FALK SYMPOSIUM 160
Edited by I. Ferkolj, Medical Centre Ljubljana, Slovenia P.R. Galle, University of Mainz, Germany A. Gangl, University of Vienna, Austria B. VUcelic, University Hospital Rebro, Zagreb, Croatia
Pathogenesis and Clinical Practice in Gastroenterology
FALK SYMPOSIUM 160
Pathogenesis and Clinical Practice in Gastroenterology Edited by
I. Ferkolj Department of Gastroenterology Medical Centre Ljubljana Slovenia
P.R. Galle University of Mainz Mainz Germany
A. Gangl University of Vienna Vienna Austria
B. Vucelic University Hospital Rebro Zagreb Croatia
Proceedings of the Falk Symposium 160 held in Portoroz, z Slovenia, June 15 16 2007
Library of Congress Cataloging-in-Publication Data is available.
ISBN-13 978-1-4020-8766-0
Published by Springer, PO Box 17, 3300 AA Dordrecht, The Netherlands Sold and distributed in North, Central and South America by Springer, 101 Philip Drive, Norwell, MA 02061 USA In all other countries, sold and distributed by Springer, PO Box 322, 3300 AH Dordrecht, The Netherlands
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Contents List of principal contributors
ix
List of chairpersons
xiii
Preface
xv
SECTION I: DYSPHAGIA AND REFLUX Chair: A Gangl, M Kozelj 1
2
3
4
Physiology of swallowing and anti-reflux mechanisms: anything new from a radiologist's view? C Kulinna-Cosentini
3
Oropharyngeal dysphagia, achalasia and other oesophageal motility problems: clinical relevance and management NJ Shaheen
7
Outcomes of different treatment approaches for gastrooesophageal reflux disease J Labenz
17
Barrett's oesophagus: screening, surveillance, treatment: are all questions answered? F Schreiber
28
SECTION II: HELICOBACTER PYLORI, NSAID, GASTRIC CANCER Chair: M Banic, B Tepes 5
Helicobacter pylori infection: diagnosis, treatment and risks of untreated infection K Monkemuller, H Neumann, LC Fry, P Malfertheiner
v
33
CONTENTS
SECTION III: INFLAMMATORY BOWEL DISEASE SECTION Chair: I Ferkolj, B Vucelic 6
7
8
Genetics, immunology and biomarkers in clinical practice: do they assist in clinical management? PL Lakatos
43
Standard therapy for ulcerative colitis and Crohn's disease: the ECCO guidelines A Ignjatovic, SPL Travis
76
Biologics, novel therapeutic alternatives in inflammatory bowel disease W Reinisch
90
SECTION IV: LOWER GASTROINTESTINAL TRACT (COELIAC DISEASE, INFECTION AND MALIGNANCY) Chair: A Gangl, B Stabuc 9
Recent advances in coeliac disease S Kolacek
10
Gastrointestinal manifestations of AIDS J Tomazic
110
11
Prevention and screening for colorectal cancer B Stabuc, S Plut
113
12
Lymphomas of the gastrointestinal tract E Zucca
118
97
SECTION V: CLINICAL CHALLENGES IN LIVER DISEASES Chair: C Trautwein, G Paumgartner 13
Do we still need liver biopsies? C Flechtenmacher, T Longerich, P Schirmacher
127
14
Inflammation and human cancer SP Hussain, XW Wang, CC Harris
137
SECTION VI: VIRAL HEPATITIS Chair: F Negro, S Zeuzem 15
New insights in the immunology of viral hepatitis B and C A Bertoletti vi
149
CONTENTS
16
New aspects of treatment of chronic hepatitis C B Kronenberger, S Zeuzem
161
SECTION VII: METABOLIC AND AUTOIMMUNE LIVER INJURY Chair: AJ Czaja, CP Day 17
Non-alcoholic steatohepatitis: metabolic syndrome of the liver CP Day
171
18
Autoimmune hepatitis S Kanzler
186
19
Immunology of primary biliary cirrhosis and primary sclerosing cholangitis A Lleo, C Selmi, P Invernizzi, M Podda, ME Gershwin
191
Alcoholic hepatitis H Tilg
208
20
SECTION VIII: COMPLICATIONS OF CIRRHOSIS Chair: M Colombo, M Schuchmann 21
Hepatorenal syndrome in cirrhosis AL Gerbes
221
22
Variceal bleeding T Sauerbruch, J Trebicka
233
23
Surveillance and prevention of hepatocellular carcinoma M Colombo
239
24
Algorithms of diagnosis and options of hepatocellular carcinoma therapy A Forner, JM Llovet
252
Surgical therapy of liver cancer: resection and transplantation G Otto, M Hoppe-Lotichius, M Heise
266
Index
275
25
vii
List of principal contributors PR Galle Innere Medizin I Klinikum der Universitat Langenbeckstr. 1 55131 Mainz Germany
A Bertoletti Singapore Institute for Clinical Sciences Brenner Centre for Molecular Medicine Agency of Science Technology and Research (A*STAR) 30 Medical Drive Singapore 117609
AL Gerbes Department of Medicine II Klinikum der Universitat Munchen-Grosshadern Marchioninistr. 15 81377 Munchen Germany
M Colombo First Division of Gastroenterology Fondazione IRCCS Maggiore Hospital Policlinico, Mangiagalli and Regina Elena University of Milan Via F. Sforza 35 20122 Milan Italy
ME Gershwin Division of Rheumatology, Allergy and Clinical Immunology University of California at Davis School of Medicine Genome and Biomedical Sciences Facility 451 E Health Sciences Drive, Suite 6510 Davis, CA 95616 USA
CP Day School of Clinical Medical Sciences Floor 4, William Leech Building The Medical School Framlington Place Newcastle upon Tyne NE2 4HH United Kingdom
CC Harris Laboratory of Human Carcinogenesis National Cancer Institute National Institutes of Health 37 Convent Drive Bethesda, MD 20892 USA
A Forner Barcelona Clinic Liver Cancer (BCLC) Group Liver Unit Hospital Clinic IDIBAPS CIBERehd University Barcelona Villarroel 170 08036 Barcelona Spain
S Kanzler Department of Gastroenterology and Hemato-Oncology Leopoldina Hospital Gustav Adolf Strasse 8 97422 Schweinfurt Germany ix
LIST OF PRINCIPAL CONTRIBUTORS
s Kolacek
WRelnlsch
University Children's Hospital Zagreb Division of Paediatrics Referral Centre for Paediatric Gastroenterology and Nutrition Klaiceva 16 10000 Zagreb Croatia
University Klinik for Internal Medicine Ill Department of Gastroenterology and Hepatology Wahringer Gurtel18-20 1090 Vienna Austria T Sauerbruch
Medizinische Klinik und Poliklinik I University of Bonn Sigmund-Freud-Strasse 25 53105 Bonn Germany
C Kulinna-Cosenlini
Medizinische Universitat Wien Universitatsklinik fur Radiodiagnostik Wahringer Gurtel18-20 1090 Wien Austria
P Schirmacher
Institute of Pathology University Hospital lm Neuenheimer Feld 220 69120 Heidelberg Germany
J Labenz
Department of Internal Medicine and Gastroenterology Jung-Stilling Hospital Academic Teaching Hospital of the University of Bonn Wichernstr. 40 57074 Siegen Germany
FSchreiber
Internal Department Medical University of Graz Division of Gastroenterology and Hepatology Auenbruggerplatz 15 8036 Graz Austria
PL Lakatos
1st Department of Medicine Semmelweis University Koranyi S 2A 1083 Budapest Hungary
NJ Shaheen
Division of Digestive Diseases and Nutrition University of North Carolina at Chapel Hill, CB #7080 Chapel Hill, NC 27599-7080 USA
K Monkemuller
Division of Gastroenterology, Hepatology and Infectious Diseases Otto-von-Guericke University Universitatsklinikum Magdeburg Leipziger Strasse 44 39116 Magdeburg Germany
B Stabuc
Department of Gastroenterology University Medical Center Ljubljana 1525 Ljubljana Slovenia H Tllg
GOHo Department of Transplantation and Hepatobi liopancreatic Surgery University of Mainz Langenbeckstr. 1 55101 Mainz Germany
Department of Medicine Division of Gastroenterology and Hepatology Christian Doppler Research Laboratory for Gut Inflammation University Hospital lnnsbruck Anichstrasse 35 6020 lnnsbruck Austria X
J Tomazic Medical Centre Ljubljana Department of Infectious Disease Japljeva 1525 Ljubljana Slovenia
E Zucca IOSI - Oncology Institute of Southern Switzerland Ospedale San Giovanni 6500 Bellinzona Switzerland
SPL Travis Gastroenterology Unit John Radcliffe Hospital Oxford OX3 9DU United Kingdom
S Zeuzem Medizinische Klinik I Theodor-Stern-Kai 7 60590 Frankfurt am Main Germany
xi
List of chairpersons A Gangl Medizinische Universitat Wien Gastroenterologie/Hepatologie Wahringer Gurtel 18-20 1090 Wien Austria
M Banic Clinical Hospital `Dubrava' Department of Medicine Division of Gastroenterology Av. Gojka Suska 6 10000 Zagreb Croatia
M Kozelj Medical Centre Ljubljana Gastroenterologijo Japljeva 2 1525 Ljubljana Slovenia
M Colombo First Division of Gastroenterology Fondazione IRCCS Maggiore Hospital Policlinico, Mangiagalli and Regina Elena University of Milan Via F. Sforza 35 20122 Milan Italy
F Negro Hoªpital Cantonal Gastro-enterologie et Hepatologie Rue Micheli-du-Crest 24 1211 Geneve Switzerland
AJ Czaja Mayo Clinic Department of Internal Medicine Gastroenterology Unit, Box 0001 200 First Street SW Rochester, MN 55905 USA
G Paumgartner Innere Medizin II Klinikum der Universita«t Munchen-Grosshadern Marchioninistrasse 15 81377 Munchen Germany
CP Day School of Clinical Medical Sciences Floor 4, William Leech Building The Medical School Framlington Place Newcastle upon Tyne NE2 4HH United Kingdom
M Schuchmann Innere Medizin I Klinikum der Universitat Langenbeckstr. 1 55131 Mainz Germany
I Ferkolj KOGE Department of Gastroenterology Japljeva 2 1525 Ljubljana Slovenia
B Sítabuc Department of Gastroenterology University Medical Center Ljubljana 1525 Ljubljana Slovenia
xiii
LIST OF CHAIRPERSONS
B Vucelic University Hospital Rebro Division of Gastroenterology Department of Medicine Kispaticeva 12 10000 Zagreb Croatia
B Tepes Medical Centre Rogaska Zdraviliski trg 9 3250 Rogaska Slatina Slovenia C Trautwein Medical Clinic III RWTH University Hospital Pauwelsstrasse 30 52074 Aachen Germany
S Zeuzem Medizinische Klinik I Theodor-Stern-Kai 7 60590 Frankfurt am Main Germany
xiv
Preface The translation of new molecular understanding of disease into clinical practice in gastroenterology is a particular challenge. The Falk Symposium 160 in Portoroz, Slovenia, provided a stimulating framework to bridge discussions from bench to bedside. The unifying theme of the Symposium, `Pathogenesis and Clinical Practice in Gastroenterology', was the growing understanding of in£ammation as a driving force in chronic disease leading to disability and malignancy. Aspects of early detection endoscopically or via molecular markers were covered as well as in-depth discussions of the validation of new ¢ndings in clinical practice. Whilst on a molecular basis gastroenterologists and hepatologists try to shed light on the same intracellular pathways, clinical implications such as surveillance of cancer in IBD or chronic hepatitis with cirrhosis have to be tailored to speci¢c needs. New therapeutic agents approved or on the horizon of early clinical studies will dramatically change the options of patients with chronic diseases such as Crohn's disease, viral hepatitis or hepatocellular carcinoma. The present book may not be able to convey the Mediterranean atmosphere of the marvellous location, but will summarize the intriguing ideas exchanged to bring forward medicine in the ¢eld of gastroenterology and hepatology. I. Ferkolj, Ljublana P. R. Galle, Mainz A. Gangl, Vienna B. Vucelic, Zagreb
xv
Section I Dysphagia and reflux Chair: A GANGL and M KOZELJ
1 Physiology of swallowing and antireflux mechanisms: anything new from a radiologist's view? C. KULINNA-COSENTINI
INTRODUCTION In the evaluation of oesophageal function the barium swallow/video£uoroscopy procedure competes very favourably with other modalities, such as manometry, endoscopy or 24-h pH-metry1,2. Furthermore the radiological swallow technique is easier to interpret and does not require additional expensive equipment or specialized training. Video£uoroscopy has become a widely accepted method, which allows precise assessment and documentation of oral, pharyngeal, and oesophageal phases of swallowing. The following short overview will pr imar ily cove r the use of video£uoroscopy in evaluating patients with pharyngeal and oesophageal diseases. Our purpose is not to provide a comprehensive discussion of all aspects of diseases but to emphasize the most important points through the normal physiology of oesophageal swallowing, and to review pathological signs of frequent diseases. The use of newer imaging techniques, like magnetic resonance imaging (MRI), in the evaluation of oesophageal diseases will be discussed.
INDICATIONS The sensitivity and speci¢city of video£uoroscopy varies greatly depending on suspected conditions, so indications should be chosen carefully: (a) globus sensation, (b) dysphagia, (c) aspiration, (d) non-cardiac chest pain, (e) suspected gastro-oesophageal re£ux disease (GORD) or motility disorders.
TECHNIQUE The technique described in this section should detect anatomical and functional abnormalities of swallowing. Swallowing is a dynamic process, so 3
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
all studies should include storage of dynamic assessment, either by videotape recording (S-VHS format) or by frame-grabbing. The use of videotaping is not only for recording of functional units of swallowing, but also for education of patients when treatment options are discussed and the patient subsequently trained. The recorder should have an automatic on/o¡ function coupled to the £uoroscopy pedal, which means that the patient does not have to swallow any extra barium for the functional evaluation. Another mandatory feature is the jog-shuttle function which allows frame-by-frame analysis. We use 25 frames/s (1 3 Lp/mm) for a standard examination.
Standard examination: example dysphagia 1. Lateral position centred on mouth, soft palate and pharynx; have patient swallow single boluses of 15 ml high-density (HD) barium. 2. Lateral position centred on the pharyngo-oesophageal (PE) sphincter: boluses of 30 ml HD ? function of PE sphincter and PE segment. 3. Anteroposterior (AP) view of pharynx: bolus of 15 ml HD ? tongue and pharynx. 4. AP or slightly oblique view, upright position passage of whole oesophagus: after a swallow of 10 ml low-density (LD) barium have the patient open the mouth to inhibit additional swallow that would superimpose the peristaltic wave (`single swallow technique'). 5. Prone right anterior oblique position: three single boluses of 10 ml LD ? peristaltic wave. 6. Prone right anterior oblique position and in supine slightly left posterior oblique position: have the patient turn from a supine to right posterior oblique and right lateral position ? spontaneous re£ux. Valsalva manoeuvre ? induced re£ux.
NORMAL SWALLOWING PATTERN Swallowing can be divided into three stages: oral, pharyngeal and oesophageal phases. The voluntary part of the oral phase includes components such as oral ¢lling, chewing, mixing with saliva, loading the food on the tongue and voluntary shift of food towards the posterior section of the tongue. The involuntary part of the oral phase involves glossopalatal expulsion and clearing of the food bolus into the pharynx. As soon as the food enters the pharynx pharyngeal contraction starts, which means the pharyngeal phase is involuntary. This includes nasopharyngeal closure, hyoid bone upward and anterior movement, laryngeal elevation, closure of the airways including the vocal cords and pharyngeal peristalsis including pharyngeal constrictor activity. The involuntary oesophageal phase needs 10 times longer than the oropharyngeal phase (together 1 s)3, and includes transportation through the oesophagus, and opening and closure of the lower oesophageal sphincter (LOS). 4
SWALLOWING AND ANTI-REFLUX MECHANISMS
SEVEN FUNCTIONAL UNITS (SEE FIGURE BELOW) Function unit
Patholo ical function
1. Oral cavity Control of bolus, transfer of the bolus onto the back of the tongue, then backwards sweeping movement of the tongue for transportation of bolus into pharynx
·Pathological tongue movement -Impaired bolus control ·Leaking ·Weakness of the tongue ·Tumour, post-op defect
2. Soft palate The soft palate abuts the posterior portion of the tongue to prevent leakage from the back of the mouth into the oral cavil or naso ha nx 3. Epiglottis Epiglottis tilted down to cover the entrance to the laryngeal aditus
·Incomplete elevation ·Nasal regurgitation ·Leaking ·Tumour. post-op defect
·lncomplete/assymetric tilt ·Tumour, post-op defect
4. Hyoid/larynx Cranial and ventral movement of hyoid and larynx to open the way for bolus and to close the airwa s 5. Pharyngeal constrictors Activity of p.c. is seen as a peristaltic wave as posterior indentation during bolus passage in lateral view.
·Insufficient elevation/movement ·Penetration ·Aspiration ·Tumour, ost-o defect ·Delayed swallowing reflex ·Retention/weakness ·Pouches ·Diverticulum (Zenker, Kilian-Jamieson) ·Tumour, post-op defect
6. PE Segment Relaxation of the pharyngooesophageal sphincter during swallowing, closed between swallows; progressive forward movement of the posterior pharyngeal wall
·Cricopharyngeal bar ·Gaping ·Web, stricture. tumour
7. Oesophagus Transportation of the bolus through the oesophagus in a maximum of 10 s; peristaltic wave; opening and closure of the LOS
·Delayed transport (support level, proximal escape) ·Non-propulsive contractions ·Hernia. Reflux ·Web, stricture, diverticulum, tumour, post-op complications
5
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
DYNAMIC MR SWALLOWING The projectional nature of video£uoroscopy in conjunction with limited softtissue contrast has generated growing interest in new techniques. Dynamic MRI has several advantages: lack of ionizing radiation, variable slice orientation without changing patient's position, and excellent soft-tissue contrast. Recently published reports have described the promising use of kinematic MR £uoroscopy on oropharyngeal swallowing4,5. In the present study we evaluated the feasibility of dynamic MR swallowing focused on oesophageal swallow and the gastro-oesophageal junction. In 28 patients we compared our results with the results of manometry and 24-h pH-metry and found a correlation of 85% in diagnosis of GORD and of 75% in motility disorders6. There are still some limitations in MR swallowing such as lower temporal resolution (one image per second) than videocinematography, and problems in depiction of a curved oesophagus7, but preliminary studies have shown dynamic MRI to be a suitable tool for evaluation of patients with motor disorders or re£ux, and warrant further studies
References 1. 2. 3. 4. 5. 6. 7.
Hewson EG, Ott DJ, Dalton CB, Chen YM, Wu WC, Richter JE. Manometry and radiology. Complementary studies in the assessment of esophageal motility disorders. Gastroenterology. 1990;98:626 32. Fuller L, Huprich JE, Theisen J JA et al. Abnormal esophageal body function: radiographic manometric correlation. Ann Surg. 1999;65:911 14. Schima W, Stacher G, Pokieser P et al. Esophageal motor disorders: video£uoroscopic and manometric evaluation prospective study in 88 symptomatic patients. Radiology. 1992;185:487 91. Barkhausen J, Goven M, von Winterfeld F et al. Visualization of swallowing using real time TrueFISP MR £uoroscopy. Eur Radiol. 2002;12:129 33. Hartl DM, Albiter M, Kolb F, Luboinski B, Sigal R. Morphologic parameters of normal swallowing events using single shot fast spin echo dynamic MRI. Dysphagia. 2003;18:255 62. Kulinna Cosentini C, Schima W, Lenglinger J, Bischof G, Cosentini EP. Comparison of dynamic MR swallowing with manometry and ph metry in dysphagic patients ¢rst experience. ECR. 2005 (Abstract). Kulinna Cosentini C, Schima W, Cosentini EP. Dynamic MR imaging of the gastroesophageal junction in healthy volunteers during bolus passage. J Magn Reson Imaging. 2007;25:749 54.
6
2 Oropharyngeal dysphagia, achalasia, and other oesophageal motility problems: clinical relevance and management N. J. SHAHEEN
INTRODUCTION Dysphagia is among the most common gastrointestinal symptoms in the elderly, with a population prevalence in the elderly in excess of 10%. While mechanical obstruction accounts for some portion of this burden, the majority of these subjects do not have strictures or cancer. Motility disorders account for a large portion of the remainder of these patients. The aim of this chapter is to describe the impact and clinical management of oropharyngeal dysphagia and oesophageal dysmotilities. We will concentrate on the epidemiology, common clinical presentations, diagnostic work-up and treatment of oropharyngeal dysphagia, and the most common oesophageal dysmotilities, including achalasia, di¡use oesophageal spasm and ine¡ective oesophageal motility.
OROPHARYNGEAL DYSPHAGIA Oropharyngeal dysphagia, also called transfer dysphagia, may result from diseases of the mouth, pharynx, and upper oesophagus (including the upper oesophageal sphincter, or UOS). A variety of neurological, myopathic and structural disorders may cause oropharyngeal dysphagia, including stroke, multiple sclerosis, Parkinson's disease, cancer and Zenker's diverticulum (Table 1). Oropharyngeal dysphagia is prevalent in the general population1,2, but is especially common in certain at-risk populations, such as the elderly, the mentally ill, and those who are status post cerebrovascular accident (CVA)3^5. In these subpopulations the point prevalence of oropharyngeal dysphagia may exceed 30%. 7
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY Table 1
Causes of oropharyngeal dysphagia
Neurological Cerebrovascular accident Multiple sclerosis Parkinson's disease Alzheimer's disease Tumours Head trauma Cerebral palsy Huntington's disease Guillian Barre disease Poliomyelitis Tardive dyskinesia Amyotrophic lateral sclerosis Friedrich's ataxia
Iatrogenic Medication induced Anticholinergics Phenthiazines Antihistamines Chemotherapy Post surgical changes Post radiation changes Infectious Botulism Syphilis Lyme disease Diphtheria
Myopathic Myasthenia gravis Polymyositis Dermatomyositis Myotonic dystrophy Oculopharyngeal dystrophy
Metabolic Thyrotoxicosis Amyloid Cushing's disease
Structural lesions Zenker's diverticulum Cricopharyngeal bar Carcinoma of the upper aerodigestive tract Extrinsic compression from thyroid or spine Oesophageal webs Congenital abnormalities
The patient with oropharyngeal dysphagia almost always points to the neck when describing symptoms. The feeling of dysphagia occurs immediately after swallowing. In addition to the feeling that food does not move in an orderly fashion down the oesophagus, other commonly associated symptoms include coughing, the feeling that one is su¡ocating or choking, drooling, and the need to spit out or regurgitate the food. Evaluation of the patient with oropharyngeal dysphagia often necessitates a multi-disciplinary approach, involving gastroenterologists, neurologists, speech pathologists, otolaryngologists, and radiologists. Appropriate work-up of the subject with oropharyngeal dysphagia begins with a thorough history and physical examination. Special attention is paid to historical elements suggesting a potential aetiology for dysphagia, including cerebrovascular disease, muscular disorders, and history of degenerative neurological processes. On examination, evidence of neurological disease when examining the cranial nerves, as well as symmetry and function of the palate, is assessed. Laboratory analysis may be performed based on this evaluation, assessing for metabolic and neurological causes of dysphagia. Additionally, computed tomography or magnetic resonance imaging of the head may be considered if focal neurological de¢cits are isolated. Endoscopy may be considered, either 8
OESOPHAGEAL MOTILITY PROBLEMS
transnasal or oropharyngeal, if elements of the history suggest a likelihood of structural abnormality. The next step in evaluation may include either a modi¢ed barium swallow, or a ¢breoptic endoscopic evaluation of swallowing (FEES). Both of these examinations allow visualization of functional elements of swallowing. The FEES examination allows for real-time endoscopic visualization of the surface contours of the pharynx during swallowing, and is a sensitive test to identify isolated dysfunction of muscles or vocal cord apposition6,7. It does not require radiation, and can be performed at the bedside, making it a useful test in the subject with mobility or positioning issues. It does not demonstrate submucosal structures, such as cervical osteophytes, in the way that a modi¢ed barium swallow can. Additionally, FEES requires the dedication of a trained and interested speech pathologist to be optimally utilized8,9. Treatment of oropharyngeal dysphagia rests on addressing the underlying disease process. If a tumour or metabolic disorder is present, medical and surgical therapies are employed to decrease functional compromise because of these conditions. In rare cases of cricopharyngeus hypertrophy, myotomy may cause resolution of symptoms10,11. In most cases, where cerebrovascular accident or neurodegenerative disorders are the aetiology of the symptoms, the cornerstones of therapy are rehabilitation and retraining by speech pathologists. This may require multiple sessions, and special techniques for eating, such as chin tuck, head rotation to the a¡ected side, or head tilt to the stronger side, are employed.
ACHALASIA Achalasia is a disease of unknown aetiology that is manifest clinically by failure of relaxation of the lower oesophageal sphincter in response to deglutition, in conjunction with the absence of primary peristalsis. Histologically, there is neuronal degeneration of the myenteric plexus 12 . This degeneration preferentially a¡ects the inhibitory, nitric oxide-producing cells, which are necessary for physiological relaxation of the lower oesophageal sphincter (LOS)13. The opposing cholinergic neurons responsible for tonic contraction are relatively spared, leading to an increased baseline tone, and inability to relax in response to swallows14. Achalasia appears to a¡ect approximately 0.5 1 in 100 000 people, with substantial variation among cultures, which may in part be related to di¡erences in availability of medical resources to make the diagnosis15,16. Men and women are approximately equally a¡ected. It is a disease generally with adult onset, and there is a broad range of reported onsets between ages 25 and 70, with onset prior to puberty uncommon. Common presenting symptoms are dysphagia and chest pain. Although the dysphagia is most commonly thought of as the cardinal feature, chest pain is surprisingly common and a¡ects the majority of patients17. Classic achalasia dysphagia is to both solids and liquids; however, early forms of the disease may present with only solid dysphagia. A gastro-oesophageal re£ux disease (GORD)-like constellation of symptoms, with substernal chest burning, is 9
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
also common18. Weight loss prior to diagnosis may be substantial, as much as 20% of the total body mass. Diagnosis of achalasia relies primarily on radiography and manometry. The classic `bird's beak' radiograph (Figure 1), composed of a hypertonic LOS and a dilated oesophageal body, often with retained food contents, is a common ¢nding in late-stage disease, and is highly speci¢c for the condition in the appropriate clinical setting. Early-stage disease and vigorous achalasia
Figure 1
Achalasia barium oesophogram
10
OESOPHAGEAL MOTILITY PROBLEMS
(achalasia with signi¢cant residual oesophageal body tone and simultaneous contractions of 460 mmHg) are less likely to show this radiographic appearance. Oesophageal manometry con¢rms the diagnosis suspected by clinical presentation and radiography. Classic manometric tracings exhibit an elevated resting LOS tone (usually 445 mmHg), and incomplete relaxation on swallowing (residual pressure 48 mmHg). Additionally, the body is usually aperistaltic, with low-amplitude simultaneous contraction in response to swallows. However, as noted above, higher-amplitude simultaneous waves may occasionally be seen in the `vigorous achalasia' variant. The primary role for endoscopy in patients with achalasia is to rule out secondary causes of achalasia, such as a tumour at the gastro-oesophageal junction. Retained food contents are often visualized on this examination. The LOS is sometimes resistant to passage of the endoscope on this examination, but this ¢nding is variable, and the clinician should not be dissuaded from the diagnosis based on the absence of this ¢nding. The appropriate management of achalasia is controversial. Medical therapy with nitrates and calcium channel blockers is inadequate to bring durable, highquality sustained relief to most patients. The ¢nding that substantial relief of dysphagia could be obtained in response to injections of intrasphincteric injections of botulinum toxin (Botox) has been tempered by the observation that such relief is short-lived, and that repeat injections are necessary, making this a sub-optimal method for long-term treatment19,20. Other options include graded pneumatic dilation and surgical myotomy, often accompanied by a surgical anti-re£ux procedure21. Advocates of each approach have arguments in their favour as initial therapy for achalasia. Advocates for repeat Botox injections note the relatively non-invasive nature of their approach, and the ease of administration. Proponents of pneumatic dilation note good 1-year response rates, and the same-day, outpatient nature of the procedure. Surgical proponents point to literature suggesting the best long-term outcomes. The comparative data on the three approaches are scant. The most voluminous literature consists of data comparing pneumatic dilation to Botox injections. These studies, on balance, suggest that pneumatic dilation is a more durable, de¢nitive therapy. Bansal et al. randomized 34 patients to receive either 80 IU of Botox divided into four injections in each of four quadrants of the LOS, or pneumatic balloon dilation with a Witzel balloon22. At 1 year, only 38% of those treated with Botox were in clinical remission, compared to 89% in the pneumatic dilation group. Ghoshal and colleagues found that initial response to Botox was similar to that of pneumatic dilation, but that the Botox e¡ect underwent a relatively quick decay, such that at 1 year there was a signi¢cantly di¡erent rate of relapse between the dilation and Botox groups23. A third study randomizing 40 patients to either Botox or pneumatic dilation also found that those undergoing treatment with pneumatic dilation were more likely to remain in clinical remission than those receiving Botox therapy (53% vs 15%, p50.01). When relapsing subjects were treated a second time with the same modality, 1-year remission rates were even more striking 100% of those in the pneumatic dilation arm demonstrated clinical remission, as opposed to 60% in the Botox arm (p50.01)24. Other work also suggests that Botox 11
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
provided good early sympton relief, with the need for subsequent re-treatment to retain the e¡ect25. Considerably less data are available on the perhaps more interesting and pertinent question for most subjects with achalasia: What is the most e¤cacious and desirable treatment when comparing Heller myotomy to pneumatic dilation? In a seminal randomized controlled trial, Csendes et al. compared pneumatic dilation with a Mosher bag to open anterior myotomy26. Patients undergoing surgery had a more durable response to therapy symptomatically, and 50% of patients undergoing pneumatic dilation subsequently required either a second dilation or surgery. Late follow-up of these patients demonstrated that those undergoing pneumatic dilation also had a greater incidence of late failure requiring further therapy27. There was a 95% success rate in the surgical arm at relieving symptoms at a median follow-up of 62 months, versus a 65% success rate in the dilation group at a median of 58 months. The applicability of these interesting results in 2007, given the changes in both the surgical and endoscopic approaches to the disease, is unclear. More recently, Felix and colleagues randomized 40 patients with achalasia to either hydrostatic balloon dilation or open abdominal oesophagocardiomyotomy with an oesophagofundopexy28. They found that subjects in both groups had an excellent symptomatic response to their assigned therapy. Additionally, radiographic clearance of contrast on barium study was similar in the groups. Manometric data suggested that the surgical myotomy group had a more profound drop in resting LOS pressure, and pH data demonstrated a somewhat higher degree of acid re£ux on 24-h pH monitoring in the dilation group than in the surgery group, although both groups had acid exposures within normal limits. Based on these data the authors concluded that both modalities of therapy were good choices for treatment of patients with achalasia, but due to the superior manometric outcomes and the decrease in acid re£ux associated with the surgical procedure, this approach might be preferred in the surgical candidate. Because this study consisted of subjects with achalasia due to Chagas disease, and because the surgical approach was di¡erent than that generally used in most centres, its generalizability is unclear. Until a de¢nitive trial of pneumatic dilation versus surgery is available, it is likely that local expertise, patient preference, and physician biases will in large part decide initial therapy of the disease. In many centres initial therapy is performed with pneumatic dilation, with surgical rescue used for nonresponders to therapy. In our centre the good surgical candidate is o¡ered surgical consultation. After hearing the pros and cons of each approach, most patients opt for laparoscopic Heller myotomy with partial fundoplication. Given the excellent short- and mid-term results with this approach, as well as the low morbidity associated with the laparoscopic approach, this seems to be a rational approach to therapy. In our centre Botox injections are generally reserved for two groups of patients: those with a limited life expectancy, and those with a clinical picture and manometric ¢ndings indeterminant for, but suggestive of, achalasia. In the elderly patient with multiple comorbidities two or three rounds of Botox injection may be all that is necessary to allow patients to live out the full extent of their life expectancy. In the subject with somewhat con£icting clinical 12
OESOPHAGEAL MOTILITY PROBLEMS
¢ndings (for instance, the subject with a clinical picture and barium examination suggestive of achalasia, but a manometric study with ¢ndings not supportive of the diagnosis), Botox administration may be used as a diagnostic test. Because the mechanism of action of Botox is similar to the e¡ect seen with surgical myotomy or pneumatic dilation, a good response to Botox injections is predictive of a good response to the more durable therapies. Therefore, it is common for such a patient to undergo an initial round of Botox injections, then to have subsequent therapy with either surgery or pneumatic dilation. Despite initial concerns to the contrary, data suggest that outcomes of patients who undergo surgery after Botox injection are excellent and similar to those of Botox-naive patients.
DIFFUSE OESOPHAGEAL SPASM Di¡use oesophageal spasm (DOS) is de¢ned as appropriate symptomatology with manometric ¢ndings of 20% or more of swallows eliciting simultaneous contractions. Other common manometric ¢ndings are repetitive contractions in response to a swallow, intermittent peristalsis, and prolonged contractions of greater than 6 s of duration. Data on the aetiology of the condition are scant, but it is thought that this may represent a similar pathophysiology to achalasia in some respects, with a relative de¢ciency of myenteric plexus neurons elaborating nitric oxide29,30. The primary clinical manifestation of DOS is chest pain. This pain is generally intermittent, intense in nature, and may be associated with the ingestion of very hot or cold liquids. A less common manifestation is dysphagia. Although DOS is commonly invoked in the di¡erential of subjects with functional chest pain not of cardiac origin, it is actually a relatively rare cause of this symptom. In one study only about 3% of subjects with non-cardiac chest pain met criteria for oesophageal spasm as an aetiology of their pain31. The work-up of this condition usually commences with an upper endoscopy to rule out in£ammation or structural causes of the symptoms. This is followed by oesophageal manometry. While most centres perform stationary manometry, 24-h ambulatory manometry may increase the yield of the examination. As noted above, manometry displays at least 20% of swallows eliciting simultaneous contractions. In addition, waveforms are often tripeaked or more, a distinctly unusual ¢nding in normal individuals. Contraction of duration of 46 s is often seen, but not necessary for diagnosis. The primary therapy in DOS is medical. Nitrates, anticholinergic agents, peppermint oil and long-acting calcium channel blockers have all been used in uncontrolled trials, and may provide enough symptomatic relief to avoid more invasive therapy32^34. Centrally acting agents, such as the tricyclic agents trazadone and imipramine, have been used successfully in these patients, with substantial relief of symptoms35. An interesting report of nine patients treated with Sildena¢l, the phosphodiesterase inhibitor, demonstrated a decrease in oesophageal tone in this patient population36. If medical therapy fails, Botox injections may be tried. An uncontrolled trial of 29 patients with spastic oesophageal motility disorders treated with 100 IU 13
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
of intrasphincteric Botox demonstrated at least 50% reduction of pain in 70% of patients; about half had complete resolution of symptoms37. In our centre we have also treated DOS patients by performing injections of 200 IU of Botox spiraled up the distal one-third of the oesophagus in 20 IU increments with good e¡ect. Surgical myotomy is reserved for subjects with disabling symptoms that are unresponsive to all of the above therapies38,39. Given the need for a long myotomy, a thoracic, not laparoscopic, approach is advisable. This approach should be avoided as long as possible, given the overall good prognosis of this condition and the uncertain response to therapy. A passing mention of `nutcracker oesophagus' should be made. This condition, de¢ned as chest pain and manometric ¢ndings of peristaltic waves in the distal oesophagus consistently in excess of 180 mmHg, may be found in some patients complaining of chest pain or dysphagia. However, the manometric ¢ndings are so poorly predictive of the disease that most clinicians do not use this disease categorization.
INEFFECTIVE OESOPHAGEAL MOTILITY Ine¡ective oesophageal motility is de¢ned manometrically as 30% or greater non-transmitted waves in the distal oesophagus with waves generally of 30 mmHg in amplitude or less. This condition may be seen as an idiopathic state, but may also be seen in association with other conditions, such as severe longstanding oesophageal re£ux, amyloid or systemic sclerosis40,41. The underlying condition often explains the pathophysiology of the disorder. For instance, in subjects with severe erosive GORD, distal smooth muscle is probably damaged by chronic re£ux and replaced by collagen, leading to poor contractility. Similarly, in in¢ltrative diseases such as amyloid and systemic sclerosis, smooth muscle is replaced, leading to inability to sustain distal peristalsis. Treatment of this condition is primarily with promotility agents. Metoclopramide and erythromycin are often used; if erythromycin is to be used, drug holidays must be utilized to avoid the tachyphylaxis associated with this agent. Tegaserod has oesophageal promotility e¡ects, but was withdrawn from the market in March 2007 because of concerns of cardiac toxicity. A recent report also demonstrated preliminary data on the e¤cacy of bethanachol as a treatment agent42. Future agents that take advantage of the promotility e¡ects of serotonin may become available in the future.
CONCLUSIONS Dysphagia is among the most common gastrointestinal complaints seen in the general population, and familiarity with the diagnosis and management of this condition is important for the general internist and essential for the gastroenterologist. This chapter has attempted to outline the aetiology, clinical presentation, diagnostic work-up and management of common causes of dysphagia. Oropharyngeal dysphagia must be distinguished from thoracic dysphagia, as the work-up and management are distinctly di¡erent. Modi¢ed 14
OESOPHAGEAL MOTILITY PROBLEMS
barium swallow and FEES study have great utility in these patients, and the primary therapy is rehabilitative for most patients. Among the idiopathic oesophageal motility disorders, achalasia, di¡use oesophageal spasm and ine¡ective oesophageal motility present distinct diagnostic and therapeutic challenges. The appropriate management of achalasia is debated, and patient comorbidities, the local expertise of the centre and patient preferences all weigh in the decision as to what therapy to pursue. Conservative medical therapy of DOS is desirable. Therapy of ine¡ective oesophageal motility rests on promotility agents, and will probably evolve over the next 5 10 years as new agents become available.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Drossman DA, Li Z, Andruzzi E et al. U.S. householder survey of functional gastrointestinal disorders. Prevalence, sociodemography, and health impact. Dig Dis Sci. 199338:1569 80. Achem SR, Devault KR. Dysphagia in aging. J Clin Gastroenterol. 2005;39:357 71. Lin LC, Wu SC, Chen HS, Wang TG, Chen MY. Prevalence of impaired swallowing in institutionalized older people in taiwan. J Am Geriatr Soc. 2002;50:1118 23. Davis LA, Thompson SS. Characteristics of dysphagia in elderly patients requiring mechanical ventilation. Dysphagia. 2004;19:7 14. Pilotto A, Di Mario F, Malfertheiner P, Valerio G, Naccarato R. Upper gastrointestinal diseases in the elderly: report of a meeting held at Vicenza, Italy, on 20 March 1998. Eur J Gastroenterol Hepatol. 1999;11:801 8. Leder SB, Acton LM, Lisitano HL, Murray JT. Fiberoptic endoscopic evaluation of swallowing (FEES) with and without blue dyed food. Dysphagia. 2005;20:157 62. Hiss SG, Postma GN. Fiberoptic endoscopic evaluation of swallowing. Laryngoscope. 2003;113:1386 93. Langmore SE. Evaluation of oropharyngeal dysphagia: which diagnostic tool is superior? Curr Opin Otolaryngol Head Neck Surg. 2003;11:485 9. Kidder TM, Langmore SE, Martin BJ. Indications and techniques of endoscopy in evaluation of cervical dysphagia: comparison with radiographic techniques. Dysphagia. 1994;9:256 61. Yip HT, Leonard R, Kendall KA. Cricopharyngeal myotomy normalizes the opening size of the upper esophageal sphincter in cricopharyngeal dysfunction. Laryngoscope. 2006;116:93 6. Colombo Benkmann M, Unruh V, Krieglstein C, Senninger N. Cricopharyngeal myotomy in the treatment of Zenker's diverticulum. J Am Coll Surg. 2003;196:370 7. Hirano I. Pathophysiology of achalasia. Curr Gastroenterol Rep. 1999;1:198 202. Takahashi T. Pathophysiological signi¢cance of neuronal nitric oxide synthase in the gastrointestinal tract. J Gastroenterol. 2003;38:421 30. Verne GN, Sallustio JE, Eaker EY. Anti myenteric neuronal antibodies in patients with achalasia. A prospective study. Dig Dis Sci. 1997;42:307 13. Mayberry JF. Epidemiology and demographics of achalasia. Gastrointest Endosc Clin N Am. 2001;11:235 48. Podas T, Eaden J, Mayberry M, Mayberry J. Achalasia: a critical review of epidemiological studies. Am J Gastroenterol. 1998;93:2345 7. Eckardt VF, Stauf B, Bernhard G. Chest pain in achalasia: patient characteristics and clinical course. Gastroenterology. 1999;116:1300 4. Anderson SH, Yadegarfar G, Arastu MH, Anggiansah R, Anggiansah A. The relationship between gastro oesophageal re£ux symptoms and achalasia. Eur J Gastroenterol Hepatol. 2006;18:369 74. Pasricha PJ, Ravich WJ, Hendrix TR, Sostre S, Jones B, Kalloo AN. Intrasphincteric botulinum toxin for the treatment of achalasia. N Engl J Med. 1995;332:774 8. Pasricha PJ, Rai R, Ravich WJ, Hendrix TR, Kalloo AN. Botulinum toxin for achalasia: long term outcome and predictors of response. Gastroenterology. 1996;110:1410 15.
15
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 21. Pehlivanov N, Pasricha PJ. Achalasia: botox, dilatation or laparoscopic surgery in 2006. Neurogastroenterol Motil. 2006;18:799 804. 22. Bansal R, Nostrant TT, Scheiman JM et al. Intrasphincteric botulinum toxin versus pneumatic balloon dilation for treatment of primary achalasia. J Clin Gastroenterol. 2003;36:209 14. 23. Ghoshal UC, Chaudhuri S, Pal BB, Dhar K, Ray G, Banerjee PK. Randomized controlled trial of intrasphincteric botulinum toxin A injection versus balloon dilatation in treatment of achalasia cardia. Dis Esophagus. 2001;14:227 31. 24. Mikaeli J, Fazel A, Montazeri G, Yaghoobi M, Malekzadeh R. Randomized controlled trial comparing botulinum toxin injection to pneumatic dilatation for the treatment of achalasia. Aliment Pharmacol Ther. 2001;15:1389 96. 25. Annese V, Basciani M, Perri F et al. Controlled trial of botulinum toxin injection versus placebo and pneumatic dilation in achalasia. Gastroenterology. 1996;111:1418 24. 26. Csendes A, Velasco N, Braghetto I, Henriquez A. A prospective randomized study comparing forceful dilatation and esophagomyotomy in patients with achalasia of the esophagus. Gastroenterology. 1981;80:789 95. 27. Csendes A, Braghetto I, Henriquez A, Cortes C. Late results of a prospective randomised study comparing forceful dilatation and oesophagomyotomy in patients with achalasia. Gut. 1989;30:299 304. 28. Felix VN, Cecconello I, Zilberstein B, Moraes Filho JP, Pinotti HW, Carvalho E. Achalasia: a prospective study comparing the results of dilatation and myotomy. Hepatogastroenterology. 1998;45:97 108. 29. Konturek JW, Gillessen A, Domschke W. Di¡use esophageal spasm: a malfunction that involves nitric oxide? Scand J Gastroenterol. 1995;30:1041 5. 30. Kahrilas PJ. Esophageal motility disorders: current concepts of pathogenesis and treatment. Can J Gastroenterol. 2000;14:221 31. 31. Dalton CB, Castell DO, Hewson EG, Wu WC, Richter JE. Di¡use esophageal spasm. A rare motility disorder not characterized by high amplitude contractions. Dig Dis Sci. 1991;36:1025 8. 32. Pimentel M, Bonorris GG, Chow EJ, Lin HC. Peppermint oil improves the manometric ¢ndings in di¡use esophageal spasm. J Clin Gastroenterol. 2001;33:27 31. 33. Pandol¢no JE, Howden CW, Kahrilas PJ. Motility modifying agents and management of disorders of gastrointestinal motility. Gastroenterology. 2000;118(Suppl. 1):S32 47. 34. Short TP, Thomas E. An overview of the role of calcium antagonists in the treatment of achalasia and di¡use oesophageal spasm. Drugs. 1992;43:177 84. 35. Handa M, Mine K, Yamamoto H et al. Antidepressant treatment of patients with di¡use esophageal spasm: a psychosomatic approach. J Clin Gastroenterol. 1999;28:228 32. 36. Lee JI, Park H, Kim JH, Lee SI, Conklin JL. The e¡ect of sildena¢l on oesophageal motor function in healthy subjects and patients with nutcracker oesophagus. Neurogastroenterol Motil. 2003;15:617 23. 37. Miller LS, Pullela SV, Parkman HP et al. Treatment of chest pain in patients with noncardiac, nonre£ux, nonachalasia spastic esophageal motor disorders using botulinum toxin injection into the gastroesophageal junction. Am J Gastroenterol. 2002;97:1640 6. 38. Leconte M, Douard R, Gaudric M, Dumontier I, Chaussade S, Dousset B. Functional results after extended myotomy for di¡use oesophageal spasm. Br J Surg 2007; E pub ahead of print. 39. Kuwano H, Miyazaki T, Masuda N, Kato H, Kusano M. Long myotomy of the esophagus and gastric cardia with a complete fundic patch procedure for di¡use esophageal spasm. Hepatogastroenterology. 2004;51:1729 31. 40. Lemme EM, Abrahao Junior LJ, Manhaes Y, Shechter R, Carvalho BB, Alvariz A. Ine¡ective esophageal motility in gastroesophageal erosive re£ux disease and in nonerosive re£ux disease: are they di¡erent? J Clin Gastroenterol. 2005;39:224 7. 41. Ho SC, Chang CS, Wu CY, Chen GH. Ine¡ective esophageal motility is a primary motility disorder in gastroesophageal re£ux disease. Dig Dis Sci. 2002;47:652 6. 42. Agrawal A, Hila A, Tutuian R, Mainie I, Castell DO. Bethanechol improves smooth muscle function in patients with severe ine¡ective esophageal motility. J Clin Gastroenterol. 2007;41:366 70.
16
3 Outcomes of different treatment approaches for gastro-oesophageal reflux disease J. LABENZ
INTRODUCTION Gastro-oesophageal re£ux disease (GORD) is a common condition a¡ecting around 20% of the adult population of industrialized countries1. In a recent population-based study from Sweden as much as 45% of the adult population su¡ered from re£ux symptoms, and 15% had erosive oesophagitis on endoscopy2. A systematic review of the literature clearly demonstrated a signi¢cant increase of re£ux symptoms and GORD in US and European populations3. Based on these epidemiological data, optimizing the initial and long-term management of this disease has beyond doubt high priority.
DEFINITION AND CLASSIFICATION OF GORD The spectrum of GORD is a continuum with occasional re£ux symptoms without impairment of the quality of life at the one end and serious, sometimes life-threatening complications such as bleeding and cancer at the other end. Moreover, in recent years many extra-oesophageal signs and symptoms have been attributed to GORD, in many cases with rather weak evidence. Even more confusing is the lack of a diagnostic gold standard. A global de¢nition and classi¢cation of GORD using rigorous methodology was therefore eagerly awaited. Very recently, an international consensus group attempted to develop such a gobally acceptable de¢nition and classi¢cation of GORD that would be useful for patients, physicians, and regulatory agencies. The consensus group succeeded despite huge di¡erences in teminology and language, prevalence, and manifestations of the disease in di¡erent countries, and published their evidence-based consensus paper on the Montreal De¢nition and Classi¢cation of GORD4. GORD has been de¢ned as a condition which develops when the re£ux of gastric content causes troublesome symptoms or complications. The clinical presentations were divided into oesophageal syndromes with or 17
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
without oesophageal injury and extra-oesophageal syndromes which were dichotomized based on scienti¢c evidence in those with established association and those with only proposed association (Figure 1).
Figure 1
Overall Montreal de¢nition of GORD (after ref. 4)
GOALS OF THERAPY Treatment of GORD consists of an initial phase and long-term management (Figure 2). Since GORD is a highly prevalent benign disorder, any treatment must be e¡ective, safe, widely available, and cost-e¡ective. The most important goal of the initial management is to succeed with the initial therapy in order to con¢rm the diagnosis, to achieve su¤cient symptom control as quickly as possible, to reassure the patient as to the benign and treatable nature of re£ux disease, and to heal the eventually underlying oesophagitis. Moreover, the therapy should not harm the patient. During the initial phase the need for ongoing therapy must be determined. In the long run the goals of treatment are the maintenance of symptomatic and endoscopic remission and, if possible, the prevention of complications. Treatment modalities must be safe and coste¡ective. 18
DIFFERENT TREATMENT APPROACHES TO GORD
Figure 2
Options for the initial and long-term management of GORD
INITIAL THERAPY OF GORD Initially GORD should be treated with lifestyle advice and anti-re£ux medication irrespective of the severity of the disease and the stage of diagnosis (with or without endoscopy). Patients with the typical re£ux syndrome and without alarm features who do not belong to a risk group for the development of cancer in the upper gastrointestinal tract can safely be treated without endoscopy5. A 4-week course with a standard dose of a proton pump inhibitor (PPI) is su¤cient to decrease the symptom load to an acceptable level in over 90% of the patients 6 . Other drugs such as H 2 -receptor antagonists or prokinetics cannot be recommended since these types of drugs are clearly less e¡ective than a PPI. GORD patients in whom an endoscopy has been performed can be devided in those with non-erosive re£ux disease (NERD), re£ux oesophagitis which means visible mucosal breaks, and complicated re£ux disease such as stricture formation, Barrett's metaplasia or even Barrett's cancer. Patients with NERD are sometimes di¤cult to treat and are clearly less responsive to acid-lowering PPI than are patients with re£ux oesophagitis7. This is the case because patients with this entity frequently do not have true acid re£ux. The expected response to PPI therapy over 4 weeks is 60 70% even with the most e¡ective PPI8. In patients not responding to double-dose PPI a pH-metry during PPI therapy is 19
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
recommended to exclude phamacological resistance. If persistent acid re£ux could be excluded, the refractory patient belongs to the group of so-called functional heartburners. In spite of lack of sound scienti¢c evidence, an individualized approach is recommended in these patients, e.g. a trial with a tricyclic antidepressant at bedtime or a serotonin reuptake inhibitor such as citalopram, which has been shown to a¡ect the oesophageal sensitivity thresholds9. Patients with refractory NERD without proven acid re£ux should not unconditionally be referred for anti-re£ux surgery or other invasive procedures. Patients with erosive oesophagitis and troublesome re£ux symptoms can be e¡ectively managed with a PPI. A meta-analysis published in 1997 showed an overall response to a PPI after 8 weeks of treatment of around 80%10. Large, well-controlled and carefully designed studies have clearly established that healing of oesophagitis and su¤cient symptom control is today possible in 490% of patients a¡ected by erosive oesophagitis11^13. The success rates depend on several factors, the most important being the severity of oesophagitis according to the Los Angeles classi¢cation system, the magnitude of acid control, and the presence or absence of Helicobacter pylori infection12. Patients with concurrent Barrett's metaplasia are pobably the most di¤cult to treat14. Patients with re£ux oesophagitis who do not adequately respond to a standard dose of a PPI for 8 weeks should be treated with a PPI given twice daily for another 4 8 weeks, and in the case of treatment failure should be referred for further investigation(s)15. Short-term treatment with a PPI for up to 6 12 months is remarkably safe. In well-controlled studies the frequency and pro¢le of adverse events while on a PPI resembles closely that occuring during intake of placebo. This favourable drug safety has been con¢rmed in a large observational study in private practices16. Drug interactions may occur as a class e¡ect since all PPI are metabolized by the cytochrome P450 (CYP), predominantly by the CYP2C19 subunit. However, according to the FDA's database, the frequency of reported drug interactions with a PPI as primary or secondary suspect drug was low17. Rebound acid hypersecretion may occasionally occur after PPI treatment for 8 or more weeks in H. pylori-negative patients18. The clinical relevance of this phenomenon, however, is unproven.
LONG-TERM MANAGEMENT OF GORD PPI therapy Efficacy In patients with NERD responding to initial therapy with a PPI, medication should be discontinued, as 25% (or more) of these patients may remain in remission over prolonged periods of time19, and NERD does not seem to warrant measures aimed at preventing complications. In the event of a relapse, indicating the need for long-term management, a number of options are available: 20
DIFFERENT TREATMENT APPROACHES TO GORD *
Continuous maintenance therapy, starting with a PPI and subsequent attempts to step down to lower dosages of the PPI or even less potent drugs20.
*
Intermittent courses of treatment for 2 4 weeks with the initially successful PPI21.
*
Patient-controlled on-demand therapy with a PPI22.
Until recently, daily use of a PPI was the recommended regimen. A growing number of controlled studies have demonstrated that on-demand therapy with a PPI is e¡ective in achieving acceptable symptom control and maintenance of the restored quality of life of patients with NERD23,24. This newly developed strategy seems to be as e¡ective as continuous treatment with a PPI, while being more cost-e¡ective due to cost savings that result from 450% less drug consumption24. After responding well to initial therapy, re£ux oesophagitis shows a strong tendency to relapse. Up to 90% of patients will relapse within the next 6 months19. Patients with mild oesophagitis (Los Angeles A/B) often have a longer relapse-free interval than patients with severe oesophagitis (Los Angeles C/D) who frequently su¡er a relapse within days of discontinuing the successful initial treatment25,26. In the light of these observations it is recommended that, in patients with mild oesophagitis, therapy should ¢rst be discontinued and the further course of the disease kept under surveillance, while in severe oesophagitis the initial successful therapy should be followed, a priori, by maintenance treatment15. Established options for long-term management are: *
Intermittent treatment for some weeks.
*
Maintenance therapy with an attempt to reduce the daily dosage of the PPI (step-down principle).
On-demand therapy has, to date, been investigated only in a small number of studies. Satisfactory control of the symptoms was achieved in the vast majority of patients, but continuous therapy proved to be superior with respect to maintenance of remission of erosive oesophagitis, so that an evidence-based recommendation is currently not possible27.
Safety Prospective long-term studies of patients on PPI have shown few sidee¡ects28,29. Database studies have, however, shown an association between long-term PPI use and community-acquired pneumonia and hip fractures30,31. There may also be a slightly increased risk for enteral infections32. At the present time it is not possible to o¡er accurate risk estimates for these associations or to determine if the associations are causal33. Some patients may develop vitamin B12 de¢ciency after prolonged intake of a PPI34.
21
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Endoscopic therapy During recent years many endoscopic anti-re£ux devices have been developed and tested within the frame of clinical studies. These include suturing devices such as Endocinch, ESD and full-thickness plication (Plicator), the Stretta procedure (radiofrequency application) and injection of polymers into the submucosa or muscle layer, e.g. Gatekeeper and Enteryx. However, in none of the studies a level of e¤cacy has been reached comparable to that with PPI therapy or anti-re£ux surgery. Moreover, safety concerns have been raised35.
Efficacy None of the devices has been shown to be highly e¤cacious in erosive re£ux oesophagitis or among patients with hiatal hernias. Initial reports derived from open-label trials reported e¤cacy in 80% or more of treated patients. However, sham-controlled studies as a proof of principle suggested that Stretta, Endocinch and Enteryx have merely the potential of amelioration of symptoms, but they did not a¡ect oesophageal acid exposure36^38. Endocinch and ESD have the problem of durability as up to 90% of sutures were lost after 1 year39,40. Very recently the Plicator device has been shown to a¡ect both symptoms and acid re£ux in a randomized sham-controlled study over 3 months41. Complete cessation of PPI therapy was possible in 50% of the patients randomized to the Plicator as compared to 24% in the sham group (p ( = 0.002). The durability of the e¡ect has not been fully elucidated, but preliminary data of an open uncontrolled series suggest fewer problems compared to other suturing devices42.
Safety Virtually all the procedures were associated with serious complications that included perforation, bleeding, pleural e¡usions, pericardial e¡usions, pneumonia, severe chest pain, and persistent dysphagia. Deaths have been reported in association with Stretta and Enteryx. In the study by Rothstein et al.41, four out of 78 patients in the Plicator arm required hospitalization after the procedure, and one underwent laparotomy to remove the plication. Until now, endoscopic procedures for GORD remain experimental and should not be used outside the realm of clinical trials. There are no properly designed studies comparing these new devices with medical therapy or antire£ux surgery. Given the devices are being marketed as an alternative to medical therapy, some evidence that they are better than, or at least as e¡ective as, medical therapy is essential. It is currently also unclear whether the claimed bene¢ts of the procedures outweigh the risks. Last but not least, the cost-e¡ectiveness of endoscopic therapy is unclear.
22
DIFFERENT TREATMENT APPROACHES TO GORD
Anti-reflux surgery The conventional criteria for considering anti-re£ux surgery in patients with GORD are: * A con¢rmed diagnosis of GORD with necessity of long-term treatment. *
Good response to PPI therapy.
*
Availability of an experienced and skilled surgeon who has demonstrated good outcomes in performing the procedure.
*
A healthy patient able to withstand the physiological stresses of surgery23.
Efficacy When a skilled surgeon performs surgery, using appropriate candidates, 85% or more of patients can be expected to achieve a good symptomatic outcome43. Importantly, a poor response to pharmacological therapy predicts a poor response to surgical therapy, as this suggests that the symptoms may be due to causes other than acid re£ux. In a community study by Vakil et al. only 61% of patients were satis¢ed with anti-re£ux surgery44. In this study 46% of surgically treated patients had pursued surgery because of inadequate symptom relief with medication. Outcomes also seem poorer in NERD, especially in female patients45. Open and randomized studies have consistently shown surgery to be at least as e¡ective as medical therapy. However, for many patients, surgery is not a permanent solution to their re£ux symptoms. In long-term follow-up of patients from randomized studies 30% or more of the surgically treated patients were back on antisecretory therapy33,46.
Risks of surgery A number of complications of fundoplication have been reported, with the risks varying between centres and surgeons. However, serious complications and side-e¡ects have been described in expert hands. These include perioperative complications with an operative mortality between 0.2% and 1.9%, and postoperative complications, the most prevalent of which are lasting dysphagia, gas bloat syndrome, rectal £atulence, and diarrhoea33,46.
Cost-effectiveness Long-term costs of medical therapy constitute a frequently cited argument for anti-re£ux surgery, especially in young patients with good response to medical therapy. Hypothetical Markov models seem to support this notion47. However, in a randomized controlled study comparing anti-re£ux surgery with omeprazole, 5-year direct medical costs were signi¢cantly lower for medical therapy than surgery. When indirect costs were also included, the cost of surgical treatment increased substantially and exceeded the cost of medical therapy further48. The cost of PPI has decreased signi¢cantly as these models 23
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY Table 1 ref. 23)
Comparison of alternative strategies for the long-term management of GORD (after
PPI E¤cacy Safety Cost-e¡ectiveness
Surgery
Endoscopic therapy
+++
+++
+ (Short-term)
No issues
Measurable morbidity and mortality
Measurable morbidity and mortality
Proven
5PPI
?
Operator-dependent
No
Yes
Yes
Ability to adjust
Yes
No
Probably yes
Learning curve
No
Yes
Probably yes
were created. Recalculating the cost of medical therapy using the price of generic PPI will result in further cost advantages for medical therapy. Considering e¤cacy, safety, and cost-e¡ectiveness, only a few carefully selected patients should undergo fundoplication for re£ux disease. Patients who are well maintained on medical therapy have more to lose with surgical intervention than to gain, and should not be o¡ered surgery. The notion that surgery may prevent oesophageal cancer is a hypothesis that is not supported by current evidence49. The only clear-cut candidates for surgery include: *
Patients with anatomical abnormalities such as a large hiatus hernia.
*
Patients with persistent regurgitation that causes troublesome symptoms despite medical therapy.
*
Carefully selected patients with extra-oesophageal disorders who have symptoms of re£ux disease such as heartburn and regurgitation, an incomplete response to medical therapy and persistent plus demonstrable re£ux on pH or impedance testing that is associated with their symptoms.
Patients should be made aware of the high likelihood of needing continued acid-inhibitory therapy following surgery and the possibility of side-e¡ects, which in some cases dwarf the symptoms that originally led to the operation33.
COMPARISON OF THE AVAILABLE TREATMENT OPTIONS Drug therapy with a PPI is and remains the mainstay of management of patients with GORD. PPIs are highly e¡ective and safe. The cost of PPI therapy has decreased over recent years. Drug therapy is not operatordependent without a learning curve, and the dose can be adjusted to the patient's need. Surgery in experienced hands is similarly e¡ective as PPI therapy, but it has the disadvantages of a higher rate of morbidity and mortality and a loss of e¡ect over time. Moreover, surgery does not seem to be as cost-e¡ective as PPI therapy; therefore, surgery should be o¡ered only to a 24
DIFFERENT TREATMENT APPROACHES TO GORD
few carefully selected patients. Endoscopic therapy is still experimental and should be performed within the frame of clinical studies only. The Plicator device has the potential that a limited number of patients may bene¢t from this procedure in the long run. However, carefully designed studies comparing endoscopic anti-re£ux therapy with the current standard of GORD management in terms of e¤cacy, safety, durability and economics are lacking, making any recommendations on its putative place in the management of GORD currently impossible (Table 1).
References 1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14.
15. 16.
Locke GR, Talley NJ, Fett SL et al. Prevalence and clinical spectrum of gastroesophageal re£ux: a population-based study in Olmsted county. Gastroenterology. 1997;112:448 56. Ronkainen J, Aro P, Storskrubb T et al. High prevalence of gastroesophageal re£ux symptoms and esophagitis with or without symptoms in the general adult Swedish population: a Kalixanda study report. Scand J Gastroenterol. 2005;40:275 85. El-Serag H. Time trends of gastroesophageal re£ux disease: a systematic review. Clin Gastroenterol Hepatol. 2007;5:17 26. Vakil NV, van Zanten S, Kahrilas P et al. The Montreal de¢nition and classi¢cation of gastroesophageal re£ux disease: a global evidence-based consensus. Am J Gastroenterol. 2006;101:1900 20. Morgner-Miehlke A, Koop H, Blum AL et al. Abklarung und Therapie von Re£uxbeschwerden. Z Gastroenterol. 2006;44:399 410. Hansen NA, Bergheim R, Fagertun H et al. A randomised prospective study comparing the e¡ectiveness of esomeprazole treatment strategies in clinical practice for 6 months in the management of patients with symptoms of gastroesophageal re£ux disease. Int J Clin Pract. 2005;59:665 71. Dean BB, Gano Jr AD, Knight K et al. E¡ectiveness of proton pump inhibitors in nonerosive re£ux disease. Clin Gastroenterol Hepatol. 2004;2:656 64. Armstrong D, Talley NJ, Lauritsen K et al. The role of acid suppression in patients with endoscopy-negative re£ux disease: the e¡ect of treatment with esomeprazole or omeprazole. Aliment Pharmacol Ther. 2004;20:413 21. Broekaert D, Fischler B, Sifrim D et al. In£uence of citalopram, a selective serotonin reuptake inhibitor, on oesophageal hypersensitivity: a double-blind, placebo controlled study. Aliment Pharmacol Ther. 2006;23:365 70. Chiba N, De Gara CJ, Wilkinson JM, Hunt RH. Speed of healing and symptom relief in grade II to IV gastroesophageal re£ux disease: a meta-analysis. Gastroenterology. 1997;112:1798 810. Castell DO, Kahrilas PJ, Richter JE et al. Esomeprazole (40 mg) compared with lansoprazole (30 mg) in the treatment of erosive esophagitis. Am J Gastroenterol. 2002;97:575 83. Labenz J, Armstrong D, Lauritsen K et al. A randomised comparative study of esomeprazole 40 mg versus pantoprazole 40 mg for healing erosive oesophagitis: the EXPO study. Aliment Pharmacol Ther. 2005;21:739 46. Richter JE, Kahrilas PJ, Johanson J et al. E¤cacy and safety of esomeprazole compared with omeprazole in GERD patients with erosive esophagitis: a randomized controlled trial. Am J Gastroenterol. 2001;96:656 65. Malfertheiner P, Lind T, Willich S et al. Prognostic in£uence of Barrett's oesophagus and Helicobacter pylori infection on healing of erosive gastro-oesophageal re£ux disease (GORD) and symptom resolution in non-erosive GORD: report from the ProGORD study. Gut. 2005;54:746 51. Labenz J, Malfertheiner P. Treatment of uncomplicated re£ux disease. World J Gastroenterol. 2005;11:4291 9. Martin RM, Dunn NR, Freemantle S, Shakir S. The rates of common adverse events reported during treatment with proton pump inhibitors use in general practice in England: cohort studies. Br J Pharmacol. 2000;50:366 72.
25
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 17. Labenz J, Petersen KU, Rosch W, Koelz R. A summary of Food and Drug Administrationreported adverse events and drug interactions occurring during therapy with omeprazole, lansoprazole and patoprazole. Aliment Pharmacol Ther. 2003;17:1015 19. 18. Hunfeld HGM, Geus WP, Kuipers EJ. Systematic review: rebound acid hypersecretion after therapy with proton pump inhibitors. Aliment Pharmacol Ther. 2007;25:39 46. 19. Carlsson R, Dent J, Watts R et al. Gastro-oesophageal re£ux disease in primary care: an international study of di¡erent treatment strategies with omeprazole. International GORD Study Group. Eur J Gastroenterol Hepatol. 1998;10:119 24. 20. Dent J. Management of re£ux disease. Gut. 2002;50(Suppl. IV):iv67 71. 21. Bardhan KD, MÏller-Lissner S, Bigard MS et al. Symptomatic gastro-oesophageal re£ux disease: double-blinded controlled study of intermittent treatment with omeprazole or ranitidine. Br Med J. 1999;318:502 7. 22. Bytzer P, Blum AL. Personal view: rationale and proposed algorithms for symptom-based proton pump inhibitor therapy for gastro-oesophageal re£ux disease. Aliment Pharmacol Ther. 2004;20:389 98. 23. Fennerty MB. Review article: Alternative approaches to the long-term management of GERD. Aliment Pharmacol Ther. 2005;22(Suppl. 3):39 44. 24. Labenz J, Morgner-Miehlke A. An update on the available treatments for non-erosive re£ux disease. Expert Opin Pharmacother. 2006;7:47 56. 25. Johnson DA, Benjamin SB, Vakil NB et al. Esomeprazole once daily for 6 months is e¡ective therapy for maintaining healed erosive esophagitis and for controlling gastroesophageal re£ux disease symptoms: a randomized, double-blind, placebocontrolled study of e¤cacy and safety. Am J Gastroenterol. 2001;96:27 34. 26. Vakil NB, Shaker R, Johnson DA et al. The new proton pump inhibitor esomeprazole is e¡ective as a maintenance therapy in GERD patients with healed erosive esophagitis: a 6month, randomized, double-blind, placebo-controlled study of e¤cacy and safety. Aliment Pharmacol Ther. 2001;15:927 35. 27. Sjostedt S, Befrits R, Sylvan A et al. Daily treatment with esomeprazole is superior to that taken on-demand for maintenance of healed erosive oesophagitis. Aliment Pharmacol Ther. 2005;22:183 91. 28. Hassall E, Kerr W, El-Serag HB. Characteristics of children receiving proton pump inhibitors continuously for up to 11 years duration. J Pediatr. 2007;150:262 7. 29. Klinkenberg-Knol E, Nelis F, Dent J et al. Long-term omeprazole treatment in resistant gastroesophageal re£ux disease: e¤cacy, safety, and in£uence on gastric mucosa. Gastroenterology. 2000;118:661 9. 30. Laheij RJ, Sturkenboom MC, Hassing RJ et al. Risk of community-acquired pneumonia and use of gastric acid-suppressive drugs. J Am Med Assoc. 2004;292;1955 60. 31. Yang Y-X, Lewis JD, Epstein S, Metz DC. Long-term proton pump inhibitor therapy and risk of hip fracture. J Am Med Assoc. 2006;296:2947 53. 32. Leonard J, Marshall JK, Moayyedi P. Systematic review of the risk of enteric infection in patients taking acid suppression. Am J Gastroenterol. 2007;102:1 10. 33. Vakil N. Review article: The role of surgery in gastro-oesophageal re£ux disease. Aliment Pharmacol Ther. 2007;25:1365 72. 34. Laine L, Ahnen D, McClain C et al. Review article: Potential gastrointestinal e¡ects of long-term acid suppression with proton pump inhibitors. Aliment Pharmacol Ther. 2000;14:651 68. 35. Shaheen NJ. The rise and fall (and rise?) of endoscopic anti-re£ux procedures. Gastroenterology. 2006;131:952 4. 36. Corley DA, Katz P, Wo JM et al. Improvement of gastroesophageal re£ux symptoms after radiofrequency energy: a randomized sham-controlled trial. Gastroenterology. 2003;125:668 76. 37. Deviere J, Costamagna G, Neuhaus H et al. Nonresorbable copolymer implantation for gastroesophageal re£ux disease: a randomized sham-controlled multicenter trial. Gastroenterology. 2005;128:532 40. 38. Schwartz MP, Wellink H, Gooszen HG et al. Endoscopic gastroplication for the treatment of gastro-oesophageal re£ux disease: a randomised, sham-controlled trial. Gut. 2007;56:20 8. 39. Schiefke I, Zabel-Langhenning A, Neumann S et al. Long-term failure of endoscopic gastroplication (EndoCinch). Gut. 2005;54:752 8.
26
DIFFERENT TREATMENT APPROACHES TO GORD 40. Schiefke I, Neumann S, Zabel-Langhenning A et al. Use of an endoscopic suturing device (the `ESD') to treat patients with gastroesophageal re£ux disease, after unsuccessful EndoCinch endoluminal gastroplication: another failure. Endoscopy. 2005;37:700 5. 41. Rothstein R, Filipi C, Caca K et al. Endoscopic full-thickness plication for the treatment of gastroesophageal re£ux disease: a randomized, sham-controlled trial. Gastroenterology. 2006;131:704 12. 42. Pleskow D, Rothstein R, Lo S et al. Endoscopic full-thickness plication for the treatment of GERD: 12-month follow-up for the North American open-label trial. Gastrointest Endosc. 2005;61:643 9. 43. Kahrilas PJ. Laparoscopic antire£ux surgery: silver bullet or the emperor's new clothes? Am J Gastroenterol. 1999;94:1721 3. 44. Vakil N, Shaw M, Kirby R. Clinical e¡ectiveness of laparoscopic antire£ux fundoplication in a U.S. community. Am J Med. 2003;114:1 5. 45. Thibault R, Coron E, Sebille V et al. Antire£ux surgery for non-erosive and erosive re£ux disease in community praxis. Aliment Pharmacol Ther. 2006;24:621 32. 46. Lundell L, Miettinen P, Myrvold HE et al. Seven-year follow-up of a randomized clinical trial comparing proton-pump inhibition with surgical therapy for re£ux oesophagitis. Br J Surg. 2007;94:198 203. 47. Bojke L, Hornby E, Sculpher M. A comparison of the cost e¡ectiveness of pharmacotherapy or surgery (laparoscopic fundoplication) in the treatment of GORD. Pharmacoeconomics. 2007;25:829 41. 48. Myrvold HE, Lundell L, Miettinen P et al. The cost of long term therapy for gastrooesophageal re£ux disease: a randomised trial comparing omeprazole and open antire£ux surgery. Gut. 2001;49:488 94. 49. Tran T, Spechler SJ, Richardson P et al. Fundoplication and the risk of esophageal cancer in gastroesophageal re£ux disease: a Veterans a¡airs cohort study. Am J Gastroenterol. 2005;100:1002 8.
27
4 Barrett's oesophagus: screening, surveillance, treatment: are all questions answered? F. SCHREIBER
INTRODUCTION Barrett's oesophagus today is understood to be a complication of gastrooesophageal re£ux disease (GORD), but unlike GORD, it is seldom seen in daily clinical practice. In 1957 the surgeon NR Barrett described an oesophagus lined with a too-short inner mucosal layer. Over the course of many years, specialists collected a vast amount of information on the aetiopathology, endoscopic and histopathological diagnosis, surveillance and treatment of this condition. It now seems clear that the de¢nition of Barrett's oesophagus has evolved over the past two decades from the columnar-lined lower oesophagus to intestinal metaplasia in the oesophagus without speci¢cation of length and circumferential extent.
DIAGNOSIS, SCREENING, SURVEILLANCE Patients with chronic GORD are those most likely to have Barrett's oesophagus. It is well accepted that the duration of GORD correlates directly with the prevalence of Barrett's oesophagus. Though the epidemiology of Barrett's oesophagus is described incompletely, it is a fact that the highest yield of intestinal metaplasia is found in white males with symptoms of chronic GORD. The most e¡ective tool for detecting Barrett's oesophagus is upper gastrointestinal endoscopy, which therefore is absolutely indicated for GORD patients. The recognition of Barrett's oesophagus in asymptomatic individuals still remains a problem, and underscores the need to assess the distal oesophagus carefully in all patients undergoing upper gastrointestinal e n d o s c o p y r e g a r d l e s s o f i n d i c a t i o n. T h e i d e n t i ¢ c at i o n o f t h e squamocolummnar junction during every endoscopy is essential for recognition of Barrett's mucosa. Oesophagitis or erythema alone may be confused with Barrett's mucosa macroscopically, so systematic and multiple 28
BARRETT'S OESOPHAGUS: SCREENING, SURVEILLANCE, TREATMENT
biopsy is essential to establish the diagnosis. To select biopsy sites more accurately, chromoscopy with various stains has been introduced into routine clinical endoscopy for patients with suspected Barrett's oesophagus. Methylene blue staining seems to be the most useful and promising technique as evidenced by the fact, that stain-targeted biopsies produce a higher yield of detected metaplastic and above all dysplastic areas. For individuals with established Barrett's oesophagus the rationale for surveillance is based on the increased risk of developing adenocarcinoma. Therefore the grade of dysplasia determines the endoscopy interval as dysplasia is the best current indicator of the risk of developing cancer. It is important to realise that dysplasia is the ¢rst step in the neoplastic process and that any grade of dysplasia may be accompanied by coexisting carcinoma. It must be emphasized that every diagnosis of dysplasia, regardless of histological grading, warrants a repeat endoscopy with extensive biopsies under chromoscopy.
THERAPEUTIC MANAGEMENT As the treatment goals for Barrett's and GORD are the same, the diagnosis of Barrett's oesophagus does not lead to a speci¢c therapeutic regime. The control of GORD symptoms is the best and most e¡ective treatment of Barrett's oesophagus without any dysplastic lesions. Therapy with proton-pump inhibitors is accepted as the standard therapy in this group of patients. Individuals for whom surgery is indicated may elect antire£ux surgery since fundoplication e¡ectively controls speci¢c symptoms. If dysplastic or premalignant epithelium exists, neither conservative nor surgical treatment is able to eliminate the risk of an adenocarcinoma. This group requires special attention with regard to any resective procedure, either endoscopic or surgical. Because dysplasia tends to be multifocal, endoscopic mucosal resection ratherly should preferably be performed as a one-piece resection endoscopic submucosal dissection rather than with piecemeal endoscopic mucosal resection, though strictures occur more commonly after complete circumferential resection than after partial resection. Other or recently developed endoscopic therapeutic modalities such as photodynamic therapy or the HALO1 system seem to be promising, as they are e¡ective and rarely involve complications. Oesophagectomy performed at a high-volume institution remains a reasonable strategy in the surgically ¢t patient; the given morbidity and mortality, especially in low-volume institutions, suggest reserve and caution under those circumstances.
CONCLUSION As Barrett's oesophagus is a well-known and now well-de¢ned pathological entity, it's diagnosis and management, as far as speci¢c surveillance and therapeutic procedures are concerned, should be carried out in highly specialised institutions. A close interdisciplinary approach among gastroenterologists, pathologists and gastrointestinal surgeons is the only guarantee for the maximum range of diagnostic and therapeutic management. 29
Section II Helicobacter pylorii, NSAID, gastric cancer Chair: M BANIC and B TEPES
5 Helicobacter pylori infection: diagnosis, treatment and risks of untreated infection K. MO«NKEMU«LLER, H. NEUMANN, L. C. FRY and P. MALFERTHEINER
INTRODUCTION Helicobacter pylori infection is associated with various gastroduodenal pathologies ranging from gastritis and peptic ulcer disease to gastric cancer. The most current guidelines for the diagnosis, indications for therapy and treatment strategies of H. pylori are outlined in the third Maastricht Consensus conference which was convened by the European Helicobacter Study Group (EHSG)1. The diagnosis of H. pylori infection is established using invasive or non-invasive methods2^6. Non-invasive tests are the urea-breath test, stoolantigen tests and serological kits with a high accuracy7. Invasive tests include endoscopy with gaining of gastric biopsies for urea test (HUT1, Helicobacter urease test) or histology (e.g. Whartin Starry silver staining). Eradication of H. pylori infection is recommended in patients with gastroduodenal pathologies such as peptic ulcer disease and low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma, atrophic gastritis, ¢rst-degree relatives of gastric cancer patients, unexplained iron de¢ciency anaemia and chronic idiopathic thrombocytopenic purpura8^10. Triple therapy using a proton pump inhibitor (PPI) with amoxicillin and clarithromycin or metronidazole given twice daily remains the recommended ¢rst-choice therapy1. Bismuth containing quadruple therapy is also a ¢rst-choice treatment option. Rescue therapy should be based on antimicrobial susceptibility1. The risk of untreated H. pylori infection appears to be highest in patients who require long-term treatment with nonsteroidal anti-in£ammatory agents11^13. Untreated H. pylori infection also leads to precancerous and cancerous stomach lesions 14^17 . Therefore, eradication of H. pylori has the potential to reduce the risk of gastric cancer development18.
33
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
DIAGNOSIS Non-invasive tests for the diagnosis of H. pylori infection include: the breath and blood 13 C-urea-breath tests (UBT); stool-antigen tests (polyclonal antibody, monoclonal antibody, and o¤ce-based); and immunological tests (laboratory- and o¤ce-based tests and tests on saliva and urine)2^6. The UBT is an accurate, practical, and readily available test with an accuracy of 95%2. The stool-antigen test also has high speci¢city and a sensitivity of 91%, but the latter decreases signi¢cantly if the stool is stored at room temperature. However, handling of stool specimens can be cumbersome, and when not analysed immediately it is necessary to store stool samples at 208C before testing3. Although serology such as UBT or HUT1 is a widely available, inexpensive and non-invasive test, its diagnostic accuracy is low (around 80%)5,6. Therefore it is better to employ tests that detect active infection. Nevertheless, serology plays an important role in patients taking PPI, which can result in falsenegative invasive and non-invasive diagnostic tests. Thus, PPI should be stopped for at least 2 weeks before performing invasive or non-invasive H. pylori testing. Serological tests are also recommended to assess H. pylori in patients with a bleeding ulcer and conditions associated with a low bacterial density (e.g. extensive mucosal atrophy and MALT lymphoma)19,20. The rapid urease test, culture, and histology, as well as UBT, have shown a limited sensitivity in patients presenting with acute bleeding peptic ulcer1. Polyclonal stool-antigen tests have a low speci¢city owing to crossreactivity with blood products. O¤ce-based serological tests or near-patient tests are extremely convenient to use, but they are not accurate and are currently not recommended for the diagnosis of H. pylori infection5.
TREATMENT Eradication of H. pylori infection is recommended in patients with gastroduodenal pathologies such as peptic ulcer disease, low-grade gastric MALT lymphoma (stage-I low-grade MALT lymphoma), atrophic gastritis, ¢rst-degree relatives of gastric cancer patients, unexplained iron de¢ciency anaemia, chronic idiopathic thrombocytopenic purpura, and H. pylori-positive and dyspeptic patients1. A `test-and-treat' strategy is recommended in adult patients under the age of 45 years presenting with persistent dyspepsia, although the cut-o¡ age may vary between countries, depending on the geographical prevalence of gastric cancer21. Economic modelling suggests that this bene¢t is cost-e¡ective showing that 12 15 infected patients need to be treated to cure one patient with non-ulcer dyspepsia22. Furthermore, eradication of H. pylori infection is carried out once and leads to long-term symptom improvement; it also reduces the risk of developing peptic ulcer disease, atrophic gastritis and, possibly, gastric cancer. Recurrent abdominal pain in children is another indication for a `test-and-treat' strategy if other causes are excluded. Current data support the notion that H. pylori eradication may prevent peptic ulcer in patients who are naive non-steroidal anti-in£ammatory drug 34
HELICOBACTER PYLORI INFECTION
(NSAID) users11,12. Nevertheless, H. pylori eradication is less e¡ective than PPI treatment in preventing ulcer recurrence in long-term NSAID users13. Triple therapy using a PPI with amoxicillin and clarithromycin or metronidazole given twice daily remains the recommended ¢rst-choice therapy. Bismuth-containing quadruple therapy, if available, is also a ¢rstchoice treatment option1. Rescue therapy should be based on antimicrobial susceptibility. Eradication of H. pylori infection has the potential to reduce the risk of gastric cancer development1.
RISK OF UNTREATED INFECTION Untreated H. pylori infection will always lead to the development of gastritis. The gastritis may progress to gastric atrophy, intestinal metaplasia and cancer18^20. In addition, gastroduodenal pathology such as duodenal and gastric ulcer will develop in at least 5% of H. pylori-infected subjects1 . Therefore, based on available evidence, it does not make any sense to knowingly leave a patient infected with H. pylori. Nevertheless, the data on H. pylori eradication in NSAID users or in the prevention of gastric cancer are still under intense scrutiny. The relationship between H. pylori infection and NSAID in gastroduodenal pathology is complex because both H. pylori and NSAID independently increase the risk of peptic ulcer bleeding signi¢cantly. If both factors are present, the risk of ulcer bleeding is increased by 6-fold11. Although results of H. pylori eradication in NSAID users are con£icting, current data demonstrate a bene¢t of H. pylori eradication in NSAID-naive patients who will require long-term NSAID therapy13. Epidemiological data demonstrate an association of H. pylori with gastric cancer precursor lesions and gastric cancer 15^17 . H. pylori infection is associated with diverse pathological gastric conditions such as chronic gastritis and intestinal metaplasia which can progress in a subgroup of patients to gastric cancer. Environmental factors, including tobacco smoking, alcohol and dietary risk factors such as ingestion of nitroso compounds, salt, and smoked foods, also play a role in the development of gastric cancer23. Nevertheless, H. pylori infection and the genetic predisposition of the host, including gene errors and abnormal gene expression, account for the most important predisposing factors for gastric cancer. H. pylori infection leads to ongoing gastric in£ammation a¡ecting the biology, growth, and death of gastric epithelial and cancer cells17. There are numerous studies describing the molecular and genetic changes of gastric cancer cells obtained from established tumours. Nearly all cancers have reduction of cellular adhesion due to mutations in E-cadherin, a-catenin, and b-catenin, microsatellite instability and increased telomerase activity24. The most common genetic abnormalities are inactivation of tumour-suppressor genes such as p5323. Activation of oncogenes such as c-Met and Her2/Neu is relatively frequent, while K-ras mutations appear to be less common. Interestingly, some genetic changes can occur in H. pylori-associated gastritis even before the onset of intestinal metaplasia23. 35
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
BENEFIT OF H. PYLORI ERADICATION TO PREVENT GASTRIC CANCER At present there are several clinical studies which have provided some information on the incidence of preneoplastic gastric lesions and gastric cancer after H. pylori eradication14^29 (Tables 1 and 2). A population-based prospective study, which included 1630 healthy subjects with a 7-year follow-up period, established a reduction in the incidence of gastric cancer after H. pylori eradication but only in the subgroup of patients without atrophy/intestinal metaplasia at the initial endoscopy25. Uemura et al. performed a prospective study in 1526 patients with gastroduodenal pathology (gastroduodenal ulcer, non-ulcer dyspepsia and gastric hyperplasia). After 7.8 years of follow-up, gastric cancer developed in 2.9% of H. pylori-positive patients. None of the H. pylori-negative patients had malignant transformation17. In another 8-year follow-up population-based study in China, which included 1006 subjects, the persistence of H. pylori infection was associated with an incidence of gastric cancer of 1.6% versus 0.4% in H. pylori-negative patients26. A bene¢t of H. pylori eradication upon the future incidence of recurrent or metachronous gastric cancer may also exist even in patients with early gastric cancer who underwent removal of the neoplasm. In a prospective non-randomized study, in patients whose early gastric cancer was removed by endoscopic resection, 6/67 patients who were not treated for the H. pylori infection developed a metachronous cancer during the 3-year follow-up period whereas no cancer developed in 65 patients who received eradication therapy27. The data suggest that H. pylori eradication reduces the incidence of gastric cancer in patients without pre-existing gastric atrophy and/or intestinal metaplasia (Table 1). Therefore, intervention strategies aimed at reducing the risk of gastric cancer need to be applied before this `point of no return' is reached. However, even if no reversibility of premalignant changes such as atrophic gastritis or intestinal metaplasia occurs, H. pylori eradication may hamper the progression of these lesions (Table 2). Ito et al., after a 5-year follow-up of 26 patients with moderate to severe atrophy who received eradication therapy, described reversibility of gastric atrophy and intestinal metaplasia in some patients 28 . In a 12-year cohort study, Mera et al. randomized 795 patients with preneoplastic lesions to receive H. pylori eradication therapy and/or anti-oxidants 29 . At the end of 6 years of intervention, those who did not receive H. pylori eradication were o¡ered it. Ninety-seven per cent of subjects were H. pylori-positive at baseline and 53% were positive at 12 years. Regression of precancerous lesions occurred in 66% of H. pylori-negative patients and in 14% of patients who remained H. pyloripositive29. In addition, H. pylori-negative subjects showed more regression and less progression than H. pylori-positive patients. Healing was also more pronounced in less advanced lesions. In summary, since precancerous lesions may not progress further, or may even regress, after H. pylori eradication, and regression of lesions occurs when they are less advanced, treatment of infection should be considered even in patients without precancerous lesions. Despite H. pylori eradication, progression of atrophic gastritis and intestinal metaplasia into gastric cancer 36
1526
1630
Uemura (2001)17
Wong (2004)25
37
1131
Take (2007)30
a
3.9
8
7
7.8
3
Prospective, not randomized
Population-based, prospective, randomized, placebo-controlled
Population-based, prospective, randomized, placebo-controlled
Prospective, placebocontrolled
Prospective, placebocontrolled
Study
Preneoplastic lesions gastric atrophy, intestinal metaplasia, dysplasia.
neg, negative; pos, positive.
