal models, in particular in the cottontop tamarin [66]. In earlier studies it was also helpful in treating CD and UC [67]. Natalizumab at a dose of two 3 mg/kg infusions at 4-week intervals produced significantly higher clinical response rates than placebo at 2, 4, 6, 8, and 12 weeks (P<0.05) [68]. A very large study of 905 patients with active CD was performed using 300 mg natalizumab or placebo to induce clinical response (>70 points decrease in CDAI at week 10) or remission. Patients were randomized using a 4:1 ratio to receive natalizumab or placebo. In patients who had received prior anti-TNF therapy, 54% in the natalizumab group compared with 35% in the placebo group demonstrated clinical response at week 10.

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There was a trend towards clinical remission (33% versus 22%, respectively, in this subgroup of 360 patients). In a smaller subgroup (n=266) with prior anti-TNF therapy and active inflammation (defined by a CRP level above the upper limit of normal), response rates were 54% versus 31% with the drug versus placebo, and remission was obtained in 36% and 20% of patients, respectively. Both differences were significant. The authors concluded that natalizumab might offer a novel therapeutic option for CD patients in whom anti-TNF therapies have not been effective [69]. A Phase III, double-blind, placebo-controlled study on maintenance of clinical response and remission in CD was performed in 339 CD patients who achieved response or remission after receiving three infusions of natalizumab. Patients were re-randomized to natalizumab or placebo for up to 12 additional monthly infusions. The primary endpoint was the proportion of patients not losing a response at every time point for an additional 6 consecutive months. At 6 months, 61% of natalizumab-treated patients continued to meet the criteria for clinical response versus 29% of subjects re-randomized to receive placebo. In the natalizumab group, 44% of patients maintained remission at 6 months compared with 26% in the placebo group. Both differences were significant. Of all treated patients, 55% could be withdrawn from steroids compared with 25% of those on placebo [70]. In a study of 79 patients with active CD who were receiving infliximab, patients were randomized to receive either natalizumab (300 mg) or placebo every 4 weeks for a total of three infusions, in addition to infliximab (5 mg/kg) every 8 weeks for a total of two infusions. The primary endpoint was the safety and tolerability of this combination. The incidence of adverse events in patients receiving both natalizumab and infliximab was similar to those receiving infliximab alone [71]. An open-label Phase III study is currently ongoing and will provide long-term safety data on this combination.

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Outcome

Placebo (n=58)

α4β β7 Antibody (mg/kg) 0.5 (n=62)

2.0 (n=65)

15

33*

34*

Normal mucosa (%)

8

29*

14

Response (%)

33

66*

57*

Clinical remission at day 43 (%)

Table 6. Effect of α4β7 integrin antibodies for ulcerative colitis [72]. Dosing weeks 0 and 4. *Significant vs placebo.

However, natalizumab was withdrawn from the market in February 2005, due to cerebral complications of progressive multifocal leukoencephalopathy in multiple sclerosis and CD treatment. As a consequence, studies of this agent are currently on hold. β7 integrin Antibodies against α4β The humanized antibody MLN-02/LDP-02 blocks the interaction between α4β7 integrin and its ligand MAdCAM-1 that mediates the selective homing of leukocytes to the gut mucosa. Treatment with such antibodies has been effective in animal models. In addition, a previous dose-finding study in 28 patients with UC has been reported. Meanwhile two larger trials have been performed. A placebo-controlled trial in 185 patients given either an α4β7 antibody (0.5 mg/kg or 2 mg/kg twice at 4-weekly intervals) or placebo found clinical remission at day 43 in 33% and 34% of patients in both dose groups versus 15% with placebo. Response was demonstrated in 66% and 57% of patients treated with the antibody versus 33% of patients treated with placebo (Table 6) [72]. An identical study performed in CD patients found clinical remission at day 57 in 37% of patients given 2 mg/kg α4β7 antibody, 29% of patients given 0.5 mg/kg, and 21% of patients given placebo. Response rate ranged from 53% to 41%, and there was no significant difference between antibody and placebo [73].

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Outcome

μg/kg) Repifermin (μ (n=60)

Placebo (n=28) 1

5

10

25

50

Remission (%)

11

11

9

0

0

0

Response (%)

36

46

18

33

42

29

Table 7. Effect of intravenous repifermin (keratinocyte growth factor) in 88 patients with moderate ulcerative colitis at 4 weeks [75].

Antisense oligonucleotides against ICAM-1 After the failure of alicaforsen (ISIS-2302, a synthetic antisense molecule to ICAM-1) in CD, a randomized controlled doubleblind study of alicaforsen enemas in active UC was reported [74]. A total of 40 patients with mild to moderately active distal UC were assigned to four dosing cohorts of 10 patients each. In each cohort, eight patients were actively treated and two were treated with placebo. Patients received 0.1, 0.5, 2, or 4 mg/mL of alicaforsen or placebo once daily for 28 consecutive days. At day 29 there was a dose-dependent improvement in the disease activity index (DAI): 4 mg/mL improved DAI by 70% compared with a placebo response of 28% (P=0.004). At 3 months, the 2 mg/mL and 4 mg/mL doses improved this status by 72% and 68% compared with placebo (11.5%). In a further study, 12 patients with pouchitis were treated with 240-mg enemas nightly for 6 weeks. This induced a significant decrease of the pouchitis DAI, with nine patients responding [75]. Larger trials evaluating higher IV doses are also underway.

Growth factors and hormones A number of growth factors have been shown in vitro to improve wound healing and the epithelial barrier in the intestinal tract. Of these, keratinocyte growth factor 2 (repifermin) was studied in patients with active UC, but did not show any significant effect (Table 7) [76].

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Placebo

6 μg/kg/day Sargramostim

Remission (%)

19

40

Response (CDAI ↓ >100) (%)

26

48*

Outcome

Table 8. Effect of subcutaneous sargramostim (granulocyte macrophagecolony stimulating factor) in active Crohn’s disease at week 6 [82]. Effect independent from C-reactive protein, some mucosal healing. CDAI: Crohn’s disease activity index. *Significant vs placebo.

Epidermal growth factor (EGF) was used in the form of an enema in a placebo-controlled trial in 24 patients [77]. However, there were problems with concomitant medication, and also with the use of a disease activity score that was not validated as a primary endpoint. The study showed that, at 2 weeks, 83% of the EGF enema group achieved remission compared with 8% of the control group according to the St Marks score (P<0.001), or 33% versus 0%, respectively, using the DAI (P=0.09). Based on the St Marks score, remission lasted for several weeks. Concerns have been raised regarding the role of EGF in the up-regulation of protooncogenes in a condition with known carcinogenic potential. Human growth hormone has been shown to improve mucosal healing and decrease permeability in animal models. The only positive study with 37 patients demonstrated some success, but this has not been repeated thus far [78].

Immunostimulating factors Some immune deficiency disorders can imitate CD in their presentation. This might indicate that immune deficiency is a major cause of the abnormalities found in CD [79]. This view is supported by studies of the function of NOD2/CARD15 as a recognition protein in the innate immune system – this protein can be mutated in CD (see Chapter 3). A number of pilot studies have shown that both granulocytecolony stimulating factor (filgrastim) and GM-CSF (sargramostim)

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Outcome

Clinical remission at week 12 (%)

Placebo (n=20)

40

α Pegylated interferon-α μg/mL) (μ 0.5 (n=19)

1.0 (n=21)

58

38

Table 9. Pegylated interferon-α treatment (weekly dosing) in active ulcerative colitis [83].

are effective in patients with CD [80,81]. A more recent study investigated the use of sargramostim 6 μg/kg/day or placebo for 8 weeks in 124 patients with moderately to severely active CD. At the end of treatment, response was achieved in 48% of sargramostim-treated patients versus 26% of placebo-treated patients, and remission in 40% versus 19% of patients, respectively (Table 8). Those outcomes were maintained through the followup period of 30 days, despite cell counts returning to baseline levels [82]. However, earlier studies indicate that continuation of treatment is necessary over prolonged periods of time.

Immunomodulators IFNs play a significant role in regulating the immune response, and are used successfully in a number of disorders such as chronic viral hepatitis and multiple sclerosis. Both IFN-α and IFN-β have been used in the treatment of CD and UC. While in uncontrolled studies IFN-α has been claimed to be effective in CD, this has not been found in all studies. A randomized placebocontrolled trial of pegylated IFN-α in active UC demonstrated no effect with two different doses (0.5 μg/kg or 1.0 μg/kg once weekly) during 12 weeks of treatment (Table 9) [83]. IFN-β1a has been tested in a number of studies. The last study was in 97 steroid-refractory patients with UC, the drug was given in doses of 1 or 3 million units three times weekly for 8 weeks. This study showed 56% remission in the higher-dose group and 30% remission in the lower-dose group compared with 38%

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remission in the placebo group [84]. Another placebo-controlled study of recombinant IFN-β1a using doses of 22, 44, or 88 μg SC three times per week demonstrated clinical response in 50% of patients and remission in 30% of patients in the IFN group, compared with 14% and 0% in the placebo group [85]. A further unpublished study showed a lack of efficacy in CD, although this was mainly due to an exceptionally high placebo response in the female study participants. Finally, IL-11, a pleiotropic cytokine that has many biological activities including barrier repair and inhibition of inflammatory signals, was studied intensively in earlier years. A recent double-blind, double-dummy controlled study of 1 mg IL-11 SC once weekly versus prednisolone (60 mg/day tapered) in 51 patients for 12 weeks demonstrated remission rates of 4% for IL-11 versus 46% for prednisolone at week 4, and 19% versus 50%, respectively, at week 6. However, after 12 weeks, 22% of patients with IL-11 and 21% of patients with prednisolone maintained remission [86].

What has been achieved? Clinically relevant results for the innumerable clinical studies that have been performed in the last few years remain rather limited (Table 10). Although a number of concepts have proven biological activity, clinically relevant effects compared with conventional treatments have only been described for infliximab and GM-CSF in larger controlled studies. There are a number of reasons for this disappointing result. Many studies included the wrong patients: it is obvious that patients without inflammation cannot reasonably be treated with an anti-inflammatory medication. When the complaints are due to stricturing stenosis or chologenic diarrhea, even the most potent biological will not be able to relieve symptoms. In several trials, subgroups with initially elevated CRP levels were found to respond much better than the remaining patients, which supports this notion.

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Substance

CD

UC

Inhibitors of proinflammatory cytokines IL-1 RA Infliximab CDP870 Adalimumab RNF-R 75 and RNF-R 55 MAP kinase inhibitor NF-κB p65 antisense

(–) + (+) (+) – (–) (+)

? + ? ? ? ? (+)

Inhibitors of Th1 polarization and T-cell proliferation Anti-CD4 Anti-IL-12 Anti-IFN-γ Anti-IL-2R IL-10

+ (+) (–) ? (–)

? ? ? (–) ?

Inhibitors of leukocyte trafficking Anti-α4 integrin Anti-α4β7 integrin Anti-ICAM-1

(+) (–) –

? (+) (+)

Growth factors and hormones EGF KGF Growth hormone

? ? (+)

(–) – ?

Immunostimulators and immunomodulators IFN-α IFN-β1a G-CSF/GM-CSF IL-11

(–) (–) (+) (–)

– (+) ? ?

Table 10. ‘Biological’ treatments for IBD. CD: Crohn’s disease; EGF: epidermal growth factor; ICAM: intercellular adhesion molecule; IL: interleukin; IFN: interferon; G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; KGF: keratinocyte growth factor; MAP: mitogen-activated protein; NF-κB: nuclear factor-κB; Th1: T helper cell type 1; UC: ulcerative colitis; ?: unknown; (–): some biological effect, not clinically relevant as yet; (+): potential, not yet completely clear; +: clinically relevant effects; –: known to be ineffective.

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Additionally, inhibition of single mediators is probably not very effective, as has been shown previously (eg, for leukotrienes and platelet-activating factor). If one considers the inflammatory cascades in the intestinal mucosa as an orchestra, the elimination of a single instrument is probably insufficient to silence the action. Since a conductor has not yet been found, we still need to eliminate several mediators or the cells producing them. The latter effect seems to be the case for infliximab, explaining its exceptional activity. However, since systemic therapies that eliminate immune cells bear risks for immune deficiency, this approach might not be the ultimate choice. Otherwise, the anti-CD4 antibody might have been the better solution.

What does the future hold? On the basis of our new understanding of homeostasis in the intestinal milieu, the balance between luminal flora and the intestinal mucosal immune system, and the components of the innate immune system that might be disturbed in IBD, it appears that novel ‘microbiological’ and biological treatments modulating these pattern recognition systems could lead the way for IBD treatments into the future. For example, administering DNA motifs of intestinal bacteria could be an interesting approach to down-regulating immune responses to ‘aggressive’ luminal bacteria. Molecules interacting locally with the signal transduction of the innate pattern recognition receptors might be interesting candidates. Finding the solution will be more complicated due to the fact that IBD consists of a number of genotypes and phenotypes – these might need differentiated treatment, and will most likely not respond to a single strategy [87].

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6 Crohn’s perianal fistulas: current treatment paradigms David A Schwartz and William J Sandborn Introduction Perianal fistulas are a cause of significant morbidity for patients with Crohn’s disease (CD). Pain, scarring, and fecal incontinence associated with perianal fistulas have a significant impact on the quality of life of these patients. In the past, perianal CD was primarily treated with surgery. More recently, as knowledge regarding the pathogenesis and anatomic considerations of the disease process have improved, additional medical and surgical options have become available. At some centers, the focus of treatment has shifted from isolated surgical or medical treatment to a multidisciplinary approach. The aim of this chapter is to update the reader on the current concepts related to fistulizing CD and the advances in medical and surgical therapy.

