EMJ Gastroenterology 12.1 2023 by European Medical Journal

Volume 12.1 November 2023

Gastroenterology

Review of

UEG Week 2023 Feature

Challenges in Management of Immune Checkpoint Inhibitor-Associated Hepatitis

Interviews Jeanin van Hooft, Patrizia Burra, and Gideon Hirschfield discuss recent innovations and patient-led care

emjreviews.com

Contents 4

Editorial Board

Welcome

Foreword

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Congress Review Review of the 31ˢᵗ United European Gastroenterology (UEG) Week, 14ᵗʰ–17ᵗʰ October 2023

Congress Features Microbiota in Clinical Practice: What is New and What is True? Anaya Malik

Rise of the Machines in Digestive Diseases Evan Kimber

Abstract Review Deleted in Colorectal Cancer Salivary Methylation Analysis is a Non-invasive and Useful Method for Screening Patients with Superficial Hypopharyngeal Cancer Hirai et al.

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Abstract Highlights Congress Interviews Jeanin van Hooft Patrizia Burra

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Interview Gideon Hirschfield

Infographic Preventive Medicine in Gastroenterology

Features Challenges in Management of Immune Checkpoint Inhibitor-Associated Hepatitis Cunningham

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Let’s Start at the Beginning: A Healthy Gut From Day 1 Shamir

Articles A Rare Complication of Valproate-Induced Acute Pancreatitis in an Adult Patient with Bipolar Disorder: A Case Report Kaur et al.

SAPHO Syndrome in Crohn's Disease Successfully Treated with Ustekinumab: Case Report and Review of the Literature Caporuscio et al.

Is Intestinal Cell Death in Necrotising Enterocolitis Assorted and Multifarious? A Special Focus on Risk Factors and Their Pathogenic Mechanisms Kanuri et al.

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Editorial Board Editor-in-Chief Prof Sorin Barbu

Iuliu Hațieganu University of Medicine and Pharmacy, Romania

Editorial Board Prof Dan Dumitrascu

Iuliu Hațieganu University of Medicine and Pharmacy, Romania

Dr Oliver Grundmann

University of Florida, USA

Prof Christoph Gubler

Stadtspital Zürich, Switzerland

Dr Hasan Haboubi

Swansea University, UK

Prof Najib Haboubi

Spire Hospital, UK

Dr Waseem Hamoudi

Royal Hospital, Jordan

Dr Devika Kapuria

Washington University, USA

Dr Panagiotis Kasapidis

Central Clinic of Athens, Greece

Prof Milan Lukáš

Charles University, Czechia

Dr Venkata Pawan Kumar Lekharaju

Central Clinic of Athens, Greece

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Aims and Scope EMJ is an online only, peer-reviewed, open access general journal, targeted towards readers in the medical sciences. We aim to make all our articles accessible to readers from any medical discipline. EMJ allows healthcare professionals to stay abreast of key advances and opinions across Europe. EMJ aims to support healthcare professionals in continuously developing their knowledge, effectiveness, and productivity. The editorial policy is designed to encourage discussion among this peer group. EMJ is published quarterly and comprises review articles, case reports, practice guides, theoretical discussions, and original research. EMJ also publishes 18 therapeutic area journals, which provide concise coverage of salient developments at the leading European congresses. These are published annually, approximately 6 weeks after the relevant congress. Further details can be found on our website: www.emjreviews.com Editorial Expertise EMJ is supported by various levels of expertise: • • • •

Guidance from an Editorial Board consisting of leading authorities from a wide variety of disciplines. Invited contributors are recognised authorities from their respective fields. Peer review, which is conducted by EMJ’s Peer Review Panel as well as other experts appointed due to their knowledge of a specific topic. An experienced team of editors and technical editors.

Peer Review On submission, all articles are assessed by the editorial team to determine their suitability for the journal and appropriateness for peer review. Editorial staff, following consultation with either a member of the Editorial Board or the author(s) if necessary, identify three appropriate reviewers, who are selected based on their specialist knowledge in the relevant area. All peer review is double blind. Following review, papers are either accepted without modification, returned to the author(s) to incorporate required changes, or rejected.

We are always keen to hear from healthcare professionals wishing to discuss potential submissions, please email: editorial.assistant@emjreviews.com To submit a paper, use our online submission site: www.editorialmanager.com/e-m-j Submission details can be found through our website: www.emjreviews.com/contributors/authors Reprints All articles included in EMJ are available as reprints (minimum order 1,000). Please contact hello@emjreviews.com if you would like to order reprints. Distribution and Readership EMJ is distributed through controlled circulation to healthcare professionals in the relevant fields across Europe. Indexing and Availability EMJ is indexed on DOAJ, the Royal Society of Medicine, and Google Scholar®; selected articles are indexed in PubMed Central®. EMJ is available through the websites of our leading partners and collaborating societies. EMJ journals are all available via our website: www.emjreviews.com Open Access This is an open-access journal in accordance with the Creative Commons Attribution-Non Commercial 4.0 (CC BY-NC 4.0) license. Congress Notice Staff members attend medical congresses as reporters when required. This Publication ISSN 2054-6203 EMJ Gastroenterology is published once a year. For subscription details please visit: www.emjreviews.com

We welcome contributions from professionals, consultants, academics, and industry leaders on relevant and topical subjects.

All information obtained by EMJ and each of the contributions from various sources is as current and accurate as possible. However, due to human or mechanical errors, EMJ and the contributors cannot guarantee the accuracy, adequacy, or completeness of any information, and cannot be held responsible for any errors or omissions. EMJ is completely independent of the review event (UEG 2023) and the use of the organisations does not constitute endorsement or media partnership in any form whatsoever.

We seek papers with the most current, interesting, and relevant information in each therapeutic area and accept original research, review articles, case reports, and features.

Front cover and contents photograph: Copenhagen, Denmark home of the UEG 2023 © OliverFoerstner / stock.adobe.com

Editorial staff have final discretion over any proposed amendments. Submissions

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Welcome letter Editor Evgenia Koutsouki Editorial Manager Anaya Malik Copy Editors Noémie Fouarge Kirsty Hewitt

Evgenia Koutsouki

Editorial Co-ordinator Natasha Meunier-McVey

Editor

Editorial Assistants Lucy Anderson, Victoria Antoniou, Abigail Craig, Ada Enesco, Evan Kimber, Darcy Richards

Dear Readers, Welcome to the 2023 issue of EMJ Gastroenterology, showcasing key highlights from United European Gastroenterology (UEG) Week 2023. This year’s meeting unfolded in Copenhagen, Denmark, centring around compelling themes, such as the role of artificial intelligence in healthcare, and challenges in gastroenterology.

Design Manager Stacey Rivers Senior Designer Roy Ikoroha

Among findings presented was a noteworthy study examining the use of the gut microbiome for predicting colorectal cancer, demonstrating the potential of the microbiome in improving existing tests, thereby advancing early detection methods for pre-cancerous lesions and colorectal cancer. In addition, results from the ATLANTIS trial demonstrate that amitriptyline, a drug commonly used for depression and chronic pain, among other conditions, can improve symptoms of irritable bowel syndrome.

Designer Steven Paul Junior Designers Dillon Benn Grove, Shanjok Gurung Head of Sales Robert Hancox Senior Project Manager Max Roy

In this issue, we are delighted to present interviews with experts discussing key topics, such as artificial intelligence and liver transplantation, as well as diversity and inclusion in gastroenterology. Within our peer-reviewed content is a highly engaging feature addressing the key issues of digestive health and emphasising the importance of introducing preventative measures in national health agendas to effectively improve digestive health. Another article of interest focuses on the challenges in the management of immunerelated hepatitis, particularly in areas where decision-making for the clinician is challenging due to a lack of evidence.

Director of Performance Keith Moule Chief Operating Officer Dan Scott Chief Commercial Officer Dan Healy Founder and Chief Executive Officer Spencer Gore

I would like to close by extending our gratitude to our Editorial Board, and to the contributors and peer reviewers of this journal, who have provided this outstanding content, upholding the highest standards of quality. Until the next issue, I hope you enjoy our content over the next year, and our team looks forward to your valuable submissions.

Contact us Editorial enquiries: editor@emjreviews.com Sales opportunities: salesadmin@emjreviews.com Permissions and copyright: accountsreceivable@emjreviews.com

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Reprints: info@emjreviews.com Media enquiries: marketing@emjreviews.com

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Foreword Dear Colleagues, It is with great pleasure that I introduce to you the latest edition of EMJ Gastroenterology. In this issue, you will find peer-reviewed articles, interviews with specialty leaders, and a review of the United European Gastroenterology (UEG) Week 2023, which took place in Copenhagen, Denmark, between the 14th–17th October. Experts from all over the world gathered at this hybrid event to share updates across a broad variety of subspecialties, presenting research findings across multiple disciplines within gastroenterology. In line with UEG’s motto, “Ingest the Best,” I urge you to peruse and absorb the interesting content straight from the specialists in this release, including interviews with both Jeanin van Hooft and Patrizia Burra. Alongside these, you will find a thought-provoking infographic on preventive medicine, which aims to change perspectives and encourage clinicians to foster better relationships with their patients, introducing

countermeasures and lifestyle changes to combat disease earlier in life. There are two features included in this release, and following the theme of preventive medicine, Shamir raises awareness for healthy nutrition practices, and discusses changes in the early years of life to prevent the development of noncommunicable diseases. Meanwhile, Cunningham describes challenges in the management of immune checkpoint inhibitor-associated hepatitis, defining severity, providing information about diagnostic hurdles, and detailing steroid use. With regard to the articles, Kundra et al. deliver a case report on valproate-induced acute pancreatitis from the unusual position of a patient with bipolar disorder. This is just one of the high-quality papers included. I would like to take this opportunity to thank all of the contributors in this issue, including peer reviewers and the Editorial Board, for their support. I hope you enjoy reading the journal!

Sorin T. Barbu Professor of Surgery, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

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UEG 2023 Review of United European Gastroenterology (UEG) Week 2023

Location:

Copenhagen, Denmark, and online

Date:

14th–17th October 2023

Citation:

EMJ Gastroenterol. 2023;12[1]:8-14. DOI/10.33590/emjgastroenterol/10300148. https://doi.org/10.33590/emjgastroenterol/10300148.

WONDERFUL Copenhagen, Denmark, was the setting for the 31st United European Gastroenterology (UEG) Week, with its slogan of ‘Ingest the Best’. More than 11,000 participants from 130 nations joined in person, and many other colleagues from around the world watched the livestreamed event. Denmark’s chic capital city, home to colourful Nyhavn, and Tivoli Gardens, the second oldest amusement park in the world, provided the perfect backdrop for this year’s UEG congress. In the largest city in Scandinavia, there was plenty for attendees to do between sessions, from viewing the city from the top of the Rundetårn, to eating traditional smørrebrød.

a role model, and announcing the organisation's efforts in promoting equality and inclusion… [and] empowering women and minority groups.” Cortez-Pinto thanked everyone involved in putting on “a large number of activities during and through the year” on the UEG’s behalf, 300 of whom are volunteers. She highlighted: “This meeting is more than just science. It is the place where we meet to make change, network, and develop.”

The annual UEG Week is a main strategic focus of the organisation, and this year, it featured 450 invited faculty members, and a choice of more than 2,600 abstract presentations. Multidisciplinary work is key for UEG, and worldclass sessions covered areas of interest across the entire field of gastroenterology.

Cortez-Pinto also addressed some of the current major challenges in the gastroenterology space, with the unpopularity of some healthcare policies, the impact of pharmaceutical companies promoting “unhealthy products,” and the prohibitive cost of carrying out research when part of a smaller group. CortezPinto also discussed the impossibility of balancing privacy with public practice, which promotes further inequalities in access to healthcare. The help or hindrance posed by artificial intelligence was also touched upon.

In the opening plenary session, the presidential address was given by Helena Cortez-Pinto, who stressed how proud she is to be the first female President of UEG: “I am proud to be serving as

A session entitled ‘Hospitals in Crisis’ was led by Marcel Levi, President of the Dutch Research Council (NWO), and Professor of Medicine at the University of Amsterdam, the Netherlands;

"Multidisciplinary work is key for UEG, and world-class sessions covered areas of interest across the entire field of gastroenterology."