1006
Zhou (2005)26
Uemura (1997)
132
No. of Follow-up patients (years)
H. pylori-eradication and gastric cancer incidence
27
Reference
Table 1
Incidence of gastric cancer after H. pylori eradication
Incidence of gastric cancer after H. pylori eradication
Incidence of gastric cancer after H. pylori eradication
Development of gastric cancer in H. pylori-infected patients
Incidence of gastric cancer, reversibility of preneoplastic lesions
End point
H. pylori-neg: 9/953 (0.9%) H. pylori-pos: 4/178 (2.2%) p = 0.04
H. pylori-neg: 1/1968 (0.4%) H. pylori-pos: 6/2448 (1.6%) After H. pylori eradication: signi¢cant reduction of atrophy in corpus p = 0.001
Patients without preneoplastic lesionsa H. pylori-neg: 0 vs. H. pylori-pos: 6 patients p = 0.02 No di¡erence in patients with preneoplastic lesions.
H. pylori-neg: 0/280 (0.0%) H. pylori-pos: 36/1246 (2.9%) p50.001
H. pylori-neg: 0/65 (0%) H. pylori-pos: 6/67 (9%) p = 0.011
Incidence of gastric cancer
HELICOBACTER PYLORI INFECTION
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY Table 2
In£uence of H. pylori eradication on preneoplastic gastric lesions No. of patients
Reference Tepes (1999)
31
Follow-up (years)
Atrophy
Intestinal metaplasia
63
4
Regression
No change
587
5
Prevents progression
Prevents progression
54
8
Improvement
Improvement
22
5
Reversibility in some patients
Reversibility in some patients
Zhou (2003)34
552
5
No regression
Regression in antrum (p ( = 0.032), no change in corpus (p ( = 0.151)
Lu (2005)35
179
3
Reduction (p ( 50.01)
Progression (n.s.)
21
1
Improvement (n.s.)
Improvement (n.s.)
795
12
Improvement
Improvement
92
1
Sung (2000)
32
Rocco (2002) Ito (2002)
33
28
Salih (2005)
36
Mera (2005)
29
Arkkila (2006)
37
Regression in antrum (p ( 50.05) Not evaluated Regression in corpus (n.s.)
n.s., non-signi¢cant (p ( 40.05).
has been demonstrated, suggesting the existence of a point of no return where genetic changes already exist and become irreversible despite elimination of the triggering carcinogen (e.g. H. pylori). i At the present time clinical evidence seems to indicate that H. pylori eradication improves or prevents the progression of gastric conditions which are potential precursors of gastric cancer.
References 1. 2. 3. 4. 5. 6. 7. 8.
Malfertheiner P, Megraud F, O'Morain C et al. Current concepts in the management of Helicobacter pylori infection the Maastricht III Consensus Report. Gut. 2007, Jan 17; Epub ahead of print. Gisbert JP, Pajares JM. Review article: C urea breath test in the diagnosis of Helicobacter pylori infection a critical review. Aliment Pharmacol Ther. 2004;20:100 17. Perri F, Manes G, Neri M et al. Helicobacter pylori antigen stool test and 13C urea breath test in patients after eradication treatments. Am J Gastroenterol. 2002;97:2756 62. Vakil N, Rhew D, Soll A et al. The cost e¡ectiveness of diagnostic testing strategies for Helicobacter pylori. Am J Gastroenterol. 2000;95:1691 8. Loy CT, Irwig LM, Katelaris PH et al. Do commercial serological kits for Helicobacter pylori infection di¡er in accuracy? A meta analysis. Am J Gastroenterol. 1996;91:1138 44. Vaira D, Holton J, Menegatti M et al. Review article: Invasive and non invasive tests for Helicobacter pylori infection. Aliment Pharmacol Ther. 2000;14(Suppl. 3):13 22. Dzierzanowska Fangrat K, Lehours P, Megraud F et al. Diagnosis of Helicobacter pylori infection. Helicobacter. 2006;11(Suppl. 1):6 13. Moayyedi P, Deeks J, Talley NJ et al. An update of the Cochrane systematic review of Helicobacter pylori eradication therapy in nonulcer dyspepsia: resolving the discrepancy between systematic reviews. Am J Gastroenterol. 2003;98:2621 6.
38
HELICOBACTER PYLORI INFECTION 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
DuBois S, Kearney DJ. Iron de¢ciency anemia and Helicobacter pylori infection: a review of the evidence. Am J Gastroenterol. 2005;100:453 9. Gasbarrini A, Franceschi F, Tartaglione R et al. Regression of autoimmune thrombocytopenia after eradication of Helicobacter pylori. Lancet. 1998;352:878. Huang JQ, Sridhar S, Hunt RH. Role of Helicobacter pylori infection and nonsteroidal anti in£ammatory drugs in peptic ulcer disease: a meta analysis. Lancet. 2002;359:14 22. Hawkey CJ, Tulassay Z, Szczepanski L et al. Randomised controlled trial of Helicobacter pylori eradication in patients on non steroidal anti in£ammatory drugs: HELP NSAIDs study. Helicobacter Eradication for Lesion Prevention. Lancet. 1998;352:1016 21. Chan FK, To KF, Wu JC et al. Eradication of Helicobacter pylori and risk of peptic ulcers in patients starting long term treatment with non steroidal antiin£ammatory drugs: a randomised trial. Lancet. 2002;359:9 13. Fischbach W, Dragosics B, Kolve Goebeler ME et al. Primary gastric B cell lymphoma: results of a prospective multicenter study. German Austrian Gastrointestinal Lymphoma Study Group. Gastroenterology. 2000;119:1191 202. Ye W, Held M, Lagergren J et al. H. pylori infection and gastric atrophy: risk of adenocarcinoma and squamous cell carcinoma of the esophagus and adenocarcinoma of the gastric cardia. J Natl Cancer Inst. 2004;96:388 96. Helicobacter and Cancer Collaborative Group. Gastric cancer and Helicobacter pylori: a combined analysis of 12 case control studies nested within prospective cohorts. Gut. 2001;49:347 53. Uemura N, Okamoto S, Yamamoto S et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med. 2001;345:784 9. Malfertheiner P, Sipponen P, Naumann M et al. H. pylori Gastric Cancer Task Force. H. pylori eradication has the potential to prevent gastric cancer: a state of the art critique. Am J Gastroenterol. 2005;100:2100 15. Lehours P, Ruskone Fourmestraux A, Lavergne A et al. Which test to use to detect Helicobacter pylori infection in patients with low grade gastric mucosa associated lymphoid tissue lymphoma? Am J Gastroenterol. 2003;98:291 5. Megraud F. Update on therapeutic options for Helicobacter pylori related diseases. Curr Infect Dis Rep. 2005;7:115 20. Chey WD, Fendrick AM. Noninvasive Helicobacter pylori testing for the `test and treat' strategy: a decision analysis to assess the e¡ect of past infection on test choice. Arch Intern Med. 2001;161:2129 32. Moayyedi P, Soo S, Deeks J et al. Systematic review and economic evaluation of Helicobacter pylori eradication treatment for non ulcer dyspepsia. Dyspepsia Review Group. Br Med J. 2000;321:659 64. Malfertheiner P, Fry LC, Monkemuller K. Can gastric cancer be prevented by Helicobacter pylori eradication? Best Pract Res Clin Gastroenterol. 2006;20:709 19. Nardone G, Rocco A, Malfertheiner P. Review article: Helicobacter pylori and molecular events in precancerous gastric lesions. Aliment Pharmacol Ther. 2004;20:261 70. Wong BC, Lam SK et al. Helicobacter pylori eradication to prevent gastric cancer in a high risk region of China: a randomized controlled trial. J Am Med Assoc. 2004;291:187 94. Zhou LY, Lin SR, Ding SG et al. The changing trends of the incidence of gastric cancer after Helicobacter pylori eradication in Shandong area. Chin J Dig Dis. 2005;6:114 15. Uemura N, Mukai T, Okamoto S et al. E¡ect of Helicobacter pylori eradication on subsequent development of cancer after endoscopic resection of early gastric cancer. Cancer Epidemiol Biomarkers Prev. 1997;6:639 42. Ito M, Haruma K et al. Helicobacter pylori eradication therapy improves atrophic gastritis and intestinal metaplasia: a 5 year prospective study of patients with atrophic gastritis. Aliment Pharmacol Ther. 2002;16:1449 56. Mera R, Fontham ET, Bravo LE et al. Long term follow up of patients treated for Helicobacter pylori infection. Gut. 2005;54:1536 40. Take S, Mizuno M, Ishiki K et al. Baseline gastric mucosal atrophy is a risk factor associated with the development of gastric cancer after Helicobacter pylori eradication therapy in patients with peptic ulcer diseases. J Gastroenterol. 2007;42(Suppl. 17):21 7. Tepes B, Kavcic B, Zaletel LK et al. Two to four year histological follow up of gastric mucosa after Helicobacter pylori eradication. J Pathol. 1999;188:24 9.
39
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 32. Sung JJ, Lin SR, Ching JY et al. Atrophy and intestinal metaplasia one year after cure of H. pylori infection: a prospective, randomized study. Gastroenterology. 2000;119:7 14. 33. Rocco A, Suriani R, Cardesi E, Venturini I, Mazzucco D, Nardone G. Gastric atrophy and intestinal metaplasia changes 8 years after Helicobacter pylori eradication. A blind, randomized study. Minerva Gastroenterol Dietol. 2002;48:175 8. 34. Zhou L, Sung JJ, Lin S et al. A ¢ve year follow up study on the pathological changes of gastric mucosa after H. pylori eradication. Chin Med J. 2003;116:11 14. 35. Lu B, Chen MT, Fan YH, Liu Y, Meng LN. E¡ects of Helicobacter pylori eradication on atrophic gastritis and intestinal metaplasia: a 3 year follow up study. World J Gastroenterol. 2005;11:6518 20. 36. Salih BA, Abasiyanik MF, Saribasak H, Huten O, Sander E. A follow up study on the e¡ect of Helicobacter pylori eradication on the severity of gastric histology. Dig Dis Sci. 2005;50:1517 22. 37. Arkkila PE, Seppala K, Farkkila MA, Veijola L, Sipponen P. Helicobacter pylori eradication in the healing of atrophic gastritis: a one year prospective study. Scand J Gastroenterol. 2006;41:782 90.
40
Section III Inflammatory bowel disease section Chair: I FERKOLJ and B VUCELIC
6 Genetics, immunology and biomarkers in clinical practice: do they assist in clinical management? P. L. LAKATOS
INTRODUCTION In recent decades there has been a change in the epidemiology of in£ammatory bowel diseases (IBD) in North America, as well as Western, and some Eastern European countries1^3. Both Crohn's disease (CD) and ulcerative colitis (UC) possibly stem from common mechanisms, while the exact aetiology remains unknown4. Nowadays a generally accepted hypothesis is that UC and CD may develop from a dysregulated response of the mucosal immune system towards microbial antigens in genetically predisposed individuals. IBD are identi¢ed as multifactorial, polygenic diseases with probable genetic heterogeneity. According to this hypothesis, di¡erent genetic backgrounds may explain the various clinical patterns of the disease4,5. In addition to genetic predisposition, various environmental and host factors (e.g. genetic, epithelial, immune, and non-immune) play a major role in the pathogenesis of IBD. Extensive heterogeneity is observed in terms of presentation, extraintestinal manifestations, and location of CD, while behaviour and response to treatment are heterogeneous in both CD and UC6. Furthermore, it is now undisputed that enteric bacterial £ora plays a key role in the pathogenesis of IBD, both in UC and CD. Nonetheless, the exact mechanism by which the intestinal mucosa loses tolerance for its bacterial neighbours remains elusive. According to the traditional view on the pathogenesis of IBD, intestinal in£ammation is mediated by the acquired immune system. The chronic in£ammation could result from an overly aggressive activity of e¡ector lymphocytes and proin£ammatory cytokines, which may overcome the control mechanisms. Alternatively, IBD may result from a primary failure of regulatory lymphocytes and cytokines, such as interleukin 10 (IL-10) and transforming growth factor beta (TGF-b), to control in£ammation and 43
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
e¡ector pathways. In addition, an important mechanism in CD is the resistance of T cells to apoptosis after activation. Recently, this hypothesis has been challenged, in part, based on the results of genetic studies. The equally important role of the host's genetic regulation of the innate immune response in the pathogenesis of CD has been brought to sharp focus by the identi¢cation of the role of the NOD2 (CARD15) gene by three independent groups in 2001. As a consequence, novel genetic determinants of susceptibility and phenotype are currently being widely investigated by genome-wide scanning and studies of positional, and possibly functional, candidate genes in identi¢ed susceptibility regions of certain chromosomes (loci)7. Furthermore, currently no simple diagnostic laboratory tool exists for diagnosing IBD. Biological markers that are potentially useful in IBD include proteins of in£ammation such as C-reactive protein (CRP), fecal calprotectin, and several antibodies. Nonetheless, these biomarkers have many limitations. The role of the various antibodies in the current IBD diagnostic algorithm is often questioned due to their limited sensitivity8. In contrast, the association of serological markers with disease behaviour and phenotype is becoming increasingly well established, while other markers (e.g. CRP, ESR, calprotectin) are mainly helpful in assessing disease activity and predicting relapse. Finally, the sequence of immunological events underlying the in£ammatory reaction in IBD is extremely complex and involves both the innate and antigendriven adaptive immune systems. Evidence indicates that dysregulation of mucosal immunity in the gut of IBD patients leads to altered production of in£ammatory cytokines (e.g. tumour necrosis factor alpha (TNF-a), a IL-1, -2, 4, -5, -10, -12, and more recently IL-17 and IL-23) and tra¤cking of e¡ector leukocytes into the bowel (see Figure 1). The latter mechanism culminates in uncontrolled intestinal in£ammation. Novel biological therapies are directed against several key players in this cascade. Major targets for such treatment include in£ammatory cytokines and their receptors, as well as adhesion molecules. Blockade of T-cell proliferation and activation, and inhibition of Tcell-derived cytokines, has been most extensively targeted by clinical trials in humans in both CD and UC. Inhibition of adhesion molecules and the use of selected growth factors also seem to have a therapeutic potential. In addition, restoration of regulatory T-cell and dendritic-cell functions is still pending further investigation in clinical trials. Thus, recent advances in understanding the immunological events of IBD have led to the discovery of novel targets of biological therapy directed against several key participants involved in the in£ammatory cascade. Since a detailed presentation of the mechanisms involved would go beyond the scope of this review, and the clinical importance in everyday practice is discussed above, we will focus on emerging data concerning the genetics, genotype phenotype associations as well as data available on biomarkers for the diagnosis and prediction of disease course in IBD. By assessing these markers we strive for an objective measurement of disease activity as symptoms may often be subjective; and secondly, to possibly avoid invasive (endoscopic) procedures that are often a burden on the patient. In this review we attempt to answer the following questions: 44
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE Bacterial antigens
Flagellin
I
TLR9
TLR3
~.:--
,, '
~,\
e
\ \
''
~
\lf Figure 1
CpG DNS
LPS
IL-4 IL-5 IL-10
IL·23 IL17
Pathogenesis of IBD (modi¢ed after ref. 5)
45
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
1. Is genetic investigation and detection of biomarkers helpful in the diagnosis of symptomatic IBD patients? 2. Is genetic testing indicated in clinically una¡ected family members to identify patients at higher risk who might require `preventive measures'? 3. Do genetic tests and biomarkers help in further classifying indeterminate colitis patients? 4. Can genetic tests and biomarkers help in predicting disease phenotype and progression (e.g. location, behaviour in CD, extraintestinal manifestations, need for surgery)? 5. Do genetic tests and biomarkers help in predicting relapse or response and side-e¡ects of medical therapy, that is, do they help in `individualizing' the treatment?
GENETICS To this date, a total of nine loci have been found to be associated with IBD, having speci¢c linkage requirements, and were subsequently labeled as IBD1-95 (see Table 1). Furthermore, it is clear that some loci correlate with either UC or CD, while others are involved in the pathogenesis of both IBD forms. Table 1 Locations of nine major loci showing linkage to in£ammatory bowel disease (modi¢ed after ref. 5) IBD locus IBD1 IBD2
Chromosome 16q13 12q14
Identi¢ed genes
Disease
NOD2/CARD15 Not known
CD
(VDR, NRAMp2, MMP18, b2 integrin)
UC
IBD3
6p
Not known (HLA, TNF)
IBD
IBD4
14q11 12
Not known (TCR, LTB4 receptor)
CD
IBD5
5q31 33
SLC22A4/A5 (IRGM, IL 4, 6, CD14)
CD
IBD6
19p13
Not known (ICAM1, C3, TBXA2)
IBD
IBD7
1p36
Not known (TNF R family, IL 23R)
IBD, UC
IBD8
16p12
Not known
CD
IBD9
3p26
Not known (CCR5, CCR9, hMLH1) DLG5 ATG16L1
IBD
10q23 2q37
46
IBD CD
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
Genes involved in the recognition of bacteria NOD2/CARD15 Gene^protein function: In 2001 the identi¢cation of NOD2 (nucleotide oligomerization domain)/CARD15 (caspase activation recruitment domain) on chromosome 16 in the IBD1 region, as a candidate gene for CD9,10, has stimulated further research focusing on novel genetic determinants of susceptibility and phenotype in IBD. The identi¢cation of NOD2/CARD15 is of great importance in understanding the pathogenesis of IBD, especially CD, as NOD2/CARD15 is an intracellular element responsible for the indirect recognition of bacterial peptidoglycan through the binding of muramyl dipeptide11,12. The function of the immune system enables it to recognize a vast number of antigens that are present in our environment (and in the gut), to mount an appropriate response as well as limit the in£ammatory and immune responses elicited by di¡erent pathogens. NOD2/CARD15 serves as direct evidence that dysregulation in the immune system and recognition of bacteria can lead to immune-mediated diseases through uncontrolled in£ammatory processes. The most intriguing question that remains to be answered concerns the mechanism whereby mutations in the NOD2/CARD15 gene predispose towards the chronic intestinal in£ammation characteristic of CD. Gain-offunction and loss-of-function mechanisms were both reported (e.g. increased permeability, activation of NF-kB, regulation of Paneth cells' degranulation, regulation of TLR2 stimulation by peptidoglycans). Interestingly, mice lacking NOD2 or possessing mutated NOD2 variants do not spontaneously develop CD. Together, these data suggest that NOD2 mutations, rather than having a direct causative role in disease development, create an intestinal environment favourable for IBD13, for instance, via decreased expression of a- and bdefensins, thus in£uencing the innate immunity. Disease susceptibility and genotype^phenotype association: Three major mutations were identi¢ed within NOD2/CARD15: one frameshift (3020insC, SNP13) and two missense mutations (R702W-SNP8 and G908R-SNP12). The presence of a NOD2/CARD15 mutation increases the risk for CD by 1.4 4.3-fold in heterozygous patients and 17.6 44.0-fold in homozygous and compound heterozygous patients. Of importance, reports exist of homozygous individuals who are disease-free14. It is estimated that any of these three common mutations, involving NOD2/CARD15, are present in a heterozygous form in 30 50% of CD patients and 7 20% of controls from North America and Europe15^18. However, various geographical di¡erences were noted: a much lower prevalence of these mutations was reported in, for example, African American, Chinese 19 and Japanese 20 populations. The prevalence was also lower in other northern European countries 21. New evidence from association studies in healthy populations and disease indices, however, suggests that CARD15/NOD2 rather interacts with other, unknown risk cofactors22. 47
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
The three common NOD2/CARD15 mutations were associated with ileal disease and ¢brostenosing behaviour. On the contrary, in colonic and ¢stulous disease, they were relatively less frequent10,23,24. The mutations were not associated with UC. In CD the population attributable risk for ileal disease is determined, in 40%, by NOD2/CARD15 and in 20% by HLA genes; the numbers are similar for ileocolonic disease (NOD2/CARD15: 30%, HLA: 40%), while colonic disease is thought to be associated with HLA and other, still unknown, genes23.
NOD1/CARD4 A protein that is structurally and functionally similar to NOD2/CARD15, the NOD1/CARD4, is located on chromosome 7p and translates into an intracellular bacterial pathogen-associated molecular pattern25. Of particular interest is its location in the midst of a region with strong IBD correlation, being translated. Con£icting data are available concerning NOD1/CARD4. Zouali et al. investigated the role of protein in 63 IBD patients. Eleven exons of the NOD1/CARD4 gene were screened for polymorphisms. Indeed, several variants were identi¢ed, none of which, according to the researchers, proved any association with IBD 26 . In contrast, a more recent publication by McGovern et al.25 located a complex insertion/deletion allele on NOD1, associating it with an early onset of IBD as well as extraintestinal manifestations.
Toll-like receptors and CD14 Gene^protein function: Toll-like receptors (TLR) expressed in myeloid cells play a major role both in detecting microbes (lipopolysaccharide) and in initiating the innate immune response. Accordingly, a disturbance in their function predisposes to infections with Gram-negative bacterial pathogens27,28. TLR may also in£uence the nature of the immune response, in particular by skewing T cells towards a Th1 or Th2 pro¢le29. Disease susceptibility and genotype^phenotype association: IBD is characterized by an altered expression pattern of TLR on the surface of intestinal epithelium, with TLR4 expression found to be up-regulated in patients with CD. In contrast, the expression of TLR2 and TLR5 is unchanged, while TLR3, which recognizes viral replication, is down-regulated30. The D299G (Asp299Gly) polymorphism of the TLR4 gene is associated with LPS hyporesponsiveness31 and was found to also be associated with CD (OR 2.45 2.80) and UC (OR 2.05) in the study by Franchimont et al.27, as well as in Greek32 and German studies33. However, results are con£icting, as other studies failed to replicate this association18,34. A recent study by a Belgian group35 investigated 35 SNP in the TLR1 10 genes. Although none of the SNP were involved in disease susceptibility to either CD or UC, a positive association between TLR1 R80T and pancolitis in UC (OR 2.8) was found. The TLR2 R753G SNP was also associated with 48
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
pancolitis (OR 4.7). Additionally, there was a negative association between TLR6 S249P and UC with proctitis only (OR 0.22). In CD a negative association was found between ileal disease involvement and TLR1 S602I (OR 0.52). Con£icting data have emerged on the association between the bacterial receptor CD14 and IBD. Klein et al. found the 159TT genotype to be associated with CD but not with UC36. In a Japanese study37 the same genotype was associated with UC (OR 1.96) but not with CD, and an additional study investigating Caucasian patients failed to demonstrate any association38.
Mucosal integrity and transport SLC22A4/OCTN1, SLC22A5/OCTN2, and OCTN3 Gene^protein function: One of the novel ¢ndings in IBD genetics in 2004 was the identi¢cation of SLC22A4/OCTN1 and SLC22A5/OCTN2 genes coding for integral membrane proteins. The proteins' functions were found to be multispeci¢c and served as bidirectional transmembrane transporters of carnitine and organic cations39. Variant alleles are associated with functional changes: the SLC22A4 variant decreases carnitine, but augments organic cation transporter activity, while the SLC22A5 variant impairs heat shock protein-driven promoter transcriptional activation42. Another possibility is that the enhanced cation transporter activity of the OCTN1 L503F variant may provoke disease by allowing aberrant uptake of toxic substrates. Disease susceptibility and genotype^phenotype association: The association between 5q31 and CD was ¢rst noted in genome-wide screens (the genes for Th2 cytokines e.g. IL-3, -4, -5, -9, -13, and IRF1 map to this region). The risk haplotype is associated with only a moderate risk for CD (OR 1.4 1.5). The e¡ect of NOD2/CARD15 was additive; however, the 5q31 haplotype was found to be an independent risk factor40,41. In a German study it was also associated with UC. Nonetheless, data support signi¢cant geographical heterogeneity as no association was found between the IBD5 haplotype and clinical presentation, nor was it correlated with IBD in a Japanese study41. Based on data available in the literature, IBD5 mainly increases the overall risk for IBD, whereas NOD2/CARD15 mutations are primarily responsible for the determination of phenotype. The SLC22A4 C1672T and the SLC22A5 promoter G207C variants increased the risk for CD by 2 2.5-fold when present as a single copy and by 4-fold in homozygous carriers42,43. The elevated risk, attributed to the OCTN TC haplotype and NOD2/CARD15 mutations, was additive with an odds ratio of 7.3 10.5 in double carriers. In a more recent Japanese study of several di¡erent SLC22A4/A5 and DLG5 polymorphisms and CD, a weak, or even no, association44 was found with very low prevalence of variant alleles, concurring with the Ashkenazi population45, where the frequency of the allele was also low, indicating racial di¡erences. 49
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
In contrast, in a recent study by Vermeire et al.46 , encompassing 981 patients, no association was found between any OCTN/DLG5 variants and IBD. In this study the OCTN TC risk haplotype was associated with penetrating disease (OR 1.474, 95% CI 1.028 2.114; p = 0.035). In a German study43 the TC haplotype was associated with non-¢stulizing non-¢brostenotic disease (OR 1.57), colonic disease (OR 1.31), an earlier age at the onset of disease, and reduced need for surgery (OR 1.38).
DLG5 DLG5 is a member of the MAGUK (membrane-associated guanylate kinase) gene family, which encodes cell sca¡olding proteins and is also involved in the maintenance of epithelial integrity and regulation of cell growth47. This latter role is potentially impaired by expression of the CD-associated DLG5 variants causing increased permeability and disease. The impact on the overall risk of developing IBD is much smaller compared to NOD2/CARD15. The association between DLG5 (Drosophilia ( discs large homolog 5) at chromosome 10q22 23 was ¢rst reported by Stoll et al.48, who showed that the DLG R30Q allele was overtransmitted to individuals with IBD (transmitted/non-transmitted, 90/73; p = 0.09) and CD (transmitted/nontransmitted, 58/43; p = 0.065). In a joint Canada UK study Daly et al.49 could replicate the association between R30Q and IBD in a Canadian/Italian case control cohort; however, the association was not found in another case control cohort from the UK. Nonetheless, the risk attributed to the variant allele was estimated to be modest (in the vicinity of 1.25). An interaction between DLG5 and NOD2/CARD15 was also detected. This interaction, however, was not replicated by the same group in English and Scottish CD patients or by recent publications from Germany 43,50 , Belgium46, Norway21) and by our group that investigated 773 Hungarian IBD patients51. In the Belgian study, overtransmission of the wild-type allele and undertransmission of the variant 113A allele was reported. The same tendency was observed in the Hungarian patients: a protective e¡ect for the 113A variant allele carriers (OR 0.67, 95% CI 0.45 1.013; p = 0.06), Thus, the contribution of this gene to disease susceptibility, if any, is relatively minor. No genotype phenotype associations were found in the studied populations50,51.
CYTOKINES, MULTIDRUG RESISTANCE AND OTHER GENE POLYMORPHISMS Although much is known about the immune mechanisms involved in the interaction between non-pathogenic, commensal bacteria and pathogenic species, and the control of local in£ammatory changes, a lack of reproducibility has challenged many of the other reported genotype phenotype associations in IBD, such as the association between interleukin polymorphisms and IBD. The association between a low-producing allele, allele 2 of the interleukin (IL)-1RA gene and UC (OR 1.3), extensive colitis, and the need for 50
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
colectomy52,53 was reported in a Caucasian population. On the contrary, another study investigating 529 IBD patients failed to con¢rm these results54. Results on IL-10 and IL-18 polymorphisms are also contradictory. In addition, an association was reported between microsatellite loci of TNFa and CD55. Recently the TNF receptor (TNFR) SF1A was reported to be associated with CD56 and was less prevalent in stricturing (OR 0.38) and colonic (OR 0.41) disease. Similarly, di¡erent TNF superfamily member 15 (TNFS15) haplotypes were associated with CD in Japanese patients (OR 2.15 3.23)57. In 2003 Brant et al. described a suggestive linkage on chromosome 7q, containing the multidrug resistance (MDR)-1 gene, in association with the appearance of UC and CD58. This particular gene is a membrane transport protein with several documented human polymorphisms having e¡ects on intestinal absorption and drug pharmacokinetics. In a German study the C3435T mutation, previously associated with a 50% decreased protein secretion, but not the G2677T mutation, correlated with UC (OR 1.6 2.0). The association was strongest in extensive UC (OR 2.64) and severe disease (OR 1.67), yet it showed no manifestation in CD59,60. In a recent English study61 six intronic variant SNP of MDR1 (especially the intron 3 G-A SNP), showed a very signi¢cant association to UC, particularly in patients with extensive disease. However, data are con£icting, since a Spanish group reported that the C3435T CC genotype was associated with CD (OR 1.58)62 and no association was found in eastern European patients63.
A DIFFERENT APPROACH The most recent studies used di¡erent methodology: instead of linkage and candidate gene analyses, broad SNP associations were investigated in genomewide scans. The new data identi¢ed additional pieces in the puzzle of IBD. The importance of altered bacterial sensation is underlined by the ¢nding that an independent association scan of 19 779 non-synonymous SNP in 735 German CD cases and 368 controls has identi¢ed a signi¢cant association of an SNP in the autophagy-related 16-like 1 gene ATG16L1 on chromosome 2q37.1 with CD64. This SNP (rs2241880, T300A), which was associated with CD in two German panels and one British panel of CD patients (ORheterozygous 1.35 1.45; OR homozygous 1.71 1.77), was associated with CARD15. The association was not observed in a German panel of UC cases or trios, suggesting that it is speci¢c to CD. The ATG16L1 protein is involved in the autophagocytosis pathway, which is known to mediate resistance to intracellular pathogens such as bacteria and viral particles65. Subsequent reports66,67 have con¢rmed the association between the variant allele and CD as well as with ileal disease in one study66; however, no evidence of interaction with CARD15, IL-23R, or IBD5 was found. Recently, an English group also reported an association between two SNP in the autophagy-inducing IRGM gene on chromosome 5q33.1 and CD in 2930 cases and 4962 controls. Variant SNP were associated with an OR of 1.36 1.3868. 51
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Recently, an association was reported between rare SNP of the IL-23R gene located on 1q3169 and CD. The variant Arg381Gln allele was associated with decreased susceptibility for CD (OR 0.26). Similar results were reported in con¢rmatory studies (OR 0.38 0.65 and 0.63 0.78) for both CD and UC from the UK70,71. Preliminary evidence suggests an epistatic interaction with the IBD5 risk haplotype. No phenotype genotype associations were discovered.
PHARMACOGENETICS ^ THE ROLE OF GENETIC FACTORS IN PREDICTING THE SUCCESS OF THERAPY AND DRUG TOXICITY Individual response to drugs is a major problem in clinical practice. Pharmacogenetic research in IBD is developing in two directions: (1) the identi¢cation and design of possible new therapeutic targets by exploiting current knowledge of susceptibility genes an area that is rapidly developing; and (2) examination of certain genomic areas that may be valuable in elucidating variations resulting in the lack of therapeutic bene¢t or adverse drug reactions, leading to a more personalized/individualized therapy. The fundamental principle underlying pharmacogenetic investigations is that variations in drug response may re£ect functional di¡erences in a gene product encoded by di¡erent alleles of the same gene. Factors may in£uence either pharmacodynamic (relating drug target responsiveness at ¢xed drug concentrations) or pharmacokinetic (determining drug and metabolite concentrations, including absorption, distribution, metabolism, and elimination) characteristics. At the present time only limited information is available, much of it regarding the genetic basis of drug metabolism and elimination. The genetic regulation of azathioprine/6-mercaptopurine (AZA/6-MP) metabolism has been widely studied and provides perhaps the best example of the clinical application of pharmacogenetics in IBD. Thiopurine Smethyltransferase (TPMT) limits the production of 6-thioguanines (6-TGN) by converting 6-MP to 6-thioruric acid and 6-methylmercaptopurine72, which may lead to myelotoxicity. Approximately 5% of the white population carries one or more variant TPMT alleles, with more than 10 variant alleles reported73. The functional consequences of alleles *2, *2A, *3B, and *3C, accounting together for more than 90% of mutant alleles, have been extensively characterized. Population studies have shown that the distribution of TPMT activity is trimodal: 0.3 0.5% of the population has low to absent activity (TPMTL/ TPMTL), around 10% have intermediate activity (TPMTL/TPMTH), and approximately 90% inherit normal to high enzyme activity (TPMTH/ TPMTH) 74 ; as previously mentioned, a correlation has been observed between the TPMT genotype and enzyme activity. Carriage of two variant alleles was associated with de¢cient TPMT activity, whereas heterozygosity was associated with intermediate enzyme activity. These data suggest that genotyping might identify patients at high risk of haematological toxicity. Unfortunately, a number of factors currently limit the incorporation of TPMT genotyping into clinical practice. The principal limitation is that genotyping 52
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
does not predict TPMT activity in all patients. Homozygosity or compound heterozygosity for TPMT variants is a well-known contraindication to AZA at conventional doses, as it causes early and profound bone marrow suppression. Nevertheless, it is clear that there are many other causes of myelotoxicity. This was accurately demonstrated by Colombel et al.75, who found that only 27% of patients had documented low TPMT activity. Other confounding genetic and environmental factors include, for instance, the patient's age, renal function, AZA formulation, coadministration of mesalazine (a reversible TPMT inhibitor) and allopurinol (XO inhibitor). Thus, the determination of TPMT activity is not an exclusive test to rely on. It may be helpful only in identifying a certain group of high-risk patients but, as the negative predictive value is rather low, it is not bene¢cial in ruling out possible side-e¡ects. Also, as the prevalence of double carriage of variant TPMT alleles is as low as 1/300, continuous monitoring of red blood cell counts remains mandatory in clinical practice. The clinical usefulness of arylamine-N-acetyltransferase 1 (NAT1), glucocorticoid receptor (GCR), MDR1 gene, DLG5, TNF, Fas-ligand, NOD2/CARD15, or 5q31 in predicting response to 5-aminosalicylate, glucocorticoids or in£iximab is even more controversial.
Biomarkers Serological markers Anti-neutrophil cytoplasmic antibody: The classical anti-neutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor the in£ammatory activity in primary small vessel vasculitides. On the basis of an international consensus statement, ANCA testing is performed with serum samples by indirect immuno£uorescence (IIF) on normal peripheral blood neutrophils. Atypical p-ANCA are most commonly seen in patients with IBD, especially UC, and some autoimmune liver diseases such as autoimmune hepatitis and primary sclerosing cholangitis. Atypical p-ANCA is present in the sera of 40 80% of patients with UC76,77 and to a lesser extent in CD (5 25%)78. The prevalence of the antibody is also high in patients with primary sclerosing cholangitis (88%)79 and autoimmune hepatitis type I (81%)80, but is detected in only 1 3% of healthy control subjects. Some sera with atypical ANCA reactivity are positive for antibodies to elastase, lactoferrin, cathepsin G, lysozyme or bactericidal permeabilityincreasing protein (BPI). However, since they are detected in only a few atypical p-ANCA-positive sera, these antigens do not appear to be the primary targets of atypical p-ANCA reactivity. The exact target antigen(s) of atypical p-ANCA has not yet been identi¢ed. An alternative method for the identi¢cation of atypical p-ANCA reactivity developed by Targan and colleagues uses a three-step process that includes enzyme-linked immunosorbent assay (ELISA) analysis, IIF assay on methanol¢xed neutrophils, followed by another IIF testing on deoxyribonuclease (DNase)-treated neutrophils. DNase-sensitive p-ANCA (i.e. not detectable on DNase-treated neutrophils) is present in the sera of 60 80% of the patients with 53
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
UC and in approximately 10 30% of CD patients. In approximately 70% of UC sera there is ablation of the p-ANCA pattern and antigen recognition after DNase digestion of the substrate cells81. This suggests that the epitope recognized by the UC-associated atypical p-ANCA is a protein DNA complex, or that the presence of intact DNA is necessary for maintaining the integrity of the epitope. In up to 30% of the sera there is conversion to homogeneous cytoplasmic staining, while in 3% of the sera the p-ANCA pattern was retained after DNase treatment of the substrate. The overall speci¢city of atypical p-ANCA is 84 95%, with a sensitivity of 48 63%, a positive predictive value (PPV) of 69%, and a negative predictive value (NPV) of 89%8. Anti-Saccharomyces cerevisiae antibodies: The other `conventional' markers, anti-Saccharomyces cerevisiae antibodies (ASCA) are antibodies directed primarily against a 200 kDa phosphopeptidomannan cell wall component of the common baker's or brewer's yeast S. cerevisiae82, eliciting the formation of both IgA and IgG antibodies. Separate and polyvalent ELISA con¢gurations are available for ASCA IgG and IgA detection. ASCA are more frequently found in CD patients (50 80%) compared to patients with UC (2 14%) and normal healthy subjects (1 7%)83^85. Approximately two-thirds of CD patients with ASCA IgG are also positive for ASCA IgA, yet 0 19% of the patients have only ASCA IgA antibodies. This ¢nding suggests that both ASCA IgG and IgA antibodies should be measured. In CD up to 90% speci¢city has been reported in specimens positive for both ASCA IgG and IgA antibodies, especially when the titre of both ASCA antibodies, IgG and IgA, is high86. Sensitivity of ASCA testing ranges from 41% to 76%, with a PPV of 88% and NPV of 68%87. ASCA IgG and IgA levels in CD patients are highly variable85,86. The prevalence of ASCA is much higher in cases of sporadic CD and in families with only CD (63%) compared to families with both CD and UC (33%). The familial trait to ASCA is obvious, but it is questionable whether this is due to the genetic background or the e¡ect of environmental agents during childhood predisposing to the disease. Newly discovered serological markers: Anti-OmpC antibody is directed against the outer membrane porin C transport protein of Escherichia coli. The detection of the IgA antibody is done with ELISA. Anti-OmpC has been reported in 30 55% of CD patients85,88, but in children and young adults it was reported in only 24%89. The prevalence of anti-OmpC was insigni¢cant in UC patients and in healthy subjects (5 11% and 5%, respectively). Anti-OmpC may be of value to aid diagnosis of ASCA-negative CD patients. The prevalence of anti-OmpC among ASCA-negative patients is 5 15%. A fragment of bacterial DNA (I2), a homologue of the tetR bacterial transcriptional factor family, has been identi¢ed from lamina propria mononuclear cells in active CD and shown to be associated with Pseudomonas £uorescens90,91. IgA anti-I2 antibodies have been detected by ELISA in IBD patients with a seroprevalence of 54% in CD and 10% in UC. Anti-I2 antibodies were also found in patients with other in£ammatory enteritis (19%) as well as in healthy subjects (4%)78. 54
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
Serological expression cloning was used by Lodes et al. to identify commensal bacterial proteins in colitic mice. The dominant antigens were found to be £agellins. Intense B-cell and CD4+ T-cell responses were observed against one of these £agellins (anti-CBir1). Colitis was induced when the T-cell line speci¢c for CBir1 was transferred into naive severe combined immunode¢cient mice. Approximately 50% of patients with CD have IgG serum reactivity to CBir1 versus 6% of UC patients and 8% of healthy subjects. CBir1 is the ¢rst bacterial antigen to induce colitis in animal IBD models and also leads to a pathological immune response in IBD patients92. Among the population of CD patients positive for atypical p-ANCA, but who do not react to other known antigens, 40 44% are positive for anti-CBir1, in contrast to UC patients who are positive for atypical p-ANCA. In this latter group it has been found in only 4% of individuals. Serum responses to CBir1 may be of help in di¡erentiating between atypical p-ANCA-positive CD and UC patients, independently of ASCA93. Anti-pancreatic antibodies (PAB) are directed against the exocrine pancreatic tissue. The exact target antigen(s), however, have not yet been identi¢ed. These antibodies are detected using IIF on human pancreas substrate. The reported prevalence of PAB is approximately 30% in CD patients compared to 2 6% of UC patients and 0 2% of control subjects94. The relevance of PAB in the pathogenesis of CD is unclear, and whether the presence of PAB identi¢es a CD subgroup also remains to be determined95. Patients with CD express antibodies to cell wall carbohydrate epitopes present in di¡erent pathogenic bacteria and fungi. Using a glycan array (GlycoChip) and ELISA, antiglycan antibodies have been detected in CD patients, including anti-laminaribioside carbohydrate antibody (ALCA) (18 38%), anti-chitobioside carbohydrate antibody (ACCA, 21 36%), and antimannobioside carbohydrate antibody (AMCA, 28%)96,97. Importantly, 24 44% of ASCA-negative CD patients, in one study96, were found to be positive for one or more of the antiglycan antibodies. Patients with CD who were positive for at least one of ALCA, ACCA, or gASCA (very similar to conventional ASCA) could be di¡erentiated from UC patients, with 77% sensitivity and more than 90% speci¢city. The PPV and NPV were 91% and 77%, respectively. When the same criteria were applied to di¡erentiate CD from control patients, the speci¢city fell to 70.3%. As one might expect, combining two or more antiglycan antibodies resulted in a higher speci¢city and PPV in di¡erentiating CD from UC, but with a loss of sensitivity, NPV, and e¤ciency. The prevalence of di¡erent serological markers in IBD and their association with the disease phenotype is summarized in Table 2.
DIAGNOSTIC VALUE OF SEROLOGICAL MARKERS IN IBD The role of atypical p-ANCA and ASCA as diagnostic markers for IBD appears to be limited because of their moderate sensitivity and presence in other conditions. Atypical p-ANCA can also be observed in other forms of colitis; for instance collagenous or eosinophilic colitis, and in various autoimmune liver diseases such as autoimmune hepatitis (AIH) and primary 55
56
41^76
24^55
54
ASCA
Anti-Omp (IgA)
Anti-I2 (IgA)
2^28
Crohn's disease (%)
10
5^24
5^15
45^82
Ulcerative colitis (%)
4
5^20
5
1^7
Healthy subjects (%)
In£ammatory enteritis (19%) Stricturing form Early need for surgery
Identi¢es ASCA^ CD patients Penetrating disease Faster disease progression Early need for surgery
Assists in di¡erentiating between CD and UC: Atypical p-ANCA+/ASCA^: UC Atypical p-ANCA^/ASCA+: CD CD: ASCA+: ileal involvement, complicated disease course, early need for surgery Atypical p-ANCA+: left-sided colitis, good therapeutic response, uncomplicated disease course UC: Atypical p-ANCA+: severe left-sided colitis, refractory to medical therapy, early need for surgery
Clinical signi¢cance
Prevalence of di¡erent serological markers in IBD and their association with disease phenotype (modi¢ed after ref. 8)
Atypical p-ANCA
Table 2
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
(continued)
57
36
27^39
PAB (IIF)
50
Crohn's disease (%)
Antiglycan antibodies (ALCA, ACCA, AMCA)
Anti-CBir1 (IgG)
Table 2
2^6
3^10
6
Ulcerative colitis (%)
0^2
0^6
8.
Healthy subjects (%)
High speci¢city, low sensitivity Signi¢cance? Distinct CD subgroup?
44% in ASCA^ patients ALCA^ more penetrating; ACCA-stenosing form (small di¡erences) Complicated behaviour, surgery?
Flagellin (CBir1) induces colitis in animal models of IBD Leads to a pathological immune response in IBD patients Di¡erentiation between atypical p-ANCA+ CD and UC Small bowel involvement Penetrating and stenosing form
Clinical signi¢cance
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
sclerosing cholangitis (PSC)98,99. ASCA has been found in autoimmune hepatitis (20%) and gastrointestinal disorders such as coeliac disease100. The combination of atypical p-ANCA and ASCA, however, may be of help in patients in whom distinction between CD and UC is not obvious with the classic diagnostic tools (patient history, radiological examination, endoscopy, and biopsy). The ASCA+/atypical p-ANCA serological pattern is mainly characteristic of CD, while the ASCA /atypical p-ANCA+ is characteristic of UC. Several independent studies found that these combinations had sensitivities ranging from 30% to 64%, speci¢city of more than 90%, and a PPV from 77% to 96%84,85,101,102. It must be emphasized that neither ASCA nor atypical p-ANCA negativity rules out IBD. Similarly, the presence of these antibodies does not con¢rm the diagnosis of IBD. Several groups have studied whether atypical p-ANCA and ASCA are subclinical markers of familial IBD. Some studies103,104 showed that atypical p-ANCA occurred frequently in healthy ¢rst-degree relatives of UC patients, whereas other studies were unable to con¢rm this observation105,106. ASCA positivity was obviously found at a higher rate in una¡ected ¢rst-degree relatives of CD patients than in the general population (20 25% vs 5%)107,108. Further studies are necessary to clarify whether the una¡ected, ASCA-positive, family members face an increased risk of disease development. In addition, Israeli et al.109 demonstrated that the presence of ASCA and atypical p-ANCA in healthy subjects can predict for the occurrence of IBD before the emergence of overt clinical manifestations. Serum samples were systematically obtained and stored from 5% of all military recruits. ASCA were detected in 31% of CD patients before clinical diagnosis. The mean interval between ASCA detection and diagnosis was 38 months. There was no ASCA positivity in the control population. Atypical p-ANCA was present in 25% of patients with available sera before the diagnosis of UC. None of their 24 matched controls was positive. At last, serological evaluation may be of help in patients with indeterminate colitis (IC) to increase the diagnostic accuracy. Ninety-seven patients with IC were enrolled, analysed for atypical p-ANCA and ASCA, and followed up prospectively in a multicentre study by Joossens et al. At the end of the 1-year follow-up a de¢nitive diagnosis was reached in 31 of 97 patients (37%). In IC patients, ASCA+/atypical p-ANCA results correlated with CD in 80%, whereas ASCA /atypical p-ANCA+ correlated with UC in 63%. The remaining ASCA /atypical ANCA+ patients were eventually determined to su¡er from CD, but clinically showed a UC-like CD phenotype. Remarkably, during the 9.9 years of follow-up, 48.5% of the patients did not demonstrate ASCA or atypical p-ANCA. In 85% of these seronegative patients the diagnosis remained indeterminate. In contrast, 48% of the seropositive patients were declared to have CD or UC on follow-up110. Adding anti-OmpC and anti-I2 to the serological panel in patients with IC did not add diagnostic clari¢cation111. Recently, a sophisticated, computer-aided system was developed from a sequential analysis of serological assay results using two-stage statistical classi¢ers including a neural network. In the validation cohort the overall accuracy of the panel for IBD diagnosis was as high as 92% (sensitivity 93%, speci¢city 95%)112. 58
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
ASSOCIATION WITH DISEASE PHENOTYPES AND PROGRESSION The occurrence of atypical p-ANCA in UC is associated with a characteristic clinical appearance and represents a distinct subgroup that is often characterized by speci¢c HLA markers. These patients have a higher probability to develop a severe left-sided UC, which is more resistant to treatment than the usual case. The disease has a more aggressive pattern requiring surgery earlier in the course of the disease113. Some authors suggest that pouchitis develops more frequently after ileal pouch anastomosis114, whereas others were unable to con¢rm this observation. The presence of atypical p-ANCA identi¢es a subgroup of CD patients characterized by `UClike' colitis; the in£ammation usually involves the left side of the colon and the response to therapy is generally good. The atypical p-ANCA in CD patients is associated with a later age of onset and a relatively lower incidence of complications, such as strictures and/or perforations115^117. The phenotype and the disease course of CD depend heavily on the presence and extent of the serological response targeted against various microbial antigens. In patients with an ASCA+ (IgG and/or IgA)/atypical p-ANCA phenotype, small bowel involvement (with or without colonic disease) is more typical than the pure colonic disease (68 76% vs 34 46%)84,85,118,119. ASCA positivity predicts a more aggressive disease course with a higher rate of complications. ASCA have been associated with stricturing (70%) and penetrating (51%) forms of disease as opposed to the in£ammatory one (14%) and a higher risk of small bowel resection113,116,120. Several studies suggest that ASCA positivity is associated with an earlier onset of disease115. ASCA IgA positivity in children may represent a higher risk for relapses (OR 2.9, 95% CI 1.33 6.33)121. The presence of anti-OmpC in adult CD patients is associated with increased prevalence of the penetrating form only87,116,117, while in children both the penetrating and stenosing forms122 are more frequent. Moreover, antibody positivity may lead to a more aggressive course of disease and a higher risk for surgical interventions. Like ASCA and anti-OmpC, anti-I2 also appears to be associated with an increased risk for complications in adult CD patients. It is an independent risk factor for the development of the stenosing form and the need for surgical interventions85,87,116,117. Recent research has shown that the anti-CBir1 antibody is associated with ileal involvement in adult CD patients independent of other serological markers, and it predisposes to the development of both stenosing and penetrating forms92. Among the antiglycan antibodies in CD, ALCA is more often positive in the penetrating form (34% vs 25%) and ACCA in the stenosing one (29% vs 18%) when compared to the in£ammatory type. Nonetheless, the di¡erences are small. No correlation was found between antiglycan positivity and the need for small bowel resections. The number of antibodies produced against microbial antigens in CD shows a positive correlation with the severity of the disease. Mow et al. 120 retrospectively analysed 303 patients and found that the simultaneous 59
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
presence of three antibodies (ASCA, anti-OmpC, and anti-I2) resulted in an increased risk of complications (stenosing form 72% vs 23%, penetrating form 58.7% vs 27.9%, and the need for surgical intervention 72% vs 23%, as compared to the seronegative group. When all three antibodies were present, the OR was 8.6 (95% CI 4.0 18.9). In addition to qualitative correlations, quantitative correlations with serological responses are also present. Patients expressing high titres of serological markers are more likely to have a complicated course of small bowel CD. In a prospective paediatric cohort (n ( = 196), Dubinsky et al.122 120 found, as did Mow et al. , that the presence and magnitude of immune responses to microbial antigens (ASCA, anti-OmpC, anti-I2, and anti-CBir1) are signi¢cantly associated with more aggressive disease phenotype. The risk of developing penetrating and/or stricturing CD was increased 11-fold in those individuals with immune responses to all four microbial antigens compared to seronegative cases (95% CI 1.5 80.4). Moreover, they demonstrated that the duration required to develop a disease complication during the 18 months of follow-up period was signi¢cantly shorter in those children who had a serological response against at least one antigen. A di¡erence exists between cohort studies performed in children and adults, indicating variations with regard to which immune response has the greatest e¡ect on disease course in children and adults. The explanation for this variability is not yet known. The presence and elevated titre of several antiglycan antibodies also predisposes to the development of complicated ileal CD97. In patients positive for more than two antiglycan antibodies (ALCA, ACCA, gASCA), ileal involvement can be seen in 93% of the cases, as compared to seronegative patients (60%) (OR 9.0, 95% CI 3.3 24.5). High ALCA titres seem to correlate with small bowel involvement and the development of stenosing and penetrating disease forms. ACCA titres, on the other hand, demonstrated no such associations. One should be aware of the fact that blatantly striking di¡erences in serological response are demonstrated only in a minority of patients. About one in four of the patients are positive for several antibodies and have markedly, and simultaneously, elevated antibody titres. The proportion of seronegative patients, or patients positive for only one antigen with a low antibody titre, is approximately equal. The remaining 50% of all CD patients have an intermediate phenotype based on the serological assessments. The aim is to ¢nd new serological markers which will be utilized to identify certain homogeneous groups of patients in this `grey zone' of disease progression and response to therapy.
SEROLOGICAL MARKERS IN THE FOLLOW-UP AND TREATMENT OF IBD In patients with UC no correlation was found between the presence and titre of atypical p-ANCA and the activity of the disease. The titre of atypical p-ANCA remains unchanged even after a colectomy123. Similarly, the presence of ASCA in CD patients is relatively constant during the course of the disease and seems to be independent of disease activity115,118. As a consequence, neither atypical 60
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
p-ANCA nor ASCA is suitable for monitoring of the disease. The ALCA and ACCA titres also seem to be independent of disease activity, but rather depend on disease duration97. The prevalence of ASCA, anti-I2, anti-OmpC, and the presence of multiple serological responses are more frequent when the disease persists for a long period of time116,124. Landers et al. discovered that, following anti-TNF-a treatment, the prevalence and titre of various antibodies (ASCA, atypical p-ANCA, anti-I2, and anti-OmpC) remained unchanged in the majority of patients 88 . Mesalamine treatment also leaves the ASCA level unchanged in active CD patients125. ASCA positivity remains even after steroid treatment, yet the antibody titre declines126. The role of the serological response in the prediction of therapeutic e¡ectiveness is yet to be determined. A Belgian study involving 279 CD patients failed to ¢nd any correlation between the atypical p-ANCA or ASCA positivity and the rate of response of patients given anti-TNF-a treatment. The investigators observed a generally poorer responsiveness in the case of atypical p-ANCA+/ASCA status, but the di¡erence was insigni¢cant127. Patients exhibiting serological responses directed against various microbial antigens (OmpC and I2) should expect a higher remission rate if the budesonide treatment was supp lemented with cip ro£oxacin and metronidazole, while in the anti-OmpC/I2 seronegative group budesonide treatment alone proved to be more e¡ective128. The study raises the possibility that certain antibiotics are more e¡ective in those CD patients who present with a marked immunological response against microbial antigens. This group of patients may be the one that can be most e¡ectively treated through manipulation of the bacterial £ora.
SEROLOGICAL MARKERS AND RECEPTORS PARTICIPATING IN INNATE IMMUNITY Genetic heterogeneity may be responsible for the di¡erences found in the serological response. Literature references are divided regarding the possible correlation between antibody production in patients with CD and the NOD2/ CARD15 status. Several studies found serological response (atypical p-ANCA, anti-I2, anti-OmpC, ASCA IgG/A) to be independent of the NOD2/CARD15 status 117,120 . Belgian and Hungarian work groups, on the other hand, con¢rmed the proposed association in large (n = 913 and n = 658, respectively) cohorts of patients. When more than one NOD2/CARD15 mutation was present, the investigators found the rate of ASCA, gASCA (glycanASCA) positivity and their titres to be signi¢cantly higher. Moreover, they found a positive correlation between the number of mutations and the prevalence of ASCA, gASCA, and ALCA, which may indicate a gene dose e¡ect85,97. Investigating the association of the serological response with the toll-like receptor (TLR)-4 gene D299G (Asp299Gly) polymorphism led to results contrasting those found with the NOD2/CARD15 assessments. If the patient had a variant TLR4 genotype the rates of ACCA and Omp positivity were less 61
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
frequent compared to the wild type, which again suggests a gene dose e¡ect. Polymorphisms may also play a role in the development of serological responses against the Gram-negative E. coli membrane protein. The Belgian work group also found a correlation between anti-Omp positivity and TLR4 gene polymorphism in patients with ulcerative colitis (n ( = 272)127. Anti-Omp positivity was signi¢cantly lower among patients with a TLR4 variant genotype and prevalence of the antibody showed an inverse correlation with the number of variants. This was, however, not con¢rmed by ¢ndings of the Hungarian group for either ASCA or Omp.
SERUM AND FECAL MARKERS C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and other laboratory markers ESR and CRP are traditional non-speci¢c markers for in£ammation. ESR is de¢ned as the rate at which erythrocytes migrate through the plasma. Inevitably, ESR will depend on the plasma concentration and on the number and size of the erythrocytes. Conditions such as anaemia, polycythemia, and thalassaemia a¡ect ESR. Compared with CRP, ESR will peak much less rapidly and may also take several days to decrease, even if the clinical condition of the patient or the in£ammation is ameliorated. Increases in ESR with age have also been described129. CRP is a pentameric protein consisting of ¢ve monomers and is one of the most important acute-phase proteins in humans 1 3 0 . Under normal circumstances CRP is produced by hepatocytes in low quantities (51 mg/L). However, following an acute-phase stimulus, such as in£ammation, hepatocytes rapidly increase the production of CRP and may reach peak levels of 350 400 mg/L. Generally, CRP levels of 10 40 mg/L are found in cases of mild in£ammation or viral infections131,132. Although CRP is non-speci¢cally up-regulated in most in£ammatory diseases, including IBD, there is remarkable heterogeneity in the CRP response between CD and UC. Whereas CD is associated with a strong CRP response, UC has only a modest or no CRP response in both adult and paediatric IBD populations133,134. This is an important fact to keep in mind when using CRP in clinical practice. There is no good explanation for this heterogeneity. Recent studies have suggested that polymorphisms in the CRP gene, located on the long arm of chromosome 1 (1q23 24), account for interindividual di¡erences in baseline CRP production in humans. Results are, however, con£icting, and one recent study investigating CRP polymorphisms in IBD patients showed no clear association with serum CRP levels135. More generally used laboratory markers include white blood cell count, platelets, and albumin. The white blood cell count will increase as part of the acute-phase response. Increased leukocytosis is therefore not a speci¢c feature of IBD and may also be seen in other in£ammatory conditions and stressful events. White blood cell count is also in£uenced by some treatments used in 62
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
IBD, such as glucocorticoids (increased) or azathioprine and 6-mercaptopurine (decreased). Platelet count will also increase and is therefore an indication, without being a speci¢c marker, of in£ammation. Albumin is a typical example of a negative acute-phase reactant and decreased levels may be found during in£ammation. However, other conditions such as malnutrition and malabsorption may also lead to low albumin levels. Other acute-phase reactants include sialic acid, a1-acid glycoprotein or orosomucoid, ¢brinogen, lactoferrin, b 2 -microglobulin, serum amyloid A, a 2 -globulin, and a 1 antitrypsin, but these have not been widely studied in IBD. A number of potentially important new serum markers could be identi¢ed by using advanced technology. Recently, protein sera pro¢les of CD, UC, in£ammatory and healthy controls were obtained with a surface enhanced laser desorption ionization time of £ight mass spectrometer (SELDI-TOFMS). This technology and the use of the multiple decision trees method led to protein biomarker patterns analysis and allowed for the selection of potential individual biomarkers with good sensitivity and speci¢city (minimum 80%). Four biomarkers showed important diagnostic value (PF4, MRP8, FIBA, and Hpa2)136.
Faecal calprotectin and other faecal markers A number of neutrophil-derived proteins present in stool have been studied, including faecal lactoferrin, lysozyme, elastase, myeloperoxidase, and calprotectin137,138. Calprotectin, a 36 kDa calcium- and zinc-binding protein, is probably the most promising marker for various reasons. In contrast with other neutrophil markers, calprotectin represents 60% of cytosolic proteins in granulocytes. The presence of calprotectin in faeces can therefore be seen as directly proportional to neutrophil migration to the gastrointestinal tract. Although calprotectin is a very sensitive marker for detecting in£ammation in the gastrointestinal tract, it is not a speci¢c marker and increased levels are also found in neoplasia, IBD, infections, and polyps. Faecal calprotectin is a very stable marker (stable for more than 1 week at room temperature) and is resistant to degradation, which makes it an attractive option. Early studies using faecal calprotectin in IBD have shown a good correlation with In-labelled leukocyte excretion and intestinal permeability139. Interestingly, increased faecal calprotectin levels have been reported after the use of non-steroidal anti-in£ammatory drugs as well as with increasing age. Further, potentially interesting markers are faecal S100A12 (calgranulin C)140 and lactoferrin141. Calgranulin C is a member of the S100 family of calcium-binding proteins speci¢cally expressed by granulocytes. Extracellular S100A12 exhibits proin£ammatory functions, including potent chemotactic activity, comparable with other chemotactic agents. In addition, bovine S100A12 is a ligand for the receptor for advanced glycation end-products (RAGE), which is found on macrophages, endothelium, and lymphocytes. Binding of S100A12 to RAGE mediates its proin£ammatory properties.
63
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Diagnostic value of biomarkers in IBD Only a few studies have investigated the value of laboratory markers in identifying individuals at risk for IBD. An early study from Shine et al. investigated 82 adults referred with abdominal symptoms142. In all patients a clinical examination, as well as a rectal biopsy, were performed, and ESR and CRP were determined. Of these markers, CRP was increased in all patients who subsequently were diagnosed with CD ((n = 19), in 50% of patients diagnosed with UC (n ( = 22), yet in none of the 41 patients with functional bowel symptoms. In a paediatric study by Beattie et al., children underwent extensive blood analysis (including haemoglobin, leukocyte count, platelet count, ESR, albumin, and CRP), ileocolonoscopy, and small bowel followthrough133. Twenty-six children were ¢nally diagnosed with CD, 13 with UC, eight with polyps, seven with various gastrointestinal disorders and 37 had a normal investigation. The best laboratory marker in di¡erentiating IBD from controls was CRP. Similar to the study by Shine and colleagues, 100% of CD patients but only 60% of UC patients had increased CRP compared with none of the children with polyps or those with a normal investigation. ESR proved to be the second-best marker. Finally, a more recent, larger study in 203 individuals referred for symptoms suggestive of lower bowel disease also showed that CRP was a good marker in di¡erentiating IBD from irritable bowel syndrome (IBS)143. Together, these studies seem to suggest that CRP is the most sensitive marker in detecting IBD, but values range between 50% and 60% for UC and between 70% and 100% for CD, and also depend on the cut-o¡ value used. In addition, high-sensitivity CRP assays have been studied in other in£ammatory conditions, such as atherosclerotic heart disease. Faecal calprotectin has been shown to enable the diagnosis of IBD. In this respect a cut-o¡ value of 30 mg/g had 100% sensitivity in discriminating active CD from IBS in the study by Tibble and colleagues137. In a paediatric study by Fagerberg and colleagues 144 , 36 children with symptoms and suspected in£ammation of the colon were subjected to stool analysis for faecal calprotectin and an ileocolonoscopy. Twenty-two patients showed in£ammation on endoscopy (of whom 20 were later diagnosed with IBD), and calprotectin levels were much higher in these patients than in children without in£ammation on endoscopy. The authors concluded that faecal calprotectin is helpful in the detection of colonic in£ammation in children with gastrointestinal symptoms suggestive of IBD, and that a positive test may prioritize endoscopy. Interestingly, similarly to serological markers, increased faecal calprotectin has been described in healthy ¢rst-degree relatives of patients with CD145. In a recent meta-analysis by von Roon et al.146 the authors concluded that calprotectin possessed a good diagnostic value in distinguishing IBD from non-IBD diagnoses; however, at a cut-o¡ value of 100 mg/g.
64
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
DO BIOMARKERS PREDICT DISEASE ACTIVITY, COURSE, AND EFFICACY OF TREATMENT IN IBD? IBD follows an alternating disease course and both CD and UC are characterized by periods of remission and relapses. However, disease £are-ups occur in a random way and are often unpredictable. CRP has been shown to be a good marker for predicting disease course and outcome in a number of diseases, thus its alteration is non-speci¢c for IBD. Its most well-known association is with cardiovascular disease and poor outcome after myocardial infarction147, and a poor prognosis in multiple myeloma148. In general, patients with severe disease more often have abnormal in£ammatory markers, compared with patients without or with only lowgrade in£ammation. This has been shown in a prospective study by Tromm and colleagues149, who investigated laboratory markers, including ESR, serum albumin, a1-proteinase inhibitor, cholinesterase, CRP, and haematocrit, and correlated them with endoscopic activity. One of the early studies in IBD showed a good correlation between ESR and clinical activity 150 . The correlation was, however, dependent on disease location, and ESR did not correlate as well with UC restricted to the rectum and with CD restricted to the upper small bowel. The study by Fagan et al. showed that both CRP and ESR correlated well with disease activity but the correlation was better for CRP151. In addition, the correlation of laboratory markers with disease activity has been shown to be much stronger for CD than for UC. However, a wide range of CRP values was observed and no clear cut-o¡ values exist between mild to moderate (10 50 mg/L), moderate to severe (50 80 mg/L), and severe disease (480 mg/L). Therefore, the comparison of individual CRP values with previous values in any given patient is of great clinical importance. Lastly, CRP showed acceptable correlation with endoscopic and histological activity in CD152. For UC, again, this correlation was weaker. In contrast, recent data from Australia suggested that a subgroup of patients with active ileal disease, low body mass index (BMI), and previous ileocaecal resection was characterized by low CRP153. In CD a number of studies have investigated a panel of laboratory markers in predicting clinical relapse. In a prospective study by Brignola et al. 41 CD patients with clinically inactive disease (CD activity index 5150) were followed-up for 6 months using a panel of in£ammatory markers (ESR, white blood cells, haemoglobin, albumin, a 2 -globulin, serum iron, CRP, a 1 glycoprotein, and a2-antitrypsin)154. A total of 17/41 patients relapsed. ESR, a2-globulin, and a1-glycoprotein proved best at distinguishing relapsers from non-relapsers. Based on these markers a prognostic index (PI) was calculated. Using this threshold all patients with a PI 40.35 relapsed over a period of 18 months, compared with 5/29 patients with a PI 50.35. Later, Boirivant et al. prospectively followed 101 outpatients with CD155. Half of the patients had an elevated CRP value and this correlated well with clinical activity. Approximately one-third of CD patients presented with active disease despite normal CRP and one-third had high CRP levels but clinically inactive disease. The likelihood of relapse after 2 years was higher in the patients with an increased CRP compared with those who had normal CRP. 65
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
More recently, the GETAID group156 proposed a simple biological score for predicting short-term relapse in CD. Multivariate analysis selected two markers predictive of relapse: CRP 420 mg/L and an ESR 415 mm/h. The relative risk of short-term relapse for patients with a positive score compared to those with a negative score was 8.0 (95% CI 2.8 22.9). Sensitivity of the score was 89% and speci¢city was 43% with a negative predictive value of 97%, suggesting that normal CRP and ESR could almost certainly rule out a relapse in the next 6 weeks. Much less data exist on the value of laboratory markers in assessing disease course and outcome in UC. The well-known prospective study from Oxford evaluated 49 severe UC patients treated with hydrocortisone and/or cyclosporin (n ( = 49). On day 3 a daily stool frequency of 48 or 3 8 with increased CRP (445 mg/L) predicted with 85% certainty the need for colectomy157. Only one relatively small study investigated the role of hs-CRP in IBD158. Ninety CD, 70 UC patients, and 160 controls were investigated. The coe¤cient of correlation between hs-CRP and the disease activity score was weak but similar in both UC (0.26) and CD (0.36). More recently, faecal calprotectin was shown to predict relapse of CD. In a study by Tibble et al. calprotectin levels of 50 mg/g or more predicted a 13-fold increased risk for relapse159. In another study160, 38 CD and 41 UC patients were investigated. All patients were in remission for a mean duration of 5 months. A baseline level of calprotectin of 150 mg/g or more was predictive for a relapse in the next year. Although sensitivity was high for both CD (87%) and UC (89%), speci¢city was much lower in the case of CD (43%) compared to UC (82%). Similarly, in a recent study, faecal calprotectin and lactoferrin were able to di¡erentiate organic colorectal disease and were associated with active disease at endoscopy, both in UC (78% and 75%, respectively) and in CD (87% and 82%, respectively)161; however, speci¢city was overall low. It is thus di¤cult to conclude from cut-o¡ values based on these studies. In addition to serum markers, faecal calprotectin also correlates well with endoscopic and histological activity in patients with UC and CD, and elevated calprotectin levels normalize once the in£ammation has resolved162. The role of further possible markers (e.g. b2-microglobulin) is con£icting. A change in CRP, following therapy, serves as a good parameter in assessing the e¡ectiveness of the drug on the underlying in£ammation. A decrease in CRP, in response to therapy, is objective evidence that the drug has a bene¢cial e¡ect on intestinal in£ammation, even in patients with small changes in their symptoms. On the other hand, persistently raised CRP indicates failure of the therapy to control mucosal in£ammation. This was clearly demonstrated by the di¡erent response rates to anti-TNF-a antibodies in patients with CD. A high baseline (5 10 mg/L) CRP value before the start of therapy was associated with a higher response compared to patients with lower CRP163,164. These ¢ndings raise the question of whether CRP should be included in patient selection for future clinical trials, at least for selected drugs. Furthermore, high CRP (420 mg/L) was found to be an independent predictor for relapse after azathioprine withdrawal in patients on azathioprine therapy for longer than 42 months165. Lastly, calprotectin, at a cut-o¡ value of 200 mg/L, at 3 months post-surgery, was useful in predicting endoscopic post-surgical recurrence in 66
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE
asymptomatic patients with a sensitivity of 63% and a speci¢city of 75%166. In contrast, in a recent Italian study167, only lactoferrin correlated signi¢cantly with CRP and showed a reliable threshold value for systemic in£ammation following ileocolonic resection.
CONCLUSIONS In conclusion, the answer to the question of whether genetic, immunological or biomarkers are useful in the current everyday clinical practice, is: maybe. Some of the markers (e.g. CRP, ESR, p-ANCA or ASCA) are already widely or increasingly used, while accepting their limitations. Further prospective clinical studies are needed to establish the clinical role for other (e.g. genetic or additional serological) tests in IBD. In the future, speci¢c diagnostic and prognostic panels that include various genetic, serological and other biomarkers, as well as clinical and environmental factors (e.g. smoking) could be the most likely approaches to diagnosis, prediction of disease course, and response to therapy in IBD; however, we have to note that adding genetic aspects will not replace proper clinical judgement by the physician, who relies on the patient's symptoms and clinical phenotype. Finally, recent advances in understanding the immunological events of IBD have led to the discovery of novel biological therapies directed against several key players involved in the in£ammatory cascade.
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GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE 79. Terjung B, Worman HJ. Anti neutrophil antibodies in primary sclerosing cholangitis. Best Pract Res Clin Gastroenterol. 2001;15:629 42. 80. Terjung B, Bogsch F, Klein R et al. Diagnostic accuracy of atypical p ANCA in autoimmune hepatitis using ROC and multivariate regression analysis. Eur J Med Res. 2004;9:439 48. 81. Vidrich A, Lee J, James E, Cobb L, Targan S. Segregation of pANCA antigenic recognition by DNase treatment of neutrophils: ulcerative colitis, type 1 autoimmune hepatitis, and primary sclerosing cholangitis. J Clin Immunol. 1995;15:293 9. 82. Main J, McKenzie H, Yeaman GR et al. Antibody to Saccharomyces cerevisiae (bakers' yeast) in Crohn's disease. Br Med J. 1988;297:1105 6. 83. Quinton JF, Sendid B, Reumaux D et al. Anti Saccharomyces cerevisiae mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in in£ammatory bowel disease: prevalence and diagnostic role. Gut. 1998;42:788 91. 84. Peeters M, Joossens S, Vermeire S, Vlietinck R, Bossuyt X, Rutgeerts P. Diagnostic value of anti Saccharomyces cerevisiae and antineutrophil cytoplasmic autoantibodies in in£ammatory bowel disease. Am J Gastroenterol. 2001;96:730 4. 85. Papp M, Istvan Altorjay I, Norman GL et al. Sero reactivity to microbial components in Crohn's disease is associated with ileal involvement, non in£ammatory disease behaviour and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patient. In£amm Bowel Dis. 2007;13:984 92. 86. Norman GL. Anti Saccharomyces cerevisiae antibodies in in£ammatory bowel disease. Clin Applied Immunol Rev. 2001;2:45 63. 87. Vermeire S, Joossens S, Peeters M et al. Comparative study of ASCA (anti Saccharomyces cerevisiae antibody) assays in in£ammatory bowel disease. Gastroenterology. 2001;120:827 33. 88. Landers CJ, Cohavy O, Misra R et al. Selected loss of tolerance evidenced by Crohn's disease associated immune responses to auto and microbial antigens. Gastroenterology. 2002;123:689 99. 89. Zholudev A, Zurakowski D, Young W, Leichtner A, Bousvaros A. 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. 90. Sutton CL, Kim J, Yamane A et al. Identi¢cation of a novel bacterial sequence associated with Crohn's disease. Gastroenterology. 2000;119:23 31. 91. Wei B, Huang T, Dalwadi H, Sutton CL, Bruckner D, Braun J. Pseudomonas £uorescens encodes the Crohn's disease associated I2 sequence and T cell superantigen. Infect Immun. 2002;70:6567 75. 92. Lodes MJ, Cong Y, Elson CO et al. Bacterial £agellin is a dominant antigen in Crohn disease. J Clin Invest. 2004;113:1296 306. 93. Targan SR, Landers CJ, Yang H et al. Antibodies to CBir1 £agellin de¢ne a unique response that is associated independently with complicated Crohn's disease. Gastroenterology. 2005;128:2020 8. 94. Lawrance IC, Hall A, Leong R, Pearce C, Murray K. A comparative study of goblet cell and pancreatic exocine autoantibodies combined with ASCA and pANCA in Chinese and Caucasian patients with IBD. In£amm Bowel Dis. 2005;11:890 7. 95. Stocker W, Otte M, Ulrich S et al. Autoimmunity to pancreatic juice in Crohn's disease. Results of an autoantibody screening in patients with chronic in£ammatory bowel disease. Scand J Gastroenterol Suppl. 1987;139:41 52. 96. Dotan I, Fishman S, Dgani Y et al. Antibodies against laminaribioside and chitobioside are novel serologic markers in Crohn's disease. Gastroenterology. 2006;131:366 78. 97. Ferrante M, Henckaerts L, Joossens M et al. New serological markers in in£ammatory bowel disease are associated with complicated disease behaviour. Gut. 2007;Apr 24 (Epub ahead of print). 98. Czaja AJ, Shums Z, Donaldson PT, Norman GL. Frequency and signi¢cance of antibodies to Saccharomyces cerevisiae inautoimmune hepatitis. Dig Dis Sci. 2004;49:611 18. 99. Reddy KR, Colombel JF, Poulain D, Krawitt EL. Anti Saccharomyces cerevisiae antibodies in autoimmune liver disease. Am J Gastroenterol. 2001;96:252 3. 100. Vernier G, Sendid B, Poulain D, Colombel JF. Relevance of serologic studies in in£ammatory bowel disease. Curr Gastroenterol Rep. 2004;6:482 7.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 101. Linskens RK, Mallant Hent RC, Groothuismink ZM et al. Evaluation of serological markers to di¡erentiate between ulcerative colitis and Crohn's disease: pANCA, ASCA and agglutinating antibodies to anaerobiccoccoid rods. Eur J Gastroenterol Hepatol. 2002;14:1013 18. 102. Koutroubakis IE, Petinaki E, Mouzas IA et al. Anti Saccharomyces cerevisiae mannan antibodies and antineutrophil cytoplasmic autoantibodies in Greek patients with in£ammatory bowel disease. Am J Gastroenterol. 2001;96:449 54. 103. Seibold F, Slametschka D, Gregor M, Weber P. Neutrophil autoantibodies: a genetic marker in primary sclerosing cholangitis and ulcerative colitis. Gastroenterology. 1994;107:532 6. 104. Shanahan F, Duerr RH, Rotter JI et al. Neutrophil autoantibodies in ulcerative colitis: familial aggregation and genetic heterogeneity. Gastroenterology. 1992;103:456 61. 105. Lee JC, Lennard Jones JE, Cambridge G. Antineutrophil antibodies in familial in£ammatory bowel disease. Gastroenterology. 1995;108:428 33. 106. Folwaczny C, Noehl N, Endres SP, Loeschke K, Fricke H. Antineutrophil and pancreatic autoantibodies in ¢rst degree relatives of patients with in£ammatory bowel disease. Scand J Gastroenterol. 1998;33:523 8. 107. Sendid B, Quinton JF, Charrier G et al. Anti Saccharomyces cerevisiae mannan antibodies in familial Crohn's disease. Am J Gastroenterol. 1998;93:1306 10. 108. Seibold F, Stich O, Hufnagl R, Kamil S, Scheurlen M. Anti Saccharomyces cerevisiae antibodies in in£ammatory bowel disease: a family study. Scand J Gastroenterol. 2001;36:196 201. 109. Israeli E, Grotto I, Gilburd B et al. Anti Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies as predictors of in£ammatory bowel disease. Gut. 2005;54:1232 6. 110. Joossens S, Reinisch W, Vermeire S et al. The value of serologic markers in indeterminate colitis: a prospective follow up study. Gastroenterology. 2002;122:1242 7. 111. Joossens S, Colombel JF, Landers C et al. Anti outer membrane of porin C and anti I2 antibodies in indeterminate colitis. Gut. 2006;55:1667 9. 112. Lichtenstein GR, Carroll S, Eggleston L, Neri B, Lois A. Validation of a computer aided analysis of in£ammatory bowel disese (IBD) serologic markers: a novel method to improve the accuracy of predicting IBD, Crohn's disease (CD), and ulcerative colitis (UC). Gastroenterology. 2007;132:A175 (S1106). 113. Sandborn WJ, Landers CJ, Tremaine WJ, Targan SR. Association of antineutrophil cytoplasmic antibodies with resistance to treatment of left sided ulcerative colitis: results of a pilot study. Mayo Clin Proc. 1996;71:431 6. 114. Sandborn WJ, Landers CJ, Tremaine WJ, Targan SR. Antineutrophil cytoplasmic antibody correlates with chronic pouchitis after ileal pouch anal anastomosis. Am J Gastroenterol. 1995;90:740 7. 115. Vasiliauskas EA, Plevy SE, Landers CJ et al. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease de¢ne a clinical subgroup. Gastroenterology. 1996;110:1810 19. 116. Vasiliauskas EA, Kam LY, Karp LC, Gaiennie J, Yang H, Targan SR. Marker antibody expression strati¢es Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics. Gut. 2000;47:487 96. 117. Klebl FH, Bataille F, Bertea CR et al. Association of perinuclear antineutrophil cytoplasmic antibodies and anti Saccharomyces cerevisiae antibodies with Vienna classi¢cation subtypes of Crohn's disease. In£amm Bowel Dis. 2003;9:302 7. 118. Vermeire S, Peeters M, Vlietinck R et al. Anti Saccharomyces cerevisiae antibodies (ASCA), phenotypes of IBD, and intestinal permeability: a study in IBD families. In£amm Bowel Dis. 2001;7:8 15. 119. Walker LJ, Aldhous MC, Drummond HE et al. Anti Saccharomyces cerevisiae antibodies (ASCA) in Crohn's disease are associated with disease severity but not NOD2/CARD15 mutations. Clin Exp Immunol. 2004;135:490 6. 120. Mow WS, Vasiliauskas EA, Lin YC et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology. 2004;126:414 24. 121. 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.