Epidemiology The cumulative frequency of perianal fistulas in patients with CD ranges from 17% to 43% in reports from referral centers [1–10]. Population-based studies from Europe and the US have shown similar rates of perianal fistulas in patients with CD, at 23% and 21%, respectively [11,12]. Perianal fistulas frequently develop prior to the onset of luminal disease. In the population-based study from the US, 45% of patients developed a fistula before or at the time of diagnosis. This underscores the difficulty in making a diagnosis of CD in patients who present with only perianal pathology. The cumulative frequency of perianal fistulas in this population-based study was 12% at 1 year, 21% at 10 years,

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Cumulative incidence of perianal fistulas (%)

100

80

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0 0

5

10

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Figure 1. Cumulative incidence of perianal fistulas among 176 Minnesota residents diagnosed with Crohn’s disease between 1970 and 1993. Modified with permission from The American Gastroenterological Society (Gastroenterology 2002:122;875–80 [12]).

and 26% at 20 years (Figure 1). The risk of developing Crohn’s perianal fistulas increases if the disease involves the distal bowel. Hellers et al. showed that 92% of patients with proctitis developed a perianal fistula, compared with only 15% of patients with isolated ileal disease [11].

Anatomy An understanding of perianal anatomy is required in order to comprehend how perianal fistulas develop, to more accurately classify the fistula, and to appreciate the theories of surgical management. The anal canal consists of: • the internal anal sphincter (IAS), which is formed from the continuation of the circular smooth muscle of the rectum • the external anal sphincter (EAS), which is formed from the downward extension of skeletal muscle from the puborectalis [13]

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Puborectalis Rectal columns Dentate line Intersphincter space External anal sphincter Internal anal sphincter

Figure 2. Schematic diagram of anatomic relationships in the perianal region. Modified with permission from Mayo Clinic © 2000.

A potential space called the intersphincteric plane lies between the two sphincters (Figure 2) [14]. The dentate (or pectinate) line marks the point of transition from the transitional and columnar epithelium of the rectum to the squamous epithelium of the anus. The dentate line is usually located in the middle portion of the IAS. Anal crypts, which contain anal glands at their bases, are present at the dentate line. These glands occasionally penetrate into the intersphincteric space and can be one source of perianal fistula development (see Figure 2) [15].

Diagnosis Successful management of Crohn’s perianal fistulas begins by determining the activity of the luminal CD and delineating the fistulizing process. Endoscopy and modern imaging techniques help to guide therapy by providing a complete assessment of the disease process at its onset. An examination under anesthesia (EUA) then confirms these findings and institutes treatment. A blinded study has shown that a combination of either magnetic resonance imaging (MRI) or endorectal ultrasound

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(EUS) with EUA can assess Crohn’s perianal fistulas with 100% accuracy [16]. An imaging modality should, in essence, provide a virtual roadmap so that the surgeon can establish drainage of any abscess that might be present and control fistula healing. This helps to prevent abscess formation or ramification from a fistula tract during treatment. Imaging can have a major impact on outcomes, because failure to recognize occult lesions (abscesses or fistula branches) might result in recurrent fistulas or abscesses, and/or convert a simple fistula into a complex fistulizing process, thus reducing the chance of healing [17–24]. The goal of therapy should be to try and prevent this progression from occurring by optimizing the medical and surgical treatment at disease onset. Potential diagnostic modalities include fistulography, computed tomography (CT), pelvic MRI, EUS, and EUA. Fistulography and CT Previously, fistulography was the most common method of defining fistula anatomy. This involves placing a small catheter into the cutaneous opening of a fistula tract and injecting contrast under pressure. However, the overall accuracy of fistulography is poor (16–50%) [25–29]. CT was also utilized in the past. However, because of its poor spatial resolution in the pelvis, CT has proven to be relatively unreliable in assessing perianal disease with an accuracy ranging from 24% to 60% [30–36]. MRI and EUS Both MRI and rectal EUS have been shown to have excellent accuracy when used to evaluate Crohn’s perianal fistulas [16,30,31,37–45]. A prospective blinded study compared the accuracy of MRI, EUS, and EUA in 34 patients with suspected Crohn’s perianal fistulas [16]. In this study, all three methods demonstrated good agreement with the consensus gold-standard (EUS 91%, MRI 87%, EUA 91%). In addition, a combination of

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any of the imaging modalities with EUA provided 100% accuracy in these patients and was the most cost-effective approach. MRI and EUS are now being used in some centers to monitor fistula response to medical therapy [45–49]. Recently, one investigator demonstrated that utilizing EUS to guide therapy (ie, medication changes and the placement/removal of setons) resulted in improved long-term fistula healing rates (76% of patients maintained long-term cessation of drainage) [50]. EUA The purpose of EUA is to provide a careful examination of the perianal process in a controlled setting. Given the nature of perianal CD, it is usually too painful to do this comfortably in the office setting where sedation is not available. The perianal fistulizing process is defined at EUA by visual inspection, palpation, and the passage of malleable probes into the fistulas when the patient is under general anesthesia. Occasionally, hydrogen peroxide and/or methylene blue are used to help detect internal openings or connections to other structures. Therapeutic procedures can also be performed at the same time. These include incision and drainage of a perianal abscess and placement of a noncutting seton. EUA is highly accurate at delineating perianal fistulas (91% in one study) [16]. However, because perianal CD leads to induration and scarring, significant lesions can be missed. BeetsTan et al. found that MRI provided useful additional information to the results of EUA in 40% of patients with Crohn’s perianal fistulas [51].

Classification schemes Classification systems describing perianal fistulizing disease are useful to facilitate communication between clinicians and surgeons, and for identifying patients who might benefit from surgical intervention. Several fistula classification systems exist.

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Milligan and Morgan’s classification An early classification scheme developed by Milligan and Morgan classified fistulas according to their relationship to the anorectal ring [52]. Their system described four types of fistulas: • low anal (below the dentate line) • high anal (above the dentate line, but below the puborectalis) • anorectal below the levator ani muscle • anorectal above the levator ani muscle Because most fistulas-in-ano arise below the dentate line, the scheme was simplified to divide fistulas into either low or high, based on their relationship to the dentate line [53]. Hughes’ classification A TNM (primary Tumor, regional lymph Nodes, and distant Metastasis) approach to classifying perianal CD was initially proposed by Hughes in 1978, with subsequent modification in 1992 [54,55]. Each major manifestation of perianal CD (ulceration, fistula, and stricture) is graded on a scale of 0–2 (0=absent, 2=severe). Fistulas are also classified as being ‘low’ (not extending above the dentate line) or ‘high’ (extending above the dentate line, sometimes to the levator ani muscles). The 1992 modification added a description of associated anal conditions, the intestinal location of other sites of CD, and a global assessment of the activity of the disease. The TNM classification system has never gained widespread acceptance because it is perceived by clinicians to have little clinical utility [56]. Parks’ classification The most anatomically precise fistula classification system is Parks’ classification. This system uses the external sphincter as a

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E C

A

B

D

External anal sphincter

Figure 3. Parks’ classification. A superficial fistula tracks below both the internal anal sphincter and external anal sphincter complexes (A). An intersphincteric fistula tracks between the internal anal sphincter and the external anal sphincter in the intersphincteric space (B). A transsphincteric fistula tracks from the intersphincteric space through the external anal sphincter (C). A suprasphincteric fistula leaves the intersphincteric space over the top of the puborectalis and penetrates the levator muscle before tracking down to the skin (D). An extrasphincteric fistula tracks outside of the external anal sphincter and penetrates the levator muscle into the rectum (E). Modified with permission from Mayo Clinic © 2000.

central point of reference [57]. Park’s classification describes five types of perianal fistulas (Figure 3): • intersphincteric • transsphincteric • suprasphincteric • extrasphincteric • superficial

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Special note is also made of any horseshoeing (crossing the midline either anteriorly or posteriorly) and its location (intersphincteric, infralevator, or supralevator). There are several limitations to this system – eg, it does not identify any associated perianal manifestations, such as skin tags or anal strictures. In addition, associated abscesses and/or connections to other structures such as the vagina or bladder are clinically important, but are not part of this system. American Gastroenterological Association classification In 2003, an American Gastroenterological Association technical review on perianal CD proposed a more clinically relevant approach to classifying the manifestations of fistulas [58]. The authors recommended dividing fistulas into either simple or complex. A simple fistula is: • superficial, intersphincteric, or low transsphincteric • possessing one opening • not associated with an abscess and /or • not connected to an adjacent structure such as the vagina or bladder In contrast, a complex fistula is: • involved with more of the anal sphincters (ie, high transsphincteric, extrasphincteric, or suprasphincteric) • possessing multiple openings • exhibiting horseshoeing • associated with a perianal abscess

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and/or • connected to an adjacent structure such as the vagina or bladder This is an important distinction clinically because several studies have shown better outcomes for patients with ‘simple’ fistula tracts [23,24,59,60].

Outcome measures CD activity index The CD activity index (CDAI) is the primary tool for quantifying the activity of luminal CD [61]. Unfortunately, this index was not designed to be sensitive to the type of morbidity experienced by patients with perianal fistulas [62]. Attempts at developing a universally accepted measure for quantifying perianal disease activity have not been accomplished to date. The therapeutic goals score was one of the first attempts at developing an index to measure perianal disease activity [63]. This assigned a baseline fistula activity score of 0 to every patient. Fistula activity was then graded at follow-up on a 7-point scale (–3 to +3) based on the initial baseline assessment. Positive numbers indicated improvement and negative numbers indicated worsening of fistula activity. Due to the subjective nature of this index, it was never widely used. Perianal disease activity index The perianal disease activity index (PDAI) was developed as the perianal equivalent to the CDAI. The PDAI is a comprehensive measure of the morbidity caused by perianal CD and evaluates five categories affected by fistulas: • discharge • pain/restriction of activities

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• restriction of sexual activity • type of perianal disease • degree of induration Each category is scored on a spectrum from 0 (no symptoms) to 4 (severe symptoms) (Table 1). Higher scores indicate more active disease. The PDAI index was initially validated in patients taking metronidazole (MTZ) for perianal fistulas [64]. It was subsequently used as a secondary outcome measure in the initial trial by Present et al. evaluating infliximab for fistulizing CD, and has now been validated in a prospective open-label study examining the use of antibiotics and azathioprine (AZA) in the treatment of perianal fistulas [65,66]. Fistula drainage assessment The fistula drainage assessment is the instrument most commonly used in clinical trials. This assessment characterizes fistulas as open (ie, actively draining) or closed. A fistula is considered open if an investigator can express purulent material from the fistula with the application of gentle pressure to the tract [65]. This measure might be somewhat misleading in that several studies utilizing either MRI or EUS have demonstrated persistent fistula activity even after fistula drainage ceases [45,47,48,67]. In addition, fistula drainage will commonly recur once therapy is discontinued. Therefore, the term ‘cessation of drainage’ rather than ‘closed’ might be the more appropriate term to use when describing a fistula in which purulent material cannot be expressed [58,68]. The term ‘closed’ should be reserved for fistulas that appear to be fibrotic or show no inflammatory activity on imaging with either MRI and/or EUS. MRI-based score An imaging-based activity measure using MRI has now been developed [47]. The MRI-based score rates the severity of the perianal fistulizing process based on the findings of MRI (Table 2).

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Categories affected by fistulas

Score

Discharge No discharge Minimal mucous discharge Moderate mucous or purulent discharge Substantial discharge Gross fecal soiling

0 1 2 3 4

Pain/restriction of activities No activity restriction Mild discomfort, no restriction Moderate discomfort, some activity limitation Marked discomfort, marked limitation Severe pain, severe limitation

0 1 2 3 4

Restriction of sexual activity No restriction Slight restriction Moderate limitation Marked limitation Unable to engage in

0 1 2 3 4

Type of perianal disease No perianal disease/skin tags Anal fissure or mucosal tear <3 Perianal fistulas ≥3 Perianal fistulas Anal sphincter ulceration or fistulas with significant undermining of skin Degree of induration No induration Minimal induration Moderate induration Substantial induration Gross fluctuance/abscess

0 1 2 3 4

0 1 2 3 4

Table 1. Perianal Crohn’s disease activity index. Reproduced with permission from the American College of Physicians.

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Categories

0 1 2 3

Location Extra- or intersphincteric Transsphincteric Suprasphincteric

1 2 3

Extension Infralevatoric Supralevatoric

1 2

Hyperintensity on T2-weighted images Absent Mild Pronounced

0 4 8

Collections (cavities >3 mm diameter) or abscesses Absent Present

0 4

Rectal wall involvement Normal Thickened

0 2

Table 2. Magnetic resonance imaging-based score.

The variables evaluated include: • number of fistula tracks • fistula location • fistula extension • hyperintensity on T2-weighted images • collections or abscesses • rectal wall involvement

152

Score

Number of fistula tracks None Single, unbranched Single, branched Multiple

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B A

Figure 4. Proposed mechanism for fistula development (1). Fistulas develop as ulcers (A) that extend over time as feces are forced into the ulcer (B). Reproduced with permission from Blackwell Scientific Publications [69].

The MRI-based score has the advantage of being a more objective measure of fistula activity than previous indices, but has yet to be fully validated.