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"This meeting is more than just science. It is the place where we meet to make change, network, and develop.” and University College London (UCL), UK. Levi prefaced his talk by referencing the COVID-19 pandemic, and underscored that the “sense of crisis in hospitals has actually remained not only in certain countries, but almost in every country” since the acute stage of the pandemic has waned. Levi gave myriad reasons for this “global crisis,” from a shortage of staff and financial resources, to logistical issues. Other showcased sessions during the opening plenary session covered results from a trial of resmetirom in non-alcoholic steatohepatitis (presented by Jörn Schattenberg, Universitätsmedizin Mainz, Germany), the academic versus industry approach in drug development (Arthur Kaser, University of Cambridge, UK), and early-stage gastrointestinal lesions (Michael Bretthauer, University of Oslo, and Oslo University Hospital, Norway). Awards were given to five authors, selected from the 4,000 abstracts submitted to UEG this year. The panel of reviewers chose the following recipients: Johan Hardvik Åkerström, Karolinska Institutet, Solna, Sweden, on anti-reflux surgery versus anti-reflux medication; Paul Lopatta, University of Ulm, Germany, on oncogenic

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GNAS and KRAS signalling in cystic pancreatic neoplasia; Bruce Sands, Mount Sinai Hospital, New York, USA, on a 2-year trial of mirikizumab; Jörn Schattenberg on the aforementioned trial of resmetirom in non-alcoholic steatohepatitis; and Peter Sinonquel, Katholieke Universiteit (KU) Leuven, Belgium, on the use of a computer-aided detection model in colorectal polyp detection. The recipient of the coveted UEG Lifetime Achievement Award 2023 for outstanding accomplishment was announced as Jan Tack, Head of the Department of Gastroenterology and Hepatology at KU Leuven; Professor of Internal Medicine and Chairman of the Gastrointestinal Motility and Sensitivity Research Group within the Translational Research Center for Gastrointestinal Disorders (TARGID); and President of the Rome Foundation, for his work on a wealth of diverse topics, from gastrointestinal motility to the psychology and physiology of the nervous system. For now, we hope you enjoy our wealth of content from UEG Week 2023, and we cannot wait for next year’s congress in Vienna, Austria. See you there! ●

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Babies with Low Birthweight More Likely to Develop Fatty Liver Disease RESEARCH presented at UEG Week has found that there is a significant association between birthweight and non-alcoholic fatty liver disease, now more often known as metabolic dysfunctionassociated steatotic liver disease (MASLD), in young people. The results showed that babies with low birthweight were four times more likely to be diagnosed with MASLD in childhood, adolescence, or young adulthood. Fahim Ebrahimi, Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden, and colleagues, used the nationwide ESPRESSO cohort in order to conduct a population-based case-control study of 165 people aged 25 years and younger, who had been diagnosed with biopsy-proven MASLD between January 1992–April 2017. Each individual with MASLD was matched up to five controls from the general population, based on age, sex, calendar year, and country. Results showed that children born with a low birthweight (<2,500 g/5 lbs 8 oz) were four times more likely to develop MASLD than those born with a normal birthweight. Babies born as small for gestational age, falling below the 10th

percentile, were over three times more likely to go on to develop MASLD early in life compared to those with an adequate birthweight (10th–90th percentiles). Those born small for gestational age or with low birthweight also had a higher relative risk of developing more severe stages of MASLD, in the form of liver fibrosis or cirrhosis. MASLD has become the most common cause of chronic liver disease worldwide, affecting over 25% of adults in Europe alone, and it is only getting more prevalent with escalating obesity rates. It is also one of the fastest-growing causes of end-stage liver disease, primary liver cancer, and liver transplantation. The team therefore concluded that it is important to develop proactive and effective strategies, such as early and targeted screening, to identify at-risk individuals, and reduce the burden of MASLD. They added: “Further research is needed to fully understand the underlying immunological and metabolic mechanisms. Several studies suggest that both overnutrition and undernutrition during pregnancy can lead to lasting epigenetic changes, that can affect an individual’s metabolism for a lifetime.” ●

"MASLD has become the most common cause of chronic liver disease worldwide, affecting over 25% of adults in Europe alone."

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Can Gut Microbiome Variations Predict Colorectal Cancer Risk? SIGNIFICANT variations in the gut microbiome have been identified in patients who developed pre-cancerous colonic lesions, according to new research presented at UEG Week 2023.

as those who developed lesions after a sample was taken, between 2015–2022. These results were compared with the general population, and those who had normal colonoscopy findings.

Colorectal cancer typically develops from precancerous lesion in the gut. Removing these lesions is an effective strategy to prevent colorectal cancer, but existing non-invasive detection techniques lead to unnecessary colonoscopies, due to a high number of false positives.

The results showed that gut microbiomes were varied, depending on the type of lesion, and the composition and function of the microbiome were different among patients with pre-existing and future lesions. Furthermore, those who developed colonic lesions after faecal sampling had increased diversity in gut microbiome compared with those who did not develop lesions. Gacesa noted: “Our findings suggest that the microbiome could act as a valuable tool to improve existing tests, advancing early detection methods for pre-cancerous lesions and colorectal cancer.”

Ranko Gacesa, University Medical Center Groningen (UMCG), the Netherlands, and colleagues, performed a large-scale prospective study that involved 8,208 patients. They correlated data from the Dutch nationwide pathology database and the Dutch Microbiome Study to identify recorded cases of colonic biopsies from the last 50 years. The researchers analysed the composition and function of gut microbiomes in patients who developed pre-cancerous lesions between 2000– 2015, before a faecal sample was taken, as well

The researchers also examined specific bacterial strains to gain more knowledge into the role of the gut microbiome, and see the functions of these bacteria within the gut. They noted that bacteria from the Lachnospiraceae family, Roseburia, and Eubacterium were associated with developing lesions in the future. ●

"Existing non-invasive detection techniques lead to unnecessary colonoscopies due to a high number of false positives."

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Amitriptyline Beneficial in Irritable Bowel Syndrome AMITRIPTYLINE, a cheap and widely available prescription drug that is commonly used for a range of health concerns, including depression and chronic pain, has been shown to improve symptoms of irritable bowel syndrome (IBS), according to data from the ATLANTIS trial presented at UEG Week 2023. IBS, which affects approximately one in 20 people worldwide, and causes abdominal pain and changes to bowel movement, can significantly impact quality of life, and available treatments only have a modest effect. While National Institute for Care and Excellence (NICE) guidelines state that lowdose amitriptyline should be considered for IBS, evidence for its benefits has been limited.

after 6 months, compared to those on placebo. They were also twice as likely to experience overall improvement in IBS symptoms. Anxiety and depression scores were monitored and found to be unaltered, showing that the benefits of the treatment were linked to the gut, and not due to the antidepressant effect of the medication.

For this trial, participants were randomly assigned to one of two groups, receiving either amitriptyline or placebo. The patients had moderate-to-severe symptoms, and an average duration of disease of 10 years. A patient dose adjustment document was developed for the trial, allowing patients to decrease or increase dosage based on the severity of their symptoms and side effects. Those taking amitriptyline reported a bigger improvement in IBS symptoms

"The results of this study are hugely encouraging. It shows that a drug already widely available to treat a number of other conditions appears to be safe and effective for people with IBS. The findings the research team have shared around the adjustment of dosages can be tremendously helpful to general practitioners in guiding them when treating patients,” stated Andrew Farmer, National Institute for Health and Care Research (NIHR), Oxford, UK. ●

Mild side effects, such as dry mouth in the morning, were reported, but no safety concerns were identified. The trial found a clear benefit of taking amitriptyline, showing that general practitioners can offer it to patients with IBS if first-line treatments do not improve their symptoms.

"Benefits of the treatment were linked to the gut, and not due to the antidepressant effect of the medication."

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Tackling Liver Disease Through Alcohol Consumption and Obesity Interventions INTRODUCTION of a 1 EUR minimum unit pricing (MUP) system on alcohol, and a set of complementary public health policies to deliver this, alongside sugar-sweetened beverage (SSB) tax, and a volumetric tax on alcohol, could lead to a significant reduction of up to 7% in incidence of chronic liver disease (CLD) and lower cancer in Europeans before 2030. A presentation delivered at UEG Week 2023, in Copenhagen, Denmark, provided updates on this important topic. Researchers employed data sourced from online databases and published literature to project static and dynamic trends in alcohol consumption and BMI, from 2022–2030, utilising a validated and peer-reviewed microsimulation model. Incidence of CLD and liver cancer were measured in France, the Netherlands, and Romania, aiming to estimate the impact of policy interventions targeting alcohol and obesity. The policies modelled were a 1 EUR MUP on alcohol; a combination of 0.7 EUR MUP and a SSB tax; and a combination of 0.7 EUR MUP, SSB tax, and a volumetric tax on alcohol. The study discovered all policy scenarios had a substantial impact, leading to reduction in annual incidence of CLD and liver cancer, ranging from 2–7%. The 1 EUR MUP policy exhibited the most significant predicted impact, potentially

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preventing 11,550 cases of CLD, and resulting in 7,921 fewer cases of liver cancer by 2030. The policy interventions combining a 0.7 EUR MUP, a SSB tax, and a volumetric tax on alcohol were also highly effective, potentially preventing 7,317 cases of CLD and 5,390 cases of liver cancer by 2030, when compared to an inaction scenario.

"Currently, Europe has the largest burden globally of diagnosed liver disease." This research highlights the focal points for implementing change, and presents the importance of targeting multiple drivers of obesity and alcohol consumption to produce change to the burden caused by CLD and liver cancer. Currently, Europe has the largest burden globally of diagnosed liver disease, with almost 30 million people alone estimated to be living with a chronic liver condition. This study is expected to guide Europeans down a path toward preventing development of disease earlier in the life course. This investigation successfully estimates the future burden of liver disease, and highlights the potential impact of policy interventions. ●

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Microbiota in Clinical Practice: What is New and What is True? Authors:

Anaya Malik, EMJ, London, UK

Citation:

EMJ Gastroenterol. 2023;12[1]:15-16. DOI/10.33590/emjgastroenterol/10304704. https://doi.org/10.33590/emjgastroenterol/10304704.

INCREASING interest in the microbiome can be linked to its great diagnostic and treatment potential, the extent of which is still largely unknown. The session ‘The Healthy Microbiota: Does It Exist?’, presented at the 31ˢᵗ United European Gastroenterology (UEG) Week in Copenhagen, Denmark, gave an in-depth review of the current landscape of the microbiome in clinical practice.

THE ‘HEALTHY’ MICROBIOME Jeroen Raes, Katholieke Universiteit (KU) Leuven, Belgium, opened the session by addressing the surge of interest in the microbiome in recent years. The first big wave of attention was approximately 10 years ago and included research into diseases of the microbiome, but lacked an understanding of what can be considered a ‘healthy’ microbiome, Raes recalled. He suggested that key features of the research, for example, variation in a clinically relevant population, temporal variation and biomarker stability, factors influencing gut flora composition, clinical endpoints, and effects of the environment in relation to the microbiome, were all largely unknown back in 2012 during this initial wave. Faecal samples can be considered as a snapshot of a maturing ecosystem and have emerged as one of the most valuable tools in microbiome research. Recent advances suggest transit time is the most important confounder of microbiome studies and should not be underestimated in clinical trials. Transit time should be taken into consideration in any trial as it is the main factor explaining variation in the microbiome between individuals, and should

therefore be controlled for. Slow transit times have been associated with a shift from saccharolytic to proteolytic metabolism, which can have significant implications for gut health. The diversity, composition, and variety of the microbiome in an individual can be determined by several host factors. Factors may include genetics, diet, age, sex, and use of antibiotics. Additionally, the present microbiome may have been affected by historical variables. While experts may not be able to categorically state what should be considered a healthy microbiome, there is knowledge on what is not indicative of a healthy microbiome. The Bacteroides 2 enterotype can be identified in approximately 10% of the population. It is characterised by dysbiosis, low cell count, low diversity, low anti-inflammatory taxa, high pro-inflammatory taxa/pathobionts, and higher temporal instability. Population studies are essential in mapping ‘normal’ variation. There are multiple physiological and lifestyle factors influencing variation, including transit time, diet, low-grade inflammation, age, sex, and drugs.

"Transit time is the most important confounder of microbiome studies and should not be underestimated in clinical trials."

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THE FUTURE OF MICROBIOME THERAPEUTICS Haggai Bar-Yoseph, Rambam Health Care Campus, Haifa, Israel, shared the future of microbiome therapeutics with the audience. There is a well-established fascination with the association between the microbiome and disease or health patterns from healthcare professionals and laymen alike. Healthcare professionals must consider where best to intervene to change the natural course of disease towards a more beneficial course.

“Westernisation is causing mass extinction of our microbiota.”

Advances in microbiome research have a major impact on the continuously growing understanding of healthy microbiota. Steering away from the dysbiotic Bact2 enterotype towards more balanced gut microbiota should be a key target for future microbiome interventions.

THE MICROBIOTA SIGNATURE AND PERSONALISED TREATMENT Gianluca Ianiro, Gemelli Hospital, Rome, Italy, presented the concept of the microbiota signature, and whether it could be used to guide personalised treatment. The gut microbiome is modifiable and is mainly influenced by the environment. Making changes to environmental factors, such as drugs, diet, lifestyle, or other therapeutics, can advantageously or disadvantageously alter the microbiota in a rapid fashion. Ianiro shared the effects of the modern lifestyle on the microbiome, stating that “Westernisation is causing mass extinction of our microbiota.” Ecologically speaking, the diversity of the microbiome is dropping tremendously, driving the prevalence of chronic disorders. Reassuringly, Ianiro remarked on the ease with which some microbiome interventions can be addressed at the bedside. The current standard for microbiome diagnostics lacks regulation and is even chaotic, Ianiro reported, and international consensus is needed to establish guidelines and standards for microbiome testing and analysis.

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Faecal microbiota transplant is a very robust intervention that is relied upon in some clinical scenarios, and can be manipulated through procedure-, recipient-, or donor-related factors. Bar-Yoseph focused on the promise of therapeutics beyond faecal microbiota transplant and other treatments on the horizon. Researchers are exploring microbial consortia, next-generation probiotics, phage targeting, and synthetic biology as potential tools to manipulate the microbiome. These interventions are being actively tested; however, they are not robust enough for the clinic. Next-generation probiotics offer potential that may be greater than traditional, single-agent interventions or ‘old probiotics’ historically consumed in food, beverages, or taken as supplements. Also referred to as live biotherapeutics, they do not have a clear history of use, are challenging in strain cultivation, may be genetically manipulated, and are undergoing testing for medical use rather than for dietary supplementation. There remains much to be discovered in this vast, evolving field of study and the associated health implications. Some interventions are yet to demonstrate the robustness required for clinical implementation, but the exciting possibilities for therapeutic intervention suggest a wealth of forthcoming developments is still to come. ●

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Rise of the Machines in Digestive Diseases Authors:

Evan Kimber, EMJ, London

Citation:

EMJ Gastroenterol. 2023;12[1]:17-19. DOI/10.33590/emjgastroenterol/10301023. https://doi.org/10.33590/emjgastroenterol/10301023.