72
GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE 122. Dubinsky MC, Lin YC, Dutridge D et al.; Western Regional Pediatric IBD Research Alliance. Serum immune responses predict rapid disease progression among children with Crohn's disease: immune responses predict disease progression. Am J Gastroenterol. 2006;101:360 7. 123. Sandborn WJ, Landers CJ, Tremaine WJ, Targan SR. Association of antineutrophil cytoplasmic antibodies with resistance to treatment of left sided ulcerative colitis: results of a pilot study. Mayo Clin Proc. 1996;71:431 6. 124. Arnott ID, Landers CJ, Nimmo EJ et al. Sero reactivity to microbial components in Crohn's disease is associated with disease severity and progression, but not NOD2/ CARD15 genotype. Am J Gastroenterol. 2004;99:2376 84. 125. Oshitani N, Hato F, Matsumoto T et al. Decreased anti Saccharomyces cerevisiae antibody titer by mesalazine in patients with Crohn's disease. J Gastroenterol Hepatol. 2000;15:1400 3. 126. Teml A, Kratzer V, Schneider B et al. Anti Saccharomyces cerevisiae antibodies: a stable marker for Crohn's disease during steroid and 5 aminosalicylic acid treatment. Am J Gastroenterol. 2003;98:2226 31. 127. Esters N, Vermeire S, Joossens S et al.; Belgian Group of In£iximab Expanded Access Program in Crohn's Disease. Serological markers for prediction of response to anti tumor necrosis factor treatment in Crohn's disease. Am J Gastroenterol. 2002;97:1458 62. 128. Mow WS, Landers CJ, Steinhart AH et al. High level serum antibodies to bacterial antigens are associated with antibiotic induced clinical remission in Crohn's disease: a pilot study. Dig Dis Sci. 2004;49:1280 6. 129. Gabay C, Kushner I. Acute phase proteins and other systemic responses to in£ammation. N Engl J Med. 1999;340:448 54. 130. Tillet WS, Francis T. Serological reactions in pneumonia with a non protein somatic fraction of the pneumococcus. J Exp Med. 1930;52:561 71. 131. Tall AR. C reactive protein reassessed. N Engl J Med. 2004;350:1450 2. 132. Ballou SP, Kushner I. C reactive protein and the acute phase response. Adv Intern Med. 1992;37:313 36. 133. Beattie RM, Walker Smith JA, Murch SH. Indications for investigation of chronic gastrointestinal symptoms. Arch Dis Child. 1995;73:354 5. 134. Saverymuttu SH, Hodgson HJ, Chadwick VS, Pepys MB. Di¡ering acute phase responses in Crohn's disease and ulcerative colitis. Gut. 1986;27:809 13. 135. Willot S, Vermeire S, Ohresser M et al. No association between C reactive protein gene polymorphisms and decrease of C reactive protein serum concentration after in£iximab treatment in Crohn's disease. Pharmacogenet Genom. 2006;16:37 42. 136. Meuwis MA, Marianne Fillet M, Geurts P et al. Biomarker discovery for in£ammatory bowel disease, using proteomic serum pro¢ling. Biochem Pharmacol. 2007;73:1422 33. 137. Tibble J, Teahon K, Thjodleifsson B et al. A simple method for assessing intestinal in£ammation in Crohn's disease. Gut. 2000;47:506 13. 138. Sugi K, Saitoh O, Hirata I, Katsu K. Fecal lactoferrin as a marker for disease activity in in£ammatory bowel disease: comparison with other neutrophilderived proteins. Am J Gastroenterol. 1996;91:927 34. 139. Roseth AG, Schmidt PN, Fagerhol MK. Correlation between faecal excretion of indium 111 labelled granulocytes and calprotectin, a granulocyte marker protein, in patients with in£ammatory bowel disease. Scand J Gastroenterol. 1999;34:50 4. 140. Foell D, Kucharzik T, Kraft M et al. Neutrophil derived human S100A12 (EN RAGE) is strongly expressed during chronic active in£ammatory bowel disease. Gut. 2003;52:847 53. 141. Walker TR, Land ML, Kartashov A et al. Fecal lactoferrin is a sensitive and speci¢c marker of disease activity in children and young adults with in£ammatory bowel disease. J Pediatr Gastroenterol Nutr. 2007;44:414 22. 142. Shine B, Berghouse L, Jones JE, Landon J. C reactive protein as an aid in the di¡erentiation of functional and in£ammatory bowel disorders. Clin Chim Acta. 1985;148:105 9. 143. Poullis AP, Zar S, Sundaram KK et al. A new, highly sensitive assay for Creactive protein can aid the di¡erentiation of in£ammatory bowel disorders from constipation and diarrhoea predominant functional bowel disorders. Eur J Gastroenterol Hepatol. 2002;14:409 12.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 144. Fagerberg UL, Loof L, Myrdal U, Hansson LO, Finkel Y. Colorectal in£ammation is well predicted by fecal calprotectin in children with gastrointestinal symptoms. J Pediatr Gastroenterol Nutr. 2005;40:450 5. 145. Thjodleifsson B, Sigthorsson G, Cariglia N et al. Subclinical intestinal in£ammation: an inherited abnormality in Crohn's disease relatives? Gastroenterology. 2003;124:1728 37. 146. von Roon AC, Karamountzos L, Purkayastha S et al. Diagnostic precision of fecal calprotectin for in£ammatory bowel disease and colorectal malignancy. Am J Gastroenterol. 2007;102:803 13. 147. Danesh J, Wheeler JG, Hirsch¢eld GM et al. C reactive protein and other circulating markers of in£ammation in the prediction of coronary heart disease. N Engl J Med. 2004;350:1387 97. 148. Bataille R, Boccadoro M, Klein B, Durie B, Pileri A. C reactive protein and beta 2 microglobulin produce a simple and powerful myeloma staging system. Blood. 1992;80:733 7. 149. Tromm A, Tromm CD, Huppe D, Schwegler U, Krieg M, May B. Evaluation of di¡erent laboratory tests and activity indices re£ecting the in£ammatory activity of Crohn's disease. Scand J Gastroenterol. 1992;27:774 8. 150. Sachar DB, Smith H, Chan S, Cohen LB, Lichtiger S, Messer J. Erythrocytic sedimentation rate as a measure of clinical activity in in£ammatory bowel disease. J Clin Gastroenterol. 1986;8:647 50. 151. Fagan EA, Dyck RF, Maton PN et al. Serum levels of C reactive protein in Crohn's disease and ulcerative colitis. Eur J Clin Invest. 1982;12:351 9. 152. Solem CA, Loftus EV Jr, Tremaine WJ, Harmsen WS, Zinsmeister AR, Sandborn WJ. Correlation of C reactive protein (CRP) with clinical, radiographic, and endoscopic activity in in£ammatory bowel disease (IBD). In£amm Bowel Dis. 2005;11:707 12. 153. Florin TH, Paterson EW, Fowler EV, Radford Smith GL. Clinically active Crohn's disease in the presence of a low C reactive protein. Scand J Gastroenterol. 2006;41:306 11. 154. Brignola C, Campieri M, Bazzocchi G, Farruggia P, Tragnone A, Lanfranchi GA. A laboratory index for predicting relapse in asymptomatic patients with Crohn's disease. Gastroenterology. 1986;91:1490 4. 155. Boirivant M, Leoni M, Tariciotti D, Fais S, Squarcia O, Pallone F. The clinical signi¢cance of serum C reactive protein levels in Crohn's disease. Results of a prospective longitudinal study. J Clin Gastroenterol. 1988;10:401 5. 156. Consigny Y, Modigliani R, Colombel JF, Dupas JL, Lemann M, Mary JY (GETAID). A simple biological score for predicting low risk of short term relapse in Crohn's disease. In£amm Bowel Dis. 2006;12:551 7. 157. Travis SP, Farrant JM, Ricketts C et al. Predicting outcome in severe ulcerative colitis. Gut. 1996;38:905 10. 158. Zilberman L, Maharshak N, Arbel Y et al. Correlated expression of high sensitivity C reactive protein in relation to disease activity in in£ammatory bowel disease: lack of di¡erences between Crohn's disease and ulcerative colitis. Digestion. 2006;73:205 9. 159. Tibble JA, Sigthorsson G, Bridger S, Fagerhol MK, Bjarnason I. Surrogate markers of intestinal in£ammation are predictive of relapse in patients with in£ammatory bowel disease. Gastroenterology. 2000;119:15 22. 160. Costa F, Mumolo MG, Ceccarelli L et al. Calprotectin is a stronger predictive marker of relapse in ulcerative colitis than in Crohn's disease. Gut. 2005;54:364 8. 161. D'Inca R, Dal Pont E, Di Leo V et al. Calprotectin and lactoferrin in the assessment of intestinal in£ammation and organic disease. Int J Colorectal Dis. 2007;22:429 37. 162. Roseth AG, Aadland E, Grzyb K. Normalization of faecal calprotectin: a predictor of mucosal healing in patients with in£ammatory bowel disease. Scand J Gastroenterol. 2004;39:1017 20. 163. Louis E, Vermeire S, Rutgeerts P et al. A positive response to in£iximab in Crohn disease: association with a higher systemic in£ammation before treatment but not with 308 TNF gene polymorphism. Scand J Gastroenterol. 2002;37:818 24. 164. Schreiber S, Rutgeerts P, Fedorak RN et al; CDP870 Crohn's Disease Study Group. CDP870 Crohn's Disease Study Group. A randomized, placebo controlled trial of certolizumab pegol (CDP870) for treatment of Crohn's disease. Gastroenterology. 2005;129:807 18.
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GENETICS, IMMUNOLOGY AND BIOMARKERS IN CLINICAL PRACTICE 165. Lemann M, Mary JY, 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. 166. Orlando A, Modesto I, Castiglione F et al. The role of calprotectin in predicting endoscopic post surgical recurrence in asymptomatic Crohn's disease: a comparison with ultrasound. Eur Rev Med Pharmacol Sci. 2006;10:17 22. 167. Scarpa M, D'Inca R, Basso D et al. Fecal lactoferrin and calprotectin after ileocolonic resection for Crohn's disease. Dis Colon Rectum. 2007;50:861 9.
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7 Standard therapy for ulcerative colitis and Crohn's disease: the ECCO guidelines A. IGNJATOVIC and S. TRAVIS
INTRODUCTION Crohn's disease (CD) and ulcerative colitis (UC) are complex in£ammatory disorders whose management can vary between clinicians from di¡erent countries. Guidelines have been published by individual countries (UK1, Germany 2 , United States 3 ) and the European Crohn's and Colitis Organization (ECCO) has reached a consensus on management across Europe4. ECCO is a forum for specialists in in£ammatory bowel disease from 23 European countries. The aim of the Consensus was to promote a European perspective on the dilemmas associated with the management of UC and CD. It is considered important because an increasing number of therapeutic trials recruit from Central and Eastern European countries where practice guidelines have yet to be published. The aim of this chapter is to summarize the guidance. Guidelines aspire to bring consistency to the management of patients, but individual cases must continue to be managed according to the clinical situation, in discussion with the patient concerned.
CROHN'S DISEASE When treating patients with CD the activity of the disease, site (ileal, ileocolonic, colonic or other), behaviour (in£ammatory, structuring, ¢stulating) and course of the disease should be considered, as well as previous medication, side-e¡ects, extraintestinal manifestations and the patient's wishes5. Patients should be encouraged to participate in treatment decisions, balancing the impact of symptoms, drug potency and potential side-e¡ects. All medical treatment has to be placed in the context of a high likelihood of needing surgery. In 592 patients followed over 13 years, 91% of those with ileocolonic disease, 72% with pancolonic, 65% with isolated small bowel, and 29% with segmental colonic disease came to surgery6; therefore, surgery should 76
STANDARD THERAPY FOR UC AND CD
always be considered as an option. Both the indication and timing are important interdisciplinary issues. With the advent of anti-tumour necrosis factor (TNF) therapy, a conservative option has emerged for cases with severe in£ammatory activity, and it is in these that primary surgery will usually be inappropriate. Thus, neither a conservative nor surgical option should be given precedence over the other, but in these di¤cult cases the best approach should be tailored to the individual.
Treatment according to the site of disease and behaviour An alternative explanation for symptoms other than active disease should always be considered, including bacterial overgrowth and bile salt malabsorption where diarrhoea predominates, or ¢brotic strictures, dysmotility and gallstones for abdominal pain. Evidence of activity is most simply con¢rmed by measuring in£ammatory markers such as C-reactive protein (CRP), but endoscopy, a white cell scan for patients not on steroids, or magnetic resonance enteroclysis may be needed in cases of doubt7.
Mild or moderately active localized ileocaecal CD The guidelines advocate budesonide 9 mg daily as the preferred treatment, and are notable for dismissing mesalazine for the treatment of active CD. This is because budesonide is superior to both placebo (OR 2.85, 95% CI 1.67 4.87)8 and mesalazine 4 g/day (OR 2.8, 95% CI 1.50 5.20)9. Budesonide achieves remission in 51 60% over 8 10 weeks 8 . Budesonide is preferred to prednisolone for mild or moderately active CD because it is associated with fewer side-e¡ects, although a Cochrane systematic review has shown budesonide to be somewhat less e¡ective (pooled OR for the ¢ve trials 0.69, 95% CI 0.51 0.95)8. Corticosteroid-related adverse e¡ects on budesonide were no di¡erent to placebo (OR 0.98, 95% CI 0.58 1.67). Unless septic complications are suspected there is no role for antibiotics. Adding cipro£oxacin and metronidazole to budesonide has shown no advantage over budesonide alone in active CD10. The Consensus considered that there was no role for primary treatment with nutritional therapy, although it was important always to consider nutrition in the treatment of CD and to use nutritional support as adjunctive therapy when appropriate.
Severe ileal disease Prednisolone or intravenous hydrocortisone are appropriate for patients with severe ileal disease. Azathioprine (or mercaptopurine) should be added for those who have relapsed, because it has a steroid-sparing e¡ect (NNT 3) and is e¡ective at maintaining remission11. Methotrexate should be considered as an appropriate alternative if thiopurines cannot be tolerated, but has speci¢c contraindications, such as pregnancy12. In£iximab (IFX) is best reserved for patients not responding to initial therapy and for whom surgery is considered inappropriate. This does not mean that surgery takes precedence over IFX. Both the indication and timing are joint decisions between patient, physician 77
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
and surgeon. IFX should be considered in addition for steroid- and immunomodulator-refractory disease although surgery is an option. Many specialists in ECCO still advocate surgery for localized ileal or ileocaecal disease in preference to IFX, particularly for obstructive symptoms, or when there is a palpable right iliac fossa mass, since this may indicate a localized enteroenteral ¢stula or sealed perforation. Others advocate resection if medical therapy is not e¡ective within 2 6 weeks. It may be di¤cult to distinguish between active disease and a septic complication, but antibiotics should be reserved for patients with a temperature or focal tenderness, or in whom imaging has indicated an abscess.
Crohn's colitis Although sulphasalazine 4 g/day is e¡ective in treatment of colonic CD it cannot be recommended as ¢rst-line due to its side-e¡ects. Systemic c o r ti c o s te roi d s ( p re d n is olo n e o r e qu ivalent) are e¡e ctive 1 3 an d immunomodulators are appropriate steroid-sparing agents for those who have relapsed. In its current formulation oral budesonide has no role in therapy of colonic disease, unless it primarily a¡ects the proximal colon (with or without ileal involvement). Metronidazole 10 20 mg/kg per day induces a response (change in CDAI 97 points for 20 mg/kg, 67 for 10 mg/kg vs 1 for placebo, p = 0.002) for colonic disease, but not remission14. It is consequently not recommended as ¢rst-line therapy and has a high incidence of side-e¡ects, but has a role in selected patients with colonic disease who wish to avoid steroids. Interestingly, IFX appears to be twice as e¡ective for isolated colitis (OR 1.91, 95% CI 1.01 3.60) as it is for isolated small bowel disease (ileitis), and four times more e¡ective in steroid-refractory Crohn's colitis (OR 4.9, 95% CI 2.2 11.0)15. IFX should certainly be considered for those patients with steroid- or immunomodulator-refractory disease, although surgery is an option. Some experts advocate the use of topical mesalazine in distal CD, although there have been no trials of topical therapy in CD.
Extensive small bowel disease The in£ammatory burden is greater in extensive (4100 cm) than in localized small bowel disease, so it is generally more severe, with nutritional consequences. The traditional approach has been to use corticosteroids, but these do not induce mucosal healing or change the pattern of disease. Early introduction of immunomodulators is appropriate for their steroid-sparing e¡ect, because of the greater burden of disease. Nutritional support should be given as an adjunct to other treatment. IFX is e¡ective at inducing remission for steroid-refractory active CD, although trials have failed to distinguish between those with extensive and more localized disease. In the Consensus panel some advocated a lower threshold for IFX in extensive disease, because of the associated severe nutritional consequences and because extensive resection risks creating a short bowel. Resection risks creating a short bowel, but nutritional support prior to multiple stricturoplasty is a valid strategy for managing extensive, stricturing small bowel disease. 78
STANDARD THERAPY FOR UC AND CD
Proximal intestinal disease Oesophageal, gastroduodenal and jejunal CD are (fortunately) uncommon, and associated with a worse prognosis16. There are no controlled trials of treatment, so recommendations are based on patient cohorts17. The Consensus advocates adding a proton pump inhibitor to conventional induction of remission and early introduction of immunomodulators. Some have a lower threshold for anti-TNF therapy and the trend to earlier use of biological therapy since publication of the Consensus supports this approach.
Perianal disease For simple perianal ¢stulas it is important to know if they are symptomatic. If they are not, nothing has to be done. Only when simple ¢stulas are symptomatic are the options of a loose seton or ¢stulotomy recommended. Antibiotics, metronidazole (750 1500 mg/day) or cipro£oxacin (1000 mg/day), should be added 18 . For complex ¢stulas (Table 1), antibiotics and/or azathioprine/6-mercaptopurine should be used as the ¢rst choice of therapy in combination with surgical therapy, in spite of a lack of clinical trials. The presence of a perianal abscess should be ruled out and, if present, it should be drained. IFX should generally be used as a second-line treatment. It was the ¢rst agent shown to be e¡ective in a randomized controlled trial for inducing closure of perianal ¢stulas and for maintaining this response for 1 year, without increasing the risk of abscess formation20. Perianal Crohn's is often associated with active disease elsewhere in the gut and experience suggests that ¢stulas will not heal unless this is treated. Despite the e¡ect of IFX, however, discharging ¢stulas will recur in a majority of patients when IFX is stopped. Consequently a combination of IFX and seton drainage is advocated. First an examination under anaesthetic is performed and a seton placed, then two doses of IFX 5 mg/kg are given a fortnight apart, then the seton is removed and a third infusion given. Only case series report this approach, but it makes practical sense21.
Maintenance of remission The risk of relapse depends on whether remission was induced medically or surgically, and the site of the disease. In clinical trials designed for the maintenance of remission, relapse rates among patients receiving placebo Table 1
Classi¢cation of perianal ¢stulas19
Simple
Complex
Super¢cial
Trans sphincteric
Inter sphincteric
Supra sphincteric Extra sphincteric
79
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
range from 30% to 60% at 1 year, and from 40% to 70% at 2 years13. The probability of relapse during the ¢rst 3 years correlates well with that observed during the following years. This is a helpful clinical point for patients. Approximately 70 80% of patients with active disease during 1 year of followup have active disease in the following year; conversely, 80% of patients in remission had no £are in the following year22. Smoking cessation reduces the risk of relapse23, so all smokers should be advised to stop, and be given help to do so.
Maintaining remission after first presentation Despite the common use of mesalazine for maintenance of remission in CD, there is no consistent evidence that it works, and a meta-analysis indicates that there is no bene¢t 24 . The odds ratio for six studies of mesalazine as maintenance therapy in which participants were followed up for 12 months was 1.00 (95% CI 0.80 1.24). No treatment is an option for patients who have had mild or moderately active disease of limited extent. For others who have had more severe disease, or have poor prognostic factors (perianal disease, extensive or proximal small bowel disease, or steroids given at ¢rst presentation), primary prophylaxis with a thiopurine (azathioprine or mercaptopurine) should be considered. The impetus for early treatment with thiopurines comes from a trial in children with newly diagnosed CD who had remission induced by steroids, showing that only 9% treated with mercaptopurine relapsed within 18 months compared to 47% of controls (p ( = 0.007)25. Corticosteroids are ine¡ective at preventing relapse and have no place in the maintenance of remission26 .
Maintaining remission after relapse requiring steroid therapy Taking into account the high risk of relapse and steroid dependence, azathioprine (AZA) is recommended. The most recent meta-analysis 27 analysed ¢ve clinical trials involving 319 patients. The 1-year remission rate was 67% for AZA and 52% for placebo (OR 2.16, 95% CI 1.35 3.47; NNT to prevent one relapse = 7). There was a dose response e¡ect (OR 1.20, 95% CI 0.60 2.41 for 1 mg/kg per day; OR 3.17, 95% CI 1.33 7.59 for 2 mg/kg per day; and OR 4.13, 95% CI 1.59 10.71 for 2.5 mg/kg per day). A further study has compared AZA withdrawal (replaced by a placebo) with its continuation for patients in remission on AZA after more than 3.5 years28. The clinical relapse rate after 18 months was higher in those who discontinued AZA (21% and 8%, respectively), but after 3 years it was 53% in those who had discontinued therapy, suggesting a bene¢t of continuing therapy 29 . The balance between bene¢t and risk should be discussed with individual patients. For patients who cannot tolerate thiopurines, weekly methotrexate is considered appropriate.
80
STANDARD THERAPY FOR UC AND CD
Maintaining remission induced by anti-TNF therapy At the time of the Consensus, only IFX was licensed for use in CD. Two placebo-controlled trials have evaluated the e¡ectiveness of repeated infusions of IFX for the maintenance of IFX-induced response in non-¢stulating CD. The largest trial (ACCENT 1) recruited 573 patients30. Responders to an initial infusion of 5 mg/kg (n ( = 335) received IFX (5 mg/kg) or a placebo at weeks 2 and 6, and then infusions of placebo, IFX 5 mg/kg or IFX 10 mg/kg every 8 weeks. The median times to loss of response were 38, 54 and 19 weeks respectively. The di¡erence was signi¢cant between IFX 5 mg/kg and placebo ( 50.002), and between 10 mg/kg and placebo (p (p ( 50.001). Steroid-free remission rates were 24%, 32% and 9% in the 5 mg/kg, 10 mg/kg and placebo groups respectively. This supports the use of continued IFX for the maintenance of remission, but patients (and their doctors) should have realistic expectations. The other option is episodic dosing combined with immunomodulators. This European view di¡ers from that commonly held in the USA, where maintenance anti-TNF therapy is the norm once started. The French GETAID group have studied 113 patients with moderately active, steroiddependent CD randomized to receive three doses of IFX (5 mg/kg at 0, 2 and 6 weeks) or placebo, in addition to AZA31. The primary endpoint was disease remission (CDAI 5150) o¡ steroids 6 months' later (week 24). The intentionto-treat analysis showed twice the steroid-free remission rate in the IFX and AZA group (57%) compared to the placebo/AZA group (29%, p = 0.003). Induction therapy with IFX consistently doubled the remission rate at every time point: from 38% to 75% at week 12 (p ( 50.001), 29% to 57% at week 24 and 22% to 40% at week 52 (p ( = 0.04). Those naive to AZA fared markedly better than the AZA-refractory (or `failure') group. For those randomized to IFX the 12-, 24- and 52-week remission rates in the AZA-naive group were 83%, 63% and 52%, compared to 64%, 50% and 27% respectively in the AZA-refractory group. The trial is not directly comparable to ACCENT 1, because not all patients in that trial were steroid-dependent, although this di¡erence should work in ACCENT 1's favour. Nevertheless it does show that induction IFX followed by AZA contrasts favourably with maintenance IFX32.
Maintenance of remission after surgery Surgery is not curative, as disease inexorably recurs. The postoperative recurrence rate depends on whether the de¢nition is clinical, endoscopic, radiological, or surgical. Data from endoscopic follow-up of patients after resection of ileocaecal disease have shown that, in the absence of treatment, the endoscopic recurrence rate is around 65 90% within 12 months and 80 100% within 3 years of the operation. The clinical recurrence without therapy is about 20 25% per year18,33. Available data do not demonstrate a robust protective e¡ect for any medical therapy, and this remains a contentious area. The consensus recommends that all patients should stop smoking and that medical prophylaxis is started after small intestinal resection, ideally within 2 weeks of resection. The choice lies between mesalazine and AZA. 81
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
A European cooperative study34 showed that mesalazine 4.0 g/day did not signi¢cantly a¡ect clinical postoperative recurrence overall. However, a substantial subgroup of 124 patients who had had isolated resection of small bowel CD did bene¢t. A meta-analysis35 included this large European trial and concluded in favour of treatment with mesalazine. A subsequent meta-analysis that is only available as an abstract included one further negative study in a total of 1141 patients36. The conclusion was that mesalazine reduced the rate of endoscopic recurrence by 18% and clinical recurrence by 15% (NNT = 6.6), which is clinically relevant. Nevertheless, given mesalazine's limited e¡ect, no prophylactic treatment is an option in some asymptomatic or low-risk patients. For `high-risk' patients, such as those having a second or extensive resection, AZA appears to be preferable, and this is recommended by the Consensus. The data are, however, open to interpretation. In a comparative trial there was a trend for mercaptopurine 50 mg/daily to be more e¡ective than placebo or mesalazine 3 g/day in preventing clinical relapse 37 . Observed rates of endoscopic recurrence at 2 years for placebo, mesalazine and 6mercaptopurine were 64%, 63% and 43% respectively. The problem was that the clinical recurrence rate (based on physician global assessment) was higher than endoscopic recurrence, which is unique. Furthermore, only 57 out of 131 patients completed the trial. A further prospective study randomized 142 patients to receive AZA 2 mg/kg per day or mesalazine 3 g/day for 24 months. This showed comparable rates of clinical (OR 2.04, 95% CI 0.89 4.67) and surgical recurrence. Subgroup analysis showed a favourable e¡ect of AZA for patients who had had a previous resection (OR 4.83, 95% CI 1.47 15.8)38.
ULCERATIVE COLITIS The ECCO Consensus meeting on the management of UC was presented at UEGW 2006. The recommendations were agreed by 61 in£ammatory bowel disease specialists from across Europe, but the supporting text is in the process of publication. As with CD, decisions on therapy should be made in conjunction with the patients after taking into account the severity, distribution (proctitis, distal, or extensive) and pattern of disease (frequency of relapse). Similarly, the response to previous medication, potential side-e¡ects and extraintestinal manifestations should be considered. The severity of UC is easier to assess than CD, simply by assessing bloody stool frequency, systemic in£ammation and by con¢rming activity by £exible sigmoidoscopy. Clinically it is most important to distinguish between those patients who have severe disease and need hospital admission and those who have mild or moderate disease. The simplest, best validated and most widely used index for identifying severe UC remains that of Truelove and Witts39: any patient who has a bloody stool frequency 56/day and a tachycardia (490 bpm), or temperature 437.88C, or anaemia (haemoglobin 510.5 g/dl), or an elevated ESR (430 mm/h) has severe UC. This index has been used in 20/32 studies of intensive intravenous treatment for severe UC40. By contrast, the simplest measure to distinguish moderate from mild colitis is mucosal friability (bleeding on light contact at sigmoidoscopy). 82
STANDARD THERAPY FOR UC AND CD
Proctitis Proctitis means active colitis limited to the rectum, and should ¢rst be treated topically. Suppositories are more appropriate than enemas, because suppositories target the site of in£ammation, while only 40% of foam enemas and 10% of liquid enemas can be detected in the rectum after 4 h41. In a metaanalysis of 11 trials in 778 patients, topical mesalazine (5-ASA) induced remission in active proctitis and distal colitis in 31 80% (median 67%) compared to 7 11% given placebo42. Topical mesalazine is at least twice as e¡ective as topical steroids, whether for symptoms (OR 2.42, 95% CI 1.72 3.41), endoscopy (OR 1.89, 95% CI 1.29 2.76), or histology (OR 2.03, 95% CI 1.28 3.20)43. There is no dose response to topical therapy and 1 g mesalazine is optimal. Clinical (and endoscopic) remission occurred in 64% (52%) within 2 weeks on Pentasa suppositories, compared to 28% (24%) on Claversal suppositories ((p50.01)44. The combination of oral and topical mesalazine is better than either alone for colitis 550 cm from the anal verge45. There have been no trials on combination therapy for proctitis alone. Combining topical mesalazine and steroids also helps46. Patients who fail to improve on topical mesalazine and topical corticosteroids should be treated with oral prednisolone, as if the colitis were more extensive or severe (below).
Distal colitis Distal colitis, according to the Montreal classi¢cation47, includes colitis extending up to the splenic £exure. This has often been termed left-sided colitis, but the term distal is used for consistency. The Consensus recommends that patients should initially be managed with oral mesalazine 42 g/day combined with topical aminosalicylates. In a meta-analysis of oral 5aminosalicylic acid compounds for active colitis48, mesalazine was more than twice as e¡ective as placebo (OR 0.40, 95% CI 0.30 0.53), but not signi¢cantly better than sulphasalazine (OR 0.83, 95% CI 0.60 1.13) for the failure to induce global clinical improvement or remission. Mesalazine was better tolerated than sulphasalazine. This is a modest bene¢t (NNT to induce remission = 10 (95% CI 7 21), and NNT = 4 to induce response or remission (95% CI 3 6)49). A further placebo-controlled trial of a multimatrix mesalazine (MMx) formulation for mild moderate UC has since reported 50 . This randomized 280 patients to either MMx 4.8 g once daily, MMx 1.2 g twice daily, or placebo for 8 weeks. The primary endpoint was remission at the end of the 8 weeks. Once- and twice-daily dosing produced similar results. Remission rates were 29% and 34% respectively, compared to 13% on placebo (p ( 50.01). As far as the dose of mesalazine is concerned, meta-analysis shows a dose response for improvement from 52.0 g, 2.0 2.9 g and 43.0 g daily ((p = 0.002), but not for remission48. The speed of response can be judged by the time to cessation of rectal bleeding. In a trial of mesalazine 2.4 g vs 4.8 g/day for patients with moderately active UC in 268 patients51 the higher dose reduced the median time to cessation of bleeding from 16 to 9 days ((p 50.05). Consequently, if rectal bleeding persists beyond 10 14 days, then the response can be said to be slow and therapy augmented, usually with steroids. 83
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Extensive UC The approach is similar to that described for distal, with the important caveat that there should be a lower threshold for decisive treatment with systemic steroids. Oral mesalazine (Pentasa1) 4 g/day with a 1 g mesalazine enema in 116 patients induced clinical remission by 8 weeks in 64% compared to 43% on oral mesalazine alone (p ( = 0.03)52. This con¢rms the added bene¢t of topical mesalazine for extensive colitis. Failure of mild or moderately active disease to respond within 2 weeks to mesalazine is an indication to consider oral prednisolone. If a patient is already on mesalazine 42 g/day or immunomodulators as maintenance therapy when the relapse occurs, then decisive treatment with steroids is considered appropriate. Prednisolone should be reduced gradually, usually over a period of 8 weeks, as more rapid reduction is associated with early relapse. An appropriate regimen for moderate disease is 40 mg/day for 1 week, then 30 mg/day for 1 week, then 20 mg/day for 1 month before reducing by 5 mg/day per week. The reason for this proactive approach is the risk of complications (including toxic dilation) in patients with extensive disease who are under-treated. IFX is also e¡ective for treating UC. Systematic review of the e¤cacy of IFX for treating patients with m o d e r a t e t o s e v e r e U C r e f r a c t o r y t o c o r t i c o s t e r o i d s a n d/o r immunomodulators, concluded that it was e¡ective for inducing clinical remission, clinical response, promoting mucosal healing, and reducing the need for colectomy in the short term53. The review took the description of `severe' at face value and failed to discriminate between outpatients and inpatients with acute severe colitis. Nevertheless, in seven randomized controlled trials, IFX (three intravenous infusions at 0, 2, and 6 weeks) was more e¡ective than placebo in inducing clinical remission (RR 3.22, 95% CI 2.18 4.76); inducing endoscopic remission (RR 1.88, 95% CI 1.54 2.28); and in inducing clinical response (RR 1.99, 95% CI 1.65 2.41) at 8 weeks.
Acute severe UC The Consensus recommends that patients with acute severe colitis with signs of systemic toxicity should be treated in hospital, with intravenous steroids (such as methylprednisolone 60 mg or hydrocortisone 400 mg daily). It also states that monotherapy with intravenous cyclosporin (to achieve a minimum therapeutic concentration) is an option for patients intolerant of intravenous steroids and that patients are best cared for jointly by a gastroenterologist and colorectal surgeon. Treatment with corticosteroids should not be delayed awaiting microbiological results for possible infective causes. In a systematic review of 32 trials of steroid therapy for acute severe colitis involving 1991 patients from 1974 to 2006 the overall response to steroids was 67% (1429/ 1991, 95% CI 65 69%)40 29% (565/1991, 95% CI 28 31%) came to colectomy. Mortality was 1% (22/1991, 95% CI 0.7 1.6%) and none of these outcomes changed between 1974 and 2006 (R ( 2 = 0.07, p = 0.8). Cyclosporin monotherapy (CsA, 4 mg/kg per day intravenously) was as e¡ective as intravenous methylprednisolone 40 mg/day for acute severe colitis in a randomized trial (response in 10/15 versus 8/15 respectively54. Monotherapy with IFX for 84
STANDARD THERAPY FOR UC AND CD
inpatients with acute severe colitis (as opposed to outpatients with severe, treatment-refractory UC) has not yet been subject to trial, although it has a place as `rescue therapy' for those who are not responding to intravenous steroids.
Intravenous steroid-resistant severe colitis The response to intravenous steroids is best assessed objectively on or about the third day. Surgical options should be considered and discussed at this stage or earlier. The objective measures include stool frequency and CRP. A stool frequency 48/day on day 3 of intensive treatment predicted colectomy in 85% on that admission (`Oxford index')55. The combination has been validated: a frequency 44 and CRP 425 mg/L on day 3 (or when the stool frequency 6 0.14 CRP 58 on day 3: `Sweden index') predicted colectomy in 75%56. Indices exist to be applied as a threshold for triggering appropriate action at an early stage. This means surgical consultation and assessment by a stomatherapist in addition to augmenting medical treatment. Second-line therapy with either cyclosporin (CsA) or IFX or tacrolimus is e¡ective in up to 70% in the short term, although long-term results are less good. In nine studies that used CsA as rescue therapy in a systematic review of severe colitis, only 100/622 (16%) received CsA40. Concerns about early toxicity have been partly addressed by low-dose (2 mg/kg) intravenous induction therapy. In the largest randomized study of CsA to date, 73 patients were randomized to either 2 mg/kg or 4 mg/ kg of intravenous CsA57. Response rates at 8 days were similar in both groups (83% and 82% respectively), with 9% coming to colectomy in the 2 mg/kg group and 13% in the 4 mg/kg group. In£iximab as a single dose (5 mg/kg) may also be e¡ective rescue therapy. A Swedish Danish study treated 45 patients (24 IFX and 21 placebo with continued intravenous betamethasone)58; 7/24 in the IFX group and 14/21 in the placebo group had a colectomy within 3 months (p ( = 0.017; OR 4.9, 95% CI 1.4 17). No patient died. Two di¡erent scores were used to identify patients before randomization to IFX or placebo and it was those with less active disease after 5 7 days of intravenous steroids who bene¢ted most from IFX. A recommendation on the best choice between CsA and IFX is not possible until there has been a comparative, randomized controlled trial. There is concern that IFX, which has a half-life of 8 10 weeks, may increase the severity of any postoperative septic complication should an emergency colectomy be necessary.
Colectomy If there is clinical deterioration or no response to second-line therapy within 4 7 days, then colectomy is recommended. The timing of colectomy remains a di¤cult decision. No individual patient wants a colectomy, but delayed decision-making by physicians increases the risk and severity of postoperative complications. In general only a single attempt at rescue therapy with a CsA, tacrolimus, or IFX should be considered before colectomy, after careful discussion between the patient, gastroenterologist and colorectal surgeon about the options and potential outcomes. If doubt persists, specialist advice 85
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
should be sought at an early stage from a referral centre. It is early and timely surgery that has reduced mortality from acute severe colitis from 24% in 1955 to 51% in specialist centres today59.
Maintenance of remission Maintenance therapy to prevent relapse is recommended, although the potential of mesalazine to have a chemopreventive action against colorectal cancer is an added incentive. Individual mesalazine derivatives all show comparable e¤cacy to sulphasalazine60. Oral mesalazine (1 2 g daily) or balsalazide 2.5 g daily should be considered as ¢rst-line therapy. Patient adherence to treatment, and not the choice of aminosalicylate, is probably the most important factor for maintaining remission. The risk of relapse increases 5-fold (OR 5.5, 95% CI 2.3 13.2) when patients take 580% of the prescribed mesalazine. For this reason, once-daily dosing may be an advantage61. AZA 1.5 2.5 mg/kg per day or mercaptopurine 0.75 1.5 mg/kg per day are both e¡ective at maintaining remission in UC. A study from Milan has shown that steroid-free clinical and endoscopic remission was achieved in 53% of AZA-treated patients, compared to 21% of those on 5-ASA (OR 4.78, 95% CI 1.57 14.5) in patients with active, steroid-dependent UC62. For arbitrary but practical purposes, AZA is considered appropriate for: 1. patients who have had a severe relapse; 2. those who require two or more corticosteroid courses within a calendar year; 3. those whose disease relapses as the dose of prednisolone is reduced below 15 mg; 4. relapse within 3 months of stopping steroids. Patients with gastrointestinal intolerance of AZA may be cautiously tried on mercaptopurine before being considered for other therapy or surgery. Steroids are ine¡ective at maintaining remission, and steroid-dependent UC in spite of AZA is an indication for surgery, after options including anti-TNF therapy have been discussed.
CONCLUSIONS Guidelines are popular, but need implementation. The ECCO Consensus on Crohn's Disease4 has been the most downloaded paper from Gut in the past 3 years. The Consensus on UC will be published in the ECCO's new Journal of Crohn's and Colitis. Changing practice is much more di¤cult. ECCO is establishing a series of regional workshops to use case-led discussions based on the guidelines to try to achieve this. Clinicians often know the treatment options, but not the likelihood of success, which can be very modest. When the steroid-free remission rate on biological therapy for either CD or UC is only 20 25%, there is a large therapeutic gap to achieve outcomes that matter to 86
STANDARD THERAPY FOR UC AND CD
patients which must include steroid-free remission, avoiding hospitalization and surgery. Earlier use of immunomodulators, appropriate selection of patients for biological therapy and early decision making about rescue therapy or colectomy for acute severe colitis are most likely to change the pattern of disease and improve outcome.
References 1. 2.
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Carter MJ, Lobo AJ, Travis SPL. British Society of Gastroenterology Guidelines for in£ammatory bowel disease 2004. Gut. 2004;53(Suppl. V):v1 16. Ho¡mann JC, Zeitz M, Bischo¡ SC et al. Diagnosis and therapy of ulcerative colitis: results of an evidence based consensus conference by the German Society of Digestive and Metabolic Diseases and the competence network on in£ammatory bowel disease. Z Gastroenterol. 2004;42:979 83. Kornbluth A, Sachar DB. Ulcerative colitis practice guidelines in adults (update): American College of Gastroenterology, Practice Parameters Committee. Am J Gastroenterol. 2004;99:1371 85. Stange EF, Travis SPL for the European Crohn's and Colitis Organisation (ECCO). European evidence based Consensus on the diagnosis and management of Crohn's disease. Gut. 2006;55(Suppl. 1):i1 58. Stange EF, Travis SPL for the European Crohn's and Colitis Organisation (ECCO). European evidence based consensus on the diagnosis and management of ulcerative colitis. J Crohn's Colitis. 2008 (In press). Farmer RG, Whelan G, Fazio VW. Long term follow up of patients with Crohn's disease. Relationship between the clinical pattern and prognosis. Gastroenterology. 1985;88:1818 25. Stange EF, Travis SP, Vermeire S et al. European evidence based consensus on the diagnosis and management of Crohn's disease: de¢nitions and diagnosis. Gut. 2006;55(Suppl. 1):i1 15. Otley A, Steinhart AH, Otley A. Budesonide for induction of remission in Crohn's disease. Cochrane Datbase Syst Rev. 2005;4:CD000296. Thomsen OO, Cortot A, Jewell DP et al. A comparison of budesonide and mesalamine for active Crohn's disease. International Budesonide Mesalamine Study Group. N Engl J Med. 1998;339:370 4. Steinhart AH, Feagan B, Wong CJ et al. Combined budesonide and antibiotic therapy for active Crohn's disease: a randomized controlled trial. Gastroenterology. 2002;123:33 40. Pearson DC, May GR, Fick GR, Sutherland LR. Azathioprine for maintaining remission of Crohn's disease. Cochrane Database Syst Rev. 2000;CD000067. Fraser AG. Methotrexate: ¢rst or second line immunomodulator? Eur J Gastroenterol Hepatol. 2003;15:225 31. Malchow H, Ewe K, Brandes JW et al. European co operative Crohn's disease study (ECCDS): results of drug treatment. Gastroenterology. 1984;86:249 66. Sutherland LR, Singleton J, Sessions J et al. Double blind, placebo controlled trial of metronidazole in Crohn's disease. Gut. 1991;32:1071 5. Vermeire S, Louis E, Carbonez A et al. Demographic and clinical parameters in£uencing the short term outcome of anti tumor necrosis factor (in£iximab) treatment in Crohn's disease. Am J Gastroenterol. 2002;97:2357 63. Jess T, Winther KV, Munkholm P, Langholz E, Binder V. Mortality and causes of death in Crohn's disease: follow up of a population based cohort in Copenhagen County, Denmark. Gastroenterology. 2002;122:1808 14. Tremaine WJ. Gastroduodenal Crohn's disease: medical management. In£amm Bowel Dis. 2003;9:127 8. Caprilli R, Gassull MA, Escher JC et al. European evidence based consensus on the diagnosis and management of Crohn's disease: special situations. Gut. 2006;55(Suppl. 1): i36 58. Sandborn WJ, Fazio VW, Feagan BG, Hanauer SB. AGA technical review on perianal Crohn's disease. Gastroenterology. 2003;125:1508 30.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 20. Sands BE, Anderson FH, Bernstein CN et al. In£iximab maintenance therapy for ¢stulizing Crohn's disease. N Engl J Med. 2004;350:876 85. 21. Hyder SA, Travis SPL, Jewell DP, George BD. Fistulating anal Crohn's disease: results of combined surgical and in£iximab treatment. Dis Colon Rectum. 2006;49:1837 41. 22. Munkholm P, Langholz E, Davidsen M, Binder V. Disease activity courses in a regional cohort of Crohn's disease patients. Scand J Gastroenterol. 1995;30:699 70. 23. Cosnes J, Carbonnel F, Beaugerie L, Le Quintrec Y, Gendre JP. E¡ects of cigarette smoking on the long term course of Crohn's disease. Gastroenterology. 1996;110:424 31. 24. Akobeng AK, Gardener E. Oral 5 aminosalicylic acid for maintenance of medically induced remission in Crohn's disease. Cochrane Database Syst Rev. 2005;CD003715. 25. Markowitz J, Grancher K, Kohn N, Lesser M, Daum F. A multicenter trial of 6 mercaptopurine and prednisone in children with newly diagnosed Crohn's disease. Gastroenterology. 2000;119:895 902. 26. Steinhart AH, Ewe K, Gri¤ths AM, Modigliani R, Thomsen OO. Corticosteroids for maintaining remission of Crohn's disease. Cochrane Database Syst Rev. 2003;(4): CD000301. 27. Pearson DC, May GR, Fick G, Sutherland LR. Azathioprine for maintaining remission of Crohn's disease (Cochrane Review). In: The Cochrane Library, 4, 2001. Oxford: Update Software. 28. Lemann M, Mary JY, Colombel J F 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. 29. Treton X, Bouhnik Y, Mary JY et al. Azathioprine withdrawal in patients with Crohn's disease maintained on prolonged remission under treatment is associated with a high risk of relapse. Gastroenterology. 2004;126(Suppl. 2):A113. 30. Hanauer SB, Feagan BG, Lichtenstein GR et al. Maintenance in£iximab for Crohn's disease: the ACCENT 1 randomised trial. Lancet. 2002;359:1541 9. 31. Lemann M, Mary J Y, Duclos B. In£iximab plus azathioprine for steroid dependent Crohn's disease patients. A randomized placebo controlled trial. Gastroenterology. 2006;130:1054 61. 32. Travis SPL. In£iximab and azathioprine: bridge or parachute? Gastroenterology. 2006;130:1354 7. 33. Rutgeerts P, Geboes K, Vantrappen G et al. Predictability of the postoperative course of Crohn's disease. Gastroenterology. 1990;99:956 63. 34. Lochs H, Mayer M, Fleig WE et al. and ECCDS. Prophylaxis of post operative relapse in Crohn's disease with mesalamine: European Cooperative Crohn's Disease Study VI. Gastroenterology. 2000;118:264 73. 35. Cottone M, Camma C. Mesalamine and relapse prevention in Crohn's disease. Gastroenterology. 2000:118:597. 36. Camma C, Viscido A, Latella G, Caprilli R, Cottone M. Mesalamine in the prevention of clinical and endoscopic post operative recurrence of Crohn's disease: a meta analysis. Dig Liver Dis. 2002;34:A86 (Abstract). 37. Hanauer SB, Korelitz BI, Rutgeerts P et al. Post operative maintenance of Crohn's disease remission with 6 mercaptopurine, mesalamine or placebo: a 2 year trial. Gastroenterology. 2004;127:723 9. 38. Ardizzone S, Maconi G, Sampietro GM et al. Azathioprine and mesalamine for prevention of relapse after conservative surgery for Crohn's disease. Gastroenterology. 2004;127:730 7. 39. Truelove SC, Witts LJ. Cortisone in ulcerative colitis: ¢nal report on a therapeutic trial. Br Med J. 1955; ii:1041 8. 40. Turner D, Walsh C, Steinhart AH, Gri¤ths AM. Response to corticosteroids in severe ulcerative colitis: a systematic review of the literature and a meta regression. Clin Gastroenterol Hepatol. 2007;5:103 10. 41. Van Bodegraven AA, Boer RO, Lourens J, Tuynman HARE, Sindram JW. Distribution of mesalazine enemas in active and quiescent ulcerative colitis. Aliment Pharmacol Ther. 1996;10:327 32. 42. Marshall JK, Irvine EJ. Rectal aminosalicylate therapy for distal ulcerative colitis: a meta analysis. Aliment Pharmacol Ther. 1995;9:293 300.
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STANDARD THERAPY FOR UC AND CD 43. Marshall JK, Irvine EJ. Rectal corticosteroids versus alternative treatment in ulcerative colitis: a meta analysis. Gut. 1997;40:775 81. 44. Gionchetti P, Rissole F, Ventura A et al. Comparison of mesalazine suppositories in proctitis and distal proctosigmoiditis. Aliment Pharmacol Ther. 1997;11:1053 7. 45. DeMicco M, Sninsky C et al. A double blind comparison of oral versus rectal mesalamine versus combination therapy in the treatment of distal ulcerative colitis. Am J Gastroenterol. 1997;92:1867 71. 46. Mulder CJJ, Fockens P, Meijer JWR et al. Beclomethasone dipropionate (3 mg) versus 5 aminosalicylic acid (2 g) versus the combination of both (3 mg/2 g) as retention enemas in active ulcerative proctitis. Eur J Gastroenterol Hepatol. 1996;8:549 53. 47. Silverberg MS, Satsangi J, Ahmad T et al. Toward an integrated clinical, molecular and serological classi¢cation of in£ammatory bowel disease: Report of a working party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol. 2005;19(Suppl. A):5A 36. 48. Sutherland L, MacDonald JK. Oral 5 aminosalicylic acid for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2006;CD000543. 49. Bebb JR, Scott BB. How e¡ective are the usual treatments for ulcerative colitis? Aliment Pharmacol Ther. 2004; 20:143 9. 50. Lichtenstein GR, Kamm MA, Boddu P et al. E¡ect of once or twice daily MMX mesalamine (SPD476) for the induction of remission of mild to moderately active ulcerative colitis. Clin Gastroenterol Hepatol. 2007;5:95 102. 51. Hanauer SB, Sandborn WJ, Kornbluth A et al. Delayed release oral mesalamine at 4.8 g/ day (800 mg tablet) for the treatment of moderately active ulcerative colitis: the ASCEND II trial. Am J Gastroenterol. 2005;100:2478 85. 52. Marteau P, Probert CS, Lindgren S et al. Combined oral and enema treatment with Pentasa (mesalazine) is superior to oral therapy alone in patients with extensive mild/moderate active ulcerative colitis: a randomised, double blind, placebo controlled study. Gut. 2005;54:960 5. 53. Lawson MM, Thomas AG, Akobeng AK. Tumour necrosis factor alpha blocking agents for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2006;3: CD005112. 54. D'Haens G, Lemmens L, Gebboes K et al. Intravenous cyclosporine versus intravenous corticosteroids as single therapy for severe attacks of ulcerative colitis. Gastroenterology. 2001;120:1323 9. 55. Travis SPL, Farrant JM, Ricketts C et al. Predicting outcome in severe ulcerative colitis. Gut. 1996;38:905 10. 56. Lindgren SC, Flood LM, Kilander AF et al. Early predictors of glucocorticoid treatment failure in severe and moderately severe attacks of ulcerative colitis. Eur J Gastroenterol Hepatol. 1998;10:831 5. 57. Van Assche G, D'Haens G, Noman M et al. Randomized, double blind comparison of 4 mg/kg versus 2 mg/kg intravenous cyclosporine in severe ulcerative colitis. Gastroenterology. 2003;125:1025 31. 58. Jarnerot G, Hertervig E, Friis Liby I et al. In£iximab as rescue therapy in severe to moderately severe ulcerative colitis: a randomized, placebo controlled study. Gastroenterology. 2005;128:1805 11. 59. Jakobovits S, Travis SPL. The management of acute severe ulcerative colitis. Br Med Bull. 2006;75 76:131 44. 60. Bebb JR, Scott BB. Systematic review: How e¡ective are the usual treatments for ulcerative colitis? Aliment Pharmacol Ther. 2004;20:143 9. 61. Kane S, Huo D, Aikens J, Hanauer S. Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis. Am J Med. 2003;114:39 43. 62. Ardizzone S, Maconi G, Russo A et al. Randomised controlled trial of azathioprine and 5 aminosalicylic acid for treatment of steroid dependent ulcerative colitis. Gut. 2006;55:47 53.
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8 Biologics, novel therapeutic alternatives in inflammatory bowel disease W. REINISCH
INTRODUCTION Biologics refer to novel exogenous protein-based molecules designed to recognize and bind speci¢c antigens, including monoclonal antibodies or derivatives of monoclonal antibodies, in order to treat disease. In in£ammatory bowel disease (IBD) biologics are largely directed at cytokines and receptors involved in T-cell activation as well as selective adhesion molecule blockers. Particularly, the success of the intravenously administered chimeric anti-tumour necrosis factor alpha (TNF-a) a IgG1 antibody, in£iximab, has set new treatment standards for IBD and substantially aided to drag Crohn's disease (CD) and ulcerative colitis (UC) out of the attic into the spotlight of internal medicine. In£iximab has demonstrated e¤cacy in the induction and maintenance of remission in luminal and ¢stulizing CD both in adults and children, and patients with moderate to severe UC. Adalimumab, a fully human monoclonal antibody anti-TNF-a, has proved to be e¡ective and safe in patients with CD after subcutaneous administration, both naive and refractory to in£iximab. Certolizumab pegol, a subcutaneous pegylated antiTNF-a Fab fragment, and natalizumab, a monoclonal 4-integrin antibody, suggested e¤cacy for luminal CD. Some evidence supports the theory that biologics not only control symptoms of CD but also may potentially alter the natural course of disease. However, economic costs and the risks of severe opportunistic infections associated with biologic therapy have still not settled the appropriate role for these agents in the clinical care of patients with IBD.
ANTI-TNF-a ANTIBODIES In£iximab was the ¢rst biologic therapy for CD approved by the FDA and EMEA. In£iximab neutralizes circulating and membrane-bound TNF, lyses activated T cells and macrophages and induces T-cell apoptosis1. The ability of 90
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in£iximab to induce remission in patients with refractory CD was shown in a small phase 2 randomized, double-blind, placebo-controlled trial, in which 108 patients received either a single in£iximab infusion of 5 mg/kg, 10 mg/kg, or 20 mg/kg or placebo 2 . After 4 weeks, 81% of patients receiving 5 mg/kg demonstrated a signi¢cant clinical response, de¢ned as a reduction of 570 points in the Crohn's Disease Activity Index (CDAI), compared with a 17% response among those who received placebo. In ACCENT I patients who had responded (reduction in the CDAI by 470 points) to in£iximab at week 2 were shown to maintain responses and clinical remissions (CDAI 150) for up to 54 weeks when continuing in£iximab dosing every 8 weeks3. At week 54 the remission rates were 28% among those receiving in£iximab 5 mg/kg, 38% among those receiving in£iximab 10 mg/kg, and 14% among those receiving placebo maintenance. Furthermore, the steroid-sparing potential of in£iximab could be demonstrated in ACCENT I. The REACH study con¢rmed the safety and e¤cacy of in£iximab to induce and maintain response and remission in paediatric patients aged 6 17 years with moderate to severe luminal CD who were failing immunomodulator therapy4. At 54 weeks in£iximab dosing at 8week intervals maintained response (64%) and remission (56%) signi¢cantly better than infusions every 12 weeks. First evidence on a more bene¢cial outcome of earlier treatment with in£iximab in the course of CD has been provided lately5. Among adult patients with CD-related enterocutaneous ¢stulas in£iximab showed its potential to induce and maintain cessation of ¢stula drainage in the ACCENT II trial. At week 54, 36% of patients in the 5 mg/kg in£iximab maintenance group had continued complete absence of draining ¢stula compared with 19% of patients maintained on placebo6. The ACT studies were two randomized placebo-controlled trials that showed the e¤cacy of in£iximab to induce response (primary endpoint) and remission among outpatients with UC 7. In£iximab treatment was associated with mucosal healing and corticosteroid withdrawal in UC. In a study among hospitalized patients experiencing acute severe UC unresponsive to intravenous steroids, those randomized to receive in£iximab had signi¢cantly lower colectomy rates at 90 days (29%) in contrast to those receiving placebo (67%)8. Adalimumab is a recombinant fully human IgG1 monoclonal antibody that binds TNF. In the CLASSIC I trial, CD patients naive to anti-TNF experienced signi¢cant response (50%) and remission (36%) rates at week 4 when given adalimumab subcutaneously (160/80 mg) at an interval of 2 weeks9. The CHARM trial showed that adalimumab, when given every other week to adalimumab responders, can maintain remission in CD patients naive or refractory to in£iximab. Additionally, adalimumab resulted in higher rates of corticosteroid-free remission and ¢stula closure at 56 weeks10. The GAIN trial showed that adalimumab is superior to placebo to induce remission at week 4 among patients with moderately to severely active CD who were intolerant of or unresponsive to in£iximab11. Certolizumab pegol is a pegylated Fab fragment of an anti-TNF monoclonal antibody, which is administered subcutaneously and does not elicit T-cell apoptosis12. In the PRECiSE 1 study patients with moderate to severe CD, 91
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de¢ned as a CDAI of 220 450, were strati¢ed based on a C-reactive protein (CRP) level 510 mg/L or 510 mg/L and baseline use of corticosteroids and/ or immunosuppressants. Response rates were signi¢cantly better for certolizumab pegol-treated patients than placebo-treated patients at weeks 6 and 26, regardless of baseline CRP levels13. Similar to the study design of ACCENT I and CHARM, maintenance of remission in patients responding to open-label certolizumab pegol at weeks 0, 2, and 4 was studied in PRECiSE 2 14 . Week 26 remission rates were 47.9% among patients receiving certolizumab pegol every 4 weeks compared with 28.6% in the placebo group. Anti-TNF antibody (in£iximab, adalimumab, certolizumab pegol) toxicities include infusion reactions (in£iximab only), delayed-type hypersensitivity reactions (in£iximab only), injection site reactions (adalimumab and certolizumab pegol only), formation of autoantibodies (mainly in£iximab), demyelination (optic neuritis, multiple sclerosis), drug-induced lupus, worsening of congestive heart failure, reactivation of latent tuberculosis, serious infections (both sepsis and opportunistic infections), non-Hodgkin's lymphoma, and possibly solid tumour malignancies15.
ANTI-ADHESION ANTIBODIES Natalizumab is a humanized monoclonal antibody targeted against 4b1 and 4b7 integrins, and inhibits the migration and activation of T cells. Natalizumab has been approved for treatment of CD by the FDA, but not by EMEA. A large phase 3 study of natalizumab, ENACT-I, for induction and maintenance treatment in CD, failed to show e¤cacy at the pre-speci¢ed time point of week 10, whereas, in ENCORE, patients with elevated CRP levels had signi¢cantly better outcome with natalizumab compared to placebo at all time points 16,17 . In ENACT-2 initial responders to natalizumab e¡ectively maintained remission. However, the study and marketing of natalizumab were voluntarily suspended in February 2005 due to three reports of JC polyoma virus-related progressive multifocal leukoencephalopathy (PML) among natalizumab-treated patients, two with multiple sclerosis and and one with CD18. Subsequent worldwide safety evaluation revealed no further cases of PML, yielding an incidence of less than 0.1%. A phase 2 study with MLN02, a monoclonal antibody that targets 4b7integrin, showed e¤cacy in patients with active UC19.
MODULATION OF OTHER CYTOKINES Phase 2 trials in CD with fontolizumab, a humanized interferon-g antibody, did not meet the primary endpoint, but a subgroup of patients given multiple higher doses did bene¢t 20 . Interleukin 12 and 23 were targeted by two humanized interleukin 12/23 antibodies, ABT-874 and CNTO 1275, as well as a small molecule apilimod mesylate (STA-5326). A phase 2 study of ABT874 in patients with active CD suggested clinical bene¢t21. Phase 2 and 3 studies of recombinant human tenovil (rHIL-10) in patients with active and steroid-dependent CD did not demonstrate e¤cacy, possibly owing to inability to achieve su¤cient mucosal concentrations when given subcutaneously22. 92
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BLOCKADE OF T CELLS Phase 1 and 2 studies with visilizumab, a humanized anti-CD3 antibody, in patients with severe UC refractory to intravenous corticosteroids, have shown an initial clinical bene¢t. Studies with monoclonal antibodies targeting the interleukin-2 receptor a-chain (CD25) on the surface of activated T cells have generated contradictory results23,24. The blockade of the co-stimulatory signal required for T-cell activation with CTLA-4-Ig (abatacept) is an interesting concept, which is approved for rheumatoid arthritis. Studies in patients with IBD are ongoing.
CONCLUSION With biologics speci¢c targeting of pathogenetic pathways involved in IBD became reality and helped to substantiate mechanisms suggested by in vitro studies. However, particularly costs of treatment and the delicate balance between over- and undertreatment with biologics remain major challenges to be addressed for a continuously growing community of IBD patients.
References 1.
Van den Brande JM, Bratt H, van den Brink GR et al. In£iximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn's disease. Gastroenterology. 2003;124:1774 85. 2. Targan SR, Hanauer SB, van Deventer SJH et al. A short-term study of chimeric monoclonal antibody CA2 to tumor necrosis factor alpha for Crohn's disease. N Engl J Med. 1997;337:1029 35. 3. Hanauer SB, Feagan BG, Lichtenstein GR et al. Maintenance in£iximab for Crohn's disease: the ACCENT I randomised trial. Lancet. 2002;359:1541 9. 4. Hyams JS, Crandall W, Kugathasan S et al. Induction and maintenance in£iximab therapy for the treatment of moderate-to-severe Crohn's disease in children. Gastroenterology. 2007;132:863 73. 5. D'Haens G, Baert F, van Assche G et al. Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn's disease: an open randomised trial. Lancet. 2008;371:660 7. 6. Sands BE, Anderson FH, Bernstein CN et al. In£iximab maintenance therapy for ¢stulizing Crohn's disease. N Engl J Med. 2004;350:876 85. 7. Rutgeerts P, Sandborn WJ, Feagan BG et al. In£iximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med. 2005;353:2462 76. 8. Jarnerot G, Hertervig E, Friis-Liby I et al. In£iximab as rescue therapy in severe to moderately severe ulcerative colitis: a randomized, placebo-controlled study. Gastroenterology. 2005;128:1805 11. 9. Hanauer SB, Sandborn WJ, Rutgeerts P et al. Human anti-tumor necrosis factor monoclonal antibody (adalimumab) in Crohn's disease: the CLASSIC-I trial. Gastroenterology. 2006;130:323 33. 10. Colombel JF, Sandborn WJ, Rutgeerts P et al. Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial. Gastroenterology. 2007;132:52 65. 11. Sandborn WJ, Rutgeerts P, Enns R et al. Adalimumab induction therapy for Crohn disease previously treated with in£iximab: a randomized trial. Ann Intern Med. 2007;146:829 38. 12. Fossati G, Nesbitt A. E¡ect of the anti-TNF agents, adalimumab, etanercept, in£iximab, and certolizumab PEGOL (CDP870) on the induction of apoptosis in activated peripheral blood lymphocytes and monocytes. Am J Gastroenterol. 2005;100(Suppl.):S298 9.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 13. Sandborn WJ, Feagan BG, Stoinov S et al. PRECISE 1 Study Investigators. Certolizumab pegol for the treatment of Crohn's disease. N Engl J Med. 2007;357:228 38. 14. Schreiber S, Khaliq-Kareemi M, Lawrance IC et al. Maintenance therapy with certolizumab pegol for Crohn's disease. N Engl J Med. 2007;357:239 50. 15. Baumgart DC, Sandborn WJ. In£ammatory bowel disease: clinical aspects and established and evolving therapies. Lancet. 2007;369:1641 57. 16. Sandborn WJ, Colombel JF, Enns R et al. Natalizumab induction and maintenance therapy for Crohn's disease. N Engl J Med. 2005;353:1912 25. 17. Targan SR, Feagan B, Fedorak R et al. Natalizumab induces sustained response and remission in patients with active Crohn's disease: results from the Encore trial. Gastroenterology. 2006;130(Suppl. 2):A108 (Abstract). 18. Van Assche G, Van Ranst M, Sciot R et al. Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn's disease. N Engl J Med. 2005;353:362 8. 19. Feagan BG, Greenberg GR, Wild G et al., Treatment of ulcerative colitis with a humanized antibody to the a4b7 integrin. N Engl J Med. 2005;352:2499 507. 20. Reinisch W, Hommes DW, van Assche G et al. A dose escalating, placebo controlled, double blind, single dose and multidose, safety and tolerability study of fontolizumab, a humanised anti-interferon gamma antibody, in patients with moderate to severe Crohn's disease. Gut. 2006;55:1138 44. 21. Mannon PJ, Fuss IJ, Mayer L et al. Anti-interleukin-12 antibody for active Crohn's disease. N Engl J Med. 2004;351:2069 79. 22. Schreiber S, Fedorak RN, Nielsen OH et al. and the Crohn's Disease IL-10 Cooperative Study Group. Safety and e¤cacy of recombinant human interleukin 10 in chronic active Crohn's disease. Gastroenterology. 2000;119:1461 72. 23. van Assche G, Sandborn WJ, Feagan BG et al. Daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), for the treatment of moderately to severely active ulcerative colitis: a randomised, double blind, placebo controlled, dose ranging trial. Gut. 2006;55:1568 74. 24. Creed TJ, Norman MR, Probert CS et al. Basiliximab (anti-CD25) in combination with steroids may be an e¡ective new treatment for steroid-resistant ulcerative colitis. Aliment Pharmacol Ther. 2003;18:65 75.
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Section IV Lower gastrointestinal tract (coeliac disease, infection and malignancy) Chair: A GANGL and B SíTABUC
9 Recent advances in coeliac disease S. KOLACíEK
INTRODUCTION Coeliac disease is a genetically determined, immune-mediated chronic noninfectious enteropathy. Though possessing a common multifactorial aetiology embracing genes and environment, the unique feature of coeliac disease is a well-de¢ned external cause a wheat constituent named gluten, and similar storage polypeptides from rye and barley. The disorder is caused by the interaction of gluten with the immune-competent cells, whereby di¡erent genes and multiple environmental factors may confer either increased or reduced risk for acquiring the disease. It could be described as a piece of music directed by gluten and performed by the orchestra of immune cells of both innate and acquired immunity, on a stage set by genes and environmental factors, with a resulting synthesis of proin£ammatory cytokines which change a velvety small bowel mucosa into a £at moon surface. However, many melodies in this symphony are far from being unravelled into simple tunes. It is not only the aetiology and pathogenesis that are still not fully clari¢ed. Coeliac disease was once thought to occur mainly in childhood, presenting mostly with the clinical picture of malabsorption and chronic diarrhoea. However, it seems that it can be diagnosed at any age, provided that a wide spectrum of di¡erent clinical manifestations a¡ecting various organ systems are anticipated and included into the di¡erential diagnosis. Until recently being considered as a rare condition, studies have proved that the estimated prevalence is as high as 0.5 1%, therefore a¡ecting at least 2.5 million European citizens. However, for each diagnosed patient, 5 10 remain undiagnosed. Despite the fact that coeliac disease is far from being completely understood, an impressive amount of research, both basic and applied, has enabled the clari¢cation of many facets of this complex disease. It is therefore the aim of this chapter to present the most recent developments concerning the epidemiology, pathogenesis, and clinical presentation, in cluding complications, and ¢nally the modes of treatment.
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ENVIRONMENTAL FACTORS As mentioned above, gluten is a major exogenous factor a `conditio sine qua non' required for the development of coeliac disease. However, as was described in the recent editorial, its quantity, time of introduction into the infant diet, and relation to breast-feeding are still not clearly elucidated1. Besides infant nutrition there are certainly other exposures that might in£uence immune reactions and result in developing either intolerance or tolerance to food constituents such as gluten.
Influence of infant feeding practice In the 1950s it was suggested that breast-feeding may postpone development of coeliac disease2, while several very recent studies shed further light on the e¡ect of early infant nutrition. In an observational case-referent study, Ivarsson and co-workers showed that longer duration of breast-feeding, smaller initial amounts of gluten and the introduction of gluten-containing foods while the child was still receiving mother's milk were associated with a reduced risk for coeliac disease3. These very important observations were also con¢rmed by a recent meta-analysis by Akobeng and colleagues4. Furthermore, another prospective observational study has shown that there might be a time frame in early infancy with an increased ability to develop oral tolerance to newly introduced food antigens5. According to these results the introduction of gluten by the age of 3 months was associated with a 5-fold increased risk for coeliac disease compared with exposure at the age of 4 6 months. Interestingly, exposure at the age of 6 months and above was again associated with a slightly increased risk5. Going back to the quantity of gluten ingested, the abovementioned Swedish study elucidated for the ¢rst time a dose-related e¡ect of gluten, i.e. a larger initial amount of gluten-containing foods was an independent risk factor for coeliac disease development3,6. Therefore, at the present time it seems that there are feeding patterns contributing to development of coeliac disease, such as the early introduction of gluten, higher amounts of gluten-containing foods and gluten introduction at the time when the child is not receiving mother's milk. However, it is still not clear if those feeding patterns are truly increasing the risk of coeliac disease, or are merely provoking its earlier and more severe presentation. Expressed in other words, long-term follow-up studies are urgently required to clarify if the bene¢cial feeding patterns such as introduction of small amounts of gluten within the favourable time frame, and while the child is still receiving breast milk, are an e¡ective prevention strategy or are merely postponing the development of the disease. If the bene¢cial e¡ects can be con¢rmed, development of preventive strategies based simply on promotion of favourable feeding patterns should be the next step7.
Viral infections A potential contribution of viral infections to the development of coeliac disease is not a very recent hypothesis. Moreover, a molecular mimicry due to 98
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a similar amino-acid sequence among gluten and some adenoviruses was suggested to be a responsible trait8. Yet another pathophysiological link between infection and coeliac disease may be an easier penetration of undigested gliadin required to initiate an immune response, through damaged, and therefore more permeable mucosa9. Further support for the triggering role of viral infection is in observations on the seasonal distribution of coeliac disease10, and also two recent papers relating rotaviral infections and coeliac disease11,12.
GENETIC FACTORS A high concordance rate for coeliac disease among monozygotic twins (approximately 75%)13, and a high prevalence among ¢rst-degree relatives (10%)14 indicate an important role for genetic factors. Development of coeliac disease is associated with certain type II HLA gene products, namely HLADQ2 or HLA-DQ8 proteins15,16. Though virtually all coeliac disease patients carry either the ¢rst or second HLA-DQ molecules, their presence is not su¤cient, as the same sequence is carried by 25 35% of the population, most of whom do not have coeliac disease17. However, it is not only that the incriminating alleles encoding HLA-DQ2 or DQ8 molecules are required for coeliac disease to develop, there is also a clear gene dose e¡ect with a 4-fold higher T cell response if gluten is presented by the immune cells homozygous for the HLA DQ-218. Moreover, this is translated in a similarly increased risk for coeliac disease development in homozygote, compared to heterozygote patients19. Much less is known about the other candidate genes. Outside the HLA region the region most probably involved is the 2q33 genomic region containing the CTLA4 gene. CTLA4 are co-stimulatory molecules upregulated upon T cell activation, and therefore even more likely to be one of the inherited factors involved in coeliac disease pathogenesis 20 . Other promising loci such as 5q and 19p and regions harbouring IL-2 and IL-21 require further linkage disequilibrium mapping studies to identify other nonHLA genes responsible for the development of coeliac disease21.
PATHOMECHANISMS (FIGURE 1) In the pathogenesis of coeliac disease three factors seem to be crucial: (a) gluten as the environmental trigger; (b) HLA-DQ2/8 as a genetic background; and (c) mucosal immune responses. The last component embraces both innate and adaptive immunity. Moreover, the adaptive immunomechanisms consist of a cellular and humoral component, the later one being an antibody response to autoantigen which is the enzyme tissue transglutaminase (tTG)15,22,23. According to present knowledge there is a component of gluten resistant to proteolysis, which therefore remains unchanged in the upper part of the small intestine and passes intact through the epithelium24, preferentially if the mucosal barrier is a¡ected by an infection11. Under the same circumstances 99
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Figure 1
Pathogenesis of coeliac disease (adapted from ref. 23)
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of damaged or in£amed mucosa the intracellular enzyme tTG is released to the extracellular compartment, converting glutamine residues into negatively charged glutamic acid25. Negative charges highly facilitate gluten peptide binding into the positively charged HLA-DQ2/DQ8 restricted groove on antigen-presenting cells. The adaptive immune response is then mediated by a subset of CD4+ T lymphocytes reactive to gliadin peptides with a consequent release of proin£ammatory cytokines, namely interferon gamma26 . This triggers an in£ammatory cascade resulting in villous atrophy and crypt hyperplasia the classical ¢nding in coeliac mucosa. However, there is also a third part of the classical pathological picture in coeliac disease, a massively increased number of intraepithelial lymphocytes (IEL) pointing in the direction of activated innate immunity. It has recently been shown that the gliadin peptide, due to its unique degradation-resistant structure, could damage the epithelial barrier and stimulate over-expression of IL-1527. This is followed by the expansion of CD8+ IEL, cytotoxic to damaged enterocytes expressing a stress protein (MIC-A) on their surface28, and adding further weight to the development of villous atrophy. Finally, a crosslinked complex of tTG and gliadin peptide, within the HLA-DQ2/DQ8 pocket on the antigen-presenting cell, will not only generate a cellular response, but will also stimulate B cells to secrete tTG-speci¢c antibodies29. Whether this autoimmune reaction is actively involved in coeliac disease pathogenesis, and whether the resultant tTG autoantibody is more than a useful serological marker of coeliac disease, is yet unknown. The molecular basis of coeliac disease, described above, is presented in Figure 1, which was adapted from ref. 23.
EPIDEMIOLOGY At the time when the diagnosis of coeliac disease was based on small intestinal biopsy of individuals who presented with chronic diarrhoea and malabsorption, the disease was considered rare, with a prevalence as low as 0.03%30. The advent of highly e¡ective serological diagnostic tests enabled mass screening of di¡erent populations, followed by an increased awareness of a wide spectrum of atypical presentations of coeliac patients, with a plethora of potential extraintestinal symptoms. Suddenly coeliac disease emerged as a serious public health problem with a 20 30-fold higher prevalence compared to the one estimated 50 years ago, approaching 1% of the population and in Europe a¡ecting approximately 2.5 million people31^33. However, coeliac disease is not reserved only for Europe it also occurs in North and South America, as well as in other regions worldwide, and the prevalence is similar34^ 36 . Though ascribed to better disease recognition, the increased prevalence, particularly in the past two decades, may also re£ect a true rise, similar to that of other autoimmune/hyperimmune diseases such as diabetes mellitus, multiple sclerosis, atopy, etc. A rapid rise in the prevalence of coeliac disease in Finland from 0.03% in the 1970s to 0.52% in the year 2000 was simultaneously followed by a signi¢cantly increased prevalence of earlier unrecognized cases from 1.03% to 1.47%. Therefore, it was concluded that the increased prevalence cannot be attributed to better recognition only; neither could it be a 101
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consequence of changes in the population genome37. The authors speculated that the rapid changes in coeliac disease frequency are rather due to environmental factors, such as infant feeding practice or decreased rate of infectious diseases, which should be addressed in future studies37.
CLINICAL ASPECTS AND COMPLICATIONS Clinical presentations Coeliac disease has traditionally been considered as a chronic enteropathy a¡ecting the small intestine, and therefore presenting as a malabsorption syndrome with chronic diarrhoea and wasting. If only the proximal small intestine is involved, the distal small bowel can compensate and the patient may not develop diarrhoea. However, patients will have signs of mineral and vitamin de¢ciency such as iron, folate, calcium, and fat-soluble vitamins. In infants and small children the disease typically presents with chronic diarrhoea, abdominal distension and failure to thrive. Vomiting, constipation, and irritability are also common. In contrast to this classical presentation, it is now commonly accepted that coeliac disease can present at any age, with a highly variable clinical picture involving various organs. Extradigestive manifestations such as bone disease, dental enamel defects, anaemia, ataxia, polyneuropathy, infertility, elevated liver enzymes and depression are wellrecognized constituents of the clinical spectrum of coeliac disease, particularly in older children and adults38^43. To make it even more complicated, it should be acknowledged that: (a) there are clinically silent patients with a typical fullblown mucosal atrophy; (b) there is no correlation between the degree of villous atrophy and the severity of clinical presentation44; (c) persistent villous atrophy despite a strict gluten-free diet, even in the presence of a silent clinical picture, still carries a risk of subsequent severe complications45; (d) patients may su¡er from gluten-dependent symptoms before developing enteropathy while being in the latent phases of coeliac disease46. Therefore, many more studies are required to clarify the pathomechanisms underlying the di¡erent manifestations of coeliac disease, both in children and in adults.
Complications Complications of coeliac disease are commonly divided into malignant and non-malignant, and both of these are responsible for the increased morbidity and mortality. While non-malignant complications are much more common, malignancy is the most serious problem with an overall risk of cancer that is almost twice that in the general population47. These ¢gures are lower than previously thought once it became clear how prevalent coeliac disease is, the risk for non-Hodgkin's lymphoma, for example, dropped from 40 into the range of 3 648,49. Patients who are unresponsive to a gluten-free diet can be particularly suspected of malignancy, and should be re-evaluated. Though dietary non-adherence is the most likely cause of their persistent mucosal 102
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atrophy, approximately 5% of them may have refractory coeliac disease a condition particularly prone to development of enteropathy-associated T-cell lymphoma (EATL) 50 . More about that severe condition can be found elsewhere9,51^53.
DIAGNOSIS OF COELIAC DISEASE (FIGURES 2 AND 3) Due to the wide spectrum of presenting symptoms the ¢rst important step in diagnosing coeliac disease is to include it into di¡erential diagnosis. The second prerequisite is to perform diagnostic procedures while the patient is still on a gluten-containing diet. The diagnostic armamentarium consists of the combination of histology, serology, the positive e¡ect of a gluten-free diet, and in dubious cases testing for HLA-DQ2/DQ8 haplotypes. The current recommendations for adults, from the WGO-OMGE Practice Guidelines, and the still-valid diagnostic criteria developed by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), are presented in Figures 2 and 3. More about the diagnostic problems can be found in the suggested literature30,46,54^57.
Figure 2
Current diagnostic criteria for paediatric patients56
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Figure 3 ref. 54)
Diagnostic algorithm for adult patients suspected for coeliac disease (adapted from
Who should be screened for coeliac disease? At ¢rst sight coeliac disease meets all ¢ve WHO criteria for the implementation of mass-screening of the general population: (a) coeliac disease is common; (b) reliable serological tests are available; (c) the number of undiagnosed patients is high; (d) treatment is well established; and (e) if untreated the disease could result in increased mortality. However, the current view is that there is insu¤cient evidence at present to support a decision to carry out mass screening of the general population due to the following: (a) long-term health consequences and the risks of undetected coeliac disease should be more precisely assessed; and (b) the economic costs of screening and treatment versus morbidity prevented, need further calculation58,59. Opposite to mass 104
RECENT ADVANCES IN COELIAC DISEASE
screening, active case-¢nding in primary care is a feasible and successful strategy for detecting coeliac disease patients, and can considerably improve awareness of the disease among health professionals60,61.
TREATMENT AND NATURAL HISTORY If gluten is the `conditio sine qua non' for disease development, elimination of all gluten-containing foods (containing wheat, rye and barley) is a logical therapeutic option, and is so far the only accepted treatment modality for coeliac disease. However, there are drawbacks to this dietetic approach, while some new therapeutic options are appearing on the horizon, and need to be considered and explored.
Adherence problems A life-long commitment to the very restrictive gluten-free diet (GFD) is far from easy. Studies have identi¢ed that patients without major health problems, mainly those detected by mass screenings, tend to be less compliant62. On the contrary, patients presenting with clear clinical symptoms, who were regularly followed, and who understood the principles of GFD, exhibit a high correlation with the adherence63. Another important issue is an ability to follow the GFD outside home at social events, when travelling, etc.64. Finally, to make the puzzle even more complicated, paediatric patients with con¢rmed coeliac disease were described, who could tolerate reintroduction of a normal glutencontaining diet after achievement of long-term clinical and histological remission on the GFD65.
Oats toxicity Whether this condition exists or not is yet another problem. Though patients with T cells reactive to avenin from oats have been detected66, oats is generally considered safe for adults and for children with coeliac disease67. A major problem concerning GFD with oats comprises di¤culties in guaranteeing that commercially available oat products are free of contamination with other toxic grains, which occurs during harvesting and milling procedures68.
Gluten threshold for gluten-free food According to the results of a recently published randomized prospective study, coeliac disease patients, in order to stay free of intestinal in£ammation, should ingest less than 50 mg of gluten per day69. Concerning international regulations on food labelling, the Codex Alimentarius Committee on Nutrition and Foods for Special Dietary Use (CCNFSDU) is now in the process of adopting 20 ppm as an acceptable level of gluten in products claiming to be `gluten-free', and 100 ppm for food rendered gluten-free (wheat starch-based products)70.
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New treatment modalities Concerning all disadvantages of lifelong GFD, it is no surprise that there is a considerable interest in exploring other therapeutic options. The most advanced and most promising non-dietary treatment is based on the use of oral enzymes proteases which can degrade toxic gliadin peptides in the gastrointestinal lumen before coming into contact with the intestinal mucosa. In that respect studies are in progress and are showing promising results71.
Natural history If coeliac disease remains unrecognized it can increase the risk of lifethreatening complications, with a consequent mortality rate exceeding that in the general population by a factor of 2 4. A gluten-free diet reduces excess mortality, lending further support to the importance of early diagnosis and treatment of coeliac disease54.
CONCLUDING REMARKS Coeliac disease is a genetically determined, immune-mediated, chronic noninfectious enteropathy. The disease develops as a consequence of interaction of gluten from wheat and of similar storage polypeptides from rye and barley with the innate and adaptive immune mechanisms, whereby HLA-DQ2/DQ8 and non-HLA genes, jointly with some environmental factors such as viral infections and infant feeding patterns, confer either increased or reduced risk for acquiring the disease. Coeliac disease a¡ects as much as 1% of populations worldwide, and is one the most common food intolerances in Europe. It can be diagnosed at any age, provided that a wide spectrum of di¡erent clinical manifestations from various organ systems are anticipated and included into the di¡erential diagnosis. The high prevalence, and the fact that for each diagnosed patient, 5 10 remain undiagnosed, makes coeliac disease an important public health problem. A restrictive lifelong gluten-free diet is presently the only accepted therapeutic modality. However, new non-dietetic treatments, such as enzymes which can degrade toxic peptides before reaching the intestinal mucosa, are promising options requiring further exploration. Finally, if coeliac disease remains unrecognized, and therefore untreated, the risk of life-threatening complications such as intestinal malignancies is signi¢cantly increased.
References 1. 2. 3.
Farrell RJ. Infant gluten and coeliac disease: too early, too late, too much, too many questions. J Am Med Assoc. 2005;293:2410 12. Andersen DH, Di Sant'Agnese PA. Idiopathic celiac disease: mode of onset and diagnosis. Pediatrics. 1953;11:207 22. Ivarsson A, Hernell O, Stenlund H, Persson LA. Breast-feeding protects against celiac disease. Am J Clin Nutr. 2002;75:914 21.
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Akobeng AK, Ramanan AV, Buchan I, Heller RF. E¡ects of breast-feeding on risk of CD: a systematic review and meta-analysis of observational studies. Arch Dis Child. 2006;91:39 43. Norris JM, Barriga K, Ho¡enberg EJ et al. Risk of celiac disease: autoimmunity and timing of gluten introduction in the diet of infants at increased risk of disease. J Am Med Assoc. 2005;293:2343 51. Ivarsson A, Persson LA, Nystrom L et al. Epidemic of coeliac disease in Swedish children. Acta Paediatr. 2000;89:165 71. Ivarsson A. The Swedish epidemic of coeliac disease explored using an epidemiological approach some lessons to be learnt. Best Pract Res Clin Gastroenterol. 2005;425 40. Kagno¡ MF, Paterson YJ, Kumar PJ et al. Evidence for the role of a human intestinal adenovirus in the pathogenesis of coeliac disease. Gut. 1987;28:995 1001. Green PHR, Cellier C. Celiac disease. N Engl J Med. 2007;357:1731 43. Ivarsson A, Hernell O, Nystrom L, Persson LA. Children born in the summer have an increased risk for coeliac disease. J Epidemiol Commun Health. 2003;57:36 9. Stene LC, Honeyman MC, Ho¡enberg EJ et al. Rotavirus infection frequency and risk of celiac disease in early childhood: a longitudinal study. Am J Gastroenterol. 2006;101:2333 40. Zanoni G, Navone R, Lunardi C et al. In celiac disease a subset of autoantibodies against transglutaminase binds to toll-like receptor 4 and induces activation of monocytes. PLOS Med. 2006;3:1637 53. Greco L, Romino R, Coto I et al. The ¢rst large population based twin study of coeliac disease. Gut. 2002;50:624 8. Ellis A. Coeliac disease: previous family study. In: McConnell RB, editor. Genetics of Coeliac Disease. Lancaster: MTP Press, 2003:197 200. Sollid LM, Markussen G, Ek J et al. Evidence for a primary association of coeliac disease to a particular HLA-DQ heterodimer. J Exp Med. 1989;169:345 50. Sollid LM, Thorsby E. HLA susceptibility genes in celiac disease: genetic mapping and role in pathogenesis. Gastroenterology. 1993;105:910 22. Koning F, Schuppan D, Cerf-Bensussan N, Sollid LD. Pathomechanisms in celiac disease. Best Pract Res Clin Gastroenterol. 2005;19:373 87. Vader W, Stepniak D, Kooy Y et al. The HLA-DQ2 gene dose e¡ect in celiac disease is directly related to the magnitude and breadth of gluten-speci¢c T-cell response. Proc Nat Acad Sci USA. 2003;100:12390 5. Mearin ML, Biemond I, Pena A et al. HLA-DR phenotypes in Spanish coeliac disease children: their contribution to the understanding of the genetics of the disease. Gut. 1983;24:532 7. van Heel DA, Hunt K, Greco L, Wijmenga C. Genetics in coeliac disease. Best Pract Res Clin Gastroenterol. 2005;19:323 39. van Heel DA, Franke L, Hunt KA et al. A genome-wide association study for coeliac disease identi¢es risk variants in the region harboring IL2 and IL21. Nature Genet. 2007;25:1 4. Koning F. Celiac disease: caught between a rock and a hard place. Gastroenterology. 2005;129:1294 301. Hourigan CS. The molecular basis of celiac disease. Clin Exp Med. 2006;6:53 9. Shan L, Molberg Q, Parrot I et al. Structural basis for gluten intolerance in celiac sprue. Science. 2002;297:2275 9. Aeschlimann D, Thomazy V. Protein cross-linking in assembly and remodelling of extracellular matrices: the role of transglutaminases. Connect Tissue Res. 2000;41:1 27. Molberg Q, McAdam S, Lundin KEA et al. T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol. 2001;31:1317. Mention JJ, Ben Ahmed M, Begue B et al. Interleukin 15: a key to disrupted intraepithelial lymphocyte homeostasis and lymphomagenesis in celiac disease. Gastroenterology. 2003;125:730 45. Hue S, Mention JJ, Monteiro RC et al. A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity. 2004;21:376 7. Sollid LM, Molberg Q, McAdam S, Lundin KEA. Autoantibodies in celiac disease: tissue transglutaminase guilt by association? Gut. 1997;41:851 2.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 30. Green PH, Jabri B. Coeliac disease. Lancet. 2003;362:383 91. 31. West J, Logan RF, Hill PG et al. Seroprevalence, correlates, and characteristics of undetected coeliac disease in England. Gut. 2003;52:960 5. 32. Maki M, Mustalahti K, Kokkonen J et al. Prevalence of celiac disease among children in Finland. N Engl J Med. 2003;348:2517 24. 33. Bingley PJ; Williams AJ, Norcross AJ et al. Undiagnosed coeliac disease at age seven: population-based prospective birth cohort study. Br Med J. 2004;328:322 3. 34. Fasano A, Berti I, Gerarduzzi T et al. Prevalence of celiac disease in at-risk and non-at-risk groups in the United States. Arch Intern Med. 2003;163:286 92. 35. Sood A, Midha V, Sood N, Malhotra V. Adult celiac disease in northern India. Indian J Gastroenterol. 2003;22:124 6. 36. Gomez JC, Selvaggio GS, Viola M et al. Prevalence of celiac disease in Argentina: screening of an adult population in the La Plata area. Am J Gastroenterol. 2001;96:2700 4. 37. Lohi S, Mustalahti K, Kaukinen K et al. Increasing prevalence of celiac disease over time. Aliment Pharmacol Ther. 2007;26:1217 25. 38. Fasano A. Clinical presentation of celiac disease in the pediatric population. Gastroenterology. 2005;128(Suppl. 1):S68 73. 39. D'Amico MA, Holmes J, Stavropoulos SN et al. Presentation of pediatric celiac disease in the United States: prominent e¡ect of breast feeding. Clin Pediatr. 2005;44:249 58. 40. Wiernik CD, van Diermen DE, Aartman IHA, Heymans HSA. Dental enamel defects in children with coeliac disease. Int J Paediatr Dent. 2007;17:163 8. 41. Harper JW, Holleran SF, Ramakrishnan R et al. Anemia in celiac disease is multifactorial in etiology. Am J Hematol. 2007;82:996 1000. 42. Green PHR, Stavropoulos SN, Panagi SG et al. Characteristics of adult celiac disease in the USA: results of a national survey. Am J Clin Gastroenterol. 2001;96:126 31. 43. Green PH. The many faces of celiac disease: clinical presentation of celiac disease in an adult population. Gastroenterology. 2005;128(Suppl. 1): S74 8. 44. Brar P, Kwon GY, Egbuna II et al. Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease. Dig Liver Dis. 2007;39:26 9. 45. Kaukinen K, Peraaho M, Lindfors K et al. Persistent small bowel mucosal villous atrophy without symptoms in coeliac disease. Aliment Pharmacol Ther. 2007;25:1237 45. 46. Kaukinen K, Collin P, Maki M. Latent coeliac disease or coeliac disease beyond vollous atrophy. Gut. 2007;56:1339 40. 47. West J, Logan RF, Smith CJ et al. Malignancy and mortality in people with coeliac disease: population based cohort study. Br Med J. 2004;329:716 19. 48. Smedby KE, Akerman M, Hildebrand H et al. Malignant lymphomas in coeliac disease: evidence of increased risk for lymphoma types other than enteropathy-type T cell lymphoma. Gut. 2005;54:54 9. 49. Howdle PD, Jalal PK, Holmes GK, Houlston RS. Primary small-bowel malignancy in the UK and its association with coeliac disease. Q J Med. 2003;96:345 53. 50. Trier JS. Celiac sprue. N Engl J Med. 1991;325:1709 19. 51. Al-toma A, Verbeek WHM, Hadithi M et al. Survival in refractory coeliac disease and enteropathy-associated T-cell lymphoma: retrospective evaluation of single-centre experience. Gut. 2007;56:1373 8. 52. Ciccocioppo R, Perfetti V, Corazza GR. Treating ETTCL: a matter of early diagnosis and chemotherapy strategies. Dig Liver Dis. 2007;39:642 5. 53. Al-toma A, Verbeek WHM, Visser OJ et al. Disappointing outcome of autologous stem cell transplantation for enteropathy-associated T-cell lymphoma. Dig Liver Dis. 2007;39:634 41. 54. Bai J, Zeballos E, Fried M et al. WGO-OMGE Practice Guideline: celiac disease. World Gastroenterol News. 2005;10:S1 8. 55. National Institutes of Health Consensus Development Conference Statement on Celiac Disease, June 28 30, 2004. Gastroenterology. 2005;128:S1 9. 56. Walker-Smith JA, Guandalini S, Schmitz J et al. Revised criteria for diagnosis of coeliac disease. Arch Dis Child. 1990;65:909 11. 57. van Heel DA, West J. Recent advances in coeliac disease. Gut. 2006;55:1037 46. 58. Mearin ML, Ivarsson A, Dickey W. Coeliac disease: is it time for mass screening? Best Pract Res Gastroenterol. 2005;19:441 52.