Etiology The specific pathogenesis of Crohn’s perianal fistulas is unknown, but two mechanisms have been proposed. Firstly, fistulas might begin as deep penetrating ulcers in the anus or rectum [54]. With the increased pressure associated with defecation, feces are forced into these ulcers, causing them to extend over time (Figure 4) [69]. A second theory is that the fistulas result from an infection or abscess of the anal glands that exist at the base of the anal crypts (Figure 5). Some of these glands penetrate into the intersphincteric space and can easily ramify from this location [15]. From the intersphincteric space, a fistula can then: penetrate through the EAS to become a transsphincteric fistula; track downward to the skin to become an intersphincteric fistula

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Figure 5. Proposed mechanism for fistula development (2). Fistulas begin as anal gland abscesses that ramify within the intersphincteric space. Reproduced with permission from Blackwell Scientific Publications [69].

or superficial fistula; or track upward in the intersphincteric space to become a suprasphincteric fistula. This theory would not account for the development of extrasphincteric fistulas, but some authors believe that these are caused by previous surgical trauma (ie, probing the tracts too vigorously) or complication from previous fistulizing disease [70,71].

Medical therapy The number of medical options available for treating Crohn’s perianal fistulas continues to increase. The agents with probable or proven efficacy include antibiotics, 6-mercaptopurine (6-MP) and AZA, methotrexate (MTX), cyclosporine (CYA), tacrolimus (TAC), and infliximab. Antibiotics Antibiotics are the most commonly used agent for the treatment of Crohn’s perianal fistulas. Various antibiotics have been used for perianal fistulas in the past – currently, the most common antibiotics employed are MTZ at a dose of 750–1,000 mg/day or ciprofloxacin (CIP) at a dose of 1,000–1,500 mg/day. However, this choice of treatment is largely based on uncontrolled case

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series that have suggested a benefit [72–79]. One of the larger case series was conducted by Bernstein et al [78]. This was an open-label study of MTZ (20 mg/kg/day) in 21 consecutive patients with perianal fistulas and CD. Fistula closure occurred in 83% of patients. Clinical improvement is usually seen after 6–8 weeks of therapy. Unfortunately, fistulas frequently recur after MTZ is discontinued [73,75]. A controlled trial of MTZ in patients with fistulizing CD has not yet been performed. Adverse events associated with MTZ include a metallic taste, glossitis, nausea, and distal peripheral sensory neuropathy. Because of the high incidence of adverse events associated with MTZ, clinicians began using CIP to treat CD fistulas in the early 1990s [80]. Although CIP is now widely used, efficacy has only been found in case studies [77]. Similar to treatment with MTZ, fistulas frequently recur once therapy is discontinued. Adverse events associated with CIP are uncommon, but can include headache, nausea, diarrhea, and rash. A prospective open-label trial of 52 patients has examined the use of antibiotics, as a bridge to immunosuppressive therapy with AZA, for patients with Crohn’s perianal fistulas [66]. Patients were given either MTZ or CIP for 8 weeks, and a subset of patients was then initiated on AZA (2–2.5 mg/kg/day). A third subset of patients was already on a stable dose of AZA (2–2.5 mg/kg/day) at study entry. These patients were allowed to continue on this medication while they were started on an antibiotic. In this study, patients who were maintained on AZA after antibiotics were discontinued had a higher response rate at week 20 compared with those who were not bridged to AZA (48% vs 15%, P=0.03). AZA and 6-MP The purine analogs AZA and 6-MP are frequently used to treat perianal fistulas in patients with CD. However, there have been

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Study

No. of patients

Site

Response

Comment

Rhodes et al., 1971 [83]

6

PA=1 EC=4 EV=1

2/6 (33%)

Given AZA 4 mg/kg/day for 10 days then 2 mg/kg/day. Duration of therapy was 2 months. Crossover study

Willoughby et al., 1971 [84]

2

NS

0/2

Given AZA 2 mg/kg/day for 6 months. Double-blinded placebo-controlled trial

Klein et al., 1974 [81]

10

PA=8 EC=1 EV=1

4/5 (80%) Placebo 2/5

Given AZA 3 mg/kg/day for 4 months. Placebo-controlled trial

Rosenberg et al., 1975 [82]

2

PA

0/1 Placebo 1/1

Given AZA 2 mg/kg/day for 6 months. Placebo-controlled trial

36 (40 fistulas)

NS

16/29 (55%) Placebo 4/17 (24%)

Given 6-MP 1.5 mg/kg/day or placebo for 1 year and then crossed over to other arm of study for an additional year

Controlled trials

Present et al., 1980 [63]

Open trial Korelitz and Present, 1985 [86]

34 PA=18 22/34 (65%) (41 EC=8 fistulas) RV=6 EE=7 Vulva=2

Given 6-MP 1.5 mg/kg/day for at least 6 months. Reported the fistula patients from previous study by Present [63]

Table 3. Azathioprine (AZA)/6-mercaptopurine (6-MP) studies for luminal Crohn’s disease, with fistula closure as a secondary endpoint. EC: enterocutaneous; EE: enteroenteric; EV: enterovesicular; NS: not specified; PA: perianal; RV: rectovaginal. Reproduced with permission from Blackwell Scientific Publications [69].

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no controlled trials with fistula closure as the primary endpoint. Fistula closure as a secondary endpoint was investigated in five controlled trials using AZA and/or 6-MP for luminal CD (Table 3) [63,81–84]. A meta-analysis published by Pearson et al. summarized the results of these five studies [85]. Pearson’s analysis demonstrated that 22 of 42 (54%) patients with perianal CD who received AZA/6-MP responded versus only 6 of 29 (21%) patients who received placebo (odds ratio [OR]=4.44). These results should be interpreted with caution since the primary goal of these studies was to treat patients with symptoms of active inflammatory CD not perianal fistulas, and only one of the five studies stratified the patients for the presence of fistulas prior to randomization. Uncontrolled case series have also been reported (one in children) [86,87]. The largest series included 34 patients with perianal fistulas. Patients received 6-MP at a dose of 1.5 mg/kg/day for a minimum of 6 months. Of the 34 patients, 13 (39%) had complete fistula closure and 9 (26%) had obvious clinical improvement of their fistulas [86]. It is important to optimize the doses of AZA and 6-MP to achieve the maximal effect. Doses of 2.0–2.5 mg/kg of AZA and 1.0–1.5 mg/kg of 6-MP have been shown to be the most efficacious [85]. Therefore, these doses should ideally be used in patients with fistulizing CD. Adverse events including leucopenia, allergic reactions, infection, pancreatitis, and drug-induced hepatitis are reported to occur in 9–15% of patients receiving AZA/6-MP for inflammatory bowel disease [85,88]. Cyclosporine Ten uncontrolled case series have reported on the use of IV CYA to treat fistulizing CD in a total of 62 patients (Table 4) [89–98]. The overall initial response rate in these studies was 83%. CYA is typically administered via a continuous IV infusion

157

158 No. of

O’Neill et al., 1997 [95]

8

2

Abreu-Martin et al.,

1996 [94]

1

Markowitz et al., 1990 [93]

1/1

Used 4 mg/kg/day IV CYA

EV=1

EE=1

EC=1

RV=2

PA=5

NS

PA

EV=2

EC=4

7/8

2/2

0/1

0/8

1/2

0/1

Used 4 mg/kg/day IV CYA

Used 2.5 mg/kg/day IV CYA

Used 4 mg/kg/day IV CYA

Used 4 mg/kg/day IV CYA

AZA/6-MP 9/16

2/5

Used 4 mg/kg/day IV CYA

Used 8 mg/kg/day of oral CYA

EC=3 PA=10

14/16

1/1 NS

Comments

4 patients treated with overlapping

5/5

6/10

Sustained response

PA=3

EV=5

Initial response

12:44

1994 [92]

Present and Lichtiger,

16

5

Hanauer and Smith,

EC=2

EV=1

PA=7

EC

Site

20/4/06

1993 [91]

1 10

Lichtiger, 1990 [90]

patients

Fukushima et al., 1989 [89]

Study

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3

62

Gurudu et al., 1999 [98]

Total

RV=1

EC=1

EE=1

53/62 (85%)

2/3

19/50 (38%)

NS

Used 4 mg/kg/day IV CYA

AZA/6-MP

Used 4 mg/kg/day IV CYA

Patients treated with overlapping AZA

Used 5 mg/kg/day IV CYA

EV=2

2/8

4/9

Patients treated with overlapping

7/9

9/9

EC=2

PA=7

EV=1

EC=2

PA=3

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Table 4. Case series reporting on the use of IV cyclosporine (CYA) to treat fistulizing CD. 6-MP: 6-mercaptopurine; AZA: azathioprine; EC: enterocutaneous; EE: enteroenteric; EV: enterovesicular; IV: intravenous; NS: not specified; PA: perianal; RV: rectovaginal.

9

7

Egan et al., 1998 [97]

1997 [96]

Hinterleitner et al.,

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(because of poor oral bioavailability) at a dose of 4 mg/kg/day. In the case series described, clinical improvement occurred rapidly, usually within 1 week and responding patients were converted to oral CYA. Relapse rates were relatively high when oral CYA was discontinued. For this reason, most clinicians use IV CYA as a ‘rescue therapy’ to induce fistula closure, followed by oral CYA for 4–6 months as a ‘bridge’ therapy while treatment with other, slower-acting immune modifier agents such as AZA/6-MP are introduced [91,96,97,99]. Adverse events associated with CYA include renal insufficiency, hirsutism, hypertension, paresthesia, headache, seizure, tremor, gingival hyperplasia, hepatotoxicity, and an increased incidence of infection, especially when CYA is combined with other immune modifiers such as corticosteroids or AZA/6-MP [100]. Tacrolimus One placebo-controlled trial and three case series have been reported on the use of TAC in patients with fistulizing CD [101–104]. In the placebo-controlled trial, 48 patients were randomized to receive a standard dose of 0.20 mg/kg/day of TAC or placebo for 10 weeks. The primary endpoint of the study was fistula improvement. In the TAC-treated patients, 43% had fistula improvement (closure of ≥50% of the draining fistulas for ≥4 weeks) compared with 8% in the placebo-treated patients (P=0.004). The OR was 8.62 in favor of fistula healing. However, fistula remission was similar between the two groups (10% vs 8%, P=0.86). Fistula closure in the treatment group was not improved with concomitant immunosuppressant therapy with AZA/6-MP (38% closure with therapy vs 50% closure without therapy, P=0.31). The mean number of adverse events was higher in the TAC-treated cohort (5.2 vs 2.3, P=0.009) and included headache, elevated creatinine, insomnia, paresthesias, and tremors. Of the 15 patients in the TAC arm who had been treated with infliximab in the past, 7 (47%) improved on TAC.

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Therefore, in patients who are intolerant or refractory to infliximab, TAC might offer an alternative therapy to colectomy to control fistula symptoms. Other authors have suggested that longer courses of treatment (>6 months) might improve the fistula healing rates seen with TAC [105]. Methotrexate Two uncontrolled case series have suggested that MTX might be effective in fistulizing CD [106,107]. In a small retrospective case series, 21/33 (56%) patients treated with MTX had complete or partial closure of their fistulas [106]. Schroder et al. utilized infliximab (three doses) as a bridge to MTX in order to maintain long-term fistula closure (>6 months) in 3/12 (33%) patients intolerant to AZA [107]. Further trials are needed before MTX can be recommended for use in fistulizing CD. α antibodies Anti-tumor necrosis factor-α Infliximab was the first agent in this class. It is a murine/human chimeric monoclonal antibody directed towards soluble and membrane-bound tumor necrosis factor (TNF)-α [108]. There have been two placebo-controlled trials demonstrating infliximab’s efficacy for fistulizing CD [65,109]. The initial trial was conducted in 94 patients with fistulizing CD, of whom 85 (90%) had perianal fistulas [65]. Patients were randomized to receive IV infusions of placebo or infliximab, 5 or 10 mg/kg, at dose intervals of 0, 2, and 6 weeks. The primary endpoint for the study was a reduction of ≥50% in the number of draining fistulas. Of the patients who received infliximab (5 and 10 mg/kg doses combined), 62% achieved the primary endpoint compared to 26% of the placebo group. In addition, 55% of the patients who received the 5 mg/kg dose and 38% of patients who received the 10 mg/kg dose had complete closure of all of their fistulas. The rate of perianal abscess formation was 11%, possibly due to closure of the cutaneous end of the fistula tract before the rest of the fistula tract had closed.