“MACHINES are taking over,” warned Mahdi Al-Taher, Maastricht University Medical Center, the Netherlands, “but we should not be afraid of them, as they are still under our control.” He was describing image-guided surgery techniques in the setting of digestive diseases, as a part of an entertaining and educational session at United European Gastroenterology (UEG) Week, hosted in Copenhagen, Denmark, between the 14th–17th October 2023. Natazja Pilonis, University of Cambridge, UK, moderated this session, which also included a discussion on machine learning-based approaches to digestive diseases, and artificial intelligence (AI) in endoscopy.

MACHINE LEARNING-BASED APPROACHES TO DIGESTIVE DISEASES The human microbiome was integral to the presentation delivered by Edoardo Pasolli, University of Naples Federico II, Italy. Although he focused on the bacterial composition of the gut, many of the components discussed have applications across wider aspects of gastroenterology. Pasolli gave an overview of the shotgun metagenomics workflow that is central to much of his work, running through the steps involved in profiling the microbiome. From a data analysis perspective, the main steps involved in producing a complete catalogue are data acquisition, sequence analysis, post-processing, and validation. Building large microbial genome catalogues is a topic at the forefront of this field, and multiple ongoing efforts to drive this initiative were referenced in his talk. In one study that compared Westernised and nonWesternised lifestyles, where Pasolli was the lead investigator, the researchers were able to reconstruct 154,723 genomes from 9,428 metagenomes, identifying 4,930 species, of which 77% were newly discovered.

Large-scale applications of work such as this can be seen in the emerging integrative approaches employed by human, environmental, animal, and food classifications. Progressing to discuss strain-resolved metagenomics, Pasolli drew attention to the challenges we experience when applying this sequencing tool from a computational perspective. Difficulties arise with the strain identification, tracking, and functional characterisation steps involved in this process. The final product of this biological and clinical interpretation, is biomarker discovery, and receiving microbiome-based prediction tools. Pasolli shared data from recent research that has been completed, combining multiple cohorts in an attempt to generalise prediction capabilities, and detect biomarkers across populations.

ARTIFICIAL INTELLIGENCE IN ENDOSCOPY Acknowledging that the use of AI in endoscopy is no longer a novel technology. Yuichi Mori, University of Oslo, Norway, and Showa University, Japan, began his section by explaining that he would focus on the very latest updates in this field. He delivered a clinical perspective and focused on the applications of AI

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in clinical practice, centring on colonoscopy, and upper gastrointestinal endoscopy. Computer-aided detection and computeraided diagnosis are the main applications for AI in colonoscopy, and Mori urged clinicians to understand the pros and cons of using these technologies in patient care. He highlighted the importance of maintaining a high adenoma detection rate (ADR) when issuing treatment to patients, focusing his talk on some of the difficulties doctors are faced with in achieving this. Describing a meta-analysis he was involved with, which compared ADR in screening colonoscopies with and without AI, Mori stated, “there is a huge benefit of using AI in terms of incremental ADR,” supported by the 24% increased detection rate discovered in this study. He did, however, also present contrasting results from trials that did not agree, highlighting the importance of taking different perspectives on the effectiveness of AI. “This type of data can’t be ignored,” Mori explained, and hinted at a requirement for future research: “We need a clinical trial which is focused on a more rigid end-point, such as cancer incidence.” Turning to computer-aided diagnosis, similarly, the key message from this field was the lack of existing evidence on AI as a diagnostic tool, and our need for more studies revealing effectiveness.

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"The use of AI in endoscopy is no longer a novel technology." Similar themes were presented on the use of AI in upper gastrointestinal endoscopy, which is another exciting, emerging field that is not exempt from challenges. Much of the work being done in this field is preliminary, and is yet to receive regulatory approval. Nonetheless, Mori highlighted devices which are ready for use in clinical practice; cloud-based computing is an initiative that is assisting diagnosis of conditions such as Barrett’s neoplasia, gastric cancer, and oesophageal squamous cell carcinoma. The scarcity of data on the effectiveness of different approaches is a familiar barrier in this specialty at present. Mori concluded with discussion of the randomised control trials of AI in both gastroscopy and colonoscopy, addressing the existing limitations with data. Currently, certain establishments are leading the way and producing multiple encouraging studies, but these are in desperate need of support from other institutions to be confirmed on a wider scale.

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IMAGE-GUIDED SURGERY TECHNIQUES Optical imaging surgery is the new normal for surgeons, and has made life far easier in everyday practice. Al-Taher began by emphasising this, and the knock-on benefits experienced by patients as a result of the rise in robotic aspects to medical procedures. He highlighted the paradigm shift that has taken place, substituting open for laparoscopic surgery. Where this minimally invasive approach has its advantages, Al-Taher underscored how the advances in current technology are working to restore the tactile feedback associated with performing surgery historically. “Improving the surgical eye, the surgical hand, and the surgical brain,” was how he described the benefits of new augmented surgeries.

"Optical imaging surgery is the new normal for surgeons."

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Three of the most promising techniques, already in use, were spotlighted in a section titled ‘seeing the invisible’: fluorescence imaging, hyperspectral imaging, and laser speckle contrast imaging. Al-Taher guided the audience through each of these, and provided insight on how they are helpful tools for clinicians, both in real-time and predictively.

CONCLUSIONS Presentations in this session provided visual, and directly applicable learnings for doctors to incorporate into their own practice. Consistently throughout this symposium, the speakers emphasised that this is no longer a novel field, and that only the latest updates were being shared. Gastroenterologists were encouraged to go away and think hard about the way they are using technology, as more and more devices continue to appear, requiring confirmation in research on a large scale. ●

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Abstract

Abstract Review Featuring insightful research from the United European Gastroenterology (UEG) Week 2023, the following abstract presents a study on the diagnostic accuracy of screening for hypopharyngeal cancer, using salivary DNA promoter methylation analysis.

Deleted in Colorectal Cancer Salivary Methylation Analysis Is a Non-invasive and Useful Method for Screening Patients with Superficial Hypopharyngeal Cancer Authors: Ryosuke Hirai,1 *Kinugasa Hideaki,1 Soichiro Ako,1 Koichiro Tsutsumi,1 Makoto Abe,2 Koji Miyahara,2 Masahiro Nakagawa,2 Motoyuki Otsuka1

Disclosure: The authors have declared no conflicts of interest. Acknowledgements: The authors would like to thank all patients for their participation in the study. The authors thank Kiyoshi Misawa (Hamamatsu University School of Medicine, Japan) for the technical support. Keywords: DCC, hypopharyngeal cancer, liquid biopsy, promoter methylation, saliva. Citation: EMJ Gastroenterol. 2023;12[1]:20-22. DOI/10.33590/emjgastroenterol/10300651. https://doi.org/10.33590/emjgastroenterol/10300651.

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With the progress in upper gastrointestinal endoscopic technology, the usefulness of endoscopic early diagnosis and treatment of hypopharyngeal cancer has been reported.1 However, it is unfeasible to perform detailed pharyngeal observation requiring sedation for all subjects undergoing upper gastrointestinal endoscopy. There is an urgent need to establish a minimally invasive and simple evaluation method for high-risk patients with hypopharyngeal cancer. The aim of this study was to verify the diagnostic accuracy of superficial hypopharyngeal cancer by salivary DNA promotor methylation analysis.

MATERIALS AND METHODS

1. Department of Gastroenterology, Okayama University Hospital, Japan 2. Department of Internal Medicine, Hiroshima City Hospital, Japan *Correspondence to gyacy14@gmail.com

BACKGROUND AND AIMS

In total, 61 patients with superficial hypopharyngeal cancer who underwent endoscopic resection at Okayama University Hospital, Japan (derivation cohort), 23 patients at Hiroshima City Hospital, Japan (validation cohort), and 51 healthy controls (control cohort) were recruited. The authors assessed DNA methylation values of four genes (DCC, PTGDR1, EDNRB, and ECAD) in tissue and saliva samples by quantitative methylation-specific PCR for bisulfite converted DNA. First, DNA was extracted from the superficial hypopharyngeal cancer area and the surrounding normal mucosal area in tissue samples resected at Okayama University Hospital. The accuracy of the discrimination between cancerous and normal mucosa was examined using DNA promoter methylation. Second, in a derivation study of saliva samples, DNA methylation values

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Abstract

Figure 1: Comparison of the diagnostic accuracy for superficial hypopharyngeal cancer by the methylation analyses of the genes in the saliva.

The black points in each chart indicate the position of the cut-off value. The AUC of the ROC analysis of DCC, EDNRB, and ECAD for superficial hypopharyngeal cancer diagnosis were 0.917 (95% CI: 0.864–0.970), 0.680 (95% CI: 0.576–0.784), and 0.639 (95% CI: 0.441–0.838), respectively. The diagnostic accuracy of DCC methylation in saliva samples was significantly higher than that of EDNRB and ECAD methylation (p<0.001, log-rank test). The cut-off values for the normalised methylation value of DCC, EDNRB, and ECAD were ≥0.163, ≥0.256, and ≥0.655, respectively. AUC: area under the curve; CI: confidence interval; ROC: receiver operating characteristic.

were analysed using the salivary DNA of the derivation cohort and control cohort. After establishing the cut-off value for differentiating between patients and healthy subjects, the diagnostic accuracy was evaluated. Third, as

a validation study, the authors analysed DNA methylation levels in the salivary DNA of the validation cohort. They determined the detection rate in patients using the cut-off value calculated in the derivation study.

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RESULTS

CONCLUSION

In the resected tissue samples, DCC (methylation value in normal mucosa: 3.94; in cancer: 12.10; p=0.003), EDNRB (11.84; 29.90; p=0.001), and ECAD (1.09; 4.73; p=0.043), showed significant differences in the amount of methylation between cancer and non-cancer site, except PTGDR1 (3.30; 4.33; p=0.47). When performing receiver operating characteristic analysis of salivary methylation for superficial pharyngeal cancer diagnosis in the derivation cohort, the area under the curve of DCC was 0.917 (95% confidence interval [CI]: 0.864–0.970), while the area under the curve of EDNRB and ECAD were 0.680 (95% CI: 0.576–0.784) and 0.639 (95% CI: 0.441–0.838), respectively (Figure 1).

In patients with hypopharyngeal cancer, salivary DNA methylation analysis of DCC showed high diagnostic accuracy even at the stage of superficial cancer. Because saliva can be collected easily and non-invasively, this method could be a useful tool for screening patients who are at high risk for hypopharyngeal cancer. ●

References 1.

Muto M et al. Early detection of superficial squamous cell carcinoma in the head and neck region and esophagus by narrow band imaging: a multicenter randomized controlled trial. J Clin Oncol. 2010;28(9):1566-72.

With the cut-off for the DCC methylation value in saliva samples setting at ≥0.163%, the sensitivity to detect hypopharyngeal cancer was 83.6% with a specificity of 90.2%. Applying this cut-off value to 23 cases of the validation cohort, 18 cases (78.3%) of patients with superficial hypopharyngeal cancer were detectable.

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Abstract Highlights

Abstract Highlights The following stories summarise a selection of the top abstracts presented at United European Gastroenterology (UEG) Week 2023. These highlights span several important topics, from artificial intelligence in colonoscopy, to endoscopy, irritable bowel syndrome, and inflammatory bowel disease. Citation:

EMJ Gastroenterol. 2023;12[1]:23-29. DOI/10.33590/emjgastroenterol/10300965. https://doi.org/10.33590/emjgastroenterol/10300965.

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Artificial Intelligence: The Latest Generation of 4K Colonoscopy IN MODERN colonoscopy practice, the human eye is enhanced by high-definition white-light visualisation, along with advanced imaging technology. However, quality is limited by the detection rate of suspicious lesions. With 4K resolution and computer-aided detection (CADe), based on artificial intelligence (AI), the next generation of endoscopes may be the answer. A research team led by Tomasz Gach, Jagiellonian University, Kraków, Poland, therefore sought to assess the effect of AI implementation in the latest generation of endoscopes and 4K visualisation, using a retrospective analysis. Results were presented at UEG Week 2023. Specifically, the Olympus Endo-Aid CADe AI system (Olympus, Tokyo, Japan) was used, together with the latest X1 series endoscope set using LED lighting and 4K ultra high-resolution technology. Included patients were over 18 years old, and had undergone a colonoscopy in the past 5 years. The study population was divided into two groups, with Group 1 consisting of 1,000 consecutive tests performed using Endo-Aid CADe (Olympus), and Group 2 consisting of 1,000 consecutive tests without the CADe system. A total of 2,000 participants were included in the analysis. The overall polyp detection rate (PDR) was similar in both test groups, yielding rates of 46.7% and 44.9%, respectively, for Group 1 and Group 2 (p=0.419). Furthermore, the adenoma

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(p=0.694) and advanced adenoma (p=0.861) detection rate changed unremarkably when AI was used. However, the mean polyp per patient score (MPP) was significantly higher in Group 1, increasing from 0.85 to 1.26 following the introduction of AI (p<0.001). Further analysis investigating each segment of the bowel revealed that PDR increased significantly in the left colon (29.3 versus 18.0%; p<0.001), with no difference identified in the other segments. Regarding MPP, a significant difference was identified exclusively in the right colon (0.33 versus 0.23; p=0.032) and the left colon (0.47 versus 0.28; p<0.001) when AI was used. Following adjustment to bowel preparation, the PDR and MPP were consistently higher in the AI group (29.3 versus 19.0%; p<0.001; and 0.48 versus 0.30; p<0.001, respectively).