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RECENT ADVANCES IN COELIAC DISEASE 59. Shamir R, Hernell O, Leshno M. Cost-efectiveness analysis of screening for celiac disease in the adult population. Med Decision Making. 2006;26:282 93. 60. Berti I, Della Vedova R, Paduano R et al. Coeliac disease in primary care: evaluation of a case-¢nding strategy. Dig Liver Dis. 2006;38:461 7. 61. Catassi C, Kryszak D, Louis-Jacques O et al. Detection of celiac disease in primary care: a multicenter case-¢nding study in North America. Am J Gastroenterol. 2007;102;1454 60. 62. Fabiani E, Taccari LM, Ratsch IM et al. Compliance with gluten-free diet in adolescents with screening-detected celiac disease: a 5-year follow-up study. J Pediatr. 2000;136:841 3. 63. Jadresin O, Misak Z, Kolacek S et al. Adherence to gluten-free diet of children with coeliac disease. J Paediatr Gastroenterol Nutr. 2008 (In press) 64. Le¥er DA, Edwards-George J, Deenis M et al. Factors that in£uence adherence to a gluten-free diet in adults with celiac disease. Dig Dis Sci. 2008;53 (In press). 65. Matysiak-Butnik T, Malamut G, Patey-Mariaud de Serre N et al. Long-term follow-up of 61 celiac patients diagnosed in childhood: evolution toward latency is possible on a normal diet. Gut. 2007;56:1379 86. 66. Arentz-Hansen H, Fleckenstein B, Molberg O et al. The molecular basis for oats intolerance in patients with celiac disease. PLOS Med. 2004;1:84 92. 67. Peraaho M, Kaukinen K, Mustalahti K et al. E¡ct of an oats-containing gluten-free diet on symptoms and quality of life in coeliac disease: a randomized study. Scand J Gastroenterol. 2004;39:27 31. 68. Hernando A, Mujico JR, Juanas D et al. Con¢rmation of the cereal type in oat products highly contaminated with gluten. J Am Diet Assoc. 2006;106:665 6. 69. Catassi C, Fabiani E, Iacono G et al. A prospective, double-blind, placebo-controlled trial to establish a safe gluten threshold for patients with celiac disease. Am J Clin Nutr. 2007;85:160 6. 70. Codex Alimentarius Committee on Nutrition and Food for Special Dietary Uses. Revised Codex Standard for Foods for Special Dietary Use for Persons intolerant to Gluten. Roma, 2007, Alinorm 08/31/26. 71. Siegel M, Bethun MT, Gass J et al. Rational design of combination enzyme therapy for celiac sprue. Chem Biol. 2006;13:649 58.
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10 Gastrointestinal manifestations of AIDS J. TOMAZíICí
Many HIV-infected patients have gastrointestinal complications. Major causes of anorexia, nausea and vomiting are medications (especially antiretrovirals, antibiotics, opiates and non-steroid anti-in£ammatory drugs), depression, intracranial pathology, gastrointestinal diseases, hypogonadism, pregnancy, lactic acidosis, etc. Treatment of the underlying condition is important. Thrush is presented as white painless plaques on the oral mucosa that can easily be scraped o¡. Because oral candidiasis itself is an opportunistic infection, it predicts disease progression and development of other AIDSrelated infections. The cause of aphthous ulcers is unknown. In di¡erential diagnosis herpes simplex virus (HSV), cytomegalovirus (CMV) and druginduced ulcers should be considered. Anaerobic bacteria cause gingivitis (from linear gingival erythema, necrotizing gingivitis, necrotizing peridontitis to necrotizing stomatitis). Oral hairy leukoplakia (caused by Epstein Barr virus) is a raised, white lesion of the oral mucosa, usually seen on the lateral margin of the tongue that cannot be scraped o¡ and will not respond to antifungal therapy. Other oral lesions: the purple-red lesions of Kaposi sarcoma, non-Hodgkin lymphoma (either swelling or ulcers), oral warts, salivary glands may be enlarged by in¢ltration with CD8+ cells. The diagnosis of Candida oesophagitis is based on the presence of thrush, odynophagia, less than 100 CD4 cells/mm3 and good response to treatment. Endoscopy is recommended with atypical presentation or failure to respond to empiric treatment. Beside Candida infection, oesophageal diseases in patients with HIV infection could be caused by CMV, HSV or aphthous ulcers. Diarrhoea is de¢ned as at least three loose or watery stools a day. It is helpful to divide diarrhoeal illnesses into acute, where most patients present almost immediately with symptoms, and chronic, where the symptoms have been present for at least 1 month and often continue in variable form for many months. It is also helpful to divide the patients with diarrhoea into those with a relatively preserved immune system (4200 CD4 cells/mm3) and those without; in those with 4200 CD4 cells/mm3, most of the causes of virulent infection are easy to diagnose on stool analysis and are treatable, or may improve 110
GASTROINTESTINAL MANIFESTATIONS OF AIDS
spontaneously. Diarrhoea in those with very reduced CD4 counts (5200 CD4 cells/mm3) and severe immunosuppression is much more likely to be caused by organisms of very limited virulence, is often associated with profound symptoms and weight loss, may not be diagnosed by simple stool analysis and responds poorly to treatment. Diarrhoea is medication-related (especially protease inhibitors) or pathogen-related (bacterial: Salmonella, Shigella, Campylobacter jejuni, Vibrio, Yersinia, Escherichia coli 0157, Clostridium di¤cile, non-tuberculous mycobacteriosis; viral: CMV, adenovirus, astrovirus, picornavirus, calicivirus; protozoal: cryptosporidiosis, microsporidiosis, isosporiasis, cyclosporiasis). The most common identi¢ed microbial cause of cholangiopathy (relatively rare diseases, seen primarily in late stage AIDS) is Crytposporidium, followed by Microsporidia, CMV and Cyclospora. Major causes of pancreatitis are drugs (especially didanosine or didanosine plus zalcitabine; NRTI-associated mitochondrial toxicity; PI-associated hypertriglyceridaemia), opportunistic infections (CMV, less common nontuberculous and tuberculous mycobacteriosis, cryptosporidiosis) and conditions that cause pancreatitis in the general population (especially alcoholism; less common gallstones and hypertriglyceridaemia). Hepatic abnormalities are quite common in the HIV-infected population and may be due to viral hepatitis, HIV-related opportunistic infections, medication toxicity (all antiretroviral drugs), alcohol, non-alcoholic fatty liver disease or malignancy (Kaposi sarcoma, non-Hodgkin lymphoma, hepatocellular carcinoma). Communities and populations at high risk for HIV infection are also likely to be at risk for co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). HIV, HBV and HCV are blood-borne pathogens transmitted through similar routes (via injection drug use, sexual contact, from mother to child during pregnancy or birth). Patients infected with HIV are less likely to clear hepatitis C viraemia, have high HCV RNA loads and experience more rapid progression of HCV-related liver disease than those without HIV infection. In the era of highly active antiretroviral therapy (HAART), HCV-related liver disease is currently, and will continue to be, a major cause of hospital admissions and deaths among HIV-infected persons. As such, e¡ective HCV treatment strategies are needed. Treatment with peginterferon plus ribavirin may be e¡ective, particularly for patients with HCV genotype 2 and 3 or those with HCV genotype 1 and a low levels of hepatitis C viral load. Early monitoring of HCV RNA response at treatment weeks 4 and 12 can e¡ectively identify persons with virological nonresponse to therapy preventing unnecessary exposure to toxicity. HIV infection can accelerate progression of HBV-related liver disease; therefore, treatment of chronic HBV infection is generally recommended for all HBV/HIV-coinfected patients. The best strategy for the management of HBV infection has not been de¢ned, particularly for individuals with chronic HBV infection who do not yet require anti-HIV therapy. For HBV/HIVcoinfected individuals for whom HIV treatment is indicated, most experts recommend the use of an antiretroviral regimen that includes the use of two agents active against HBV (e.g. tenofovir plus emtricitabine or lamivudine). 111
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HBV vaccination is indicated for all children and adults who are at increased risk of HBV infection, including HIV-infected patients, patients with multiple sexual partners, men who have sex with men, and patients who engage in IDU. Various patterns of abdominal pain are particularly common in HIVinfected patients. A syndrome of right upper quadrant pain is often associated with thickening or dilation of the bile duct seen on computed tomography or ultrasonography and a raised alkaline phosphatase level. The most common cause of this syndrome is an AIDS-related sclerosing cholangitis, the pathology of which is very similar to that of idiopathic sclerosing cholangitis. It is not clear that any treatment helps, although sphincterotomy has sometimes been performed. The pain tends to improve over months or years. Epigastric pain may be due to a non-HIV-related condition such as peptic ulcer, but a number of patients with this syndrome have Kaposi's sarcoma or CMV infection of the distal oesophagus or stomach lining. Lymphoma of the stomach will also produce this pain. Lower abdominal pain is often associated with severe constipation and is most frequently caused by opiate use. Di¡use abdominal pain often associated with rebound abdominal tenderness may be found in bacterial or CMV causes of diarrhoea. Loin pain has become a common problem in the era of indinavir use. In up to 4% of patients taking this drug, renal stones occur, composed most commonly of indinavir crystals. Sometimes no stone is found but the patient usually has haematuria or indinavir crystals in the urine. Wasting syndrome is de¢ned as involuntary loss of at least 10% of original body weight accompanied by persistent diarrhoea (at least two bowel movements daily for more than 30 days) or extreme fatigue and/or fever without apparent infectious aetiology. Wasting syndrome is an exclusion diagnosis. Weight loss remains an independent risk factor for mortality, even in the era of HAART, and every patient should be weighed regularly! The risk for apparent infections is signi¢cantly elevated and there is also cognitive impairment in these patients.
112
11 Prevention and screening for colorectal cancer B. SíTABUC and S. PLUT
INTRODUCTION Colorectal cancer is one of the most commonly diagnosed cancers, and a leading cause of cancer deaths in the developed regions of the world. According to the International Agency for Research on Cancer (IARC) estimates 1 023 152 new cases of colorectal cancer were diagnosed and 529 978 colorectal deaths were registered worldwide in 20021. Colorectal cancer is the fourth most common cancer diagnosed and fourth most common cause of cancer mortality in men worldwide, whereas in women it was the third most common cancer diagnosed and the fourth most common cause of cancer mortality. However, mortality re£ects not only prevalence, but also quality of health system (availability of screening programmes, diagnostic services and adequate therapy). In some developed countries the incidence and mortality rates are stable and in some countries even declining2. This decline could be attributed to primary prevention (rise in awareness and healthier lifestyle), screening programmes (earlier detection enables treatment of precancerous adenomatous polyps and non-invasive cancer) and improved treatment. However, in countries where socioeconomic status is improving, the incidence of colorectal cancer has been rising (Eastern Europe, Asia, Latin America)3. The most important risk factors for colorectal cancer are older age, male gender, diet and poor physical exercise habits, a personal history of in£ammatory bowel disease, certain genetic syndromes, and a family history of colorectal cancer or adenomatous polyps. The incidence of colorectal cancer rises dramatically from the age of 50 years. About 50% of all colorectal cancer cases are diagnosed after age of 65 years4. Males are more a¡ected, presumably because of di¡erent dietary and lifestyle habits, tobacco and alcohol use and body stores of iron. The e¡ect of diet is still under heavy debate; however, a consistently high caloric intake, obesity and metabolic syndrome are associated with risk for colorectal cancer. Eating frequent high fat-containing meals promotes carcinogenic bile acid secretion. Grains, fruit, vegetables and dairy product contain protective dietary ¢bre, minerals and vitamins5,6. Su¤cient recreational activity from youth throughout adult life has been shown to reduce 113
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
the risk for colorectal cancer7. Chronic in£ammatory bowel disease leads to increased turnover of epithelial cells and thus an increased risk for colorectal cancer. The longer the duration of the disease, and the more extensive the in£ammation is, the greater the risk8. By de¢nition, adenomatous epithelium is neoplastic and all adenomas display some degree of dysplasia and are associated with increased risk of cancer9. At present we can deduce that the aetiology of more than 30% of colorectal cancer is directly related to the environment; namely diet and lifestyle. Less than 15% is related to the inherited risk factors, namely family history of colorectal cancer, and the remainder is the result of interaction between these aetiologies10. Because the aetiology is multifactorial there is no single major preventable risk factor, either an individual food item or a micronutrient that causes or prevents colorectal cancer, but it seems that lifestyle and diet throughout the entire life are of vital importance. The maintenance of an ideal body weight, promotion of physical activity, reduced consumption of animal fats, and increased consumption of fruits and vegetables have salutary e¡ects beyond the primary prevention of colorectal cancer. The search for chemopreventive agents has given no useful results11. A prolonged use of non-steroidal anti-in£ammatory drugs, COX-2 inhibitors12 and aspirin13 is associated with reduced risk of colorectal cancer, but long-term studies are needed to evaluate e¡ectiveness and side-e¡ects14. It is very likely chemopreventive drugs will some day be recommended, but they are currently no substitute for screening the general population or surveillance of risk groups. Colorectal cancer screening and case-¢nding can prevent the development of colorectal cancer and reduce the risk of deaths. Four randomized trials have shown that faecal occult blood testing (FOBT) is e¡ective in lowering the colorectal cancer mortality rate (15 33%) and its incidence in individuals who undergo screening15^18. FOBT available for screening is based on two principal technologies: chemical tests and immunochemical tests. The chemical tests use guaiac resin to detect the peroxidase activity of haem. They react to any peroxidase in faeces (e.g. in plant foods and haem in red meat) and are a¡ected by certain chemicals (e.g. vitamin C). Because of the relative stability of haem these tests may detect bleeding from any part of the gastrointestinal tract, including the stomach19. While of proven e¤cacy, guaiac-based FOBT have two major shortcomings: restriction of diet and drugs is needed to optimize speci¢city and sensitivity20 and they are not selective for colorectal bleeding. The faecal immunochemical tests use antibodies speci¢c for human globin, and as such are not a¡ected by diet. Globin is rapidly degraded by digestive enzymes and this causes immunochemical FOBT to be highly sensitive for occult bleeding of colorectal origin 19 . Neoplasms exfoliate abundantly into the bowel, and DNA from those cells can be assayed using modern molecular techniques. Because of the molecular heterogeneity of cancer, a combination of markers has to be selected to achieve optimal sensitivity and speci¢city. At the current level of technology DNA testing is costly, and requires speci¢c stool sampling and processing; this prevents the use of a DNA test for screening purposes21. 114
PREVENTION AND SCREENING FOR COLORECTAL CANCER
After 18 years of follow-up in a Minnesota trial, annual FOBT screening was found to reduce colorectal cancer mortality by 33% and, when performed every other year, by 21%22, a rate consistent with the results of the biennial screening in the European trials16^18. Furthermore, in a recent FOBT screening in 478 250 residents of the pilot areas in England and Scotland, the overall positive test was 1.9% and the rate of detecting cancer was 1.62 per 1000 screened people. The positive predictive value was 10.9% for cancer and 35% for adenoma. Of 552 colorectal cancers detected by screening, 48% of all screen-detected cancers were stage I, and only 1% metastasized at the time of diagnosis23. The cost per life-year saved by FOTB screening is similar to or less than that of breast cancer screening 24; this makes FOTB an acceptable screening test. The main disadvantages are its low compliance rate for the ¢rst and repeated screening (20 70%)25, as well as its moderate sensitivity for detecting colorectal cancer and low sensitivity for polyps. A positive FOTB sample warrants further diagnostic evaluation, preferably colonoscopy. The FOTB screening performed every year, and combined with £exible sigmoidoscopy every 5 years, is more e¡ective than either method alone. However, despite the fact that FOTB may be less sensitive for distal colon lesions, both methods together do not greatly improve detection rates for proximal lesions 26 . The disadvantages of this type of screening are inconvenience, high cost and complications with an uncertain gain in e¡ectiveness. Screening sigmoidoscopy can be compared to one-breast only mammography screening. Although there are no randomized studies evaluating whether screening colonoscopy alone reduced the incidence and mortality from colorectal cancer, several guidelines have included colonoscopy as a screening option. Colonoscopy has a proven high sensitivity for detecting polyps and carcinomas of the whole colon27. It is the most complete means for examining the colon. It enables the examiner not only to con¢rm macroscopic diagnoses with histological examination of biopsies, but also allows therapeutic intervention. The disadvantages include vigorous bowel preparation and the need for trained examiners. Adequate withdrawal times of colonoscopy from caecum to anus of minimum 6 min are required to ensure su¤cient neoplastic lesion detection28. Perforation rates for diagnostic colonoscopy vary between 0.06% and 0.2%, and mortality varies between 0% and 0.06%29,30. The choice of a 10-year interval between screening colonoscopies for people at average risk is based on estimates of the sensitivity of colonoscopy and the rate at which advanced adenomas develop31. Double-contrast barium enema is less sensitive for the detection of polyps (polyps smaller than 1 mm in diameter in particular) and cancer than is colonoscopy, and involves exposing the examinee to radiation32. In addition, this procedure does not permit removal of polyps or biopsy of cancer and, if polyps and cancer are suspected, an additional colonoscopy has to be performed. Computed tomography colonography or virtual colonoscopy has been evaluated as a possible colorectal cancer screening method33. Because of the lack of therapeutic ability, requirement of full bowel cleaning, radiation exposure, long learning curve and high cost, the use of virtual colonoscopy outside of clinical trials cannot be recommended34. Magnetic resonance imaging colonography eliminates radiation exposure, but is even more expensive. 115
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In 2003 the European Commission issued recommendations for screening for breast, cervical and colorectal cancer valid in all member countries. The Republic of Slovenia adopted this programme and national guidelines for colorectal screening were published35. Following experiences of the 13 EU countries that have a well-organized screening programme for colorectal cancer, e.g. Finland, France, Great Britain and Spain, an organized screening for colorectal cancer by using an immunochemical faecal occult blood test will be launched in April of 2008. Organized colorectal cancer screening has a greater potential to reduce cancer incidence and mortality due to the higher achievable levels of population coverage, follow-up and quality compared with opportunistic screening. However, due to the low compliance for colorectal screening, only improved awareness and knowledge of the general population concerning colorectal cancer risk factors and the bene¢ts of screening can improve compliance.
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Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2002: Cancer Incidence. Mortality and Prevalence Worldwide. IARC CancerBase No. 5, version 2.0., Lyon: IARC Press 2004. http://www dep.iarc.fr/ Ries LAG, Melbert D, Krapcho M et al., editors. SEER Cancer Statistics Review, 1975 2004, National Cancer Institute. Bethesda, MD, 2006. http://seer.cancer.gov/csr/ 1975 2004/ Bonithon Kopp C, Benhamiche AM. Are there several colorectal cancers? Epidemiological data. Eur J Cancer. 1999;8(Suppl. 1):S3 S12. Shureiqi I, Cooksley CD, Morris J, Soliman AS, Levin B, Lippman SM. E¡ect of age on risk of second primary colorectal cancer. J Natl Cancer Inst. 2001;93:1264 6. Cho E, Smith Warner SA, Spiegelman D et al. Dairy foods, calcium, and colorectal cancer: a pooled analysis of 10 cohort studies. J Natl Cancer Inst. 2004;96:1724. Glade MJ. Food, nutrition, and the prevention of cancer: a global perspective. American Institute for Cancer Research/World Cancer Research Fund, American Institute for Cancer Research, 1997. Chao A, Connell CJ, Jacobs EJ et al. Amount, type, and timing of recreational physical activity in relation to colon and rectal cancer in older adults: the Cancer Prevention Study II Nutrition Cohort. Cancer Epidemiol Biomarkers Prev. 2004;13:2187 95. Munkholm P. Review article: The incidence and prevalence of colorectal cancer in in£ammatory bowel disease. Aliment Pharmacol Ther. 2003;18(Suppl. 2):1 5. O'Brien MJ, Winawer SJ, Zauber AG et al.; National Polyp Study Workgroup. Flat adenomas in the National Polyp Study: is there increased risk for high grade dysplasia initially or during surveillance? Clin Gastroenterol Hepatol. 2004;2:905 11. Gertig DM, Hunter DJ. Genes and environment in the etiology of colorectal cancer. Semin Cancer Biol. 1998;8:285 98. Corpet DE, Pierre F. From animal models to prevention of colon cancer. Systematic review of chemoprevention in min mice and choice of the model system. Cancer Epidemiol Biomarkers Prev. 2003;12:391 400. Koutsos MI, Shi¡ SJ, Rigas B. Can nonsteroidal anti in£ammatory drugs be recommended to prevent colon cancer in high risk elderly patients? Drugs Aging. 1995;6:421 5. Imperiale TF. Aspirin and the prevention of colorectal cancer. N Engl J Med. 2003;348:879 80. Jankowski JA, Hawk ET. A methodologic analysis of chemoprevention and cancer prevention strategies for gastrointestinal cancer. Nat Clin Pract Gastroenterol Hepatol. 2006;3:101 11.
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PREVENTION AND SCREENING FOR COLORECTAL CANCER 15. Mandel JS, Bond JH, Church TR et al. Reducing mortality from colorectal cancer by screening for fecal occult blood. Minnesota Cancer Control Study. N Engl J Med. 1993;328:1365 71. Erratum: N Engl J Med. 1993;329:672. 16. JÖrgensen OD, Kronborg O, Fenger C. A randomised study of screening for colorectal cancer using faecal occult blood testing: results after 13 years and seven biennial screening rounds. Gut. 2002;50:29 32. 17. Hardcastle JD, Chamberlain JO, Robinson MH et al. Randomised controlled trial of faecal occult blood screening for colorectal cancer. Lancet. 1996;348:1472 7. 18. Faivre J, Dancourt V, Lejeune C et al. Reduction in colorectal cancer mortality by fecal occult blood screening in a French controlled study. Gastroenterology. 2004;126:1674 80. 19. Young GP, Cole S. New stool screening tests for colorectal cancer. Digestion. 2007;76:26 33. 20. Sinatra MA, St John DJ, Young GP. Interference of plant peroxidases with guaiac based fecal occult blood tests is avoidable. Clin Chem. 1999;45:123 6. 21. Osborn NK, Ahlquist DA. Stool screening for colorectal cancer: molecular approaches. Gastroenterology. 2005;128:192 206. 22. Mandel JS, Church TR, Bond JH et al. The e¡ect of fecal occult blood screening on the incidence of colorectal cancer. N Engl J Med. 2000;343:1603 7. 23. UK Colorectal Cancer Screening Pilot Group. Results of the ¢rst round of a demonstration pilot of screening for colorectal cancer in the United Kingdom. Br Med J. 2004;329:133. 24. Salkeld G, Young G, Irwig L, Haas M, Glasziou P. Cost e¡ectiveness analysis of screening by faecal occult blood testing for colorectal cancer in Australia. Aust NZ J Public Health. 1996;20:138 43. 25. Cole SR, Young GP, Esterman A, Cadd B, Morcom J. A randomised trial of the impact of new faecal haemoglobin test technologies on population participation in screening for colorectal cancer. J Med Screen. 2003;10:117 22. 26. Winawer SJ, Flehinger BJ, Schottenfeld D, Miller DG. Screening for colorectal cancer with fecal occult blood testing and sigmoidoscopy. J Natl Cancer Inst. 1993;85:1311 18. 27. Burnand B, Bochud M, Froehlich F, Dubois RW, Vader JP, Gonvers JJ. Appropriateness of colonoscopy: screening for colorectal cancer in asymptomatic individuals. Endoscopy. 1999;31:673 83. 28. Barclay RL, Vicari JJ, Doughty AS, Johanson JF, Greenlaw RL. Colonoscopic withdrawal times and adenoma detection during screening colonoscopy. N Engl J Med. 2006;355:2533 41. 29. Gatto NM, Frucht H, Sundararajan V, Jacobson JS, Grann VR, Neugut AI. Risk of perforation after colonoscopy and sigmoidoscopy: a population based study. J Natl Cancer Inst. 2003;95:230 6. 30. Mandel JS, Church TR, Ederer F, Bond JH. Colorectal cancer mortality: e¡ectiveness of biennial screening for fecal occult blood. J Natl Cancer Inst. 1999;91:434 7. 31. Schoen RE. Surveillance after positive and negative colonoscopy examinations: issues, yields, and use. Am J Gastroenterol. 2003;98:1237 46. 32. Winawer SJ, Stewart ET, Zauber AG et al. A comparison of colonoscopy and double contrast barium enema for surveillance after polypectomy. National Polyp Study Work Group. N Engl J Med. 2000;342:1766 72. 33. Morrin MM, LaMont JT. Screening virtual colonoscopy ready for prime time? N Engl J Med. 2003;349:2261 4. 34. Van Gelder RE, Nio CY, Florie J et al. Computed tomographic colonography compared with colonoscopy in patients at increased risk for colorectal cancer. Gastroenterology. 2004;127:41 8. 35. Markovic S, Repse S, Hejinen S et al. Management of colorectal cancer; a national clinical guideline. Ljubljana: Ministry of Health of Republic of Slovenia, 2003.
117
12 Lymphomas of the gastrointestinal tract E. ZUCCA
The gastrointestinal tract is the most frequently involved extranodal localization, representing 30 40% of extranodal lymphomas and from 4% to 20% of all non-Hodgkin's lymphoma (NHL) cases1,2. In Western countries the most common location is the stomach (approximately 50 60%), followed by the small intestines (30%) and the large intestine (around 10%)3. Involvement of the oesophagus is very rare. These proportions can di¡er geographically, with small intestinal lymphomas being more common in the Middle East. The most common histological subtype in localized gastrointestinal tract presentations is di¡use large B-cell lymphoma which is present in approximately 60% of the gastric and 70% of the intestinal cases. Mucosaassociated lymphoid tissue (MALT) lymphoma represents about 35% of primary gastric lymphoma but less than 10% of the intestinal ones. Follicular lymphomas are very rare in the stomach but have been reported in up to 17% of intestinal cases. The other histological subtypes include T-cell lymphomas, Burkitt's lymphoma and mantle cell lymphomas (which in the gastrointestinal tract often present as a multiple lymphomatous polyposis) and are much less common and taken together comprise approximately 5% of the cases3,4. The incidence of gastrointestinal lymphoma may have increased over recent decades5,6. This is probably due to an actual increase, but more e¤cient case registration and improved diagnostic tools have also played a role. There are important geographical variations in the incidence rates of gastrointestinal tract lymphomas, perhaps correlated to the rate of Helicobacter pylori gastric infection in the examined regions 7 . Less consistent epidemiological information is available on intestinal lymphoma. Patients with coeliac disease have an increased risk of developing enteropathy-type T cell lymphoma (ETCL), although the risk in the most recent studies seems lower than previously thought8. In the past, surgery was considered an essential component of the diagnostic work-up in view of the fact that it provided adequate tumor specimens for diagnosis. However, su¤cient material for diagnosis is nowadays obtained in more than 90% of cases by endoscopic biopsy and diagnostic surgery, at least in gastric lymphomas, is no longer needed1,4,9. 118
LYMPHOMAS OF THE GASTROINTESTINAL TRACT
Imaging techniques have also signi¢cantly improved and, in the past decade, the introduction of endoscopic ultrasonography has been proven to be useful in assessing the depth of the stomach wall in¢ltration and the presence of perigastric lymph nodes and in identifying patients at high risk of bleeding and/or perforation10^12. In MALT lymphomas, deep in¢ltration of the gastric wall is associated with a greater risk of lymph-nodal positivity and a smaller chance of response to antibiotics only13,14. Until the late 1980s surgical resection with postoperative radiotherapy and/ or chemotherapy was standard. However, it has been later clearly shown by several studies that chemotherapy alone or chemotherapy followed by radiotherapy may produce similar results and that gastrectomy is thus redundant. In a recent large German study, 393 with localized primary gastric lymphoma were treated with radiotherapy and/or chemotherapy only or additional surgery15. The survival rate at 42 months for patients treated with surgery was 86% compared with 91% for patients without surgery. Hence the need for surgery for diagnostic purposes has disappeared, at least for gastric tumours, and the assumption of an increased risk of perforation and bleeding associated with front-line chemotherapy, for which `debulking surgery' was carried out preventively has not been con¢rmed in any modern series. On the contrary, several studies have reported a high degree of post-surgical complications that resulted in a delay in the start of chemotherapy, and surgical resection has been replaced by conservative therapeutic approaches for gastric lymphomas16^18. For primary intestinal lymphoma, however, there are no studies clearly demonstrating that surgery is unnecessary. Optimal treatment of gastrointestinal lymphomas depends mainly on the histological type, but also on the site and the stage of the disease.
DIFFUSE LARGE B-CELL LYMPHOMA OF THE STOMACH Di¡use large B-cell lymphoma (DLCL) represents the most common histological type among gastrointestinal lymphomas. Patients with locally advanced or disseminated aggressive gastrointestinal lymphomas appear to behave in the same manner as those with other advanced lymphomas, with comparable histology and prognostic factors. Therefore, treatment of disseminated DLCL is today based on chemotherapy combined with rituximab with or without radiotherapy. In general, the same guidelines followed for nodal DLCL can also be applied to gastrointestinal lymphomas with aggressive histologies.
MARGINAL ZONE B-CELL LYMPHOMAS OF THE GASTROINTESTINAL TRACT Gastric MALT lymphoma is often multimodal within the stomach but is characterized by an indolent natural history and prolonged con¢nement to the site of origin in most cases. Epidemiological evidence of a plausible aetiological correlation between gastric MALT lymphomas and chronic H. pylori infection 119
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
has been found19,20; clinical studies demonstrated histological regressions of gastric MALT lymphoma after eradication of H. pylori in the majority of patients who received antibiotic therapy. Nowadays, it is generally accepted that eradication of H. pylori with antibiotics should be the sole initial treatment of MALT lymphomas con¢ned to the gastric wall21. A strict follow-up after antibiotics is highly advisable, also because it is not possible to completely exclude the presence of a concomitant aggressive DLCL not demonstrated in the diagnostic gastric biopsies. Post-antibiotic molecular follow-up studies showed in about half of the cases a long-term persistence of monoclonal Bcell after histological regression of the lymphoma, but this event does not necessarily herald a relapse, and its clinical signi¢cance remains unclear. For the management of the subset of H. pylori-negative cases, and for the patients who fail antibiotic therapy, a choice can be made between conventional oncological modalities. Very good disease control using radiation therapy has been reported by several institutions. Surgery has been widely and successfully used in the past, but the precise role for surgical resection should be rede¢ned in view of the excellent results achieved with conservative approaches. Patients with systemic disease should be considered for systemic treatment but only a few anti-cancer compounds and chemotherapy regimens have been tested speci¢cally in MALT lymphomas22. The anti-CD20 monoclonal antibody rituximab is also very active and may represent an additional option for the treatment of systemic disease23. Immunoproliferative small intestinal disease (IPSID), alpha heavy chain disease, and Mediterranean lymphoma all refer to the same condition, which is presently considered a variant of low-grade MALT lymphoma, characterized by a di¡use lymphoplasmacytic/plasmacytic in¢ltrate in the small intestine. Most cases have been described in the Middle East, especially in the Mediterranean area where the disease is endemic, a¡ecting young adults. Patients usually present with poor performance status and severe malabsorption. Several authors have reported that treatment with antibiotics can produce clinical, histological and immunological remissions in early stages24.
MULTIPLE LYMPHOMATOUS POLYPOSIS This is a peculiar type of lymphoma presenting with multiple lymphomatous polyps of the gastrointestinal tract. In most cases it represents the intestinal form of mantle-cell lymphoma. The prognosis is quite poor in spite of aggressive chemotherapy, similar to its nodal counterpart. In rare instances multiple polyposis appears as a clinical syndrome produced by di¡erent histological subtypes other than mantle cell; thus the term multiple lymphomatous polyposis should not be used to de¢ne a histopathological entity1.
120
LYMPHOMAS OF THE GASTROINTESTINAL TRACT
Primary intestinal lymphomas The majority of primary intestinal lymphomas are large cell tumours of B cell lineage, but distinct histological presentations can include intestinal MALT lymphoma or IPSID, enteropathy-associated T-cell lymphoma, mantle cell lymphoma, or follicular lymphoma underscoring the importance of skilled histological diagnosis. The management of large B-cell lymphoma is based on surgery followed by chemotherapy (which is today usually combined with rituximab). In patients in whom complete tumour resection is not feasible, treatment is the same as described above for gastric localizations. The outcome reported in the literature varies depending on the extent of disease and histology. In large series of intestinal lymphomas a 60 75% 5-year survival for patients with B-cell lymphomas is reported, but only a 25% 5-year survival for those with T-cell tumours25^27.
Enteropathy-type T cell lymphoma (ETCL) According to the WHO classi¢cation, ETCL is a tumour of intraepithelial T lymphocytes; it usually occurs in the sixth or seventh decades but there have been sporadic reports of cases in young adults. Abdominal pain and/or exacerbation of enteropathy-associated symptoms (malabsorption, loss of responsiveness to a gluten-restricted diet) are the most common presentation features. Approximately 25% of cases present with an intestinal perforation. The clinical course is often very unfavourable1.
Gastrointestinal Burkitt's lymphoma A common childhood lymphoma but very rare in adults, sporadic Burkitt's lymphoma often presents with abdominal pain and intussusception. The ileocaecal region is the most common site of involvement and most cases are primarily intestinal. The histological and cytogenetic features are similar to those of the classical endemic African form, as is its association with the Epstein Barr virus28. The primary treatment modality is intensive combination chemotherapy. Local and locoregional therapy alone (i.e. surgery and radiotherapy) does not provide adequate treatment, even for patients with localized disease. Surgery remains important for establishing the diagnosis and should also be considered in the presence of a completely resectable mass1.
References 1. 2. 3.
Zucca E, Roggero E, Bertoni F, Cavalli F. Primary extranodal non-Hodgkin's lymphomas. Part 1: Gastrointestinal, cutaneous and genitourinary lymphomas. Ann Oncol. 1997;8:727 37. d'Amore F, Brincker M, Gronbaek K et al. Non-Hodgkin's lymphoma of the gastrointestinal tract: a population-based analysis of incidence, geographic distribution, clinicopathologic presentation features, and prognosis. J Clin Oncol. 1994;12:1673 84. Shenkier TN, Connors JM. Primary extranodal non-Hodgkin's lymphomas. In: Canellos GP, Lister TA, Young BD, editors. The Lymphomas, 2nd edn. Philadelphia: Saunders Elsevier; 2006:325 47.
121
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 4.
5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Koch P, del Valle F, Berdel WE et al. Primary gastrointestinal non-Hodgkin's lymphoma: I. Anatomic and histologic distribution, clinical features, and survival data of 371 patients registered in the german multicenter study GIT NHL 01/92. J Clin Oncol. 2001;19:3861 73. Gurney KA, Cartwright RA, Gilman EA. Descriptive epidemiology of gastrointestinal non-Hodgkin's lymphoma in a population-based registry. Br J Cancer. 1999;79:1929 34. Nakamura S, Matsumoto T, Iida M, Yao T, Tsuneyoshi M. Primary gastrointestinal lymphoma in Japan: a clinicopathologic analysis of 455 patients with special reference to its time trends. Cancer. 2003;97:2462 73. Doglioni C, Wotherspoon AC, Moschini A, De Boni M, Isaacson PG. High incidence of primary gastric lymphoma in northeastern Italy. Lancet. 1992;339:834 5. Catassi C, Bearzi I, Holmes GK. Association of celiac disease and intestinal lymphomas and other cancers. Gastroenterology. 2005;128(Suppl. 1):S79 86. Koch P, del Valle F, Berdel WE et al. Primary gastrointestinal non-Hodgkin's lymphoma: II. Combined surgical and conservative or conservative management only in localized gastric lymphoma results of the prospective German multicenter study GIT NHL 01/92. J Clin Oncol. 2001;19:3874 83. Nakamura S, Matsumoto T, Suekane H et al. Predictive value of endoscopic ultrasonography for regression of gastric low grade and high grade MALT lymphomas after eradication of Helicobacter pylori. Gut. 2001;48:454 60. Caletti G, Zinzani PL, Fusaroli P et al. The importance of endoscopic ultrasonography in the management of low-grade gastric mucosa-associated lymphoid tissue lymphoma. Aliment Pharmacol Ther. 2002;16:1715 22. Fischbach W, Goebeler-Kolve ME, Greiner A. Diagnostic accuracy of EUS in the local staging of primary gastric lymphoma: results of a prospective, multicenter study comparing EUS with histopathologic stage. Gastrointest Endosc. 2002;56:696 700. Steinbach G, Ford R, Glober G et al. Antibiotic treatment of gastric lymphoma of mucosaassociated lymphoid tissue. An uncontrolled trial. Ann Intern Med. 1999;131:88 95. Ruskone-Fourmestraux A, Lavergne A, Aegerter PH et al. Predictive factors for regression of gastric MALT lymphoma after anti-Helicobacter pylori treatment. Gut. 2001;48:297 303. Koch P, Probst A, Berdel WE et al. Treatment results in localized primary gastric lymphoma: data of patients registered within the German multicenter study (GIT NHL 02/96). J Clin Oncol. 2005;23:7050 9. Ferreri AJ, Cordio S, Ponzoni M, Villa E. Non-surgical treatment with primary chemotherapy, with or without radiation therapy, of stage I II high-grade gastric lymphoma. Leuk Lymphoma. 1999;33:531 41. Schmidt WP, Schmitz N, Sonnen R. Conservative management of gastric lymphoma: the treatment option of choice. Leuk Lymphoma. 2004;45:1847 52. Gobbi PG, Ghirardelli ML, Cavalli C et al. The role of surgery in the treatment of gastrointestinal lymphomas other than low-grade MALT lymphomas. Haematologica. 2000;85:372 80. Zucca E, Bertoni F, Roggero E, Cavalli F. The gastric marginal zone B-cell lymphoma of MALT type. Blood. 2000;96:410 19. Isaacson PG, Du MQ. MALT lymphoma: from morphology to molecules. Nat Rev Cancer. 2004;4:644 53. Zucca E, Cavalli F. Are antibiotics the treatment of choice for gastric lymphoma? Curr Hematol Rep. 2004;3:11 16. Bertoni F, Zucca E. State-of-the-art therapeutics: marginal-zone lymphoma. J Clin Oncol. 2005;23:6415 20. Martinelli G, Laszlo D, Ferreri AJ et al. Clinical activity of rituximab in gastric marginal zone non-Hodgkin's lymphoma resistant to or not eligible for anti-Helicobacter pylori therapy. J Clin Oncol. 2005;23:1979 83. Al-Saleem T, Al-Mondhiry H. Immunoproliferative small intestinal disease (IPSID): a model for mature B-cell neoplasms. Blood. 2005;105:2274 80. Ibrahim EM, Ezzat AA, El-Weshi AN et al. Primary intestinal di¡use large B-cell nonHodgkin's lymphoma: clinical features, management, and prognosis of 66 patients. Ann Oncol. 2001;12:53 8.
122
LYMPHOMAS OF THE GASTROINTESTINAL TRACT 26. Cortelazzo S, Rossi A, Oldani E et al. The modi¢ed International Prognostic Index can predict the outcome of localized primary intestinal lymphoma of both extranodal marginal zone B-cell and di¡use large B-cell histologies. Br J Haematol. 2002;118:218 28. 27. Domizio P, Owen RA, Shepherd NA, Talbot IC, Norton AJ. Primary lymphoma of the small intestine: a clinicopathological study of 119 cases. Am J Surg Pathol. 1993;17:429 42. 28. Ferry JA. Burkitt's lymphoma: clinicopathologic features and di¡erential diagnosis. Oncologist. 2006;11:375 83.
123
Section V Clinical challenges in liver diseases Chair: C TRAUTWEIN and G PAUMGARTNER
13 Do we still need liver biopsies? C. FLECHTENMACHER, T. LONGERICH and P. SCHIRMACHER
GENERAL CONSIDERATIONS Liver biopsies are obtained as a diagnostic measure to either evaluate di¡use liver diseases or focal liver processes. Since liver biopsy, despite technical improvements, is not without risk for the patient1, its indication has to be made carefully in a proper clinical context. The information obtained following liver biopsy has to have an impact on the therapeutic decision (therapy planning, prognosis assessment) 2 and cannot be achieved by an easier, cheaper, or less invasive procedure. Thus the application of liver biopsy is subjected to changes in diagnostic algorithms, therapeutic options, and disease management. Inevitable prerequisites for a successful diagnostic biopsy are a clear clinical indication, an optimal information exchange prior to biopsy evaluation, experienced processing and evaluation of the biopsy by the pathologist, as well as an established structure for post-biopsy work-up (recall procedures, clinico-pathological conferences). In contrast to other diagnostic procedures, the complete spectrum of morphological changes can and will be evaluated in a liver biopsy (thus giving the possibility to uncover changes not even suspected clinically) and speci¢c analyses can be repeated (on the para¤n-embedded tissue) or performed in reference centres even much later if novel information may require it. These facts are rarely considered when techniques/assays are discussed to replace liver biopsy. Optimal evaluation of a liver biopsy requires adequate starting material. Regarding focal lesions it is necessary to assess whether the lesion has been reached su¤ciently or not. In encapsulated lesions part of the ¢brous capsule may plug the needle and may result in negative tumour sampling, despite correct needle placement as demonstrated by radiological/ultrasound imaging. There are no general rules about how much of a lesion has to be biopsied. Principally diagnosis may be reached with only a few tumour cells present, but diagnostic e¤ciency is certainly a function of the amount of tumour tissue available for evaluation. If only small parts of the tumour are present, additional special analyses (immunohistology, molecular pathology) necessary to further subtype the lesion or to assess therapeutic target structures may be restricted or even impossible. In contrast, repetitive biopsies may increase the 127
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
frequency of complications (e.g. bleeding, metastatic spread). In di¡use liver diseases there are more de¢nitive experiences. There is a minimal requirement of 1.5 cm of biopsy length and a diameter of at least 1.2 mm (otherwise portal tracts tend to drop out). There is still a signi¢cant gain of information up to a biopsy length of 2.5 cm; when the needle diameter exceeds 1.8 mm the complication rate signi¢cantly increases3; thus the biopsy technique should be adjusted to these needs. Smaller biopsies (e.g. in children or by the transjugular route) will certainly be evaluated and may give important information, but assessment of grading and staging may be £awed. It is an absolute need that liver biopsy has to be accompanied by correct and su¤cient clinical information. This includes values and dynamics of the relevant serum parameter, patient history, imaging information (in focal lesions), as well as the relevant clinical question to be addressed. If this information is missing, diagnostic evaluation and di¡erential diagnostic considerations will be rudimentary or inappropriate. In order to support this information process, standard reporting sheets are helpful. Furthermore highlevel biopsy evaluation requires a regular recall structure considering unsolved cases or those cases with a discrepant clinical course, in order to further improve diagnosis and resultant decision-making.
CHRONIC HEPATITIS According to all current consensus statements liver biopsy is the gold standard in the assessment of disease activity (grading) and extent of ¢brosis and architectural distortion (staging) in chronic hepatitis, which makes it an important and highly recommended diagnostic procedure4. In recent years its application has started to shift more and more to meet the needs of individualized therapeutic approaches. Besides grading and staging5^8 (Table 1) histology contributes to the determination of the aetiology, especially addressing the question of comorbidity. Although hard to quantify, confounding morbidity (such as chronic alcohol abuse) is underestimated, especially in chronic hepatitis C, and may a¡ect as much as 20% of cases. Qualitative assessment of steatosis and ¢brosis pattern helps to uncover confounding disease, which is the major factor for rapid progression of chronic hepatitis C 9^11 . Grading and staging play decisive roles in the estimation of prognosis and the decision and urgency to treat. The theoretical basis for evaluation of the necroin£ammatory activity is the modi¢ed hepatic activity index (mHAI), but clinical practice is dominated by qualitative scores such as the Desmet/Scheuer score and the METAVIR score. Staging evaluates ¢bre deposition and also architectural distortion (see below). Since staging integrates disease development, it is the most important information with regard to course and prognosis of the hepatitis12. Liver biopsy evaluation plays an integral role in the evaluation of autoimmune hepatitis, being part of the diagnostic score13. Histology is an important factor in the di¡erential diagnosis of autoimmune hepatitis vs druginduced liver toxicity of hepatitic type. In addition the decision to terminate immunosuppressive therapy should include bioptic assessment of the residual 128
DO WE STILL NEED LIVER BIOPSIES?
in£ammatory activity. Persistence of interface hepatitis or portal in£ammation indicates a very high risk of disease recurrence after drug omission14.
STEATOSIS AND STEATOHEPATITIS In steatosis and steatohepatitis the role of liver biopsy is less well de¢ned and certainly the majority of patients will not be biopsied. There is consensus that biopsy diagnosis is the only means to truly assess the extent of in£ammation. Assessment of necroin£ammatory activity and ¢brosis allows one to determine the progressive nature of the disease and thus its prognosis. In the absence of signi¢cant treatment options it may help to motivate the patient to change behaviour and (in non-alcoholic steatohepatitis, NASH) to e¤ciently treat the metabolic causes. For NASH a qualitative sco re that descr ib es necroin£ammation (grading) and ¢brosis (staging) has been developed15. This provides some prognostic information but its true clinical value still has to be determined.
FIBROSIS Fibrosis evaluation is a hotly debated issue in hepatology. So far the gold standard in ¢brosis evaluation is analysis of the liver biopsy16. The main arguments against liver biopsy are the problems of sampling error and interindividual variability. While sampling is an issue which can be addressed only by biopsies of su¤cient size, the inter- and intra-observer reliability of ¢brosis evaluation is excellent, as for example shown in the large METAVIR trials8. In addition to other staging algorithms that are evaluated only for individual liver diseases the semiquantitative ¢brosis score (`Chevallier score')17 can be universally applied. This score is extremely helpful for the assessment of liver ¢brosis, especially in clinical trials, but its clinical application is limited. So far there have been numerous attempts to develop reliable serum assays/ surrogate markers for liver ¢brosis. Among other problems these assays measure either matrix turnover or degradation, but not the amount of ¢bre deposition; they integrate disease processes in other organs, and their discriminative power, especially in lower stages of ¢brosis, is negligible18^20. Measurement of liver sti¡ness (Fibroscan) in principle has proved to correlate with ¢bre content. Since its reproducibility is quite good it has been used to determine liver ¢brosis19,21. Nevertheless it is unable to replace liver biopsy, since it is signi¢cantly modulated by the extent of in£ammatory activity and steatosis, it is not applicable under various conditions (adipositas, ascites), and it is unable to evaluate architectural distortion. Thus, even in the evaluation of chronic ¢brotic liver disease, liver biopsy is still unsurpassed in its diagnostic value.
129
Table 1
Grading and staging of chronic viral hepatitis
Grading according to Des met and Scheuer lSI Verbal
His to/ogi£·ul features
mHAI
score Mild portal inOantmation. none or I
2
3
Minimal
1- 3
4-8
Mild
Moderate
9- 12
Ishak's modified histological activity index (mHAI) 171
minimal acinar inflammation/few sing le
cell necrosis, no interface hepatitis Mild to moderate portal inflammation. focal interface hCfl"titis. some single cell necrosis. no confluent nec-rosis Moderate to severe portal inOantmation, confluent interface hepatitis. multiple single-cell necrosis. some conOucnt
InterJace hepatitis
Severe
13- 18
130
interface hepatitis, severe acinar inflammation including confluent~
Focal. few portal tracts Focal, majoril.)'of]l
50% of;portal lracts None Focal Few zone 3-ncerosis
2 3
0 I
necrosis 4
None
I
4
necrosis. no bridging or panacinar Severe portal inllammation and
0
2 Connuenl necroses
3 4
s
tJrjdgiJlg_311() p(lnacinar_ncerosi~
6 0
META VIR-gr ading [81
I Interface hepatitis
Acinar inflammatory
Single-cell necroses
Score
foci 0
0
0 I
I 0,1
A=O
I
A= I
0,1
A=2
2
2 0,1,2
A=3
Interface hefl"titis: 0 = no. I = mild. 2 s moderate, 3 • severe 0 = no, I = mild. 2 = mode-rate. 3 = severe
2 4
2
2
Acinar necrosis:
Port-ttl innammation
3
0, 1
3
2 3 4 0
Multip_le zone 3·necrosis
Zone 3- and few portO>cemral brid\!ing necrosis Zone 3- and multiple portoccntral bridging
necrosis ranacinar/muhiacinar necrosis None I focus!MPF (objective IOx) 2 to 4 foci/MPF S to 10 foci/MI'F > I0 foci/M PF None
Mild. few or all portal tracts Moderate, few or all portal tracts Moderate, all portal tracts Severe. all ponal tracts
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Grade
Staging according to Desmet and Scheurer IS! Score
Verbal
IIistological features
No fi brosis
No portal fibrosis
I
Mild fibrosis
2
Moderate fibrosis
3
Severe fibrosis
Enlarged portal tracts without septa lncomplet.e portoportal septa. preserved archite<:turc Portal librosis with septa and architctural distonion (dinbrences in central vein portal tract distances, no evidence of complc.te cirrhotic rcmodellina) Probable or dclinite cirrhotic rcmodcll inl't
Criteriou
Score 0 I
131 4
Cirrhosis
Central vein
2
Perisinusoidal fibrosis
0 I 2
0 Portal tract
MET A VIR-staging[81 Sfll'e
FO Fl F2 F3 F4
Hi!ttolo,icul eature No portal fibrosis Portal fibrosis without septa Portal fibrosis with few septa Portal fibrosis with numerous Cirrhosis
Number of septa
septa~ no cirrhosis
I 2 3 0 I
2 3 0 I
Width of septa
2 3
Verbal Normal or absence of vein (cirrhosis) Moderately thickened (stellate aspect of vein wall) Markedly thickened wall (annular aspect of vein wall with numerous iibrous extensions between hcpai<Jcvtes) Normal Localized fibrosis Dillitse fi brosis Normal Enlarged without scota Enlarged with scota Cirrhosis None < 6 scpta/10 mm > 6 septa! I 0 mm Nodular org3niz.ation Thin and/or incomoletc Thick and loose connective matrix Very thick and dense connective matrix > 2/3 bioosv area
DO WE STILL NEED LIVER BIOPSIES?
0
Semiquantitative severity score according to Chevallier
1!11
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
BILIARY DISEASES Liver biopsy has various impacts in di¡erent chronic biliary diseases. While there is little impact in obstructive biliary disease it plays a signi¢cant role in idiopathic chronic biliary disease, especially chronic non-suppurative cholangitis such as primary biliary cirrhosis (PBC) and to a lesser extent primary sclerosing cholangitis (PSC). In PBC staging of the disease22^24 is helpful in establishing the diagnosis, evaluating the rare autoimmune overlap syndrome and determining prognosis and need for transplantation, although the last aspect has lost some impact due to the e¡ect of choleretic treatment. In PSC the limitation of bioptic evaluation is the focal nature of the disease. Thus staging of PSC is less accurate and the diagnostic lesions are found in only about 30 40% of cases. Nevertheless, biopsy is helpful in determining the need for transplantation and excluding confounding disease. In recent years we have more and more unravelled the uncharted territory of chronic idiopathic/ ductopenic biliary disease. Additionally, entities such as sclerosing pancreatitis/cholangitis, ischaemic cholangiopathy, drug-induced cholangitis, and many more have been described more accurately, and their distinction is of high clinical relevance, and impossible without biopsy.
HEREDITARY DISEASES The most important hereditary disease to be evaluated by biopsy is hereditary iron storage disease. Liver biopsy is important in grading the iron content (usually a ¢ve-grade scale from 0 to 4+ is used). Here direct morphological evaluation is superior to biochemical analysis on iron content per dry liver weight, since it is combined with comprehensive morphological analyses of all diagnostic questions. Furthermore it does not require a splitting of the biopsy material, and iron stains are largely quantitative. Only morphological evaluation is able to distinguish hepatocellular from non-hepatocellular iron storage, which is of decisive relevance for de¢ning the underlying aetiology and correct treatment. Iron content evaluation has gained more relevance, since only about 30% of individuals with proven homozygous C282Y mutation of the HFE gene will develop clinically signi¢cant disease, and an increasing number of biopsies occur in the setting of increased transferring saturation coupled with a positive mutation test. Fibrosis evaluation (in a three-grade scale) is also relevant. While lack of ¢brosis indicates restitution ad integrum after iron depletion, only the presence of cirrhosis justi¢es enrolment of patients into a hepatocellular carcinoma (HCC) surveillance programme. While lifetime HCC frequency is about 25% in cirrhotic haemochromatosis patients, it is limited to a few case reports in non-cirrhotic patients. Furthermore, without biopsy, iron storage may be uncovered, although representing a signi¢cant confounding morbidity in patients with other diseases, especially chronic alcohol abuse. In other more rare hereditary diseases biopsy is generally required to establish the diagnosis and extent of disease. 132
DO WE STILL NEED LIVER BIOPSIES?
OTHER NON-NEOPLASTIC LIVER DISEASES One of the frequent reasons for biopsy is hepatic toxicity of drugs and paramedications. In most cases it will emerge as liver enzyme elevation, hepatitis or cholestatic liver disease of unknown cause. Although drugs may give rise to almost all types of liver disease, there are several features that, in combination with the exclusion of other causes, allow one to suspect drug toxicity. Clinicopathological correlation (especially careful drug/toxin history) will allow the identi¢cation of the responsible agent in a signi¢cant number of cases. Omission or changing of the drug, combined with observation of the clinical course, may help to con¢rm the suspicion. Although protocol biopsies are rarely performed after liver transplantation, biopsy is essential in distinguishing the various potential causes for elevated serum liver enzyme concentrations in this setting. There is a multitude of reasons (rejection, ischaemic lesions, disease recurrence, post-transplant infection, drug toxicity, etc.) that in their complexity can be evaluated only by careful assessment of the biopsy in combination with the serological and clinical information. Additionally, grading of acute rejection contributes to the therapeutic decision regarding anti-rejection therapy.
FOCAL LIVER LESIONS There is certainly much to be said about the role of liver biopsy in focal liver lesions. In general the usefulness of biopsy tends to be underestimated while its complications are rather overemphasized. Certainly lesions that should not be biopsied (e.g. haemangioma, Echinococcus cysts) must be identi¢ed by careful imaging analysis. Focal nodular hyperplasia (FNH) is usually clearly de¢ned by imaging techniques and thus does not warrant liver biopsy, but the percentages of presumed FNH that do not show unambiguous features by imaging or demonstrate a suspicious clinical course vary between 10% and 30%. In these cases biopsy is helpful to ascertain the diagnosis and exclude other di¡erential diagnoses, such as adenoma or ¢brolamellar HCC. In hepatocellular adenoma besides establishing the diagnosis a new combined morphological/molecular classi¢cation has added new relevant information for decision making. It is now possible to identify those rare (bcatenin-positive) adenomas carrying an increased risk of malignant transformation25,26. Regarding hepatocellular carcinoma a consensus has been achieved that biopsy is not required beyond the size of 2 cm if two di¡erent imaging techniques (or alternatively one imaging analysis together with alpha-fetoprotein levels) are diagnostic. This may have resulted in some misinterpretation. The main argument against liver biopsy in this setting is the potential for needle-tract seeding. This risk is hard to estimate based on the numbers reported, since frequencies of up to 5% are likely to carry reporting bias and may vary dependent on the biopsy technique. There are no data demonstrating a decreased survival time following needle-tract metastasis; thus this complication should not signi¢cantly in£uence the decision to biopsy. Small (52 cm) highly di¡erentiated hepatocellular lesions (including highly 133
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Figure 1 Con¢rmation of minute HCC in hilar position by immunohistochemistry. A: Biopsy cylinder with minimal in¢ltrates of a moderately di¡erentiated microtrabecular HCC. B: Some tumour cells show strong GPC3 expression. C: HSP70 is expressed weakly by some HCC cells. D: Strong glutamine synthetase expression in nearly all tumour cells
di¡erentiated HCC) are among the most di¤cult to detect in liver pathology. In addition to meticulous morphological work-up, novel molecular algorithms have been added that improve diagnostic accuracy27. Immunohistological analysis for glypican 3 (GPC3), HSP70, and glutamine synthetase supports the diagnosis of early HCC in about 70% of cases (Figure 1). In other cases of more rare primary liver tumours biopsy is essential to establish the diagnosis, and as a rule of thumb biopsy should always be performed if other diagnostic tests are ambiguous, the clinical course di¡ers from the expectation and the result of the biopsy is likely to in£uence clinical decision-making. There are many reasons to biopsy potential metastatic lesions, the most frequent being the establishment of the diagnosis in cases of a so-far-undetected primary, and its impact for di¡erential therapy. Another steadily rising reason is the molecular analysis of therapeutic target structures or factors in£uencing chemotherapeutic regimen as well as (partial) therapeutic resistance, as exempli¢ed by de-novo c-kit mutations in liver metastases of gastrointestinal stromal tumours.
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DO WE STILL NEED LIVER BIOPSIES?
FINAL CONCLUSIONS Overall it is certainly true to state that liver biopsy has maintained its important role in the clinical diagnostic spectrum. Since biopsy as an invasive diagnostic technique has to be justi¢ed in every case, its application is subjected to changes in non-invasive diagnostic measures and therapeutic options. So far there is no non-invasive technique on the horizon to replace liver biopsy, although speci¢c indications to biopsy are constantly changing. One should always consider that morphological evaluation of a liver biopsy is the only technique that allows direct observation of the relevant changes; it can be used for speci¢c analyses including a rising number of molecular pathological evaluation tools, and analyses can be performed or repeated even years later. Finally, although not considered in detail in this brief review, liver biopsy has been a major promoter of progress in clinical research addressing chronic liver diseases, and should always be considered as a standard and major source of information in clinical trials.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Piccinino F, Sagnelli E, Pasquale G, Giusti G. Complications following percutaneous liver biopsy. A multicentre retrospective study on 68,276 biopsies. J Hepatol. 1986;2:165 73. Grant A, Neuberger J. Guidelines on the use of liver biopsy in clinical practice. British Society of Gastroenterology. Gut. 1999;45(Suppl. 4):IV1 11. Crawford AR, Lin XZ, Crawford JM. The normal adult human liver biopsy: a quantitative reference standard. Hepatology. 1998;28:323 31. Pradat P, Alberti A, Poynard T et al. Predictive value of ALT levels for histologic ¢ndings in chronic hepatitis C: a European collaborative study. Hepatology. 2002;36:973 7. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classi¢cation of chronic hepatitis: diagnosis, grading and staging. Hepatology. 1994;19:1513 20. Batts KP, Ludwig J. Chronic hepatitis. An update on terminology and reporting. Am J Surg Pathol. 1995;19:1409 17. Ishak K, Baptista A, Bianchi L et al. Histological grading and staging of chronic hepatitis. J Hepatol. 1995;22:696 9. Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996;24:289 93. Fernandez Rodriguez CM, Gutierrez ML, Serrano PL et al. Factors in£uencing the rate of ¢brosis progression in chronic hepatitis C. Dig Dis Sci. 2004;49:1971 6. Monto A, Patel K, Bostrom A et al. Risks of a range of alcohol intake on hepatitis C related ¢brosis. Hepatology. 2004;39:826 34. Walsh MJ, Vanags DM, Clouston AD et al. Steatosis and liver cell apoptosis in chronic hepatitis C: a mechanism for increased liver injury. Hepatology. 2004;39:1230 8. Marcellin P, Asselah T, Boyer N. Fibrosis and disease progression in hepatitis C. Hepatology. 2002;36(Suppl. 1):S47 56. Alvarez F, Berg PA, Bianchi FB et al. International Autoimmune Hepatitis Group Report: Review of criteria for diagnosis of autoimmune hepatitis. J Hepatol. 1999;31:929 38. Czaja AJ, Davis GL, Ludwig J, Taswell HF. Complete resolution of in£ammatory activity following corticosteroid treatment of HBsAg negative chronic active hepatitis. Hepatology. 1984;4:622 7. Kleiner DE, Brunt EM, Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313 21. Dienstag JL. The role of liver biopsy in chronic hepatitis C. Hepatology. 2002;36(Suppl. 1): S152 60. Chevallier M, Guerret S, Chossegros P, Gerard F, Grimaud JA. A histological semiquantitative scoring system for evaluation of hepatic ¢brosis in needle liver biopsy specimens: comparison with morphometric studies. Hepatology. 1994;20:349 55.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 18. Sandrin L, Fourquet B, Hasquenoph JM et al. Transient elastography: a new noninvasive method for assessment of hepatic ¢brosis. Ultrasound Med Biol. 2003;29:1705 13. 19. Castera L, Vergniol J, Foucher J et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of ¢brosis in chronic hepatitis C. Gastroenterology. 2005;128:343 50. 20. Miele L, Forgione A, Gasbarrini G, Grieco A. Noninvasive assessment of ¢brosis in non alcoholic fatty liver disease (NAFLD) and non alcoholic steatohepatitis (NASH). Transl Res. 2007;149:114 25. 21. Castera L, Forns X, Alberti A. Non invasive evaluation of liver ¢brosis using transient elastography. J Hepatol. 2008;48:835 47. 22. Scheuer P. Primary biliary cirrhosis. Proc R Soc Med. 1967;60:1257 60. 23. Ludwig J, Dickson ER, McDonald GS. Staging of chronic nonsuppurative destructive cholangitis (syndrome of primary biliary cirrhosis). Virchows Arch A Pathol Anat Histol. 1978;379:103 12. 24. Locke GR 3rd, Therneau TM, Ludwig J, Dickson ER, Lindor KD. Time course of histological progression in primary biliary cirrhosis. Hepatology. 1996;23:52 6. 25. Van der Borght S, Libbrecht L, Katoonizadeh A et al. Nuclear beta catenin staining and absence of steatosis are indicators of hepatocellular adenomas with an increased risk of malignancy. Histopathology. 2007;51:855 6. 26. Bioulac Sage P, Rebouissou S, Thomas C et al. Hepatocellular adenoma subtype classi¢cation using molecular markers and immunohistochemistry. Hepatology. 2007;46:740 8. 27. Di Tommaso L, Franchi G, Park YN et al. Diagnostic value of HSP70, glypican 3, and glutamine synthetase in hepatocellular nodules in cirrhosis. Hepatology. 2007;45:725 34.
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14 Inflammation and human cancer S. P. HUSSAIN, X. W. WANG and C. C. HARRIS
CHRONIC INFLAMMATION AND HEPATOCELLULAR CANCER (HCC) Free radicals Chronic in£ammation and infection is frequently associated with increased cancer risk, although exceptions can be cited including rheumatoid arthritis and human papillomavirus infection (Table 1)1. Infection with hepatitis B and C viruses (HBV and HCV) causes in£ammation with the release of free radicals, chemokines and cytokines resulting in DNA damage, cell proliferation, ¢brosis, and angiogenesis. The p53 pathway is a key responder to in£ammatory stress2. Free radicals, e.g. reactive nitrogen or oxygen species, can directly damage DNA and proteins and indirectly damage these macromolecules via lipid peroxidation (Figure 1). The p53 pathway responds to lower levels of DNA damage by cell cycle checkpoint arrest, facilitating DNA repair as an adapter in the formation of DNA repair protein complexes and transcriptional transactivation of DNA repair genes3. By mediating cell death due to extensive DNA damage, p53 also contributes to these processes by switching from increased expression of anti- to pro-oxidant genes. p53 can both transcriptionally transrepress pro-oxidant/nitrosative genes, e.g. NOS2, and transactivate anti-oxidant genes expressing glutathione peroxidase, aldehyde dehydrogenase, and Mn-superoxide dismutase, sestrins, and TIGAR (TP53induced glycolysis and apoptosis regulator) (Table 2). An animal model of the Li Fraumeni syndrome has provided new insights into the protective antioxidative and nitrosative function of p53. TP53 null mice are predisposed to lymphoma and leukaemia4. If these mice are fed an anti-oxidative and antinitrosative agent, i.e. N-acetylcysteine, the number of tumours was markedly 5 decreased in support . of the protective function of p53 in tumour suppression . Nitric oxide (NO ) an important bioregulatory and signalling molecule, may . play a signi¢cant role in carcinogenesis6^11. NO is catalysed by a family of enzymes known as nitric oxide synthases (NOS)12,13. The isoforms NOS1 and NOS3 are found to be constitutively expressed, e.g. in neurons (NOS1) or . endothelial cells (NOS3). They produce NO levels ranging from picomolar to nanomolar concentrations. In contrast, NOS2 (also. called iNOS) generally requires induction, but is able to produce NO concentrations in the 137
Liver Colon Colon
Haemochromatosis Crohn's disease Ulcerative colitis
219 3 6
Odds ratio
Caveats ^ Rheumatoid arthritis is an example of a chronic in£ammatory disease without an increased cancer risk, e.g. joint sarcoma ^ Oncogenic human papilloma viruses are examples of cancer-prone chronic infections without in£ammation
`18% of human cancers, i.e. 1.6 million per year, are related to infection'61
Tumour site
Urinary bladder Colon Liver Oesophagus Lung pleura Multiple sites
Parasitic Schistosoma hematobium Schistosoma japonicum Liver £uke Chemical/physical/metabolic Acid re£ux Asbestos Obesity
Gastric Ovary
Bacterial Helicobacter pylori Pelvic in£ammatory disease
Tumour site Liver Liver
Acquired disease Viral Hepatitis B Hepatitis C
Chronic in£ammation and infection can increase cancer risk61
Inherited disease
Table 1
138
50^100 410 1.3^6.5
2^14 2^6 14
11 3
88 30
Odds ratio
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
INFLAMMATION AND HUMAN CANCER
Figure 1 Overview of di¡erent reactive oxygen and nitrogen oxide species (ROS/RNOS) and their reaction products leading to DNA damage during in£ammation
Table 2
Examples of anti oxidant and anti nitrosative activities of the p53 tumour suppressor
Transcriptional transrepression of pro oxidant enzymes
Transcriptional transactivation of anti oxidant enzymes
NOS2 Human and mouse cells29 Mouse model31
GPx62 ALDH463 MnSOD64 SESN 1 and 25 TIGAR65
micromolar range (reviewed in ref. 6). NOS2 gene expression can be induced by bacterial endotoxins, proin£ammatory cytokines, or hypoxia14^16 in many cell types including macrophages17 and hepatocytes14,18,19, as well as in a variety of human tumours (reviewed in ref. 6). During chronic viral hepatitis the upregulation of certain proin£ammatory cytokines, e.g. tumour necrosis factor alpha (TNF-a) and interferon gamma (IFN-g), has been repeatedly demonstrated20,21. These proin£ammatory cytokines, TNF-a, IFN-g and interleukin 1 (IL-1), induce NOS2 gene expression, which leads to increased . NO concentrations in human hepatocytes22,23. In addition, NOS2 is also induced directly by the HBV and HCV. In vitro studies demonstrate that the HBx protein is capable of transcriptional transactivation of NOS224^26. An 139
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
induction of the NOS2 expression has also been seen after hepatocytes are exposed to woodchuck hepatitis virus surface antigen (WhsAg)27. In patients with HBV or HCV infection a consistent up-regulation of hepatic NOS2 has been shown19,28. . We and others were able to show that NO -related DNA damage leads to p53 accumulation and p53-mediated apoptosis29,30 (Figure 2). Moreover, we transrepressor of NOS2 presented results showing that p53 is a transcriptional . expression in vivo and attenuates an. excessive NO production in a regulatory negative feedback loop29,31,32. NO can also activate the arachidonic acid pathway by enhancing the enzymatic activity of cyclo-oxygenase 2 (COX2) (Figure 3). The mechanism of this activation is by NOS2 binding and nitrosylation of COX233. Increased COX2 activity results in the release of prostaglandin 2E (PGE2), which diminishes the apoptotic pathway and activates the WNT pathway34,35. The WNT signal transduction and COX2 pathways are key contributors to liver carcinogenesis36, and COX2 also predicts poor survival of patients with HCC37. The WNT pathway can also transcriptionally activate both NOS2 and COX2 by TCF4-b-catenin binding to positive response elements in their promoters38,39. Therefore, the NOS2 and COX2 are concomitantly activated by proin£ammatory cytokines, hypoxia and the WNT pathway. The WNT pathway . is frequently activated in HCC associated with high AFB1 exposure40. NO can both damage DNA to induce the anti-carcinogenic p53 stress response pathway and mutate cancer-related genes including TP53 (Figure 2). PGE2 attenuates apoptosis41, which may
Figure 2
. NO has both anti and pro carcinogenic e¡ects
140
INFLAMMATION AND HUMAN CANCER
Figure 3 Model of the interactive pathways in nitrosative stress induced TP53 mutation. NOS2 and COX2 are positively regulated by hypoxia, proin£ammatory cytokines, and the WNT signal transduction pathway. NOS2 produces a high concentration of NO that can both damage DNA to induce mutations in cancer related genes (for example, TP53), and induce p53 dependent cell cycle arrest and apoptosis, so that cells containing mutated TP53 attain selective clonal growth and survival advantages. NOS2 can physically bind COX2 and activate it by nitrosylation. COX2 can increase lipid peroxidation to produce unsaturated aldehydes that can induce TP53 mutations, and increase PGE2 concentrations. PGE2 can attenuate p53 mediated apoptosis and activate the WNT pathway, resulting in increased b catenin that, with TCF 4, transcriptionally activates NOS2 and COX2. High amounts of p53 can participate in negative feedback loops in the regulation of NOS2 and COX2. Lesser amounts of p53 can enhance COX2 accumulation
allow survival of hepatocytes with .DNA damage and increase the probability of somatic mutations. Because NO -induced apoptosis can be p53-dependent, cells with mutant TP53 have a clonal expansion advantage. Consistent with this model, we have found a signi¢cant association as well as a dose response relationship between TP53 mutations (G:C to A:T transition at CpG sites) and an increased NOS2 activity in patients with colon cancer42. Furthermore, we and others have demonstrated a positive association of NOS2 expression and a comparable TP53 mutational spectrum in lung cancer43,44. This TP53 mutation spectrum is consistent with increased rates of N2O3-induced deamination of 5methylation at CpG sites. In summary, oxidative/nitrosative stress, mediated by free radicals, which is also induced by the cytokine pro¢le of various in£ammatory hepatic disorders such as HBV and HCV infection, or haemochromatosis, can act as an endogenous carcinogen to increase TP53 mutations and provide a microenvironment allowing a clonal growth advantage for cells with TP53 mutations.
Inflammatory cytokines Analysis of HCV-associated cirrhosis revealed an up-regulation of proin£ammatory, pro-apoptotic, and pro-proliferative genes, which might re£ect groups of genes being involved in HCV-related cirrhosis progressing to 141
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
HCC45,46. Gene expression pro¢les of the HCV genotypes 1b, 2a, and 4d core proteins in HepG2 and Huh-7 cells indicated that each core protein has its own expression pro¢le and that each of them seems to be implicated not only in HCV replication, but also pathogenesis and oncogenesis47. In another study with transient HCV core gene transfected Huh-7 cells, by Fukutomi et al.48, most transcriptionally regulated genes were involved in cell growth or oncogenic signalling. Of particular interest were growth-related genes in the WNT-1 pathway. In addition, to further focus on the role of p53 in HCC, a number of TP53 mutant and TP53 wild-type HCC cases were analysed by microarrays identifying 83 p53-related genes in TP53 mutant HCC when compared with wild-type TP53 HCC49. The authors conclude that the most striking ¢nding of their study was that HCV-infected HCC with wild-type TP53 and those with mutant TP53 di¡er signi¢cantly in their gene expression patterns. Cell cycle-related genes (CCNG2, BZAP45) and cell proliferationrelated genes (SSR1, ANXA2, S100A10, and PTMA) were overexpressed in mutant TP53 tumours compared with wild-type TP53 tumours. Based on their results they assume that mutant TP53 tumours have higher malignant potentials than those with wild-type TP53. Studies such as those mentioned above may help to identify major pathways being involved in HCV-related HCC, and may help to select potential targets for new potential therapies.
Figure 4 Clonal and dual proclivity models of tumour evolution to metastasis. In the clonal evolution model a normal hepatocyte (light grey) progresses to a tumour through clonal expansion of an initiated cell with a mutation (medium grey), followed by the acquisition of sequential mutations (dark grey) that support metastasis. In the dual proclivity model multiple tumour initiating events are concurrent in premalignant lesions before tumour formation and metastasis. These events are inherent in the primary tumour and/or are modulated by the stromal environment66
142
INFLAMMATION AND HUMAN CANCER
SUMMARY Chronic viral hepatitis causes a myriad of cellular, tissue, and systemic e¡ects 50,51 . This brief review has focused on only two factors of the in£ammatory response, free radicals and cytokines, and their impact on the p53 tumour-suppressor biological pathway. The continuous release of proin£ammatory cytokines, e.g. TNF-a and IL-1b, during chronic viral hepatitis activates NOS2 that enzymatically generates a . high concentration of NO resulting in DNA damage and the activation of the p53 tumour-suppressor pathway. The transcriptionally activated p53 can transrepress NOS2 in a negative feedback loop and transactivates genes involved in cell cycle. checkpoint, apoptosis, autophagy, glycolysis and senescence52. The NO -induced direct DNA damage or indirect damage by increased lipid peroxidation and release of unsaturated aldehydes to form promutagenic DNA adducts can also cause mutations in cancer-related genes such as TP53. Etheno-DNA adducts are increased in liver diseased by chronic viral hepatitis, haemochromatosis and Wilson disease (reviewed in ref. 53). TP53 mutant hepatocytes have also been quantitatively detected in these same diseases (ref 54; and Hussain et al., unpublished results). The p53 mutation sp e c tr u m o c cu r r i ng i n HC C a s s o c i ate d w ith v i ral h e p atiti s o r haemochromatosis is consistent with oxidative and nitrosative DNA damage. Although we have shown that 4-hydroxynonenal formed by lipid peroxidation can induce p53 mutations in vitro including p53 codon 249ser mutations54, the p53 codon 249ser mutations are not detected in HCC associated with chronic viral hepatitis alone or haemochromatosis55, but are common in HCC when highly carcinogenic a£atoxin B1 exposure also occurs in the diet of people with chronic viral hepatitis56. We have recently discussed the possible mechanisms leading to p53 mutations in HCC57. The contributions of in£ammatory cytokines to carcinogenesis have been recently reviewed58,59. Wang and co-workers60 have also presented evidence suggesting that the humoral cytokine pro¢le shifts towards anti-in£ammatory responses to promote HCC metastases. They also have suggested clonal and dual proclivity models of tumour evolution to metastases (Figure 4).
Acknowledgements We thank Dorothea Dudek-Creaven for editorial and graphic assistance, Karen MacPherson for bibliographical assistance and manuscript preparation, and Mohammed Khan for technical data.
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22. 23. 24. 25.
Hussain SP, Hofseth LJ, Harris CC. Radical causes of cancer. Nat Rev Cancer. 2003;3:276 85. Staib F, Robles AI, Varticovski L et al. The p53 tumor suppressor network is a key responder to microenvironmental components of chronic in£ammatory stress. Cancer Res. 2005;65:10255 64. Sengupta S, Harris CC. p53: tra¤c cop at the crossroads of DNA repair and recombination. Nat Rev Cell Mol Biol. 2005;6:44 55. Donehower LA, Harvey M, Slagle BL et al. Mice de¢cient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992;356:215 21. Sablina AA, Budanov AV, Ilyinskaya GV, Agapova LS, Kravchenko JE, Chumakov PM. The antioxidant function of the p53 tumor suppressor. Nat Med. 2005;11:1306 13. Ambs S, Hussain SP, Harris CC. Interactive e¡ects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. FASEB J. 1997;11:443 8. Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. 1994;63:175 95. Hentze MW, Kuhn LC. Molecular control of vertebrate iron metabolism: mRNA based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci USA. 1996;93:8175 82. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109 42. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994;78:915 18. Tamir S, Tannenbaum SR. The role of nitric oxide (NO) in the carcinogenic process. Biochim Biophys Acta. 1996;1288:F31 6. Forstermann U, Kleinert H. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn Schmiedebergs Arch Pharmacol. 1995;352:351 64. Marletta MA. Nitric oxide synthase structure and mechanism. J Biol Chem. 1993;268:12231 4. Lombard DB, Guarente L. Nijmegen breakage syndrome disease protein and MRE11 at PML nuclear bodies and meiotic telomeres. Cancer Res. 2000;60:2331 4. Nussler AK, Di Silvio M, Billiar TR et al. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J Exp Med. 1992;176:261 4. Wild CP, Umbenhauer D, Chapot B, Montesano R. Monitoring of individual human exposure to a£atoxins (AF) and N nitrosamines (NNO) by immunoassays. J Cell Biochem. 1986;30:171 9. Xie QW, Cho HJ, Calaycay J et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992;256:225 8. Mowat M, Sauder M, Pereira D. The activated form of p53 is not a transactivator of the intracisternal A particle long terminal repeat promoter. Oncogene. 1990;5:241 4. Vodovotz Y, Kim PK, Bagci EZ et al. In£ammatory modulation of hepatocyte apoptosis by nitric oxide: in vivo, in vitro, and in silico studies. Curr Mol Med. 2004;4:753 62. Gonzalez Amaro R, Garcia Monzon C, Garcia Buey L et al. Induction of tumor necrosis factor alpha production by human hepatocytes in chronic viral hepatitis. J Exp Med. 1994;179:841 8. Mihm S, Hutschenreiter A, Fayyazi A, Pingel S, Ramadori G. High in£ammatory activity is associated with an increased amount of IFN gamma transcripts in peripheral blood cells of patients with chronic hepatitis C virus infection. Med Microbiol Immunol (Berl). 1996;185:95 102. de Vera ME, Shapiro RA, Nussler AK et al. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc Natl Acad Sci USA. 1996;93:1054 9. Laskin DL, Heck DE, Laskin JD. Role of in£ammatory cytokines and nitric oxide in hepatic and pulmonary toxicity. Toxicol Lett. 1998;102 3:289 93. Elmore LW, Hancock AR, Chang SF et al. Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis. Proc Natl Acad Sci USA. 1997;94:14707 12. Amaro MJ, Bartolome J, Carreno V. Hepatitis B virus X protein transactivates the inducible nitric oxide synthase promoter. Hepatology. 1999;29:915 23.
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INFLAMMATION AND HUMAN CANCER 26. Majano PL, Garcia Monzon C, Lopez Cabrera M et al. Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus induced gene upregulation. J Clin Invest. 1998;101:1343 52. 27. Liu RH, Jacob JR, Hotchkiss JH, Cote PJ, Gerin JL, Tennant BC. Woodchuck hepatitis virus surface antigen induces nitric oxide synthesis in hepatocytes: possible role in hepatocarcinogenesis. Carcinogenesis. 1994;15:2875 7. 28. Kane JM, III, Shears LL, Hierholzer C, Ambs S, Billiar TR, Posner MC. Chronic hepatitis C virus infection in humans: induction of hepatic nitric oxide synthase and proposed mechanisms for carcinogenesis. J Surg Res. 1997;69:321 4. 29. Forrester K, Ambs S, Lupold SE et al. Nitric oxide induced p53 accumulation and regulation of inducible nitric oxide synthase (NOS2) expression by wild type p53. Proc Natl Acad Sci USA. 1996;93:2442 7. 30. Messmer UK, Brune B. Nitric oxide induced apoptosis: p53 dependent and p53 independent signalling pathways. Biochem J. 1996;319:299 305. 31. Ambs S, Ogunfusika MO, Merriam WG, Bennett WP, Billiar TR, Harris CC. Upregulation of NOS2 expression in cancer prone p53 knockout mice. Proc Natl Acad Sci USA. 1998;95:8823 8. 32. Hussain SP, Hofseth LJ, Harris CC. Tumor suppressor genes: at the crossroads of molecular carcinogenesis, molecular epidemiology and human risk assessment. Lung Cancer. 2001;34(Suppl. 2):S7 15. 33. Kim SF, Huri DA, Snyder SH. Inducible nitric oxide synthase binds, S nitrosylates, and activates cyclooxygenase 2. Science. 2005;310:1966 70. 34. Castellone MD, Teramoto H, Williams BO, Druey KM, Gutkind JS. Prostaglandin E2 promotes colon cancer cell growth through a Gs axin beta catenin signaling axis. Science. 2005;310:1504 10. 35. Buchanan FG, DuBois RN. Connecting COX 2 and Wnt in cancer. Cancer Cell. 2006;9:6 8. 36. Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002;31:339 46. 37. Kondo M, Yamamoto H, Nagano H et al. Increased expression of COX 2 in nontumor liver tissue is associated with shorter disease free survival in patients with hepatocellular carcinoma. Clin Cancer Res. 1999;5:4005 12. 38. Du Q, Park KS, Guo Z et al. Regulation of human nitric oxide synthase 2 expression by Wnt beta catenin signaling. Cancer Res. 2006;66:7024 31. 39. Araki Y, Okamura S, Hussain SP et al. Regulation of cyclooxygenase 2 expression by the wnt and ras pathways. Cancer Res. 2003;63:728 34. 40. Devereux TR, Stern MC, Flake GP et al. CTNNB1 mutations and beta catenin protein accumulation in human hepatocellular carcinomas associated with high exposure to a£atoxin B1. Mol Carcinogen. 2001;31:68 73. 41. Gupta RA, DuBois RN. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase 2. Nat Rev Cancer. 2001;1:11 21. 42. Ambs S, Bennett WP, Merriam WG et al. Relationship between p53 mutations and inducible nitric oxide synthase expression in human colorectal cancer. J Natl Cancer Inst. 1999;91:86 8. 43. Ambs S, Bennett WP, Merriam WG et al. Vascular endothelial growth factor and nitric oxide synthase expression in human lung cancer and the relation to p53. Br J Cancer. 1998;78:233 9. 44. Fujimoto H, Sasaki J, Matsumoto M et al. Signi¢cant correlation of nitric oxide synthase activity and p53 gene mutation in stage I lung adenocarcinoma. Jpn J Cancer Res. 1998;89:696 702. 45. Honda M, Kaneko S, Kawai H, Shirota Y, Kobayashi K. Di¡erential gene expression between chronic hepatitis b and c hepatic lesion. Gastroenterology. 2001;120:955 66. 46. Shackel NA, McGuinness PH, Abbott CA, Gorrell MD, McCaughan GW. Insights into the pathobiology of hepatitis C virus associated cirrhosis: analysis of intrahepatic di¡erential gene expression. Am J Pathol. 2002;160:641 54. 47. Dou J, Liu P, Zhang X. Cellular response to gene expression pro¢les of di¡erent hepatitis C virus core proteins in the Huh 7 cell line with microarray analysis. J Nanosci Nanotechnol. 2005;5:1230 5.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 48. Fukutomi T, Zhou Y, Kawai S, Eguchi H, Wands JR, Li J. Hepatitis C virus core protein stimulates hepatocyte growth: correlation with upregulation of wnt 1 expression. Hepatology. 2005;41:1096 105. 49. Okada T, Iizuka N, Yamada Okabe H et al. Gene expression pro¢le linked to p53 status in hepatitis C virus related hepatocellular carcinoma. FEBS Lett. 2003;555:583 90. 50. Levrero M. Viral hepatitis and liver cancer: the case of hepatitis C. Oncogene. 2006;25:3834 47. 51. Kremsdorf D, Soussan P, Paterlini Brechot P, Brechot C. Hepatitis B virus related hepatocellular carcinoma: paradigms for viral related human carcinogenesis. Oncogene. 2006;25:3823 33. 52. Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8:275 83. 53. Bartsch H, Nair J. Chronic in£ammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair. Langenbecks Arch Surg. 2006;391:499 510. 54. Hussain SP, Raja K, Amstad PA et al. Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: oxyradical overload diseases. Proc Natl Acad Sci USA. 2000;97:12770 5. 55. Marrogi AJ, Khan MA, van Gijssel HE et al. Oxidative stress and p53 mutations in the carcinogenesis of iron overload associated hepatocellular carcinoma. J Natl Cancer Inst. 2001;93:1652 5. 56. Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 1994;54:4855 78. 57. Hussain SP, Schwank J, Staib F, Wang XW, Harris CC. TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene. 2007;26:2166 76. 58. Budhu A, Wang XW. The role of cytokines in hepatocellular carcinoma. J Leukocyte Biol. 2006;80:1197 213. 59. Lin WW, Karin M. A cytokine mediated link between innate immunity, in£ammation, and cancer. J Clin Invest. 2007;117:1175 83. 60. Budhu A, Forgues M, Ye QH et al. Prediction of venous metastases, recurrence and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell. 2006;10:99 111. 61. Stewart B, Kleihues P. World Cancer Report. IARC Press. 2003:57. 62. Tan M, Li S, Swaroop M, Guan K, Oberley LW, Sun Y. Transcriptional activation of the human glutathione peroxidase promoter by p53. J Biol Chem. 1999;274:12061 6. 63. Yoon KA, Nakamura Y, Arakawa H. Identi¢cation of ALDH4 as a p53 inducible gene and its protective role in cellular stresses. J Hum Genet. 2004;49:134 40. 64. Hussain SP, Amstad P, He P et al. p53 induced up regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis. Cancer Res. 2004;64:2350 6. 65. Bensaad K, Tsuruta A, Selak MA et al. TIGAR, a p53 inducible regulator of glycolysis and apoptosis. Cell. 2006;126:107 20. 66. Budhu AS, Zipser B, Forgues M, Ye QH, Sun Z, Wang XW. The molecular signature of metastases of human hepatocellular carcinoma. Oncology. 2005;69(Suppl. 1):23 7.