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The follow-up study examined the use of infliximab in maintaining fistula closure [109]. In this trial, the 195 patients who were considered responders at week 14 (≥50% reduction in draining fistulas) to the initial induction sequence of infliximab (5 mg/kg at 0, 2, and 6 weeks) were randomized to receive 5 mg/kg infliximab every 8 weeks or placebo. At week 54, 39% of patients who were on maintenance infliximab had complete cessation of fistula drainage compared with only 9% of patients in the placebo cohort (P=0.009). Based on the randomized controlled trial data, patients should be started on infliximab at a dose of 5 mg/kg given at 0, 2, and 6 weeks, and then every 8 weeks for maintenance. Patients who have had an initial response to infliximab but subsequently lose this response can usually regain it by escalating the dose to 10 mg/kg [110]. Recent studies have suggested advantages associated with providing concomitant immunosuppressive therapy for patients on infliximab. These advantages include [111,112]: • a decreased rate of adverse reactions related to antibody formation to infliximab • preservation of drug efficacy • even better, more prolonged, fistula healing rates More recently, a small, open-label trial on a fully humanized anti-TNF-α antibody (adalimumab) has demonstrated fistula healing rates similar to those reported with infliximab [113]. Fistula improvement was seen in 5/9 (56%) patients and fistula closure occurred in 3/9 (33%) patients. Adverse events observed in patients treated with anti-TNF therapy include infusion reactions, an increased rate of infections (including tuberculosis), delayed hypersensitivity reactions,

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formation of anti-double-stranded DNA antibodies, and, in rare cases, drug-induced lupus. Some patients treated with infliximab can develop anti-infliximab antibodies [108,114]. Miscellaneous nonsurgical therapies A variety of other miscellaneous nonsurgical therapies have been reported to be of benefit in patients with fistulizing CD including elemental diets, total parenteral nutrition, mycophenolate mofetil, thalidomide, granulocyte-colony stimulating factor, and hyperbaric oxygen [115–135]. Controlled trials are needed before these agents can be recommended for routine use. The efficacy of newer therapeutic agents such as natalizumab have yet to be tested for fistulizing CD [136]. In fact, natalizumab did not appear to be effective for Crohn’s perianal fistulas in the small number of patients included in one of the initial trials of natalizumab for CD [137]. Subsequent trials, including ENACT (Evaluation of Natalizumab As Continuous Therapy)-1 and ENACT-2, have excluded patients with active fistulas [138,139]. Therefore, natalizumab is not recommended for use in fistulizing CD until further studies are performed.

Surgical therapy The best outcomes are achieved when surgical and medical therapies are used in conjunction [23,50,60,140]. This allows for control of fistula healing during surgical treatment, and optimizes outcomes with medical therapy. Conversely, surgical therapy in isolation, without addressing the underlying disease process, leads to poor wound healing, recurrent fistulas, and even incontinence [22,141,142]. Several observational studies have highlighted the benefit of this type of multidisciplinary approach. Regueiro et al. demonstrated that patients who had an EUA prior to infliximab treatment were significantly less likely to have a recurrence of their fistula compared with patients receiving infliximab alone

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(44% vs 79%, P=0.001) [23]. Similarly, Topstad et al. reported a complete response in 67% of patients treated using this multidisciplinary approach [60]. Potential surgical treatments include: • incision and drainage of perianal abscesses • fistulotomy • placement of noncutting setons • transanal endorectal advancement flap • intestinal resection • temporary diverting colostomy or ileostomy • proctectomy or proctocolectomy The application of these various treatments for the manifestations of fistulizing CD including perianal abscesses, perianal fistulas, and rectovaginal fistulas is discussed below. Perianal abscess A perianal abscess is the most common cause of pain for patients with perianal CD. An abscess should be suspected in the setting of perianal pain, tenderness, or flocculence. This typically develops as a result of a cryptoglandular infection or an obstructed fistula tract [57,143]. The treatment of a perianal abscess is to establish surgically adequate drainage, to relieve sepsis, and prevent damage to the sphincter complex [17–20,144–154]. The specific surgical approach utilized to drain the perianal abscess depends on its location. A superficial abscess is either: • located near the skin surface, close to the anal verge

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• an intersphincteric abscess with no cephalad extension above the dentate line • an ischiorectal abscess inferior to the postnatal space Superficial perianal abscesses can be treated with a simple incision and drainage procedure. Because of the risk of poor wound healing in this setting, fistulotomy should not be performed on any associated superficial fistulas. By definition, a deep perianal abscess is one of the following: • not near the skin surface • in the deep post-anal space • above the levator ani muscle (ie, supralevator) In contrast to superficial abscesses, deep perianal abscesses, because of their distance from the skin, have a tendency to close off following incision and drainage procedures. This leads to recurrent abscesses and potentially new fistula formation. Therefore, for an isolated (no associated fistula) deep perianal abscess, a mushroom catheter should be placed in order to ensure adequate drainage as the abscess heals. For fistulas associated with a high fistula tract, incision and drainage should be performed, followed by placement of a noncutting seton. Several authors have shown that there is an improvement in outcomes for patients with deep perianal abscesses treated with indwelling setons or drains [17,151]. Perianal fistulas Fistulotomy Several factors, including the degree of rectal inflammation and the type of fistula, help determine the most appropriate surgical treatment for patients with Crohn’s perianal fistulas.

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Probe Open fistula tract

External opening

Fistula

Anus

Figure 6. In the absence of active proctocolitis, simple low transsphincteric, intersphincteric, and superficial fistulas can be treated with a fistulotomy. Reproduced with permission from Mayo Clinic © 2000.

In general, patients with low fistulas, including superficial, low intersphincteric, and low transsphincteric fistulas, can be treated with a fistulotomy (Figure 6) [6,10,18,20,22,59,141,142, 144,146,147,150,152,153,155–9]. Reported healing rates with fistulotomy in this setting vary widely in the literature from 8% to 100%. However, most studies show initial healing rates ≥80% (Table 5). The recurrence rates of fistulas initially healed with fistulotomy range in the literature from 5% to 89%, with most studies reporting rates between 0% and 20%. Incontinence is a serious concern with fistulotomy because it involves cutting open the fistula tract. Incontinence rates following fistulotomy range from 0% to 50%. In addition, the rate of proctectomy or long-term diverting stoma after fistulotomy has been reported to be as high as 60%. Although the reported rates of healing, recurrence, incontinence, and proctectomy associated with fistulotomy vary considerably because of differences in patient selection and referral center

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(a)

(b)

Figure 7. How setons prevent abscess formation during medical therapy. (a) An abscess forms in the middle of a fistula tract during medical treatment because the cutaneous openings of the fistula tract tend to close prematurely. (b) Setons allow for continual drainage from the fistula until the center of the tract becomes inactive. Reproduced with permission from Blackwell Scientific Publications [69].

bias, there is a tendency for greater healing rates in patients without evidence of active proctitis. For example, Nordgren et al. noted that healing occurred following fistulotomy in only 4/15 patients (27%) with active Crohn’s proctocolitis compared with 10/12 patients (83%) without rectal involvement [141]. For this reason, the best approach for patients with a low fistula and active proctitis is the use of a noncutting seton. Enema therapy Enema therapy is a potential means of treating active proctitis prior to surgery. However, the use of enema therapy to control active proctitis in patients with CD is not warranted. There is no evidence that either steroid or mesalamine enemas are effective for Crohn’s proctitis. Noncutting setons A noncutting seton is a suture or drain that is threaded into the cutaneous orifice of a perianal fistula, through the fistula tract, across the mucosal orifice of the fistula into the rectum, and then out through the anal canal. A noncutting seton helps to prevent

167

168 13 19 17 46 5

Fry et al., 1989 [153]

Fuhrman and Larach, 1989 [19]

Morrison et al., 1989 [146]

Levien et al., 1989 [150]

Kangas, 1991 [152]

26

Williamson et al., 1995 [18]

7 (27%)

22 (85%)

2 (40%)

5 proctocolitis 26

6 (100%)

6 ileal or ileocecal

Winter et al., 1993 [156]

Nordgren et al., 1992 [141]

3 (60%)

29 (63%)

16 (94%)

18 (95%)

13 (100%)

9 (60%)

(93%)

15

Bernard et al., 1986 [147]

1 (8%)

38 wounds

12

Keighley and Allan, 1986 [144]

18 (90%)

33 patients

20

Hobbiss and Schofield, 1982 [6]

25 (78%)

NS

3/22 (14%)

NS

2 (33%)

NS

NS

19 (41%)

2/16 (13%)

NS

NS

NS

NS

4/18 (22%)

NS

0

Recurrence

NS

0

0

0

(21%)

7 patients

NS

NS

NS

NS

NS

NS

6 (50%)

NS

NS

0

Incontinence

NS

0

3 (60%)

0

(9%)

3 patients

2 (40%)

4 (9%)

1 (6%)

2 (10%)

0

1 (7%)

NS

3 (15%)

NS

0

Proctectomy

12:44

(41 wounds)

32

Marks et al., 1981 [10]

4 (100%)

Healing

20/4/06

Williams et al., 1991 [155]

4

No. of patients

Sohn et al., 1980 [20]

Study

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27

34

35 44 33

Scott and Northover, 1996 [59]

McKee and Keenan, 1996 [157]

Sangwan et al., 1996 [142]

Platell et al., 1996 [159]

Michelassi et al., 2000 [158]

27 (82%)

40 (91%)

35 (100%)

21 (62%)

22 (81%)

10 (100%)

NS

2/40 (5%)

31 (89%)

4/22 (18%)

NS

4 (40%)

NS

0

0

NS

5 (19%)

5 (50%)

NS

0

0

10 (29%): 7 proctectomy + 3 stoma

5 (19%): 3 proctectomy + 2 stoma

1 (10%)

20/4/06 12:44

Table 5. Results of conventional fistulotomy by laying open the tract for low perianal fistulas in patients with Crohn’s disease. NS: not specified.

10

Halme and Sainio, 1995 [22]

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abscess formation by maintaining drainage from the tract while the fistula is healing (Figure 7). High fistulas involving a significant portion of the EAS (such as high transsphincteric, suprasphincteric, or extrasphincteric fistulas) necessitate a more conservative surgical approach to minimize the risk of incontinence. Noncutting setons are the treatment of choice for patients with high fistulas and active rectal inflammation, and might also be the best option for patients with high fistulas and no evidence of proctitis [7,18,22,59,142,146,149,155,157, 160–163]. A noncutting seton is also the best approach for patients with a low fistula and active proctitis. Fistulas frequently recur when the seton is removed. Because a significant portion of the anal sphincter is involved with high fistulas, fistulotomy or fistulectomy (fistula excision) in this setting are associated with a high rate of incontinence and/or nonhealing, ultimately leading to proctectomy [22,141,146,157,162]. Endorectal advancement flap An endorectal advancement flap is an alternative to fistulotomy for patients with low perianal fistulas who do not have active proctitis, or as a substitute for noncutting setons for patients with high fistulas without rectal inflammation [18,153,157,164–169] (Table 6). An advancement flap involves incising a flap of tissue around the internal opening of a fistula. The flap includes mucosa, submucosa, and circular muscle. The internal opening of the fistula tract is then excised and the flap is pulled down to cover the opening (Figure 8) [167]. This procedure produces excellent results in patients without active proctitis, with reported healing rates of up to 74% [167]. However, active rectal inflammation has been associated with a high rate of flap failure [164,165,170]. Diverting ileostomy Temporary diversion of the fecal stream with a diverting ileostomy can be used as a treatment for severely active perianal

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(a)

(b)

Dentate Line Internal opening Fistula External opening Probe Cored fistula tract

(c)

(d) Advancement flap Resectal internal opening Resectal internal opening

Figure 8. Endorectal advancement flap. (a) Probe the fistula tract to identify the internal opening of the fistula. (b) Incise the internal opening of the fistula tract. (c) Incise a flap of tissue (includes mucosa, submucosa, and circular muscle) around the site of the resected internal opening of the fistula. (d) Pull the flap down to cover the site of the resected internal opening of the fistula. Reproduced with permission from Mayo Clinic © 2000.

CD. The rationale for this therapy is two-fold. Firstly, by diverting the fecal stream, the fecal flow across the fistula tract is reduced, allowing the rectal mucosa to heal and the fistula to close. The second rationale for a diverting ileostomy is to eliminate active perianal sepsis before proceeding with a proctectomy, in order to reduce the risk of an unhealed perianal wound. In general, this practice is rarely used as primary therapy, because several studies have shown that the majority of patients who undergo a temporary diverting ileostomy for perianal CD are never able to have intestinal continuity restored [171–177]. Proctectomy Historically, the proctectomy operation rate for perianal CD has been reported to range from 10% to 18% [150,155,178]. Whether the use of noncutting setons and infliximab will be successful in significantly decreasing the rate of proctectomy

171

172 2 22 24 27 10

13 21 23 21 9 7 5 2 5 1 10

Koganei et al., 1995 [154]

Sugita et al., 1995 [160]

Williams et al., 1991 [155]

Pearl et al., 1993 [163] Williams et al., 1991 [161] McKee and Keenan, 1996 [157] Halme and Sainio, 1995 [22]

Morrison et al., 1989 [146] McKee and Keenan, 1996 [157] Halme and Sainio, 1995 [22] Nordgren et al., 1992 [141]

No. of patients

van Donegen and Lubbers, 1986 [7] Williamson et al., 1995 [18] Sangwan et al., 1996 [142] Scott and Northover, 1996 [59] White et al., 1990 [149]

Study

Healed

2 (100%) 19/22 (86%) 22/24 (92%) 23/27 (85%) 10/10 (100%) improved 0 closed Seton 10/13 (77%) improved 8 (62%) closed Seton 17/21 (81%) improved 8 (38%) closed Seton 20/23 (87%) improved 3/23 (13%) closed Seton 21/21 (100%) Seton 7/9 (78%) Seton 4/7 (57%) Seton or excision 1(20%) + fecal diversion Seton + lay open 2/2 (100%) Lay open 2/5 (40%) Lay open 0 Lay open 4/10 (40%)

Seton Seton Seton Seton Seton

Treatment

NS 3/5 (60%) 0 1/4 (25%)

0 2/9 (22%) 3/7 (43%) NS

NS 2 (40%) NS NS

0 NS NS NS

6 (26%)

1 (5%)

0 2 (40%) 0 6 (60%)

0 NS 2 (29%) 3 (60%)

5 (22%)

NS

1 (8%)

0 3 (14%) 7 (29%) 4 (15%) 0

Proctectomy

12:44

9/20 (45%)

9/17 (53%)

0

NS 14 (64%) NS 4 (15%) NS

Incontinence

20/4/06

3/10 (30%)

0 9/19 (47%) 17/24 (71%) 4/27 (15%) 2/10 (20%)

Recurrence

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10 6 3 6 20 26 6 4 2 13

Matos et al., 1993 [162]

Jones et al., 1987 [164]

Fry et al., 1989 [153]

Lewis and Bartolo, 1990 [165]

Makowiec et al., 1995 [166]

Joo et al., 1998 [167]

Robertson and Mangione, 1998 [168]

Williamson et al., 1991 [161]

McKee and Keenan, 1996 [157]

Marchesa et al., 1998 [169]

Sleeve advancement flap

Transanal advancement flap

Transanal advancement flap

8/13 (62%)

1/2 (50%)

1/4 (25%)

3/6 (50%)

19/26 (73%)

16/20 (80%)

5/6 (83%)

3/3 (100%)

2/6 (33%)

10/10 (100%)

1/2 (50%)

5/13 (38%)

1/2 (50%)

3/4 (75%)

3/6 (50%)

7/26 (27%)

4/20 (20%)

1/6 (17%)

0

4/6 (67%)

6 (60%)

NS

NS

NS

NS

NS

NS

0

NS

NS

NS

5/10 (50%)

NS

3 (23%)

1 (50%)

NS

0

2 (8%)

0

0

0

1 (17%)

0

1 (50%)

12:44

Transanal advancement flap

Transanal advancement flap

Transanal advancement flap

Transanal full-thickness advancement flap

Transanal advancement flap

Transanal full-thickness advancement flap

Excision and primary closure

Lay open and/or excision + fecal diversion

20/4/06

Table 6. Results of treatment for high or complex perianal fistulas in patients with Crohn’s disease. NS: not specified.