"Included patients were over 18 years old and had undergone a colonoscopy in the past 5 years." To conclude, recent evidence is optimistic regarding AI efficiency in improving the quality and efficacy of colonoscopy. The results discussed by this study widen the overall perspective; however, future randomised control trials should elucidate the role of AI in colonoscopy. ●

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Thigh Ultrasound Identifies High-Risk Patients with Systemic Sarcopenia HIGH-RISK patients with systemic sarcopenia in cirrhosis can be identified with thigh ultrasound, according to new research presented at this year’s UEG Week. Defined as low muscle mass and strength, sarcopenia is linked to adverse outcomes in cirrhosis, such as hepatic encephalopathy, infection, and increased hospitalisation. Reza Saeidi, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland, and colleagues, recruited 124 patients to determine whether thigh ultrasound was a low-cost tool to identify sarcopenia in cirrhosis. The researchers recruited 45 patients with alcohol liver disease, along with 15 with non-alcoholic steatohepatitis, four with viral disease, three with autoimmune diseases, 10 with a combination of the aforementioned conditions, and three with other aetiologies. A total of 44 healthy controls participated. The researchers used thigh ultrasound on the right thigh, moving from the top of the patella to the iliac crest, and bioelectric impedance analysis was performed as the validation

standard. They also recorded gait speed, sit-tostand time, handgrip, and Liver Frailty Index (LFI). The healthy controls could walk and stand faster, and had stronger handgrip. Using thigh ultrasound, the researchers were able to determine that total muscle thickness was higher in the control group than in the cirrhosis group. Thigh ultrasound-measured total muscle thickness and superficial fat thickness also strongly correlated with bioelectrical impedance analysis height-adjusted skeletal muscle mass. The researchers further discovered that total muscle mass predicted skeletal muscle mass, and that frailty and LFI scores were negatively correlated with total muscle thickness. The team concluded that thigh ultrasound is a rapid and low-cost tool to identify sarcopenia in cirrhosis, and that lower total muscle thickness is linked to reduced muscle function and increased frailty in cirrhosis. More studies are needed to see if thigh ultrasound can be used with biomarkers to determine which patients would benefit from intensive nutrition intervention. ●

"The researchers used thigh ultrasound on the right thigh, moving from the top of the patella to the iliac crest."

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A New Approach to Selecting Early Rectal Cancer for Endoscopic Intermuscular Dissection ENDOSCOPIC intermuscular dissection has been proven to be effective at removing deep submucosal invasive rectal cancers. However, a lack of knowledge and confidence in the anatomical characterisation of early rectal cancers (ERC) means that selecting which tumours are suitable for this technique is a challenge, both endoscopically and radiologically. Research presented at UEG Week in Copenhagen, Denmark, explored whether the application of a novel systemic approach for staging rectal cancer lesions using MRI can identify tumours suitable for local excision in the intermuscular plane more accurately. The study involved a 1-day training session held by an expert radiologist in the Netherlands, who had developed and validated the systematic reporting approach for assessing ERC on MRI. After this session, the 12 training radiologists, as well as the expert radiologist, reported a retrospective case series of pseudonymised MRI scans, blinded to pathological outcome data. Scans were derived from consecutive patients from two academic centres in the Netherlands who were suspected of having submucosal invasive rectal cancer at optical diagnosis between 2018–2022. All scans were matched to clinical and pathological outcome data.

Of the 246 patients (median age: 67 years; 71.1% male) included in the study, it was found that 186 would retrospectively have been suitable candidates for a local excision in the intermuscular plane based on the pathology data (≤pT2 limited to the circular muscularis propria). MRI accuracy in identifying these tumours as locally resectable was 81.7% (95% confidence interval [CI]: 76.3–86.3) in expert radiologist diagnosis, and 78.5% (95% CI: 72.7–83.5) for the study radiologists (range individual radiologists: 64.1–94.6%). The primary outcomes included sensitivity, specificity, positive predictive value, and negative predictive value, which were 86.0%, 68.3%, 89.4%, and 61.2% for expert diagnosis, and 80.5%, 72.4%, 90.0%, and 54.6% for trained study radiologists, respectively. Though previously MRI was not considered useful for the identification of ERC suitable for endoscopic local excision, this research suggests that it is accurate and reproducible. This approach may improve care by enabling better case selection for endoscopic intermuscular dissection; however, further research is needed to assess the clinical impact of this new approach. ●

"This approach may improve care by enabling better case selection for endoscopic intermuscular dissection."

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Prevalence of Irritable Bowel Disease Impacted by Changes in Definitions CHANGES in the symptom-based definitions of irritable bowel syndrome (IBS), especially the abdominal pain frequency threshold, impact the global prevalence of the disease, according to an abstract presented at UEG Week 2023. Previous data has shown that the use of the current Rome IV criteria substantially lowered IBS prevalence. Navkiran Tornkvist, Sahlgrenska University Hospital, Gothenburg, Sweden, and colleagues, analysed how different changes in diagnostic criteria influence this prevalence. The study included 54,127 individuals who completed the internet survey in the Rome Foundation Global Epidemiology Study (RFGES; 49.1% female; mean age: 44.3 years; range: 18–97 years). Rome IV and Rome III diagnostic questions, quality of life (PROMIS-10 QoL), healthcare utilisation (“ever consulted a doctor about a bowel problem?”), and IBS symptom severity scale (IBS-SSS) were used. The team applied the following different definitions to diagnostic criteria: using bloating/ abdominal distension as a proxy for discomfort; removing the requirement that abdominal pain and/or discomfort and changes in bowel habits should be associated in time; changing the frequency threshold of abdominal pain and/or discomfort; and adding discomfort to the definition.

Results showed that prevalence of IBS was 10.1% with Rome III criteria compared to 4.1% with Rome IV criteria. Prevalence rates were doubled by lowering the frequency threshold for abdominal pain and/or discomfort from ≥once per week to ≥2–3 days a month. Prevalence rate stayed similar when using the Rome III symptom definition of abdominal pain and/or discomfort with the Rome IV frequency threshold (≥once a week). Furthermore, prevalence rate was not influenced by the removal of the required association in time between changes in bowel habits and abdominal pain and/or discomfort. Finally, the team noted a female predominance, increases in healthcare utilisation, and reduction in QOL compared with the general global population, and on average, moderate symptom severity for all evaluated definitions.

"Prevalence of IBS was 10.1% with Rome III criteria compared to 4.1% with Rome IV criteria." The team concluded that relatively minor changes in symptom-based definitions of IBS substantially shape global prevalence, with alterations in abdominal pain/discomfort frequency threshold having the highest impact. This data will aid the definition of the Rome V criteria. ●

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The Link Between Physical Activity and Inflammatory Bowel Disease PHYSICAL activity (PA) in early childhood is not significantly associated with later risk of inflammatory bowel disease (IBD), according to research presented at UEG Week 2023. While PA has been inversely associated with IBD risk in adults, studies on paediatric populations have been lacking. Tereza Lerchova, University of Gothenburg, Sweden, and colleagues, used population-based cohorts that followed children from birth (1997– 2009) to 2021. Using questionnaire responses received from the ABIS and MoBa cohorts, the researchers collected data on the amount of screen time (ST) and degree of PA of children aged 36 months. Using the patient registers from Norway and Sweden, the researchers defined IBD as ≥2 diagnostic records of the disease. There was a total of 65,978 participants with data on PA and ST from both cohorts (ABIS: n=8,810; MoBa: n=57,168). During the 928,981 person-years follow-up, 266 participants were diagnosed with IBD (ABIS: n=65; MoBa: n=201). The researchers adjusted for parental IBD,

immune-mediated comorbidities, education level, maternal/paternal origin, maternal smoking during pregnancy, and the child’s sex. Children were put into high and low PA groups, and the results indicate that there was no significantly increased risk of IBD (adjusted pooled hazard ratio [HR]: 1.12; 95% confidence interval [CI]: 0.87–1.43), Crohn’s disease (adjusted pooled HR: 1.00; 95% CI: 0.67–1.48), or ulcerative colitis (adjusted pooled HR: 1.23; 95% CI: 0.66– 2.32). High versus low STs also showed no link to later IBD (adjusted pooled HR: 0.91; 95% CI: 0.71–1.17), Crohn’s disease (adjusted pooled HR: 0.98; 95% CI: 0.58–1.66), or ulcerative colitis (adjusted pooled HR: 0.75; 95% CI: 0.28–1.98). The MoBa cohort also had data for children at 18 months (n=75,254), where 256 participants developed IBD during follow-up. However, sub-analysis showed that PA and ST were not significantly linked with later IBD risk. Therefore, PA in early life was not significantly associated with later IBD risk. ●

"While PA has been inversely associated with IBD risk in adults, studies on paediatric populations have been lacking."

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Phenotypic-Genotypic Associations of Abdominal Pain in Children DESPITE abdominal pain being the most commonly experienced symptom in children, its pathophysiology is heterogenous and its mechanisms are not entirely understood. Furthermore, reports of genetic associations for abdominal pain in children are rare. New research presented at UEG Week in Copenhagen, Denmark, on the 17th October, employed robust stratification in a large sample in order to understand the determinants of abdominal pain in children.

cluster three (26.7%). The children in cluster one had allergic diseases (99.3%) and all their mothers also had allergic diseases. The second cluster contained children whose mothers’ comorbidities, such as abdominal pain (70%), allergic diseases (40%), and migraine (34%), were the highest compared to the other clusters. In cluster three, the frequencies of comorbidities, including allergic diseases and mothers' comorbidities, were very low.

The study involved analysing the frequency of comorbidities of 1,247 children who experienced abdominal pain (average age: 8.4±1.1 years old; male/female: 615/659) from the Born in Bradford birth cohort, registered between 2007–2011. They were then clustered using the unsupervised machine-learning algorithm, and genome-wide association studies were used to seek significant variants of single nucleotides polymorphisms responsible for differences between the clusters, using 6,488 children (male/female: 3,375/3,113) without abdominal pain as the control.

Genome-wide association studies showed that there were two, one, and four single-nucleotide polymorphisms with genome-wide significance (p<5.0×10-8) in clusters one, two, and three, respectively. Enrichment analysis revealed that ARPC5 and CLDN genes associated with tight junction may account for abdominal pain in cluster one. Genes associated with bile acid biosynthesis might also be associated with abdominal pain in clusters one and two.

Researchers divided the children with abdominal pain into three clusters: 137 in cluster one (10.8%), 677 in cluster two (53.1%), and 340 in

These findings suggest that there are three distinct phenotypes associated with abdominal pain in children, which may require a new therapeutic approach according to their aetiology and pathophysiology. ●

"The study involved analysing the frequency of comorbidities of 1,247 children who experienced abdominal pain."

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Stay up to date with new advancements across European healthcare Visit EMJ for our comprehensive collection of peerreviewed research articles, latest interviews, and features across a range of therapeutic disciplines. Visit EMJ

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Interview

Congress Interviews In the following interviews, experts provide insight into their careers and achievements to date within gastroenterology, giving update on a host of topics from the applications of artificial intelligence, to liver transplantation, and a closer look at the workings of the United European Gastroenterology (UEG) organisation.

Jeanin van Hooft Professor of Gastroenterology and Hepatology, Head of the Department of Gastroenterology and Hepatology, Leiden University Medical Center (LUMC), the Netherlands; Secretary General, United European Gastroenterology (UEG)

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10301328. https://doi.org/10.33590/emjgastroenterol/10301328.

What initially sparked your interest in gastroenterology, and has continued to motivate you over the years? When I was a trainee, I thought: am I going to be a surgeon or am I going to go into internal medicine? At that stage, we had a doctor join from another hospital who was a gastroenterologist, and he showed that this specialty offered the ideal mix of analytical thinking and hands-on work. He let me join the hands-on programme and, while completing my internship, he allowed me to conduct some endoscopies, which I found fascinating. I still look forward to Thursdays when I have a whole endoscopy list. On the other hand, I also still like the analytical aspects. I currently work with a great group of fellows, and I really like having the time and availability to dive into patient care with them and see what is going on. I enjoy working out the puzzle and finding out what the problems behind conditions are.

What has been your greatest achievement during your time at the Leiden University Medical Center (LUMC), the Netherlands, where you currently head the Gastroenterology and Hepatology Department? I only started here 3 years ago, but what I have tried to do is increase the team’s visibility. We have lots of talented people here, but they are all quite modest. I really empower them to get onto the podium and to show how good they are, and this is starting to pay off. Seeing this team present at large meetings makes me proud. We have a good mix of staff; both basic researchers as well as clinicians, male and female, young and slightly older. I am very proud of what they are now achieving.

"The UEG is keen on creating an energetic and innovative vibe."

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How is the United European Gastroenterology (UEG) continuing to encourage experts in this specialty to “ingest the best,” and what changes have you brought about in your role as Secretary General? The UEG is keen on creating an energetic and innovative vibe. Nowadays, we have online access with the ‘myUEG’ platform, and there are, for example, digital poster sessions at UEG Week, which I think are great. Alongside these new aspects, the congress continues to try and make connections between the specialties of gastroenterology, from pathology to motility, from liver to endoscopy. My specific role is to facilitate these connections. I am not trying to drastically change the organisation, but to focus on the team we work with and keep this positive energy. The big achievement is the way we work together.

What are you most looking forward to about UEG Week 2023?

Firstly, I am looking forward to the digital poster sessions. I really appreciate these; there will be screens and small groups of people discussing research done by other fellows. These will be highly comprehensive and provide a great opportunity to interact with others. Previously with physical posters, people did tend to just pass by, but now people sit down instead and watch a poster pitch; I am really looking forward to more of that this year. What I also always like about UEG Week is meeting the next generation and discovering their interests. Plus, it is great to see mixing of cultures. I am familiar with the Dutch field, but this might be completely different for fellows in Greece or Sweden, for example. Ultimately, we move forward together.

What challenges have you overcome co-founding Women in Gastroenterology and serving on the Equality & Diversity taskforce for the UEG? What is the immediate focus for these groups? I think that some people believe there are no longer diversity issues, and people around me

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tell me these groups are no longer needed. But I disagree; look at the editorial board of most journals. Most of the time these are not very diverse. Look at the doctors doing live demonstrations, and most of the time they have the same skin colour or are the same gender. This is the same across many specialties. Therefore, I think we are in real need of role models, considering both gender and ethnicity. I think it is important to have access to a diverse spectrum of people, and this is the future. Individuals from marginalised groups will only get involved if they see they can do it too.