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Section VI Viral hepatitis Chair: F NEGRO and S ZEUZEM
15 New insights in the immunology of viral hepatitis B and C A. BERTOLETTI
INTRODUCTION Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the two major causes of chronic liver in£ammation worldwide 1,2 . Despite distinct virological features, both viruses are preferentially hepatotropic, not directly cytopathic and elicit liver diseases that share several aspects of their natural history. HBV and HCV infections also share several features of the adaptive immune response. Multispeci¢c antiviral CD4 and CD8 responses with a T helper (Th) type 1 pro¢le of cytokine production are detectable in the blood of subjects with a self-limited infection3^16. These responses are stronger than those found in patients with chronic infection17^21. Based on these observations it has been proposed that the ability to mount an e¤cient cellular immune response is the main mechanism responsible for HBV and HCV control, whilst a defect in this response leads to chronicity (reviewed in refs 2 and 22). Since the viral and immunological events which occur in the ¢rst weeks after HBV and HCV infections are likely to in£uence substantially the ¢nal outcome of infection this chapter will discuss the mechanisms that regulate the induction of a fully integrated innate and adaptive immune response from the early stages of HBV and HCV infection.
DELAYED INDUCTION OF ADAPTIVE IMMUNITY DURING HBV AND HCV INFECTION Available studies in humans and in animal models of HBV and HCV infection appear to be consistent with the interpretation that HBV and HCV are ignored by the adaptive immune response for about 2 months after primary infection. After needlestick exposure to HCV-positive blood, patients who subsequently controlled the infection showed antiviral speci¢c CD8+ and CD4+ T cell responses only after 7 10 weeks from exposure10 (Figure 1). The appearance of HBV-speci¢c CD8+ and CD4+ T cells during the incubation time of acute hepatitis B follows the same pattern23. Activation of the virus-speci¢c cellular 149
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Figure 1 Kinetic model of serum levels of HCV RNA, HBV DNA and alanine aminotransferase (ALT) during self limited HBV and HCV infection. HCV: virus replication starts early (detectable within 1 week) and increases slightly for an additional 4 6 weeks. Signs of liver damage (ALT elevation) and of adaptive immunity are detectable 6 8 weeks after infection. Viraemia is substantially lower in HCV than in HBV infection. HBV: HBV DNA is not detectable for about 4 7 weeks after infection. After this period the HBV DNA increases vigorously and it is then rapidly controlled in self limited infection. Circulating HBV speci¢c CD4+ and CD8+ T cells can be detected during the initial rise of virus replication. Reduction of HBV replication is observed in the absence of a proportional increase of ALT
150
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immunity is followed by the humoral response which appears at least 10 12 weeks after HBV24 and HCV25 infections. These data are in line with results obtained in animal models. During primary HCV infection of chimpanzees, virus-speci¢c antibody responses are detectable 8 12 weeks after infection and HCV-speci¢c T cell responses appear with identical delays26^30. This delayed induction of the adaptive immunity to HBV and HCV contrasts with the reported kinetics of virus-speci¢c T cell responses to other virus infections. Although comparison of di¡erent studies must be interpreted with caution, because of possible sensitivity variations in the assays, it is intriguing that while T cell responses to simian immunode¢ciency virus (SIV), human immunode¢ciency virus and cytomegalovirus can be detectable at only 2 weeks after infection31,32, this ¢nding is only occasional in HCV-infected chimpanzees, since only 10 of the 12 animals reported in literature26^30,33 show a clear HCV-speci¢c T cell response by 4 weeks from infection. The pathogenetic implications of the apparent delay of the virus-speci¢c adaptive immune response are likely to be di¡erent in the two infections. Despite the methods used for viral quanti¢cation often being di¡erent, the available data show a di¡erent kinetic pattern of HBV and HCV replication (see Figure 1). While HCV-RNA levels rise rapidly within the ¢rst few days of HCV infection10,26,29,30,34 HBV-DNA and HBV antigens are not detectable in serum and liver for about 4 7 weeks after HBV infection24,35,36, even using methods with a level of sensitivity comparable to those used for HCV detection37. Thus, while during HCV infection the adaptive response ignores for several weeks a substantial quantity of virus (at least 106 copies/ml), the limited quantity of HBV antigens present in the early phases of infection is the possible cause of the delayed induction of the HBV-speci¢c adaptive immunity. In addition to the late detection, quantitative and functional defects of the adaptive immune responses are present during primary HBV and HCV infections. The peak frequency of HBV-speci¢c CD8+ T cells during the acute phase of disease in patients with self-limited infections generally does not exceed 1% of circulating CD8+ T cells38. Higher frequencies (up to 7% of CD8+ T cells) have been observed in acute HCV-infected patients, but most of these cells show impaired production of interferon gamma (IFN-g), g low perforin content and defective capacity for expansion and lysis of target cells 16,39 . These functional defects are present in the acutely infected population, irrespective of the subsequent outcome towards recovery or chronic viral persistence, and are transient only in patients who succeed in clearing the virus spontaneously39^41. Several factors are probably responsible for these defects of the adaptive immunity: intrahepatic recruitment and subsequent deletion of activated virusspeci¢c CD8+ T cells42, antigen-driven T or B cell exhaustion43, the ability of liver sinusoidal endothelial cells to suppress the expansion of Th1-type cells44 and the production of viral proteins with immunomodulatory e¡ects45^47. In addition, late appearance, reduced size, and functional T cell defects detected in both primary HBV and HCV infections might also be a consequence of the inability of the innate immune response to provide an e¤cient containment of early virus replication and to promote timely and appropriate T cell priming. 151
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THE INNATE IMMUNE RESPONSE IN HCV AND HBV INFECTIONS One of the ¢rst defence mechanisms that an infected host is able to mount against viral infections is the production of type I interferons. In addition to antiviral functions these cytokines possess important immunomodulatory e¡ects48. Changes in the transcription of IFN-a response genes have been demonstrated to occur as early as 2 days after HCV infection in the liver of a chimpanzee acutely infected with HCV, and these changes parallel the viral replication kinetics49,50. HCV starts to replicate immediately after infection and viraemia is generally detectable 1 2 weeks after inoculation in humans10 and within a few days in chimps26,29,34 (Figure 1). It is likely that HCV replication is the direct cause of type I IFN production, since double-stranded RNA is a strong IFN-a inducer51. Although HCV is sensitive in vitro to IFN52,53, recent data raise the hypothesis that the virus may have developed strategies to interfere with the IFN type I antiviral activity (reviewed in ref. 54). For example, the HCV E2 protein can inhibit in vitro the kinase activity of PKR (double-stranded RNA-activated protein kinase), one of the antiviral proteins induced by IFN55. Because of this interference the initial production of type I IFN may only be able to slow, but not block, virus replication. HCV viral kinetics is in line with this possibility. The initial rapid and early peak of viral replication can be followed by a period of about 4 6 weeks when HCV-RNA can slowly increase or remain at stable levels10,26,29,34,49,50 in the absence of virus-speci¢c T and B cell induction and liver in£ammation (see Figure 1). The coexistence of HCV replication with the absence of signs of liver in£ammation is not only indicative of the non-cytopathic nature of HCV, but also calls into question the pathogenic contribution of natural killer (NK) and NK-T cells in HCV infection. NK and NK-T cells are components of the innate immune system that respond within minutes or hours after infection by producing IFN-g and by killing infected cells 56 . Direct evidence of the activation state of NK and NK-T cells during the initial phase of HCV infection is lacking. Nevertheless, the absence of signs of liver in£ammation in the ¢rst 4 6 weeks after HCV infection 10,26,29,34,49 suggests that the contribution of NK and/or NK-T cells in this early phase may be minimal. This possibility is also supported by recent data showing that the HCV envelope protein E2 can directly inhibit NK cell functions57,58 by interacting with CD81, a tetraspannin molecule expressed on the surface of di¡erent cell lineages, that is a putative HCV receptor59. The direct inhibitory e¡ect of the HCV envelope protein on global NK cell function may contribute to the absence of liver in£ammation, despite the early signs of HCV replication and the early production of IFN-a, which has been shown to promote NK cell recruitment into the liver60. Is the pattern of innate immune response activation similar in HBV infection? As discussed earlier, available data show a di¡erent replicative pattern after HBV and HCV infections (see Figure 1). After an apparent initial quiescent phase of about 4 7 weeks24,35^37, HBV starts to replicate vigorously, reaching levels of around 109 1010 copies/ml in 1 2 weeks23,61, and infecting most hepatocytes37,62^64. In animal models of HBV infection this rapid peak of viral replication is accompanied in self-limited infection by a typical acute 152
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phase of disease with IFN-g and tumour necrosis factor alpha (TNF-a) a production, followed by the triggering of the adaptive immunity37,64. Animals that develop chronicity lack the acute-phase response and fail to prime an adequate adaptive response65^67. Thus, activation of innate components of the immune system seems to represent a key element in the control of the initial HBV burst. Available data do not allow the contribution of the individual components of the innate immunity to be dissected out. However, their overall e¤ciency is remarkable since the HBV-DNA quantity falls by more than 80% within 2 3 weeks after the peak of viral replication62,63, and before the peak of the antigen-speci¢c CD8 response and liver damage23,37,64. Is this initial IFN-g production primarily sustained by NK and NK-T cells? Although we lack direct evidence of the role of NK and NK-T cells during natural HBV infection, the experimental data are consistent with the possibility that the initial burst of IFN-g and the subsequent rapid inhibition of HBV are mainly mediated by these components of innate immunity. Activation of NK-T cells in the transgenic mouse model of HBV infection has been demonstrated and shown to inhibit virus replication through the production of IFN-g68^70. The real importance of NK-T cells during natural HBV pathogenesis is, however, questionable, since while NK-T cells are highly enriched in mouse liver (*20 30%)71, in humans classical NK-T cells only occasionally reach frequencies of higher than 1%72. It is instead possible that classical NK cells are the principal source of this IFN-g production 64 . A rapid drop in viral replication occurs in the presence of intrahepatic IFN-g production, before the massive intrahepatic recruitment of T cells. In this setting the NK cells can be the principal source of IFN-g production, causing rapid inhibition of HBV replication and recruitment of virus-speci¢c and non-speci¢c cells 69,73. A similar sequence of events has been reported in patients studied during the incubation phase of hepatitis B. An increased number of circulating NK cells precedes the peak of HBV replication, and is followed, 2 4 weeks later, by the appearance of HBV-speci¢c CD8+ T cells , when viral replication has already dropped23.
ROLE OF THE DENDRITIC CELLS IN VIRUS CONTROL Dendritic cells act as a link between innate and adaptive immunity. Signals delivered by the innate immune system (type I IFN production, interactions with NK cells) lead to the proper maturation of dendritic cells (DC), which are critical for triggering the antigen-speci¢c immune response74. The ability of DC to induce T cell responses is also in£uenced by their tissue localization, by the antigen dose and by the interaction with helper T cells74. Activation of cytotoxic T cells in lymph nodes draining the site of infection occurs hours after the initial challenge75 and is mediated by DC. CD8+ T cells (but also CD4+ T cells) stimulated for only 24 h by mature DC proliferate extensively76. Given the crucial role played by DC in T cell priming, it is obvious to raise the hypothesis that an alteration of DC function and/or maturation during primary HCV infection can contribute to the delayed appearance of HCVspeci¢c CD8+ and CD4+ T cells after infection. Although no conclusive data 153
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are available regarding the function of DC during acute or chronic HCV infection, it has been shown in vitro that the E2 protein of HCV can directly inhibit NK cell function57,58 . Furthermore, experiments performed with monocyte-derived DC indicate that these cells show an altered maturation process in chronic HCV infection77^79 which may be related to the presence of HCV79 or to the e¡ect of HCV proteins80. However, more recent data have not con¢rmed the altered functional state of DC during HCV infection81. Infection of DC by HBV is even more controversial than in HCV, and the described functional defects are minimal82^84. The delayed appearance of HBV-speci¢c immune responses after primary infection seems to depend more on the kinetics of HBV replication (i.e. HBV persist for at least 5 8 weeks in the newly infected host without detectable signs of e¤cient replication), than on immune dysfunctions. The evidence that HBV-speci¢c CD4+ and CD8+ T cells become detectable a short time after the exponential increase in HBV replication23,37 suggests that DC function is not a¡ected by the virus in the early phases of self-limited HBV infection.
THE COLLAPSE OF THE ADAPTIVE IMMUNITY The inability to control the infection and the establishment of chronicity lead to a progressive decline of the adaptive immunity with a lower number of circulating and intrahepatic virus-speci¢c CD8+ and CD4+ T cells85^88 as well as low and restricted production of virus-speci¢c antibodies25. Patients who develop chronic HCV infection show persistently defective CD8 + T cell function39^41,89. In chronic HBV patients a persistently high production of viral antigens can delete or tolerize antigen-speci¢c T90 and B43 cells. HBV is typically able to produce large quantities of non-infectious subviral particles containing only envelope antigens and a secretory form of the nucleocapsid protein, called HBeAg, that in transgenic mice induces the preferential production of Th2 cytokines by deletion of Th1 cells91,92. Chronic exposure to HBV antigens has been shown to delete HBV-speci¢c B cells43 and to cause an alteration of the hierarchy and function of HBV-speci¢c CD8+ T cells. Recent data have demonstrated that these HBV- and HCV-speci¢c exhausted T cells are characterized by the up-regulation of a programmed death 1 (PD-1) molecule, and that blocking of the PD-1 engagement with its ligand ( PD-L1) leads to an enhancement of their antiviral and proliferation functions89,93. The progressive qualitative and quantitative collapse of the adaptive immunity in both chronically HBV- and HCV-infected patients has a profound e¡ect on the composition of the virus populations infecting individual patients. Since chronic patients are unable to rapidly control the infection, progressive selection of T and B cell escape variant viruses can occur. HBV and HCV variants carrying mutations in helper94,95, cytotoxic96^98 and B cell99^102 epitopes have been detected in chronic hepatitis B and C, and this selection is postulated to take place principally during the initial phases of infections98,101,103,104. In HCV-infected chimps, viral mutants that are able to escape CD8+ T cell responses emerge early after infection105^107. Furthermore, a longitudinal study of patients with chronic hepatitis C revealed that the 154
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sequences of CD8+ T cell epitopes of the infecting viral populations did not diversify during several years of follow-up, despite the relatively high frequency of escape mutations initially present103. The selective pressure exerted by the humoral response also acts early, since mutations in B cell epitope regions found in chronic HCV patients have been shown to develop in the initial phases of infection101. At later stages T and B cell epitopes appear to be ¢xed within the viral populations. This is a further indication of the progressive collapse of the immune response in persistent HCV and HBV infections resulting in the inability of the residual T and B cell activity to exert selective pressure on the virus.
CONCLUDING REMARKS Clari¢cation of the virological and immunological events occurring during HBV and HCV infections allows us to better de¢ne the di¡erent mechanisms involved in viral persistence. In both infections, once chronicity develops, the common outcome is a progressive collapse of the immune system which makes it unlikely that the host can spontaneously reacquire the capacity to control virus replication. At present the treatment of chronic HBV and HCV infections relies on the use of drugs with direct antiviral e¡ects. However, the di¡erent vigour and quality of the virus-speci¢c immunity in self-limited versus chronic hepatitis suggests that therapeutic restoration of the antiviral immunity could lead to the control of virus replication and disease in chronically infected subjects. The real problem with the therapeutic vaccine approach is that the immune system of chronically HBV- and HCV-infected patients does not have the same functional e¤ciency and cell repertoire of subjects who have been able to control the infection. Therefore, strategies to optimize the e¤cacy of therapeutic vaccination are needed. In this perspective, e¡orts should be directed to develop vaccines designed not only to prime e¤ciently naive T and B cells76, but also to boost the antiviral e¤ciency of T cells coexisting with high antigen concentrations, or restoring the T cell functional pro¢le interfering with the PD-1/PD-L1 interaction. In addition, inhibition of viral replication by antiviral drugs may be an alternative strategy to restore the antiviral e¤ciency of the immune system. This is suggested by the observation that lamivudine treatment of chronically infected HBV patients can increase the frequency and improve the function of circulating HBV-speci¢c helper and cytotoxic T cells108,109. Data supporting the rationale of this approach were reported in animal models of HBV infection, in which the e¤cacy of therapeutic vaccination is enhanced by previous antiviral treatment110.
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NEW INSIGHTS IN THE IMMUNOLOGY OF HVB AND HVC 27. Major ME, Mihalik K, Puig M et al. Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge. J Virol. 2002;76:6586 95. 28. Bassett SE, Brasky KM, Lanford RE. Analysis of hepatitis C virus inoculated chimpanzees reveals unexpected clinical pro¢les. J Virol. 1998;72:2589 99. 29. Bassett SE, Guerra B, Brasky K et al. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology. 2001;33:1479 87. 30. Thimme R, Bukh J, Spangenberg HC et al. Viral and immunological determinants of hepatitis C virus clearance, persistence and disease. Proc Natl Acad Sci USA. 2002;99:15661 8. 31. Koup RA, Safrit JT, Cao Y et al. Temporal association of cellular immune responses with the initial control of viremia in primary human immunode¢ciency virus type 1 syndrome. J Virol. 1994;68:4650 5. 32. Rentenaar RJ, Gamadia LE, van DerHoek N et al. Development of virus speci¢c CD4(+) T cells during primary cytomegalovirus infection. J Clin Invest. 2000;105:541 8. 33. Cooper S, Erickson A, Adams E et al. Analysis of a succesful immune response against hepatitis C virus. Immunity. 1999;10:439 49. 34. Shimizu YK, Weiner AJ, Rosenblatt J et al. Early events in hepatitis C virus infection of chimpanzees. Proc Natl Acad Sci USA. 1990;87:6441 4. 35. Korba BE, Cote PJ, Wells FV et al. Natural history of woodchuck hepatitis virus infections during the course of experimental viral infection: molecular virologic features of the liver and lymphoid tissues. J Virol. 1989;63:1360 70. 36. Berquist KR, Peterson JM, Murphy BL, Ebert JW, Maynard JE, Purcell RH. Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect Immun. 1975;12:602 5. 37. Thimme R, Wieland S, Steiger C et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol. 2003;77:68 76. 38. Maini MK, Boni C, Ogg GS et al. Direct ex vivo analysis of hepatitis B virus speci¢c CD8+ T cells associated with the control of infection. Gastroenterology. 1999;117:1386 96. 39. Urbani S, Boni C, Missale G et al. Virus speci¢c CD8+ lymphocytes share the same e¡ector memory phenotype but exhibit functional di¡erences in acute hepatitis B and C. J Virol. 2002;76:12423 34. 40. Gruener NH, Lechner F, Jung MC et al. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J Virol. 2001;75:5550 8. 41. Wedemeyer H, He XS, Nascimbeni M et al. Impaired e¡ector function of hepatitis C virus speci¢c CD8+ T cells in chronic hepatitis C virus infection. J Immunol. 2002;169:3447 58. 42. Crispe IN, Dao T, Klugewitz K, Mehal WZ, Metz DP. The liver as a site of T cell apoptosis: graveyard, or killing ¢eld? Immunol Rev. 2000;174:47 62. 43. Barnaba V, Franco A, Alberti A, Benvenuto R, Balsano F. Selective killing of hepatitis B envelope antigen speci¢c B cells by class I restricted, exogenous antigen speci¢c T lymphocytes. Nature. 1990;345:258 60. 44. Klugewitz K, Blumenthal Barby F, Schrage A, Knolle PA, Hamann A, Crispe IN. Immunomodulatory e¡ects of the liver: deletion of activated CD4+ e¡ector cells and suppression of IFN gamma producing cells after intravenous protein immunization. J Immunol. 2002;169:2407 13. 45. Large M, Kittlesen D, Hahn Y. Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. J Immunol. 1999;162:931 8. 46. Kittlesen DJ, Chianese Bullock KA, Yao ZQ, Braciale TJ, Hahn YS. Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T lymphocyte proliferation. J Clin Invest. 2000;106:1239 49. 47. Vanlandschoot P, Van Houtte F, Roobrouck A, Farhoudi A, Leroux Roels G. Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte speci¢c receptor. J Gen Virol. 2002;83:1281 9. 48. Biron CA. Interferons alpha and beta as immune regulators a new look. Immunity. 2001;14:661 4. 49. Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol. 2001;75:7059 66.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 50. Su AI, Pezacki JP, Wodicka L et al. Genomic analysis of the host response to hepatitis C virus infection. Proc Natl Acad Sci USA. 2002;99:15669 74. 51. Cella M, Salio M, Sakakibara Y, Langen H, Julkunen I, Lanzavecchia A. Maturation, activation, and protection of dendritic cells induced by double stranded RNA. J Exp Med. 1999;189:821 9. 52. Blight KJ, Kolykhalov AA, Rice CM. E¤cient initiation of HCV RNA replication in cell culture. Science. 2000;290:1972 4. 53. Castet V, Fournier C, Soulier A et al. Alpha interferon inhibits hepatitis C virus replication in primary human hepatocytes infected in vitro. J Virol. 2002;76:8189 99. 54. Tan SL, Katze MG. How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. Virology. 2001;284:1 12. 55. Taylor D, Shi S, Romano P, Barber G, Lai M. Inhibition of interferon inducible protein kinase PKR by HCV E2 protein. Science. 1999;285:107 10. 56. Biron CA, Brossay L. NK cells and NKT cells in innate defense against viral infections. Curr Opin Immunol. 2001;13:458 64. 57. Tseng CT, Klimpel GR. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med. 2002;195:43 9. 58. Crotta S, Stilla A, Wack A et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med. 2002;195:35 41. 59. Pileri P, Uematsu Y, Campagnoli S et al. Binding of hepatitis C virus to CD81. Science. 1998;282:938 41. 60. Salazar Mather TP, Lewis CA, Biron CA. Type I interferons regulate in£ammatory cell tra¤cking and macrophage in£ammatory protein 1alpha delivery to the liver. J Clin Invest. 2002;110:321 30. 61. Whalley SA, Murray JM, Brown D et al. Kinetics of acute hepatitis B virus infection in humans. J Exp Med. 2001;193:847 54. 62. Jilbert A, Wu T, England J et al. Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvment. J Virol. 1992;66:1377 88. 63. Kajino K, Jilbert AR, Saputelli J, Aldrich CE, Cullen J, Mason WS. Woodchuck hepatitis virus infections: very rapid recovery after prolonged viremia and infection of virtually every hepatocyte. J Virol. 1994;68:5792 803. 64. Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV. Viral clearance without destruction of infected cells during acute HBV infection. Science. 1999;284:825 9. 65. Cote PJ, Toshkov I, Bellezza C et al. Temporal pathogenesis of experimental neonatal woodchuck hepatitis virus infection: increased initial viral load and decreased severity of acute hepatitis during the development of chronic viral infection. Hepatology. 2000;32:807 17. 66. Nakamura I, Nupp JT, Cowlen M et al. Pathogenesis of experimental neonatal woodchuck hepatitis virus infection: chronicity as an outcome of infection is associated with a diminished acute hepatitis that is temporally de¢cient for the expression of interferon gamma and tumor necrosis factor alpha messenger RNAs. Hepatology. 2001;33:439 47. 67. Menne S, Roneker CA, Roggendorf M, Gerin JL, Cote PJ, Tennant BC. De¢ciencies in the acute phase cell mediated immune response to viral antigens are associated with development of chronic woodchuck hepatitis virus infection following neonatal inoculation. J Virol. 2002;76:1769 80. 68. Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med. 2000;192:921 30. 69. Kakimi K, Lane TE, Chisari FV, Guidotti LG. Cutting edge: Inhibition of hepatitis B virus replication by activated NK T cells does not require in£ammatory cell recruitment to the liver. J Immunol. 2001;167:6701 5. 70. Vilarinho S, Ogasawara K, Nishimura S, Lanier LL, Baron JL. Blockade of NKG2D on NKT cells prevents hepatitis and the acute immune response to hepatitis B virus. Proc Natl Acad Sci USA. 2007;104:18187 92. 71. Bendelac A, Rivera MN, Park SH, Roark JH. Mouse CD1 speci¢c NK1 T cells: development, speci¢city, and function. Annu Rev Immunol. 1997;15:535 62. 72. de Lalla C, Galli G, Aldrighetti L et al. Production of pro¢brotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis. J Immunol. 2004;173:1417 25.
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NEW INSIGHTS IN THE IMMUNOLOGY OF HVB AND HVC 73. Kakimi K, Lane TE, Wieland S et al. Blocking chemokine responsive to gamma 2/ interferon (IFN) gamma inducible protein and monokine induced by IFN gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus speci¢c cytotoxic T lymphocytes. J Exp Med. 2001;194:1755 66. 74. Banchereau J, Briere F, Caux C et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767 811. 75. Mueller SN, Jones CM, Smith CM, Heath WR, Carbone FR. Rapid cytotoxic T lymphocyte activation occurs in the draining lymph nodes after cutaneous herpes simplex virus infection as a result of early antigen presentation and not the presence of virus. J Exp Med. 2002;195:651 6. 76. Kaech SM, Wherry EJ, Ahmed R. E¡ector and memory T cell di¡erentiation: implications for vaccine development. Nat Rev Immunol. 2002;2:251 62. 77. Kanto T, Hayashi N, Takehara T et al. Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus infected individuals. J Immunol. 1999;162:5584 91. 78. Bain C, Fatmi A, Zoulim F, Zarski JP, Trepo C, Inchauspe G. Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gastroenterology. 2001;120:512 24. 79. Au¡ermann Gretzinger S, Kee¡e EB, Levy S. Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection. Blood. 2001;97:3171 6. 80. Sarobe P, Lasarte JJ, Casares N et al. Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins. J Virol. 2002;76:5062 70. 81. Decalf J, Fernandes S, Longman R et al. Plasmacytoid dendritic cells initiate a complex chemokine and cytokine network and are a viable drug target in chronic HCV patients. J Exp Med. 2007;204:2423 37. 82. Wang FS, Xing LH, Liu MX et al. Dysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection. World J Gastroenterol. 2001;7:537 41. 83. Beckebaum S, Cicinnati VR, Dworacki G et al. Reduction in the circulating pDC1/pDC2 ratio and impaired function of ex vivo generated DC1 in chronic hepatitis B infection. Clin Immunol. 2002;104:138 50. 84. Lohr HF, Pingel S, Bocher WO, Bernhard H, Herzog Hau¡ S, Rose John S. Reduced virus speci¢c T helper cell induction by autologous dendritic cells in patients with chronic hepatitis B restoration by exogenous interleukin 12. Clin Exp Immunol. 2002;130:107 14. 85. Maini MK, Boni C, Lee CK et al. The role of virus speci¢c CD8+ cells in viral control and liver damage during persistent hepatitis B virus (HBV) infection. J Exp Med. 2000;191:1269 80. 86. He X S, Rehermann B, Lopez Labrador FX et al. Quantitative analysis of hepatitis C virus speci¢c CD8+ T cells in peripheral blood and liver using peptide MHC tetramers. Proc Natl Acad Sci USA. 1999;96:5692 7. 87. Rehermann B, Chang KM, McHutchison JG, Kokka R, Houghton M, Chisari FV. Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection. J Clin Invest. 1996;98:1432 40. 88. Rosen HR, Miner C, Sasaki AW et al. Frequencies of HCV speci¢c e¡ector CD4+ T cells by £ow cytometry: correlation with clinical disease stages. Hepatology. 2002;35:190 8. 89. Radziewicz H, Ibegbu CC, Fernandez ML et al. Liver in¢ltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD 1 and low levels of CD127 expression. J Virol. 2007;81:2545 53. 90. Reignat S, Webster GJ, Brown D et al. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J Exp Med. 2002;195:1089 101. 91. Milich D, Chen M, Hughes J, Jones J. The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence. J Immunol. 1998;160:2013 21. 92. Milich DR, Schodel F, Hughes JL, Jones JE, Peterson DL. The hepatitis B virus core and e antigens elicit di¡erent Th cell subsets: antigen structure can a¡ect Th cell phenotype. J Virol. 1997;71:2192 201. 93. Boni C, Fisicaro P, Valdatta C et al. Characterization of hepatitis B virus (HBV) speci¢c T cell dysfunction in chronic HBV infection. J Virol. 2007;81:4215 25.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 94. Wang H, Eckels DD. Mutations in immunodominant T cell epitopes derived from the nonstructural 3 protein of hepatitis C virus have the potential for generating escape variants that may have important consequences for T cell recognition. J Immunol. 1999;162:4177 83. 95. Frasca L, Del Porto P, Tuosto L et al. Hypervariable region 1 variants act as TCR antagonists for hepatitis C virus speci¢c CD4+ T cells. J Immunol. 1999;163:650 8. 96. Rehermann B, Pasquinelli C, Mosier SM, Chisari FV. Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection. J Clin Invest. 1995;96:1527 34. 97. Bertoletti A, Costanzo A, Chisari FV et al. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med. 1994;180:933 43. 98. Tsai S L, Chen Y M, Chen M H et al. Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity. Gastroenterology. 1998;115:954 66. 99. Weiner AJ, Geysen HM, Christopherson C et al. Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections. Proc Natl Acad Sci USA. 1992;89:3468 72. 100. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol. 1994;68:1494 500. 101. Farci P, Shimoda A, Coiana A et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science. 2000;288:339 44. 102. Booth JC, Kumar U, Webster D, Monjardino J, Thomas HC. Comparison of the rate of sequence variation in the hypervariable region of E2/NS1 region of hepatitis C virus in normal and hypogammaglobulinemic patients. Hepatology. 1998;27:223 7. 103. Chang KM, Rehermann B, McHutchison JG et al. Immunological signi¢cance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest. 1997;100:2376 85. 104. Urbani S, Amadei B, Cariani E et al. The impairment of CD8 responses limits the selection of escape mutations in acute hepatitis C virus infection. J Immunol. 2005;175:7519 29. 105. Weiner A, Erickson AL, Kansopon J et al. Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant. Proc Natl Acad Sci USA. 1995;92:2755 9. 106. Erickson AL, Kimura Y, Igarashi S et al. The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes. Immunity. 2001;15:883 95. 107. Thomson M, Nascimbeni M, Gonzales S, Murthy KK, Rehermann B, Liang TJ. Emergence of a distinct pattern of viral mutations in chimpanzees infected with a homogeneous inoculum of hepatitis C virus. Gastroenterology. 2001;121:1226 33. 108. Boni C, Bertoletti A, Penna A et al. Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B. J Clin Invest. 1998;102:968 75. 109. Boni C, Penna A, Ogg G et al. Lamivudine treatment can overcome cytotoxic T cell hyporesponsiveness in chronic hepatitis B: new perspective for immune therapy. Hepatology. 2001;33:963 71. 110. Menne S, Roneker CA, Korba BE, Gerin JL, Tennant BC, Cote PJ. Immunization with surface antigen vaccine alone and after treatment with 1 (2 £uoro 5 methyl beta l arabinofuranosyl) uracil (L FMAU) breaks humoral and cell mediated immune tolerance in chronic woodchuck hepatitis virus infection. J Virol. 2002;76:5305 14.
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16 New aspects of treatment of chronic hepatitis C B. KRONENBERGER and S. ZEUZEM
INTRODUCTION Antiviral therapy for patients with chronic hepatitis C has greatly improved during the recent years; however, only half of the patients with chronic hepatitis C can be cured, with the current standard of care of chronic hepatitis C consisting of pegylated interferon alpha (peginterferon alpha) and ribavirin. It can be anticipated that many of the patients who do not respond to antiviral therapy will develop liver cirrhosis and potentially hepatocellular carcinoma. The improvement of interferon-based antiviral therapy is therefore highly necessary, especially for `di¤cult-to-cure' patients with HCV genotype 1.
EXTENDED TREATMENT DURATION IN PATIENTS INFECTED WITH HCV GENOTYPE 1 The aim of antiviral therapy is a sustained virological response (SVR) de¢ned as undetectable HCV-RNA 24 weeks after the end of therapy. A pivotal trial investigated the association between treatment duration and SVR in patients infected with HCV genotype 1 after treatment with peginterferon alpha-2a and ribavirin1. In this trial the SVR rate was signi¢cantly higher in patients treated for 48 weeks than in patients treated for 24 weeks. Thus the treatment duration of 48 weeks is currently the standard treatment duration for patients with chronic HCV genotype 1 infection. Extended treatment duration may further improve SVR rates in patients with chronic HCV genotype 1 infection. Whether an extended treatment duration of 72 weeks improves SVR was investigated in a study by Berg et al.2. In this study treatment-naive patients with chronic HCV genotype 1 infection were randomized for treatment with peginterferon alpha-2a plus ribavirin (800 mg) for 72 weeks or 48 weeks. In this study the overall SVR rates were similar in both treatment arms (53% vs 54% in the 48 weeks vs 72 weeks treatment arm, respectively). The trial showed that an extended treatment duration cannot be generally recommended in patients infected with HCV genotype 1. 161
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In the same study a subgroup analysis of the SVR rate in patients with slow virological response (undetectable HCV-RNA between weeks 12 and 24) was performed. Patients with a slow virological response showed a signi¢cantly higher SVR rate when treated for 72 instead of 48 weeks (29% vs 17%). Thus a longer treatment duration may improve the SVR rate of patients infected with HCV genotype 1 and a slow virological response. Similar results were obtained in a trial by Sanchez-Tapias et al.3. In this trial treatment-naive patients with chronic HCV (any genotype) were treated with peginterferon alpha-2a plus ribavirin (800 mg/day). Patients with detectable HCV-RNA at week 4 were randomized for a total of 48 weeks or 72 weeks of treatment, respectively. The SVR rate in the subgroup of patients with HCV genotype 1 and slow virological response was signi¢cantly higher in patients treated for 72 weeks than in those treated for 48 weeks (44% vs 28%). Overall, the studies by Berg et al. and Sanchez-Tapias et al. indicate that extended treatment duration may improve the SVR rate in patients infected with HCV genotype 1 and slow virological response. However, both studies used a low dose of ribavirin (800 mg) instead of a weight-based standard dosing of ribavirin. An important question is whether an extended treatment duration remains superior to a standard treatment duration when patients receive weight-based ribavirin. Two studies investigated SVR after treatment with peginterferon alpha-2b plus weight-based ribavirin (800 1400 mg and 1000 1200 mg, respectively) for 48 weeks or 72 weeks in slow virological responders (all treatment-naive) (summarized in ref. 4). A slow virological response was de¢ned by at least a 2 log10 decline in baseline serum HCV-RNA but detectable viraemia at 12 weeks of therapy and with undetectable HCV-RNA at 24 weeks. In both studies SVR after treatment for 72 weeks was higher than after treatment for 48 weeks (38% vs 18% and 69% vs 52%, respectively). Both studies indicate that extended treatment duration may also improve SVR in slow virological responders treated with weight-based ribavirin4. A longer treatment duration may also be a treatment option for previous non-responders to interferon alpha-based therapy. Whether retreatment with peginterferon alpha-2a plus weight-based ribavirin (1000 1200 mg) for 72 weeks increases SVR rates in previous non-responders to peginterferon alpha2b plus ribavirin was investigated in the REPEAT trial; in this trial patients were randomized for 48 or 72 weeks of treatment with peginterferon alpha-2a induction (360 mg/week peginterferon alpha-2a for 12 weeks followed by peginterferon alpha-2a standard dose of 180 mg/week) or peginterferon alpha2a standard treatment. All patients received weight-based ribavirin (1000 1200 mg). Patients in both arms, the peginterferon alpha-2a induction and the peginterferon alpha-2a standard arm, showed higher SVR rates after 72 weeks of treatment than after 48 weeks of treatment, respectively (16% and 14% vs 7% and 9%). This trial shows that retreatment for 72 weeks is a reasonable treatment option for previous non-responders5.
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SHORTER TREATMENT DURATION Interferon alpha-based treatment is associated with a large number of adverse events and reduction in quality of life. In order to reduce the burden for patients with chronic hepatitis C, a shorter treatment duration without compromising SVR is appreciated. The pivotal trial by Hadziyannis et al.1 compared 24 weeks vs 48 weeks of treatment in HCV genotype 1-infected patients. The trial showed that a treatment duration of 24 weeks is associated with a lower SVR rate compared with a treatment duration of 48 weeks. In the trial by Zeuzem et al. treatment-naive patients with HCV genotype 1 infection and a low baseline viral load of 4600 000 IU/ml were treated with peginterferon alpha-2b and ribavirin for 24 weeks6. In this trial the end-oftreatment and sustained virological response rates were 80% and 50%, respectively. The 48-week historical control group had similar end-oftreatment (74%) but higher sustained virological response rates (71%)7. This di¡erence was due to a high virological relapse rate after 24 weeks of therapy (37%) compared with the historical control (4%). However, a subset of patients with an initial virological response at treatment week 4 achieved a similar sustained virological response rate (89%) as in the control group (85%). Jensen et al.8 reanalysed data from the trial by Hadziyannis et al.1 and noted that about a quarter of patients infected with genotype 1 had an initial virological response after 4 weeks of treatment and achieved a SVR in 89% of those randomized to 24 weeks of treatment. A low baseline viral load was independently associated with an initial virological response. Overall, these studies indicate that a shorter course of treatment may be su¤cient in patients with HCV genotype 1 infection, low baseline viral load and virological response at week 4. In patients with chronic HCV genotype 2 or 3 infection, who have a better response to interferon alpha than patients chronically infected with HCV genotype 1, the standard treatment duration is 24 weeks1. Several trials investigated whether even shorter treatment durations are possible in patients with genotype 2 or 3 infection without compromising SVR rates. The ¢rst trials showed that a shorter treatment duration of 12 16 weeks is equally e¡ective as the standard treatment duration in patients infected with HCV genotype 2 or 3 who achieve a rapid virological response after 4 weeks of therapy9,10. However, the large ACCELERATE trial, comparing 16 vs 24 weeks of treatment in patients with HCV genotype 2 or 3 infection (n ( = 1469), showed that a shorter treatment duration of 16 weeks is associated with a reduced SVR rate compared with the standard treatment duration11. In the ACCELERATE trial a shorter course of therapy over 16 weeks has been shown to be as e¡ective as a 24-week course only in those patients with genotype 2 or 3 infection who have a baseline viral load 4400 000 IU/ml and achieve an early virological response at week 411.
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NEW INTERFERONS Albinterferon is a novel long-acting form of interferon alpha which results from the genetic fusion of interferon alpha with human albumin. Albinterferon has a longer half-life than peginterferon alpha. A recent phase 2b clinical trial investigated the antiviral e¤cacy and tolerability of albinterferon in combination with ribavirin in patients with chronic hepatitis C genotype 1 infection who were naive to interferon alpha based treatment12. In this trial the sustained virological response rate was 58% and 55% in patients treated with albinterferon 900 mg and 1200 mg every 2 weeks plus ribavirin, respectively, versus 58% in patients treated with peginterferon alpha-2a 180 mg once weekly plus ribavirin. The health-related quality-of-life scores were more favourable in the albinterferon group than in the peginterferon alpha-2a group. Overall, these results indicate that albinterferon plus ribavirin may o¡er e¤cacy comparable to peginterferon alpha-2a plus ribavirin with half of the injections and the potential for less impairment of quality of life. Albinterferon is currently being investigated in two randomized open-label, active controlled, multi-centre, non-inferiority phase 3 trials to evaluate the e¤cacy, safety and impact on health-related quality of life of albinterferon in combination with ribavirin versus peginterferon alpha-2a in combination with ribavirin (ACHIEVE 1 and 2,3). Despite great success in optimization and individualization of interferon alpha-based treatment, it appears unlikely that further individualization of standard therapy will markedly improve the convenience and outcome of antiviral therapy. For those patients with chronic hepatitis C who did not respond to interferon alpha-based antiviral therapy, there is currently no approved treatment option available. `Di¤cult-to-cure' patient populations include patients infected with HCV genotype 1, HIV/HCV co-infected patients, patients with advanced liver cirrhosis and patients with recurrent hepatitis C after liver transplantation13.
SPECIFICALLY TARGETED ANTIVIRAL THERAPY FOR HEPATITIS C Recent progress in the structure determination of HCV proteins, development of a subgenomic replicon system14 and more recently of a cell culture infectious HCV clone15,16 have enabled the development of speci¢cally targeted antiviral therapies for hepatitis C (STAT-C). Many HCV-speci¢c compounds are under investigation in preclinical and clinical trials. The NS3/4A protease cleaves at four downstream sites in the polyprotein to generate the N-termini of the NS4A, NS4B, NS5A, and NS5B proteins. Due to the pivotal role of NS3/4A in the HCV replication cycle, NS3/4A appears as a promising target for a speci¢cally targeted therapy. The structure of the NS3/ 4A protease has been determined and several inhibitors have been developed. The protease inhibitors ciluprevir, boceprevir, and telaprevir were investigated in patients with chronic hepatitis C and showed a decline of HCV-RNA of 2 3 log10 copies/ml after 2 days17, 1.08 1.61 log10 IU/ml after 7 days18, and 2.0 4.4 log10 IU/ml after 14 days of treatment, respectively19. 164
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The RNA-dependent RNA polymerase is located in the NS5B protein and has a central role in the HCV replication cycle. Inhibition of NS5B appears as another promising target for STAT-C. Nucleoside type and non-nucleoside type NS5B inhibitors have been developed. The nucleoside type NS5B inhibitor valopicitabine and the non-nucleoside type NS5B inhibitor HCV-796 showed a decline of HCV-RNA of 0.15 1.21 log10 IU/ml after 14 days20 and 1.4 log10 IU/ml after 14 days21, respectively, in previous non-responders to interferon alpha-based therapy. Overall, these studies con¢rm the antiviral e¤cacy of protease inhibitors, nucleoside type and non-nucleoside type polymerase inhibitors in patients with chronic hepatitis C. Safety and tolerability are important issues in the development of new compounds. Despite antiviral e¤cacy the development of the protease inhibitor ciluprevir was stopped due to cardiotoxicity in animals. The development of the nucleoside type polymerase inhibitor valopicitabine and the non-nucleoside type polymerase inhibitor HCV-796 was stopped due to gastrointestinal adverse events and elevation of liver enzymes, respectively. Because of high levels of HCV replication and the low ¢delity of the HCV polymerase, selection of resistant isolates during therapy may occur. The development of resistance was investigated in the replicon model and in patients with chronic hepatitis C. The most detailed analysis on selection of resistant strains was performed in patients with chronic HCV-1 infection receiving telaprevir monotherapy. In these patients mutations that confer lowlevel resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36+155, 36+156) were detected and correlated with telaprevir exposure and virological response22. Combination therapy with peginterferon alpha or other direct antiviral drugs seems mandatory to avoid developing resistance. The protease inhibitors boceprevir, telaprevir, the nucleoside analogue polymerase inhibitors valopicitabine, R1626 and the non-nucleoside polymerase inhibitor HCV-796 were investigated in combination with interferon alpha-based therapy. A combination of peginterferon alpha-2b with the protease inhibitor boceprevir showed a decline of HCV-RNA of 2.45 2.86 log 10 IU/ml in genotype 1-infected non-responders after 14 days of treatment18. In treatment-naive patients infected with HCV genotype 1 the combination of telaprevir and peginterferon alpha-2a showed a decline of HCV-RNA of 5.49 log10 IU/ml after 14 days23. The nucleoside analogue R1616 in combination with peginterferon alpha-2a showed a decline of 3.6 4.5 log10 IU/ml in treatment-naive HCV genotype 1-infected patients24. The non-nucleoside analogue HCV-769 was associated with a 2.6 3.2 log10 IU/ml decline of HCV-RNA in HCV genotype 1-infected patients after 14 days of therapy25. A central question is whether STAT-C may enhance the SVR rate of patients with chronic hepatitis C. So far the only SVR results for STAT-C are available for the protease inhibitor telaprevir in combination with peginterferon alpha2a with and without ribavirin. In the PROVE-1 trial 61% of patients with chronic HCV genotype 1 infection achieved a SVR 24 weeks after treatment with telaprevir plus peginterferon alpha-2a plus ribavirin for 12 weeks and peginterferon alpha-2a plus ribavirin for an additional 12 weeks (total 165
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treatment duration 24 weeks)26. In the PROVE-2 trial 65% of patients infected with HCV genotype 1 maintained a virological response 12 weeks after treatment with telaprevir plus peginterferon alpha-2a plus ribavirin for 12 weeks followed by peginterferon alpha-2a plus ribavirin for an additional 12 weeks27. The SVR rates of the control arms of both studies in which patients received peginterferon alpha-2a plus ribavirin standard therapy for 48 weeks are pending. Historical SVR rates in treatment-naive patients treated with peginterferon alpha-2a plus ribavirin standard therapy for 48 weeks are around 42 54% in patients infected with HCV genotype 11,7. Compared with these historical SVR rates the PROVE trials suggest that the addition of highly active protease inhibitors to standard therapy may improve the SVR rate with a shorter treatment duration.
CONCLUSIONS Peginterferon alpha and ribavirin for 24 weeks and 48 weeks is the current standard treatment for patients with chronic hepatitis C infected with HCV genotype 1 and 2/3, respectively. Recent trials have focused on the individualization of treatment based on virological response. In `di¤cult-tocure' patients infected with HCV genotype 1 and slow virological response an extended treatment duration of 72 weeks has been shown to enhance SVR rates compared with the standard treatment duration of 48 weeks. In patients infected with HCV genotype 1, low pretreatment viral load and virological response at treatment week 4, a shorter treatment duration of 24 weeks has been shown to be equally e¡ective as the standard treatment duration of 48 weeks. In patients infected with HCV genotype 2/3, low pretreatment viraemia and virological response at week 4, a treatment duration of 16 weeks has been shown to be equally e¡ective as a treatment duration of 24 weeks. However, despite great success in the individualization of treatment, further improvement of SVR rates by modi¢cation of interferon alpha-based treatment appears unlikely. The NS3/4A protease and the NS5B RNA-dependent RNA polymerase are promising targets of STAT-C. Several compounds have been shown to be e¡ective in monotherapy. However, development and selection of resistant strains limit virological response in monotherapy; therefore combination of protease and polymerase inhibitors with peginterferon alpha and ribavirin seems mandatory. Two recent trials reported initial SVR results of treatment with the protease inhibitor telaprevir in combination with peginterferon alpha2a and ribavirin. The results indicate that a higher SVR with shorter treatment duration is possible. It is anticipated that HCV-speci¢c inhibitors will improve treatment opportunities of patients with chronic hepatitis C, especially in `di¤cult-to-cure' patients. However, approval of HCV-speci¢c compounds cannot be expected before 2011/2012.
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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Hadziyannis SJ, Sette H Jr, Morgan TR et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med. 2004;140:346 55. Berg T, von Wagner M, Nasser S et al. Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon-alfa-2a plus ribavirin. Gastroenterology. 2006;130:1086 97. Sanchez-Tapias JM, Diago M, Escartin P et al. Peginterferon-alfa2a plus ribavirin for 48 versus 72 weeks in patients with detectable hepatitis C virus RNA at week 4 of treatment. Gastroenterology. 2006;131:451 60. Pearlman B, Ehleben C. Improved response rates with treatment extension to 72 weeks in slow responders to peginterferon and weight-based ribavirin in chronic hepatitis C virus infection. Hepatology. 2007;46:356A. Jensen DM, Freilich B, Andreone P et al. Pegylated interferon alfa-2a plus ribavirin in prior non-responders to pegylated interferon alfa-2b/RBV: Final e¤cacy and safety outcomes of the REPEAT Study. Hepatology. 2008;46:291A. Zeuzem S, Buti M, Ferenci P et al. E¤cacy of 24 weeks treatment with peginterferon alfa2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol. 2006;44:97 103. Manns MP, McHutchison JG, Gordon SC et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet. 2001;358:958 65. Jensen DM, Morgan TR, Marcellin P et al. Early identi¢cation of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology. 2006;43:954 60. von Wagner M, Huber M, Berg T et al. Peginterferon-alpha-2a (40KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C. Gastroenterology. 2005;129:522 7. Mangia A, Santoro R, Minerva N et al. Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N Engl J Med. 2005;352:2609 17. Shi¡man ML, Suter F, Bacon BR et al. Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3. N Engl J Med. 2007;357:124 34. Zeuzem S, Benhamou Y, Bain V et al. Antiviral response at week 12 following completion of treatment with albinterferon alfa-2b plus ribavirin in genotype 1, IFN-naive, chronic hepatitis C patients. J Hepatol. 2007;46:293A. Zeuzem S. Heterogeneous virologic response rates to interferon-based therapy in patients with chronic hepatitis C: who responds less well? Ann Intern Med. 2004;140:370 81. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110 13. Wakita T, Pietschmann T, Kato T et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med. 2005;11:791 6. Lindenbach BD, Evans MJ, Syder AJ et al. Complete replication of hepatitis C virus in cell culture. Science. 2005;309:623 6. Hinrichsen H, Benhamou Y, Wedemeyer H et al. Short-term antiviral e¤cacy of BILN 2061, a hepatitis C virus serine protease inhibitor, in hepatitis C genotype 1 patients. Gastroenterology. 2004;127:1347 55. Sarrazin C, Rouzier R, Wagner F et al. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology. 2007;132:1270 8. Reesink HW, Zeuzem S, Weegink CJ et al. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study. Gastroenterology. 2006;131:997 1002. Zhou XJ, Afdhal N, Godofsky E et al. Pharmacokinetics and pharmacodynamics of valopicitabine (NM283), a new nucleoside HCV polymerase inhibitor: Results from a phase I/II dose-escalation trial in patients with HCV-1 infection. J Hepatol. 2005;42:229A. Chandra P, Raible D, Harper D, Speth J, Villano S, Bichler G. Antiviral activity of the nonnucleoside polymerase inhibitor, HCV-796, in patients with chronic hepatitis C:
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22. 23. 24. 25. 26. 27.
preliminary results from a randomized, double-blind, placebo-controlled, ascending multiple dose study. Gastroenterology. 2007;130:748A. Sarrazin C, Kie¡er TL, Bartels D et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology. 2007;132:1767 77. Forestier N, Reesink HW, Weegink CJ et al. Antiviral activity of telaprevir (VX-950) and peginterferon alfa-2a in patients with hepatitis C. Hepatology. 2007;46:640 8. Pockros PJ, Nelson D, Godofsky E et al. Robust synergistic antiviral e¡ect of R1626 in combination with peginterferon alfa-2a (400), with or without ribavirin interim analysis results of Phase 2a study. Hepatology. 2007;46:311A. Villano S, Raible D, Harper D et al. Antiviral activity of the non-nucleoside polymerase inhibitor, HCV-796, in combination with pegylated interferon alfa-2b in treatment naive patients with chronic HCV. J Hepatol. 2007;46:24A. Jacobson IM, Everson G, Gordon SC et al. Interim analysis results from a phase 2 study of telaprevir with peginterferon alfa-2A and ribavirin in treatment-naive subjects with hepatitis C. Hepatology. 2007;46:315A. Hezode C, Ferenci P, Dusheiko GM et al. PROVE2: Phase II study of VX950 (TELAPREVIR) in combination with peginterferon alfa2A with or without ribavirin in subjects with chronic hepatitis C; ¢rst interim analysis. Hepatology. 2007;46:268A.
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Section VII Metabolic and autoimmune liver injury Chair: AJ CZAJA and CP DAY
17 Non-alcoholic steatohepatitis: metabolic syndrome of the liver C. P. DAY
INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is increasingly diagnosed worldwide and considered to be the commonest liver disorder in Western countries. It comprises a disease spectrum which includes variable degrees of simple steatosis (fatty liver), non-alcoholic steatohepatitis (NASH) and cirrhosis. Simple steatosis is benign, whereas steatohepatitis (NASH) is characterized by hepatocyte injury, in£ammation and ¢brosis which can lead to cirrhosis, liver failure and hepatocellular carcinoma (HCC). NAFLD is strongly associated with obesity, insulin resistance, hypertension and dyslipidaemia, and is now regarded as the liver manifestation of the metabolic syndrome. Rapid spread of the obesity `pandemic' in adults and children, coupled with the realization that the outcomes of obesity-related liver disease are not entirely benign, has led to a rapid growth in clinical and basic studies in NAFLD. This chapter will concentrate on an update of clinical aspects of this increasingly important disease.
EPIDEMIOLOGY NAFLD is often an asymptomatic illness in which the liver blood tests may be completely normal. This has made studies on prevalence extremely di¤cult, with most relying on ultrasound which is known to be sensitive only when more than a third of the liver is a¡ected by steatosis. With this proviso the prevalence of NAFLD appears to be around 20 30% in Western adults1,2 and 15% in Asians3. Due to the lack of prospective studies the true incidence of NAFLD is not well de¢ned, although from the information available it appears to be low4. Since liver biopsy is the only method of accurately diagnosing steatohepatitis, incidence/prevalence studies of NASH are rare. According to available data NASH is much rarer than NAFLD, a¡ecting 2 3% of the general population5. NAFLD and NASH are strongly associated with the presence and severity of obesity. Studies in severely obese patients (BMI 435 kg/m2) undergoing 171
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bariatric surgery have reported prevalences of NAFLD and NASH of 91% and 37%, respectively6, while a post-mortem study reported NASH to be present in 3% of non-obese, 19% of obese and 50% of morbidly obese individuals7. A recent novel observational study in NAFLD patients has demonstrated that, while central obesity correlates with the severity of in£ammation, dorsocervical lipohypertrophy correlates with hepatocyte injury, in£ammation, and ¢brosis8. Type 2 diabetes mellitus (T2DM) is the other major association of NAFLD with a prevalence of 70% recently reported from an ultrasound survey of almost 3000 unselected Italian T2DM patients9,10. Even in the absence of obesity and T2DM, NAFLD is closely associated with other features of the metabolic syndrome, with one study of non-diabetics with NAFLD reporting that 18% of normal-weight patients and 67% of obese patients ful¢lled criteria for the metabolic syndrome11. There are no accurate data regarding temporal changes in the prevalence of NAFLD; however, the rising prevalence of obesity, diabetes and the metabolic syndrome seems likely to be re£ected in an increasing prevalence of NAFLD. This trend is of particular concern in the paediatric population, where the reported increase in obesity will undoubtedly result in a higher incidence and prevalence of paediatric and adult NAFLD in the future. To date studies in children have reported a prevalence of NAFLD of 3% in the general paediatric population and 53% in obese children12,13. Reports of toddlers with NAFLD and primary-school children with NAFLD-related cirrhosis are clearly a cause for alarm14.
NATURAL HISTORY OF NAFLD In marked contrast to alcoholic steatohepatitis, the short-term prognosis of NAFLD is good. The largest prospective histological study of the natural history of NAFLD, with a mean follow-up of 13 years, has recently been published15. Data from this and other studies suggest that the long-term hepatic prognosis of patients with NAFLD depends on the histological stage of disease at presentation16. Among patients with simple steatosis 12 40% will develop NASH with early ¢brosis after 813 years. For patients presenting with NASH and early ¢brosis around 15% will develop cirrhosis and/or evidence of hepatic decompensation over the same time-period, increasing to 25% of patients with advanced pre-cirrhotic ¢brosis at baseline. In the most recent study weight gain and the presence of portal tract ¢brosis on index biopsy were the only signi¢cant predictors of ¢brosis progression15. About 7% of subjects with compensated cirrhosis associated with NAFLD will develop a HCC within 10 years, while 50% will require a transplant or die from a liver-related cause17. The risk of HCC in NAFLD-related cirrhosis is comparable to that in cirrhosis associated with alcohol or hepatitis C18. This may partly explain the recently reported associations of HCC with high BMI and T2DM19. Liver transplantation is increasingly available to those with chronic liver failure and about 10 12% of liver transplants in the United States are for NAFLD cirrhosis20. Unfortunately the condition can recur in transplanted organs. The overall survival of patients with NAFLD is less than that of an age- and sex172
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matched population with liver disease the third leading cause of death in NAFLD patients compared to the thirteenth leading cause in a general population21.
SUSCEPTIBILITY While the vast majority of individuals with obesity, insulin resistance and the metabolic syndrome will have steatosis, only a minority will ever develop steatohepatitis, ¢brosis and cirrhosis. Potential environmental determinants of NAFLD are dietary factors and small bowel bacterial overgrowth22. Recent studies have shown that diets high in saturated fat, soft drinks and meat, and low in antioxidants and omega-3-containing ¢sh are associated with an increased risk of NAFLD/NASH23,24. With respect to alcohol intake, while there is no doubt that obesity increases the risk of cirrhosis in heavy drinkers25, emerging evidence suggests that `sensible' light alcohol intake may be protective against NAFLD/NASH26,27, an e¡ect that appears likely to be due to the bene¢cial e¡ect of light alcohol intake on insulin sensitivity. Family studies and inter-ethnic variations in susceptibility suggest that genetic factors may be important in determining disease risk. Although no genetic associations with advanced NAFLD have been replicated in large studies, preliminary data suggest that polymorphisms in the genes encoding microsomal triglyceride transfer protein, phosphatidylethanolamine transferase, superoxide dismutase 2, the CD14 endotoxin receptor, tumour necrosis alpha (TNF-a), a transforming growth factor beta (TGF-b), and angiotensinogen may be associated with an increased risk of steatohepatitis and/or ¢brosis28. With the advent of high-throughput gene analyses and the reduced cost of whole genome-wide scans it seems likely that genes contributing to inherited susceptibility to this common disease will be identi¢ed in the near future.
DISEASE ASSOCIATIONS WITH NAFLD Cardiovascular disease Given the close association between NAFLD and classical cardiovascular risk factors it is perhaps not surprising that, when compared to controls, patients with NAFLD have a higher prevalence of atherosclerosis, as shown by in creased carotid wall intimal th i ckness, in c re ased nu mb er s of atherorosclerotic plaques and increased plasma markers of endothelial dysfunction 9,29,30 . This association also extends to children, with the prevalence of coronary and aortic atheroma higher in children with fatty liver compared to controls in an autopsy-based report31. Consistent with these observations two natural history studies have reported that the increased agerelated mortality observed in patients with NAFLD is attributable to cardiovascular as well as liver-related deaths 15,17 . Although an indirect association between NAFLD and cardiovascular disease is expected, a 173
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growing body of evidence supports a direct role for NAFLD in the pathogenesis of atheromatous cardiovascular disease. A recent study of unsele cted patients with T2DM reported that the prevalen c e of cardiovascular, cerebrovascular and peripheral vascular disease was signi¢cantly greater in those with NAFLD than in those without, independent of the individual components of the metabolic syndrome10. A similar ¢nding has been observed for microvascular diseases, nephropathy and retinopathy32. The mechanism of any direct e¡ect of NAFLD on cardiovascular risk remains unclear; possibilities include the release of atherogenic in£ammatory cytokines and pro-coagulant factors from the steatotic liver33.
Polycystic ovary syndrome (PCOS) As with the association between NAFLD and the metabolic syndrome, the now well-established association between NAFLD and the PCOS seems likely to be indirect as a result of both conditions being characterized by insulin resistance. Up to 30% of females with PCOS have elevated alanine transaminase (ALT) levels34 and an NAFLD prevalence of 42% has been reported in a series of PCOS patients with a mean age of 25 years35. More recently advanced ¢brotic liver disease has been reported in patients with PCOS36, suggesting that women with this syndrome require careful hepatic evaluation.
Obstructive sleep apnoea (OSA) Chronic intermittent hypoxia, as seen in OSA, has been associated with cardiovascular disease, the metabolic syndrome and insulin resistance37. As might be expected, therefore, a proportion of patients with OSA have elevated liver enzymes and histological features of NASH independent of body weight38. The severity of histology and the associated insulin resistance both correlate with the severity of OSA, strongly implicating insulin resistance as the pathogenic mechanism linking OSA to NASH, although not entirely excluding a role for hypoxic liver injury. As with PCOS, this and other similar reports suggest that patients with OSA require hepatic evaluation, and that the diagnosis of OSA should be considered in NAFLD patients reporting daytime somnolence, sleep disturbances or any other symptoms suggesting a diagnosis of OSA.
CLINICAL PRESENTATION NAFLD is a largely asymptomatic condition that may reach an advanced stage before it is suspected or diagnosed. Symptoms such as right upper quadrant discomfort, fatigue and lethargy have been reported in up to 50% of patients, but are uncommon modes of presentation. Most patients with NAFLD are diagnosed after they are found to have hepatomegaly or, more commonly, unexplained abnormalities of liver blood tests performed as part of routine health checks or during drug monitoring (e.g. statin therapy). NAFLD is the commonest cause of incidental abnormal liver blood tests accounting for 60 174
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90% of such cases39,40. Importantly, the vast majority (around 80%) of patients with NAFLD have normal liver blood tests2, and there is no di¡erence in histological severity between those with and without abnormal tests 41 . Accordingly, NAFLD should be suspected and sought in all patients with established risk factors, including PCOS and OSA, regardless of liver blood tests. The history should concentrate on determining the presence/absence of metabolic conditions commonly associated with `primary' NAFLD syndrome components, cardiovascular disease and OSA and on excluding alternative causes of steatosis including excessive alcohol intake, previous abdominal surgery (leading to bacterial overgrowth) and drugs causing NAFLD, such as amiodarone and tamoxifen. On examination, most patients are centrally obese and dorsocervical lipohypertrophy (a `bu¡alo hump') appears to be a particular feature of the fat distribution in patients with advanced NAFLD8. Features of PCOS (hyperandrogenism) should be sought in young women with suspected NAFLD 35 , and clinical evidence of lipodystrophy should be sought in young, non-obese patients in view its association with NAFLD42.
INVESTIGATION In the absence of advanced disease routine liver blood tests are either normal or typically show mild elevations of transaminases, alkaline phosphatase and gamma glutamyl transpeptidase (GGT) 1.5 3 times the upper limit of normal. The ALT/AST ratio is greater than 1 unless there is advanced ¢brotic NAFLD or the patient is a covert heavy drinker. Other blood tests are aimed at detecting associated conditions, such as dylipidaemia, and excluding alternative causes of abnormal liver blood tests. Regarding lipids, it is worth measuring serum levels of apolipoprotein B (apoB) in patients either with no obvious risk factors for NAFLD or with low levels of low-density lipoprotein and high-density lipoprotein cholesterol, looking for evidence of hypobetalipoproteinaemia, a rare, familial cause of NAFLD43. Serum ferritin is often raised in NAFLD patients44 and has been associated with advanced ¢brosis45. HFE genotyping should be carried out when hyperferritaemia is accompanied by raised transferrin saturation. Autoantibodies associated with autoimmune hepatitis (AIH), including antinuclear antibody (ANA) and smooth muscle antibody (SMA), are often present at low titres in patients with NAFLD, and have been associated with more advanced disease in some, but not all, studies46,47. Around one in 10 of these patients have histological features of autoimmune hepatitis on biopsy and ful¢l diagnostic criteria for probable/de¢nite AIH46. Currently available imaging modalities including ultrasound, computed tomography and routine magnetic resonance imaging (MRI) are all excellent at detecting steatosis (once more than around a third of the liver is a¡ected) but none can reliably detect NASH or ¢brosis 48. Newer imaging techniques, including proton magnetic resonance spectroscopy 4 9 and transient elastography50, show promise, but require further study prior to routine use for disease staging. 175
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The role of liver biopsy Undoubtedly the most important and controversial issue to consider in the investigation of patients with suspected NAFLD is whether or not to perform a liver biopsy. For diagnosis, biopsy is not required in a `typical' patient with abnormal liver blood tests, classical risk factors for NAFLD (obesity, T2DM, dyslipidaemia) and an ultrasound showing steatosis; however, a high ferritin with HFE mutations, positive autoantibodies (ANA, SMA) or the use of medications associated with drug-induced liver injury all may justify a biopsy to exclude alternative/additional diagnoses. The main indication to perform a biopsy is, however, the accurate staging of the disease since: (a) di¡erent stages have di¡erent prognoses and therefore require di¡erent management strategies, and (b) no currently available imaging techniques can perform this role48.
Non-invasive markers for staging NAFLD The current reliance on liver biopsy for disease staging has prompted many studies aimed at de¢ning clinical or laboratory-based variables capable of acting as surrogate markers of disease stage51. Various clinical and laboratory markers have been shown to be associated with advanced ¢brosis (bridging ¢brosis or cirrhosis) in patients with NAFLD, notably advanced age (445 years), BMI 430 kg/m2, T2DM (or raised fasting blood glucose), the severity of OSA38 an AST/ALT ratio greater than 1, hyperferritinaemia45 and positive autoantibodies46. At present it would therefore seem reasonable to restrict liver biopsy to patients with at least some, if not all, of these risk factors. Some of these markers (age, BMI, T2DM, AST/ALT ratio) have recently been combined together with platelet count and serum albumin concentration, into a NAFLD ¢brosis `score' that accurately predicts the presence or absence of advanced ¢brosis in the majority of patients with NAFLD52. This score has recently been combined with the European Liver Fibrosis (ELF) panel of serum ¢brosis markers53, and shown to have an accuracy of over 90% in di¡erentiating di¡erent ¢brosis stages in NAFLD 54 . With respect to the non-invasive diagnosis of NASH rather than ¢brosis stage, serum levels of a caspase cleavage product of the hepatocyte protein cytokeratin-18 (a putative marker of hepatocyte apoptosis) have recently been shown to accurately predict the presence of NASH in a small pilot study55. Clearly this and other tests and scoring systems require further validation before they can be used in routine clinical practice, but they do appear, at last, to o¡er real potential to replace the need for liver biopsy in the majority of patients with NAFLD.
OVERALL MANAGEMENT STRATEGY FOR NAFLD Almost no large randomized controlled trials (RCT) have been published on which to establish evidence-based treatment recommendations for NAFLD. Accordingly, current management strategies are directed at treating, where present, the individual components of the metabolic syndrome, since this will reduce the risk of cardiovascular disease and may also be bene¢cial for the 176
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liver. Alcohol intake should not exceed `sensible' limits, but there is no need to advise complete abstinence as an emerging body of data suggests that light to moderate intake may actually reduce the risk of NAFLD25,26. In view of their largely benign prognosis these strategies are all that is required for patients with simple steatosis who can be managed by general or primary-care physicians with no requirement for formal hepatological follow-up. In contrast, patients with more advanced NAFLD require long-term follow-up by hepatologists in light of their increased propensity for disease progression and the resulting need for surveillance for complications including oesophageal varices and HCC. These patients will also be candidates either for emerging `second-line' therapies currently being evaluated in large RCT or for entry into these trials. The rationale for NAFLD therapies is based on a growing understanding of disease pathogenesis with a particular focus on reducing insulin resistance, hepatic free fatty acid (FFA) levels, oxidative, endoplasmic reticulum and cytokine-mediated stress and in£uencing the balance and e¡ects of pro¢brotic, proin£ammatory and anti¢brotic, anti-in£ammatory adipokines released from adipose tissue56. Current and emerging therapies for NAFLD can be divided into those directed at the metabolic syndrome components with potential liver e¡ects and those directed primarily at the liver.
TREATMENTS DIRECTED AT COMPONENTS OF THE METABOLIC SYNDROME Obesity Obesity is a rational target for NAFLD therapy since weight loss should reduce many of the putative mediators of liver injury including insulin resistance, hepatic FFA supply and proin£ammatory, pro¢brotic adipokines.
Diet and exercise Several small, largely uncontrolled, studies have shown an improvement in either ALT or steatosis following diet (with or without exercise)-induced weight loss. There is very little evidence that necroin£ammation or ¢brosis can be improved by weight loss alone, although a few small case series have shown some improvement in these parameters with drastic weight loss (reviewed in ref. 57). To date almost all studies of diet-induced weight loss have employed simple calorie restriction, with very few attempting to manipulate speci¢c dietary components. This area seems worthy of study, since intakes of both saturated fat and ¢bre are known to in£uence insulin resistance and diets high in saturated fat, soft drinks and meat and low in omega 3-containing ¢sh appear to be associated with both NAFLD and NASH23. Dietary fat intake has also been shown to correlate with liver fat content and insulin resistance in short-term studies of obese, non-diabetic women independently of changes in total-body, subcutaneous or abdominal fat58. The value of exercise in achieving and maintaining weight loss and improving insulin resistance is well established and thus far the only controlled study of weight loss that has 177
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reported histological improvement (steatosis) combined calorie restriction with increased exercise59.
Pharmacological anti-obesity agents Encouraging improvements in liver histology have been reported from pilot studies of the intestinal lipase inhibitor orlistat in patients with NASH60,61; however there is no evidence as yet that this improvement is over and above what would be expected from the resulting weight loss. Nonetheless, data from currently ongoing large RCT of orlistat are awaited with interest. The cannabinoid receptor 1 (CB1) antagonist rimonabant has been shown to be e¡ective in reducing weight and waist circumference, with improvements in several metabolic parameters including insulin resistance62. Its e¡ects on the liver in patients with NAFLD are, as yet, unknown; however, animal data demonstrating that CB1 blockade is both anti-steatotic63 and anti-¢brotic64 provide a strong rationale for forthcoming clinical trials of drugs directed at the CB1 receptor in NAFLD.
Bariatric surgery Various surgical procedures are currently in use for the treatment of obesity. Biliopancreatic diversion appears to carry a signi¢cant risk of liver failure and worsening ¢brosis, and should therefore be avoided in patients with NAFLD; however, more encouraging results have been reported for gastric bypass and gastric banding surgery65,66. To date all studies have shown improvements in metabolic parameters and steatosis with some, but not all, reporting improvements in necroin£ammation and ¢brosis65.
Type 2 diabetes mellitus and insulin resistance Evidence that insulin resistance may contribute to both in£ammation and ¢brosis in NAFLD has led to several pilot studies of metformin and other insulin-sensitizing agents in patients with NAFLD with and without diabetes. There is as yet no direct evidence that hyperinsulinaemia per se adversely a¡ects the liver; however, evidence from animal studies that insulin is a direct cause of both hepatic steatosis and ¢brosis67 might suggest that insulin or sulphonylureas should be avoided if possible. It is of interest, therefore, that a recent pilot study in patients with T2DM has shown that long-term high-dose insulin therapy results in a reduction of transaminases and hepatic steatosis, presumably re£ecting the bene¢cial e¡ects of insulin on blood glucose and adipose tissue lipolysis68. Whether or not long-term insulin therapy increases ¢brosis in patients with NAFLD is, however, as yet unknown.
Metformin Pilot studies of metformin in diabetic and non-diabetic patients with NAFLD have shown inconsistent e¡ects on liver blood tests and steatosis (determined by MRI or MR proton spectroscopy). However, the largest RCT to date, in 178
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non-diabetic NAFLD patients, has been more encouraging. In this 12-month, randomized open-label trial, metformin treatment (2 g/day) was associated with signi¢cantly higher rates of normalized aminotransferase levels and with signi¢cant decreases in liver fat, necroin£ammation and ¢brosis, compared with either vitamin E treatment or a weight-reducing diet69. The low number of patients who agreed to a second biopsy does, however, limit the strength of the conclusions that can be drawn from this study.
Thiazolidinediones (TZD) TZD act as agonists for the peroxisome proliferator-activated receptor-g (PPARg). They improve insulin sensitivity, at least in part, via anti-steatotic e¡ects in liver and muscle which may in turn result from an increase in the secretion of the anti-in£ammatory, anti-¢brotic adipokine, adiponectin, by adipocytes. Moreover, their potential as a therapy for NAFLD is further increased by evidence from animal models showing that they may also exert direct anti-¢brotic e¡ects in the liver70. Pilot studies of the second-generation TZD, pioglitazone and rosiglitazone, have consistently reported encouraging improvements in insulin sensitivity, liver blood tests and liver histology, and several large RCT are currently in progress. The ¢rst placebo-controlled RCT of pioglitazone in the treatment of patients with NASH has recently reported signi¢cant improvements in steatosis, in£ammation and ballooning necrosis associated with a non-signi¢cant decrease in ¢brosis 71. A note of caution over the use of TZD in the treatment of NASH has arisen recently as a result of several meta-analyses of trials of TZD in T2DM patients that have consistently shown that rosiglitazone increases the incidence of myocardial infarction and heart failure72. The risk of heart failure is also increased by pioglitazone, but it is associated with a lower risk of myocardial infarction and stroke compared to placebo-treated patients73. This is reassuring since a recent study suggests that pioglitazone treatment for NASH has to be continued long term, since stopping it led to a worsening of steatosis and in£ammation74.
Dyslipidaemia Hypertriglyceridaemia a¡ects 20 80% of patients with NAFLD. As with antiobesity and insulin-sensitizing drugs, there are sound scienti¢c reasons to support the use of ¢brates the conventional triglyceride-lowering agents in patients with NAFLD. Fibrates are agonists for the PPARa receptor, a transcription factor that up-regulates the transcription of genes encoding various proteins that would be expected to reduce hepatic FFA levels, and also exerts anti-in£ammatory e¡ects. As with many other potential therapies for NAFLD, studies of PPARa agonists in animal models of NASH have been encouraging75; however, the only controlled study in patients with histological follow-up reported that 1 year of clo¢brate had no e¡ect on liver biochemistry or histology76. There is less rationale for using HMG CoA reductase inhibitors (statins) to treat NAFLD; however, they can be safely prescribed for `conventional' indications, including T2DM and high cardiovascular risk. Importantly, there is no evidence that patients with pre-existing NAFLD are 179
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at increased risk of statin-induced idiosyncratic hepatotoxicity, or that statins are associated with a higher frequency of hepatic steatosis or serum ALT abnormalities in these subjects77.
Hypertension No RCT have speci¢cally examined the e¡ect of di¡erent anti-hypertensive agents on the liver in hypertensive patients with NAFLD. However, a growing body of evidence from animal models of hepatic ¢brosis and NASH suggests that therapy directed at the renin angiotensin system and a-blockers may be bene¢cial for the liver78,79. As yet only one pilot study has examined the use of angiotensin II receptor blockade in patients with NASH, and this showed a reduction in serum markers of ¢brosis 80. Newer angiotensin II receptor blockers with insulin-sensitizing e¡ects seem worthy of study in NAFLD81.
TREATMENTS DIRECTED AT THE LIVER An increased understanding of the mechanisms of progressive liver damage in NAFLD has stimulated the search for therapies speci¢cally targeting the liver rather than at the individual components of the metabolic syndrome that may have bene¢cial e¡ects.
Antioxidants Several encouraging pilot studies of various agents indicate potential bene¢cial e¡ects which may be related to their anti-oxidant e¡ects. These include probucol82, betaine83, iron depeletion through venesection84 and vitamin E85. However, a recent RCT of vitamin E combined with vitamin C in patients with NASH found no overall improvement in hepatic ¢brosis score compared with placebo86.
Anti-cytokine agents Bene¢cial e¡ects of anti-TNF-a therapies have been demonstrated in animal models of NASH, and the two pilot studies in patients with NAFLD have reported improvements in aminotransferase levels87 and histology88. Given the emerging importance of proin£ammatory cytokines in both liver pathology and insulin resistance in obesity, it seems likely that cytokines and their regulatory molecules, including NF-kB, will become major therapeutic targets in both NAFLD and T2DM in the near future.
Ursodeoxycholic acid (UDCA) Given its long history as a hepatoprotectant, and recent evidence that bile acids may act as molecular chaperones capable of reducing endoplasmic reticulum (ER) stress implicated in NASH pathogenesis56,89 it is hardly surprising that UDCA has been considered as a potential treatment for NASH. To date, 180
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however, the only large, placebo-controlled RCT in patients with NASH showed no bene¢t of UDCA (13 15 mg/kg per day) on liver histology after 2 years treatment90. More encouraging results have recently been reported from a study combining UDCA with vitamin E91.
Liver transplantation for patients with NAFLD Patients with NAFLD who progress to decompensated cirrhosis, or who develop HCC, are candidates for liver transplantation. A favourable outcome depends on removing the factors that originally caused the liver damage. Perhaps unsurprisingly, steatosis recurs in most patients within 4 years, with 50% developing NASH and ¢brosis; cases of recurrent cirrhosis are also reported92,93. Risk factors for recurrence are the presence of insulin resistance or T2DM pre- and post-transplantation, weight gain following transplantation, and a high cumulative steroid dose. These ¢ndings highlight the importance of ensuring weight and metabolic control in reducing the risk of disease recurrence, in a group of patients who will undoubtedly contribute increasing numbers to transplant programmes in the future.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 14. Molleston J, White F, Teckman J et al. Obese children with steatohepatitis can develop cirrhosis in childhood. Am J Gastroenterol. 2002;97:2460 2. 15. Ekstedt M, Franzen LE, Mathiesen UL et al. Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology. 2006;44:865 73. 16. Day CP. Natural history of NAFLD: remarkably benign in the absence of cirrhosis. Gastroenterology. 2005;129:375 8. 17. Sanyal AJ, Banas C, Sargeant C et al. Similarities and di¡erences in outcomes of cirrhosis due to nonalcoholic steatohepatitis and hepatitis C. Hepatology. 2006;43:682 9. 18. Nair S, Mason A, Eason J, Loss G, Perillo R. Is obesity an independent risk factor for hepatocellular carcinoma in cirrhosis? Hepatology. 2002;36:150 55. 19. Calle E, Rodriguez C, Walker-Thurmond K, Thun M. Overweight, obesity and mortality from cancer in a prospectively studied cohort of US adults. N Engl J Med. 2003;348:1625 38. 20. McCullough A. The clinical features, diagnosis and natural history of non alcoholic fatty liver disease. Clin Liver Dis. 2004;8:521 33. 21. Adams LA, Lymp JF, St Sauver J et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology. 2005;129:113 21. 22. De Alwis N, Day CP. Genetics of alcoholic liver disease and nonalcoholic fatty liver disease. Semin Liver Dis. 2007;27:44 54. 23. Musso G, Gambino R, De Michieli F et al. Dietary habits and their relations to insulin resistance and postprandial lipemia in nonalcoholic steatohepatitis. Hepatology. 2003;37:909 16. 24. Zelber-Sagi S, Nitzan-Kaluski D, Goldsmith R et al. Long term nutritional intake and the risk of nonalcoholic fatty liver disease (NAFLD): a population based study. J Hepatol. 2007;47:711 17. 25. Naveau S, Giraud V, Borotto E, Aubert A, Capron F, Chaput JC. Excess weight risk factor for alcoholic liver disease. Hepatology. 1997;25:108 11. 26. Dixon J, Bhathal P, O'Brian P. Non-alcoholic fatty liver disease: predictors of non-alcoholic steatohepatitis and liver ¢brosis in the severely obese. Gastroenterology. 2001;121:91 100. 27. Suzuki A, Angulo P, St Sauver J, Muto A, Okada T, Lindor K. Light to moderate alcohol consumption is associated with lower frequency of hypertransaminasemia. Am J Gastroenterol. 2007;102:1912 19. 28. De Alwis N, Day CP. Genes and nonalcoholic fatty liver disease. Curr Diab Rep. 2008 (In press). 29. Targher G, Bertolini L, Scala L, Zoppini G, Zenari L, Falezza G. Non alcoholic hepatic steatosis and its relation to increased plasma biomarkers of in£ammation and endothelial dysfunction in nondiabetic men. Role of visceral adipose tissue. Diabet Med. 2005;22:1354 8. 30. Fracanzani A, Burdick L, Raselli L et al. Risk of early atherosclerosis evaluated by carotid artery intima media thickness in patients with non alcoholic fatty liver disease: a case control study. Hepatology. 2005;42:610 11A. 31. Schwimmer J, Deutsch R, Behling C, Lavine J. Fatty liver as a determinant of atherosclerosis. Hepatology. 2005;42:610A. 32. Targher G, Bertolini L, Rodella S, Zoppini G, Lippi G, Day CP. Nonalcoholic fatty liver disease is independently associated with an increased prevalence of chronic kidney disease and proliferative/laser treated retinopathy in type 2 diabetic patients. Diabetologia. 2008 (In press). 33. Targher G, Arcaro G. Non-alcoholic fatty liver disease and increased risk of cardiovascular disease. Atherosclerosis. 2007;191:235 40. 34. Schwimmer J, Hhorram O, Chiu V, Schwimmer W. Abnormal aminotransferase activity in women with polycystic ovary syndrome. Fertil Steril. 2005;83:494 7. 35. Cerda C, Perez-Ayuso RM, Riquelme A et al. Nonalcoholic fatty liver disease in women with polycystic ovary syndrome. J Hepatol. 2007;47:412 17. 36. Setji T, Holland N, Sanders L, Pereira K, Diehl A, Brown A. Nonalcoholic steatohepatitis and nonalcoholic fatty liver disease in young women with polycystic ovary syndrome. J Clin Endocrinol Metab. 2006;91:1741 7. 37. Volk R, Somers V. Obesity related cardiovascular disease: implications of obstructive sleep apnea. Diabet Obes Met. 2005;8:250 60.