2

Morrison et al., 1989 [146]

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remains to be determined. For patients who continue to suffer with persistent perianal pain and discharge despite an adequate trial of medical therapy, proctectomy might offer a better quality of life. Rectovaginal fistulas The healing rates associated with either medical or surgical treatment of rectovaginal fistulas are universally lower than those reported for other types of perianal fistulas [49,60,179]. In general, surgical treatment of rectovaginal fistulas in patients with perianal CD should be reserved for those patients without active proctitis. Although there are reports of patients with low fistulas achieving a satisfactory outcome after fistulotomy, this procedure is associated with an increased risk of sphincter injury [56,159,180–185]. There are a number of surgical approaches available for patients with rectovaginal fistulas, including primary closure, transanal advancement flap, sleeve advancement flap, and transvaginal advancement flap (Table 7) [56,153,155,158,164,166,180–195]. Although there are reports of patients with rectovaginal fistulas being treated with the placement of noncutting setons, this procedure should be rarely used [158]. The placement of a noncutting seton in this setting tends to enlarge the cutaneous opening, making the fistula more symptomatic. One exception to this rule would be if there was a rectovaginal septal abscess associated with the fistula; in this circumstance, drainage of the abscess might be enhanced by the noncutting seton. The success rate for primary closure and advancement flaps in patients with rectovaginal fistulas range from 50% to 100%. Cancer A few case reports and small case series detail the development of adenocarcinoma, squamous cell carcinoma, or basal cell carcinoma in chronic perianal fistulas secondary to CD [196–203]. Although rare, malignant degeneration should be considered in the differential diagnosis of a chronic nonhealing fistula, especially if

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perianal and rectal CD is otherwise quiescent. If there is suspicion of cancer, patients should undergo an EUA, with curettage or biopsy of the fistulous tract and histologic review of the material. When cancer is identified in a fistula track, standard oncologic surgical principles and procedures should be followed.

Treatment algorithm It is likely that the complications associated with perianal CD can be prevented through early institution of optimal therapy from the very onset of symptoms. In general, this can be best achieved by a combination of medical and surgical therapy. Treatment must be individualized for each patient on the basis of the type of fistula present (simple or complex), the degree of rectal inflammation present, and the severity of symptoms. The treatment algorithm in Figure 9 might be useful to help guide therapy. Because the digital rectal exam can be inaccurate, treatment decisions should not be based on this alone. Imaging modalities such as EUS and MRI can provide a virtual roadmap for planning therapy, and prevent unnecessary surgery in patients found to have simple fistulas. In addition, EUA can misclassify up to 10% of patients with perianal fistulas, leading to poor outcomes. The accuracy of EUA can be improved to 100% with the addition of either MRI or EUS [16]. The data supporting this aggressive initial diagnostic approach are preliminary at best, but seem to demonstrate improved outcomes in these patients [50]. Patients should also have an endoscopy to determine the degree of rectal inflammation. Once the initial assessment has been completed, patients can be stratified into the appropriate treatment regime. Simple perianal fistulas The treatment goal for patients with simple fistulas should be cure, ideally without the requirement for long-term maintenance therapy that is administered primarily to suppress recurrence of the fistula. Potential treatment options for simple fistulas include antibiotics, fistulotomy, AZA/6-MP, and possibly infliximab (see Figure 9).

175

176 4 6

Radcliffe et al., 1988 [184]

Cohen et al., 1989 [187]

5

9

Bandy et al., 1983 [183]

1

2

Givel et al., 1982 [182]

Farkas and Gingold, 1983 [189]

2

Tuxen and Castro, 1979 [186]

Radcliffe et al., 1988 [184]

4

Hudson, 1970 [180]

3

1

O’Leary et al., 1998 [185]

2

12

Radcliffe et al., 1998 [184]

Lay open

Transanal advancement flap

Anterior advancement flap

Primary closure

Primary closure

Primary closure

Primary closure

Primary closure

Primary closure

Primary closure

Primary closure

Lay open

Lay open

Lay open

Lay open

Lay open

Lay open

Healed

1 (100%)

3 (60%)

1 (50%)

3 (100%)

3 (50%)

2 (50%)

8 (89%)

1 (50%)

1 (50%)

3 (75%)

1 (100%)

6 (50%)

9 (100%)

0

1 (100%)

0

0

3 (60%)

0

NS

0

0

1+1 ileostomy (33%)

3/15 (20%) NS

0

0

0

0

0

4 (33%)

0

1 (100%)

0

0

2 (67%)

0

Proctectomy

12:44

O’Leary et al., 1998 [185]

9

Francois et al., 1993 [56]

Type of repair Lay open

20/4/06

Wiskind and Thompson, 1992 [188]

1 1

Williams et al., 1991 [155]

Givel et al., 1982 [182]

Bandy et al., 1983 [183]

3 1

Faulconer and Muldoon, 1975 [181]

5

No. of patients

Hudson, 1970 [180]

Study

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1 1

O’Leary et al., 1998 [185]

Michelassi et al., 2000 [158]

Transanal anocutaneous flap

Seton

Transvaginal flap

Transvaginal flap

Sleeve advancement flap

Sleeve advancement flap

Linear advancement flap

Curvilinear advancement flap

7 (70%)

0

0

13 (93%)

2 (100%)

3 (60%)

3 (50%)

13 (54%)

0

0

1 ileostomy (7%)

0

1 (20%)

1 (17%)

3 (13%)

0

0

11 (69%)

0

0

0

NS

1 (10%)

Table 7. Results of surgical treatment of rectovaginal fistulas in patients with Crohn’s disease. NS: not specified.

2 14

5

Hull et al., 1996 and 1997 [191,192]

Sher et al., 1991 [194,195]

6

Hull et al., 1996 and 1997 [191,192]

4 (25%)

3 (50%)

Transanal endorectal mucosal flap 2 (67%)

Transanal advancement flap

Transanal advancement flap

10 (83%)

0

4 (67%)

6 (60%)

12:44

Simmang et al., 1998 [193]

10 24

Fry et al., 1989 [153]

Hull et al., 1996 and 1997 [191,192]

3

Michelassi et al., 2000 [158]

Transanal advancement flap

Transanal advancement flap

Transanal advancement flap

Transanal full-thickness advancement flap

20/4/06

Hesterberg et al., 1993 [190]

6 16

O’Leary et al., 1998 [185]

1 12

Makowiec et al., 1995 [166]

6

Radcliffe et al., 1988 [184]

Cohen et al., 1989 [187]

10

Jones et al., 1987 [164]

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177

178 Treat as complex fistulizing process

Continue maintenance azathioprine/6-MP ± infliximab

Treatment success

Consider tacrolimus in selected patients

Treatment failure

Continue maintenance azathioprine/6-MP ± infliximab

Treatment success

Figure 9. Treatment algorithm for perianal fistulas. 6-MP: 6-mercaptopurine; EUA: examination under anesthesia; EUS: endorectal ultrasound; MRI: magnetic resonance imaging. aData advocating imaging and EUA for all patients with fistulas is preliminary, but seems to demonstrate improved outcomes.

Continue maintenance azathioprine/6-MP ± infliximab

1. Fistulotomy 2. If fistulotomy fails, treat as complex fistulizing process

Treatment failure

1. Placement of a noncutting seton 2. Antibiotics, azathioprine/6-MP, and infliximab

12:44

Treatment success

Antibiotics, azathioprine/6-MP, and infliximab

Antibiotics and azathioprine/6-MP Consider infliximab

Complex fistula

20/4/06

Treatment failure

Simple fistula with rectal inflammation

Simple fistula without rectal inflammation

1. History and physical examination 2. Endoscopy to assess activity of Crohn’s disease 3. Imaging study (EUS or MRI) to delineate perianal disease processa 4. EUAa

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Treatment options Antibiotics are widely used to treat simple fistulas and have been recommended in previous practice guidelines and treatment algorithms, but have not been evaluated in placebo-controlled trials [204,205]. The surgical literature demonstrates a high success rate using fistulotomy for simple fistulas. The general approach to simple fistulas in the surgical literature is to try a short course of antibiotics first, and reserve fistulotomy for those patients who do not respond. However, this strategy is based on small case series. There are no controlled trials comparing fistulotomy with sham operation or medical therapy, and some patients fail to heal, leading to a complex fistulizing process, and even proctectomy. Immunosuppressive medications (AZA/6-MP) can be used to treat simple fistulas and are recommended in treatment guidelines, but have not been evaluated in placebo-controlled trials in which fistula healing or closure is the primary endpoint [205]. Furthermore, these agents are slow acting and thus might be of more utility for maintaining fistula closure than for initial induction of fistula closure. As previously mentioned, a 2003 prospective trial showed an equivocal short-term fistula healing rate in patients treated with both AZA and antibiotics, but a better long-term fistula response rate in patients continued on AZA after inducing fistula closure with antibiotics [66]. Infliximab has proven effective in placebo-controlled trials for both the reduction in the number of draining fistulas and the maintenance of that reduction. A three-dose induction regime and an 8-weekly maintenance regime are approved by the US Food and Drug Administration (FDA) for the treatment of fistulas. However, infliximab is expensive, and concomitant immunosuppressive therapy is probably required to prevent the formation of antibodies to infliximab that can lead to infusion reactions and/or loss of efficacy.

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Recommendations There are insufficient high-quality data (level 1 evidence) to make definitive recommendations regarding which treatment strategy (antibiotics, fistulotomy, AZA/6-MP, or infliximab) is preferred. It is possible that ‘curative’ fistulotomy might have a lower economic cost than induction and maintenance therapy with infliximab, but caution should be used when accepting this conclusion because there are insufficient data detailing the morbidity (incontinence and proctectomy) following fistulotomy. Similarly, there are insufficient data to determine whether long-term maintenance therapy with infliximab is required in patients with simple fistulas who respond to induction therapy. Because the only data that exists from a controlled trial for the treatment of simple fistulas utilized infliximab, a definitive, evidence-based treatment algorithm for the treatment of simple fistulas is not possible. Patients and physicians will need to decide together what treatment option is best for them, based on cost, toxicity, and the lack of controlled trial evidence for antibiotics. However, a rational approach to simple fistulas based on the current evidence available would be to first treat simple fistulas without proctitis medically, with a combination of antibiotics, immunosuppressive therapy (AZA/6-MP), and, in some cases, infliximab. After fistulas heal, immunosuppressive therapy should be continued to maintain fistula closure. If there is no response to antibiotic treatment after 8 weeks, the patient could be treated with a fistulotomy. Repeat imaging evaluation could be helpful to identify any abscess or fistula extension that might have occurred during treatment. Patients with simple fistulas and active proctitis should be treated as if they have a complex fistula, given the potentially poor outcome associated with aggressive surgical intervention in this setting.

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Complex perianal fistulas Treatment options Patients with complex fistulas are the most difficult to treat. Even with intensive medical therapy, the rate of fistula healing is lower than with simple fistulas. The treatment goal for patients with complex perianal fistulas is fistula closure followed by suppression of recurrence. Potential treatment options for complex fistulas include antibiotics, AZA/6-MP, infliximab, and surgery (dilation of anal strictures, placement of noncutting setons, endorectal advancement flaps, repair of rectovaginal fistulas, fecal diversion, and proctectomy) (see Figure 9). TAC and CYA can be rarely used in selected patients, in particular those refractory or intolerant to infliximab. Antibiotics are widely used to treat complex fistulas. They are recommended in practice guidelines and treatment algorithms, but have not been evaluated in placebo-controlled trials [204,205]. Relapse rates for complex fistulas are high after antibiotic therapy is discontinued, so they should probably be used in combination with other medical and surgical therapies, not as primary therapy, in this setting. Similarly, immunosuppressive medications (AZA and 6-MP) are recommended in treatment guidelines, but have not been evaluated in placebo-controlled trials in which fistula healing or closure is the primary endpoint [205]. Furthermore, these agents are slow acting and might be of more utility for maintaining fistula closure than for the initial induction of fistula closure. As mentioned previously, infliximab has proven effective in placebo-controlled trials for reducing the number of draining fistulas and the maintenance of that reduction. Concomitant immunosuppressive therapy is probably required, but, given the low rate of fistula closure and the increased morbidity associated with complex fistulas, this is less of a concern. In all patients treated with infliximab, AZA/6-MP or MTX should be given concomitantly, both to counteract an immunogenic reaction to infliximab and to help maintain remission.