Are there any topics or gaps in literature that you feel warrant greater attention within the field of gastrointestinal endoscopy? And where can we expect to see your research focus in the near future? When I started in the field, we had just taken on enteral, in particular colonic, stenting and we have now come full circle here. After about 10 years, we have handed this back to the surgeons and moved away. I have moved into pancreatobiliary research, with a special focus on pancreatic diseases. I have a real interest in early detection of pancreatic ductal adenocarcinoma by fluid analysis, imaging, or artificial intelligence (AI). AI is a really hot topic and I think it could help. But there are also dangers here. Referring back to diversity, lots of AI systems are trained on one specific population. If this is applied to the whole world, there may be severe shortcomings. I think that if we really want to make progress here, we must be very thoughtful and careful about the input, because you get out what you put in. I have learnt that we often focus on fixing conditions, but we should probably move more towards preventing them. Conducting procedures can make you feel like a hero, and you do not get quite the same buzz from having lengthy conversations with patients. But I think this is even more important, and maybe we should move towards talking to people even before they become patients. This way they can get their lifestyle in order, and we do not find them later in obesity endoscopy treatment, for example. ●

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Patrizia Burra University of Padua, Italy; Public Affairs Group Chair, United European Gastroenterology (UEG)

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10301072. https://doi.org/10.33590/emjgastroenterol/10301072.

Was there a particular person or event that encouraged you down your path towards specialising in gastroenterology, hepatology, and liver transplantation? Regarding my decision to undertake my studies in the field of gastroenterology, a prominent role was certainly played by my mentor, Remo Naccarato, the father of gastroenterology in Padua, Italy. Subsequently, the choice to specialise in hepatology and liver transplantation happened when I moved to England, UK, for my PhD.

Drawing upon your leadership experience in several organisations, including the International Liver Transplant Society (ILTS), European Liver and Intestine Transplant Association (ELITA), and European Association for Study of the Liver (EASL), how important is the work United European Gastroenterology (UEG) is completing? Weighing up these groups, is there anything UEG does better, or could improve in its operations? There is a huge difference between scientific societies that are specifically dedicated to liver transplantation and the UEG, in term of fields of interest. Comparing the EASL and UEG, they are both extremely strong societies, and the experiences in both favour my knowledge, expertise, and personal relationships.

"This is not a scientific session, but a patient engagement event led by the UEG Public Affairs Group."

Having authored over 350 articles, including your latest article, ‘Uniting to defeat steatotic liver disease: a global mission to promote healthy livers and healthy lives’. What was the key message in this paper, and where can we expect to see your education focus lie in the near future? Today, metabolic dysfunction-associated steatotic liver disease (MASLD), previously referred to as non-alcoholic fatty liver disease, has an enormous burden on individuals, families, communities, societies, and healthcare systems across the globe. Fortunately, MASLD is preventable and detectable, and its progressive forms and complications are increasingly manageable. We have a collective obligation to use this knowledge to deliver meaningful change, from early diagnosis to therapeutics and sound policy responses. We must leverage this unique window of opportunity to end MASLD as a global public health threat.

What are the highlights from the packed scientific programme at UEG Week 2023 for you? For me, the highlights are ‘Postgraduate Teaching: Fulminant hepatitis, liver failure and liver transplantation’, ‘Never waste a good disaster’ (hepatobiliary and pancreas), and the ‘Digestive Health Roundtable: Advancing Digestive Health: Collaborating for Better Outcomes’. This is not a scientific session, but a patient engagement event led by the UEG Public Affairs Group.

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Could you talk a bit about the Public Affair Group, which you chair within UEG, and the awareness for digestive health this group is raising? The UEG Public Affairs Group raises awareness of digestive health among specialists and decisionmakers at European Union (EU) level through policy papers, targeted campaigns, and meetings with stakeholders. We are in regular contact with Members of the European Parliament (MEP), especially through our MEP Digestive Health Group,1 as well as other health stakeholders in Brussels, Belgium, to promote digestive health on the EU agenda. Ahead of the EU elections in 2024, we are currently in the process of developing a targeted campaign to ensure (digestive) health remains a political priority for the EU, and also to maintain the strong relationships we have built over the years, and establish new ones in the next parliamentary term. Our multiple awareness-raising campaigns2 throughout the year, the biggest one being Digestive Health Month each May, address the general public, policymakers, medical experts, and patients. To strengthen our advocacy work, we also build alliances and collaborate with European and national patient organisations, working towards positive change at policy level across Europe.

How much of an impact do you believe UEG has directly on gastroenterologists, but also indirectly on their patients?

fields and career stages to connect, learn from, and work with each other. This wide range on offer impacts and benefits the expertise of thousands of digestive health professionals, and, as a result, the quality of patient care. Moreover, as mentioned earlier, UEG and the Public Affairs Group in particular work closely with patient organisations to advocate for improved patient outcomes and quality of life.

What are the most significant changes you have observed in the specialty over the course of your career to date? Over the years, I have observed a huge improvement in the technical facilities for the clinical management of patients, particularly in endoscopy and interventional radiology; the increase in knowledge in the diagnosis and treatment of gastro- and liver diseases, and the incredible increase in structures linked to teaching, training, and communication.

Are there any particular innovations within liver transplantation, or in wider gastroenterology, that you are excited about seeing translated into practice soon? Within liver transplantation, I am seeing important innovations in transplant oncology, machine perfusion, and patients and caregiver care. ●

Looking at UEG’s broad offer in terms of worldclass education, research support, fellowships, and grants, and not to mention the annual congress UEG Week, the direct impact on the career path of gastroenterologists and other digestive health experts is substantial. With the My Connect platform, UEG also offers a unique online space for digestive health experts from all

References 1.

United European Gastroenterology (UEG). MEP Digestive Health Group. Available at: https://ueg.eu/publicaffairs/mep-digestive-health-group. Last accessed: 27 September 2023.

United European Gastroenterology (UEG). Stakeholder collaboration. Available at: https://ueg.eu/public-affairs/ advocacy/stakeholder-collaboration. Last accessed: 27 September 2023.

"Ahead of the EU elections in 2024, we are currently in the process of developing a targeted campaign to ensure (digestive) health remains a political priority."

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Interview

Interview The following interview takes a deep dive into the experiences of Gideon Hirschfield, a leading hepatologist currently based in Toronto, Canada. He spoke to EMJ about patient-centred treatment across several sub-specialties, with a focus on autoimmune liver disease and transplantation.

Gideon Hirschfield Lily and Terry Horner Chair in Autoimmune Liver Disease, University of Toronto, Ontario, Canada; Honorary Professor of Autoimmune Liver Disease, University of Birmingham, UK

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10301852. https://doi.org/10.33590/emjgastroenterol/10301852.

With your many years’ experience as a consultant hepatologist, what initially sparked your interest in liver disease and has motivated you to continue researching? I was lucky. I first did my undergraduate training in Oxford, UK, and then Cambridge, UK. When I was doing my clinical training in Cambridge, I was very fortunate to be exposed as a medical student to a professorial medical surgical firm with academic physician and surgeon scientists, with a clear focus on liver disease. This included experience with Sir Roy Calne, who was, as many people know, a pioneer of liver transplantation. That combination in Cambridge meant that my very first exposure in medicine was in advanced hepatology and transplantation, and that really sparked my interest and enjoyment of looking after patients with complex liver diseases.

Before your current spell in Toronto, Canada, you studied at both Oxford and Cambridge, and educated as a professor at the University of Birmingham, UK. Where do you feel you gained your most valuable experience? That is a very hard question to answer. Each stage of my career has been positive and has facilitated the next stage. I was very lucky to go to Oxford and learn how to write. I was then very lucky to go to Cambridge, and learn how to think critically about clinical medicine. I also gained junior doctor experience in central London, UK, particularly at the Hammersmith Hospital, which is a phenomenal academic health science centre. This led to a PhD at University College London, UK, on the Royal Free Hospital, London, UK campus, providing significant critical appraisal skills in basic science. After this, I returned to Cambridge to complete my advanced training in liver disease. Then, fortunately for me, I spent some time in Toronto, Canada, and was faculty here, before returning to faculty in Birmingham. Currently, I am back in Toronto.

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Gastroenterology

Interview

I do not think that I can claim that one place has been, you know, the place. I think that what I have learned is that if you put yourself in exciting environments, with colleagues who are interested in being physician scientists and academic medics, you can bring together really high-quality clinical medicine and clinical science, often spanning laboratory work. I am now working more predominantly in clinical translational work and clinical trials. You cannot plan everything. It could have worked out that I became something else. It depends on who you are lucky enough to meet, who you are lucky enough to be mentored by, and where opportunities arise at different times. So, for anyone moving through a similar career, it is impossible to plan everything. I fundamentally enjoy looking after patients with complex liver disease, and I anchor everything around being an active clinician.

A couple of your recent publications have focused on optimising liver transplantation. Could you summarise the key points from the topical article you co-authored, called ‘Availability of living donor optimizes timing of liver transplant in high-risk waitlisted cirrhosis patients’, considering the long waiting lists many hospitals are currently experiencing? I think that is an important question, and it does have relevance both in the UK and in Canada, and also beyond. We look after patients with primary sclerosing cholangitis (PSC), a rare disease, as one of our academic and clinical interests. Unfortunately, it has a very high need for liver transplantation. More than 50% of patients will need a transplant and often these are young patients. Currently, the way liver transplants are allocated is based on something called the model for end-stage liver disease (MELD) score and derivatives of this score measure synthetic liver failure. What that means is that not everybody who needs a liver transplant, based on their blood tests or symptoms, is prioritised in the same way. Currently, prioritisation is driven by how serious your liver failure is. But this does not necessarily work for young people with PSC, whose MELD scores do not accurately reflect how sick they are. What we were interested to see is what was happening in our programme in Toronto and comparing that with the USA.

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"And I anchor everything around being an active clinician." In Toronto, we have access to live donor transplantation. This is not something that has taken off as much in the UK, but it is available elsewhere in the world. What we have noticed is that our patients with PSC were using disproportionate amounts of live donor transplantation to get the organ that they needed to prolong their life and improve their quality of life. We used this as a marker to say that the current system is not allocating livers to these patients in an equitable way, at least not in an equitable way that measures what they need. So, whilst the MELD score may be quite good for picking up 30-day or 3-month mortality, there are many reasons for liver transplantation, including PSC, where the MELD score does not reflect what is going on in your patient. In our programme, live donor transplantation is the reason why our patients are not dying on the waiting list, which reflects that the system is not perfect. Therefore, the point of the paper was to highlight this inequity, recognising that there are not enough livers and that is always going to be a challenge. But we need to think about how we allocate who gets the next liver, and this cannot only be based on who is the sickest as defined by who has the highest short-term mortality through a MELD score. It needs to look at other ways of measuring this morbidity and potential mortality. There are many people around the world thinking about how we allocate livers. This is an ongoing problem, as more and more people will need liver transplantation for other diseases. Therefore, we have a commitment, as a community of transplanters, to find ways to measure who goes next that do not only look at death, which may be skewed by patient age. This should take into account lots of factors, particularly relevant for diseases like biliary diseases, primary biliary cholangitis (PBC), and PSC. Females will often have a lower MELD score because of their body mass, and the biology of the disease means that the MELD score may be lower, and it does not always capture risk of cancer.

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Interview

As a widely recognised thought leader in liver medicine and patient-centred treatment, how have you seen this field change over the course of your career? I think our patients are much more at the centre of healthcare, particularly in the clinic. There is also much better access to their medical information. Our patients can see every blood result, every letter, every scan, every appointment, and they have live access. There is literally no difference between what I can see and what they can see in the ambulatory setting. So, I think that is a major step forward for patients. Our patients are also engaged in the design of clinical services and the design of clinical research. A major change is the amount of time we now spend with patients and patient organisations discussing their needs. Does a transplant service allocate organs in a way that is fair to them? How is their quality of life addressed? How do we widen access? How do we ensure that all patients get access to high quality healthcare? I would say that we are much better at doing that than ever, even though we are not perfect (we never will be perfect) and have a publicly funded health system where there are significant strains on resources. Many of the things we would like to do are not necessarily in our control. But, without doubt, since I began training, our patient engagement and patient participation has never been higher.

learn from some of the drugs that have been developed and from related diseases, but we really need to understand why someone gets PSC. Why do they get it at an early stage? Why is it associated with colitis? Understanding this means we may be able to use transformational therapies to really stop the disease and the inflammation. That would really, ultimately, improve quality and quantity of life. We would love to be at the forefront of treatments, but we are limited by the fact that we do not fully understand what we should target. We sort of target what we see, but what we see is a reflection of many, many different factors, such as environmental and genetic, that led to the patient presenting. What we see in the clinic are patients with a heterogeneous disease presentation, so some people have mild disease, some have severe disease, some have symptomatic disease, and others are asymptomatic. We cannot yet make it personalised. We are getting more personalised and goal driven in our therapies, and we aim for normal tests, but we use biomarkers that are incredibly crude, as well as simple liver tests. These are great, and they are very immediately applicable, but they are not very sophisticated. To really personalise care, we need better biomarkers in the clinic, not just in the research setting. We need these biomarkers to link to why someone gets the disease.

"To really personalise care, we need better biomarkers in the clinic, not just in the research setting."