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NASH: METABOLIC SYNDROME OF THE LIVER 38. Tanne F, Gagnadoux F, Chazouilleres O et al. Chronic liver injury during obstructive sleep apnea. Hepatology. 2005;41:1290 6. 39. Skelly M, James P, Ryder S. Findings on liver biopsy to investigate abnormal liver function tests in the absence of diagnostic serology. J Hepatol. 2001;35:195 9. 40. Pendino G, Mariano A, Surace P et al. Prevalence and etiology of altered liver tests: a population based survey in a Mediterranean town. Hepatology. 2005;41:1151 9. 41. Mofrad P, Contos MJ, Haque M et al. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology. 2003;37:1286 92. 42. Javor E, Ghany M, Cochran E et al. Leptin reverses nonalcoholic steatohepatitis in patients with severe lipodystrophy. Hepatology. 2005;41:753 60. 43. Ta n o l i T, Tu e P, Ya b l o n s k i y D, S c h o n f e l d G. Fa t t y l i v e r i n f a m i l i a l hypobetalipoproteinaemia: roles of the APOB defects, intra-abdominal adipose tissue, and insulin sensitivity. J Lipid Res. 2004;45:941 7. 44. Trombini P, Piperno A. Ferritin, metabolic syndrome and NAFLD: elective attractions and dangerous liaisons. J Hepatol. 2007;46:549 52. 45. Bugianesi E, Manzini P, D'Antico S et al. Relative contribution of iron burden, HFE mutations and insulin resistance to ¢brosis in nonalcoholic fatty liver. Hepatology. 2004;39:179 87. 46. Adams LA Lindor KD, Angulo P. The prevalence of autoantibodies and autoimmune hepatitis in patients with non alcoholic fatty liver disease. Am J Gastroenterol. 2004;90:1316 20. 47. Loria P, Carulli N, Lonardo A. The prevalence of autoantibodies and autoimmune hepatitis in patients with nonalcoholic fatty liver disease. Am J Gastroenterol. 2005;100:1200 1. 48. Saadeh S, Younossi ZM, Remer EM et al. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology. 2002;123:745 50. 49. Cox I, Sharif A, Cobbold J, Thomas H, Taylor-Robinson S. Current and future applications of in vitro magnetic resonance spectroscopy in hepatobiliary disease. World J Gastroenterol. 2006;12:4773 83. 50. Yoneda M, Fujita K, Inamori M, Nakajima A. Transient elastography in patients with non alcoholic fatty liver disease (NAFLD). Gut. 2007;56:1330 1. 51. Guha IN, Parkes J, Roderick PR, Harris S, Rosenberg WM. Non-invasive markers associated with liver ¢brosis in non-alcoholic fatty liver disease. Gut. 2006;55:1650 60. 52. Angulo P, Hui JM, Marchesini G et al. The NAFLD ¢brosis score: a non-invasive system that accurately identi¢es liver ¢brosis in patients with NAFLD. Hepatology. 2007;45:846 54. 53. Rosenberg WM, Voelker M, Thiel R et al. Serum markers detect the presence of liver ¢brosis: a cohort study. Gastroenterology. 2004;127:1704 13. 54. Guha I, Parkes J, Roderick P et al. Non-invasive markers of ¢brosis in non alcoholic fatty liver disease: validating the European Liver Fibrosis panel and exploring simple markers. Hepatology. 2008 (In press). 55. Wieckowska A, Zein NN, Yerian LM, Lopez AR, McCullough AJ, Feldstein AE. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology. 2006;44:27 33. 56. Day CP. From fat to in£ammation. Gastroenterology. 2006;130:207 10. 57. Harrison SA, Day CP. Bene¢ts of lifestyle modi¢cation in NAFLD. Gut. 2007;56:1760 9. 58. Westerbacka J, Lammi K, Hakkinen A, A. R, Salminen I, Aro A, Yki-Jarvinen H. Dietary fat content modi¢es liver fat in overweight nondiabetic subjects. J Clin Endocrinol Metab. 2005;90:2804 9. 59. Ueno T, Sugawara S, Sujaku K et al. Therapeutic e¡ects of diet and exercise in obese patients with fatty liver. J Hepatol. 1997;27:103 10. 60. Harrison SA, Fincke C, Helinski D, Torgerson S, Hayashi P. A pilot study of orlistat treatment in obese, nonalcoholic steatohepatitis patients. Aliment Pharmacol Ther. 2004;20:623 8. 61. Hussein O, Grosovski M, Schlesinger S, Szvalb S, Assy N. Orlistat reverses fatty in¢ltration and improves hepatic ¢brosis in obese patients with nonalcoholic steatohepatitis (NASH). Dig Dis Sci. 2007;52:2512 19. 62. Van Gaal LF, Rissanen AM, Scheen AJ et al. E¡ects of the cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk factors in overweight patients: 1year experience from the RIO-Europe study. Lancet. 2005;365:1389 97.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 63. Gary-Bobo M, Elachouri G, Galla J et al. Rimonabant reduces obesity associated hepatic steatosis and features of the metabolic syndrome in obese Zucker fa/fa rats. Hepatology. 2007;46:122 9. 64. Teixeira-Clerc F, Julien B, Grenard P et al. CB1 cannabinoid receptor antagonism: a new strategy for the treatment of liver ¢brosis. Nat Med. 2006;12:671 6. 65. Dixon J, Bhathal P, Hughes N, O'Brien P. Improvement in liver histological analysis with weight loss. Hepatology. 2004;39:1647 54. 66. Klein S, Mittendorfer B, Eagon C et al. Gastric bypass surgery improves metabolic and hepatic abnormalities associated with nonalcoholic fatty liver disease. Gastroenterology. 2006;130:1564 72. 67. Adachi M, Osawa Y, Uchinami H, Kitamura T, Accili D, Brenner D. The forkhead transcription factor FoxO1 regulates proliferation and transdi¡erentiation of hepatic stellate cells. Gastroenterology. 2007;132:1434 46. 68. Juurinen L, Tiikkainen M, Hakkinen A, Hakkarainen A, Yki-Jarvinen H. E¡ects of insulin therapy on liver fat content and hepatic insulin sensitivity in patients with type 2 diabetes. Am J Physiol Endocrinol Metab. 2006;292:E829 35. 69. Bugianesi E, Gentilcore E, Manini R et al. A randomized controlled trial of metformin versus vitamin E or prescriptive diet in nonalcoholic fatty liver disease. Am J Gastroenterol. 2005;100:1082 90. 70. Galli A, Crabb DW, Ceni E et al. Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro. Gastroenterology. 2002;122:1924 40. 71. Belfort R, Harrison SA, Brown K et al. A placebo controlled trial of pioglitazone in subjects with non-alcoholic steatohepatitis. N Engl J Med. 2006;355:2297 307. 72. Singh S, Loke Y, Furberg C. Long-term risk of cardiovascular events with rosiglitazone. A meta-analysis. J Am Med Assoc. 2007;298:1189 95. 73. Linco¡ M, Wolski K, Nicholls S, Nissen S. Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus. J Am Med Assoc. 2007;298:1180 8. 74. Lutchman G, Modi A, Kleiner D et al. The e¡ects of discontinuing pioglitazone in patients with nonalcoholic steatohepatitis. Hepatology. 2007;46:424 9. 75. Ip E, Farrell G, Hall P, Robertson G, Leclercq I. Administration of the potent PPAR alpha agonist, Wy-14,643, reverses nutritional ¢brosis and steatohepatitis in mice. Hepatology. 2004;39:1286 96. 76. Laurin J, Lindor K, Crippin J et al. Ursodeoxycholic acid or clo¢brate in the treatment of nonalcoholic induced steatohepatitis: a pilot study. Hepatology. 1996;23:1464 7. 77. Browning J. Statins and hepatic steatosis: perspectives from the Dallas Heart Study. Hepatology. 2006;44:466 71. 78. Hirose A, Ono M, Saibara T et al. Angiotensin II type 1 receptor blocker inhibits ¢brosis in rat nonalcoholic steatohepatitis. Hepatology. 2007;45:1375 81. 79. Yokohama S, Yoneda M, Haneda M et al. Therapeutic e¤cacy of an angiotensinogen II receptor antagonist in patients with nonalcoholic steatohepatitis. Hepatology. 2004;40:1222 5. 80. Oben J, Roskams T, Yang S et al. Norepinephrine induces hepatic ¢brosis in leptin de¢cient ob/ob mice. Biochem Biophys Res Commun. 2003;308:284 92. 81. Ichikawa Y. Comparative e¡ects of telmisartan and valsartan on insulin resistance in hypertensive patients with metabolic syndrome. Intern Med. 2007;46:1331 6. 82. Merat S, Malekzadeh R, Sohrabi M et al. Probucol in the treatment of nonalcoholic steatohepatitis: a double blind randomized controlled study. J Hepatol. 2003;38:414 18. 83. Abdelmalek M, Angulo P, Jorgensen R, Sylvestre P, Lindor K. Betaine, a promising new agent for patients with nonalcoholic steatohepatitis: results of a pilot study. Am J Gastroenterol. 2001;96:2711 17. 84. Facchini F, Hua N, Stoohs R. E¡ect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease. Gastroenterology. 2002;122:931 9. 85. Lavine J. Vitamin E treatment of nonalcoholic steatohepatitis in children: a pilot study. J Pediatr. 2000;136:734 8. 86. Harrison SA, Torgerson S, Hayashi P, Ward J, Schenker S. Vitamin E and Vitamin C treatment improves ¢brosis in patients with nonalcoholic steatohepatitis. Am J Gastroenterol. 2003;98:2485 90.
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NASH: METABOLIC SYNDROME OF THE LIVER 87. Adams LA, Zein C, Angulo P, Lindor K. A pilot trial of pentoxyfylline in nonalcoholic steatohepatitis. Am J Gastroenterol. 2004;99:2365 8. 88. Satapathy S, Sakhuja P, Malhotra V, Sharma B, Sarin S. Bene¢cial e¡ects of pentoxifylline on hepatic steatosis, ¢brosis and necroin£ammation in patients with non alcoholic steatohepatitis. J Gastroenterol Hepatol. 2007;22:634 8. 89. Ozcan U, Yilmaz E, Ozcan L et al. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science. 2006;313:1137 40. 90. Lindor K, Kowdley K, Heathcote E et al. Ursodeoxycholic acid for treatment of non alcoholic steatohepatitis: results of a randomized trial. Hepatology. 2004;39:770 8. 91. Dufour J, Oneta C, Gonvers J et al. Swiss Association for the Study of the Liver: randomized placebo controlled trial of ursodeoxycholic acid with vitamin E in nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol. 2006;4:1537 43. 92. Contos MJ, Cales W, Sterling RK et al. Development of nonalcoholic fatty liver disease after orthotopic liver transplantation for cryptogenic cirrhosis. Liver Transplant. 2001;7:363 73. 93. Ong J, Younossi Z, Reddy V et al. Cryptogenic cirrhosis and post-transplantation nonalcoholic fatty liver disease. Liver Transplant. 2001;7:797 801.
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18 Autoimmune hepatitis S. KANZLER
INTRODUCTION Autoimmune hepatitis (AIH) is a necroin£ammatory liver disease of unknown aetiology that occurs in children and adults of all ages (for review see ref. 1). Consensus about its de¢nition and diagnostic requirements have been formulated2. Variant, overlapping, or mixed forms of AIH that share features with other putative autoimmune liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis, also occur. The distinctions among these disorders at present are mostly descriptive3.
IMMUNOPATHOGENESIS Although the pathomechanism of the disease is still unknown, an underlying genetic predisposition has been suggested due to the fact that patients are predominantly of female gender (the women to men ratio is approximately 6:1) and the association of the disease with certain human leucocyte antigens (HLA). HLA genes reside in the major histocompatibility complex (MHC), located on the short arm of chromosome 6. The MHC is a genetic system with extensive polymorphism. Although multiple genes are probably involved, HLA genes appear to play the dominant role in a predisposition to AIH 4 . Particularly HLA B8, DR3; DR4 are found with a signi¢cantly higher frequency in di¡erent populations5,6. The challenge is to investigate whether these ¢ndings may help us to better understand the aetiology of AIH, to predict the prognosis of the disease, or to further improve therapeutic concepts.
DIAGNOSIS The presentation of AIH is very heterogeneous, and may be characterized by an undulating course with periods of decreased or increased activity; thus clinical manifestations are variable, ranging from asymptomatic disease to severe icteric hepatitis. Even fulminant hepatic failure may occur depending on the intensity of the autoimmune reaction (Figure 1). 186
AUTOIMMUNE HEPATITIS
Figure 1 Intensity of autoimmune response and variant clinical presentations of autoimmune hepatitis
Patients may present with non-speci¢c symptoms of varying severity, such as fatigue, lethargy, malaise, anorexia, nausea, abdominal pain, and itching. Arthralgia involving small joints is common. Physical examination may reveal no abnormalities, but it may also reveal hepatomegaly, splenomegaly, jaundice, and signs and symptoms of chronic liver disease7. Many patients with an acute presentation have histological evidence of chronic disease on liver biopsy, indicating that they probably have had subclinical disease for a long time (acute on chronic disease). Long periods of subclinical disease may also occur after presentation. In addition, diseases with an autoimmune background, such as Hashimoto thyroiditis, ulcerative colitis, type 1 diabetes, rheumatoid arthritis, and coeliac disease, are more frequently found in patients with AIH8. In general, hepatitis with elevation of aminotransferase elevations (AST, ALT) leads to the diagnosis of AIH. Viral and toxic hepatitis in particular must be excluded. Some cases, however, are characterized by cholestasis, with high levels of conjugated bilirubin and alkaline phosphatase. In such circumstances extrahepatic obstruction and cholestatic forms of viral hepatitis, drug-induced disease, primary biliary cirrhosis, primary sclerosing cholangitis, and variant syndromes must be considered. One characteristic laboratory feature of AIH is elevation of serum globulins, in particular, gamma globulin, with a selective IgG elevation, which are generally 1.2 3.0 times normal. The characteristic circulating autoantibodies 187
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
seen in AIH include antinuclear antibodies (ANA), smooth-muscle antibody (SMA), soluble liver antigen/liver pancreas autoantibodies (SLA/LP), and liver kidney microsome autoantibodies (LKM). In addition, pANCA and anti-LC-1 are frequently en countered in patients with AIH. Antimitochondrial antibodies (AMA) are sometimes present in patients with AIH. In these patients an overlap syndrome of AIH and primary biliary cirrhosis should be considered 9 . However, it should be noted that autoantibodies are found in various liver diseases, and their presence, by itself, is not diagnostic of AIH. With respect to the pathogenesis of AIH, there is little evidence that autoantibodies play a crucial role. Since there is no single test proving the diagnosis of AIH (an exception could be anti-SLA/LP autoantibodies) liver histology remains of central importance. Since percutaneous liver biopsy is frequently a¥icted with a high sampling error with respect to the staging of ¢brosis and cirrhosis10 we frequently perform (mini)-laparoscopic guided liver biopsy in AIH patients, particularly at initial diagnosis. The histological appearance of AIH is the same as that of chronic hepatitis of other aetiology and, although certain changes are characteristic, no ¢ndings are speci¢c for AIH11. AIH is generally characterized by a mononuclear-cell in¢ltrate invading the limiting plate (periportal in¢ltrate, also called piecemeal necrosis or interface hepatitis that progresses to lobular hepatitis). There may be an abundance of plasma cells, a ¢nding that in the past led to the use of the term `plasma-cell hepatitis'. Eosinophils are frequently present. The portal lesion generally spares the biliary tree. Fibrosis is present in all but the mildest forms of AIH. In advanced disease the ¢brosis is extensive and, with the distortion of the hepatic lobule and the appearance of regenerative nodules, this results in cirrhosis11,12.
TREATMENT If untreated, severe AIH has a very high mortality rate of up to 50% after 3 5 years of diagnosis13. Immunosuppressive therapy with corticosteroids, usually in combination with azathioprine, is considered the gold standard to induce and maintain remission, and at the same time response to immunosuppressive therapy con¢rms the diagnosis of AIH14. The therapeutic goal should be complete normalization of transaminases since progression to liver cirrhosis may occur in patients with remaining in£ammatory activity within the liver. On the other hand side-e¡ects of therapy should be acceptable. The magnitude of aminotransferase and gamma globulin elevations does not necessarily correlate with the histological extent of injury, and provides little help with respect to the initiation of treatment. Under immunosuppression the vast majority of patients achieve complete remission15. In patients who do not respond su¤ciently to immunosuppressive therapy the diagnosis of AIH should be re-evaluated. In some patients an overlap syndrome of AIH with primary biliary cirrhosis or primary sclerosing cholangitis can be a reason for insu¤cient response to immunosuppression. 188
AUTOIMMUNE HEPATITIS
Although some patients remain in remission after drug treatment is withdrawn, most require long-term maintenance therapy. Even though there is only scarce evidence for how long maintenance therapy should be given, it has been proposed that patients should be in stable remission for at least 4 years before withdrawal of immunosuppressive therapy is considered 15 . Since biochemical response and a clinical remission do not necessarily mean that there is histological evidence of resolution of AIH, repeated liver biopsy should be performed, particularly if withdrawal of immunosuppressive therapy is planned. In the very few patients who do not tolerate, or have signi¢cant side-e¡ects to, standard therapy alternative immunosuppressive therapies have been proposed, mainly on the basis of small series or case reports. Cyclosporine appeared e¡ective in a group of adult patients who were corticosteroid-resistant16. A regimen of cyclosporine for 6 months, followed by the administration of prednisone and azathioprine, was reported as successful in inducing remission in children17. Limited data are available concerning the use of tacrolimus 18, methotrexate19, cyclophosphamide20, ursodiol 2 1 , and mycophenolate mofetil 2 2 . How far budenoside, a corticosteroid with a very high ¢rst-pass e¡ect in the liver, can replace conventional corticosteroids is an open question. A small trial in patients without liver cirrhosis has shown promising results23. The ¢nal data from a large international study on the value of budenoside in patients with AIH is awaited. Although AIH runs a very chronic course with relapses under therapy (if immunosuppression is rapidly reduced), and particularly after discontinuation of immunosuppressive therapy, long-term prognosis is excellent provided there is close medical surveillance and/or treatment 24 . For some reason the development of hepatocellular carcinoma is a very rare complication in patients with AIH, even though many patients have established liver cirrhosis at the time of diagnosis, and although patients are immunosuppressed. This fact might give further insight into the pathogenesis of liver carcinogenesis.
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Krawitt EL. Autoimmune hepatitis. N Engl J Med. 2006;354:54 66. Alvarez F, Berg PA, Bianchi L et al.; International Autoimmune Hepatitis Group Report: Review of criteria for the diagnosis of autoimmune hepatitis. J Hepatol. 1999;31:929 38. Czaja AJ. Variant forms of autoimmune hepatitis. Curr Gastroenterol Rep. 1999;1:63 70. Donaldson PT. Genetics in autoimmune hepatitis. Semin Liver Dis. 2002;22:353 64. Teufel A, Worns M, Weinmann A et al. Genetic association of autoimmune hepatitis and human leucocyte antigen in German patients. World J Gastroenterol. 2006;12:5513 16. Muratori P, Czaja AJ, Muratori L et al. Genetic distinction between autoimmune hepatitis in Italy and North Amercia. World J Gastroenterol. 2005;11:1862 6. Kessler WR, Cummings OW, Eckert G, Chalasani N, Lumeng L, Kwo PY. Fulminant hepatic failure as the initial presentation of acute autoimmune hepatitis. Clin Gastroenterol Hepatol. 2004;2:625 31. Obermayer Straub P, Perheentupa J, Braun S et al. Hepatic autoantigens in patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. Gastroenterology. 2001;121:668 77. Lohse AW, zum Buschenfelde KH, Franz B, Kanzler S, Gerken G, Dienes HP. Characterization of the overlap syndrome of primary biliary cirrhosis (PBC) and
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10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
autoimmune hepatitis: evidence for it being a hepatitic form of PBC in genetically susceptible individuals. Hepatology. 1999;29:1078 84. Denzer U, Arnoldy A, Kanzler S, Galle PR, Dienes HP, Lohse AW. Prospective randomized comparison of minilaparoscopy and percutaneous liver biopsy: dignosis of cirrhosis and complications. J Clin Gastroenterol. 2007;41:103 10. Batts KP, Ludwig J. Histopathology of autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis. In: Krawitt EL, Wiesner RH, Nishioka M, editors. Autoimmune Liver Diseases, 2nd edn. Amsterdam: Elsevier, 1998:115 40. Pratt DS, Fawaz KA, Rabson A, Dellelis R, Kaplan MM. A novel histological lesion in glucocorticoid responsive chronic hepatitis. Gastroenterology. 1997;113:664 8. Soloway RD, Summerskill WH, Baggenstoss AH et al. Clinical, biochemical, and histological remission of severe chronic active liver disease: a controlled study of treatments and early prognosis. Gastroenterology. 1972;63:820 33. Johnson P, McFarlane IG, Williams R. Azathioprine for long term maintenance of remission in autoimmune hepatitis. N Engl J Med. 1995;333:958 63. Kanzler S, Gerken G, Lohr H, Galle PR, Meyer zum Buschenfelde KH, Lohse AW. Duration of immunosuppressive therapy in autoimmune hepatitis. J Hepatol. 2001;34:354 5. Fernandes NF, Redeker AG, Vierling JM, Villamil FG, Fong TL. Cyclosporine therapy in patients with steroid resistant autoimmune hepatitis. Am J Gastroenterol. 1999;94:241 8. Alvarez F, Ciocca M, Canero Velasco C et al. Short term cyclosporine induces a remission of autoimmune hepatitis in children. J Hepatol. 1999;30:222 7. Van Thiel DH, Wright H, Carroll P et al. Tacrolimus: a potential new treatment for autoimmune chronic active hepatitis: results of an open label preliminary trial. Am J Gastroenterol. 1995;90:771 6. Burak KW, Urbanski SJ, Swain MG. Successful treatment of refractory type 1 autoimmune hepatitis with methotrexate. J Hepatol. 1998;29:990 3. Kanzler S, Gerken G, Dienes HP, Meyer zum Buschenfelde KH, Lohse AW. Cyclophosphamide as alternative immunosuppressive therapy for autoimmune hepatitis report of three cases. Z Gastroenterol. 1997;35:571 8. Czaja AJ, Carpenter HA, Lindor KD. Ursodeoxycholic acid as adjunctive therapy for problematic type 1 autoimmune hepatitis: a randomized placebo controlled treatment trial. Hepatology. 1999;30:1381 6. Richardson PD, James PD, Ryder SD. Mycophenolate mofetil for maintenance of remission in autoimmune hepatitis in patients resistant to or intolerant of azathioprine. J Hepatol. 2000;33:371 5. Wiegand J, Schuler A, Kanzler S et al. Budenoside in previously untreated autoimmune hepatitis. Liver Int. 2005;25:927 34. Kanzler S, Lohr H, Gerken G, Galle PR, Lohse AW. Long term management and prognosis of autoimmune hepatitis (AIH): a single center experience. Z Gastroenterol. 2001;39:344 8.
190
19 Immunology of primary biliary cirrhosis and primary sclerosing cholangitis A. LLEO, C. SELMI, P. INVERNIZZI, M. PODDA and M. E. GERSHWIN
INTRODUCTION Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown aetiology characterized by high titre serum antimitochondrial autoantibodies (AMA), a striking female predominance, and an autoimmunemediated destruction of the small and medium-size intrahepatic bile ducts1 . PBC is a peculiar, yet representative, organ-speci¢c autoimmune disease. The presence of serum AMA and autoreactive T and B cells, in conjunction with the co-occurrence of other autoimmune diseases, implies an autoimmune pathogenesis for PBC. A complex genetic background is suggested by the incidence of the disease among ¢rst-degree relatives2, the high concordance rate among twins3, and the proposed role for sex chromosome defects4. To summarize, PBC onset recognizes two necessary components: i.e. a permissive genetic background and an environmental trigger. Primary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic in£ammation and obliterative ¢brosis of the intrahepatic and/or extrahepatic biliary tree, which leads to cholestasis, hepatic ¢brosis and, ultimately, cirrhosis. Its natural history is complicated by the predisposition to the development of cholangiocarcinoma. The aetiopathogenesis of PSC is unknown, although there is growing evidence that (auto)immune-mediated mechanisms may play a role. Lymphocytes derived from the in£amed gut appear to enter the liver via the enterohepatic circulation and determine an abnormal immune response in genetically susceptible individuals. PBC and PSC should be considered as unique diseases within the range of autoimmunity; herein we will summarize the critical immunological aspects that characterize these conditions.
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IMMUNOLOGY OF PBC B cells and autoantibodies Serum antimitochondrial antibodies (AMA) are highly speci¢c for PBC and can be detected in nearly 100% of patients, when sensitive diagnostic methodologies based on recombinant antigens are used5. They are directed against members of the 2-oxoacid dehydrogenase complexes (2-OADC) existing in the inner membrane of mitochondria. Among them, the major autoantigen is the E2 subunit of the pyruvate dehydrogenase complex (PDCE2). The epitopes for this antibody localize to three domains of the PDC-E2 component: (a) the inner/outer lipoyl domain; (b) the E3 binding domain; and (c) the catalytic and E2-binding domain6. Crossreactivity is also found against other 2-OADC, such as 2-oxo glutarate dehydrogenase (OGDC-E2) and branched-chain 2-oxo acid dehydrogenase (BCOADC-E2), involved respectively in the citric acid cycle and in amino acid catabolism. The principal autoantigens in PBC are summarized in Table 1. Anti-PDC-E2 antibodies are capable of inhibiting PDC-E2 enzyme activity. Targeting of enzymes is a common feature of autoantibodies detected in patients with autoimmune diseases, as is the inhibition of their activity by these autoantibodies. The role of lipoic acid in the epitope recognition by AMA is unclear. The pathogenic role of AMA remains debated since no clinical correlation can be found and animal models developing serum AMA do not manifest PBC-like liver lesions. In addition to AMA, PBC sera can present other disease-speci¢c autoantibodies, particularly antinuclear ones (ANA). ANA react against nuclear pore glycoproteins of the inner nuclear membrane, gp210 and p62, with a detection rate up to 50% and high prevalence among AMA-negative PBC7. Other epitopes within the inner nuclear membrane include Sp100, another nucleoprotein, and lamin B receptor; both appear speci¢c for PBC, but the prevalence is variable, being about 30% for the former and less than 1% for the latter. ANA have been shown to correlate with disease severity and progression8; analysing the overall prevalence of p62 and gp210, a higher prevalence was identi¢ed in AMA-negative subjects; Table 1
Mitochondrial and nuclear autoantigens in PBC
Mitochondrial antigens 2 OADC subunits Pyruvate dehydrogenase complex Nuclear antigens Nuclear pore complex Multiple nuclear dots
PDC E2 OGDC E2 BCOADC E2 E3BP PDC E1a gp210 nucleoporin 62 Sp100 PML
192
IMMUNOLOGY OF PBC AND PSC
secondarily, the subjects found positive for ANA show a more severe and aggressive form of the condition characterized by worse clinical and biochemical parameters when analysed in both cross-sectional and longitudinal fashions.
T cells Both CD4+ and CD8+ T cells have been demonstrated to be involved in the pathogenesis of PBC and, histologically, in¢ltration of autoreactive T cells in the liver and periductular spaces is one of the most prominent immune features. Both CD4+ and CD8+ T lymphocytes can be isolated from PBC liver sections and recognize epitopes of PDC-E29; moreover, using recombinant fragments of PDC-E2 it has been demonstrated that there is overlapping in the PDC-E2 speci¢c T (both CD4+ and CD8+) and B cell epitopes10. The minimal T-cell epitope was identi¢ed as amino acid residues 163 176 (GDLLAEIETDKATI), within the inner lipoyl domain of PDC-E2 10 . Phenotypically, the PDC-E2 163^176 T-cell clones were positive for CD4, CD45RO, and T cell receptor (TCR) ab. The human leucocyte antigen (HLA)-restriction molecules for this epitope have been identi¢ed as HLADR53 (B4*0101)11. In addition, PDC-E2163^176-speci¢c CD4+ T-cell clones recognize other mitochondrial autoantigens, including the OGDC-E2, BCOADC-E2, and E3BP12. More speci¢cally, these T-cell clones crossreacted with the amino acid residues 100 113 of OGDC-E2, residues 90 103 of BCOADC-E2, and residues 34 47 of E3BP, all located in the lipoyl domain, suggesting that OGDC-E2100^113, BCOADCE290^103, and E3BP34^47 may also be CD4+ T-cell epitopes. CD8+ T cells (CTL) from peripheral blood of patients with PBC have been studied in the context of HLA-A2.1 restriction and have been found to identify amino-acid residues 159 167 and 165 17410. Speci¢c MHC class I restricted CTL can also be generated by in vitro stimulation with antigen-pulsed dendritic cells13 from blood of patients with PBC, but not from healthy controls, evidencing the presence of speci¢c T-cell clones in the blood of patients. Interestingly, an increase in CTL precursors in the blood in the early stages of the disease compared to the advanced ones and a 10-fold increase of speci¢c liver CTL compared to peripheral blood were detected in the same study, thus supporting the role of these cells and their speci¢c recruitment, in the evolution of the disease. To summarize, all T cell subpopulations recognize overlapping epitopes within the same lipoylated regions, thus supporting the hypotheses of a common trigger mechanism, possibly molecular mimicry. CD4+CD25high natural regulatory T cells (T Treg) warrant further discussion since these have been found to play a role in several autoimmune diseases. In PBC a relative reduction of Tregg compared with controls has been reported with a lower ratio of Treg over CD8+ cells in PBC liver compared to chronic hepatitis C and autoimmune hepatitis14.
193
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY Table 2
Imunopathological characteristics of biliary epithelium in PBC Normal
PBC
Expression level of PDC E2
+
+++
Adhesion molecules ICAM 1 VCAM 1 LFA 1 E selectine
+ /+ /+
++ + + ++
Large bile ducts, few CD4+
Small bile ducts, increased CD4+CD28
Biliary intra epithelial lymphocytes Apoptosis related molecules Fas (CD95) Granzyme B Perforin bcl 2
++
+ /+ /+
BEC phagocytosis of apoptotic BEC
++
Cytokines INF g IL 2 IL 6 IL 6 receptor TNF a TNF receptor
++ ++ ++ /+ ++ ++
/+ /+
Modi¢ed from ref. 61.
The role of cholangiocytes Biliary epithelial cells (BEC) constitute a heterogeneous cell population15. In addition to morphological diversity, small and large cholangiocytes manifest di¡erences in secretory, proliferative and apoptotic responses to liver injury16,17. Table 2 summarizes the immunogenic characteristics of BEC in PBC patients. As mentioned above, PBC is characterized by the destruction of the small and medium-size intrahepatic bile ducts, lined by small cholangiocytes. The identi¢cation of speci¢c characteristics of BEC such as the di¡erential expression of cell adhesion molecules, the response to cytokines and growth factors and their potential ability to act as antigen-presenting cells (APC), may explain the strict organ speci¢city of the immune-mediated injury in PBC. The major immunogenic epitope recognized by both autoantibodies and autoreactive cells in PBC has been identi¢ed in the E2 subunit of pyruvate dehydrogenase (PDC), a molecule located on the inner mitochondrial membrane of all nucleated cells18. One of the most puzzling and critical questions in the pathogenesis of PBC is why, despite the ubiquitous expression of the molecules, the damage is highly localized and commonly involves also the salivary gland epithelial cells. In fact, patients with PBC often have 194
IMMUNOLOGY OF PBC AND PSC
concurrent Sjogren's syndrome19. BEC express cell surface adhesion molecules which lead to the adhesion and recognition by lymphocytes. Moreover, several studies have demonstrated that BEC of both healthy and diseased liver have the capacity to increase the expression of adhesion molecules, ICAM-1, MHC class I and II, tumour necrosis alpha (TNF-a), a interferon gamma (IFN-gg) and interleukin-1 (IL-1)20^22 upon stimulation with proin£ammatory cytokines23 abundant in the chronic in£ammatory milieu. Up-regulation of VCAM-1 and LFA-1 has also been demonstrated24. The other immune peculiarity attributed to BEC is their ability to act as APC, similar to other epithelial cells. In support of this concept, several studies demonstrate that BEC express HLA class II23,25 and such expression is increased in injured cholangiocytes and after stimulation with IFN-g and IL1. BEC have also been shown to express accessory molecules responsible for the secondary co-stimulatory signal to T cells, CD28, B7-1 or B7-226. These interactions with T cells might also be responsible for bile duct loss, one of the fundamental characteristics of PBC progression. Importantly, data obtained in recent years point towards apoptosis as a leading mechanism for ductopenia. Harada and colleagues demonstrated the BEC susceptibility to apoptosis via the perforine/granzyme B pathway, enhanced by CD95/ CD95L in patients with PBC27. The hypothesis is further strengthened as apoptotic BEC express CD40, Fas and FasL, and transcriptional up-regulation of the latter molecules, after stimulation with CD40L, exiting in apoptosis28. In 2001 Odin and colleagues reported that glutathionylation of the lysine lipoic acid moiety of PDC-E2 is dramatically reduced by serum AMA, and that such a mechanism is related to the expression of an anti-apoptotic and anti-oxidant protein, Bcl-229. The cell concentration of this molecule is further correlated with the persistence of immunogenicity of PDC-E2 after apoptosis, pointing to a direct involvement of Bcl-2 in glutathionylation. Interestingly, human and rat BEC express higher levels of Bcl-2 compared to other cells30,31, and should thus contain higher glutathione levels, which would then translate in lower immunogenicity; no explanation has been proposed thus far for this apparent contradiction. Nonetheless, enhanced glutathionylation, linked to the evidence that PDC-E2 is released from apoptotic BEC without caspase cleavage 32 appears fundamental in the recognition of the mitochondrial epitope by the immune system upon cell death. A possible unifying hypothesis has been proposed by Allina and colleagues, who recently demonstrated that apoptotic cells are phagocytosed by BEC and consequently are an exogenous source of autoantigens in BEC33. Importantly, these ¢ndings support the paradigm that tissue-speci¢c damage in PBC is due to cell type-speci¢c di¡erences in apoptosis and phagocytosis of apoptotic cells. Antigenicity of BEC selfmolecules or highly homologous epitopes could also be related to their role in mucosal immunity. Similar to other epithelial cells, BEC actively secrete IgA, which have been demonstrated to be reactive to PDC-E2 in almost all body £uids of patients with PBC, including saliva, urine and bile34,35. Further, Fukushima and colleagues36 detected the co-localization of such antibodies and PDC-E2, or a mimicking molecule, on the apical surface and in the cytoplasm of BEC and their presence in allografts of patients with recurrent 195
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
PBC following liver transplant. To assess the direct pathogenicity of this antibody subclass, Shuji Matsumura and colleagues incubated highly puri¢ed AMA-IgA with canine kidney cells transfected with the human polymeric Ig receptor and induced caspase up-regulation to provide evidence of a direct toxic e¡ect32.
Innate immunity in PBC Innate immunity is considered the ¢rst line of defence against infections, but its importance has been appreciated only recently and its role in autoimmunity remains to be clari¢ed. The cellular components of innate immunity, including dendritic cells (DC), and other professional APC, and natural killer T cells (NK), are known for their regulatory function in detailing the quality and quantity of subsequent adaptive immune responses including antigen-speci¢c antibody and T cell responses. The innate immunity in PBC patients is characterized by an increased response to pathogen-associated stimuli, as indicated by higher levels of proin£ammatory cytokines secreted by monocytes from patients with PBC after exposition to microorganism patterns 37 . Recently, our group has demonstrated that the elevation of serum IgM is secondary to an aberrant innate immune response, potentially induced by stimulation of toll-like receptor (TLR) 9 by bacterial CpG-B38. Given the potential causative role of microorganisms in disease development, this ¢nding should encourage a broader analysis of this branch of the immune system. NK have been linked to autoimmune diabetes and experimental autoimmune encephalomyelitis, the murine model of multiple sclerosis39 and the role of NKT in autoimmunity is attracting growing attention. Recently, Chuang and colleagues demonstrated a marked increase in the frequency and absolute number of blood and liver NK cells in PBC patients. Moreover, in the same study the cytotoxic activity and perforin expression by isolated NK cells were signi¢cantly increased in PBC patients associated with increased levels of plasma IL-8 and the expression of CD128a (IL-8 receptor) on NK cells. In contrast, the levels of IFN-g, IL-6 and IL-8 synthesized by NK cells were signi¢cantly decreased in PBC as compared to controls40. Whereas the innate immune system hyper-responsiveness, in its sole entity, is probably not su¤cient for the breakdown of tolerance, these alterations might ultimately play a role in the initiation and/or perpetuation of the autoimmune adaptive response.
Cytokines Th1 cytokine pro¢les have been reported in serum and liver from patients with PBC 41 and IFN-g, a Th1 cytokine, is transcriptionally up-regulated 42 . Moreover, BEC of patients with PBC overexpress TNF-a and its receptor, which could support the hypothesis of a paracrine e¡ect on the cells, leading to proliferation and potentially apoptosis 43 . Recent ¢ndings suggest the involvement of a cytokine cytokine receptor interaction in the pathogenesis of PBC40. Although T cells are the principal source of cytokines, endothelial cells and macrophages may also play a role in producing the observed cytokine 196
IMMUNOLOGY OF PBC AND PSC
pattern. Furthermore, types of APC, genetic background, costimulator molecules, and types and amounts of antigens may also in£uence the di¡erentiation of Th0 cells into Th1 or Th2 cells.
Immunogenetics of PBC Although the aetiology of PBC remains enigmatic, there are several pieces of data supporting the thesis that genetic predisposition contributes to the onset of PBC. This view is based on the following lines of evidence: (a) the concordance rate of PBC in monozygotic twins is 63%; (b) approximately 6% of patients with PBC have a ¢rst-degree relative who also su¡ers from PBC, and ¢nally, (c) it has suggested a signi¢cant role for X chromosome defects in PBC, based on the observation that women with PBC have a signi¢cantly enhanced monosomy X frequency in peripheral white blood cells compared to agematched healthy women 4. Haplotypes of the major histocompatibility complex loci appear to play a minor or geographically limited role in PBC susceptibility. In summary, a susceptible genetic background is considered to be necessary, yet not su¤cient, to explain PBC onset nor the female predominance, and several environmental factors have been suggested as additional players in tolerance breakdown.
Animal models The development of an animal model would be extremely helpful in elucidating the undoubtedly multifactorial causation and progression of PBC. Three mouse strains have been most recently reported to be spontaneous PBC model animals and will be brie£y described. First, a variant of the non-obese diabetic (NOD) mouse model (NOD.c3c4) has been described as presenting autoimmune cholestasis and PBC-speci¢c serology; it shows AMA positivity of 50 60% and ANA positivity of 80 90%. Histologically it presents lymphocyte in¢ltration around portal tracts with chronic non-suppurative destructive cholangitis and epithelioid granuloma formation; however, the morphological features of the bile ducts di¡er somewhat from those in human PBC44. Second, a mouse with a dominant negative form of transforming growth factor b (TGFb) receptor II (dnTGFbRII) is the second reported model of PBC-like liver disease; it shows 100% AMA positivity against PDC-E2. TGF-b receptor II is essential for signal transduction of TGF-b, which regulates activation of lymphocytes; this model suggests that a speci¢c condition of T cells with impaired TGF-b signalling in the presence or absence of B cells is involved in the pathogenesis of PBC-like disease45. The third PBC animal model is a IL2Ra knockout mouse, with 100% AMA positivity against PDC-E2, 80% ANA positivity, and lymphocyte in¢ltration around the portal tracts accompanied by cholangiocyte injury46.
Pathogenic mechanisms Based on the above-mentioned data on PBC immunology, several theories have been proposed for the pathogenesis of the immune-mediated tissue injury observed in PBC. In all cases such theories should not be regarded as 197
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IMMUNOLOGY OF PBC AND PSC
independent from other aetiological factors leading to PBC susceptibility (i.e. genetic background, environmental triggers), but rather as e¡ector mechanisms leading to the clinical manifestations. Figure 1 presents a possible pathophysiological model of PBC.
Expression of PDC-like antigen The hypothesis for the selective destruction of BEC states that the immunodominant AMA autoantigen, the PDCE2, which is normally located in the mitochondrial inner membrane, could be aberrantly expressed on the cell surface and thus be recognized by speci¢c antibodies. Several scenarios have been hypothesized. First, although in situ hybridization studies of PDC-E2 mRNA showed no signi¢cant di¡erence in the amount of PDC-E2 transcript present in PBC liver compared with other liver diseases, PDC-E2 may be selectively overexpressed in small bile duct BEC. Second, variants of PDC-E2 may cause an abnormal turnover of the molecule, leading to the accumulation of PDC-E2 in these subpopulations of cells. It is possible that toxic substances disposed by the liver may accumulate in the biliary epithelium and potentially modify the PDC-E2 molecule locally, leading to the production of such variants. Third, altered PDC-E2 mRNA could be produced by the abnormal transcription of PDC-E2. For example, it is possible that abnormal splicing during synthesis of PDC-E2 mRNA would substitute an endoplasmic reticulum targeting signal instead of a mitochondrial targeting signal. Thus, PDC-E2 may potentially be delivered into the endoplasmic reticulum and Golgi apparatus via a secretory route to be expressed on the cell surface of biliary ducts, instead of mitochondria. Although direct evidence supporting these mechanisms is currently lacking, it remains possible that the molecules expressed and identi¢ed on the ductular surface of BEC and recognized by anti-PDC-E2 antibodies may not be PDC-E2 itself, but are PDC-E2 mimics that crossreact with human PDC-E2. Some experimental data seem to support this hypothesis.
Role of IgA Another hypothesis that might explain the selective targeting of bile ducts in PBC is that the autoantigen-speci¢c IgA antibody plays a role. If AMA-IgA autoantibodies are responsible for the speci¢c destruction of BEC in PBC, it is possible that this occurs by disrupting cell metabolism of the cells whereby the AMA-IgA bound to the mitochondrial antigen induces cellular dysfunction and hence the tissue speci¢city. Interestingly, IgA from PBC patients colocalized with PDC-E2 inside the cells and on the apical membrane of BEC47. These data support the idea that both the aberrant polar expression of PDC-E2 and the tra¤cking of IgA in BEC are possible mechanisms for selective damage of BEC.
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Molecular mimicry Bacterial infection in various settings has been repeatedly invoked in the aetiopathogenesis of PBC. This aetiology is usually linked to the concept of molecular (epitope) mimicry. The crossreactivity of AMA with prokaryotic antigens has been reported for a number of microbes. This crossreactivity is not particularly surprising given the conserved sequence of PDC-E2 across all species, from eubacteria to mammals. Brie£y, we can summarize our theory on molecular mimicry in PBC as follows. The microorganism (possibly the ubiquitous Novosphingobium aromaticivorans48) enters the human system through the digestive mucosa. Bacterial mimics containing lipoic acid residues at this point might be modi¢ed by xenobiotics to form immunoreactive adducts. This modi¢cation would then be su¤cient to trigger the innate immune system to initiate a cascade of local in£ammatory events, via TLR for example, thus resulting in local dendritic cell activation and antigen processing. Mucosal APC in turn activate autoreactive T and B cells that are directed to the liver through the portal system. T cells participate directly in the autoimmune injury and/or further recruit autoreactive lymphocytes. B cells, on the other hand, secrete AMA, particularly of the IgA type. AMA IgA are then transported to the vascular side of the bile duct cell where they react with the PDC-E2-like molecules located on the luminal surface cell membrane. This binding then initiates the apoptotic signalling cascade. Ultimately, the immune complexes of post-apoptotic PDC-E2 and IgG-AMA and the direct cytopathic e¡ects of autoreactive T cells (and possibly AMA) contribute to the tissue injury observed in PBC. However, recent studies provide an alternative explanation, since microbial CpG enhances IgM production in peripheral blood mononuclear cell cultures and CD27+ memory B cells in PBC patients are responsible for this IgM production through TLR 9 signalling. CpG can also stimulate AMA production and expression of TLR9, CD86, and one of the potassium channels, KCa3.1, in B cells of PBC patients. Up-regulated expression of TLR9 and CD86 and AMA secretion induced by CpG are suppressed by a speci¢c blocker of the KCa3.1 channel, namely TRAM. These data indicate that B-cell immunity of PBC patients is a¡ected by an enhanced innate immunity response, and imply that TRAM-34 may manipulate B-cell autoimmunity in PBC38.
Tregs Several studies have demonstrated that the transfer of T cells lacking the Treg subset into athymic nude mice results in the development of various T cellmediated autoimmune diseases. Experimental data demonstrate that PBC patients displayed signi¢cantly lower frequencies of Tregs as percentages of total TCR-ab+/CD4+ T cells, which may contribute to the breakdown in tolerance in PBC14.
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IMMUNOLOGY OF PSC Despite the solid association with in£ammatory bowel diseases (IBD, particularly ulcerative colitis), the presence of serum autoantibodies, and the reported HLA susceptibility suggesting an autoimmune mechanism, the aetiopathogenesis of PSC remains unclear. It has been debated whether PSC is an atypical autoimmune disease or an immune-mediated in£ammatory disease (somehow similar to IBD). In fact, the autoimmune hypothesis fails to explain a number of important di¡erences between PSC and classical autoimmune diseases. The features of PSC that di¡er from those of a classical autoimmune disease include the predominance of males, the absence of disease-speci¢c autoantibodies, and its poor response to immunosuppressive medications. PSC recognizes individual susceptibility, as indicated by a number of association studies on HLA haplotypes.
Serum autoantibodies in PSC It is known that various autoantibodies can be simultaneously detected in patients with PSC, but there is growing evidence that only anti-neutrophil cytoplasm antibodies (ANCA) are diagnostically meaningful, even if their pathogenetic role in PSC remains unclear. There are two well-established IIF patterns of ANCA: di¡use cytoplasmic £uorescence accentuated between the nuclear lobes (c-ANCA) or homogeneous £uorescence of the perinuclear cytoplasm (p-ANCA). Between 33% and 88% of PSC patients has been reported to present with p-ANCA49. Classical p-ANCA are clearly di¡erent from so-called atypical p-ANCA, which are almost exclusively found in patients with IBD, PSC or AIH50. Atypical p-ANCA react with the nuclear envelope rather than cytoplasmic antigens, which has led to the introduction of the more accurate term, peripheral antineutrophil nuclear antibodies (pANNA)51. The antigens recognized by p-ANNA appear to be represented by the 50-kDa myeloid-speci¢c nuclear envelope protein52. Other targets of A NCA i n P S C i n clu d e h- l a mp - 2 , p rote i n a s e- 3 an d o n ly rarely myeloperoxidase. Additionally, a high frequency of autoantibodies against human BEC has been reported in PSC sera53, and the binding of these antiBEC-Ab determines ERK1/2 signalling and up-regulation of TLR. This latter induces BEC to produce cytokines/chemokines and, consequently, determines the recruitment of in£ammatory cells 5 4 . The multiple non-speci¢c autoantibodies observed in PSC are most likely epiphenomena related to chronic in£ammation and immunogenetics favouring vigorous immune responses. ANA can be detected in a substantial portion of PSC patients (53%), and SMA in 13 20%, whereas AMA are absent. Almost two-thirds of PSC patients have anti-cardiolipin antibodies. Recent evidence supports the hypothesis that BEC are a target of autoantibodies: in one study 63% of the patients had antibodies to BEC, which induced the expression and production of CD44 and IL-653.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY Table 3 Immunomodulatory roles of chemokines and cytokines produced by biliary epithelial cells in PSC after activation Function Chemokines CCL25 CCL28 VCAM 1 Cytokines INF g TNF a TGF b IL 6
References
Ligand for CCR9+ gut primed T cells T cells peribiliary recruitment Ligand for the T cell integrin receptor a1b4, express on BEC surface Proin£ammatory paracrine e¡ect Expression of IL 8 (CXCL8) induction Pro¢brotic e¡ect Proliferation of autoreactive T cells and Ig production
60 60 60
54 54, 62 54, 63 54
Cellular immune response in PSC Recent studies suggest that the lymphocytic portal tract in¢ltration in PSC contains T cells primed by antigens in the gut-associated lymphoid tissues (GALT). The presence of subclinical, microscopic colitis or clinically active IBD would increase the quantities and diversities of such recirculating gutprimed memory T cells, recirculating memory T cells primed in the GALT during episodes of viral or bacterial enteritis would also be present in individuals without IBD. Grant and collegues suggested that mucosal lymphocytes can home to both the gut and the liver because of overlapping expression of addressins between these organs55, and they showed that liverin¢ltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-speci¢c chemokine CCL2556. Moreover, BEC are not only passive targets, they play an active role, and it is now clear that they respond to PAMP with a proin£ammatory pattern with secretion of multiple chemokines, cytokines (such as TNF-a, IL-1b, IL-6, and IFN-g) g and growth factors54. This BEC activation seems to be determinant in T-cell chemotaxis and ¢brogenesis stimulation, and it might be maintained by a cytokine autocrine paracrine signalling (Table 3). Studies of circulating lymphocyte subsets in PSC have produced rather con£icting results, although there does seem to be some consensus on the ¢nding that there is a fall in CD8+ T cells as the disease progresses. The fact that this change occurs only in advanced disease, however, means that it is unlikely to be signi¢cant in the pathogenesis of the disease
Immunogenetics Susceptibility to PSC is acquired through inheriting one of a number of patterns of genetic polymorphisms which together cause a predisposition to development of the disease. Presently, the main contribution to genetic susceptibility in PSC is believed to come from genes in the human leukocyte antigen (HLA) complex. Association with HLA class I and II genes in PSC has been shown in repeated studies. Two major risk haplotypes have been 202
IMMUNOLOGY OF PBC AND PSC
identi¢ed: the HLA-DRB1*0301-DQA1*0501-DQB1*0201 and the HLADRB1*1301-DQA1*0103-DQB1*0603 haplotypes. Associations with the class I HLA-A*01, -B*08 and -Cw*0701 alleles have also been reported. Because of both linkage disequilibrium and polygenetic in£uence, the picture is complex and the primary susceptibility loci on these PSC-associated HLA haplotypes have yet to be identi¢ed57. Overall there is no particular HLA association with disease progression in PSC. None of the reports of MHC genes in PSC is able to account for all of the genetic risk and, similar to other autoimmune diseases, it is likely that immunoregulatory genes elsewhere in the human genome contribute to both disease susceptibility and progression. The recent focus in the genetics of autoimmune disease has been on cytokine genes. There are associations with cytotoxic lymphocyte antigen-4 (CTLA4), one of the di¡erentiation antigens exclusively expressed on activated CD4+ and CD8+ T cells, and there is evidence of polymorphism in the genes that regulate collagen synthesis and ¢brosis such as MMP3 or stromelysin, but these have yet to be widely con¢rmed. In contrast none of the IL-1, IL-10, TGFB1, CCR5 or TNFRSF6 genes appears to be associated with PSC58. Recently, it has also been suggested that a functional gene variant of SXR, a ligand-dependent transcription factor that mediates protection against bile acid-induced liver injury in cholestatic animal models, modi¢es disease course in PSC59.
Pathogenic mechanisms O'Mahony and Vierling have proposed a multistep model to explain the aetiopathogenesis of PSC60 in which the disease development requires the concurrent presence of: (a) HLA and non-HLA alleles that favour production of high, sustained levels of proin£ammatory cytokines, and (b) recirculating, gut-primed memory T cells. The ¢rst step in the PSC aetiopathogenesis would be the cytokine induction of an aberrant hepatic endothelial cell expression of adhesion molecules. Thus, the second step would be concurrent secretion of the gut-speci¢c chemokine CCL25 by portal dendritic cells or activated macrophages, leading to establishment of a chemokine gradient, uptake by endothelial cells and expression on the luminal surface of blood vessels. Binding of gut-primed memory T cells expressing speci¢c receptors would undergo transendothelial migration and chemoattraction along the CCL25 tissue gradient. However, in the absence of additional signals supplied by cholangiocytes, the gut-primed T cells would transiently secrete Th1 cytokines. Further, as a third step, there would be the activation of cholangiocyte gene expression by proin£ammatory cytokines (IL-1b, TNF-a, IL-6, IFN-g) g and bacterial PAMP (LPS and possibly lipoteichoic acid, peptidoglycans, and bacterial GpC). These stimuli determine a cholangiocyte phenotype responsible for chemoattraction, functional activation of gut-primed T cells to the peribiliary space and ¢brous obliterative cholangitis.
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CONCLUSIONS AND FUTURE PROSPECTIVES As indicated by the available evidence, several aspects of in£ammatory biliary diseases remain enigmatic. PBC and PSC should both be considered, in di¡erent ways, as unique diseases within the broad range of autoimmunity. Yet it appears that PBC and PSC share limited features and should be considered as separate diseases. The recent advancement in the de¢nition of potential PBC triggers has shed some light on the aetiology of the disease, but much remains to be done to explain how these mechanisms ultimately lead to cell damage. The genetic predisposition also remains to be well elucidated, alongside other mysterious features of PBC such as the susceptibility to infections, the aberrant humoral and cellular reactivity, the female predisposition and family clustering. It is crucial to determine the pathogenic role of AMA in the bile duct damage of PBC. Currently, the real possibility of an animal model appears to be the only way to provide a clear demonstration of such a pathogenic mechanism. The scenario is quite di¡erent in PSC, where research e¡orts are limited by the rare occurrence of the disease that militates against the performance of large studies. A common ¢eld in need of elucidation is the innate immune response in both PBC and PSC and the role of BEC in the initiation and perpetuation of the peribiliary in£ammatory process.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13.
Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med. 2005;353:1261 73. Gershwin ME, Selmi C, Worman HJ et al. Risk factors and comorbidities in primary biliary cirrhosis: a controlled interview based study of 1032 patients. Hepatology (Baltimore, MD). 2005;42:1194 202. Selmi C, Mayo MJ, Bach N et al. Primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment. Gastroenterology. 2004;127:485 92. Invernizzi P, Miozzo M, Battezzati PM et al. Frequency of monosomy X in women with primary biliary cirrhosis. Lancet. 2004;363:533 5. Oertelt S, Rieger R, Selmi C et al. A sensitive bead assay for antimitochondrial antibodies: chipping away at AMA negative primary biliary cirrhosis. Hepatology. 2007;45:659 65. Van de Water J, Gershwin ME, Leung P, Ansari A, Coppel RL. The autoepitope of the 74 kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med. 1988;167:1791 9. Worman HJ, Courvalin JC. Antinuclear antibodies speci¢c for primary biliary cirrhosis. Autoimmun Rev. 2003;2:211 17. Wesierska Gadek J, Penner E, Battezzati PM et al. Correlation of initial autoantibody pro¢le and clinical outcome in primary biliary cirrhosis. Hepatology. 2006;43:1135 44. Van de Water J, Ansari A, Prindiville T et al. Heterogeneity of autoreactive T cell clones speci¢c for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis. J Exp Med. 1995;181:723 33. Shimoda S, Nakamura M, Ishibashi H, Hayashida K, Niho Y. HLA DRB4 0101 restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases. J Exp Med. 1995;181:1835 45. Shimoda S, Van de Water J, Ansari A et al. Identi¢cation and precursor frequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis. J Clin Invest. 1998;102:1831 40. Shimoda S, Nakamura M, Ishibashi H et al. Molecular mimicry of mitochondrial and nuclear autoantigens in primary biliary cirrhosis. Gastroenterology. 2003;124:1915 25. Kita H, Lian ZX, Van de Water J et al. Identi¢cation of HLA A2 restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross presented by dendritic cells. J Exp Med. 2002;195:113 23.
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IMMUNOLOGY OF PBC AND PSC 14. Lan RY, Cheng C, Lian ZX et al. Liver targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis. Hepatology. 2006;43:729 37. 15. Katayanagi K, Van de Water J, Kenny T et al. Generation of monoclonal antibodies to murine bile duct epithelial cells: identi¢cation of annexin V as a new marker of small intrahepatic bile ducts. Hepatology. 1999;29:1019 25. 16. Kanno N, LeSage G, Glaser S, Alvaro D, Alpini G. Functional heterogeneity of the intrahepatic biliary epithelium. Hepatology. 2000;31:555 61. 17. Alpini G, Glaser S, Robertson W et al. Large but not small intrahepatic bile ducts are involved in secretin regulated ductal bile secretion. Am J Physiol. 1997;272:G1064 74. 18. Gershwin ME, Mackay IR, Sturgess A, Coppel RL. Identi¢cation and speci¢city of a cDNA encoding the 70 kd mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol. 1987;138:3525 31. 19. Ito M, Kojima T, Miyata M et al. Primary biliary cirrhosis (PBC) CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia) overlap syndrome complicated by Sjogren's syndrome and arthritis. Intern Med. 1995;34:451 4. 20. Leon MP, Bassendine MF, Wilson JL, Ali S, Thick M, Kirby JA. Immunogenicity of biliary epithelium: investigation of antigen presentation to CD4+ T cells. Hepatology. 1996;24:561 7. 21. Wu CT, Davis PA, Luketic VA, Gershwin ME. A review of the physiological and immunological functions of biliary epithelial cells: targets for primary biliary cirrhosis, primary sclerosing cholangitis and drug induced ductopenias. Clin Dev Immunol. 2004;11:205 13. 22. Scholz M, Cinatl J, Blaheta RA, Kornhuber B, Markus BH, Doerr HW. Expression of human leukocyte antigens class I and class II on cultured biliary epithelial cells after cytomegalovirus infection. Tissue Antigens. 1997;49:640 3. 23. Ayres RC, Neuberger JM, Shaw J, Joplin R, Adams DH. Intercellular adhesion molecule 1 and MHC antigens on human intrahepatic bile duct cells: e¡ect of pro in£ammatory cytokines. Gut. 1993;34:1245 9. 24. Yasoshima M, Nakanuma Y, Tsuneyama K, Van de Water J, Gershwin ME. Immunohistochemical analysis of adhesion molecules in the micro environment of portal tracts in relation to aberrant expression of PDC E2 and HLA DR on the bile ducts in primary biliary cirrhosis. J Pathol. 1995;175:319 25. 25. Saidman SL, Duquesnoy RJ, Zeevi A, Fung JJ, Starzl TE, Demetris AJ. Recognition of major histocompatibility complex antigens on cultured human biliary epithelial cells by alloreactive lymphocytes. Hepatology. 1991;13:239 46. 26. Tsuneyama K, Harada K, Yasoshima M, Kaji K, Gershwin ME, Nakanuma Y. Expression of co stimulatory factor B7 2 on the intrahepatic bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis: an immunohistochemical study. J Pathol. 1998;186:126 30. 27. Harada K, Ozaki S, Gershwin ME, Nakanuma Y. Enhanced apoptosis relates to bile duct loss in primary biliary cirrhosis. Hepatology. 1997;26:1399 405. 28. A¡ord SC, Ahmed Choudhury J, Randhawa S et al. CD40 activation induced, Fas dependent apoptosis and NF kappaB/AP 1 signaling in human intrahepatic biliary epithelial cells. FASEB J. 2001;15:2345 54. 29. Odin JA, Huebert RC, Casciola Rosen L, LaRusso NF, Rosen A. Bcl 2 dependent oxidation of pyruvate dehydrogenase E2, a primary biliary cirrhosis autoantigen, during apoptosis. J Clin Invest. 2001;108:223 32. 30. Charlotte F, L'Hermine A, Martin N et al. Immunohistochemical detection of bcl 2 protein in normal and pathological human liver. Am J Pathol. 1994;144:460 5. 31. Nakopoulou L, Stefanaki K, Vourlakou C, Manolaki N, Gakiopoulou H, Michalopoulos G. Bcl 2 protein expression in acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Pathol Res Pract. 1999;195:19 24. 32. Matsumura S, Van De Water J, Leung P et al. Caspase induction by IgA antimitochondrial antibody: IgA mediated biliary injury in primary biliary cirrhosis. Hepatology. 2004;39:1415 22. 33. Allina J, Hu B, Sullivan DM et al. T cell targeting and phagocytosis of apoptotic biliary epithelial cells in primary biliary cirrhosis. J Autoimmun. 2006;27:232 41. 34. Tanaka A, Nalbandian G, Leung PS et al. Mucosal immunity and primary biliary cirrhosis: presence of antimitochondrial antibodies in urine. Hepatology. 2000;32:910 15.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 35. Reynoso Paz S, Leung PS, Van De Water J et al. Evidence for a locally driven mucosal response and the presence of mitochondrial antigens in saliva in primary biliary cirrhosis. Hepatology. 2000;31:24 9. 36. Fukushima N, Nalbandian G, Van De Water J et al. Characterization of recombinant monoclonal IgA anti PDC E2 autoantibodies derived from patients with PBC. Hepatology. 2002;36:1383 92. 37. Mao TK, Lian ZX, Selmi C et al. Altered monocyte responses to de¢ned TLR ligands in patients with primary biliary cirrhosis. Hepatology. 2005;42:802 8. 38. Moritoki Y, Lian ZX, Wul¡ H et al. AMA production in primary biliary cirrhosis is promoted by the TLR9 ligand CpG and suppressed by potassium channel blockers. Hepatology. 2007;45:314 22. 39. Hammond KJ, Kronenberg M. Natural killer T cells: natural or unnatural regulators of autoimmunity? Curr Opin Immunol. 2003;15:683 9. 40. Chuang YH, Lian ZX, Tsuneyama K et al. Increased killing activity and decreased cytokine production in NK cells in patients with primary biliary cirrhosis. J Autoimmun. 2006;26:232 40. 41. Nagano T, Yamamoto K, Matsumoto S et al. Cytokine pro¢le in the liver of primary biliary cirrhosis. J Clin Immunol. 1999;19:422 7. 42. Martinez OM, Villanueva JC, Gershwin ME, Krams SM. Cytokine patterns and cytotoxic mediators in primary biliary cirrhosis. Hepatology. 1995;21:113 19. 43. Yasoshima M, Kono N, Sugawara H, Katayanagi K, Harada K, Nakanuma Y. Increased expression of interleukin 6 and tumor necrosis factor alpha in pathologic biliary epithelial cells: in situ and culture study. Lab Invest. 1998;78:89 100. 44. Irie J, Wu Y, Wicker LS et al. NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis. J Exp Med. 2006;203:1209 19. 45. Oertelt S, Lian ZX, Cheng CM et al. Anti mitochondrial antibodies and primary biliary cirrhosis in TGF beta receptor II dominant negative mice. J Immunol. 2006;177:1655 60. 46. Wakabayashi K, Lian ZX, Moritoki Y et al. IL 2 receptor alpha( / ) mice and the development of primary biliary cirrhosis. Hepatology. 2006;44:1240 9. 47. Malmborg AC, Shultz DB, Luton F et al. Penetration and co localization in MDCK cell mitochondria of IgA derived from patients with primary biliary cirrhosis. J Autoimmun. 1998;11:573 80. 48. Padgett KA, Selmi C, Kenny TP et al. Phylogenetic and immunological de¢nition of four lipoylated proteins from Novosphingobium aromaticivorans, implications for primary biliary cirrhosis. J Autoimmun. 2005;24:209 19. 49. Seibold F, Weber P, Klein R, Berg PA, Wiedmann KH. Clinical signi¢cance of antibodies against neutrophils in patients with in£ammatory bowel disease and primary sclerosing cholangitis. Gut. 1992;33:657 62. 50. Terjung B, Herzog V, Worman HJ et al. Atypical antineutrophil cytoplasmic antibodies with perinuclear £uorescence in chronic in£ammatory bowel diseases and hepatobiliary disorders colocalize with nuclear lamina proteins. Hepatology. 1998;28:332 40. 51. Terjung B, Worman HJ. Anti neutrophil antibodies in primary sclerosing cholangitis. Best Pract Res Clin Gastroenterol. 2001;15:629 42. 52. Terjung B, Spengler U, Sauerbruch T, Worman HJ. `Atypical p ANCA' in IBD and hepatobiliary disorders react with a 50 kilodalton nuclear envelope protein of neutrophils and myeloid cell lines. Gastroenterology. 2000;119:310 22. 53. Xu B, Broome U, Ericzon BG, Sumitran Holgersson S. High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6. Gut. 2002;51:120 7. 54. Karrar A, Broome U, Sodergren T et al. Biliary epithelial cell antibodies link adaptive and innate immune responses in primary sclerosing cholangitis. Gastroenterology. 2007;132:1504 14. 55. Grant AJ, Lalor PF, Salmi M, Jalkanen S, Adams DH. Homing of mucosal lymphocytes to the liver in the pathogenesis of hepatic complications of in£ammatory bowel disease. Lancet. 2002;359:150 7. 56. Eksteen B, Grant AJ, Miles A et al. Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut homing lymphocytes to the liver in primary sclerosing cholangitis. J Exp Med. 2004;200:1511 17.
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IMMUNOLOGY OF PBC AND PSC 57. Karlsen TH, Boberg KM, Vatn M et al. Di¡erent HLA class II associations in ulcerative colitis patients with and without primary sclerosing cholangitis. Genes Immun. 2007;8:275 8. 58. Donaldson PT, Norris S. Immunogenetics in PSC. Best Pract Res Clin Gastroenterol. 2001;15:611 27. 59. Karlsen TH, Lie BA, Frey Froslie K et al. Polymorphisms in the steroid and xenobiotic receptor gene in£uence survival in primary sclerosing cholangitis. Gastroenterology. 2006;131:781 7. 60. O'Mahony CA, Vierling JM. Etiopathogenesis of primary sclerosing cholangitis. Semin Liver Dis. 2006;26:3 21. 61. Gershwin ME, Nishio A, Ishibashi H, Lindor K. Primary biliary cirrhosis. In: Gershwin ME, Vierling JM, Manns MP, editors. Liver Immunology. Philadelphia: Hanley and Belfus, 2003. 62. Morland CM, Fear J, McNab G, Joplin R, Adams DH. Promotion of leukocyte transendothelial cell migration by chemokines derived from human biliary epithelial cells in vitro. Proc Assoc Am Phys. 1997;109:372 82. 63. Cameron RG, Blendis LM, Neuman MG. Accumulation of macrophages in primary sclerosing cholangitis. Clinical Biochem. 2001;34:195 201.
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20 Alcoholic hepatitis H. TILG
INTRODUCTION Severe alcoholic hepatitis is still associated with high mortality and the presence of liver failure manifested by jaundice, coagulopathy and encephalopathy is a poor prognostic indicator. The management of these patients initially includes several supportive measures such as treatment of alcohol withdrawal, administration of £uid and vitamins and admission to an intensive-care unit for the unstable patient. Glucocorticoids have for decades been the most intensively studied therapy in alcoholic hepatitis. Although glucocorticoid treatment has been reported to improve survival in certain patient groups, meta-analyses of clinical trials to date have failed to show a clear bene¢t of this therapy. The greatest bene¢t for corticosteroids might be in patients showing stable or decreasing bilirubin levels within 1 week of therapy. Nutritional supplementation is also likely to be bene¢cial. The main progress in better understanding its pathophysiology has come from cytokine studies. Various proin£ammatory cytokines such as tumour necrosis factor alpha (TNF-a) a have been proposed to play a role in this disease. This advancement has recently led to pilot studies investigating anti-TNF drugs such as pentoxifylline or in£iximab, an anti-TNF antibody, in the treatment of this disease, although a small controlled study showed no bene¢t. New treatment modalities are urgently awaited, as standard therapies for this highly fatal disorder are insu¤cient.
CLINICAL ASPECTS OF ALCOHOLIC LIVER DISEASE Chronic consumption of alcohol results in a spectrum of liver abnormalities, ranging from simple fatty liver to steatohepatitis, cirrhosis and hepatocellular carcinoma. Fatty liver is often unnoticed and reverses within a few weeks of sobriety. Only a minority of consistent, heavy drinkers with hepatic steatosis ever develop clinically relevant liver disease1. The latter observation suggests the importance of other host or environmental factors in determining the evolution of alcohol-related liver disease. Risk factors for serious liver damage in habitual alcohol drinkers include certain polymorphisms in alcohol208
ALCOHOLIC HEPATITIS
metabolizing enzymes, obesity, exposure to other hepatotoxins (e.g. acetaminophen), and infection with hepatitis C. A high prevalence of hepatitis C virus infection (up to 65%) has been recognized in alcoholics. These patients tend to have more severe disease, decreased survival and an increased risk of hepatocellular carcinoma. In many patients a speci¢c co-morbidity factor is never identi¢ed.
Alcoholic hepatitis (AH) The appearance of steatohepatitis is an important rate-limiting step in the development of progressive alcoholic liver disease. One-month mortality rates of 40 50% have been reported in patients hospitalized with acutely decompensated liver disease due to alcohol-induced steatohepatitis. Disease severity and mortality risk in patients with AH may be estimated by using a discriminant function formula (Maddrey score) calculated as follows: discriminant function = (4.66[prothrombin time control PT] + (serum bilirubin)) where PT refers to the prothrombin time and the bilirubin concentration2 is measured in units of mg/dl. A value greater than 32 is associated with a high short-term mortality, and has been used to determine the need for speci¢c treatment in patients with severe AH.
Clinical presentation and diagnosis Patients with severe AH (DF 432) typically present with fever, hepatomegaly, jaundice and anorexia (Table 1). The presence of liver failure manifested by coagulopathy, jaundice, and/or encephalopathy is a poor prognostic indicator, usually indicating the presence of limited hepatic functional reserve. Fever is typically modest (5388C) and other sources such as superimposed spontaneous bacterial peritonitis must be excluded. Approximately 40 50% of patients have ascites, and tender hepatomegaly is common. Leukocytosis is frequent and correlates with the severity of hepatic injury3. Neutrophilic in¢ltration is commonly seen on liver biopsy and these cells may play an important role in the hepatic injury. Although the diagnosis can be con¢rmed by a liver biopsy, clinical and laboratory features are often adequate for establishing the diagnosis. The absolute values for serum SGOT and SGPT are almost always less than 400 IU/L, and higher concentrations should raise the suspicion of concurrent liver injury due to viral or other aetiologies. Table 1
Clinical and laboratory signs of severe alcoholic hepatitis
Fever Hepatomegaly Jaundice Anorexia Coagulopathy Encephalopathy Leukocytosis SGOT/SGPT 5400 IU/L
209
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PATHOPHYSIOLOGICAL ASPECTS IN AH Role of TNF-a in AH Recently, various proin£ammatory cytokines have been proposed to play a role in this disease4^6. Among these cytokines the proin£ammatory cytokine TNF-a has emerged as a key cytokine in the in£ammatory process. The involvement of TNF-a has been especially demonstrated in acute AH. Indeed, AH was one of the ¢rst diseases shown to exhibit increased circulating TNF-a levels7^9. In addition, serum concentrations of various TNF-inducible cytokines such as interleukin-1 (IL-1) and IL-8 are also increased in patients with acute AH10,11. Furthermore, plasma levels of both TNF and soluble TNF receptors are correlated with endotoxaemia and stage of liver disease12 . This ¢nding, coupled with evidence that long-term ingestion of alcohol increases intestinal permeability and that patients with the highest serum concentrations have the highest in-hospital mortality, indicates that intestinally derived endotoxin and endotoxin-regulated cytokines, such as TNF-a, have a role in the pathogenesis of alcoholic steatohepatitis13,14. Furthermore, Mookerjee et al. demonstrated that TNF-a is the key mediator of portal and systemic haemodynamic derangements in severe AH15.
Animal models highlighting the role of TNF-a Human data supporting a key role for TNF-a in alcohol-related liver diseases are further substantiated by data from animal experiments16,17. Several studies in rats, mice, and tissue culture have focused on the role of cytokines, especially TNF-a, in experimental models of alcoholic liver disease6,16,17. Perhaps the most compelling evidence supporting a key role for this cytokine comes from studies using mice in which the type-1 TNF receptor gene has been disrupted as these mice are resistant to alcohol-induced liver disease17. Anti-TNF antibody treatment has also been successfully used to prevent liver injury in alcohol-fed rats16. Furthermore, alcohol-associated liver injury is inhibited when the animals are treated with poorly absorbed oral antibiotics or Lactobacillus to decrease endotoxaemia, supporting the hypothesis that gut-derived bacterial products such as endotoxin might be important in activation of Kup¡er cells and/or other cell types in the liver. This is in accordance with the recent observation that chronic ethanol feeding causes more severe liver injury in wild-type mice than in CD14 knockouts18. These results further support the notion that gut-derived endotoxin acting via its cellular receptor CD14 plays a major role in the development of early alcohol-induced liver injury.
Apoptosis and AH Apoptosis, a major form of cell death, has been noted in a diverse spectrum of liver diseases. In experimental models and isolated hepatocytes, alcohol has been shown to induce liver cell apoptosis. Recently, two reports demonstrated that enhanced apoptosis is a prominent feature of AH19,20. Apoptosis was detected using the TUNEL assay and by demonstrating caspase activation. 210
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Interestingly, the extent of apoptosis correlated with clinical disease activity. Therefore, therapeutic strategies aimed at inhibiting apoptosis in AH (e.g. antiTNF treatments) should be of interest in the near future.
STANDARD THERAPY OF ACUTE AH Hospitalized patients with acute AH are often severely ill, and the acute hospital mortality is high in patients with poor prognostic signs. Several supportive measures are routinely required in these patients.
General management of patients with severe AH One of the ¢rst therapeutic aspects is to treat alcohol withdrawal with benzodiazepines. The administration of £uid, calories, vitamins and minerals is usually required. Overhydration should be avoided, as this may increase ascites, lower the plasma sodiu m conc entration, and precipitate gastrointestinal haemorrhage from varices. Vitamin K is usually administered to patients with a prolonged prothrombin time, even though this regimen is typically ine¡ective because coagulopathy re£ects more underlying liver failure. Correction of the coagulopathy with fresh frozen plasma is not recommended in the absence of active haemorrhage, as it might increase the risk of variceal haemorrhage in a patient with portal hypertension. Admission to an intensivecare unit should be considered in the unstable patient. Airway protection should be assured in the encephalopathic patient (Table 2). Table 2
General management of patients with severe alcoholic hepatitis
Treatment of alcohol withdrawal Administration of £uid, calories, vitamins (vitamin K, thiamine, folate) and minerals Airway protection (encephalopathy) Management of ascites (paracentesis, cultures) Prompt antibiotic therapy in case of proven infection Fresh frozen plasma (only if active haemorrhage) Nutritional therapy, correction of pre existing protein calorie malnutrition
Corticosteroids Glucocorticoids have been the most intensively studied treatment for AH2,21,22. The e¤cacy of corticosteroids in AH has been evaluated in several controlled trials, which have produced variable results. Although glucocorticoid treatment has been reported to improve survival, meta-analyses of the clinical trials to date have failed to show a clear bene¢t of such therapy23,24. One of the reasons for such variable results in many clinical trials in that this disorder may be due to the heterogeneity among the studies. A bene¢t of corticosteroid treatment is most likely in patients who have severe disease, which can be 211
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
estimated using the discriminant function (Maddrey score). A reduction in 30day mortality has been observed in studies that strati¢ed patients according to the severity of disease. Similarly, in one meta-analysis, the bene¢t of corticosteroids was limited to patients who had hepatic encephalopathy (an indication of severe disease)24. Mortality is high (approximately 40%) even in patients treated with corticosteroids. Patients who are eligible for corticosteroids should be treated with prednisolone (40 mg daily for 4 weeks followed by a taper). Prednisolone is preferred to prednisone, because the latter requires conversion to prednisolone (the active form) in the liver, a process that may be impaired in AH. Careful monitoring for evidence of infection, gastrointestinal bleeding, glucose intolerance, or renal failure is essential while the patient is on steroid therapy. Despite 13 randomized controlled trials, and several meta-analyses, the debate over the use of steroids continues. Individual analyses of the last three randomized controlled trials including only patients with a Maddrey score 432 showed a signi¢cant bene¢t of steroids vs placebo (85% vs 65%; 102 on placebo, 113 on steroids)25. It needs to be mentioned that in two trials patients with gastrointestinal haemorrhage were excluded and pretreament histology was available in only one study. Even though, as suggested by these authors, we might be able to de¢ne subgroups of patients who show a treatment bene¢t on steroids, mortality despite treatment remains too high. Therefore, alternative strategies have been, and continue to be, sought.
Nutritional therapy The e¤cacy of nutritional therapy in AH has been evaluated in multiple clinical trials. Although various results have been reported, the majority of studies have demonstrated improvement in liver function and histology. However, there has been no consistent reduction in mortality associated with nutritional therapy. Enteral feeding is preferred to the intravenous route. Although protein ingestion is a theoretical risk factor for the development of hepatic encephalopathy, protein feeding is well tolerated, and protein should not be routinely restricted in patients with AH. In patients who develop encephalopathy associated with protein feeding, use of branched-chain amino acids may be helpful.
Nutritional therapy versus corticosteroids The e¡ectiveness of nutritional therapy versus glucocorticoids treatment was assessed in a controlled trial involving 71 patients with severe AH who were randomly assigned to prednisolone (40 mg/day) or enteral tube feeding for 28 days followed by 1 year of follow-up26. Early mortality during treatment was similar in both groups (25 vs 31%), although deaths occurred signi¢cantly earlier with enteral feeding (median 7 vs 23 days). Mortality was higher in the steroid group during follow-up (37 vs 8%).
212
ALCOHOLIC HEPATITIS
NEW THERAPIES FOR AH Pentoxifylline The observation that TNF-a levels are increased in patients with AH provided a rationale for the study of pentoxifylline (an inhibitor of TNF synthesis) in AH. So far only one study has been performed. In this study, 101 patients with severe AH were included. Mortality at 4 weeks was signi¢cantly decreased in the group randomized to pentoxifylline (25% vs 46%)27. The bene¢t appeared to be related to a signi¢cant reduction in the risk of developing hepatorenal syndrome. These results are encouraging but need to be con¢rmed by others before this therapy can be routinely recommended. The study did not include a treatment arm with corticosteroids, although the survival bene¢t was higher than observed in several studies with corticosteroids.
Anti-TNF antibody treatment of acute AH Based on studies of alcohol-related liver disease in animals alternative therapies for these diseases have been proposed. In animals, treatment with various antioxidants (Table 3) decreases alcohol-induced liver damage and improves the fatty liver that develops in rats fed choline- and methionine-de¢cient diets, but so far the bene¢ts have been inconsistent in the small groups of patients with alcoholic liver disease. Evidence that TNF-a is important in animals with steatohepatitis suggests that TNF-a is another potential therapeutic target in patients with these diseases. Recently various TNF-neutralizing agents have been successfully used in the treatment of chronic in£ammatory disorders such as Crohn's disease, ulcerative colitis, rheumatoid arthritis and psoriasis. Based upon those experiences, and the convincing preclinical and clinical data on the role of TNF-a in AH, pilot trials with in£iximab, a TNF-a-neutralizing antibody, have been performed and recently reported. Table 3
Potential bene¢cial treatments in alcoholic steatohepatitis
Rationale
Therapy
Prevent gut derived endotoxaemia
Antibiotics, Lactobacillus
Inhibit TNF a activity
Anti TNF antibodies, TNF receptor antagonists, pentoxifylline
Generalized inhibition of in£ammation
Glucocorticoids, MARS
Decrease reactive oxygen species production
Glutathione prodrugs (S adenosyl methionine, betaine, choline), vitamin E, silymarin, propylthiouracil
Reduce liver lipid content
Ursodeoxycholic acid
TNF, tumour necrosis factor.
213
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Spahr et al. have randomized 20 patients with biopsy-proven severe alcoholic hepatitis and treated them with prednisone 40 mg/day for 28 days and either in£iximab 5 mg/kg body weight or placebo28. In£iximab was administered once on day 0 and was well tolerated. At day 28 the Maddrey score improved signi¢cantly only in the prednisone/in£iximab group. Furthermore, in£iximab therapy was associated with decreased circulating levels of IL-6 and IL-10 at day 10 of treatment, whereas histology was not a¡ected after a median time of 10 days (8 12 days). Even though the authors observed changes in prothrombin time and serum bilirubin, this pilot study gave no details on survival, due to the limited sample size of included patients. We recently reported results of a similar pilot study with in£iximab29. In contrast to the trial of Spahr et al. we did not use steroids, and we treated 12 patients with biopsy-con¢rmed AH and a Maddrey score 432. We also used a single dose of 5 mg/kg in£iximab. All patients were hospitalized and received standard treatment including treatment of alcohol withdrawal with benzodiazepines, administration of £uid, calories, vitamins and minerals and management of ascites. In four patients we did a follow-up biopsy within 28 days of treatment. We observed a signi¢cant decrease in bilirubin levels, Maddrey score, neutrophil counts and C-reactive protein levels. Changes in circulating cytokine levels were only transient (IL-6, IL-8). More importantly, we observed remarkable changes in the expression of a mainly TNF-driven chemokine, namely IL-8, in the liver, and also histological improvements with decreased fat content and reduced neutrophil in¢ltration in the liver. These changes suggest that this novel treatment could also alter histological features of the disease. Two patients, however, died of septicaemia within 28 days, whereas 10 of 12 patients were alive at a median of 15 months. To further understand the e¡ects of in£iximab in severe AH Mookerjee and colleagues tested, in in£iximab-treated patients, the hypothesis that TNF-a is an important mediator of the circulatory disturbances in AH15. Cardiovascular haemodynamics, hepatic venous pressure gradient (HVPG) and hepatic and renal blood £ow were measured before in£iximab, 24 h after in£iximab, and prior to hospital discharge. Of the 10 reported patients, nine were alive at 28 days. Mean HVPG decreased signi¢cantly at 24 h with a sustained reduction prior to discharge. Mean arterial pressure and systemic vascular resistance increased signi¢cantly, paralleled by a reduction in cardia index. Furthermore, hepatic and renal blood £ow also increased signi¢cantly, suggesting that TNFa is one of the key mediators in the observed circulatory disturbances in AH.
The French infliximab study A French multicentre in£iximab trial in AH was stopped in October 2002 by the French drug agency. In this study the authors compared prednisolone (40 mg/day) either with placebo or in£iximab given intravenously (10 mg/kg three times: week 0, 2 and 4). The mean end-point was 2-month mortality. After including 36 patients, an interim analysis revealed higher mortality in the in£iximab group, and the study was stopped30. It needs to be mentioned, however, that in this study unusually high doses of in£iximab, combined with short treatment intervals, were used. 214
ALCOHOLIC HEPATITIS
So, what can we learn from these con£icting results with in£iximab? The main issue treating patients with advanced AH is their high risk of severe infections. This aspect may be true using either steroids and/or in£iximab. We now know from many clinical trials using in£iximab in patients with various diseases that, despite its potential to reactivate tuberculosis, this treatment is associated with a remarkable ben¢t/risk ratio also with respect to infections. Certainly patients with AH may have an increased risk of developing tuberculosis or fungal infections compared to patients with rheumatoid arthritis or Crohn's disease. This may, however, also be true for treatment with steroids. Therefore, it is probably more important to challenge our current concepts in treating AH as waiting in a smouldering disease until the patient ful¢ls a Maddrey score 432. In our daily practice we often observe the patient over days using a watch-and-wait strategy until we ¢nally decide to treat him or her, e.g. with steroids. This concept may be challenged as we might think about earlier treatment dealing with a patient who is less immunocompromised using more selective treatment such as a neutralizing antibody. Certainly we need randomized trials with in£iximab, but also new concepts in better de¢ning the patient population with AH who need early treatment, a concept which is practised in most diseases.
CONCLUSIONS Endotoxin-inducible cytokines, such as TNF-a, TNF-regulated cytokines, and cytokines that modulate the synthesis and biological actions of TNF, are produced by many cells within the liver and other tissues. This extensive cytokine network mediates various aspects of liver injury and repair, especially in AH. Based on these new aspects in the pathophysiology of AH new treatment approaches are devoloped, as `standard' therapies such as glucocorticoids are not e¡ective in many patients. Pentoxifylline, a TNF inhibitor, has recently been shown to provide encouraging results in the treatment of this disorder. Other TNF-neutralizing therapies such as in£iximab have been tested with con£icting results in pilot studies and in a single controlled study. Besides the search for new treatments we also have in the future to better de¢ne the patient who needs speci¢c treatment, as our current defensive strategy treating only the sickest patients may be wrong. As intensive research over recent decades has not revealed any universally e¡ective treatment, novel therapeutic strategies based on the better understanding of its pathophysiology are urgently awaited.
References 1. 2. 3.
Tilg H, Day CP. Management strategies in alcoholic liver disease. Nat Clin Pract Gastroenterol Hepatol. 2007;4:24 34. Maddrey WC, Boitnott JK, Bedine MS, Weber FL Jr, Mezey E, White RI Jr. Corticosteroid therapy of alcoholic hepatitis. Gastroenterology. 1978;75:193 9. Orrego H, Blake JE, Blendis LM, Medline A. Prognosis of alcoholic cirrhosis in the presence and absence of alcoholic hepatitis. Gastroenterology. 1987;92:208 14.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27.