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TAC and CYA can rarely be considered in selected patients who fail multimodality treatment with other medical and surgical therapies, including infliximab. This practice is based on uncontrolled case series with CYA and one small, short-term, placebo-controlled trial that showed a reduction in the number of draining fistulas with TAC. However, nephrotoxicity and other side effects frequently occur, so these drugs should be used with caution. In addition, the trials examining TAC and CYA were of short duration and were therefore unable to determine whether maintenance therapy after initial fistula closure is safe and effective. Surgical therapy is largely palliative and complementary to medical therapy. Perianal abscesses should be drained and anal strictures dilated. Patients with complex fistulas or rectovaginal fistulas without rectal inflammation can be treated with an endorectal advancement flap. However, there is a high recurrence rate following endorectal advancement flap procedures. Fistulotomy is contraindicated due to the increased risk of nonhealing and incontinence. The most conservative approach (ie, noncutting setons) is probably the best strategy in this setting. Noncutting setons should be placed in all patients with macroscopic evidence of rectal inflammation, and in most patients with complex fistulas. This guarantees resolution of perianal sepsis and allows for control of fistula healing during medical therapy. Several case series have now demonstrated an improved outcome in patients treated with noncutting setons [23,50,60]. The exact length of time the setons should be left in place during therapy remains controversial. Most patients will have cessation of drainage after one or two doses of infliximab, and setons are commonly removed at that point. However, although cessation of drainage occurs quickly, true fistula inactivity (on EUS or MRI) can take months to occur. Therefore, a longer period of time before seton removal might be beneficial.

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Recommendations The best outcomes occur when a combination of medical and surgical therapy is utilized. Based on this observation and the information above, a rationale approach to the treatment of complex fistulas involves, firstly, controlling perianal sepsis and establishing control of fistula healing with seton placement. After this has been accomplished, medical therapy should be started with antibiotics, AZA/6-MP, and infliximab. The optimal timing for seton removal has yet to be determined, but leaving the seton in place for longer than 2 weeks might be helpful. After a fistula closes, patients should be continued on immunosuppressive therapy and infliximab for maintenance of closure. As a last resort, fecal diversion or proctectomy can be undertaken. Rectovaginal fistulas In general, rectovaginal fistulas have a lower rate of healing than any other type of perianal fistula. Therefore, the treatment goals are mainly to decrease fistula drainage to a minimal level that is ‘acceptable’. Complete fistula closure, suppression of recurrence, and even cure can be achieved in a small subset of patients. Potential treatment options for rectovaginal fistulas include both medical and surgical therapy. AZA/6-MP, infliximab, CYA, and TAC have been used to treat rectovaginal fistulas in uncontrolled series [86,91,92,97,101,206]. Surgical treatment of rectovaginal fistulas can only be performed in the absence of active rectal inflammation. Thus, for luminal inflammatory disease, aggressive treatment with AZA/6-MP, MTX, and/or infliximab is necessary. Other perianal disease should be treated as outlined above. If the rectovaginal fistula persists after the patient has received therapy to treat both the fistula and the proctitis, and there is no evidence of an anorectal stricture or active proctitis, then surgical repair with transanal or transvaginal advancement flaps, or laparotomy with primary closure or sleeve advancement, can be performed. Temporary diverting ileostomy or colostomy can be performed simultaneously to divert the fecal stream and decrease the risk of

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nonhealing. If advancement flap procedures (which involve coring out the fistula) fail, the rectovaginal fistula might actually increase in size and cause worsening of symptoms. Because of this risk, advancement flap surgery should be reserved for patients with disabling symptoms including stool per vagina, recurrent vaginitis, and vulvar excoriations. As a last resort, fecal diversion or proctectomy can be performed. Some women with persistent but reduced symptoms following medical/surgical therapy might choose to accept residual drainage, rather than undergo a proctectomy with an ostomy, to optimize their overall quality of life.

Conclusion Perianal fistulas are a common manifestation of CD that can lead to significant morbidity, and even proctectomy. Treatment should begin with diagnostic evaluations including endoscopy to assess rectal disease activity, and imaging studies (EUS or MRI) to fully delineate any fistulas or abscesses that might be present. A combined medical and surgical approach will be needed for most patients, although a minority of patients with simple fistulas might heal with either medical or surgical treatment (fistulotomy) alone. The remainder of patients should have surgical drainage established with noncutting setons and then be started on antibiotics, AZA/6-MP, and infliximab. TAC or CYA can rarely be considered in selected patients who fail multimodality treatment with other medical and surgical therapies, before proceeding to fecal diversion or proctectomy.

References 1.

2. 3.

184

Rankin GB, Watts HD, Melnyk CYA, et al. National Cooperative Crohn’s Disease Study: extraintestinal manifestations and perianal complications. Gastroenterology 1979;77:914–20. Farmer RG, Hawk WA, Turnbull RB Jr. Clinical patterns in Crohn’s disease. A statistical study of 615 cases. Gastroenterology 1975;68:627–35. Williams DR, Coller JA, Corman ML, et al. Anal complications in Crohn’s disease. Dis Colon Rectum 1981;24:22–4.

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4. 5.

6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16.

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167. Joo JS, Weiss EG, Nogueras JJ, et al. Endorectal advancement flap in perianal Crohn’s disease. Am Surg 1998;64:147–50. 168. Robertson WG, Mangione JS. Cutaneous advancement flap closure: alternative method for treatment of complicated anal fistulas. Dis Colon Rectum 1998;41:884–6. 169. Marchesa P, Hull TL, Fazio VW. Advancement sleeve flaps for treatment of severe perianal Crohn’s disease. Br J Surg 1998;85:1695–8. 170. Mizrahi N, Wexner SD, Zmora O, et al. Endorectal advancement flap: are there predictors of failure? Dis Colon Rectum 2002;45:1616–21. 171. Harper PH, Kettlewell MG, Lee EC. The effect of split ileostomy on perianal Crohn’s disease. Br J Surg 1982;69:608–10. 172. Zelas P, Jagelman DG. Loop illeostomy in the management of Crohn’s colitis in the debilitated patient. Ann Surg 1980;191:164–8. 173. Grant DR, Cohen Z, McLeod RS. Loop ileostomy for anorectal Crohn’s disease. Can J Surg 1986;29:32–5. 174. Sher ME, Bauer JJ, Gorphine S, et al. Low Hartmann’s procedure for severe anorectal Crohn’s disease. Dis Colon Rectum 1992;35:975–80. 175. Yamamoto T, Allan RN, Keighley MR. Effect of fecal diversion alone on perianal Crohn’s disease. World J Surg 2000;24:1258–62; discussion 1262–3. 176. Orkin BA, Telander RL. The effect of intra-abdominal resection or fecal diversion on perianal disease in pediatric Crohn’s disease. J Pediatr Surg 1985;20:343–7. 177. Edwards CM, George BD, Jewell PD, et al. Role of a defunctioning stoma in the management of large bowel Crohn’s disease. Br J Surg 2000;87:1063–6. 178. Wolff BG, Culp CE, Beart RW, et al. Anorectal Crohn’s disease. A long-term perspective. Dis Colon Rectum 1985;28:709–11. 179. Penninckx F, Moneghini D, D’Hoore A, et al. Success and failure after repair of rectovaginal fistula in Crohn’s disease: analysis of prognostic factors. Colorectal Dis 2001;3:406–11. 180. Hudson CN. Acquired fistulae between the intestine and the vagina. Ann R Coll Surg Engl 1970;46:20–40. 181. Faulconer HT, Muldoon JP. Rectovaginal fistula in patients with colitis: review and report of a case. Dis Colon Rectum 1975;18:413–15. 182. Givel JC, Hawker P, Allan RN, et al. Enterovaginal fistulas associated with Crohn’s disease. Surg Gynecol Obstet 1982;155:494–6. 183. Bandy LC, Addison A, Parker RT. Surgical management of rectovaginal fistulas in Crohn’s disease. Am J Obstet Gynecol 1983;147:359–63. 184. Radcliffe AG, Ritchie JK, Hawley PR, et al. Anovaginal and rectovaginal fistulas in Crohn’s disease. Dis Colon Rectum 1988;31:94–9. 185. O’Leary DP, Milroy CE, Durdey P. Definitive repair of anovaginal fistula in Crohn’s disease. Ann R Coll Surg Engl 1998;80:250–2. 186. Tuxen PA, Castro AF. Rectovaginal fistula in Crohn’s disease. Dis Colon Rectum 1979;22:58–62. 187. Cohen JL, Stricker JW, Schoetz DJ Jr, et al. Rectovaginal fistula in Crohn’s disease. Dis Colon Rectum 1989;32:825–8. 188. Wiskind AK, Thompson JD. Transverse transperineal repair of rectovaginal fistulas in the lower vagina. Am J Obstet Gynecol 1992;167:694–9. 189. Farkas AM, Gingold BS. Repair of rectovaginal fistula in Crohn’s disease by rectal mucosal advancement flap. Mt Sinai J Med 1983;50:420–3. 190. Hesterberg R, Schmidt WU, Muller F, et al. Treatment of anovaginal fistulas with an anocutaneous flap in patients with Crohn’s disease. Int J Colorectal Dis 1993;8:51–4. 191. Hull TL, Fazio VW. Surgical approaches to low anovaginal fistula in Crohn’s disease. Am J Surg 1997;173:95–8.

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192. Ozuner G, Hull TL, Cartmill J, et al. Long-term analysis of the use of transanal rectal advancement flaps for complicated anorectal/vaginal fistulas. Dis Colon Rectum 1996;39:10–14. 193. Simmang CL, Lacey SW, Huber PJ Jr. Rectal sleeve advancement: repair of rectovaginal fistula associated with anorectal stricture in Crohn’s disease. Dis Colon Rectum 1998;41:787–9. 194. Sher ME, Bauer JJ, Gelernt I. Surgical repair of rectovaginal fistulas in patients with Crohn’s disease: transvaginal approach. Dis Colon Rectum 1991;34:641–8. 195. Bauer JJ, Sher ME, Jaffin H, et al. Transvaginal approach for repair of rectovaginal fistulae complicating Crohn’s disease. Ann Surg 1991;213:151–8. 196. Ky A, Sohn N, Weinstein MA, et al. Carcinoma arising in anorectal fistulas of Crohn’s disease. Dis Colon Rectum 1998;41:992–6. 197. Church JM, Weakley FL, Fazio VW, et al. The relationship between fistulas in Crohn’s disease and associated carcinoma. Report of four cases and review of the literature. Dis Colon Rectum 1985;28:361–6. 198. Chaikhouni A, Regueyra FI, Stevens JR. Adenocarcinoma in perineal fistulas of Crohn’s disease. Dis Colon Rectum 1981;24:639–43. 199. Ying LT, Hurlbut DJ, Depew WT, et al. Primary adenocarcinoma in an enterocutaneous fistula associated with Crohn’s disease. Can J Gastroenterol 1998;12:265–9. 200. Korelitz BI. Carcinoma arising in Crohn’s disease fistulae: another concern warranting another type of surveillance. Am J Gastroenterol 1999;94:2337–9. 201. Wong NA, Shirazi T, Hamer-Hodges DW, et al. Adenocarcinoma arising within a Crohn’s-related anorectal fistula: a form of anal gland carcinoma? Histopathology 2002;40:302–4. 202. Kulaylat MN, Gallina G, Bem J, et al. Carcinoma arising in anorectal fistulas of Crohn’s disease. Dis Colon Rectum 1999;42:826–8. 203. Moore-Maxwell CA, Robboy SJ. Mucinous adenocarcinoma arising in rectovaginal fistulas associated with Crohn’s disease. Gynecol Oncol 2004;93:266–8. 204. Schwartz DA, Pemberton JH, Sandborn WJ. Diagnosis and treatment of perianal fistulas in Crohn disease. Ann Intern Med 2001;135:906–18. 205. Hanauer SB, Sandborn W. Management of Crohn’s disease in adults. Am J Gastroenterol 2001;96:635–43. 206. Ricart E, Panaccione R, Loftus EV, et al. Infliximab for Crohn’s disease in clinical practice at the Mayo Clinic: the first 100 patients. Am J Gastroenterol 2001;96:722–9.