Where do you see the gaps in research at current in the field of autoimmune liver disease, and what are the next steps for implementing personalised therapy for patients with immune and inflammatory mediated liver diseases? Fundamentally, we would like to know the cause of the disease. If we knew the cause of the disease, we would be at a point where we can move beyond controlling the disease to curing the disease. In diseases like PBC, we have made enormous headways into controlling the disease. We have new and emerging therapies that are highly effective at stopping disease progression and improving symptoms. That said, they do not yet cure the patient. For PSC, we have not made as much progress. We hope that we will

This is a journey, and the barrier to that going as fast as we would like is that, fortunately, or unfortunately, depending on how you look at it, these are rare diseases. Working together takes time to bring all the samples and all the patients to get our science up to scratch. We are pleased that the burden of disease is limited by how rare the diseases are, but at the same time, we are frustrated by the fact that because it is rare, the pace of change is inevitably a bit slower.

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Gastroenterology

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How does practising and researching in Canada differ to the UK? Are there any large similarities and/or differences? Medicine has a lot of similarities in Canada and the UK. I always think of Canada as nesting between Europe and the USA, in terms of how we practice. We are a publicly funded health service. We have absolutely no private health care for anything that is reimbursed by the government. That is actually quite distinct to the UK, where there is a dual model of healthcare. We have the same opportunities to provide healthcare to everyone, and the same challenges. Healthcare needs are growing at a rate that funding cannot keep up with, due to reasons such as ageing populations. Medicine is more complex than it ever has been, but there are opportunities for new and emerging therapies. We live many of the similar problems that the UK has, particularly in terms of waiting times and access to care. But, equally, we focus on delivering healthcare for everyone who needs it, just based on residency requirements. There are lots of similarities, and lots of similarities in practice; however, Canada is very close geographically to the USA, so naturally some of our approaches start to look more like the medicine in the USA, in terms of patient choice, physician independence, and hospitals.

Where can we expect to see your research focus lie in the near future?

I hope that over the next five years we'll be continuing our work to understand what happens to patients living with autoimmune liver disease, particularly PBC, PSC, and autoimmune hepatitis. By that I mean better cohort studies, and using those cohort studies to understand biomarkers that we can use in clinical trials to predict outcomes early, so that our patients can have effective trial interventions without waiting a long time for clinical trial results. I hope that we will be able to work alongside the development of new therapies that improve quantity and quality of life, and that we will be able to prove that those therapies are really

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helping our patients. Finally, I hope that if we educate our colleagues, and also educate other stakeholders in hospital and primary care, about the importance of rare liver diseases, particularly autoimmune, this will raise awareness. This would mean we get earlier diagnoses, patients are referred to specialist centres earlier, and are more likely to get early and effective treatment, both targeting quantity and quality of life. In 5–10 years’ time, I hope to see a field where the diseases are being chipped away at, and we need liver transplantation less than less. We are already doing this for PBC (there are some very exciting new therapies coming soon), and we already have reasonable approaches, notably for autoimmune hepatitis, but we would like to improve them. Our real high priority is to make a difference now in PSC.

What advice would you give to a young hepatologist/gastroenterologist making their way into practice and research? Always choose good mentors, show curiosity in your patients, and be compassionate. Always think: why did your patient get the illness they have? Think about this whatever illness you choose to study or find interesting. If you understand the basic science and the unmet needs, you will be at the cutting edge of getting your patients the best care. Wherever you practise, if you set your goal as providing the best care for your patients, your patients will appreciate that, and you will have a more satisfying career. Ultimately, if you are delivering state-of-theart treatments that are effective and novel, you can be part of great quality of care. Everyone can take part in clinical research. It does not matter where you find yourself or what sector of medicine you are in, everyone can contribute to understanding what happens to our patients, and how to treat them better. So, being an active clinician and being involved in research is not restricted to academics. It is beholden on everyone who practises medicine to aspire to practise at the highest level. It is a lot more interesting and a lot of fun. ●

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EMJ Podcasts The EMJ Podcast aims to provoke conversations around the latest trends and innovations in healthcare, provide engaging and educational content for healthcare professionals, and hosts conversations with physician entrepreneur, Jonathan Sackier. Listen today www.emjreviews.com

Preventive Medicine in Gastroenterology

Citation: EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10302591. https://doi.org/10.33590/emjgastroenterol/1030

A Preventive Approach

Colorectal Cancer

Aims to decrease rates of morbidity and mortality, also reducing overall healthcare costs.

Third most common cancer type

Early discussion, education, and emphasis on downstream consequences of inadequately treated disease can better inform patients. Family history genetic tests create personalised prevention programmes by detecting malignancies and risks, to inform changes, and prevent development of disease at an earlier, curable stage. Interventions focus on lifestyle adaptation, and encourage patients to be proactive with their approach to healthcare. Identifying which preventive measures are better delivered at primary, secondary, and tertiary care levels is crucial to delivery of quality care. Barriers:

Highest rates currently seen in A year, and Japan 60,000.

2020

~2 millio

2040

~1.6 millio

Mortality can be reduced through Improved colonoscopy has contr

FOBT, sigmoidoscopy, and colon the most used screening techniq

• Focus on symptom control ahead of prevention • Infrequent healthcare visits • Confusion over where and when preventive services should be offered

IBD 4.9 million cases globally in 2019 ~2.2 million Americans will live with IBD by 2025 Lifestyle modification significantly reduces risk, improving QoL and patient outcomes. Primary care providers are often not comfortable providing routine preventive care to patients with IBD, particularly those on immunosuppressants. Patients often consider their gastroenterologist their primary provider of care. Clinicians must communicate comorbidities with the patient, and co-ordinate with other specialists to improve quality of care.

Key: FIT: faecal immunochemical test; FOBT: faecal occult blood test; IBD: inflammatory bowel disease; QoL: quality of life. References: 1. Weaver KN, Long MD. Preventive medicine in inflammatory bowel disease. Clinical Gastroenterol Hepatol. 2019;17(5):824-8. 2. Farraye FA et al. ACG clinical guideline: preventive care in inflammatory bowel disease. Am J Gastroenterol. 2017;112(2):241-58. 3. Kanth P, Inadomi JM. Screening and prevention of colorectal cancer. BMJ. 2021;374:n1855. 4. National Institutes of Health (NIH); National Cancer Institute (NCI). Colorectal cancer prevention (PDQ®)–patient version. 2022. Available at: https://www.cancer.gov/types/colorectal/patient/ colorectal-prevention-pdq. Last accessed: 22 May 2023. 5. Nolfo F et al. Pharmacological and dietary prevention for colorectal cancer. BMC Surg. 2013;13(Suppl 2):S16. 6. World Health Organization (WHO); International Agency for Research on Cancer (IARC). Colorectal cancer awareness month 2022. 2022. Available at: https://www.iarc.who.int/featured-news/ colorectal-cancer-awareness-month-2022/. Last accessed: 22 May 2023. 7. Binefa G et al. Colorectal cancer: from prevention to personalized medicine. World J Gastroenterol. 2014;20(22):6786-808. 8. Chiu H-M et al. Long-term effectiveness of faecal immunochemical test screening for proximal and distal colorectal cancers. BMJ. 2021;70(12):2321-9. 9. GI Doctors. Gastrointestinal cancer prevention. 2022. Available at: https://gidoctors.co.uk/tests/cancer-prevention/. Last accessed: 23 May 2023. 10. Abegunde AT et al. Preventive health measures in inflammatory bowel disease. World J Gastroenterol. 2016;22(34):7625-44.

11. 12. 13. 14. 15. 16. 17.

Lopes EW UChicago 23 May 20 Stanford accessed Kouzu K e Wang R e Study 20 World He cancer-aw Bowel Ca that,meat

02591.

worldwide, growing annually.

Asia; China reports 280,000 deaths per

on cases were diagnosed

FIT continues to prove an effective and sustainable tool for populationbased screening. Primary prevention interventions involve dietary measures such as limiting red and processed meat. Recent developments in drug therapy are promising.

on deaths are forecast

h screening and early detection. ributed to improvement in recent years.

noscopy, which is the ‘gold standard’, are ques.

Protective factors to decrease risk:

Physical activity

Aspirin

Combination hormone replacement therapy

Polyp removal

Gastrointestinal Cancer Cessation of smoking and treating Helicobacter pylori infection are protective factors that decrease risk. Risk assessment is recommended in families with cancer predisposition, eg., Lynch syndrome, hereditary diffuse gastric cancer, and polyposis syndromes. Gut microbiota plays an important role in dictating carcinogenesis. Ongoing investigations focus on providing preventive treatment through modification of this gut microbiome. Preventive recommendations:

Regular physical activity

Maintenance of a healthy weight

High fibre diet with vitamins A and C

W et al; EPIC-IBD Investigators. Lifestyle factors for the prevention of inflammatory bowel disease. Gut. 2022;72(6):1093-100. o Medicine. Gastrointestinal cancer risk & prevention. 2023. Available at: https://www.uchicagomedicine.org/cancer/types-treatments/gastrointestinal-cancers/gi-cancer-risk. Last accessed: 023. Medicine Health Care. Stomach cancer prevention. 2023. Available at: https://stanfordhealthcare.org/medical-conditions/cancer/stomach-cancer/stomach-cancer-prevention.html. Last d: 23 May 2023. et al. Bacterial translocation in gastrointestinal cancers and cancer treatment. Biomedicines. 2022;10(2):380. et al. Global, regional and national burden of inflammatory bowel disease in 204 countries and territories from 1990 to 2019: a systematic analysis based on the Global Burden of Disease 019. BMJ. 2023;13(3):e065186. ealth Organisation (WHO), International Agency for Research on Cancer. Colorectal Cancer Awareness Month 2022. 2022. Available at: https://www.iarc.who.int/featured-news/colorectalwareness-month-2022/. Last accessed: 25 May 2023. ancer UK. Diet. 2019. Available at: https://www.bowelcanceruk.org.uk/about-bowel-cancer/risk-factors/reducing-your-risk/diet/#:~:text=There%20is%20strong%20evidence%20 ts%20as%20much%20as%20possible. Last accessed: 14 August 2023.

Limiting alcohol intake

Feature

Challenges in Management of Immune Checkpoint Inhibitor-Associated Hepatitis Authors:

*Morven Cunningham1 1. Toronto Centre for Liver Disease, University Health Network, Ontario, Canada *Correspondence to morven.cunningham@uhn.ca

Disclosure:

Cunningham has received honoraria from Abbvie.

Received:

22.05.23

Accepted:

29.08.23

Keywords:

Checkpoint inhibitor, hepatitis, immunotoxicity, immune-related adverse events.

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10303980. https://doi.org/10.33590/emjgastroenterol/10303980.

INTRODUCTION Immune checkpoint inhibitors (ICI) are a novel class of anticancer therapy that have been transformative in treating a diverse range of cancers, extending survival in some patients, and producing significant and durable tumour responses. ICI enhance immunological responses against tumour cells by inhibiting receptor-ligand interactions in immune checkpoint pathways, which may be subverted by tumour cells to prevent their destruction by cytotoxic T cells. The two classes of ICI currently in routine clinical use are monoclonal antibodies targeting CTLA-4 and PD-L1.1,2 Use of ICI is associated with a novel range of side effects, termed immune-related adverse events (IRAE). These can affect any organ system, and are thought to arise from immunemediated attack on healthy host tissues following reduction in self-tolerance due to immune checkpoint blockade. Immune-related hepatitis (IRH), usually manifesting as elevation of hepatocellular enzymes, may occur in 2–25% of patients, depending on ICI class or combination.1,2

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Management of IRAE represents a particular clinical challenge. Management guidelines, such as those from the European Society for Medical Oncology (ESMO),1 were initially developed based on clinical trial protocols and case reports/series, and have been revised to reflect expertise gained from increased experience in managing IRAE. While these management algorithms appear generally effective for most patients, they have never been evaluated prospectively, and many areas of uncertainty remain. This article will review some of the challenges in management of patients with IRH, focusing on areas where hepatologists are often asked to assist with management, but where little evidence exists to guide decision-making (Figure 1). Specifically, this includes diagnosis and role of liver biopsy; management of corticosteroids (CS) and other immunosuppressive therapies (IST); risks of ICI rechallenge in patients with IRH; and ICI treatment in special populations, such as those with viral hepatitis.

DIAGNOSTIC CHALLENGES Several biomarkers associated with increased risk for IRAE have been identified, including

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Feature

Figure 1: Summary of key challenges in management of patients with immune checkpoint inhibitorassociated hepatitis.

Does this patient need a biopsy?

Does this patient need corticosteroids?

What is the best choice of second line therapy?

What is the optimal dose of corticosteroids?

How should IST be tapered/stopped?

Can ICI therapy be restarted? ICI: immune checkpoint inhibitor; IST: immunosuppressive therapy.

cellular markers, such as neutrophil to lymphocyte ratio; cytokine/chemokine expression, including IL-6, IL-17, CXCL 9, CXCL 10, and CXCL 11; single nucleotide polymorphisms; and gut microbiota. However, no single biomarker reliably predicts IRAE development.3 The association between IRAE and anticancer efficacy limits the identification of a diagnostic biomarker, such that a toxicity biomarker may also indicate cancer response.4 Therefore, IRH currently remains a diagnosis of exclusion. Initial investigation and management are usually conducted by the patient’s primary oncologist. However, early involvement of a hepatologist is desirable, both to facilitate consideration of alternate causes for liver injury before initiation of IST, which complicates the clinical picture, and to build expertise in managing these complex patients. The role of biopsy in diagnosis has been a topic of debate. Biopsy is often deferred, due to concerns that it will delay initiation of therapy. Histopathological findings can vary widely, with no pathognomonic features of IRH described. Conversely, a biopsy may be helpful to exclude an alternate/concurrent disease process. A

consensus view is that biopsy can usually be reserved for atypical presentations, or patients who fail to respond to treatment with IST (Figure 2).5

HOW SEVERE IS 'SEVERE'? Reflecting the development of management guidelines from clinical trial protocols, severity of IRH is graded using Common Terminology Criteria for Adverse Events (CTCAE; Table 1). Under these criteria, severity may be adjudicated biochemically rather than clinically, based on the extent of liver enzyme elevation regardless of liver function. This contrasts with other severity assessment tools, such as the Drug Induced Liver Injury Network (DILIN) severity index, which incorporates markers of liver function into severity grading (Table 2).6 For example, an asymptomatic patient with ALT elevation 5x upper limit of normal, but normal bilirubin and international normalised ratio (INR; a relatively common presentation of IRH) would be adjudicated as high grade (Grade 3) by CTCAE criteria, but only mild by DILIN criteria. Using CTCAE to manage IRAE is

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embedded within guidelines and clinical practice, but may overestimate liver injury and lead to overtreatment. Whether adopting alternate DILIN severity assessments to stratify liver injury and guide therapy would improve outcomes in patients with IRH remains unknown.