McClain CJ, Barve S, Barve S, Deaciuc I, Hill DB. Tumor necrosis factor and alcoholic liver disease. Alcohol Clin Exp Res. 1998;22(5 Suppl.):248S 52. McClain CJ, Barve S, Deaciuc I, Kugelmas M, Hill D. Cytokines in alcoholic liver disease. Semin Liver Dis. 1999;19:205 19. Tilg H, Diehl AM. Cytokines in alcoholic and nonalcoholic steatohepatitis. N Engl J Med. 2000;343:1467 76. Bird GL, Sheron N, Goka AK, Alexander GJ, Williams RS. Increased plasma tumor necrosis factor in severe alcoholic hepatitis. Ann Intern Med. 1990;112:917 20. McClain CJ, Cohen DA. Increased tumor necrosis factor production by monocytes in alcoholic hepatitis. Hepatology. 1989;9:349 51. Khoruts A, Stahnke L, McClain CJ, Logan G, Allen JI. Circulating tumor necrosis factor, interleukin 1 and interleukin 6 concentrations in chronic alcoholic patients. Hepatology. 1991;13:267 76. Hill DB, Marsano LS, McClain CJ. Increased plasma interleukin 8 concentrations in alcoholic hepatitis. Hepatology. 1993;18:576 80. Sheron N, Bird G, Koskinas J et al. Circulating and tissue levels of the neutrophil chemotaxin interleukin 8 are elevated in severe acute alcoholic hepatitis, and tissue levels correlate with neutrophil in¢ltration. Hepatology. 1993;18:41 6. Hanck C, Rossol S, Bocker U, Tokus M, Singer MV. Presence of plasma endotoxin is correlated with tumour necrosis factor receptor levels and disease activity in alcoholic cirrhosis. Alcohol Alcohol. 1998;33:606 8. Felver ME, Mezey E, McGuire M et al. Plasma tumor necrosis factor alpha predicts decreased long term survival in severe alcoholic hepatitis. Alcohol Clin Exp Res. 1990;14:255 9. Adachi Y, Moore LE, Bradford BU, Gao W, Thurman RG. Antibiotics prevent liver injury in rats following long term exposure to ethanol. Gastroenterology. 1995;108:218 24. Mookerjee RP, Sen S, Davies NA, Hodges SJ, Williams R, Jalan R. Tumour necrosis factor alpha is an important mediator of portal and systemic haemodynamic derangements in alcoholic hepatitis. Gut. 2003;52:1182 7. Iimuro Y, Gallucci RM, Luster MI, Kono H, Thurman RG. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and in£ammation caused by chronic exposure to ethanol in the rat. Hepatology. 1997;26:1530 7. Yin M, Wheeler MD, Kono H et al. Essential role of tumor necrosis factor alpha in alcohol induced liver injury in mice. Gastroenterology. 1999;117:942 52. Yin M, Bradford BU, Wheeler MD et al. Reduced early alcohol induced liver injury in CD14 de¢cient mice. J Immunol. 2001;166:4737 42. Natori S, Rust C, Stadheim LM, Srinivasan A, Burgart LJ, Gores GJ. Hepatocyte apoptosis is a pathologic feature of human alcoholic hepatitis. J Hepatol. 2001;34:248 53. Ziol M, Tepper M, Lohez M et al. Clinical and biological relevance of hepatocyte apoptosis in alcoholic hepatitis. J Hepatol. 2001;34:254 60. Carithers RL Jr, Herlong HF, Diehl AM et al. Methylprednisolone therapy in patients with severe alcoholic hepatitis. A randomized multicenter trial. Ann Intern Med. 1989;110:685 90. Ramond MJ, Poynard T, Rue¡ B et al. A randomized trial of prednisolone in patients with severe alcoholic hepatitis. N Engl J Med. 1992;326:507 12. Christensen E, Gluud C. Glucocorticoids are ine¡ective in alcoholic hepatitis: a meta analysis adjusting for confounding variables. Gut. 1995;37:113 18. Imperiale TF, McCullough AJ. Do corticosteroids reduce mortality from alcoholic hepatitis? A meta analysis of the randomized trials. Ann Intern Med. 1990;113:299 307. Mathurin P, Mendenhall CL, Carithers RL Jr et al. Corticosteroids improve short term survival in patients with severe alcoholic hepatitis (AH): individual data analysis of the last three randomized placebo controlled double blind trials of corticosteroids in severe AH. J Hepatol. 2002;36:480 7. Cabre E, Rodr|guez Inglesias P, Caballer|a J et al. Short and long term outcome of severe alcohol induced hepatitis treated with steroids or enteral nutrition: a multicenter randomized trial. Hepatology. 2000;32:36 42. Akriviadis E, Botla R, Briggs W, Han S, Reynolds T, Shakil O. Pentoxifylline improves short term survival in severe acute alcoholic hepatitis: a double blind, placebo controlled trial. Gastroenterology. 2000;119:1637 48.
216
ALCOHOLIC HEPATITIS 28. Spahr L, Rubbia Brandt L, Frossard J et al. Combination of steroids with in£iximab or placebo in severe alcoholic hepatitis: a randomized controlled pilot study. J Hepatol. 2002;37:448. 29. Tilg H, Jalan R, Kaser A et al. Anti tumor necrosis factor alpha monoclonal antibody therapy in severe alcoholic hepatitis. J Hepatol. 2003;38:419 25. 30. Naveau S, Thabut D, Chryssostalis A et al. A double blind randomized controlled trial of in£iximab associated with prednisolone in acute alcoholic hepatitis Hepatology. 2004;39:1390 7.
217
Section VIII Complications of cirrhosis Chair: M COLOMBO and M SCHUCHMANN
21 Hepatorenal syndrome in cirrhosis A. L. GERBES
INTRODUCTION De¢nition and diagnostic criteria for hepatorenal syndrome (HRS) established in 19941 were based on the following three concepts: 1. Renal failure in HRS is functional and caused by marked intrarenal arteriolar vasoconstriction. 2. HRS occurs in the setting of a systemic circulatory dysfunction caused by extrarenal vasodilation. 3. Plasma volume expansion does not improve renal failure. Four new concepts have emerged since then: 1. Extrarenal arterial vasodilation mainly occurs in the splanchnic bed, whereas other major vascular beds, such as the brain and the liver, may be vasoconstricted. This may contribute to some of the frequent extrarenal complications, i.e. progressive hepatic failure and encephalopathy. 2. Cardiac output in patients with HRS may be low, normal, or high, but all the same it is insu¤cient because of the reduced peripheral vascular resistance. This may aggravate the systemic circulatory dysfunction of HRS. 3. The most frequent trigger of type 1 HRS is bacterial infection, mainly spontaneous bacterial peritonitis (SBP). 4. HRS is not always a terminal event as it can be improved by medical treatment, and the improvement is associated with improved survival.
221
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
BACKGROUND FOR THE NEW CONCEPTS The ¢rst of these concepts was discovered during investigations carried out using Doppler ultrasonography or plethysmography both before and after 1994. These studies were performed in patients with varying severity of cirrhosis, and reveal arterial vasodilation in the splanchnic circulation as well as arterial vasoconstriction in other areas such as the brain, kidneys, and liver2^9, whereas cutaneous and muscular blood £ow is low, normal, or even increased9^12. The dilation of the splanchnic vessels is mainly caused by local release of potent vasodilators such as nitric oxide (NO)13. These vasodilators also make the splanchnic circulation resistant to the vasoconstrictive e¡ect of angiotensin II, noradrenalin, vasopressin, and endothelin14-20. Consequently, the homeostasis of arterial pressure in cirrhosis depends on the e¡ect exerted by the endogenous vasoconstrictor systems in extrasplanchnic organs. As arterial vasodilation increases with progression of cirrhosis, the role of vasoconstrictors in maintaining haemodynamic stability becomes critical. This explains why cirrhotic patients with HRS are predisposed to develop renal, hepatic, and cerebral vasoconstriction. Regarding the second new concept, insu¤cient cardiac output contributing to renal hypoperfusion in patients with HRS was ¢rst suggested by Tristani and Cohn21, but two more recent studies have provided further evidence22,23. The ¢rst study showed that the cardiac output of cirrhotic patients with SBP who developed progressive renal failure was relatively low, in spite of infection resolution, compared to the cardiac output measured before renal failure and to that of patients with SBP who did not develop renal failure22. The second study compared non-azotaemic cirrhotic patients who developed HRS with similar patients who did not, and showed that low cardiac output and high plasma renin activity (PRA) were independent predictors of HRS23. Moreover, in patients developing HRS, the progression of circulatory dysfunction leading to arterial hypotension and renal failure occurred in the setting of continued decrease in cardiac output and increase in PRA. These ¢ndings support the hypothesis that hyperdynamic circulation is essential to maintain central blood volume and renal perfusion in cirrhosis. Therefore, when cardiac output decreases, e¡ective hypovolaemia occurs, leading to renal hypoperfusion and HRS. Why cardiac output becomes relatively insu¤cient in patients developing HRS is unknown. The role of so-called `cirrhotic cardiomyopathy' is a reliable hypothesis that needs to be validated. HRS can be triggered by precipitating events. The most important are infections, bleeding, and large-volume paracentesis without albumin administration24^27. The role of SBP has recently been emphasized. Changes in circulatory function, endogenous vasoactive systems, and renal function in patients developing renal failure triggered by SBP are identical to those observed in patients with HRS unrelated to infection, suggesting that the pathogenesis of progressive renal failure in cirrhotic patients with infection is the same as HRS. The most important concept of HRS, however, arises out of studies exploring new therapeutic strategies28. Since type 1 HRS is often associated with a rapid deterioration of liver function with increased levels of bilirubin 222
HEPATORENAL SYNDROME IN CIRRHOSIS
and prothrombin time, it has traditionally been viewed as a manifestation of terminal hepatic failure. In fact, type 1 HRS was known as `terminal functional renal failure'. The corollary of this was that patients with type 1 HRS died because of irreversible liver failure, and that liver transplant (LT) was the only e¡ective treatment. Therefore, the demonstration that type 1 HRS can be improved by vasoconstrictors32 or by transjugular intrahepatic portosystemic shunt (TIPS)33,34, and that reversal of type 1 HRS is associated with improved survival, represents a major change in the notion of the syndrome. In conclusion, the main pathogenetic mechanism in type 1 HRS is a potentially reversible deterioration of systemic circulatory function, mostly due to splanchnic vasodilation and renal vasoconstriction, and often triggered by a precipitating event (Figure 1). In addition to renal failure the syndrome may be associated with other organ dysfunctions, such as decreased cardiac output, hepatic failure, and encephalopathy. Altogether, portal hypertension, peripheral vasodilation and markedly decreased centrally e¡ective blood volume are the triggers for HRS. Thus, any novel concepts for treatment will have to counteract these changes.
Figure 1
Pathogenetic mechanisms of hepatorenal syndrome
223
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
NEW DEFINITION OF HRS HRS is a potentially reversible syndrome that mainly occurs in patients with cirrhosis, ascites, and liver failure, and less frequently in patients with acute liver failure or acute alcoholic hepatitis. It is characterized by impaired renal function, marked alterations in cardiovascular function and overactivity in the endogenous vasoactive systems. Intrarenal vasoconstriction causes low glomerular ¢ltration rate (GFR), whereas in systemic circulation vascular resistance decreases due to splanchnic and peripheral arterial vasodilation. There are two types of HRS. Type 2 HRS is characterized by moderate renal failure (serum creatinine from 133 to 226 mmol/L or 1.5 to 2.5 mg/dl), with a steady or slowly progressive course. It appears spontaneously, but can also follow a precipitating event. Type 2 HRS is typically associated with refractory ascites. Survival time of patients with type 2 HRS is shorter than that of nonazotaemic cirrhotic patients with ascites, but better than that of patients with type 1 HRS. Type 1 HRS is characterized by rapid progressive renal failure de¢ned by doubling of the initial serum creatinine concentrations to a level greater than 226 mmol/L (2.5 mg/dl) in less than 2 weeks. It may appear spontaneously, but often develops after a precipitating event, particularly SBP. Type 1 HRS usually occurs within the setting of an acute deterioration of circulatory function characterized by arterial hypotension and activation of endogenous vasoconstrictor systems, and may be associated with impaired cardiac and liver functions as well as encephalopathy. The natural prognosis of type 1 HRS is very poor. The main di¡erences with the de¢nition reported in 19961 are: 1. the potential reversibility of HRS without LT; 2. the dominant role of the splanchnic bed in arterial vasodilation; 3. the frequent role of SBP as an event precipitating type 1 HRS; 4. the concept that, in addition to renal failure, the function of other organs is frequently impaired.
Revised diagnostic criteria of HRS As there are no speci¢c hallmarks of HRS, the diagnosis is based on the exclusion of other types of renal failure. The criteria necessary to diagnose HRS are reported in Table 1. The main di¡erences between these criteria and those previously established1 are: 1. Creatinine clearance has been excluded because it is more complicated than simple serum creatinine for routine purposes, and it does not increase the accuracy of renal function estimation in cirrhotic patients. 2. Renal failure in the setting of ongoing bacterial infection, but in the absence of septic shock, is now considered HRS. This means that treatment of HRS can be started without waiting for complete recovery from the infection. 224
HEPATORENAL SYNDROME IN CIRRHOSIS Table 1 1. 2. 3.
4. 5. 6.
New diagnostic criteria of hepatorenal syndrome
Cirrhosis with ascites Serum creatinine 4133 mmol/L (1.5 mg/dl) No improvement of serum creatinine (decrease to a level of 133 mmol/L or less) after at least 2 days with diuretic withdrawal and volume expansion with albumin; the recommended dose of albumin is 1 g/kg of body weight per day up to a maximum of 100 g/day Absence of shock No current or recent treatment with nephrotoxic drugs Absence of parenchymal kidney disease as indicated by proteinuria 4500 mg/day, microhaematuria (450 red blood cells per high power ¢eld), and/or abnormal renal ultrasonography
3. Plasma volume expansion should be performed with albumin rather than saline. Members of the panel agreed that albumin causes a greater and more sustained expansion than saline. 4. Minor diagnostic criteria have been removed as they are not essential.
TREATMENT OF HRS New treatments of HRS are designed to expand the central blood volume by simultaneously increasing the total plasma volume and reducing intense peripheral vasodilation. This strategy is not entirely new, as in 1967 Tristani and Cohn21 showed that dextran infusion improved cardiac output and renal perfusion in oliguric cirrhotic patients, and 18 years later Shapiro et al.32 showed that the urine, water and sodium excretion in cirrhotic patients with ascites was improved by the administration of norepinephrine combined with head-out water immersion, a manoeuvre aimed at expanding central blood volume. However, clinically relevant results have only been obtained more recently with the use of albumin and various vasoconstrictors. The mechanism by which vasoconstrictors and albumin improve GFR in patients with HRS has yet to be completely clari¢ed. Nevertheless, it is known that a 2-day administration of terlipressin to patients with HRS induces a vasopressor e¡ect associated with a signi¢cant decrease in PRA and increase in GFR 33 , indirectly indicating correction of circulatory dysfunction. It is conceivable that vasopressin analogues cause vasoconstriction of the splanchnic bed allowing redistribution of the total blood volume from this bed to some of the extrasplanchnic organs including the kidneys. In addition, the inhibition of the sympathetic nervous and renin angiotensin systems shifts the autoregulatory curve to the left, making renal blood £ow and GFR more responsive to changes in blood pressure. Albumin is traditionally considered to improve circulatory function in cirrhosis by expanding central blood volume and increasing cardiac output38. Moreover, recent studies have shown that the administration of albumin to cirrhotic patients with SBP causes arterial vasoconstriction and blood pressure increase36, probably attributable to the 225
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
ability of albumin to bind vasodilators. It is therefore conceivable that an improvement of renal function in patients with HRS treated with vasoconstrictors and albumin is due to the additive e¡ects that the two compounds have on cardiac function and peripheral arterial circulation.
Vasoconstrictors and albumin Lenz et al.34 showed that GFR may be moderately improved by ornipressin infusion in patients with HRS, but the drug was given for only 4 h, therefore precluding assessment of its long-term e¡ects. Two more studies demonstrated that a long-term (1 2 weeks) infusion of ornipressin, combined with albumin or dopamine, normalized serum creatinine concentrations in many patients with type 1 HRS29,38. Interestingly, recurrence of renal failure rarely occurred after treatment withdrawal, and in the few cases where it did recur a second course of therapy was successful. However, there was a drawback with ornipressin: the frequent occurrence of ischemic complications29,38. Widespread use of vasoconstrictors in patients with HRS has therefore become reality only after the use of safer compounds such as terlipressin39^41, a vasopressin analogue with longer activity, and the a2-agonist midodrine combined with octreotide42,43. Table 2 summarizes the data available on the use of terlipressin in type 1 HRS. They include three randomized controlled trials (RCT)33,41,46, and many pilot or retrospective studies. These studies show that: (a) although GFR rarely reaches normal levels, a short period of treatment with terlipressin can improve renal function in up to 65% of patients with type 1 HRS; (b) the e¡ectiveness of terlipressin is probably enhanced by albumin 41 ; (c) HRS recurs after treatment withdrawal in approximately 20% of patients, but re-treatment is often e¡ective; (d) in most cases dilutional hyponatraemia associated with HRS improves with terlipressin treatment40,41; (e) severe side-e¡ects of the treatment are rare (5 10%). With regard to survival, patients who experienced a complete reversal of type 1 HRS by terlipressin seem to have an improved short-term survival. Therefore, the long-term survival of patients with type 1 HRS treated with terlipressin merits further investigation. Nevertheless, using terlipressin to improve renal function is an important support resource for patients included on an LT waiting list who develop type 1 HRS45. The initial dose of terlipressin in many studies ranged from 0.5 to 1 mg every 4 6 h. This regimen was maintained until reversal of HRS, which usually occurred within the second week of treatment. In other studies the initial dose in cases without an early response was increased up to 2 mg every 4 6 h. The daily dose of albumin was generally 20 40 g, preceded in some studies by a load of 1 g/kg of body weight. Some refer to central venous pressure to establish and titrate albumin doses and to prevent £uid overload. Experience of using midodrine in patients with type 1 HRS is more limited. To date there are only two pilot studies42,43. In both of these studies midodrine was combined with octreotide to enhance the e¡ect of splanchnic vasoconstriction, but doses and routes of administration were quite di¡erent. Angeli et al.42 used 7.5 12.5 mg t.i.d. of oral or intravenous midodrine plus 100 200 mg t.i.d. of subcutaneous octreotide, whereas Wong et al.43 used 2.5 mg 226
227
RCT RCT
RCT
Moreau, 200239 Uriz, 200040 Mulkay, 200148 Angeli, 200642 Ortega, 200245
Hadengue, 199833 Solanki, 200344
Sanyal, 2006 (abstract)52 120/229
53/99 4/6 12/12 12/19 T+A: 8/9 T^A: 1/7 6/9 T: 5/12 P: 0/12 T+A: 19/56 A: 7/56 2^12
4^8
2 2
3.2+1.2 3^6 1^6 2^12 2
Dose (mg/day)
2^27
14
2 14
11+12 5^15 8^14 415 4^14
Duration (days)
93/208
27/56{ 27/56{
37/99 4/6 3/12 13/19 8/9 1/7 n.d. n.d.
Survival at 4 weeks{
34/154
0/9 5/12
23/99 1/6 4/12 n.d. 1/16
Adverse events
Survival was reported at 60 days.
{
Successful treatment means partial or complete response in renal function improvement. T+A = terlipressin + albumin; T^A = terlipressin without albumin; T = terlipressin; P = placebo; A = albumin.
{
*R = retrospective; PU = prospective uncontrolled; RCT = randomized controlled trial.
Total
R PU PU PU PU
Author, year (ref.)
Success rate of treatment{
Characteristics and results of major studies reporting the e¡ect of terlipressin in patients with cirrhosis and type 1 HRS
Type of study*
Table 2
HEPATORENAL SYNDROME IN CIRRHOSIS
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
t.i.d. of oral midodrine plus an intravenous infusion of octreotide (25 mg/h after a bolus of 25 mg). The dose of midodrine was adjusted to increase mean arterial pressure to 90 mmHg. Albumin was also given in these studies. The results are similar to those obtained using terlipressin, although response was slower. Since octreotide alone had no impact on GFR in patients with HRS46, it is likely that midodrine plays the main role in improving GFR. A pilot study explored the e¡ect of norepinephrine infusion (0.5 3 mg/h) combined with albumin and furosemide in type 1 HRS47. The doses were titrated to increase mean arterial pressure by 10 mmHg. Reversal of HRS was achieved in 10 out of 12 cases and was associated with improvement in urinary sodium excretion and decrease in PRA. Norepinephrine is cheaper and more widely available than terlipressin, but it can induce arrhythmias more often. Therefore, the role of norepinephrine in patients with type 1 HRS should still be established on the basis of future comparisons with terlipressin or midodrine/octreotide. Only a few patients with type 2 HRS have been speci¢cally treated using terlipressin and albumin31. In most cases normalization of serum creatinine was observed, but in contrast to type 1 HRS, renal failure invariably recurred after treatment withdrawal.
TIPS Only a few studies have assessed the role of TIPS in HRS 91 patients in total. Most were prospective but uncontrolled studies 30,31,43,49,50 . Three were performed in patients with type 1 HRS30,31,43, one in patients with type 1 and patients with type 2 HRS49, and the last speci¢cally investigated type 2 HRS50. The following can be observed: 1. Signi¢cant suppression of the endogenous vasoactive systems, particularly the renin angiotensin system33, and a decrease of creatinine levels were recorded after TIPS in most patients with type 1 HRS. The rate of the creatinine decrease was slower than is usually obtained using terlipressin plus albumin. 2. Recurrence of HRS was rare, provided that there was no shunt malfunction. 3. Hepatic encephalopathy was a frequent complication of TIPS but was adequately managed by medical treatment. 4. TIPS almost always induced a reduction of ascites volume. 5. Resolution of type 1 HRS by TIPS can improve survival. 6. Sequential treatment with vasoconstrictors and albumin followed by TIPS could be used as an alternative approach to increasing the probability of long-term success43. 7. Although TIPS may improve renal function and refractory ascites in patients with type 2 HRS, its e¡ect on survival is still unde¢ned.
228
HEPATORENAL SYNDROME IN CIRRHOSIS
However, since almost all studies excluded patients with a history of severe encephalopathy, or serum bilirubin levels over 85 mmol/L (5 mg/dl), or Child Pugh score over 12, the applicability of TIPS may be rather limited in patients with HRS who frequently show jaundice, encephalopathy, and high Child Pugh scores51. There has been little investigation into the mechanism through which TIPS exerts bene¢cial e¡ects in patients with HRS. Nevertheless, as TIPS functions as a side-to-side portacaval shunt, it is expected to relieve portal hypertension, which plays a pivotal role in the pathogenesis of splanchnic arterial vasodilation51. Moreover, TIPS insertion is associated with an increase in cardiac output and an expansion in central blood volume 53,54 . The simultaneous e¡ects on the splanchnic and systemic circulation may represent the mechanism by which TIPS improves renal perfusion, GFR, urine sodium and water excretion, and hyponatraemia.
Extracorporeal albumin dialysis (ECAD) This procedure uses a cell-free albumin-containing dialysate that is recirculated and perfused through charcoal and anion exchange columns (Molecular Adsorbent Recycling System, MARS). The system is also connected to a haemodialysis or haemoperfusion apparatus. ECAD enables the removal of albumin-bound substances, including bilirubin, bile acids, aromatic amino acids, medium-chain fatty acids, and cytokines55. There are few data available for ECAD in cirrhotic patients with HRS, and these data are controversial56^58. ECAD decreases serum creatinine levels, but it is not de¢nitively known whether or not this e¡ect is due to a true improvement of renal function or simply to the ¢ltration process. A few studies reported that systemic haemodynamics improved during ECAD, indicated by increases of arterial pressure and systemic vascular resistances and decreases of cardiac output, PRA, and norepinephrine levels. However, studies regarding the e¡ect of ECAD on survival in patients with type 1 HRS included too few patients to draw any de¢nite conclusions. Moreover, ECAD is a very expensive procedure and should still therefore be considered experimental59,60.
References 1. 2. 3. 4. 5. 6.
Arroyo V, Gines P, Gerbes A et al. De¢nition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis. Hepatology. 1996;23:164 76. Fernandez Seara J, Prieto J, Quiroga J et al. Systemic and regional hemodynamics in patients with liver cirrhosis and ascites with and without functional renal failure. Gastroenterology. 1989;97:1304 12. Maroto A, Gines P, Arroyo V et al. Brachial and femoral artery blood £ow in cirrhosis: relationship to kidney dysfunction. Hepatology. 1993;17:788 93. Rivolta R, Maggi A, Cazzaniga M et al. Reduction of renal cortical blood £ow assessed by Doppler in cirrhotic patients with refractory ascites. Hepatology. 1998;28:1235 40. Guevara M, Bru C, Gines P et al. Increased cerebrovascular resistance in cirrhotic patients with ascites. Hepatology. 1998;28:39 44. Sacerdoti D, Bolognesi M, Merkel C, Angeli P, Gatta A. Renal vasoconstriction in cirrhosis evaluated by duplex Doppler ultrasonography. Hepatology. 1993;17:219 24.
229
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Sugano S, Yamamoto K, Atobe T et al. Postprandial middle cerebral arterial vasoconstriction in cirrhotic patients. A placebo, controlled evaluation. J Hepatol. 2001;34:373 7. Dillon JF, Plevris JN, Wong FC et al. Middle cerebral artery blood £ow velocity in patients with cirrhosis. Eur J Gastroenterol Hepatol. 1995;7:1087 91. Moller S, Henriksen J. The systemic circulation in cirrhosis. In: Gines P, Arroyo V, Rodes J, Schrier RW, editors. Ascites and Renal Dysfunction in Liver Disease, 2nd edn. Oxford: Blackwell, 2005:139 55. Luca A, Garcia Pagan JC, Feu F et al. Noninvasive measurement of femoral blood £ow and portal pressure response to propranolol in patients with cirrhosis. Hepatology. 1995;21:83 8. Piscaglia F, Zironi G, Gaiani S et al. Relationship between splanchnic, peripheral and cardiac hemodynamics in cirrhosis of di¡erent degrees of severity. Eur J Gastroenterol Hepatol. 1997;9:799 804. Wong F, Logan A, Blendis L. Hyperinsulinemia in preascitic cirrhosis: e¡ects on systemic and renal hemodynamics, sodium homeostasis, forearm blood £ow and sympathetic nervous activity. Hepatology. 1996;23:414 22. Mitamura K, Kawauchi A, Sasaki K et al. Measurement of renal artery blood £ow velocity by Doppler ultrasonography in chronic liver disease. Hepatol Res. 1999;15:201 14. Domenicali M, Ros J, Fernandez Varo G et al. Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats: role of cannabinoid and vanilloid receptors. Gut 2005;54:522 7. Castro A, Jimenez W, Claria J et al. Impaired response to angiotensin II in experimental cirrhosis: role of nitric oxide. Hepatology. 1993;18:367 72. Heller J, Schepke M, Gehnen N et al. Altered adrenergic responsiveness of endothelium denuded hepatic arteries and portal veins in patients with cirrhosis. Gastroenterology. 1999;116:387 93. Hartleb M, Moreau R, Cailmail S, Gaudin C, Lebrec D. Vascular hyporesponsiveness to endothelin 1 in rats with cirrhosis. Gastroenterology. 1994;107:1085 93. Michielsen PP, Boeckxstaens GE, Sys SU, Herman AG, Pelckmans PA. The role of increased nitric oxide in the vascular hyporeactivity to noradrenaline in long term portal vein ligated rats. J Hepatol. 1995;23:341 7. Islam M, Williams B, Madhavan K, Hayes P, Hadoke P. Selective alteration of agonist mediated contraction in hepatic arteries isolated from patients with cirrhosis. Gastroenterology. 2000;118:765 71. Helmy A, Newby DE, Jalan R et al. Nitric oxide mediates the reduced vasoconstrictor response to angiotensina II in patients with preascitic cirrhosis. J Hepatol. 2003;38:44 50. Tristani FE, Cohn JN. Systemic and renal hemodynamics in oliguric hepatic failure: e¡ect of volume expansion. J Clin Invest 1967;46:1894 6. Ruiz del Arbol L., Urman J, Fernandez J et al. Systemic, renal, and hepatic hemodynamic derangement in cirrhotic patients with spontaneous bacterial peritonitis. Hepatology. 2003;38:1210 18. Ruiz del Arbol L, Monescillo A, Arocena C et al. Circulatory function and hepatorenal syndrome in cirrhosis. Hepatology. 2005;42:439 47. Follo A, Llovet JM, Navasa M et al. Renal impairment after spontaneous bacterial peritonitis in cirrhosis: incidence, clinical course, predictive factors and prognosis. Hepatology. 1994;20:1495 501. Terra C, Guevara M, Torre A et al. Renal failure in patients with cirrhosis and sepsis unrelated to spontaneous bacterial peritonitis: value of MELD score. Gastroenterology. 2005;129:1944 53. Cardenas A, Gines P, Uriz J et al. Renal failure after upper gastrointestinal bleeding in cirrhosis: incidence, clinical course, predictive factors, and short term prognosis. Hepatology. 2001;34:671 6. Salerno F, Badalamenti S. Drug induced renal failure in cirrhosis. In: Gines P, Arroyo V, Rodes J, Schrier RW, editors. Ascites and Renal Dysfunction in Liver Disease, 2nd edn. Oxford: Blackwell, 2005:372 82. Moreau R, Lebrec D. The use of vasoconstrictors in patients with cirrhosis: type 1 HRS and beyond. Hepatology. 2006;43:385 94.
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HEPATORENAL SYNDROME IN CIRRHOSIS 29. Guevara M, Gines P, Fernandez Esparrach G et al. Reversibility of hepatorenal syndrome by prolonged administration of ornipressin and plasma volume expansion. Hepatology. 1998;27:35 41. 30. Brensing KA, Textor J, Perz J et al. Long term outcome after transjugular intrahepatic portosystemic stent shunt in non transplant cirrhotics with hepatorenal syndrome: a phase II study. Gut. 2000;47:288 95. 31. Guevara M, Gines P, Bandi JC et al. Transjugular intrahepatic portosystemic shunt in hepatorenal syndrome: e¡ects on renal function and vasoactive systems. Hepatology. 1998;28:416 22. 32. Shapiro MD, Nicholls, Groves BM KM et al. Interrelationship between cardiac output and vascular resistance as determinant of e¡ective arterial blood volume in cirrhotic patients. Kidney Int. 1985;28:206 11. 33. Hadengue A, Gadano A, Moreau R et al. Bene¢cial e¡ects of the two day administration of terlipressin in patients with cirrhosis and hepatorenal syndrome. J Hepatol. 1998;29:565 70. 34. Lenz K, Hortnagl H, Druml W et al. Ornipressin in the treatment of functional renal failure in decompensated cirrhosis: e¡ects on renal hemodynamics and atrial natriuretic factor. Gastroenterology. 1991;101:1060 7. 35. Fernandez J, Navasa M, Garcia Pagan JC et al. E¡ect of intravenous albumin on systemic and hepatic hemodynamics and vasoactive neurohormonal systems in patients with cirrhosis and spontaneous bacterial peritonitis. J Hepatol. 2004;41:384 90. 36. Bevers LM, van Faassen EE, Vuong TD et al. Low albumin levels increased endothelial NO production and decrease vascular NO sensitivity. Nephrol Dial Transplant. 2006;21:3443 9. 37. Sort P, Navasa M, Arroyo V et al. E¡ect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis. N Engl J Med. 1999;5:403 9. 38. Gulberg V, Bilzer M, Gerbes AL. Long term therapy and retreatment of hepatorenal syndrome type 1 with ornipressin and dopamine. Hepatology. 1999;30:870 5. 39. Moreau R, Durand F, Poynard T et al. Terlipressin in patients with cirrhosis and type 1 hepatorenal syndrome: a retrospective multicenter study. Gastroenterology. 2002;122:923 30. 40. Uriz J, Gines P, Cardenas A et al. Terlipressin plus albumin infusion: an e¡ective and safe therapy of hepatorenal syndrome. J Hepatol. 2000;33:43 8. 41. Ortega R, Gines P, Uriz J et al. Terlipressin therapy with and without albumin for patients with hepatorenal syndrome: results of a prospective nonrandomized study. Hepatology. 2002;36:941 8. 42. Angeli P, Guarda S, Fasolato S et al. Switch therapy with cipro£oxaxin vs. intravenous ceftazidime in the treatment of spontaneous bacterial peritonitis in patients with cirrhosis: similar e¤cacy at lower cost. Aliment Pharmacol Ther. 2006;23:75 84. 43. Wong F, Pantea L, Sniderman K. Midodrine, octreotide, albumin, and TIPS in selected patients with cirrhosis and type 1 hepatorenal syndrome. Hepatology. 2004;40:55 64. 44. Solanki P, Chawla A, Garg R et al. Bene¢cial e¡ects of terlipressin in hepatorenal syndrome: a prospective, randomized placebo controlled clinical trial. J Gastroenterol Hepatol. 2003;18:152 6. 45. Ortega R, Gines P, Uriz J et al. Terlipressin therapy with and without albumin for patients with hepatorenal syndrome: results of a prospective nonrandomized study. Hepatology. 2002;36:941 8. 46. Pomier Layrargues G, Paquin SC, Hassoun Z et al. Octreotide in hepatorenal syndrome: a randomized, double blind, placebo controlled, crossover study. Hepatology. 2003;38:238 43. 47. Duvoux C, Zanditenas D, Hezode C et al. E¡ects of noradrenalin and albumin in patients with type 1 hepatorenal syndrome: a pilot study. Hepatology. 2002;36:374 80. 48. Mulkay JP, Louis H, Donckier V et al. Long term terlipressin administration improves renal function in cirrhotic patients with type 1 hepatorenal syndrome: a pilot study. Acta Gastroenterol Belg. 2001;64:15 19. 49. Testino G, Ferro C, Sumberaz A et al. Type 2 hepatorenal syndrome and refractory ascites: role of transjugular intrahepatic portosystemic stent shunt in eighteen patients with
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50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.
advanced cirrhosis awaiting orthotopic liver transplantation. Hepatogastroenterology. 2003; 50:1753 5. Gerbes AL, Gulberg V. Bene¢t of TIPS for patients with refractory ascites: serum bilirubin may make the di¡erence. Hepatology. 2005;41:217. Jalan R, Forrest EH, Redhead DN, Dillon JF, Hayes PC. Reduction in renal blood £ow following acute increase in the portal pressure: evidence for the existence of a hepatorenal re£ex in man? Gut. 1997;40:664 70. Sanyal A, Boyer T, Garcia Tsao G et al. A prospective randomized double blind, placebo controlled trial of terlipressin for type 1 hepatorenal syndrome (HRS). Hepatology. 2006;44 (Suppl. 1):694A (Abstract). Salerno F, Cazzaniga M, Pagnozzi G et al. Humoral and cardiac e¡ects of TIPS in cirrhotic patients with di¡erent `e¡ective' blood volume. Hepatology. 2003;38:1370 7. Schwartz JM, Beymer C, Althaus SJ et al. Cardiopulmonary consequences of transjugular intrahepatic portosystemic shunts: role of increased pulmonary artery pressure. J Clin Gastroenterol. 2004;38:590 4. Stange J, Ramlow W, Mitzner S, Schmidt R, Klinkmann H. Dialysis against a recycled albumin solution enables the removal of albumin bound toxins. Artif Organs. 1993;17:809 13. Sen S, Davies NA, Mookerjee RP et al. Pathophysiological e¡ects of albumin dialysis in acute on chronic liver failure: a randomized controlled study. Liver Transplant. 2004;10:1109 19. Mitzner SR, Stange J, Klammt S et al. Improvement of hepatorenal syndrome with extracorporeal albumin dialysis MARS: results of a prospective, randomized, controlled clinical trial. Liver Transplant. 2000;6:277 86. Heemann U, Treichel U, Loock J et al. A dialysis in cirrhosis with superimposed acute liver injury: a prospective, controlled study. Hepatology. 2002;36:949 58. Gerbes AL. The patient with refractory ascites. Best Pract Res Clin Gastroenterol. 2007;21:551 60. Gerbes AL, Gulberg V. Progress in treatment of massive ascites and hepatorenal syndrome. World J Gastroenterol. 2006;12:516 19.
232
22 Variceal bleeding T. SAUERBRUCH and J. TREBICKA
INTRODUCTION Variceal bleeding is caused by portal hypertension, which can be assessed by measurement of the gradient between the wedged hepatic venous pressure and the free hepatic venous pressure (HVPG). A threshold of 10 mmHg is associated with the formation of collaterals to bypass splanchnic venous blood through the liver1,2. Some of these collaterals, the so-called oesophageal varices, are covered only by a thin layer of squamous epithelium in the distal oesophagus so that they may rupture and bleed at this speci¢c anatomical site (see Figure 1). The risk of variceal bleeding depends on the vessel's wall tension, which is determined by the diameter of the vessel and the transmural pressure gradient3, i.e. large super¢cial varices with a high blood pressure carry the highest risk of rupture and bleeding. The mean pressure of such varices3 is around 20+5
Figure 1 Large oesophageal varices with thin wall parts (red colour signs, arrow) have a very high risk of bleeding (30 40%)
233
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
mmHg. Bleeding of varices below a HVPG of 12 mmHg is extremely rare4. Thus, reduction of portal hypertension below this threshold is a major aim. This is di¤cult to achieve using drugs4, but feasible by creating an intrahepatic shunt (TIPS) or arti¢cial extrahepatic portal venous or splenorenal collaterals (open surgical shunt) 5 . Since shunts may induce or aggravate hepatic encephalopathy and worsen liver function they are not the ¢rst choice in the treatment of variceal bleeding. It is common opinion that variceal bleeding occurs less frequently, and can be better managed, than 20 or 30 years ago; however, it still has a high mortality of 20 40%6,7. Four circumstances recommending treatment of variceal bleeding can be distinguished: 1. preprimary prophylaxis to prevent formation of varices, 2. primary prophylaxis to prevent bleeding from varices that have already formed, 3. treatment of acute bleeding, and 4. prophylaxis of rebleeding.
PREPRIMARY PROPHYLAXIS Although treatments exist to prevent variceal formation in the way that they prevent the development of liver cirrhosis and portal hypertension (e.g. successful antiviral therapy) there are no convincing approaches to prevent variceal formation once cirrhosis of the liver has taken place2,8. This may change in the future through the establishment of novel pharmaceutical approaches, including anti¢brotic therapies such as statins9^11, angiotensin-II type 1 (AT 1)-receptor antagonists12^14, interferons15 or urotensin II receptor antagonists16. In animal models it has been shown that statins decrease intrahepatic resistance in cirrhosis with portal hypertension9, and this has been con¢rmed in the ¢rst human studies 11 . Furthermore, it has been indicated that statins may decrease ¢brosis if applied early10. Recent studies have shown that AT 1-receptor blockers and urotensin II receptor antagonists lower portal pressure in cirrhosis via a decrease in portal blood £ow12,13,16. There is evidence for an anti¢brotic e¡ect of AT 1-receptor antagonists14 and interferons15.
PRIMARY PROPHYLAXIS Prevention of ¢rst bleeding is indicated when certain endoscopic criteria are ful¢lled, namely varices larger than 5 mm in diameter with or without so-called red colour signs17. Both indicate a high wall tension, especially when occurring together18. These varices bear a risk of ¢rst bleeding of 30 40% within 2 years after their assessment (see Figure 1). 234
VARICEAL BLEEDING
The current strategy to prevent ¢rst bleeding is to apply drugs that reduce portal pressure or to ligate the varices. It has been shown that non-selective b-blockers lead to an adequate reduction of portal pressure (de¢ned as at least 20%) in about 50% of patients by reducing portal venous in£ow via lowering of the heart rate and increasing splanchnic vascular arterial resistance 19,20 . Since there are many nonresponders for medical treatment, only half of the patients receive adequate protection, i.e. the bleeding rate within 2 years is reduced from 30 40% to around 20%. In the past decade a number of controlled trials have shown that ligation of varices is at least equally e¡ective21; however, it may provoke lifethreatening bleeding in a small percentage of patients and might exert ligationspeci¢c lethal complications such as bleeding ulcers22. This is the main reason why most centres still use non-selective b-blockers as ¢rst choice for primary prevention, and refer to ligation only in cases in which patients do not tolerate the drug, are non-compliant or have contraindications. Unfortunately, we have no trials that test the clinical e¡ect of a haemodynamically controlled medical treatment, i.e. application of a nonselective b-blocker only in those patients in whom the drugs achieve an adequate reduction of HVPG. Recently, a trial was presented comparing carvediol, a non-selective bblocker, with an additional intrinsic a1-adrenergenic activity. Interestingly, carvediol was signi¢cantly more e¡ective than ligation, mainly because there was more early bleeding in the ligation group, partly induced by the endoscopic procedure itselff23.
ACUTE VARICEAL BLEEDING All patients with acute variceal bleeding, de¢ned as endoscopically proven bleeding from a varix (see Figure 2) or fresh blood in the oesophagus or stomach with varices and no other bleeding source, should receive antibiotics and vasoactive treatment with a vasopressin analogue or a somatostatin analogue even in the case of clinical suspicion prior to endoscopy24,25. These drugs curb splanchnic perfusion in addition to £ow and pressure of blood in the varices. Several trials have shown that this approach increases the rate of acute haemostasis and leads to a borderline improvement of survival as compared to endoscopic haemostasis alone 26,27 . The best endoscopic tool for acute hemostasis is ligation, provided it is technically feasible 25,28 ; however, injection of a sclerosant or glue may also be appropriate in acute situations, especially if heavy bleeding does not allow interruption of the procedure in order to mount the ligation device onto the endoscope. Ligation then may follow immediately after the successful interruption of acute bleeding by injection29. All patients with acute variceal bleeding should receive antibiotics as soon as possible for at least 5 days, regardless of whether there are signs of infection or not. This measure improves the survival rate of patients with liver cirrhosis and acute variceal bleeding24,30. 235
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
A
B
Figure 2
Acute variceal bleeding from oesophageal varices (A) and from gastric varices (B)
PREVENTION OF RECURRENT BLEEDING There is a 60 70% chance of variceal rebleeding in patients who were successfully treated for an acute bleeding episode. In other words, rebleeding prophylaxis is always necessary31,32. Non-selective b-blockers reduce the risk of rebleeding by only 20% and mortality by around 5%; however, if the patients are haemodynamic responders, i.e. if the HVPG can be decreased by at least 20% or below 12 mmHg, the rebleeding risk is reduced to 15 20%. The combination with nitrates may be somewhat more potent, but is not standard33,34. According to current trials the most promising initial therapy for rebleeding prophylaxis is continuation of ligation until eradication of varices with concomitant application of a non-selective b-blocker, although adjuvant medical treatment has been questioned by a very recent controlled study. Patients who rebleed under this regimen may receive a TIPS or a distal splenorenal shunt35.
References 1. 2. 3. 4. 5.
Le Moine O, Hadengue A, Moreau R et al. Relationship between portal pressure, esophageal varices, and variceal bleeding on the basis of the stage and cause of cirrhosis. Scand J Gastroenterol. 1997;32:731 5. Groszmann RJ, Garcia Tsao G, Bosch J et al. Beta blockers to prevent gastroesophageal varices in patients with cirrhosis. N Engl J Med. 2005;353:2254 61. Rigau J, Bosch J, Bordas JM et al. Endoscopic measurement of variceal pressure in cirrhosis: correlation with portal pressure and variceal hemorrhage. Gastroenterology. 1989;96:873 80. Garcia Tsao G, Groszmann RJ, Fisher RL, Conn HO, Atterbury CE, Glickman M. Portal pressure, presence of gastroesophageal varices and variceal bleeding. Hepatology. 1985;5:419 24. Barton R, Rosch J, Saxon R, Lakin P. TIPS: short and long term results; a survey of 1750 patients. Semin Intervent Radiol. 1995:364 7.
236
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20. 21. 22. 23. 24. 25. 26.
McCormick PA, O'Keefe C. Improving prognosis following a ¢rst variceal haemorrhage over four decades. Gut. 2001;49:682 5. Carbonell N, Pauwels A, Serfaty L, Fourdan O, Levy VG, Poupon R. Improved survival after variceal bleeding in patients with cirrhosis over the past two decades. Hepatology. 2004;40:652 9. Merkel C, Marin R, Angeli P et al. A placebo controlled clinical trial of nadolol in the prophylaxis of growth of small esophageal varices in cirrhosis. Gastroenterology. 2004;127:476 84. Trebicka J, Hennenberg M, Laleman W et al. Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho kinase and activation of endothelial nitric oxide synthase. Hepatology. 2007;46:242 53. Trebicka J, Neef M, Hennenberg M et al. Atorvastatin inhibits ¢brogenesis through inhibition of activation of hepatic stellate cells. J Hepatol. 2007;46:S134. Zafra C, Abraldes JG, Turnes J et al. Simvastatin enhances hepatic nitric oxide production and decreases the hepatic vascular tone in patients with cirrhosis. Gastroenterology. 2004;126:749 55. Schepke M, Werner E, Biecker E et al. Hemodynamic e¡ects of the angiotensin II receptor antagonist irbesartan in patients with cirrhosis and portal hypertension. Gastroenterology. 2001;121:389 95. Croquet V, Moal F, Veal N et al. Hemodynamic and anti¢brotic e¡ects of losartan in rats with liver ¢brosis and/or portal hypertension. J Hepatol. 2002;37:773 80. Sookoian S, Fernandez MA, Castano G. E¡ects of six months losartan administration on liver ¢brosis in chronic hepatitis C patients: a pilot study. World J Gastroenterol. 2005;11:7560 3. Rincon D, Ripoll C, Lo Iacono O et al. Antiviral therapy decreases hepatic venous pressure gradient in patients with chronic hepatitis C and advanced ¢brosis. Am J Gastroenterol. 2006;101:2269 74. Trebicka J, Leifeld L, Hennenberg M et al. Hemodynamic e¡ects of urotensin II and its speci¢c receptor antagonist palosuran in cirrhotic rats. Hepatology. 2008;47:1264 76. North Italian Endoscopic Club for the Study and Treatment of Esophageal Varices. Prediction of the ¢rst variceal hemorrhage in patients with cirrhosis of the liver and esophageal varices. A prospective multicenter study. N Engl J Med. 1988;319:983 9. Kleber G, Sauerbruch T, Fischer G, Paumgartner G. Pressure of intraoesophageal varices assessed by ¢ne needle puncture: its relation to endoscopic signs and severity of liver disease in patients with cirrhosis. Gut. 1989;30:228 32. Poynard T, Cales P, Pasta L, Ideo G, Pascal JP, Pagliaro L, Lebrec D. Beta adrenergic antagonist drugs in the prevention of gastrointestinal bleeding in patients with cirrhosis and esophageal varices. An analysis of data and prognostic factors in 589 patients from four randomized clinical trials. Franco Italian Multicenter Study Group. N Engl J Med. 1991;324:1532 8. Garcia Tsao G. Current management of the complications of cirrhosis and portal hypertension: variceal hemorrhage, ascites, and spontaneous bacterial peritonitis. Gastroenterology. 2001;120:726 48. Khuroo MS, Khuroo NS, Farahat KL, Khuroo YS, So¢ AA, Dahab ST. Meta analysis: endoscopic variceal ligation for primary prophylaxis of oesophageal variceal bleeding. Aliment Pharmacol Ther. 2005;21:347 61. Schepke M, Kleber G, Nurnberg D et al. Ligation versus propranolol for the primary prophylaxis of variceal bleeding in cirrhosis. Hepatology. 2004;40:65 72. Banares R, Moitinho E, Piqueras B et al. Carvedilol, a new nonselective beta blocker with intrinsic anti alpha1 adrenergic activity, has a greater portal hypotensive e¡ect than propranolol in patients with cirrhosis. Hepatology. 1999;30:79 83. Ioannou GN, Kowdley KV. Review: Antibiotic prophylaxis reduces mortality and bacterial infection in cirrhosis and gastrointestinal bleeding. ACP J Club. 2002;137:94. de Franchis R. Evolving consensus in portal hypertension. Report of the Baveno IV consensus workshop on methodology of diagnosis and therapy in portal hypertension. J Hepatol. 2005;43:167 76. Sung JJ, Chung SC, Yung MY et al. Prospective randomised study of e¡ect of octreotide on rebleeding from oesophageal varices after endoscopic ligation. Lancet. 1995;346:1666 9.
237
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 27. Cales P, Masliah C, Bernard B et al. Early administration of vapreotide for variceal bleeding in patients with cirrhosis. N Engl J Med. 2001;344:23 8. 28. Garcia Pagan JC, Bosch J. Endoscopic band ligation in the treatment of portal hypertension. Nat Clin Pract Gastroenterol Hepatol. 2005;2:526 35. 29. Gross M, Schiemann U, Muhlhofer A, Zoller WG. Meta analysis: e¤cacy of therapeutic regimens in ongoing variceal bleeding. Endoscopy. 2001;33:737 46. 30. Bernard B, Grange JD, Khac EN, Amiot X, Opolon P, Poynard T. Antibiotic prophylaxis for the prevention of bacterial infections in cirrhotic patients with gastrointestinal bleeding: a meta analysis. Hepatology. 1999;29:1655 61. 31. Bernard B, Lebrec D, Mathurin P, Opolon P, Poynard T. Beta adrenergic antagonists in the prevention of gastrointestinal rebleeding in patients with cirrhosis: a meta analysis. Hepatology. 1997;25:63 70. 32. de la Pena J, Brullet E, Sanchez Hernandez E et al. Variceal ligation plus nadolol compared with ligation for prophylaxis of variceal rebleeding: a multicenter trial. Hepatology. 2005;41:572 8. 33. Study Group Spanish Cooperative Variceal Rebleeding. Multicenter RCT comparing drug therapy vs combination of drug therapy + endoscopic variceal ligation in the prevention of rebleeding in patients with cirrhosis. Hepatology. 2006;44:202A. 34. Villanueva C, Minana J, Ortiz J et al. Endoscopic ligation compared with combined treatment with nadolol and isosorbide mononitrate to prevent recurrent variceal bleeding. N Engl J Med. 2001;345:647 55. 35. Henderson JM, Boyer TD, Kutner MH et al.; DIVERT Study Group. Distal splenorenal shunt versus transjugular intrahepatic portal systematic shunt for variceal bleeding: a randomized trial. Gastroenterology. 2006;130:1643 51.
238
23 Surveillance and prevention of hepatocellular carcinoma M. COLOMBO
INTRODUCTION Prevention of hepatocellular carcinoma (HCC) is feasible since the tumour occurs in the context of well-recognized, potentially preventable risk factors1. Strategies of prevention di¡er in the various geographical areas, in accordance with the geographical variability of risk factors. In this chapter primary prevention means preventing the onset of cancer, by lowering an individual's exposure to known risk factors such as hepatitis viruses, alcohol or progressive liver disease. Secondary prevention aims at counteracting HCC severity, i.e. reducing tumour recurrence after eradication. In patients chronically exposed to risk factors for HCC, surveillance is the only practical approach to improve treatment outcome2. Surveillance is the process by which diagnostic tests and recall procedures have been standardized to provide a clinically meaningful de¢nition of early cancer1. Both prevention and surveillance are expected to reduce HCC-related mortality2.
WHO SHOULD BE UNDER SURVEILLANCE? Chronic carriers of the hepatitis B virus (HBV) and patients with compensated cirrhosis of any aetiology are the target population for surveillance (Table 1)1. Since HCC risk increases in parallel with ageing, level of viraemia and severity of the underlying liver disease, di¡erent categories of risk are established with di¡erent priorities for screening1. In Africa, where infection with HBV is often perinatally transmitted from infected mothers and is potentiated by exposure to the oncogenic dietary a£atoxin B, HCC frequently develops early in life prior to development of cirrhosis. In this epidemiological context, surveillance should be instituted earlier in life than in Western carriers1. Cirrhosis is the single, most important risk factor for HCC, which transforms into liver cancer at annual rates of approximately 3%3. This rate, however, may increase in the presence of co-factors known to accelerate progression of HCC such as a£atoxin B 1 , alcohol consumption, iron and overweight, and in livers 239
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY Table 1
Population to be screened1
Patients with cirrhosis of any aetiology Hepatitis B carriers Asian males 540 years Asian females 550 years Family history of HCC African Americans over age 20
expressing histopathological markers of increased cell proliferation, liver cell dysplasia and regenerative macronodules4^9. By combining sex, age and serum alphafetoprotein (AFP) levels, patients with compensated cirrhosis can be strati¢ed in categories at increasing risk for the tumour. The lowest risk group (1.5% rate per year) included women younger than 53 years of age with normal AFP levels compared to men older than 53 year of age with high AFP levels who are at the highest risk (10%) for developing HCC10. In alcoholic patients11, sex, age, disease severity and liver cell dysplasia identi¢ed a subset of patients with an exceedingly high (72%) risk of developing HCC during a follow-up of 3 years. In Spain, similar criteria of disease aetiology and severity (excluding cell dysplasia) led to the identi¢cation of patients at high risk (30.1%) and those at low risk (2.3%) of developing HCC during 4 years of surveillance 12 . Interestingly, patient strati¢cation by clinical and histological scores does not change the time intervals for surveillance, since the growth rates of tumours is the only variable which in£uences the outcome of screening. Patients with severe co-morbidities and those with advanced liver disease (Child Pugh C class patients), who do not ¢t criteria for curative therapies, should not be under surveillance. Surveillance programmes should be restricted to individuals below 30 years and those above 75 years of age, due to the low risk of cancer in younger people, while the older ones would not have signi¢cant bene¢t if diagnosed with a HCC. However, these criteria for selection are not evidence-based and may not apply in all geographical areas.
SURVEILLANCE TESTS Abdominal ultrasound (US) is the method of choice, and 6 months has been selected by most as the ideal interval of surveillance2. The serum assay AFP is no longer considered for surveillance, due to the high rates of false-positive and false-negative results in patients with chronic liver disease2. However, AFP may still have an ancillary role in the few patients with a di¤cult-to-diagnose tumour, such as a tumour that does not appear at US as an expanding node, but silently in¢ltrates the liver1. Tumours with this pattern, however, are encountered quite rarely, can be visualized by spiral computed tomography or magnetic resonance and, most importantly, are not bene¢ted by early diagnosis. The serological marker des-gamma carboxyprothrombin (DCP or PIVKAII), an abnormal prothrombin protein elaborated by the neoplastic liver cells, is 240
SURVEILLANCE AND PREVENTION OF HCC
of limited impact in screening since its serum levels are in£uenced by tumour size and invasiveness13; it is therefore better indicated for assessing disease severity. Other serum markers are under evaluation but none of them have proved to be sensitive or speci¢c enough to be used for screening of patients at risk for HCC1.
THE RECALL POLICY A liver node detected during surveillance needs to be diagnosed either by a USguided liver biopsy or imaging studies (Table 2). Liver biopsy is indicated in patients who are candidated to curative treatments and with a smaller than 1 cm node that escapes diagnosis by imaging techniques. The diagnostic accuracy of US-guided liver biopsy is approximately 70% for nodes between 1 and 2 cm in size, largely depending on operator expertise, and the size and location of the tumour14. Overall, the biopsy procedure is associated with a negligible risk of morbidity, mortality, and seeding15. Table 2
Criteria applied for con¢rming HCC in patients with a node detected by ultrasound
Histological criteria (liver biopsy) Non invasive criteria (restricted to cirrhotic patients) Lesion has nodular con¢guration Lesion is at least 1 cm in longest diameter* Lesion shows arterial hypervascularization: Hyper enhanced nodule in the arterial phase by two imaging techniques** Hyper enhanced nodule in the arterial phase and as hypo enhanced nodule in the portal venous or delayed phase by one imaging technique** *Apply to lesions emerged during US surveillance. For lesions detected at ¢rst imaging examination, lesion diameter should be at least 2 cm to allow non-invasive diagnosis of HCC. **Imaging techniques include: contrast-enhanced US, contrast-enhanced spiral CT, and gadoliniumenhanced MRI.
The diagnostic algorithm based on imaging applies only to patients with cirrhosis. The cirrhotic liver is mainly supplied by portal venous blood compared to HCC which is nourished by arteries. The applicability of imaging, however, is limited in nodes 51 cm due to the low number of unpaired arteries (Figure 1). The applicability of imaging is better for nodes 41 cm since the number of unpaired arteries increases in parallel with increasing size of the tumour16. Most nodules 51 cm are not detected by liver biopsy either, but require monitoring with US every 3 months (enhanced follow-up) for ¢nal diagnosis. Nodules 51 cm identi¢ed during surveillance, and those of at least 2 cm detected at ¢rst imaging examination, need to be investigated with two coincident techniques among contrast-US, spiral CT and MR. However, a single imaging technique is enough to diagnose a HCC, wh e n eve r wa sh- ou t of c o ntra s t m e d iu m fol low i ng ar te r i al hypervascularization can be demonstrated. Expert radiologists can rule out 241
I Mass on surveillance ultrasound (US) in a cirrhotic liver I +
+
1-2 em
I Repeat US at 3-4 1
I Two dynamic imaging studies I
..
+
month intervals
J
1 Stable over 24 1 months
+ Coincidental typical vascular pattern on dynamic imaging
I Enlarging I
+
+
+ Atypical vascular pattern with both techniques
~
242
Non diagnostic
Proceed according to lesion size
~ Typical vascular pattern on dynamic imaging or AFP > 200 ng/ml
I
I
+ I Repeat biopsy or imaging follow-up
r-
I Other diagnosis I
+
I Change in size/profile I + Repeat imaging and/or biopsy
I Positive I Figure 1
.--L Atypical vascular pattern
Biopsy
I Diagnosis of HCC I
I
I One dynamic imaging technique I
Typical vascular pattern with one technique
I
1 Return to standard surveillance protocol (6 monthly)
I
> 2 em
•
I Negative
Treat as hepatocellular carcinoma
Diagnostic algorithm for hepatocellular carcinoma (HCC) developing in patients with cirrhosis (ref. 1)
I
•
I
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
I
I<1 cml
SURVEILLANCE AND PREVENTION OF HCC
false-positive results due to arteriovenous shunts or macroregenerative nodules with dysplastic liver cells17 and false-negative results with imaging that may occur when searching for very early tumours. Indeed, 20% of all HCC 1 2 cm in size appear as hypoattenuated nodes with contrast imaging techniques18, and close follow-up of these nodes will demonstrate that two-thirds of them will convert to hyperattenuation in 3 years time, transforming into distinctly nodular tumours, often through a nodule-in-nodule process19. High-grade dysplastic macronodules (HGDN) can be con¢dently discriminated from an early HCC by expert pathologists following exclusion of microscopic stromal invasion20. Structural and functional analysis of the genetic pro¢le of the nodules may be of diagnostic help since they reveal the existence of a subset of progression-associated genes, which distinguishes dysplastic nodules from early HCC, with great accuracy21. Early cancer can also be di¡erentiated from HGDN for overexpression of the gene GPC3 and surviving together with underexpression of LYVE-3, or by immunochemistry for GPC-322. Interestingly, most HGDN with carcinogenetic potential already show an oncogene-speci¢c gene expression signature found in mature HCC23.
DOES SURVEILLANCE REDUCE LIVER-RELATED MORTALITY? One randomized controlled study24 based on semi-annual investigations with US and AFP in 18 816 Shanghai urban residents with chronic hepatitis, demonstrated a 37% increase in survival for the individuals under surveillance. Reanalysis of several clinic-based studies suggested, but did not prove, a reduction in liver-speci¢c mortality rates in patients with an HCC detected during surveillance compared to patients with an incident cancer2. These studies, however, were somehow biased by the retrospective selection of patients and di¡erences in tumour staging, and type of treatments. In a reanalysis of 112 patients with a HCC detected during surveillance in Milan, patients treated for a liver cancer during the last 5 years of surveillance survived longer than those treated previously (90% vs 55%, p = 0.0009)10. Increased survivals could be con¢dently attributed to a signi¢cant reduction in the mortality rates of treated patients (from 34% to 5%, p = 0.003), due to wider application of curative treatments and improved selection of patients undergoing ablative treatments10. Surveillance appears to be cost-e¡ective, i.e. the costs of detection, con¢rmatory studies and treatment are outbalanced by the number of life-years gained. In one study the standard threshold for coste¡ectiveness was calculated to be US$ 50 000 per quality-adjusted life year (qualy)25. In other studies in patients with compensated cirrhosis, an increase in the mean life expectancy with cost-e¡ectiveness ratios between US$ 26 000 and 55 000 per qualy, was calculated26^28. A retrospective study of patients with compensated cirrhosis in Italy showed an incremental cost-e¤cacy ratio of surveillance vs no surveillance to be US$ 112 993 per life-year saved, the cost of surveillance being increased by surgery applied to 15 patients with a HCC detected during surveillance29.
243
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
PRIMARY PREVENTION Several public health measures may reduce the population's exposure to the known aetiological agents of HCC. These include also the use of arti¢cial substances or dietary interventions aimed at preventing liver disease, altering susceptibility to HCC, and slowing or preventing progression to cirrhosis.
Preventing liver disease In developed countries, improved infection control standards, improvements in the screening of blood donors, and the hepatitis B vaccination programmes started in the mid-1980s have greatly reduced the burden of chronic hepatitis among the general population 30. In Taiwan, HBV transmission in early childhood was interrupted by mass vaccination of all newborns, that was started in the 1980s and caused a reduction of the incidence for liver cancer in children of 6 14 years of age (from 0.57 to 0.36 cases per 100 000) 31 . Investments in educational programmes might e¤ciently counteract alcoholrelated HCC, which accounts for up to 40% of all HCC. In developing countries, post-transfusion hepatitis is still a major medical problem, since approximately half of blood units are not screened for hepatitis viruses (and HIV) and transmission of viral hepatitis in the community is boosted by poor sanitary conditions, shortage of disposable medical instruments and sharing of personal instruments32. Spread of HBV in many countries is also facilitated by economic constraints halting the HBV vaccine being incorporated into national immunization programmes. Further, in vast regions of Asia and Africa, the risk of HCC is boosted by diet contaminated with a£atoxins, i.e. mycotoxins produced by Aspergillus £avus fungi, which increase the risk of HCC in patients with chronic HBV infection33. Improved crop storage and handling to limit fungal growth or genetic engineering of crops to become resistant to fungal infection or toxin biosynthesis, however, require important economic investments.
Altering susceptibility to HCC Chemopreventive agents such as the antischistosomial drug oltipraz and chlorophyllin, a¡ect both the metabolism and disposition of a£atoxin and can be used to alter individuals' susceptibility to HCC. In a phase II randomized, placebo-controlled, double-blind study in adults with detectable serum a£atoxin albumin adduct levels, 1 month therapy with weekly oltipraz led to a signi¢cant decrease in a£atoxin M1 excreted in urine, whereas sustained daily low-dose oltipraz increased conjugation of a£atoxin33. The cheaper and safer chlorophyllin tested in a randomized, double-blind, placebo-controlled study in Qidong, showed a 55% reduction in urinary excretion of a£atoxin biomarkers34.
244
SURVEILLANCE AND PREVENTION OF HCC
Slowing progression of liver disease Antiviral treatments, aimed at stopping hepatitis progression by attenuating liver cell in£ammation, are expected to prevent or delay appearance of the tumour.
Hepatitis B-related HCC In HBeAg seropositive patients, interferon a therapy may lead to anti-HBe seroconversion and attenuation of disease progression29. The clinical bene¢t of anti-HBe seroconversion is not universal, however, since the risk of HCC largely depends on disease severity at the time of anti-HBe seroconversion35. In anti-HBe seropositive chronic hepatitis B the goal of therapy is long-term suppression of viraemia and disease activity and, eventually, anti-HBs seroconversion. Twelve to 24 months of interferon therapy, or the inde¢nite administration of nucleos(t)ide analogues, may serve the purpose. In two studies in Europe, HCC developed less frequently in interferon responders than in non-responders36,37, but this was not replicated by others38. All interferon studies, however, were somehow £awed by methodological problems such as heterogeneity of the patients enrolled, which prevents comparison between treatment subgroups, and lack of randomization which prevents control for disease severity, which is a relevant independent predictor for HCC. In a retrospective multicentre study of 656 Italian patients treated with lamivudine (46% with cirrhosis), HCC developed in 31 during 4 years of follow-up, with preference for cirrhotic patients who broke through during treatment39 (Figure 2). One disputed point is whether sustained response to
Figure 2 A retrospective cohort study of HBeAg negative cirrhotics treated with lamivudine in Italy. Hepatocellular carcinoma (HCC) developed more frequently in patients with a virological breakthrough than in those with sustained virus suppression
245
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Figure 3 A randomized controlled study of lamivudine in Asian patients with advanced chronic hepatitis B. Hepatocellular carcinoma (HCC) was detected less frequently in lamivudine treated patients than in controls
analogues helps prevent HCC. A prospective study in 124 cirrhotic patients (19% decompensated) under chronic treatment with lamivudine or lamivudine + adefovir, showed HCC to develop in 26% long-term responders, independently of baseline characteristics40. To what extent lamivudine therapy may prevent HCC in patients with advanced ¢brosis is also questioned by a large randomized controlled study enrolling both HBeAg-positive and HBeAgnegative Asian patients (Figure 3). During 34 months of study a statistical di¡erence between 7.4% and 3.9% in HCC incidence between untreated and treated patients, was observed that, however, would be nulli¢ed if patients with a HCC detected during the ¢rst year of study are removed from analysis41.
Hepatitis C-related HCC A meta-analysis of 17 trials originally designed to test the virological response in patients with hepatitis C virus (HCV)-related cirrhosis42, showed a slight preventive e¡ect of interferon on HCC development in cirrhotic patients compared to non-cirrhotic patients. The interferon seemed more active in preventing HCC among patients with a sustained biochemical response to therapy, which however represent a small fraction of all cirrhotic patients. Since 1990 a prospective cohort study of patients with chronic hepatitis C was established in Fukuoka, Japan, to assess the long-term e¤cacy of interferon alpha and beta in preventing HCC43. The risk of HCC in treated patients was 1.4% per year compared to historical controls (3 6%), and it was higher in nonresponders to interferon alpha than in responders (11.4% vs 0.8%, p50.05). While no di¡erences were noted between responders and non-responders to either interferon, the risk of HCC appeared to depend upon the ALT change after therapy (p ( = 0.01). More recently the Tokyo Chiba Hepatitis Research 246
SURVEILLANCE AND PREVENTION OF HCC
Figure 4 A prospective cohort study comparing 271 patients with HCV cirrhosis treated with interferon to 74 untreated patients, showing reduced rates of hepatocellular carcinoma (HCC) among treated patients
Group44 reported results of a prospective study in 271 patients with HCVcirrhosis treated with interferon for 26 88 weeks and 74 patients who declined treatment (Figure 4). During a median follow-up of 6.8 years, HCC developed more frequently in untreated than in treated patients (47% vs 31%), and, among the latter patients, in those who did not respond to therapy than in responders (35.3% vs 17.2%). Mortality rates were also better in treated than in untreated patients (17% vs 32%). The optimistic results of this study, however, should be accepted with caution, since the study did not address the reasons for refusing therapy with interferon while prognostic factors other than interferon might have contributed to the survival di¡erences between groups. Retrospective studies in Japan45, Taiwan46, and Italy47, suggested an increase in HCC incidence and liver-related mortality in patients with chronic hepatitis C who were left untreated, as well as in non-responders to interferon alpha. In 920 Italian patients with HCV-cirrhosis, achievement of a sustained virological response after interferon alpha monotherapy was associated with a reduction of liver-related mortality, as a consequence of the reduced risk of decompensation and HCC47.
Secondary prevention HCC recurs in 75% of patients within 5 years following ablation1,2. Tumour recurrences detected shortly after treatment probably represent microscopic metastases from the primary tumours, substantially re£ecting a failure of the treatment of controlling local recurrence. Conversely, recurrences appearing 247
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
late after ablation of a HCC probably represent second primary tumours induced by multicentric carcinogenesis. Adjuvant therapy therefore di¡ers according to the nature of the recurrent tumour. While e¡ective chemotherapy is not available to prevent recurrences related to microscopic metastases from a primary HCC, treatment with polyprenoic acid, transhepatic arterial embolization with 131I lipiodol and adoptive immunotherapy have been e¡ective in preventing second primary recurrences 2 . In a randomized controlled study, survival rates at 6 years were 74% in the treated group vs 46% in the untreated group48 (p ( = 0.04). Intra-arterial lipiodol 131I irradiation of the remnant liver after hepatic resection was associated with a 50% decrease in HCC recurrence and mortality rates during an average follow-up of 35 months in one study49, whereas a second study showed less impressive changes in the survival ¢gures (3-year survival of 92% in treated vs 71% in untreated)50. Adoptive immunotherapy with autologous lymphocytes caused a 41% reduction of the risk of recurrence and a signi¢cant prolongation of the HCCfree survival51. There is little evidence that interferon prevents recurrence of HCC. In a study of 150 Caucasian patients with HCV-related HCC treated with curative resection, 48 weeks therapy with interferon alpha did not reduce recurrence-free survival52; however, there was a 50% reduction rate of late recurrence in the treatment arm and a signi¢cant bene¢t on late recurrences in HCV patients lacking markers of previous HBV infection.
CONCLUSIONS In resource-rich countries prevention of viral hepatitis through screening of blood donors and universal vaccination against HBV of all newborns, probably resulted in a reduction of HCC risk. This remains a di¤cult-to-reach objective in resource-poor countries, where 50% of all donations are not screened for hepatitis B and C and 25 million newborns every year do not receive basic immunization. Current evidence does not support the theory that interferon and anti-HBV analogues prevent HCC, especially in patients who have already developed cirrhosis, i.e. those who are at greatest risk of developing liver cancer. US surveillance every 6 months favours early detection of HCC, which in turn may increase the application of curative treatments. Though e¤cacy of surveillance to reduce liver-speci¢c mortality is not evidence-based, studies of decision analysis indicate that surveillance may bene¢t HCC patients in terms of survival. The cost-e¡ectiveness of surveillance could be improved by more accurate selection of at-risk patients by means of newly developed molecular tests for screening.
References 1. 2. 3.
Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology. 2005;42:1208 36. Bruix J, Sherman M, Llovet JM et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona 2000 EASL Conference. J Hepatol. 2001;35:421 30. Colombo M, de Franchis R, Del Ninno E et al. Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl J Med. 1991;325:675 80.
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Sun Z, Lu P, Gail MH et al. Increased risk of hepatocellular carcinoma in male hepatitis B surface antigen carriers with chronic hepatitis who have detectable a£atoxin metabolite M1. Hepatology. 1999;30:379 83. Makino Y, Shiraki K, Sugimoto K et al. Histological features of cirrhosis with hepatitis C virus for prediction of hepatocellular carcinoma development; a prospective study. Anticancer Res. 2000;20:3709 16. Donato MF, Arosio A, Del Ninno E et al. Liver cell proliferation predicts hepatocellular carcinoma and long term survival in patients with compensated cirrhosis. Hepatology. 2001;34:523 8. Borzio M, Bruno S, Roncalli M et al. Liver cell dysplasia is a major risk factor for hepatocellular carcinoma in cirrhosis: a prospective study. Gastroenterology. 1995;108:812 17. Kobayashi M, Ikeda K, Hosaka T et al. Dysplastic nodules frequently develop into hepatocellular carcinoma in patients with chronic viral hepatitis and cirrhosis. Cancer. 2006;106:636 47. Park NY, Roncalli M. Large liver cell dysplasia: a controversial entity. J Hepatol. 2006;45:734 43. Sangiovanni A, Del Ninno E, Fasani P et al. Increased survival of cirrhotic patients with a hepatocellular carcinoma detected during surveillance. Gastroenterology. 2004;126:1005 14. Ganne Carrie N, Chastang C, Chapel F et al. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in western patients with cirrhosis. Hepatology. 1996;23:1112 18. Velazquez RF, Rodriguez M, Navascues CA et al. Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology. 2003;37:520 7. Marrero JA, Su GL, Wei W et al. Des gamma carboxyprothrombin can di¡erentiate hepatocellular carcinoma from nonmalignant chronic liver disease in American patients. Hepatology. 2003;37:1114 21. Borzio M, Borzio F, Macchi R et al. The evaluation of ¢ne needle procedures for the diagnosis of focal liver lesions in cirrhosis. J Hepatol. 1994;20:117 21. Buscarini L, Fornari F, Bolondi L et al. Ultrasound guided ¢ne needle biopsy of focal liver lesions: techniques, diagnostic accuracy and complications. A retrospective study on 2091 biopsies. J Hepatol. 1990;11:344 8. Kojiro M. The evolution of pathologic features of hepatocellular carcinoma. In: Tabor E, editor. Viruses and Liver Cancer: Perspectives in Medical Virology. Amsterdam: Elsevier, 2002:113 22. Fracanzani AL, Burdick L, Borzio M et al. Contrast enhanced Doppler ultrasonography in the diagnosis of hepatocellular carcinoma and premalignant lesions in patients with cirrhosis. Hepatology. 2001;34:1109 12. Bolondi L, Gaiani S, Celli N et al. Characterization of small nodules in cirrhosis by assessment of vascularity: the problem of hypovascular hepatocellular carcinoma. Hepatology. 2005;42:27 34. Takayasu K, Muramatsu Y, Mizuguchi Y et al. CT evaluation of the progression of hypoattenuating nodular lesions in virus related chronic liver disease. Am J Roentgenol. 2006;187:454 63. Kojiro M, Roskams T. Early hepatocellular carcinoma and dysplastic nodules. Semin Liver Dis. 2005;25:133 42. Nam SW, Park JY, Ramasamy A et al. Molecular changes from dysplastic nodule to hepatocellular carcinoma through gene expression pro¢ling. Hepatology. 2005;42:809 18. Llovet JM, Chen Y, Wurmbach E et al. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology. 2006;131:1758 67. Thorgeirsson SS, Lee JS, Grisham JW. Molecular prognostication of liver cancer: end of the beginning. J Hepatol. 2006;44:798 805. Zhang BH, Yang BH, Tang ZY. Randomized controlled trial of screening for hepatocellular carcinoma. J Cancer Res Clin Oncol. 2004;130:417 22. Drummond MF, O'Brien B, Stoddart GL et al. Cost e¡ectiveness analysis. In: Drummond MF, O'Brien B, Stoddart GL, Torrance GW, editors. Methods for the Economic Evaluation of Health Care Programmes, 2nd edn. New York: Oxford University Press, 1997:96 138.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 26. Sarasin FP, Giostra E, Hadengue A. Cost e¡ectiveness of screening for detection of small hepatocellular carcinoma in western patients with Child Pugh class A cirrhosis. Am J Med. 1996;101:422 34. 27. Arguedas MR, Chen VK, Eloubeidi MA et al. Screening for hepatocellular carcinoma in patients with hepatitis C cirrhosis: a cost utility analysis. Am J Gastroenterol. 2003;98:679 90. 28. Lin OS, Kee¡e EB, Sanders GD et al. Cost e¡ectiveness of screening for hepatocellular carcinoma in patients with cirrhosis due to chronic hepatitis C. Aliment Pharmacol Ther. 2004;19:1159 72. 29. Bolondi L, So¢a S, Siringo S et al. Surveillance programme of cirrhotic patients for early diagnosis and treatment of hepatocellular carcinoma: a cost e¡ectiveness analysis. Gut. 2001;482:251 9. 30. Spada E, Mele A, Ciccozzi M et al. Changing epidemiology of parenterally transmitted viral hepatitis: results from the hepatitis surveillance system in Italy. Dig Liver Dis. 2001;33:778 84. 31. Chang MH, Chen CJ, Lai MS et al. Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. Taiwan Childhood Hepatoma Study Group. N Engl J Med. 1997;336:1855 9. 32. Prati D. Transmission of hepatitis C virus by blood transfusions and other medical procedures: a global review. J Hepatol. 2006;45:607 16. 33. Wang JS, Shen X, He X, et al. Protective alterations in phase 1 and 2 metabolism of a£atoxin B1 by oltipraz in residents of Qidong, People's Republic of China. J Natl Cancer Inst. 1999;91:347 54. 34. Egner PA, Wang JB, Zhu YR et al. Chlorophyllin intervention reduces a£atoxin DNA adducts in individuals at high risk for liver cancer. Proc Natl Acad Sci USA. 2001;98:14601 6. 35. Yang HI, Lu SN, Liaw YF et al. Hepatitis B e antigen and the risk of hepatocellular carcinoma. N Engl J Med. 2002;347:168 74. 36. Papatheodoridis GV, Manesis E, Hadziyannis SJ. The long term outcome of interferon alpha treated and untreated patients with HBeAg negative chronic hepatitis B. J Hepatol. 2001;34:306 13. 37. Brunetto MR, Oliveri F, Coco B et al. Outcome of anti HBe positive chronic hepatitis B in alpha interferon treated and untreated patients: a long term cohort study. J Hepatol. 2002;36:263 70. 38. Lampertico P, Del Ninno E, Vigano M et al. Long term suppression of hepatitis B e antigen negative chronic hepatitis B by 24 month interferon therapy. Hepatology. 2003;37:756 63. 39. Di Marco V, Marzano A, Lampertico P et al. Clinical outcome of HBeAg negative chronic hepatitis B in relation to virological response to lamivudine. Hepatology. 2004;40:883 91. 40. Lampertico P, Vigano M, Manenti E et al. Five years of sequential Lam to Lam+Adv therapy suppresses HBV replication in most HBeAg negative cirrhotics, preventing decompensation but not hepatocellular carcinoma. J Hepatol. 2006;43;S38. 41. Liaw YF, Sung JJY, Chow WC et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med. 2004;351:1521 31. 42. Crax| A, Camma C. Prevention of hepatocellular carcinoma. Clin Liver Dis. 2005;9:329 46. 43. Kashiwagi K, Furusyo N, Kubo N et al. A prospective comparison of the e¡ect of interferon alpha and interferon beta treatment in patients with chronic hepatitis C on the incidence of hepatocellular carcinoma development. J Infect Chemother. 2003;9:333 40. 44. Shiratori Y, Ito Y, Yokosuka O et al. Antiviral therapy for cirrhotic hepatitis C: association with reduced hepatocellular carcinoma development and improved survival. Ann Intern Med. 2005;142:105 14. 45. Yoshida H, Arakawa Y, Sata M et al. Interferon therapy prolonged life expectancy among chronic hepatitis C patients. Gastroenterology. 2002;123:483 91. 46. Yu ML, Lin SM, Chuang WL et al. A sustained virological response to interferon or interferon/ribavirin reduces hepatocellular carcinoma and improves survival in chronic hepatitis C: a nationwide, multicentre study in Taiwan. Antivir Ther. 2006;11:985 94.
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SURVEILLANCE AND PREVENTION OF HCC 47. Bruno S, Stro¡olini T, Colombo M et al. Sustained virological response to interferon alpha is associated with improved outcome in HCV related cirrhosis: a retrospective study. Hepatology. 2007;45:579 87. 48. Muto Y, Moriwaki H, Ninomiya M et al. prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. N Engl J Med. 1996;334:1561 7. 49. Lau WY, Leunt TWT, Ho SKW et al. Adjuvant intra arterial lipiodol iodine 131 for resectable hepatocellular carcinoma: a prospective randomised trial. Lancet. 1999;353:797 801. 50. Boucher E, Corbinais S, Rolland Y et al. Adjuvant intra arterial injection of iodine 131 labeled lipiodol after resection of hepatocellular carcinoma. Hepatology. 2003;38:1237 41. 51. Takayama T, Sekine T, Makuuchi M et al. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet. 2000;356:802 7. 52. Mazzaferro V, Romito R, Schiavo M et al. Prevention of hepatocellular carcinoma recurrence with alpha interferon after liver resection in HCV cirrhosis. Hepatology. 2006;44:1543 54.
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24 Algorithms of diagnosis and options of hepatocellular carcinoma therapy A. FORNER and J. M. LLOVET
INTRODUCTION In the past two decades hepatocellular carcinoma (HCC) has experienced a progressive increase in its incidence, at present constituting the sixth most common cancer worldwide, the third cause of cancer-related mortality1 and the main cause of death among cirrhotic patients2. These facts are mostly due to the long-term impact of chronic hepatitis C virus (HCV) infection, along with the increase in early detection3. In developed areas cirrhosis underlies HCC in the majority of cases, and this preneoplastic entity is considered the main risk factor for HCC. The available potential curative treatments (surgical resection, liver transplantation and percutaneous ablation) are applicable in only a relatively small proportion of asymptomatic early-stage cases. For this reason surveillance for HCC in cirrhotic patients is recommended, and relies on biannual abdominal ultrasound in patients who would bene¢t from e¡ective treatment if HCC were diagnosed4,5.
DIAGNOSIS An adequate diagnosis of liver nodules detected by screening ultrasound is a major clinical challenge. Pathological analysis has been the only conventional way to achieve a conclusive HCC diagnosis. However, its accuracy is far from optimal, particularly in nodules smaller than 2 cm, where high rates of falsenegative results due to sampling error or misdiagnosis between high-grade dysplasic nodules and HCC have been reported. During recent years improvements in imaging techniques have made possible to identify a speci¢c vascular pattern of HCC during dynamic imaging studies characterized by an intense contrast uptake during the arterial phase followed by washout in the delayed venous phase (Figure 1). Taking these premises into account, the American Association for the Study of Liver Diseases (AASLD) has recently published a strategy for non-invasive diagnosis of nodules detected by ultrasound (US) screening in cirrhotic patients 4, which is summarized in Figure 2. 252
DIAGNOSIS AND OPTIONS OF HCC THERAPY
Figure 1 MRI scan of a patient a¡ected with liver cirrhosis and HCC. The upper panel shows an intense vascular intake during the arterial phase after contrast administration. The lower panel illustrates the speci¢c contrast washout during the delayed phase
Brie£y, HCC diagnosis can be con¢dently established in cirrhotic patients if liver nodules present a contrast uptake in the arterial phase followed by washout in venous/delayed phases in dynamic imaging studies (contrastenhanced ultrasound (CEUS), dynamic computed tomography (CT) or magnetic resonance imaging (MRI) with gadolinium). In nodules between 1 and 2 cm this speci¢c vascular pattern should be demonstrated coincidentally in two imaging techniques. Lesions showing an atypical vascular pattern need histological con¢rmation. Finally, nodules smaller than 1 cm are di¤cult to diagnose, and thus a closer follow-up is recommended; if growth is detected the recall strategy according to size will be applied. These recommendations, based 253
&Urveilance ptOtoocl
(6-12 monthly)
Traa! as llepa1ociAJiar cardnama
Figure 2 Diagnostic strategy to apply upon US detection of a hepatic nodule in patients with cirrhosis. Reproduced with permission from ref. 4
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
254 Return 10 auondard
DIAGNOSIS AND OPTIONS OF HCC THERAPY
on several cohort and case control studies, have recently been validated in a prospective study evaluating the diagnostic accuracy of CEUS and MRI in solitary nodules between 5 and 20 mm detected by US screening6. In this study the coincidental ¢nding of the speci¢c vascular pattern by CEUS and MRI was associated with a speci¢city and positive predictive value of 100%, thus validating the AASLD proposal. This non-invasive criterion, though, was met in only 33% of cases, and thus in the majority of cases the conclusive HCC diagnosis is based on pathological ¢ndings. Even in expert hands a 30% falsenegative rate of HCC has been reported, highlighting the di¤culty of diagnosis of HCC in non-referral centres. For that reason a more standardized diagnosis of early HCC is needed, considering the switch in diagnosis experienced recently in the West, where it is expected that in coming years 20 30% of tumours will be less than 2 cm in size by the time of diagnosis. Recent studies based on gene expression pro¢les have identi¢ed a three-gene set (Glypican-3, survivin and LYVE1) able to discriminate between dysplastic nodules and HCC 52 cm with an accuracy of 85 90%, unparalleled by conventional means7,8. In addition, positive immunostaining of Glypican-3 was able to discriminate between the two entities, con¢rming previous reported results.