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Abbreviations

6-MMP 6-MP 6-TG 6-TGN 6-TIMP 6-TU AA AB ACCENT

ACT AF ASCA AZA BMDC BMDM cAMP CARD CBC CD CD14 CDAI CI CIP cox2 CRP CT CTLA4 CYA DAI DEFCR DLG DR EAS EC EE EGF EGFR

6-methyl mercaptopurine 6-mercaptopurine 6-thioguanine 6-thioguanine nucleotide 6-thioinosine monophosphates 6-thiouric acid African American antibody A Crohn’s disease Clinical trial Evaluating infliximab in a New long term Treatment regimen Active Ulcerative Colitis allele frequency anti-Saccharomyces cerevisiae antibody azathioprine bone marrow-derived dendritic cells bone marrow-derived macrophages cyclic adenosine monophosphate caspase recruitment domain complete blood count Crohn’s disease monocyte differentiation antigen gene Crohn’s disease activity index confidence interval ciprofloxacin cytochrome oxidase subunit 2 C-reactive protein computed tomography cytotoxic T lymphocyteassociated 4 cyclosporine disease activity index defensin-related cryptidin discs, large homolog down-regulator of transcription external anal sphincter enterocutaneous enteroenteric epidermal growth factor epidermal growth factor receptor

EIM ELISA ENACT EPHX EPXH ESR EUA EUS EV FDA GETAID

G-CSF GM-CSF GSTM GSTP GSTT GTP H2 HD HEK HGPRT HLA HMPAO I2

IAS IBD IBS ICAM IFN Ig IG IL IL1RN IM

extraintestinal manifestations enzyme-linked immunoabsorbent assay test Evaluation of Natalizumab as Continuous Therapy microsomal epoxide hydrolase microsomal epoxide hydrolase gene erythrocyte sedimentation rate exam under anesthesia endorectal ultrasound enterovesicular Food and Drug Administration Group d’Etude Therapeutique des Affections Inflammatoires du tube Digestif granulocyte-colony stimulating factor granulocyte-macrophage colony stimulating factor glutathione S-transferase μ glutathione S-transferase π glutathione S-transferase θ guanosine triphosphate IBD5 haplotype human defensin human embryonic kidney hypoxanthine guanine phosphoribosyltransferase human leukocyte antigen hexa-methyl-propyleneamine-oxime Crohn’s disease related sequence from Pseudomonas fluorescens internal anal sphincter inflammatory bowel disease irritable bowel syndrome intercellular adhesion molecule interferon immunoglobulin intragastric interleukin IL-1 receptor antagonist intramuscular

195

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Abbreviations

IMPDH iNOS IP IRF ITPA IV KGF LCT LRR MadCAM MAP MCP MDP MDR MIF mip2 MMP MP MRI MTX MTZ N2 ci N2 wt NA NACHT NF NFKB NJ NNT NOD NPV NS OCTN ompC OR PA pANCA PAI

196

inosine monophosphate dehydrogenase inducible nitric oxide synthase intraperitoneal interferon regulatory factor inosine triphosphate pyrophosphatase intravenous keratinocyte growth factor lactase phlorizin hydrolase leucine-rich repeat mucosal adressin cell adhesion molecule mitogen-activated protein membrane cofactor protein muramyl dipeptide multidrug resistance protein macrophage migration inhibitory factor chemokine ligand 2 matrix metalloproteinase mercaptopurine magnetic resonance imaging methotrexate metronidazole mutant Leu1007fs nod2 wild-type nod2 not available NOD2 nucleotide-binding domain nuclear factor nuclear factor-κB non-Jewish number needed to treat nucleotide-binding oligomerization domain negative predictive value not significant organic cation transporter Escherichia coli outermembrane porin protein C odds ratio perianal perinuclear anti-neutrophil cytoplasmic antibody plasminogen activator inhibitor

Pc PC PDAI PDT PPARG PPV PSCD Rac1 RANTES

RC RCT RIP2 RR RV SA SBCR SC SCYA SEEK SNP STAT TAC TC TDT Th TLR TNF TNM TPMT UC UGIE US VCAM VDR VNTR WBCC XO

corrected P-value platelet count perianal disease activity index pedigree disequilibrium test peroxisome proliferatoractivated receptor γ positive predictive value primary systemic carnitine deficiency ras-related C3 botulinum toxin substrate regulated upon activation, normal T-cell expressed and secreted rectovaginal randomized controlled trial receptor interacting protein-2 relative risk rectovaginal serum albumin small-bowel contrast radiography subcutaneous small inducible cytokine psoriasis susceptibility protein single nucleotide polymorphism signal transducer and activator of transcription tacrolimus C1672T/G207C haplotype transmission disequilibrium test T helper cell toll-like receptor tumor necrosis factor primary tumor, regional lymph nodes, and distant metastasis thiopurine methyl transferase ulcerative colitis upper gastrointestinal endoscopy ultrasound vascular adhesion molecule vitamin D receptor variable number tandem repeat white blood cell count xanthine oxidase

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Index

Page numbers in bold refer to figures. Page numbers in italics refer to tables. A abbreviations, 195–199 abscess(es) drainage, 144 perianal abscess, 165 rectovaginal fistulas, 174 fistulas formation, 153 perianal, 164–165 rectovaginal septal, 174 setons preventing formation, 167 Active Ulcerative Colitis I, 117 Active Ulcerative Colitis II, 117 adalimumab, 121–122, 122 fistulas, 162 adenocarcinoma, 174 alicaforsen, 130–131 American Gastroenterological Association classification, 148–149 anal canal, 143, 143–145 anal cancer, 174–175 anal fistulas see perianal fistulas antibiotics, perianal fistula management, 154 complex fistulas, 181, 183 simple fistulas, 179 antibodies autoantibodies, 5, 121, 121 predicting disease activity/outcome, 39 predicting natural history/monitoring complications, 12 therapeutic see biological therapy see also specific antibodies anti-CD3 antibodies, 126 anti-CD4 antibodies, 126 anti-CD40 ligand antibodies, 126 anti-inflammatory medication, 134 anti-infliximab antibodies, 118 anti-α4 integrin antibodies, 128–129 anti-α4β7 integrin antibodies, 130, 130 anti-interferon-γ antibodies, 124–125 anti-interleukin-2 receptor antibodies, 127 anti-interleukin-6 receptor antibodies, 125–126 anti-interleukin-12 antibodies, 124, 125 anti-interleukin-18 antibodies, 124 anti-ompC, Crohn’s disease vs ulcerative colitis, 35 anti-Saccharomyces cerevisiae antibody (ASCA) combined with pANCA, 7 Crohn’s disease vs ulcerative colitis, 34–35

differentiating between UC and CD, 6–8 pediatric patients, 34 predicting disease activity/outcome, 38–39 predicting natural history/monitoring complications, 12 response to infliximab, 13, 15 screening, 5–6 antisense oligonucleotides against ICAM-1, 130–131 anti-tumor necrosis factor (TNF) antibodies, 115–122 see also infliximab; specific antibodies anus, squamous epithelium, 143 ASCA see anti-Saccharomyces cerevisiae antibody (ASCA) Ashkenazi Jews NOD2 risk variant, 46 autoantibodies/autoimmunity infliximab-induced, 121, 121 screening, 5 see also specific antibodies azathioprine (AZA), 15–19, 93–105 adverse effects, 99–102, 157 Crohn’s disease, 95–96 remission induction, 95 remission maintenance, 95–96 dosage, 99, 103 duration therapy, 104 efficacy in postoperative setting, 98–99 efficacy monitoring, 103–106 fistulas, 155–157, 156, 158–159 complex perianal, 181, 183 rectovaginal, 183 infliximab and, 118 metabolism/mechanism of action, 93–94, 94 studies, 95 6-thioguanine nucleotide level measurement, 18–19 TPMT activity, 17 ulcerative colitis, 96, 97, 98 B bacteria intestinal, DNA motifs, 136 NOD2 mutations, defective response to, 49 basal cell carcinoma, 174 basiliximab, 127 biochemical therapy monitoring, 109–110 biological therapy, 113–140, 116–117, 135 achievements, 134, 136 cell migration/adhesion, inhibitors, 116, 127–131

197

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Index

future, 136 immunodulators, 117, 133–134 immunostimulating, 117, 132–133 proinflammatory cytokines, inhibitors, 115–124, 116 T-cell proliferation, inhibitors, 124–127, 166 T helper cell type 1 polarization, inhibitors, 116, 124–127 see also specific treatments biopsy liver, methotrexate, 109 scintigraphy vs, 30–31 birth defects, 6-MP/AZA treatment, 102 Blau syndrome, 57 blood tests, 14 complete blood count, 3–4 diagnostic, 1–8 disease monitoring, 9–10 erythrocyte sedimentation rate (ESR), 2–3, 10, 11 hemoglobin count, 4, 10, 103 nutritional deficiencies monitoring, 12–13 platelet counts, 4, 9–10, 11, 103 predicting natural history/monitoring complications, 11–13 serum albumin (SA) level, 2–3, 10 treatment monitoring AZA/6-MP, 103 response to infliximab, 15 white cells, 3, 9–10, 11 bone marrow suppression/toxicity, 16 6-mercaptopurine/azathioprine treatment, 100–101 TMPT activity, 17–18 C calcium deficiency, monitoring, 12 calprotectin, 20 cancer 6-mercaptopurine/azathioprine treatment and, 102 infliximab and, 119 perianal fistulas, 174–175 candidate genes, 59, 65, 66–74, 75–83 capsule endoscopy, 32–33 CARD15 gene see NOD2 gene catheter, mushroom, 165 CD14 gene, 78–79 CDP571, 120–121 CDP870, 121 cell migration/adhesion, inhibition, 127–131 chemokines, muramyl dipeptide stimulation, 52 C-insertion double-mutant mice/human cell lines, NOD2, 55 ciprofloxacin (CIP), fistulas, 154–155 classification schemes, Crohn’s perianal fistulas, 145–149 C-nucleotide frameshift mutation, 56 colonic inflammation, differentiating between UC and CD, 7 colonoscopy, 30, 31 colostomy, rectovaginal fistulas, 183 complete blood count (CBC), diagnostic, 3–4 computed tomography (CT), Crohn’s perianal fistulas, 144

198

corticosteroid-dependent Crohn’s disease, methotrexate, 107 corticosteroids, 109–110, 113 AZA/6-MP, 96 see also specific drugs C-reactive protein (CRP) level, 2–3 monitoring disease, 9–10 predicting natural history/monitoring complications, 11 Crohn’s disease (CD) anti-ompC, 35 drug therapy azathioprine, 95–96 biological see biological therapy methotrexate, 106–108 see also specific treatments ileal, 7, 47–48, 52, 53 immunoglobulin (IgA) subtype, 6, 12, 39 immunoglobulin (IgG) subtype, 6 incidence, pediatric, 27–28 pediatric see pediatric IBD perianal fistulas see Crohn’s perianal fistulas risk factors, 29 ASCA association, 7 CD14 allele, 79 NOD2 gene see NOD2 gene toll-like receptors, 79 see also genetics ulcerative colitis vs, 6–8, 34–36 Crohn’s disease activity index (CDAI), 149 A Crohn’s disease Clinical trial Evaluating infliximab in a long term Treatment regimen (ACCENT), 115–116 Crohn’s perianal fistulas, 141–193 anatomy, 142–143, 143 classification schemes, 145–149 diagnosis, 143–145 disease activity index, 151 epidemiology, 141–142 etiology, 153, 153–154, 154 incidence, 142 medical therapy, 154–163 nonsurgical therapies, 163 outcome measures, 149–153 surgical therapy, 163–175, 172–173 diverting ileostomy, 170–171 endorectal advancement flap, 170 enema therapy, 167 fistulotomy, 165–167, 166, 179, 180, 182 noncutting setons, 167, 170 proctectomy, 171, 184 see also individual techniques treatment algorithm, 175, 178, 179–184 cyclosporine adverse effects, 160 complex perianal fistulas, 182 fistulas, 157, 158–159, 160 cytokines IBD pathophysiologic mechanisms, 114–115 muramyl dipeptide stimulation, 52 proinflammatory, inhibition, 115–124, 116 see also individual types