DOES EVERYONE NEED STEROIDS? The cornerstone of management for high-grade IRH is stopping ICI and treatment with high-dose CS. Figure 2 describes a proposed management algorithm. Little natural history is reported on

Figure 2: Suggested management algorithm for patients with high-grade immune checkpoint inhibitorassociated hepatitis.

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Ä˱ʡͮʡǵͮɟ ͮ͝ɟ̿́ʡɎ‫ͮڂ‬ǵ̵ɟ̿‫ڂ‬΢ʎɟ˱‫ڂ‬ǵͮ‫ڂ‬ ̿ǵɎɟ‫ڂ‬ԆՄԇ Ɖˍ́΢‫ͮ͝ڂ‬ɟ̿́ʡɎ‫ͮڂ‬ǵ̵ɟ̿‫́ڂ‬Ξɟ̿‫ڂ‬Ԋ–ԍ‫ڂ‬ ΢ɟɟ˅͝

żɟȻ́ͽ˱Ɏ‫ڂ‬ɿˍǵ̿ɟ‫ڂ‬ʡ˱‫ڂ‬ ˍʡΞɟ̿‫ڂ‬ɟ˱φΰ˪ɟ͝

żɟɂ́˱͝ʡɎɟ̿‫ڂ‬ǵˍͮɟ̿˱ǵͮɟ‫ڂ‬Ɏʡǵʄ˱́͝ʡ͝ H́˱͝ʡɎɟ̿‫ˍڂ‬ʡΞɟ̿‫ڂ‬Ȼʡ̵́͝ΰ Ėʡ˪ʡͮ‫ͮ͝ڂ‬ɟ̿́ʡɎ‫ڂ‬Ɏ́͝ʡ˱ʄ‫ڂ́ͮڂ‬Ԇձԋ‫˪ڂ‬ʄՄ˅ʄ ^ǵ̿ˍΰ‫ڂ‬ǵɎɎʡͮʡ́˱‫́ڂ‬ɿ‫͝ڂ‬ɟɂ́˱Ɏ‫ˍڂ‬ʡ˱ɟ ÄƉƗհ‫ͽ͝ڂ‬ɂʎ‫ڂ‬ǵ͝ ġġ

ICI: immune checkpoint inhibitor; IST: immunosuppressive therapy.

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Table 1: Common Terminology Criteria for Adverse Events (CTCAE) for bilirubin and liver enzyme elevations.

CTCAE Classification

Total bilirubin

ALT/AST

ALP

Grade 1

>ULN: 1.5 x ULN

>ULN: 3.0 x ULN

>ULN: 2.5 x ULN

> 1.0–1.5 x baseline if baseline was abnormal

1.5–3.0 x baseline if baseline was abnormal

2.0–2.5 x baseline if baseline was abnormal

1.5–3.0 x ULN

3.0–5.0 x ULN

2.5–5.0 x ULN

1.5–3.0 x baseline if baseline was abnormal

3.0–5.0 x baseline if baseline was abnormal

2.5–5.0 x baseline if baseline was abnormal

>3.0–10.0 x ULN

>5.0–20.0 x ULN

>3.0–10.0 x baseline if baseline was abnormal

5.0–20.0 x baseline if baseline was abnormal

>10.0 x ULN

>20.0 x ULN

>10.0 x baseline if baseline was abnormal

>20.0 x baseline if baseline was abnormal

Grade 2

Grade 3

Grade 4

Version 5.0, November 2017. ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP, alkaline phosphatase; CTCAE: Common Terminology Criteria for Adverse Events; ULN: upper limit of normal.

Table 2: Drug Induced Liver Injury Network (DILIN) severity index for adjudicating the severity of drug induced liver injury.6

DILIN Score

Grade

Definition

Mild

• • •

ALT/ALP elevation above ULN; and Bilirubin <2.5 mg/dL; and INR <1.5

Moderate

• •

ALT/ALP elevation above ULN; and Bilirubin ≥2.5 mg/dL or INR ≥1.5

Moderate to severe

• •

Elevated ALT, ALP, bilirubin, and/or INR; and Hospitalisation due to DILI, or prolongation of an existing hospitalisation due to DILI

Severe

• A. B.

Elevated ALT and/or ALP and bilirubin ≥2.5 mg/dL and at least one of: Liver failure (INR ≥1.5, ascites, or hepatic encephalopathy) Other organ failure believed to be due to DILI event

•

Death/transplant due to DILI event

Fatal

ALT: alanine aminotransferase; ALP: alkaline phosphatase; DILI: drug-induced liver injury; DILIN: Drug Induced Liver Injury Network; INR: international normalised ratio; ULN: upper limit of normal.

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evolution of IRH in the absence of CS. However, even high-grade IRH may resolve in some patients after simply holding ICI.7 Management strategies have been proposed where CS are reserved for patients with synthetic dysfunction and/or severe histological inflammation,7 but these have proven challenging to implement, perhaps due to concerns over delaying CS treatment in patients who do not improve. ESMO guidelines emphasise prompt diagnosis of IRAE, based on a broad consensus that early treatment is important to improve outcomes.1 However, the default position of high-dose CS for all patients with high-grade IRH probably overtreats some patients, and will make it harder to conduct research into biomarkers associated with spontaneous resolution.

WHEN THE STEROIDS DON’T WORK, WHAT NEXT?

STEROID DOSING: BENEFITS VERSUS HARMS

Mycophenolate mofetil (MMF) is the most frequently used second-line agent for IRH, likely due to its ease of use, favourable toxicity profile, and demonstrated efficacy in case reports/series. However, MMF-refractory cases are reported. Tacrolimus has been successfully used as third-line therapy in these cases. However, whether an alternate agent would provide a more consistent response as second-line therapy is unknown.5 Plasma exchange and anti-thymocyte globulin have been used with some success in patients with liver failure.1 Azathioprine is suggested in some practice guidelines,1,2 presumably extrapolated from management of idiopathic autoimmune hepatitis. However, there is very little supporting data, and its delayed onset of action seems counterintuitive in acute IRH. Anti-TNF blockade with infliximab has shown efficacy in cases of IRH. Still, societal recommendations that infliximab is contraindicated in IRH will likely limit more widespread evaluation.1,2 This is based on the rare association between infliximab and immune-mediated hepatitis, usually associated with more prolonged infliximab exposure than in IRAE management.

ESMO guidelines recognise the risk for steroidassociated adverse events in patients treated with very high doses of CS, and emphasise CS treatment at the lowest dose and for the shortest period.1 Specific management guidance for IRH recommends a “step up” approach, stratified according to grade of liver injury, starting with lower doses (0.5–1.0 mg/kg) in Grade 2 IRH, or higher doses (1-2 mg/kg) in Grade 3 or 4 IRH, and increasing the dose if necessary. In practice, there is considerable variation in approach to CS dosing amongst individual physicians, with some preferring a “top-down” strategy, starting with high CS doses (up to 2 mg/kg at initiation), whilst others prefer to “step up”, starting with lower doses (0.5–1.0 mg/kg), and increase the dose if response is poor. A recent large retrospective study suggested that very high doses of CS (>1.5 mg/kg) do not confer any additional advantage in terms of time to resolution of liver injury, or need for additional second-line IST in patients with high-grade IRH, but were associated with increased rates of steroid-associated side effects.8 Therefore, the management strategy described in Figure 2 avoids escalating steroids to doses >1.5 mg/ kg, and proposes earlier introduction of secondline IST in patients who do not respond to lower doses. This approach should be prospectively evaluated for effect on resolution of hepatitis and reduction of treatment-associated harms.

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Most patients with IRH resolve their liver injury with CS treatment. However, retrospective series suggest that 10–30% of patients develop resistant hepatitis, where liver enzymes initially respond to CS but then flare with CS taper; or refractory hepatitis, where there is no response to CS treatment.8,9 These patients often require additional IST, and prompt recognition of CS resistance/refractoriness with introduction of second-line treatment may ultimately reduce exposure to high-dose CS and associated side effects. ESMO guidelines recommend that a second agent is initiated after 48–72 hours in patients with high-grade IRAE, not responding to CS alone (Figure 2).1

Although management of IRAE is a priority in patients who are ICI-treated, concerns that broad spectrum IST may compromise the cancer response to ICI have led to proposals for individualised treatment regimens, targeting key inflammatory components implicated in specific IRAE.10 Anti-cytokine therapies, such as tocilizumab, may potentially treat IRAE without

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compromising anti-tumour immunity. There are increasing case reports of tocilizumab in treatment of IRH, with many, although not all, reporting favourable outcomes.1,11 Rational development of a selective IST approach for IRH is desirable, ideally guided by further translational research providing greater insights into its pathophysiology. Meanwhile, earlier use of tocilizumab may be an attractive strategy to treat IRH without compromising cancer response, reserving more broadly immune-suppressing treatments as second- or third-line, and warrants further evaluation. A Phase II study of ipilimumab, nivolumab, and tocilizumab for advanced melanoma is currently underway, to investigate whether addition of tocilizumab is associated with a decrease in IRAE and/or increased anticancer efficacy.12 As up to 25% of patients treated with ipilimumab/nivolumab may develop IRH,1 the study’s results may help further define the role of tocilizumab in its prevention and treatment.

EXIT STRATEGIES: HOW TO MANAGE THE TAPER Most cases of IRH resolve with IST without evolving into chronic hepatitis. There are significant variations in practice regarding initiation and rate of CS dose taper. It is not clear if resistant hepatitis may be associated with early initiation or rapid rate of taper. There are clear benefits to avoiding steroid resistance, as this prolongs exposure to IST, increases potential for CS-associated side effects, and may delay resumption of anticancer therapy.

symptomatic Grade 3 or any Grade 4 IRH, due to risk of recurrent severe toxicity.2 However, other treatment options may be limited, and especially as IRAE may be associated with improved cancer response to ICI therapy, patients may benefit from re-treatment. ESMO guidelines suggest consideration of three possible scenarios for ICI resumption: class switch from anti-CTLA-4 to anti-PDL1, or vice versa; resumption of the same class agent or same molecule; or resumption of ICI with prophylactic immunosuppressive therapy. Data to support any of these options is limited, and individualised discussion of rechallenge by a multidisciplinary team is recommended.1 A recent prospective study confirmed the overall safety of rechallenge in patients with highgrade IRH, reporting a recurrence rate of 35%. Most patients in this study developed IRH after treatment with anti-PDL1 monotherapy, and most (78%) were re-treated with the same drug.13 In general, severity of the recurrent episode of IRH was similar to the index episode, although one patient developed severe toxicity with liver failure. At the time of ICI rechallenge, 34% were still receiving prednisone (5–10 mg/day) for the previous IRAE, but this did not appear to reduce the risk for recurrence of IRH.13 Based on limited data, ICI rechallenge appears generally safe in patients with high-grade IRH. There is currently no proven benefit to prophylaxis with CS or other IST.

VIRAL HEPATITIS

ESMO guidelines recommend that in Grade 2 IRH, CS should be tapered over 2 weeks after liver injury has improved to Grade 1. For Grade 3 or 4 IRH, tapering over 4–6 weeks is recommended once liver injury has improved to Grade 2.1 Although helpful in their specificity, these tapering regimens are empirical, and would benefit from prospective evaluation to establish best practices in CS management.

All patients should be screened for hepatitis B (HBV) and hepatitis C (HCV) infection before starting ICI.14,15 Most ICI registration trials excluded patients with chronic viral hepatitis, except trials of ICI for hepatocellular carcinoma. Data from these studies, and subsequent realworld data, confirm the overall safety of ICI in patients with chronic viral hepatitis without an adverse impact on cancer outcomes, or risk for IRAE.1,15

CAN IMMUNE CHECKPOINT INHIBITORS BE RESTARTED?

In theory, blocking the PD-1/PD-L1 axis in patients with chronic viral hepatitis may partially restore the function of virus-specific CD8 T cells, leading to enhanced viral recognition, and potentially bystander liver injury due to the

Societal guidelines have recommended permanent discontinuation of ICI following

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enhanced antiviral immune response. Damage to hepatocytes may also cause release of virus into the circulation, resulting in viral reactivation.15 In patients with chronic HBV infection, reactivation risk may be as high as 21%, but is substantially reduced by treatment with prophylactic antivirals. Therefore, all patients with chronic HBV should receive antiviral prophylaxis during ICI treatment, and 6–12 months after.14,15 There is little data on risk of reactivation in patients with resolved HBV infection, and current recommendations are for careful monitoring without routine use of antiviral prophylaxis.14 It is important to recognise that if patients require CS to treat any IRAE, this may increase risk for HBV reactivation, and the need for antiviral prophylaxis should be re-evaluated.14 Some studies have observed a fall in HCV viral load in patients with chronic HCV treated with ICI. HCV reactivation has rarely been described, manifesting as an elevation in liver enzymes.15 There are now several reports of successful HCV treatment with direct-acting antivirals (DAA) concomitant with ICI therapy, so chronic HCV infection is not a contraindication to proceeding with ICI therapy.1,15 Whether there is a benefit to initiating DAA therapy as prophylaxis and optimal DAA treatment in the event of HCV reactivation would benefit from further investigation.