PROGNOSIS Prognosis prediction is key to determining the best therapeutic option and to giving correct information to patients and their relatives. HCC is a complex disease. Prognosis assessment in these patients requires taking into account variables related to the tumour stage and the liver function status, because cirrhosis is present in the majority of patients. Unidimensional systems that consider only tumour stage, such as tumour node metastasis (TNM), or liver dysfunction, such as Child Pugh score, are inaccurate in classifying HCC patients. In the past decade several prognosis prediction systems have been proposed9. The Barcelona Clinic Liver Cancer (BCLC) system has been endorsed by the AASLD and the European Association for the Study of the Liver (EASL)10,11 and validated by European and American cohorts, and it is the only one that links prognostic predictors to treatment indication. Brie£y, the BCLC system considers ¢ve main prognostic groups (Figure 3): 1. Very early stage (stage 0): patients with single HCC less than 2 cm, with well-preserved liver function without signi¢cant portal hypertension; candidates to surgical resection ¢t into the so-called very early stage (BCLC 0). In this particular case the expected 5-year survival exceeds 75%. 2. Early stage (BCLC A): patients with well-preserved liver function and an asymptomatic single HCC or up to three nodules each less than 3 cm. Such patients will bene¢t from curative therapies, including resection, liver transplantation (LT), and percutaneous ablation, and the expected 5-year survival is 50 75%.
255
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
3. Intermediate stage (BCLC B): patients with HCC exceeding these limits, but who have preserved liver function and are free of cancer-related symptoms and vascular invasion or extrahepatic spread. Such patients may bene¢t from palliation with chemoembolization, with an expected 2-year survival of 60%. 4. Advanced stage (BCLC C): patients with mild cancer-related symptoms and/ or vascular invasion or extrahepatic spread, but with maintained liver function. In this stage no treatment until now has conclusively shown survival bene¢t, and these patients have been candidates to evaluate new agents. Recently, sorafenib, an orally available multikinase inhibitor, has shown survival improvement in a randomized placebo-controlled trial (RCT), and it is expected that sorafenib will be considered as the standard of care for advanced HCC among scienti¢c societies and regulatory agencies. 5. Terminal stage (BCLC D): this includes patients with great tumour burden (Okuda III) or severe cancer-related symptoms. Child Pugh class C patients with tumours beyond the transplantation threshold also ¢t into terminal stage. The expected survival is less than 3 months; unnecessary su¡ering should be avoided and palliative treatments should be recommended. In the coming years e¡orts will be directed to de¢ning a molecular-based prognostic system that will allow more accurate patient classi¢cation and will help in the treatment decision process12.
TREATMENT As mentioned previously, the treatment decision depends on tumour staging. Taking into account the proposed BCLC approach, patients with early disease may bene¢t from potential curative treatments: surgical resection, percutaneous ablation and liver transplantation. Disappointingly, despite the wide use of screening programmes in developed countries, less than 50% of patients are diagnosed at an early stage and only palliative treatment can be o¡ered. In well-selected patients with intermediate HCC the only treatment that has shown bene¢t in terms of survival is transarterial chemoembolization (TACE). Patients with advanced disease may bene¢t from emerging therapies, and until now the only one that has shown survival bene¢t is sorafenib. Other new agents, mainly acting through disrupted molecular pathways, are currently under evaluation.
Surgical resection Hepatic resection is the treatment of choice for HCC in non-cirrhotic patients (5% of cases in the West, 40% in Asia), in whom major resections can be performed with low rates of life-threatening complications. However, in cirrhotic patients hepatic resection is not always feasible and selection of ideal 256
R CC
STAGEO
STAGE A-C
STAGE D
Child A: PST 0
Okuda 1·2: Child A·B: PS 0·2
Okuda 3: Child C: PS >2
I
Single<2cm
Carcinoma in situ
*
EARLY STAGE A
*
INTERMEDIATE STAGE
Single or 3 nodules
Multinodular. f>ST 0
*
l
ADVANCED STAGE C Porml invasion. Nl. MI . PST 1-2
TERMINAL STAGED
257
1~ 1
l
S in<>le
3 nodules ~3 em
Portal pressure bi lirubin
+ Norma l ---+ Inc reased ---+
No
Associated diseases 4
1
~ Yes CHEMOEMBOLIZATION
NEW AGENTS
CURATIVE TREATMENT (30%)
RANDOM IZED CONTROLLED TRIALS (30%)
SYMPTOMATIC lTO (30%)
5-year survival: 50-70%
3-year survival: 20-40%
1-year survival: I0%
Figure 3 Strategy for staging and treatment assignment in patients diagnosed with HCC according to the BCLC proposal. Adapted with permission from ref. 4. TTO, treatment
DIAGNOSIS AND OPTIONS OF HCC THERAPY
1
VERY EARLYSTAG EO
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
candidates involves an adequate assessment of the liver functional reserve and tumour extension. In that sense surgical resection should be reserved for optimal candidates presenting a solitary HCC with compensated cirrhosis, normal bilirubin levels and absence of clinically signi¢cant portal hypertension (platelet count 5100 000/mm 3 , splenomegaly, oesophageal varices, and hepatic vein pressure gradient 510 mmHg)4,13. As a result of advances in patient selection, surgical technique and postoperative management, the modern standards for resection for HCC in cirrhotic patients include perioperative mortality below 3%, transfusion rate less than 10%, and a 5-year survival rate of at least 50%13. The main drawback of resection is the high rate of recurrence during follow-up, which may reach 70% at 5 years14. From genomic investigations it has been determined that 60 70% of recurrences are intrahepatic metastases undetected at the time of resection, appearing during the ¢rst 2 years, whereas 30 40% are de novo tumours, which occur later, usually more than 2 years after resection. Some pathological characteristics, such as vascular invasion, satellites and poor di¡erentiation degree, can predict early recurrence 15, and we have proposed enlisting the patient for liver transplantation before the appearance of recurrence when analysis of the resected specimen reveals vascular invasion and/or additional satellites16. S everal approaches to reduce the recurrence rate13, su ch as chemoembolization, immunotherapy with activated lymphocytes, internal radiation, retinoids, and interferon administration17, have been evaluated but, despite promising results with all of them, data lack su¤cient strength to introduce them in routine clinical practice and their preventive value and survival impact need further investigation.
Liver transplantation Two decades ago the recruitment of patients with far-advanced disease was associated with unacceptable results that brought into debate the adequacy of orthotopic liver transplantation (OLT) as a treatment for HCC. However, the ¢nding that patients with incidental asymptomatic tumours had the same outcome as patients with non-malignant disease encouraged re-evaluation of liver transplantation as an e¤cient treatment in well-selected patients. In this respect in 1996 Mazzaferro and collaborators reported a pivotal study showing a 4-year survival rate of 74% and a recurrence rate of 8% when OLT was performed in patients with a single HCC 45 cm or up to three nodules each 53 cm 18 . Other groups have con¢rmed these results, and early HCC accomplishing these restrictive criteria has become an unquestionable transplantation indication. However, the situation has changed radically. The shortage of donors has led to a progressive increase in waiting time, with dropout rates exceeding 20% and reduced survival according to the intentionto-treat principle 14 . Several approaches to improve outcome after liver transplantation according to the intention-to-treat principle have been proposed. First, increasing the pool of donors by the use of living donors, domino/split liver transplantation, or high-risk donors. Living donor liver transplantation (LDLT) has become the most feasible alternative to cadaveric liver transplantation and a cost-e¡ectiveness study has shown that it is 258
DIAGNOSIS AND OPTIONS OF HCC THERAPY
bene¢cial when the waiting time is expected to exceed 7 months19. However, its real low applicability, and concerns about the donors' risk and postoperative complications, mainly of biliary origin, temper the potential bene¢t of this approach. Second, prioritizing those patients with a high probability of progression and dropout; however, there are still no good proposals for this issue, and the model for end-stage liver disease (MELD) system, widely applied in the USA and Europe, has failed to properly prioritize HCC patients with preserved liver function. Finally, several adjuvant therapies while waiting have been evaluated. Chemotherapy has failed to show bene¢t and locoregional treatments (such as percutaneous ablation and chemoembolization) may reduce progression and, despite the absence of robust scienti¢c evidence of a survival improvement, they are frequently used. A cost-e¡ectiveness analysis using a Markov model suggested a survival bene¢t for adjuvant treatments when waiting times go beyond 6 months20. Future strategies could be based on the development of molecular targeted agents that, while they can be safely applied in all patients, would prevent, or at least delay, tumour progression. Finally, taking into account the pivotal role of mammalian target of rapamycin (mTOR) activation in cellular proliferation, blocking this target may be an attractive approach to decrease the probability of recurrence after OLT during follow-up21, and several mTOR inhibitors such as rapamycin are currently under evaluation.
Percutaneous ablation Percutaneous ablation is a minimally invasive and e¡ective treatment that has become the ¢rst-line treatment for patients a¡ected with early HCC who are not candidates for surgical resection or liver transplantation. This approach is based on tumour ablation by injection of substances, mainly ethanol (percutaneous ethanol injection, PEI) or by changes in intra-tumour temperature, particularly increasing it through radiofrequency (RF)22. PEI obtains its ablative capacity with the injection of absolute ethanol directly into the tumours through a ¢ne needle under US guidance. It has low cost, it is widely available and it is a well-tolerated procedure with few sidee¡ects. In expert hands this procedure can obtain complete necrosis in 70 80% of single tumours 43 cm and in Child Pugh A patients may achieve a 5-year survival rate of 50%22. The ablative capability of PEI is worse in tumours between 3 and 5 cm, due to the presence of intratumoral septa that prevent the spread of the injected ethanol, achieving complete necrosis in less than 50%. RF produces thermal injury in tissue via an alternating electric current in the RF range (460 500 kHz) through a device (single or multiple cooled-tip electrodes or single electrodes associated with J-hook needles) placed by US guidance. RF has greater ablative capability than PEI, it achieves complete necrosis in signi¢cantly fewer sessions and it provides a better rate of response in tumours 43 cm23. The main drawbacks are the high cost of the equipment, and the higher rate and severity of adverse e¡ects, with a reported complication rate of up to 12% and mortality of 0.5%22,24. Although initial studies have failed to show di¡erences in terms of survival25,26, a recent Japanese RCT directly comparing RF with PEI in 238 Child Pugh A B patients with up to 259
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
three nodules 53 cm each showed a 4-year survival signi¢cantly higher in RF than in PEI (74% vs 57%) with similar incidences of adverse events27. As surgical resection the main disadvantage of percutaneous ablation is recurrence despite the initial complete necrosis, exceeding 70% at 5 years. Finally, some groups have compared resection versus percutaneous ablation in tumours 53 cm28,29. Although no di¡erences have been found between these two approaches, methodological issues of these investigations warrant future research.
Transarterial procedures The predominant arterial vascularization of HCC is the pathological basis for the e¡ectiveness of transarterial procedures, and this approach is indicated in patients with intermediate HCC. From all transarterial procedures evaluated in HCC, the only one that has shown survival bene¢t is transarterial chemoembolization (TACE). This technique consists in intra-arterial infusion of a chemotherapeutic agent (doxorubicin, cisplatin, mitomycin) suspended in a carrier substance, frequently lipiodol, followed by a blockade of feeding arterial branches with an embolizing agent, habitually gelatin sponge prepared as 0.5 1 mm cubes. To avoid injury to the surrounding non-tumoral liver, and to reduce the rate of side-e¡ects, the embolization procedure should be as selective as possible. Despite good selection of patients and an accurate technique this procedure is not innocuous, and is usually associated with adverse events. The majority of patients su¡er the postembolization syndrome, consisting of transient abdominal pain, ileus, and fever. Serious complications related to the procedure are infrequent (510%) but may be severe, such as ischaemic cholecystitis, biliary strictures and hepatic abscess, and death related to treatment may occur in up to 4% of cases30. TACE should be reserved for patients with preserved liver and renal function, normal clotting parameters, preserved portal vein £ow, and no vascular invasion/extrahepatic spread. Although the e¤cacy of TACE in terms of inducing objective responses is clear, achieving partial or complete tumour necrosis in approximately 35 43% of cases31, its e¤cacy in terms of survival improvement was not con¢rmed until the recent publication of two RCT31,32 and a cumulative meta-analysis33. However, despite initial response, in almost all cases tumours recur during follow-up. This is due to the tumour capacity to recover its vascularization through an intensive collateral formation, which is accompanied by re-growing capacity and tumour progression. Several topics are under investigation to further increase the bene¢ts of chemoembolization. E¡orts are directed to ¢nding the best chemotherapeutic drug and embolization agent, and ¢nding how to increase intratumoral concentrations of the drug. In that respect encouraging results have been obtained in a recent phase II trial using calibrated drug-eluting beads, an embolizing device that slowly releases chemotherapy, showing an objective response rate of 70% applying the EASL WHO criteria with neglectable systemic toxicity34. These results provide the rationale to move to phase III studies assessing survival outcomes. Another issue is clarifying the best treatment scheme for repeated treatment, whether it should be undertaken at 260
DIAGNOSIS AND OPTIONS OF HCC THERAPY
a ¢xed time or on disease progression after an initial response. Finally, e¡orts are directed towards evaluating the use of adjuvant therapies to delay tumour progression after TACE. In this respect, being aware of the key role of angiogenesis in arterial collateral formation after the procedure, angiogenesis blockade is an attractive target, and several trials evaluating its potential utility are currently ongoing. Other transarterial procedures such as intra-arterial chemotherapy, proposed for use in patients in whom portal thrombosis precludes hepatic artery £ow occlusion, have failed to show survival bene¢t, and their use is not recommended. Finally, a promising technique that is currently under evaluation is internal radiation. This is based on the delivery of high radiation doses through a transarterial approach, thereby preserving the normal liver parenchyma from damaging radiation. Promising results have been obtained in preliminary studies using yttrium-90 microspheres35, but again, survival bene¢t has not yet been properly evaluated, and further robust studies are awaited before incorporating this approach in the treatment algorithm of unresectable HCC.
Targeted therapies HCC has been shown to be resistant to chemotherapy, and in the past decade several cytotoxic drugs and chemotherapeutic schemes have been evaluated in advanced HCC, with marginal responses and inadmissible rates of severe adverse e¡ects. Therefore, classical systemic chemotherapy should be discouraged in cirrhotic patients with advanced HCC. In recent years signi¢cant advances in the knowledge of the molecular pathogenesis of HCC have occurred36, and this has led to the development of new agents that act directly through some disrupted molecular pathways. Although several promising agents are currently under evaluation 37 , sorafenib is the only one that, until now, has unequivocally shown survival improvement in advanced HCC. This orally available, small molecule is a multikinase inhibitor with activity against several tyrosine kinases (VEGFR2, PDGFR, c-Kit receptors), and serine/threonine kinases (b-Raf, p38). In preclinical experiments sorafenib has shown antiproliferative and antiangiogenic activity38, and a phase II clinical trial involving 137 patients with advanced HCC demonstrated that sorafenib induced stable disease for 4 months in 34% of the patients with an overall median survival of 9.2 months39. Based on those results a large multicentre randomized phase III double-blind placebo-controlled trial to assess the e¤cacy and safety of sorafenib in advanced HCC was conducted. In this trial 602 compensated, Child Pugh A cirrhotic patients with histologically proven advanced HCC were randomized to sorafenib 400 mg twice daily (n ( = 299) versus placebo (n ( = 303). Primary endpoints were overall survival and time to symptomatic progression based on patient-reported outcome (FHSI8-TSP). Secondary endpoints included time to progression, disease control rate and safety. The median overall survival was 10.7 months with sorafenib and 7.9 months with placebo (hazard ratio (HR) for death 0.69; 95% CI, 0.55 0.87; p50.001), and the median time to progression was 5.5 months with sorafenib versus 2.8 261
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
months with placebo (HR 0.58; 95% CI 0.45 0.74; p50.001). There were no di¡erences in FHSI8-TSP assessment and disease control rate was 43% in the sorafenib group and 32% in the placebo group (p ( = 0.002). Adverse events were habitually mild and manageable, and the most frequently reported were diarrhoea, weight loss, asthenia, hand foot skin reactions, and hypophosphataemia 40. Therefore, taking these results into account, it is expected that sorafenib will be considered as the standard of care for advanced HCC among scienti¢c societies and regulatory agencies. Other targeted agents have been evaluated in phase II clinical trials in advanced HCC, particularly erlotinib 4 1 , ge¢tinib 4 2 , bevacizumab 43 , sunitinib44, bortezomib or mTOR inhibitors (rapamycin, everolimus) with promising results, but RCT are needed to evaluate their real impact on survival. Furthermore, additional studies should assess the bene¢ts of combined molecular therapy compared with a single agent, or the value of these targeted therapies as an adjuvant treatment following curative or locoregional procedures.
SUMMARY HCC is currently the main cause of death in cirrhotic patients. Early detection through biannual US surveillance is recommended. Non-invasive criteria based on cutting-edge imaging techniques have been proven to be accurate in the diagnosis of most nodules 42 cm and it is also speci¢c in one-third of HCC 52 cm. Molecular diagnosis of early HCC is available, and needs prospective testing. Prognosis evaluation is very complex and should take into account tumour burden, liver functional status and presence of symptoms. The BCLC system is endorsed by the EASL and AASLD, is externally validated in European and American cohorts, and links staging with treatment indication. Patients at stage 0 (very early) or A (early stage) will bene¢t from potentially curative therapies, including resection, liver transplantation, and local ablation, providing 5-year survival rates exceeding 50 60%. Patients with multinodular asymptomatic tumours without vascular invasion/extrahepatic spread are c at e g o r i z e d a s s t a g e B (i n t e r m e d i at e s t a g e) a n d b e n e ¢ t f ro m chemoembolization. Patients presenting with either mild cancer-related symptoms (ECOG 1 2) or an invasive tumoral pattern (vascular invasion/ extrahepatic spread) are at stage C (advanced stage). Sorafenib a multikinase inhibitor is the ¢rst agent showing survival bene¢t for these cases, and it will become the standard of care in these cases. Finally, patients with severe symptoms, liver impairment or massive tumours present at stage D (terminal stage), with dismal prognosis. Several signi¢cant advances are expected in the near future. A growing increase in the detection of early tumours will be followed by an improvement in survival as a result of the higher application of potentially curative therapies, aside from the lead-time bias e¡ect. Knowledge of the molecular pathogenesis of the disease might enable us to con¢rm the molecular diagnosis of this neoplasm at very early stages. It is also expected that the main genomic alterations at preneoplastic (gatekeepers) and neoplastic stages will be 262
DIAGNOSIS AND OPTIONS OF HCC THERAPY
unravelled. Recent advances in the identi¢cation of a molecular classi¢cation of HCC will in the near future lead to a more rational assessment of moleculartargeted therapies, leading the research towards a personalized medicine. Results from the sorafenib trial in advanced cases have paved the way for future research in the assessment of bene¢ts of molecular-targeted therapies in the adjuvant setting and combination therapies in advanced stages.
Acknowledgements This work was in part supported by grants from the Instituto de Salud Carlos III (grants PI 05/150 and PI 06/132). Alejandro Forner is partially supported by a grant from the Instituto de Salud Carlos III (PI 05/645) and a grant from the BBVA foundation. Josep M. Llovet has support from the US National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK076986-01), the Samuel Waxman Cancer Research Foundation and the Spanish National Health Institute (SAF-2007-61898).
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74 108. Sangiovanni A, Prati GM, Fasani P et al. The natural history of compensated cirrhosis due to hepatitis C virus: a 17 year cohort study of 214 patients. Hepatology. 2006;43:1303 10. El Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med. 1999;340:745 50. Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology. 2005;42:1208 36. Bruix J, Sherman M, Llovet JM et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona 2000 EASL conference. European Association for the Study of the Liver. J Hepatol. 2001;35:421 30. Forner A, Vilana R, Ayuso C et al. Diagnosis of hepatic nodules 20 mm or smaller in cirrhosis: prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma. Hepatology. 2008;47:97 104. Villanueva A, Newell P, Chiang DY, Friedman SF, Llovet JM. Genomics and signaling pathways In hepatocellular carcinoma. Semin Liver Dis. 2007;27:55 76. Llovet JM, Chen Y, Wurmbach E et al. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology. 2006;131:1758 67. Sala M, Forner A, Varela M, Bruix J. Prognostic prediction in patients with hepatocellular carcinoma. Semin Liver Dis. 2005;25:171 80. Llovet JM, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classi¢cation. Semin Liver Dis. 1999;19:329 38. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003;362:1907 17. Lee JS, Thorgeirsson SS. Genome scale pro¢ling of gene expression in hepatocellular carcinoma: classi¢cation, survival prediction, and identi¢cation of therapeutic targets. Gastroenterology. 2004;127(Suppl. 1):S51 5. Llovet JM, Schwartz M, Mazzaferro V. Resection and liver transplantation for hepatocellular carcinoma. Semin Liver Dis. 2005;25:181 200. Llovet JM, Fuster J, Bruix J. Intention to treat analysis of surgical treatment for early hepatocellular carcinoma: resection versus transplantation. Hepatology. 1999;30:1434 40. Imamura H, Matsuyama Y, Tanaka E et al. Risk factors contributing to early and late phase intrahepatic recurrence of hepatocellular carcinoma after hepatectomy. J Hepatol. 2003;38:200 7.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 16. Sala M, Fuster J, Llovet JM et al. High pathological risk of recurrence after surgical resection for hepatocellular carcinoma: an indication for salvage liver transplantation. Liver Transplant. 2004;10:1294 300. 17. Mazzaferro V, Romito R, Schiavo M et al. Prevention of hepatocellular carcinoma recurrence with alpha interferon after liver resection in HCV cirrhosis. Hepatology. 2006;44:1543 54. 18. Mazzaferro V, Regalia E, Doci R et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med. 1996;334:693 9. 19. Sarasin FP, Majno PE, Llovet JM, Bruix J, Mentha G, Hadengue A. Living donor liver transplantation for early hepatocellular carcinoma: a life expectancy and cost e¡ectiveness perspective. Hepatology. 2001;33:1073 9. 20. Llovet JM, Mas X, Aponte JJ et al. Cost e¡ectiveness of adjuvant therapy for hepatocellular carcinoma during the waiting list for liver transplantation. Gut. 2002;50:123 8. 21. Sahin F, Kannangai R, Adegbola O, Wang J, Su G, Torbenson M. mTOR and P70 S6 kinase expression in primary liver neoplasms. Clin Cancer Res. 2004;10:8421 5. 22. Beaugrand M, N'Kontchou G, Seror O, Ganne N, Trinchet JC. Local/regional and systemic treatments of hepatocellular carcinoma. Semin Liver Dis. 2005;25:201 11. 23. Lencioni R, Cioni D, Crocetti L et al. Early stage hepatocellular carcinoma in patients with cirrhosis: long term results of percutaneous image guided radiofrequency ablation. Radiology. 2005;234:961 7. 24. Livraghi T, Solbiati L, Meloni MF, Gazelle GS, Halpern EF, Goldberg SN. Treatment of focal liver tumors with percutaneous radio frequency ablation: complications encountered in a multicenter study. Radiology. 2003;226:441 51. 25. Lencioni RA, Allgaier HP, Cioni D et al. Small hepatocellular carcinoma in cirrhosis: randomized comparison of radio frequency thermal ablation versus percutaneous ethanol injection. Radiology. 2003;228:235 40. 26. Livraghi T, Goldberg SN, Lazzaroni S, Meloni F, Solbiati L, Gazelle GS. Small hepatocellular carcinoma: treatment with radio frequency ablation versus ethanol injection. Radiology. 1999;210:655 61. 27. Shiina S, Teratani T, Obi S et al. A randomized controlled trial of radiofrequency ablation with ethanol injection for small hepatocellular carcinoma. Gastroenterology. 2005;129:122 30. 28. Huang GT, Lee PH, Tsang YM et al. Percutaneous ethanol injection versus surgical resection for the treatment of small hepatocellular carcinoma: a prospective study. Ann Surg. 2005;242:36 42. 29. Chen MS, Li JQ, Zheng Y et al. A prospective randomized trial comparing percutaneous local ablative therapy and partial hepatectomy for small hepatocellular carcinoma. Ann Surg. 2006;243:321 8. 30. Bruix J, Sala M, Llovet JM. Chemoembolization for hepatocellular carcinoma. Gastroenterology. 2004;127(Suppl. 1):S179 88. 31. Llovet JM, Real MI, Montana X et al. Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma: a randomised controlled trial. Lancet. 2002;359:1734 9. 32. Lo CM, Ngan H, Tso WK et al. Randomized controlled trial of transarterial lipiodol chemoembolization for unresectable hepatocellular carcinoma. Hepatology. 2002;35:1164 71. 33. Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival. Hepatology. 2003;37:429 42. 34. Varela M, Real M, Burrel M et al. Chemoembolization of hepatocellular carcinoma with drug eluting beads. E¤cacy and doxorubicin pharmacokinetics. J Hepatol. 2007;46:474 81. 35. Kulik LM, Carr B, Mulcahy MF et al. Safety and e¤cacy of 90Y radiotherapy for hepatocellular carcinoma presenting with or without portal vein thrombosis. Hepatology. 2008;47:71 81. 36. Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002;31:339 46. 37. Roberts LR, Gores GJ. Hepatocellular carcinoma: molecular pathways and new therapeutic targets. Semin Liver Dis. 2005;25:212 25.
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DIAGNOSIS AND OPTIONS OF HCC THERAPY 38. Liu L, Cao Y, Chen C et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/ PRF/5. Cancer Res. 2006;66:11851 8. 39. Abou Alfa GK, Schwartz L, Ricci S et al. Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol. 2006;24:4293 300. 40. Llovet J, Ricci S, Mazzaferro V et al. Randomized phase III trial of sorafenib versus placebo in patients with advanced hepatocellular carcinoma (HCC). J Clin Oncol. 2007;25 (18S):LBA1. 41. Philip P, Mahoney M, Allmer C et al. Phase II study of erlotinib (OSI 774) in patients with advanced hepatocellular cancer. J Clin Oncol. 2005;23:6657 63. 42. O'Dwyer P, Giantonio B, Levy D, Kauh J, Fitzgerald D, Benson A III. Ge¢tinib in advanced unresectable hepatocellular carcinoma: results from the Eastern Cooperative Oncology Group's Study E1203. J Clin Oncol. 2006;24(Suppl.):4143. 43. Schwartz J, Schwartz M, Lehrer D et al. Bevacizumab in unresectable hepatocellular carcinoma (HCC) for patients without metastasis and without invasion of the portal vein. J Clin Oncol. 2006;24(Suppl.):4144. 44. Zhu A, Sahani D, di Tomaso E et al. A phase II study of sunitinib in patients with advanced hepatocellular carcinoma. J Clin Oncol. 2007;25(Suppl.):4637.
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25 Surgical therapy of liver cancer: resection and transplantation G. OTTO, M. HOPPE-LOTICHIUS and M. HEISE
INTRODUCTION Hepatocellular carcinoma (HCC) is the ¢fth most common cancer worldwide and is estimated to cause approximately half a million deaths per year. Most tumours (80%) develop in cirrhotic livers caused by viral hepatitis C or B and alcoholic liver disease. In the natural course survival depends on the stage of the disease. At an early stage 3-year survival is 65% without treatment, in the intermediate stage between 10% and 50% of patients are reported to be alive after 2 years, and in the ¢nal stage median survival only rarely exceeds 6 months. Surgical treatment is capable of doubling survival. The results of local ablative treatment in early carcinoma are very similar to surgical treatment1. Patients with ¢nal stage disease are usually not amenable to any form of treatment. Therefore, in the Western world only a minority of patients with HCC may be treated by surgery. The number of patients amenable to liver transplantation is even lower. In selected cohorts of patients surgical treatment amounts to 40%, but regarding all patients with HCC this ¢gure does not exceed 10%2.
DIAGNOSTIC WORKUP With regard to the surgical approach, diagnostic workup is an important step in the therapeutic strategy. It is commonly accepted that magnetic resonance imaging is superior to computerized tomography in view of diagnostic accuracy. Nevertheless, detailed assessment of the tumour remains unreliable. Studies comparing the size and number of lesions in the specimen after liver transplantation (LT) with the preoperatively detected nodules resulted in a very limited accuracy of preoperative imaging. The most important obstacle for exact preoperative assessment is the underlying cirrhosis. Lesions of less than 1 cm in diameter are usually missed (Table 1). Diagnostic workup before liver surgery includes an assessment of the functional impairment of the liver. One of the most reliable tools to judge 266
SURGICAL THERAPY OF LIVER CANCER Table 1 staging
Liver transplantation: assessment of the number of nodules: reliability of preoperative
Sotiropoulos et al. 20053
DDLT accuracy 63%; LCLT accuracy 57%
Kaihara et al. 20034
Correct diagnosis of nodules 53 cm preoperatively: 21/56 (37.5%)
Krinsky et al. 20035
4% of lesions 51 cm detected in MRI
Own results
Diagnostic accuracy in CT scan: 60%
MRI, magnetic resonance imaging; CT, computed tomography.
perioperative risk is the assessment of bilirubin level and portal hypertension. In patients without portal hypertension and normal bilirubin the perioperative risk is only slightly elevated. If one of the two parameters is positive the risk is moderate, and surgery should be avoided in cases with elevated bilirubin and portal hypertension. In addition to cirrhosis-related functional impairment, up to 100% of patients experience recurrent disease within 5 years after hepatic resection. Even though long-term survival is mainly in£uenced by tumour recurrence, cirrhosis substantially impairs long-term survival: more than 30% of patients die from cirrhosis and not from recurrence. Five-year survival following liver resection ranges from 10% to 50% depending on the underlying disease and the respective tumour features6,7.
SURGICAL THERAPY LT is generally not indicated in patients with HCC in a non-cirrhotic liver, as comparable survival may be achieved by resectional surgery. In the European Liver Transplant Registry, 5-year survival in patients with LT for HCC in noncirrhotic livers is 41%. This ¢gure is comparable to the results following liver resection. LT was assumed to be superior to conventional surgery in patients with liver cirrhosis; however, this is not the case for all patients. Early results including patients with advanced disease did not exceed a 5-year survival of 20 25%. The improvement in the results of LT over recent decades was due to the exclusion of patients with advanced tumours. This has led to 5-year survival rates reaching 70%8. An important advance occurred when the in£uence of tumour size and number on these results was recognized9. At present the Milan criteria de¢ning the indication of LT in HCC (one nodule 45 cm or three or more nodules 43 cm) are generally accepted10. Based on these criteria a 5-year survival of 60 70% may be achieved. Results in LT are hampered by several factors (Figure 1). Regarding perioperative mortality, in LT the impact of the surgical approach is less important than the functional impairment in liver resection being caused by the underlying cirrhosis. Functional impairment is the leading cause of mortality following liver resection, reaching 10% even in Child A patients. 267
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Figure 1
Prognosis for HCC after LT; causes of postoperative mortality
Whereas patients with more than one small nodule are obviously best treated by LT, the indication for resection versus LT in patients with small and singular nodules remains a controversial issue. After all, survival after liver resection is comparable to LT in patients with singular nodules and Child A cirrhosis11,12. Figure 2 shows comparable results of 32 patients with liver resection compared to 66 patients with LT treated at the University of Mainz. Taking into account that the dropout of patients during waiting time before transplantation may be as high as 40%, intention-to-treat analyses result in superior survival for patients who primarily undergo conventional surgery. These considerations lead to important and interesting questions: is primary resection the ¢rst choice for small singular tumours in Child A cirrhosis, and may transplantation be applicable as a second-line treatment for those patients who experience tumour recurrence after resection? Unfortunately, only a small percentage of patients primarily treated by resection has been amenable to LT if recurrence occurred, and the results were inferior compared to the results after LT as a ¢rst treatment option13,14. Nevertheless, liver resection is performed in many centres if feasible. We prefer to perform LT as the ¢rst treatment option even in singular tumours. Due to the lack of donor organs, patients with HCC who are candidates for LT have to wait until an organ is available. During waiting time the tumour may exceed the listing criteria and such patients have to be delisted (drop out). There are several approaches to bridging this period: radiofrequency ablation, ethanol injection, transarterial chemoembolization (TACE) or even liver resection. Despite the substantial body of literature covering this issue, so far there is no evidence that one of these approaches deserves preference. 268
SURGICAL THERAPY OF LIVER CANCER
Figure 2 Resection (LR) versus transplantation (LT): survival in patients with singular HCC (55 cm) and Child A cirrhosis after LR versus LT; own results
In our opinion it is not only necessary to bridge the waiting time before transplantation, but it is more important to select patients with tumours suitable for LT. In the face of organ shortage this would contribute considerably to a reasonable allocation of liver grafts in patients with HCC. Even if tumours with vascular invasion, and poorly di¡erentiated tumours, are usually excluded from LT, reliable biological markers capable of selecting appropriate tumours are urgently needed. Hepatic resection was performed in 105 patients; in 65 of these patients HCC occurred in a non-cirrhotic liver. Five-year survival in non-cirrhotic patients was 49% compared to 40% in cirrhotic patients (Figure 3). In 119 patients LT was performed. In the majority of these patients (n ( = 69) HCC was identi¢ed during the preoperative workup. The rate of incidentalomas was 28%. Crude survival following LT was 57%; the recurrence rate after 5 years was 22.5%.
269
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Figure 3 results
Results of liver resection for HCC in patients with versus without cirrhosis; own
LIVING DONOR LIVER TRANSPLANTATION Living donor liver transplantation (LDLT) is considered a treatment option for patients with HCC. If a living donor is available the problems related to waiting time for a deceased donor liver transplantation (DDLT) are avoided. Most importantly, dropout during waiting time by exceeding the listing criteria may be prevented. In patients with macrovascular in¢ltration LDLT should not be performed. Other exclusion criteria may be histological grading, multinodular disease, and less important tumour size. These criteria, however, cannot be reliably assessed during the diagnostic workup. Even histological grading may be misleading due to sampling error when liver biopsy is performed. Waiting time per se seems to be a useful tool to select patients suitable for LDLT. In a study comprising 92 patients, relative to DDLT recipients, LDLT recipients had 270
SURGICAL THERAPY OF LIVER CANCER
Figure 4 TACE protocol and procedures performed in HCC before LT; University of Mainz, Germany
a shorter time from listing to transplant (mean 160 vs 469 days, p50.0001) but a higher rate of HCC recurrence within 3 years (29% vs 0%, p = 0.002), whereas there was no di¡erence in mortality or the combined outcome of mortality or recurrence15. Therefore, as long as tumour criteria re£ecting the aggressive biological behaviour are lacking, waiting time may be an option to select patients suitable for LDLT. Moreover, it might be advisable to comply with the generally accepted listing criteria for patients with HCC scheduled for LDLT in order to avoid unjusti¢ed living donation. Nevertheless, some centres advocate a moderate exceeding of the listing criteria in LDLT16.
TACE AS A BRIDGE TO TRANSPLANTATION We have used TACE as a bridge to transplantation 17 . Thereby, TACE pretreatment was repeatedly performed (on average 5.7 cycles). In Figure 4 the principles of our protocol are shown. Usually patients meeting the Milan criteria are immediately listed and TACE pretreatment is started, whereas patients beyond the listing criteria are listed after downstaging, i.e. after three cycles of TACE. Out of 96 patients exceeding or meeting the Milan criteria 60 were ¢nally transplanted (Figure 5); 24 of them met the Milan criteria whereas 36 exceeded the listing criteria. Patients who had stable disease after listing during pretreatment before LT experienced a very low recurrence rate (6.5%) compared to patients with minimal tumour progress (61.5%; p = 0.0001; Figure 6). Repeatedly performed TACE pretreatment may therefore not only bridge the waiting time but may select biologically favourable tumours suitable for LT. 271
PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY
Figure 5 Survival of 96 patients after inclusion in the TACE protocol. The di¡erence in 5 year survival in responders (transplanted) and non responders (not transplanted) is highly signi¢cant ( 50.0001) (intention to treat analysis) (p
SUMMARY *
Resection is the preferable treatment option in non-cirrhotic livers.
*
Cirrhosis is the crucial prognostic factor in liver resection: cirrhosis hampers the results after resection due to the limited functional capacity of the organ, and cirrhosis is a premalignant lesion.
*
Liver resection in Child A cirrhosis with singular HCC results in similar survival as transplantation. Only a small proportion of patients is amenable to LT if recurrence occurs after liver resection.
*
Liver transplantation is the treatment of choice for patients with small oligonodular tumours in cirrhosis. 272
SURGICAL THERAPY OF LIVER CANCER
Figure 6
Recurrence of HCC after LT in patients pretreated by repeatedly performed TACE
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol. 2005;40:225 35. El Serag HB, Mason AC, Key C. Trends in survival of patients with hepatocellular carcinoma between 1977 and 1996 in the United States. Hepatology, 2001;33:62 5. Sotiropoulos GC, Malago M, Molment E et al. Liver transplantation for hepatocellular carcinoma in cirrhosis: is clinical tumor classi¢cation before transplantation realistic? Transplantation. 2005;79:483 7. Kaihara S, Kiuchi T, Ueda M et al. Living donor liver transplantation for hepatocellular carcinoma. Transplantation. 2003;75(Suppl. 3):S37 40. Krinsky G. Imaging of dysplastic nodules and small hepatocellular carcinomas: experience with explanted livers. Intervirology. 2004;47:191 8. Llovet JM, Fuster J, Bruix J. Intention to treat analysis of surgical treatment for early hepatocellular carcinoma: resection versus transplantation. Hepatology. 1999;30:1434 40. Arii S, Yamaoka Y, Agawa S et al. Results of surgical and nonsurgical treatment for small sized hepatocellular carcinomas: a retrospective and nationwide survey in Japan. Hepatology. 2000;32:1224 9. Yoo HY, Patt CH, Geschwind JF, Thuluvath PJ. The outcome of liver transplantation in patients with hepatocellular carcinoma in the United States between 1988 and 2001: 5 year survival has improved signi¢cantly with time. J Clin Oncol. 2003;21:4329 35. Bismuth H, Chiche L, Adam R et al. Liver resection versus transplantation for hepatocellular carcinoma in cirrhotic patients. Ann Surg. 1993;218:145 51.
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PATHOGENESIS AND CLINICAL PRACTICE IN GASTROENTEROLOGY 10. Mazzaferro V, Regalia E, Doci R et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med. 1996;334:693 9. 11. Margarit C, Escart|n A, Castells L et al. Resection for hepatocellular carcinoma is a good option in Child Turcotte Pugh class A patients with cirrhosis who are eligible for liver transplantation. Liver Transplant. 2005;11:1242 51. 12. Poon RT, Fan ST, Lo CM et al. Di¡erence in tumor invasiveness in cirrhotic patients with hepatocellular carcinoma ful¢lling the Milan criteria treated by resection and transplantation: impact on long term survival. Ann Surg. 2007;245:51 8. 13. Adam R, Azoulay D. Is primary resection and salvage transplantation for hepatocellular carcinoma a reasonable strategy? Ann Surg. 2005;241:671 2. 14. Belghiti J. Transplantation for liver tumors. Semin Oncol. 2005;32(Suppl. 8):29 32. 15. Freise CE et al. Hepatocellular carcinoma recurrence and death following living and deceased donor liver transplantation. Am J Transplant. 2007;7:1601 8. 16. Malago M, Sotiropoulos GC, Nadalin S et al. Living donor liver transplantation for hepatocellular carcinoma: a single center preliminary report. Liver Transplant. 2006;12:934 40. 17. Otto G, Herber S, Heise M et al. Response to transarterial chemoembolization as a biological selection criterion for liver transplantation in hepatocellular carcinoma. Liver Transplant. 2006;12:1260 7.
274
Index abdominal pain, HIV-infected patients 111 AC see alcoholic hepatitis achalasia 9 13 barium oesophogram 10(¢g.) adalimumab 90, 91 adaptive immunity 149 51 decline 154 5 adenocarcinoma 9 a£atoxins 244 AFP see alphafetoprotein AIDS, gastrointestinal complications 110 12 AIH see autoimmune hepatitis albinterferon 164 albumin 63, 226 8 alcohol abuse 132 alcoholic hepatitis (AC) 208 15 apoptosis 210 11 clinical presentation 209(table) pathophysiology 210 11 therapy 211 15, 211(table), 213(table) alphafetoprotein (AFP) 240 AMA see antimitochondrial autoantibodies ANA see antinuclear antibody ANCA see anti-neutrophil cytoplasmic antibody anti-cytokine agents 180 anti-neutrophil cytoplasmic antibody (ANCA) 53, 59, 201 anti-obesity agents 178 anti-OmpC antibody 54, 59 anti-pancreatic antibodies (PAB) 55 anti-re£ux mechanisms 3 6 anti-re£ux surgery 23 4 anti-Saccharomyces cerevisiae antibodies (ASCA) 54 antigen-presenting cells (APC) 194, 195 antimitochondrial autoantibodies (AMA) 191, 192, 192(¢g.) antinuclear antibody (ANA) 175, 188 antioxidants 180
APC see antigen-presenting cells apoptosis 140, 195 alcoholic hepatitis 210 11 arterial pressure 222 arthralgia 187 ASCA see anti-Saccharomyces cerevisiae antibodies atrophic gastritis 34 autoantibodies, PBC 192 3 autoimmune hepatitis (AIH) 186 9, 187 (¢g.) azathioprine (AZA) 80 azathioprine/6-mercaptopurine (AZA/6MP) 52 B cells, PBC 192 3 Barcelona Clinic Liver Cancer (BCLC) system 255, 262 bariatric surgery 178 Barrett's cancer 19 Barrett's oesophagus 28 9 BCLC see Barcelona Clinic Liver Cancer system BEC see biliary epithelial cells benzodiazepine 211 b-blockers 235 betaine 180 biliary diseases 132 biliary epithelial cells (BEC) 194 biologics 90 3 biomarkers 53 4 see also serological markers biopsy 29 boceprevir 165 Botox 11, 12 13 breast-feeding 98 budesonide, localized ileocaecal CD 77 Burkitt's lymphoma 121 C-reactive protein (CRP) calgranulin C 63
275
44, 62, 64, 65, 77
INDEX Candida oesophagitis 110 cardiac output 222 cardiovascular disease, NAFLD 173 4 carvediol 235 CD see Crohn's disease cellular immune response, PSC 202 certolizumab pegol 90, 91 chemoembolization 260 chemokines 137 PSC 202(table) chemotherapy 259 chest pain 13 Chevallier score 129 Child-Pugh score 255 chlorophyllin 244 cholangiocytes, PBC 194 6 cholangiopathy, HIV-infected patients 111 chronic hepatitis 128 9, 130 1(table) chronic in£ammatory bowel disease, colorectal cancer 114 cirrhosis HCC surveillance 239 hepatorenal syndrome 221 9 citalopram 20 co-infection, HIV-infected patients 111 coeliac disease 97 106 clinical presentation 102 complications 102 3 diagnosis 103, 104(¢g.) environmental factors 98 9 epidemiology 101 2 genetic factors 99 paediatric 103(¢g.) pathomechanisms 99 101, 100(¢g.) screening 104 5 treatment 105 6 colectomy 85 6 colonoscopy 115 colorectal cancer 113 16 corticosteroids 84, 211 12 Crohn's colitis 78 Crohn's disease (CD) 43, 76 biomarkers 65 6 colonic 78 ileocaecal 77 maintenance therapy 79 82 serological markers 58, 59 60 severe 77 8 therapy 76 82 CRP see C-reactive protein cyclosporine 189 cytokines 44, 50 1, 92 coeliac disease 101
HCC 137 in£ammatory 141 2 PBC 196 7 PSC 202(table) DC see dendritic cells DCP see des-gamma carboxyprothrombin dendritic cells (DC) 153 4, 196 des-gamma carboxyprothrombin (DCP) 240 diabetes 178 80 diarrhoea, HIV-infected patients 110 11 diet colorectal cancer 113 NAFLD 173, 177 8 di¡use large B-cell lymphoma (DLCL) 119 di¡use oesophageal spasm (DOS) 13 14, 15 distal colitis 83 DLCL see di¡use large B-cell lymphoma DLG5 50 DNA damage 140, 143 dorsocervical lipohypertrophy 175 DOS see di¡use oesophageal spasm double-contrast barium enema 115 ductopenia 195 dynamic MR swallowing 6 dyslipidaemia 179 80 dysphagia 4, 7, 14 15 ECAD see extracorporeal albumin dialysis ECCO see European Crohn's and Colitis Organization endoscopy achalasia 11 anti-re£ux devices 22 variceal bleeding 235 enteropathy-type T-cell lymphoma (ETCL) 103, 118, 121 erosive oesophagitis 20 erythema 29 erythrocyte sedimentation rate (ESR) 62 ESR see erythrocyte sedimentation rate ETCL see enteropathy-type T-cell lymphoma European Crohn's and Colitis Organization (ECCO) guidelines 76 87 extracorporeal albumin dialysis (ECAD) 229 faecal calprotectin 63, 64 faecal markers 63 faecal occult blood testing (FOBT) fat distribution 175
276
114 15
INDEX ¢breoptic endoscopic evaluation of swallowing (FEES) 9, 15 ¢brosis 129 FNH see focal nodular hyperplasia FOBT see faecal occult blood testing focal lesions 127 focal liver lesions 133 4 focal nodular hyperplasia (FNH) 133 fontolizumab 92 free radicals 137 43 in£ammation 139(¢g.) functional heartburners 20 fundoplication 23, 24
post-transfusion 244 hepatitis B virus (HBV) 111, 149 55 adaptive immunity 149 51, 150(¢g.), 154 5 dendritic cells 153 4 HCC surveillance 239 immunization 244 in£ammation 137 innate immunity 152 3 hepatitis C virus (HCV) adaptive immunity 149 51, 150(¢g.), 154 5 albinterferon 164 alcoholics 209 dendritic cells 153 4 genotype 1 infection 161 2 in£ammation 137, 141 2, 149 55 innate immunity 152 3 interferon alpha treatment 163 STAT-C 164 6 treatment 161 6 hepatocellular carcinoma (HCC) 132, 134 (¢g.), 239, 252, 266 diagnosis 242(¢g.), 252 5, 254(¢g.), 266 7 hepatitis B-related 245 6 hepatitis C-related 246 7, 252 in£ammation 137 43 mortality 243 MRI 253(¢g.) NASH 171 prevention 244 8 prognosis 255 6 surgical therapy 266 73 surveillance 239 43, 240(table), 241 (table) susceptibility 244 targeted therapy 261 2 treatment 256 62, 257(¢g.) hepatorenal syndrome (HRS) 221 9 diagnostic criteria 225(table) pathogenesis 223(¢g.) terlipressin studies 227(table) treatment 225 8 hereditary iron storage disease 132 high-grade dysplastic macronodules (HGDN) 243 hip fractures 21 HLA see human leukocyte antigen HRS see hepatorenal syndrome human leukocyte antigen (HLA) complex 202 hyperandrogenism 175 hyperferritaemia 175
GALT see gut-associated lymphoid tissues gamma globulin 187 gastric cancer, Helicobacter pylori 33, 35, 36, 37(table), 38(table) gastritis, Helicobacter pylori 35 gastro-oesophageal re£ux disease (GORD) 9, 17 25 anti-re£ux surgery 23 4 Barrett's oesophagus 28 9 de¢nition 17 18 endoscopic therapy 22 management 19(¢g.) Montreal de¢nition 18(¢g.) treatment 18 19 gene polymorphisms 50 1 genetics bacterial recognition 47 9 mucosal integrity 49 50 predisposition 43 GFD see gluten-free diet glomerular ¢ltration rate (GFR) 224, 225 glucocorticoids 208, 211 12 gluten 97, 99 gluten-free diet (GFD) 105 GORD see gastro-oesophageal re£ux disease gut-associated lymphoid tissues (GALT) 202 HALO 29 HBV see hepatitis B virus HCC see hepatocellular carcinoma HCV see hepatitis C virus heartburn 24 Helicobacter pylori 20, 33 8, 120 1 Heller myotomy 12 hepatitis AIH 186 9 alcoholic 208 15 liver biopsy 128 9
277
INDEX hypertension 180 hypertriglyceridaemia 179 hypobetalipoproteinaemia 175
liver biopsies 127 35, 176, 241 liver disease chronic 132 hepatitis 111, 128 9 hereditary iron storage disease 132 HIV-infected patients 111 prevention 244 liver transplantation (LT) 133, 181, 258 9, 267(table) HCC 267 9, 268(¢g.), 269(¢g.) living donor 258, 270 1 liver-kidney microsome autoantibodies (LKM) 188 living donor liver transplantation (LDLT) 258, 270 1 LKM see liver-kidney microsome autoantibodies LOS see lower oesophageal sphincter lower oesophageal sphincter (LOS) 9 LT see liver transplantation lymphomas 118 21
IBD see in£ammatory bowel disease IEL see intraepithelial lymphocytes IFN see interferon IFX see in£iximab IgA antibodies 199 IL see interleukin ileal pouch anastomosis 59 immunomodulators 81 immunoproliferative small intestinal disease (IPSID) 120 immunosuppressive therapy 188 indeterminate colitis (IS), serological markers 58 ine¡ective oseophageal motility 14 infant feeding practice 98 in£ammation 43, 62 cancer 137 43 cancer risk 138(table) in£ammatory bowel disease (IBD) 43, 45 (¢g.) biologics 90 3 biomarkers 64 genetics 46(table) pharmacogenetics 52 serological markers 55 8, 56 7(table), 60 1 in£ammatory cytokines 141 2 in£iximab (IFX) 77 8, 81, 90 2, 214 15 innate immunity 61 2, 152 3 PBC 196 insulin resistance 177, 178 80 interferon gamma (IFN-g) g 139 interferon (IFN), HCV 152 interleukin 1b (IL-1b ) 139, 143 interleukin 10 (IL-10) 43 internal radiation 261 intraepithelial lymphocytes (IEL) 101 intravenous steroid-resistant severe colitis 85 IPSID see immunoproliferative small intestinal disease IS see indeterminate colitis
Maddrey score 212 major histocompatibility complex (MHC) 186 MALT see mucosa-associated lymphoid tissue mesalazine 80, 82 metformin 178 9 methotrexate 77 mHAI see modi¢ed hepatic activity index MHC see major histocompatibility complex modi¢ed hepatic activity index (mHAI) 128 molecular mimicry 99, 200 MRI, swallowing 6 mucosa-associated lymphoid tissue (MALT) lymphoma 118, 119 mucosal friability 82 mucosal immune system 43, 50 1 mucosal integrity, genetics 49 50 multidrug resistance 50 1 myelotoxicity 53 NAFLD see non-alcoholic fatty liver disease NASH see non-alcoholic steatohepatitis natalizumab 92 natural killer (NK) cells 152, 196 NERD see non-erosive re£ux disease NHL see non-Hodgkin's lymphoma nitric oxide (NO) 137, 140(¢g.), 141(¢g.), 143, 222 NK see natural killer cells NO see nitric oxide NOD1/CARD4 48
lactoferrin 63 LDLT see living donor liver transplantation leukocytosis 62 Li-Fraumeni syndrome 137 lifestyle, colorectal cancer 114 ligation 235
278
INDEX NOD2/CARD15 44, 47 non-alcoholic fatty liver disease (NAFLD) 171 cardiovascular disease 173 4 clinical presentation 174 5 diabetes 178 80 diagnosis 175 epidemiology 171 2 hypertension 180 management 176 7 obesity 177 8 OSA 174 PCOS 174 prognosis 172 3 susceptibility 173 non-alcoholic steatohepatitis (NASH) 129, 171 81 non-erosive re£ux disease (NERD) 19 non-Hodgkin's lymphoma (NHL) 118 non-steroidal anti-in£ammatory drug (NSAID) 34 5 NS5B inhibitors 165 NSAID see non-steroidal anti-in£ammatory drug nutritional therapy 78, 212 13
PEI see percutaneous ethanol injection pentoxifylline 213 peptic ulcer, NSAIDs 34 percutaneous ablation 259 60 percutaneous ethanol injection (PEI) 259, 268 perianal disease 79, 80 perianal ¢stulas 79(table) pharmacogenetics 52 5 photodynamic therapy 29 plasma renin activity (PRA) 222, 225 pneumatic dilation 12 pneumonia 21 polycystic ovary syndrome (PCOS), NAFLD 174 polyps 115 portal hypertension 233 post-transfusion hepatitis 244 PPI see proton pump inhibitor PRA see plasma renin activity prednisolone, severe ileal disease 77 primary biliary cirrhosis (PBC) 132, 191 animal models 196 biliary epithelium 194(table) cholangiocytes 194 6 cytokines 196 7 immunogenetics 196 immunology 192 200 innate immunity 196 pathogenesis 197 200, 198(¢g.) primary intestinal lymphomas 121 primary sclerosing cholangitis (PSC) 132, 191 immunogenetics 202 3 immunology 201 3 pathogenesis 203 probucol 180 proctitis 83 programmed death 1 (PD-1) molecule 154 promotility agents 14 proton pump inhibitor (PPI) 19, 20 2 Barrett's oesophagus 29 proximal intestinal disease 79 PSC see primary sclerosing cholangitis
oats toxicity 105 obesity 171, 173, 177 8 obstructive sleep apnoea (OSA), NAFLD 174 oesophageal cancer 24 oesophageal manometry 11, 13 oesophageal motility problems 7 15 oesophageal varices 233(¢g.), 236(¢g.) oesophagectomy 29 oesophagitis 28 OLT see orthotopic liver transplantation oltipraz 244 oral hairy leukoplakia 110 orlistat 178 ornipressin 226 oropharyngeal dysphagia 7 9 causes 8(table) orthotopic liver transplantation (OLT) 258 OSA see obstructive sleep apnoea
re£ux oesophagitis 19, 21 regurgitation 24 resection 78, 119, 256 7 ribavirin 161 2, 166 rimonabant 178
p53 gene 35, 137, 139(table) PAB see anti-pancreatic antibodies pancreatitis, HIV-infected patients 111 PBC see primary biliary cirrhosis PCOS see polycystic ovary syndrome PD-1 see programmed death 1 molecule peginterferon alpha 163, 166
SBP see spontaneous bacterial peritonitis serological markers
279
INDEX disease progression 59 61 IBD 55 8, 56 7(table) innate immunity 61 2 serum markers 62 3 Sjogren's syndrome 195 smooth muscle antibody (SMA) 175, 188 sorafenib 261 speci¢cally targeted antiviral therapies for hepatitis C (STAT-C) 164 6 spontaneous bacterial peritonitis (SBP) 221 STAT-C see speci¢cally targeted antiviral therapies for hepatitis C statins 174, 179, 234 steatohepatitis 129, 209 steatosis 129 steroid dependence 80 stool-antigen test 33 sulphasalazine 78 suppositories 83 surgery, remission maintenance 81 sustained virological response (SVR) 161 swallowing functional units 5(¢g.) physiology 3 6
transarterial chemoembolization (TACE) 256, 260, 268, 271 2, 271 (¢g.) transfer dysphagia see oropharyngeal dysphagia b 43 transforming growth factor beta (TGF-b) transjugular intrahepatic protosystemic shunt (TIPS) 223, 228 9, 234 tumour evolution 142(¢g.) a 90 2 tumour necrosis factor alpha (TNF-a) alcoholic hepatitis 210, 213 14 cancer 139, 143 see also in£iximab TZD see thiazolidinediones UBT see urea-breath test UC see ulcerative colitis UDCA see ursodeoxycholic acid ulcerative colitis (UC) 43, 65, 82 6 acute severe 84 6 extensive 84 maintenance therapy 86 serological markers 58, 59 ultrasound (US) 240, 252 urea-breath test (UBT) 33, 34 ursodeoxycholic acid (UDCA) 180 1 US see ultrasound
T cell priming 151 T cells PBC 193 4 Tregs 200 TACE see transarterial chemoembolization terlipressin 226 TGF-b see transforming growth factor beta thiazolidinediones (TZD) 179 thiopurine S-methyltransferase (TPMT) 52 thrombocytosis 63 thrush 110 TIPS see transjugular intrahepatic protosystemic shunt TLR see toll-like receptors TNF-a see tumour necrosis factor alpha toll-like receptors (TLR) 48, 196 TPMT see thiopurine S-methyltransferase
variceal bleeding 233 6 prophylaxis 234 5 vasoconstrictors 226 8 video£uoroscopy 3 5 viral infections 98 9 visilizumab 93 vitamin B12 de¢ciency 21 vitamin E 180 vitamin K 211 wasting syndrome, HIV-infected patients 111
280
Falk Symposium Series 43. Reutter W, Popper H, Arias IM, Heinrich PC, Keppler D, Landmann L, eds.: Modulation ofLiver Cell Expression. Falk: Symposium No. 43. 1987 ISBN: 0-85200-677-2* 44. Boyer JL, Bianchi L, eds.: Liver Cirrhosis. Falk: Symposium No. 44. 1987 ISBN: 0-85200-993-3* 45. PaumgartnerG,StiehlA,GerokW,eds.:BileAcidsandtheLiver.FalkSymposiumNo. 45. 1987 ISBN: 0-85200-675-6* 46. Goebell H, Peskar BM, Malchow H, eds.: Inflammatory Bowel Diseases - Basic Research & Clinical Implications. Falk: Symposium No. 46. 1988 ISBN: 0-7462-0067-6* 47. Bianchi L, Holt P, James OFW, Butler RN, eds.: Aging in Liver and Gastrointestinal Thlct. Falk:SymposiumNo. 47.1988 ISBN: 0-7462-0066-8* 48. Heilmann C, ed.: Calcium-Dependent Processes in the Liver. Falk: Symposium No. 48. 1988 ISBN: 0-7462-0075-7* 50. Singer MV, Goebell H, eds.: Nerves and the Gastrointestinal Thlct. Falk: Symposium No. 50.1989 ISBN: 0-7462-0114-1 51. BannaschP, Keppler D,WeberG, eds.: Liver Cell Carcinoma. Falk:SymposiumNo. 51. 1989 ISBN: 0-7462-0111-7 52. Paumgartner G, Stiehl A, Gerok W, eds.: Trends in Bile Acid Research. Falk: ISBN: 0-7462-0112-5 Symposium No. 52.1989 53. Paumgartner G, Stiehl A, Barbara L, Roda E, eds.: Strategies for the Treatment of Hepatobiliary Diseases. Falk: Symposium No. 53. 1990 ISBN: 0-7923-8903-4 54. Bianchi L, Gerok W, Maier K-P, Deinhardt F, eds.: Infectious Diseases of the Liver. Falk Symposium No. 54. 1990 ISBN: 0-7923-8902-6 55. Falk Symposium No. 55 not published 55B. Hadziselimovic F, Herzog B, Biirgin-WolffA, eds.: Inflammatory Bowel Disease and Coeliac Disease in Children. International Falk: Symposium. 1990 ISBN 0-7462-0125-7 56. Williams CN, eds.: Trends in Inflammatory Bowel Disease Therapy. Falk: Symposium ISBN: 0-7923-8952-2 No. 56.1990 57. Bock KW, Gerok W, Matern S, Schmid R, eds.: Hepatic Metabolism and Disposition of Endo- and Xenobiotics. Falk Symposium No. 57. 1991 ISBN: 0-7923-8953-0 58. Paumgartner G, Stiehl A, Gerok W, eds.: Bile Acids as Therapeutic Agents: From Basic ISBN: 0-7923-8954-9 Science to Clinical Practice. Fallc: Symposium No. 58. 1991 59. Halter F, Garner A, Tytgat GNJ, eds.: Mechanisms of Peptic Ulcer Healing. Falk Symposium No. 59.1991 ISBN: 0-7923-8955-7 60. Goebell H, Ewe K, Malchow H, Koelbel Ch, eds.: Inflammatory Bowel DiseasesProgress in Basic Research and Clinical Implications. Falk Symposium No. 60. 1991 ISBN: 0-7923-8956-5 61. Falk Symposium No. 61 not published 62. Dowling RH, Folsch UR, LOser Ch, eds.: Polyamines in the Gastrointestinal Tract. Falk Symposium No. 62. 1992 ISBN: 0-7923-8976-X 63. Lentzc MJ, Reichen J, eds.: Paediatric Cholestasis: Novel Approaches to Treatment. Falk Symposium No. 63. 1992 ISBN: 0-7923-8977-8 64. Demling L, Friihmorgen P, eds.: Non-Neoplastic Diseases of the Anorectum. Falk: Symposium No. 64. 1992 ISBN: 0-7923-8979-4 64B. Gressner AM, Ramadori G, eds.: Molecular and Cell Biology of Liver Fibrogenesis. International Falk: Symposium. 1992 ISBN: 0-7923-8980-8 •These titles were published under the MTP Press imprint.
Falk Symposium Series 65. Hadziselimovic F, Herzog B, eds.: Inflammatory Bowel Diseases and Morbus Hirschprung. Falk Symposium No. 65. 1992 ISBN: 0-7923-8995-6 66. Martin F, McLeod RS, Sutherland LR, Williams CN, eds.: Trends in Inflammatory Bowel Disease Therapy. Fal.k. Symposium No. 66. 1993 ISBN: 0-7923-8827-5 67. SchOlmerich J, Kruis W, Goebell H, Hohenberger W, Gross V, eds.: Inflammatory Rowe/Diseases-Pathophysiology as BasisofTTeatment. Fal.k. Symposium No. 67. 1993 ISBN: 0-7923-8996-4 68. Paumgartner G, Stiehl A. Gerok W, eds.: Bile Acids and The Hepatobiliary System: From Basic Science to Clinical Practice. Fal.k. Symposium No. 68. 1993 ISBN: 0-7923-8829-1 69. Schmid R, Bianchi L, Gerok W, Maier K-P, eds.: Extrahepatic Manifestations in Liver Diseases. Fal.k. Symposium No. 69. 1993 ISBN: 0-7923-8821-6 70. Meyer zum Biischenfelde K-H, Hoofnagle J, Manns M, eds.: Immunology and Liver. Falk Symposium No. 70. 1993 ISBN: 0-7923-8830-5 71. Surrenti C, Casini A, Milani S, Pinzani M , eds.: Fat-Storing Cells and Liver Fibrosis. Falk Symposium No. 71. 1994 ISBN: 0-7923-8842-9 72. Rachmilewitz D, ed: Inflammatory Bowel Diseases-1994. Fal.k. Symposium No. 72. 1994 ISBN: 0-7923-8845-3 73. Binder HJ, Cummings J, Soergel KH, eds.: Short Chain Fatty Acids. Falk Symposium No. 73.1994 ISBN: 0-7923-8849-6 73B. Mollmann HW, May B, eds.: Glucocorticoid Therapy in Chronic Inflammatory Bowel Disease:from basicprinciples to rational therapy. International Falk Workshop. 1996 ISBN 0-7923-8708-2 74. Keppler D, JungermannK, eds.: Transport in the Liver. Falk Symposium No. 74.1994 ISBN: 0-7923-8858-5 74B. Stange EF, ed: Chronic Inflammatory Rowe/Disease. Falk Symposium. 1995 ISBN: 0-7923-8876-3 75. van Berge Henegouwen GP, van Hoek B, De Groote J, Matern S, Stockbriigger RW, eds.: Cholestatic Liver Diseases: New Strategies for Prevention and Treatment of Hepatobiliary and Cholestatic Liver Diseases. Falk Symposium 75. 1994. ISBN: 0-7923-8867-4 76. Monteiro E, Tavarela Ve1oso F, eds.: Inflammatory Bowel Diseases: New Insights into Mechanisms of Inflammation and Challenges in Diagnosis and Treatment. Fal.k. Symposium 76. 1995. ISBN 0-7923-8884-4 77. Singer MV, Ziegler R, Rohr G, eds.: Gastrointestinal Tract andEndocrine System. Fal.k. Symposium 77.1995. ISBN0-7923-8877-1 78. Decker K., GerokW, AndusT, Gross V, eds.: Cytokinesandthe Liver. Falk Symposium 78. 1995. ISBN 0-7923-8878-X 79. Ho1stege A, SchOlmerichJ, Hahn EG, eds.: Portal Hypertension. Falk Symposium 79. 1995. ISBN0-7923-8879-8 80. Hofmann AF, Paumgartner G, Stiehl A, eds.: Bile Acids in Gastroenterology: Basicand Clinical Aspects. Falk Symposium 80. 1995 ISBN 0-7923-8880-1 81. Riecken EO, Stallmach A, Zeitz M, Heise W, eds.: Malignancy and Chronic Inflammation in the Gastrointestinal Tract- New Concepts. Fal.k. Symposium 81. 1995 ISBN 0-7923-8889-5 82. Fleig WE, ed.: Inflammatory Bowel Diseases: New Developments and Standards. Fal.k. Symposium 82. 1995 ISBN 0-7923-8890-6
Falk Symposium Series 82B.Paumgartner G, Beuers U, eds.: Bile Acitb in Liver Diseases. International Falk Workshop. 1995 ISBN 0-7923-8891-7 83. Dobrilla G, Felder M, de Pretis G, eds.: Advances in Hepatobiliary and Pancreatic Diseases: Special Clinical Topics. Falk Symposium 83. 1995. ISBN 0-7923-8892-5 84. Fromm H, Leuschner U, eds.: Bile Acitb- Cholestasis- Gallstones: Advances in Basic and Clinical Bile AcidResearch. Falk Symposium 84. 1995 ISBN 0-7923-8893-3 85. Tytgat GNJ, Bartelsman JFWM, van Deventer SJH, eds.: Inflammatory Bowel Diseases. Falk Symposium 85. 1995 ISBN 0-7923-8894-1 86. Berg PA, Leuschner U, eds.: Bile Acitb and Immunology. Falk Symposium 86. 1996 ISBN 0-7923-8700-7 87. Schmid R, Bianchi L, Blum HE, Gerok W, Maier KP, Stalder GA, eds.: Acute and Chronic Liver Diseases: Molecular Biology and Clinics. Falk Symposium 87. 1996 ISBN 0-7923-8701-5 88. Blum HE, Wu GY, Wu CH, eds.: Molecular Diagnosis and Gene Therapy. Falk Symposium 88. 1996 ISBN 0-7923-8702-3 88B.Poupon RE, Reichen J, eds.: Surrogate Markers to Assess Efficacy of TReatment in Chronic Liver Diseases. International Falk Workshop. 1996 ISBN 0-7923-8705-8 89. Reyes HB, Leuschner U, Arias IM, eds.: Pregnancy, Sex Hormones and the Liver. Falk Symposium 89. 1996 ISBN 0-7923-8704-X 89B. Broelsch CE, Burdelski M, Rogiers X, eds.: Cholestatic Liver Diseases in Children and Adults. International Falk Workshop. 1996 ISBN 0-7923-8710-4 90. Lam S-K, Paumgartner P, Wang B, eds.: Update on Hepatobiliary Diseases 1996. Falk Symposium90.1996 ISBN0-7923-8715-5 91. HadziselimovicF, Herzog B, eds.: Inflammatory BowelDiseases and Chronic Recurrent ISBN 0-7923-8722-8 AbdominalPain. Falk Symposium 91. 1996 91B.Alvaro D, Benedetti A, Strazzabosco M, eds.: Vanishing Bile Duct SyndromePathophysiology and Treatment. International FalkWorkshop. 1996 ISBN 0-7923-8721-X 92. Gerok W, Loginov AS. Pokrowskij VI, eds.: New Trends in Hepatology 1996. Falk Symposium92.1997 ISBN0-7923-8723-6 93. Paumgartner G, Stiehl A, Gerok W, eds.: Bile Acitb in Hepatobiliary Diseases- Basic Research and Clinical Application. Falk Symposium 93. 1997 ISBN 0-7923-8725-2 94. Halter F,WintonD,Wright NA, eds.: The GutasaModelin Cell andMolecular Biology. Falk Symposium 94. 1997 ISBN 0-7923-8726-0 94B.Kruse-Jarres JD, SchOlmerich J, eds.: Zinc and Diseases of the Digestive Tract. International FalkWorkshop. 1997 ISBN 0-7923-8724-4 95. Ewe K, Eckardt VF, Enck P, eds.: Constipation and Anorectal Insufficiency. Falk Symposium95.1997 ISBN0-7923-8727-9 96. Andus T, Goebell H, Layer P, SchOlmerich J, eds.: Inflammatory BowelDisease-from Bench to Bedside. Falk Symposium 96. 1997 ISBN 0-7923-8728-7 97. Campieri M, Bianchi-Porro G, Fiocchi C, SchOlmerich J, eds. Clinical Challenges in Inflammatory Bowel Diseases: Diagnosis, Prognosis and Treatment. Falk Symposium 97.1998 ISBN0-7923-8733-3 98. Lembcke B, Kruis W, Sartor RB, eds. Systemic Manifestations ofIBD: The Pending Challengefor Subtle Diagnosis and Treatment. Falk Symposium 98. 1998 ISBN 0-7923-8734-1 99. Goebell H, Holtmann G, Talley NJ, eds. Functional Dyspepsia and l"itahle Bowel Syndrome: Concepts and Controversies. Falk Symposium 99. 1998 ISBN 0-7923-873 5-X
Falk Symposium Series 100. Blum HE, Bode Ch, Bode JCh, Sartor RB, eds. Gut and the Liver. Falk Symposium 100.1998 ISBN0-7923-8736-8 101. Rachmilewitz D, ed. V International Symposium on Inflammatory Bowel Diseases. Falk Symposium 101. 1998 ISBN0-7923-8743-0 102. Manns MP, Boyer JL, Jansen PLM, Reichen J, eds. Cholestatic Liver Diseases. Falk Symposium 102. 1998 ISBN0-7923-8746-5 102B. Manns MP, Chapman RW, Stiehl A, Wiesner R, eds. Primary Sclerosing ISBN 0-7923-8745-7 Cholangitis. International Falk Workshop. 1998. 103. Hii.ussinger D, Jungermann K, eds. Liver and Nervous System. Falk Symposium 102. 1998 ISBN0-7924-8742-2 103B. Hii.ussinger D, Heinrich PC, eds. Signalling in the Liver. International Falk Workshop.1998 ISBN0-7923-8744-9 103C. Fleig W, ed. Normal and Malignant Liver Cell Growth. International Falk Workshop.1998 ISBN0-7923-8748-1 104. Stallmach A, Zeitz M, Strober W, MacDonald IT, Lochs H, eds. Induction and Modulation ofGastrointestinal Inflammation. Falk Symposium 104. 1998 ISBN0-7923-8747-3 105. Emmrich J, Liebe S, Stange EF, eds. I111Wvative Concepts in Inflammatory Bowel Diseases. Falk Symposium 105. 1999 ISBN 0-7923-8749-X 106. Rutgeerts P, Colombe! J-F, Hanauer SB, SchOimerich J, Tytgat GNJ, van Gossum A, eds. Advances in Inflammatory Bowel Diseases. Falk Symposium 106. 1999 ISBN 0-7923-8750-3 107. Spicak: J, Boyer J, Gilat T, Kotrlik K. Ma.reCek. Z, Paumgartner G, eds. Diseases of the Liver and the Bile Ducts - New Aspects and Clinical Implications. Falk Symposium 107. 1999 ISBN0-7923-8751-1 108. Paumgartner G, Stiehl A, Gerok W, Keppler D, Leuschner U, eds. Bile Acids and Cholestasis. Falk Symposium 108. 1999 ISBN 0-7923-8752-X 109. Schmiegel W, Scholmerich J, eds. Colorectal Cancer - Molecular Mechanisms, Premalignant State and its Prevention. Falk Symposium 109. 1999 ISBN 0-7923-8753-8 110. Domschke W, Stoll R, Brasitus TA, Kagnoff MF, eds. Intestinal Mucosa and its Diseases-Pathophysiology and Clinics. Falk:Symposium 110.1999 ISBN 0-7923-8754-6 llOB. Northfield TC, Ahmed HA, Jazwari RP, Zentler-Munro PL, eds. Bile Acids in ISBN 0-7923-8755-4 Hepatohiliary Disease. Falk Workshop. 2000 111. Rogier G, Kullmann F, Rutgeerts P, Sartor RB, Scholmerich J, eds. IBD at the End ofits First Century. Falk:Symposium 111.2000 ISBN0-7923-8756-2 112. Krammer HJ, Singer MV, eds. Neurogastroenterology: From the Basics to the Clinics. Falk Symposium 112. 2000 ISBN 0-7923-8757-0 113. Andus T, Rogier G, Schlottmann K, Frick E, Adler G, Schmiegel W, Zeitz M, Scholmerich J, eds. Cytokines and Cell Homeostasis in the Gastrointestinal Tract. Falk Symposium 113.2000 ISBN0-7923-8758-9 114. Manns MP, Paumgartner G, Leuschner U, eds. Immunology and Liver. Falk Symposium 114. 2000 ISBN 0-7923-8759-7 115. Boyer JL, Blum HE, Maier K-P, Sauerbruch T, Stalder GA, eds. Liver Ci"hosis and its Development. Falk Symposium 115. 2000 ISBN 0-7923-8760-0 116. Riemann JF, Neuhaus H, eds. Interventional Endoscopy in Hepatology. Falk Symposium 116. 2000 ISBN 0-7923-8761-9
Falk Symposium Series 116A. Dienes HP, Schirmacher P, Brechot C, Okuda K, eds. Chronic Hepatitis: New Concepts ofPathogenesis, Diagnosis and 'lreatment. Falk Workshop. 2000 ISBN 0-7923-8763-5 117. Gerbes AL, Beuers U, Jiingst D, Pape GR, Sackmann M, Sauerbruch T, eds. Hepatology 2000- Symposium in Honour ofGustav Pawngartner. Falk Symposium ISBN0-7923-8765-1 117.2000 117A. Acalovschi M, Paumgartner G, eds. Hepatobiliary Diseases: Cholestasis and Gallstones. Falk Workshop. 2000 ISBN 0-7923-8770-8 118. Friihmorgen P, Bruch H-P, eds. Non-Neoplastic Diseases of the Anorectum. Falk Symposium 118. 2001 ISBN 0-7923-8766-X 119. Fellermann K, Jewell DP, Sandborn WJ, SchOlmerich J, Stange EF, eds. Immunosuppression in Inflammatory Bowel Diseases - Standards, New Developments, Future 'lrends. Falk Symposium 119.2001 ISBN0-7923-8767-8 120. van Berge Henegouwen GP, Keppler D, Leuschner U, Paumgartner G, Stiehl A, eds. Biology ofBile Acids in Health and Disease. Falk Symposium 120. 2001 ISBN 0-7923-8768-6 121. Leuschner U, James OFW, Dancygier H, eds. Steatohepatitis (NASH and ASH). Falk Symposium 121.2001 ISBN0-7923-8769-4 121A. Matern S, Boyer JL, Keppler D, Meier-Abt PJ, eds. Hepatohiliary Transport: From Bench to Bedside. Falk Workshop. 2001 ISBN 0-7923-8771-6 122. Campieri M, Fiocchi C, Hanauer SB, Jewell DP, Rachmilewitz R, Scholmerich J, eds. Inflammatory Bowel Disease - A Clinical Case Approach to Pathophysiology, Diagnosis, and Treatment. Falk Symposium 122. 2002 ISBN 0-7923-8772-4 123. Rachmilewitz D, Modigliani R, Podolsky DK, Sachar DB, Tozun N, eds. VI International Symposium on Inflammatory Bowel Diseases. Falk Symposium 123. 2002 ISBN0-7923-8773-2 124. Hagenmiiller F, Manns MP, Musmann H-G, Riemann JF, eds. Medical Imaging in Gastroenterology and Hepatology. Falk Symposium 124. 2002 ISBN 0-7923-8774-0 125. Gressner AM, Heinrich PC, Matern S, eds. Cytokines in Liver Injury and Repair. Falk Symposium 125.2002 ISBN0-7923-8775-9 126. Gupta S, Jansen PLM, Klempnauer J, Manns MP, eds. Hepatocyte Transplantation. Falk Symposium 126.2002 ISBN0-7923-8776-7 127. Hadziselimovic F, ed. Autoimmune Diseases in Paediatric Gastroenterology. Falk ISBN0-7923-8778-3 Symposium 127.2002 127A.Berr F, Bruix J, Hauss J, Wands J, Wittekind Ch, eds. Malignant Liver Thmours: Basic Concepts and Clinical Management. Falk Workshop. 2002 ISBN 0-7923-8779-1 128. ScheppachW, Scheurlen M, eds. Exogenous Factors in Colonic Carcinogenesis. Falk Symposium 128. 2002 ISBN 0-7923-8780-5 129. Paumgartner G, Keppler D, Leuschner U, Stiehl A, eds. Bile Acids: From Genomics to Disease and Therapy. Falk Symposium 129. 2002 ISBN 0-7923-8781-3 129A. Leuschner U, Berg PA, Holtmeier J, eds. Bile Acids and Pregnancy. Falk Workshop. 2002 ISBN 0-7923-8782-1 130. Holtmann G, Talley NJ, eds. Gastrointestinal I'!flammation and Disturbed Gut Function: The Challenge ofNew Concepts. Falk Symposium 130. 2003 ISBN 0-7923-8783-X 131. Herfarth H, Feagan BJ, Folsch UR, Scholmerich J, Vatn MH, Zeitz M, eds. Targets of Treatment in Chronic Inflammatory Bowel Diseases. Falk Symposium 131. 2003 ISBN 0-7923-8784-8
Falk Symposium Series 132. Galle PR, Gerken G, Schmidt WE, Wiedenmann B, eds. Disease Progression and Carcinogenesis in the Gastrointestinal Tract. Falk Symposium 132. 2003 ISBN 0-7923-8785-6 132A. Staritz M, Adler G, Knuth A, Schmiegel W, Schmoll H-J, eds. Side-effects of Chemotherapy on the Gastrointestinal Tract. Falk Workshop. 2003 ISBN0-7923-8791-0 132B. Reutter W, Schuppan D, Tauber R, Zeitz M, eds. Cell Adhesion Molecules in Health and Disease. Falk Workshop. 2003 ISBN 0-7923-8786-4 133. Duchmann R, Blumberg R, Neurath M, SchOlm.erich J, Strober W, Zeitz M. Mechanisms ofIntestinal Inflammation: Implications for Therapeutic Intervention in IBD. Falk Symposium 133.2004 ISBN0-7923-8787-2 134. Dignass A, Lochs H, Stange E. Trends and Controversies in IBD- Evidence-Based Approach or Individual Management?Falk Symposium 134. 2004 ISBN 0-7923-8788-0 134A. Dignass A, Gross ID, BuhrV, James OFW. Topical Steroids in Gastroenterology and Hepatology. Falk Workshop. 2004 ISBN 0-7923-8789-9 135. LukaS M, Manns MP, Spicak J, Stange EF, eds. Immunological Diseases of Liver and Gut. Falk Symposium 135. 2004 ISBN 0-7923-8792-9 136. Leuschner U, Broome U, Stiehl A, eds. Cholestatic Liver Diseases: Therapeutic Options and Perspectives. Falk Symposium 136. 2004 ISBN 0-7923-8793-7 137. Blum HE, Maier KP, Rodes J, Sauerbruch T, eds. Liver Diseases: Advances in Treatment and Prevention. Falk Symposium 137. 2004 ISBN 0-7923-8794-5 138. Blum HE, Manns MP, eds. State of the Art of Hepatology: Molecular and Cell Biology. Falk Symposium 138. 2004 ISBN 0-7923-8795-3 138A. Hayashi N, Manns MP, eds. Prevention ofProgression in Chronic Liver Disease: An Update on SNMC (Stronger Neo-Minophagen C). Falk Workshop. 2004 ISBN 0-7923-8796-1 139. Adler G, Blum HE, Fuchs M, Stange EF, eds. Gallstones: Pathogenesis and Treatment. Falk Symposium 139. 2004 ISBN 0-7923-8798-8 140. Colombe! J-F, Gasche C, SchOlm.erich J, Vucelic C, eds. Inflammatory Bowel Disease: Translation from Basic Research to Clinical Practice. Falk Symposium 140. 2005. ISBN 1-4020-2847-4 141. Paumgartner G, Keppler D, Leuschner U, Stiehl A, eds. Bile Acid Biology and its ISBN 1-4020-2893-8 Therapeutic Implications. Falk Symposium 141.2005 142. Dienes H-P, Leuschner U, Lohse AW, Manns MP, eds. Autoimmune Liver Disease. Falk Symposium 142.2005 ISBN 1-4020-2894-6 143. Ammann RW, Biichler MW, Adler G, DiMagno EP, Sarner M, eds. Pancreatitis: Advances in Pathobiology, Diagnosis and Treatment. Falk Symposium 143. 2005 ISBN 1-4020-2895-4 144. Adler G, Blum AL, Blum HE, Leuschner U, Manns MP, Messner J, Sartor RB, Scholmerich J, eds. Gastroenterology Yesterday- Today- Tomorrow: A Review and Preview. Falk Symposium 144. 2005 ISBN 1-4020-2896-2 145. Henne-Bruns D, Buttenschon K, Fuchs M, Lohse AW, eds. ArtificialLiver Support. Falk Symposium 145.2005 ISBN 1-4020-3239-0 146. Blumberg RS, Gangl A, Manns MP, Tilg H, Zeitz M, eds. Gut-Liver Interactions: ISBN 1-4020-4143-8 Basic and Clinical Concepts. Falk Symposium 146. 2005 147. Jewell DP, Colombe! JF, Pefia AS, Tromm A, Warren BS, eds. Colitis: Diagnosis and Therapeutic Strategies. Falk Symposium 147.2006 ISBN 1-4020-4315-5
Falk Symposium Series 148. Kruis W, Forbes A, Jauch K-W, Kreis ME, Wexner SD, eds. Diverticular Disease: Emerging Evidence in a CommtJn Condition. Falk Symposium 148. 2006 ISBN 1-4020-4317-1 149. van Cutsem E, Rustgi AK, Schmiegel W, Zeitz M, eds. Highlights in Gastrointestinal Oncology. Falk Symposium 149. 2006. ISBN 1-4020-5108-5 150. Galle PR, Gerken G, Schmidt WE, Wiedenmann B, eds. Disease Progression and Disease Prevention in Hepatology and Gastroenterology. Falk Symposium 150. 2006 ISBN 1-4020-5109-3 151. Fraser A, Gibson PR, Hibi T, Qian J-M, SchOlmerich, eds. Emerging Issues in Inflammatory Bowel Disease. Falk Symposium 151. 2006 ISBN-13 978-1-4020-5701-4 152. Not published. 153. Dignass A, Rachmilewitz D, Stange E-F, Weinstock JV, eds. Immunoregulation in Inflammatory Bowel Diseases - Current Understanding and Innovation. Falk Symposium 153.2007 ISBN-13 978-1-4020-5888-2 154. Adler G, Fiocchi C, Lazebnik LB, Vorobiev GI, eds. Inflammatory Bowel DiseaseDiagnostic and Therapeutic Strategies. Falk Symposium 154. 2007 ISBN-13 978-1-4020-6115-8 155. Keppler D, Beuers U, Leuschner U, Stiehl A, Trauner M, Paumgartner G, eds. Bile Acids: Biological Actions and Clinical Relevance. Falk Symposium 155. 2007 ISBN 978-1-4020-6251-3 156. Blum HE, Cox DW, Hii.ussinger D, Jansen PLM, Kullak-Ublick: GA, eds. Genetics in Liver Diseases. Falk Symposium 156. 2007 ISBN 978-14020-6393-0 157. Diehl AM, Hayashi N, Manns MP, Sauerbruch T, eds. Chronic Hepatitis: Metabolic, Cholestatic, Viral and Autoimmune. Falk Symposium 157.2007 ISBN 978-1-4020-6522-4 158. Gasche G, Herrerias Gutierrez JM, Gassull M, Monterio E, eds. Intestinal Inflammation and Colorectal Cancer. Falk Symposium 158. 2007 ISBN 978-1-4020-6825-6 159. TOziin N, Mantzaris G, Dagh U, Scholmerich J, eds. IBD 2007- Achievements in Research and Clinical Practice. Falk Symposium 159. 2008 ISBN 978-1-4020-6986-4 160. Ferkolj I, Gangl A, Galle PR, Vucelic B, eds. Pathogenesis and Clinical Practice in Gastroenterology. Falk Symposium 160.2008 ISBN 978-1-4020-8766-0 161. Carey MC, Gabryelewicz A, Dite P, Keim V, Mossner J, eds. Future Perspectives in Gastroenterology. Falk Symposium 161. 2008 ISBN 978-1-4020-8832-2 162. Bosch J, Lammert F, Burroughs AK, Lebrec D, Sauerbruch T, eds. Liver Cirrhosis: From Pathophysiology to Disease Management. Falk Symposium 162. 2008 ISBN 978-1-4020-8655-7