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Index

D daclizumab, 127 defensins, NOD2 mutation, 53 diarrhea, methotrexate, 109 diet, risk factor, 29–30 digital rectal exam, fistulas, 175 DLG5 (Drosophila discs large homolog 5), 76 DNA motifs, intestinal bacteria, 136 draining, complex perianal fistulas, 181 Drosophila discs large homolog 5 (DLG5), 76 drug-induced lupus, infliximab, 119 drug toxicity, tests, 15–16 E ENACT (Evaluation of Natalizumab As Continuous Therapy) trials, 163 endorectal advancement flap, 170, 171 complex perianal fistulas, 182 endorectal ultrasound (EUS), Crohn’s perianal fistulas, 143, 144–145 endoscopy capsule, 32–33 Crohn’s perianal fistulas, 143 monitoring disease, 8–9 upper, 33 upper gastrointestinal (UGIE), 30, 31 enema therapy, Crohn’s perianal fistulas, 167 enteric flora, treatment target, 114 environmental risk factors, 29–30 genetic vs, 46 enzyme-linked immunoabsorbent assay (ELISA), 5, 36 epidermal growth factors (EGF), 131–132 Epstein–Barr virus, 126 erythrocyte sedimentation rate (ESR), 2–3 monitoring disease, 10 predicting natural history/monitoring complications, 11 etanercept, 122 ethnicity, NOD2 gene, 44, 45 examination under anesthesia (EUS), perianal fistulas, 143, 144–145, 175 drainage assessment, 150 external anal sphincter (EAS), fistulas, 170 F factor XIIIa (serum transglutaminase), monitoring disease, 10 fecal diversion, rectovaginal fistulas, 184 fecal tests, 14 calprotectin, 20 lactoferrin, 19 filgrastim (granulocyte-colony stimulating factor), 132–133 fistulas drainage assessment, 150 formation, 153, 153–154 methotrexate, 161 NOD2 and, 48 perianal see perianal fistulas rectovaginal see rectovaginal fistulas fistulography, Crohn’s perianal fistulas, 144 fistulotomy, 165–167, 166 clinical evidence, 168–169 complex perianal fistulas, 182 healing, 167

recurrence rate, 166 simple perianal fistulas, 179, 180 G G908R mutation, 47 genetics, 43–91 IBD5 haplotypes, 57–59 IBD candidate genes, 59, 65, 66–74, 75–83 linkage studies, 61–64, 63 NOD2 gene see NOD2 gene OCTN genes, 59–61 GM-CSF (sargramostim), 132–133, 134 granulocyte-colony stimulating factor (filgrastim), 132–133 granulomas, NOD2, 48 growth factors, 117, 131–132 H hemoglobin count AZA/6-MP treatment monitoring, 103 diagnostic, 4 monitoring disease, 10 hepatic complications, monitoring, 13 hepatotoxicity, AZA/6-MP treatment, 101–102 heterogeneity, NOD2 gene, 44, 46 99m Tc-hexamethyl-propylene-amine-oxime (HMPAO), 30 hormones, 117, 131–132 Hughes’ classification, fistulas, 146 hypersensitivity reactions, AZA/6-MP treatment, 102 I IBD5 haplotypes, 57–59, 58 candidate genes, 59 variants, 83 IBD5, linkage studies, 61 IL1RN ( IL-1 receptor antagonist), 82–83 ileal Crohn’s disease ASCA association, 7 NOD2 and, 47–48, 52, 53 ileostomy, diverting Crohn’s perianal fistulas, 170–171 rectovaginal fistulas, 183 imaging methods Crohn’s perianal fistulas, 143–144 pediatric patients, 30–33 immune response IBD pathophysiologic mechanisms, 114–115 NOD2 and, 49–50 innate immunity, 52–53 knockout (nod2-/-) mice, 53–54 treatment targets, 114–115, 117 immunodulators, 114–115, 117 immunoglobulin (IgA) subtypes Crohn’s, 6 predicting disease activity/outcome, 39 predicting natural history/monitoring complications, 12 ulcerative colitis, 6 immunoglobulin (IgG) subtypes anti- α4 integrin, 128 Crohn’s disease vs ulcerative colitis, 6 predicting disease activity/outcome in pediatric CD, 39

199

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Index

immunostimulating factors, 117, 132–133 immunosuppressive therapy, 118 complex perianal fistulas, 181 patients on infliximab, 162 simple perianal fistulas, 179, 180 infection, fistulas formation, 153 inflammatory bowel disease (IBD) diagnostic blood tests, 2–8 genetic susceptibility, 84 monitoring disease activity, 8–11 pathophysiologic mechanisms, 114 pediatric see pediatric IBD predicating natural history/complications, 11–13 prevalence, 6 therapy response, predicating, 13–20 see also Crohn’s disease (CD); ulcerative colitis (UC) inflammatory cascades, 134 infliximab, 13, 15, 117–120, 134 antibodies against, 118 autoantibody formation, 120, 120 complex perianal fistulas, 181, 182, 183 fistulas, 161–163 immunosuppressive therapy, 162 mechanism of action, 117, 118 pregnancy and, 119 rectovaginal fistulas, 183 safety, 119 simple perianal fistulas, 179, 180 infusion reactions, infliximab, 119 inosine triphosphate pyrophosphatase gene(ITPA), AZA side effects, 100 integrins, antibodies against, 128–129, 130, 130 intercellular adhesion molecule 1 (ICAM-1), 80–81 antisense oligonucleotides against, 130–131 interferon(s), 133–134 see also specific types interferon-α, 133, 133 interferon-β, 133 interferon- γ, antibodies against, 124–125 interleukin-1 (IL-1), inhibition, 115–122 interleukin 1 receptor antagonist (IL1RN), 82–83 interleukin-2 receptor, antibodies against, 127 interleukin-6 receptor, antibodies against, 125–126 interleukin 11 (IL-11), 134 interleukin-12, antibodies against, 125 interleukin-12 (IL-12), antibodies against, 124 interleukin-16 (IL-16), 80–81 interleukin-18 (IL-18), antibodies against, 124 intestinal bacteria, DNA motifs, 136 intestinal pneumonitis, methotrexate, 109 iron deficiencies, monitoring, 12–13 ITPA gene (inosine triphosphate pyrophosphatase), AZA side effects, 101 K knockout mice, (nod2-/-), 53–54, 55 L lactoferrin, 19 laparotomy, rectovaginal fistulas, 183

200

Leu1007fs mutation, 47 leucine-rich repeat region (LRR), 49–50 leucopenia, 16, 18 6-MP/AZA treatment, 100–101 methotrexate, 109 leukocytes counts, 3, 9–10, 11 scintigraphy, 31 trafficking, inhibition, 116 linkage studies, 61–64, 63 genetic progress, 61–64 genome screens, 84 Listeria monocytogenes, 54 liver AZA/6-MP toxicity, 101–102 AZA/6-MP treatment monitoring, 103 biopsy, methotrexate, 109 complications, monitoring, 13 enzyme monitoring, 13 M macrophages activation, muramyl dipeptide (MDP), 54 migration inhibitory factor (MIF), 80 magnetic resonance imaging (MRI), perianal fistulas, 143, 144–145 drainage assessment, 150 scoring, 150, 152, 152–153, 175 malignancy see cancer MAP kinase inhibitors, 123 MCP1 (monocyte chemoattractant protein 1), 81–82 MDR1 (multi resistance gene 1), 65, 75 MDR1 (multi-resistance gene 1) polymorphisms, 75 6-mercaptopurine (6-MP), 15–19, 93–105 6-thioguanine nucleotide level measurement, 18–19 adverse effects, 157 dosage, 103 duration therapy, 104 efficacy in postoperative setting, 98–99 efficacy monitoring, 103–106 fistulas, 155–157, 156, 158–159 complex perianal, 181, 183 rectovaginal, 183 metabolism/mechanism of action, 93–94, 94 side effects, 99–102 studies, 95 ulcerative colitis, 96, 98 mesalamine, 99 methotrexate therapy, 93, 105–110 dosage, 108–109 duration of therapy, 109 fistulas, 161 infliximab and, 118 mechanism of action, 105 remission evaluation, 107–108 remission induction, 105–106, 106 remission maintenance, 106–107 side effects, 109 studies, 106 ulcerative colitis (UC), 107 6-methyl mercaptopurine (6-MMP), 16

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metronidazole (MTZ), 150 fistulas, 154–155 MIF (macrophage migration inhibitory factor), 80 Milligan and Morgan’s classification, fistulas, 146 monocyte chemoattractant protein 1 (MCP1), 81–82 mucosal immune system, treatment target, 114 multi resistance gene 1 (MDR1), 65, 75 polymorphisms, 75 muramyl dipeptide (MDP) macrophage activation, 54 NOD2 mutation, 49–50 stimulation, 51–52 mushroom catheter, perianal abscess, 165 myelosuppression, TMPT activity, 17 N natalizumab, 128–129 fistulas, 163 nausea, methotrexate, 109 neutropenia, 6-MP/AZA treatment, 100 NFKB1 (nuclear factor-κB p50 isoform), 76–77 NOD2 gene, 43–57 animal models studies, 53–57 double-mutant mice/human cell lines, 55–57 NOD2 knockout (nod2-/-) mice, 53–54, 55 Crohn’s disease phenotype and, 47–48 defensins, 53 functions, 49 genetic epidemiology, 43–47 ethnicity, 44, 45 mutations and risk, 47 regional heterogeneity, 44, 46 variants/mutations, 46–47 innate immunity, 52–53 linkage studies, 61 mechanism of action in CD, 49–53 defective response to bacteria, 49 LRR region, 49–50 MDP stimulation, 51–52 transfection experiments, 51 mutant, 50 mutations, 12 testing, 3 other diseases and, 57 as risk allele, 79 variants, 83 wild-type, 50, 55 noncutting setons perianal fistulas, 167, 170 complex, 182 rectovaginal fistulas, 174 North American Crohn’s Study Group Investigators multicenter study, 108–109 nuclear factor-κB inhibition, 123–124 p50 isoform (NFKB1), 76–77 nucleotide level measurement, 6-thioguanine, 18–19 nutritional deficiencies, monitoring, 12–13

O OCTN genes, 59–61 demography/phenotype, 60–61 transporters, 60 onercept, 122 P pANCA see perinuclear anti-neutrophil cytoplasmic antibody (pANCA) pancreas enzymes, AZA/6-MP treatment monitoring, 103 pancytopenia, 16 Paneth cells, 53 Parks’ classification, fistulas, 146–148, 147 pediatric IBD, 27–42 diagnostic algorithm, 37, 38 diagnostic imaging methods, 30–33 epidemiology, 27–30 diet, 29–30 environmental risk factors, 28–29 screening, 6 serological markers, 33–39 perianal abscess, 164–165 formation, 161 incision/drainage, 165 perianal disease activity index (PDAI), 149–150 perianal fistulas complex, 181–183 recommendations, 183 treatment options, 181–182 simple, 175 recommendations, 180 treatment options, 179 see also Crohn’s perianal fistulas perinuclear anti-neutrophil cytoplasmic antibody (pANCA) combined with ASCA, 7 Crohn’s disease vs ulcerative colitis, 34–35 differentiating between UC and CD, 6–8 pediatric patients, 34 predicting disease activity/outcome, 38–39 predicting natural history/monitoring complications, 12 response to infliximab, 13, 15 screening, 5–6 peroxisome proliferator-activated receptor γ (PPARG), 77 platelet count AZA/6-MP treatment monitoring, 103 diagnostic, 4 monitoring disease, 9–10 predicting natural history/monitoring complications, 11 pneumonitis, intestinal, 109 PPARG (peroxisome proliferator-activated receptor γ), 76–77 prednisone, 96 pregnancy, infliximab and, 119 proctectomy Crohn’s perianal fistulas, 171, 184 rectovaginal fistulas, 184 proinflammatory cytokines, inhibition, 115–124, 116 pulmonary embolism, infliximab, 119

201

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Index

purine analog therapy, 93–112 duration of treatment, 104 metabolite, measurement, 18 R R702W NOD2 mutation, 47 RDP58, 123 rectovaginal fistulas, 174, 183–184 6-mercaptopurine (6-MP), 183 surgical treatment, 176–177, 183–184 rectovaginal septal abscess, 174 repifermin, 131 S Salmonella typhi, 51–52 SAMP1/YitFc (SAMP1/Fc) mice, 77 sargramostim (GM-CSF), 132, 132–133, 134 scintigraphy, 30–31 evaluation, 31 leukocyte, 31 sclerosing cholangitis, 13 sepsis, 57 perianal abscess, 165 serological markers, 33–39 Crohn’s disease vs ulcerative colitis, 34–36 predicting disease activity/outcome, 37–39 screening, 5–6, 36 serum albumin (SA) level diagnostic, 2–3 monitoring disease, 10 serum sickness-like disease, infliximab, 119 serum transglutaminase (factor XIIIa), monitoring disease, 10 setons, 170 abscess prevention, 167 noncutting see noncutting setons placement, complex perianal fistulas, 183 small bowel contrast radiography (SBCR), 31 social implications, 113 splenocytes, 54 squamous cell carcinoma, 174 squamous epithelium, anus, 143 99m Tc-stannous colloid leukocyte scintigraphy, 31 steroids see corticosteroids stricturing complications, NOD2, 48 surgical therapy perianal fistulas complex, 182 Crohn’s see Crohn’s perianal fistulas rectovaginal fistulas, 176–177, 183–184 see also specific techniques T tacrolimus, fistulas, 160–161 complex perianal, 182 T-cell proliferation, inhibition, 116, 124–127 tenascin C, monitoring disease, 11 thalidomide, 123 T helper cell type 1 polarization, inhibition, 116, 124–127 6-thioguanine (6-TG) AZA/6-MP treatment monitoring, 103 measuring, 18–19 therapy, 102

202

thioguanine metabolite, 18 thiopurine methyl transferase (TPMT) enzyme, 16 genotype-based testing, 18 measuring low enzyme activity, 17–18 thrombocytopenia, 6-MP/AZA treatment, 100–101 toll-like receptors, 78–79 risk allele, 79 TPMT mutations 6-mercaptopurine/azathioprine treatment, 100 predicting response to therapy, 16 transfection experiments, NOD2 gene, 51 transmission disequilibrium test (TDT), 57–61 tumor necrosis factor (TNF) IBD pathophysiologic mechanisms, 114–115 inhibition, 115–122, 128 adalimumab, 121–122, 122, 162 adverse effects, 162–163 CDP571, 120–121 CDP870, 121, 121 fistula management, 161–163 infliximab see infliximab MAP kinase inhibitors, 123 mechanisms of action, 118 onercept, 122 RDP58, 123 thalidomide, 123 monitoring disease, 11 soluble receptors, 122 U ulcerative colitis (UC) anti-ompC, 35 CD14, 79 Crohn’s disease vs, 6–8, 34–36 incidence, 28 6-mercaptopurine (6-MP), 96, 98 methotrexate therapy, 107 pediatric see pediatric IBD risk factors, 29 toll-like receptors, 79 ulcers, penetrating, 153 ultrasonography, 32 upper endoscopy, 33 upper gastrointestinal endoscopy (UGIE), 30, 31 V vitamin B12 deficiencies, monitoring, 12–13 vitamin D deficiencies, monitoring, 12 W white blood cell count (WBCC) diagnosis, 3 monitoring disease, 9–10 predicting natural history/monitoring complications, 11 Z zinc deficiencies, monitoring, 12

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