References 1.

Haanen J et al. Management of toxicities from immunotherapy: ESMO clinical practice guideline for diagnosis, treatment and followup. Ann Oncol. 2022;33(12):121738. Schneider BJ et al. Management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy: ASCO guideline update. J Clin Oncol. 2021;39(36):4073-126.

SUMMARY AND CONCLUSIONS As ICI use expands, so will the incidence of IRAE, including IRH. Diagnostic biomarkers are currently lacking, which specifically identify IRH and stratify severity. Current biorepository studies may help to identify biomarkers that fulfil this need. Increased experience in IRH management has led to refinements in successive iterations of clinical practice guidelines, although many recommendations remain empirical, and lack rigorous evaluation. Proposed management (for example, as described in Figure 2) is moving towards a strategy of therapeutic parsimony, with reduced CS dosing, and earlier introduction of IST to minimise the risk for steroid-associated side effects. At the same time, a new therapeutic paradigm is emerging in IRAE management to decouple IRAE from cancer response, through the use of more selective IST. MMF is widely used as second-line IST for IRH, with a generally good effect. There are increasing reports of successful use of tocilizumab as third-line therapy, and results from ongoing trials may indicate whether it should be used earlier in the treatment pathway. Finally, greater understanding of the pathophysiology underlying IRH may inform selective IST treatment strategies to optimise outcomes for patients treated with ICI therapies.

limiting factor to the increasing use of immune checkpoint inhibitors. JHEP Rep. 2020;2(6):100170. 6.

Fontana RJ et al. Drug-Induced Liver Injury Network (DILIN) prospective study: rationale, design and conduct. Drug Saf. 2009;32(1):55-68.

De Martin E et al. Characterization of liver injury induced by cancer immunotherapy using immune checkpoint inhibitors. J Hepatol. 2018;68(6):1181-90.

Hommes JW et al. Biomarkers of checkpoint inhibitor induced immune-related adverse events-a comprehensive review. Front Oncol. 2020;10:585311.

Li M et al. Effect of corticosteroid dosing on outcomes in highgrade immune checkpoint inhibitor hepatitis. Hepatology. 2022;75(3):531-40.

Issa M et al. Risk factors and predictors of immune-related adverse events: implications for patients with non-small cell lung cancer. Expert Rev Anticancer Ther. 2022;22(8):861-74.

Miller ED et al. Clinical characteristics and adverse impact of hepatotoxicity due to immune checkpoint inhibitors. Am J Gastroenterol. 2020;115(2):251-61.

De Martin E et al. Liver toxicity as a

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10. Martins F et al. New therapeutic perspectives to manage

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refractory immune checkpointrelated toxicities. Lancet Oncol. 2019;20(1):e54-64. 11. Ali SB et al. Tocilizumab in grade 4 hepatitis secondary to immune checkpoint inhibitor: a case report and review of the literature. Immunotherapy. 2023;15(14):1125132. 12. NYU Langone Health. A phase II study of the interleukin-6 receptor inhibitor tocilizumab in combination with ipilimumab and nivolumab in patients with unresectable stage III or stage IV melanoma. NCT03999749. https:// classic.clinicaltrials.gov/ct2/show/ NCT03999749. 13. Riveiro-Barciela et al. Retreatment with checkpoint inhibitors after a severe immune-related hepatitis: results from a prospective multicenter study. Clin Gastroenterol Hepatol. 2023;21(3):732-40. 14. Hwang JP et al. Hepatitis B virus

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screening and management for patients with cancer prior to therapy: ASCO provisional clinical opinion update. J Clin Oncol. 2020;38(31):3698-715.

15. Tapia Rico G et al. The safety and efficacy of immune checkpoint inhibitors in patients with advanced cancers and pre-existing chronic viral infections (Hepatitis

B/C, HIV): a review of the available evidence. Cancer Treat Rev. 2020;86:102011.

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Let’s Start at the Beginning: A Healthy Gut From Day 1 Authors:

*Ranaan Shamir1,2 1. Sackler Faculty of Medicine, Tel Aviv University, Israel 2. Institute of Gastroenterology, Nutrition and Liver Diseases, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel *Correspondence to raanan@shamirmd.com

Disclosure:

The author has declared no conflicts of interest.

Received:

18.05.23

Accepted:

23.05.23

Keywords:

Breastfeeding, diet, digestive health, digestive diseases, digestive cancers, food allergies, human microbiome, nutrition.

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10303345. https://doi.org/10.33590/emjgastroenterol/10303345.

Establishing healthy nutrition practices from infancy through to childhood and adolescence is essential in ensuring a healthy digestive system throughout our lives. Several studies have found that changes made during these early years can have a significantly positive effect later in life, including the prevention of non-communicable diseases, such as obesity, diabetes, inflammatory diseases, and certain types of cancer.1 In the context of today’s digestive health landscape, the importance of these early preventative measures cannot be overstated. A recent pan-European study, commissioned by United European Gastroenterology (UEG), on the burden of digestive diseases and cancers, revealed the significant health and economic toll these diseases exert on societies in terms of disability and premature mortality, as well as on health system expenditure and productivity losses.2 In 2019 alone, there were an estimated 72 million incident cases of digestive disease across Europe, with an associated direct cost of 25.3 billion EUR.2

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Effectively preventing these diseases and their burden starts early in life, so it is critical that we focus on the measures we can take right from the beginning to reverse this concerning trend, and protect the health of future generations.

THE FIRST 1,000 DAYS Our gut health and development of digestive diseases are closely tied to the human microbiome. While genetic variability is a significant contributor to the composition of the human microbiome, environmental factors, such as diet, medication, and stress, heavily influence the microbiome,3 potentially increasing an individual’s risk of developing certain digestive diseases. One of these factors, diet, comes into play early in life, particularly in the first 1,000 days.3 Numerous studies have demonstrated the pivotal role of exclusive breastfeeding, and the age of introduction of a variety of foods during infancy, in establishing a well-developed adult microbiome.3

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The European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the World Health Organization (WHO) recommend exclusive breastfeeding in the first 6 months of life, and continued breastfeeding as long as mutually desired by both mother and child. Breast milk is considered the optimal source of nutrition at this young age, as it contains a mixture of bioactive peptides, nutrients, vitamins, and minerals that are essential for driving healthy development.4,5 The benefits of breastfeeding extend well beyond infancy, and have been associated with various long-term health outcomes, including a lower incidence of inflammatory and autoimmune diseases, asthma, certain types of cancer, Type 1 diabetes, and one of the most pressing public health crises currently facing the world, obesity.5,6 Investigating this issue from a health equity perspective, breastfeeding has also been highlighted as one of the most cost-effective interventions for protecting children against malnutrition, with breastfed infants benefitting from lower infectious morbidity and mortality rates.6 However, despite this evidence, breastfeeding rates remain well below WHO recommendations in both high- and low-income countries.7 This demonstrates the urgent need for educational and promotional strategies across the globe that support and motivate a successful mother–infant breastfeeding relationship.5 Another consideration in long-term disease prevention is the introduction of allergenic foods, such as peanuts, eggs, and shellfish, at an early age. In recent years, the increasing prevalence of food allergies has become a growing health problem, affecting up to 10% of individuals in Western countries.8

References 1.

Agosti M et al. Nutritional and metabolic programming during the first thousand days of life. Pediatr Med Chir. 2017;39(2):157.

Rose TC et al. Analysis of the burden and economic impact of digestive diseases and investigation of research gaps and priorities in the field of digestive health in the European Region-White Book 2: Part 1. 2022;1-215.

A meta-analysis and systematic review from 2023 showed that the early introduction of multiple allergenic foods was associated with a lower risk of developing food allergies later in life.9 A 2017 position paper by ESPGHAN supports this, recommending the introduction of allergenic foods as early as 4 months of age, while supporting continued breastfeeding.10 While complementary foods (solids and liquids other than breast milk or infant formula) should be introduced after 4 months, whole cow’s milk should not be used as a main drink until the child reaches 12 months of age.10 Research indicates that infants who consume large volumes of cow’s milk face an increased risk of iron deficiency and iron deficiency anaemia.10 This heightened risk is likely due to the low iron content and bioavailability of iron from cow’s milk, as well as the displacement of other iron-rich foods from the diet.10

TIME FOR ACTION The worrying lack of progress seen in recent decades in confronting the burden of digestive diseases and cancers points to the need for the greater adoption of evidence-based preventative methods. In line with the theme selected by the World Gastroenterology Organisation (WGO) for World Digestive Health Day, which focuses on the importance of maintaining a healthy gut from the outset, we are exploring ways to ensure action is taken at an early stage. To push forward progress, the preventative measures discussed must be prioritised and integrated into national health agendas to ensure digestive health is effectively addressed, and considered at the very beginning. This early action will deliver tangible benefits for decades to come.

Dong TS, Gupta A. Influence of early life, diet, and the environment on the microbiome. Clin Gastroenterol Hepatol. 2019;17(2):231-42.

ESPGHAN Committee on Nutrition; Agostoni C et al. Breast-feeding: a commentary by the ESPGHAN Committee on Nutrition. J Pediatr Gastroenterol Nutr. 2009;49(1): 112-25.

World Health Organization (WHO). Infant and young child feeding. June 2021. Available at: https:// www.who.int/news-room/factsheets/detail/infant-and-youngchild-feeding. Last accessed: 17 May 2023.

Victora CG et al.; Lancet Breastfeeding Series Group. Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet. 2016;387(10017):475-90.

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Vaz JS et al. Monitoring breastfeeding indicators in high-income countries: levels, trends and challenges. Matern Child Nutr. 2021;17(3):e13137.

Loh W, Tang MLK. The epidemiology of food allergy in the global context. Int J Environ Res Public Health. 2018;15(9):2043.

Scarpone R et al. Timing of allergenic food introduction and risk of immunoglobulin E-mediated food allergy: a systematic review and meta-analysis. JAMA Pediatr. 2023;177(5):489-97.

10. Fewtrell M et al. Complementary feeding: a position paper by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) Committee on Nutrition. J Pediatr Gastroenterol Nutr. 2017;64(1): 119-32.

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A Rare Complication of Valproate-Induced Acute Pancreatitis in an Adult Patient with Bipolar Disorder: A Case Report Authors:

P. Kaur,1 D. Bhasin,2 H. Singh,1 *T.S. Kundra3 1. Department of Critical Care, Max Super Speciality Hospital, Mohali, India 2. Department of Pulmonology and Critical Care, Max Super Speciality Hospital, Mohali, India 3. Department of Anaesthesiology and Intensive Care, Government Medical College, Patiala, India *Correspondence to tvskundra@yahoo.co.in

Disclosure:

The authors have declared no conflicts of interest. Written informed consent was obtained from the patient for publication.

Received:

13.04.22

Accepted:

27.02.23

Keywords:

Acute abdomen, amylase, case report, pancreatitis, valproate.

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10308417. https://doi.org/10.33590/emjgastroenterol/10308417.

Abstract Background: Valproate-induced pancreatitis is an idiosyncratic reaction of the drug, commonly seen in the paediatric age group, between 1 week to 8 years of starting the drug. Case: The authors present a case report of a 61-year-old patient who presented with acute pancreatitis. All common causes were ruled out. The patient had a significant treatment history of taking valproate for bipolar disorder for 12 years. The patient was resuscitated with intravenous fluids and analgesics. Sodium valproate was stopped. The patient was monitored to detect early symptoms of systemic inflammatory response syndrome or any organ dysfunction or failure. The patient was sent to the ward after 4 days. The patient had a good outcome due to early diagnosis and resuscitation. Conclusion: Acute pancreatitis is a rare and potentially fatal complication in patients on valproate. After ruling out other common causes, a detailed medical and treatment history can lead to the diagnosis of this entity. Healthcare providers should be vigilant about the early signs and symptoms of pancreatitis, including acute abdomen, vomiting, and raised serum amylase and lipase.

Key Points 1. Valproate-induced pancreatitis is an idiosyncratic reaction of the drug, commonly seen in paediatric age group, between 1 week to 8 years of starting the drug.

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2. This entity is a diagnosis of exclusion. After ruling out other common causes, a detailed medical and treatment history can lead to the diagnosis of this entity. 3. Even in adults, and beyond the 8-year window, healthcare providers should be vigilant about the early signs and symptoms of pancreatitis, including acute abdomen, vomiting, and raised serum amylase and lipase in patients who are taking this drug.

INTRODUCTION Sodium valproate, or valproic acid (VPA), is a broad-spectrum antiepileptic drug, commonly used for the treatment of epilepsy, bipolar disorder, migraine headache, and diabeticneuropathy related pain.1 VPA was approved by the U.S. Food and Drug Administration (FDA) in 1978 for treating epilepsy and absence seizures.2 Since then, it has been used either as monotherapy, or in combination with other anticonvulsant agents for the treatment of mixed and complex partial seizures, acute manic episodes in bipolar disorder, and for prophylaxis of migraine headache.3 It is also the most prescribed mood stabiliser by far due to its little impact on cognitive function and central nervous system.4 VPA is also effective in treating myoclonic, simple partial, and generalised tonicclonic seizures.5 VPA is a simple eight-carbon branched-chain fatty acid, which is structurally unrelated to any other marketed drug. Several mechanisms of VPA’s therapeutic effects have been reported, including increasing levels of γ-aminobutyric acid by decreasing reabsorption and catabolism, restraining neuronal repetitive firing via suppression of ion channels, and targeting the transcriptomic system to directly inhibit Class I histone deacetylases.6-9 However, serious complications may be associated with valproate. These effects include hepatotoxicity, teratogenicity, possible polycystic ovaries with a potential sterile effect, and acute pancreatitis.10 These side effects need to be considered when prescribing this medication to certain susceptible populations, such as the paediatric age group and persons with intellectual disability and neurological deficits, including mental retardation, cerebral palsy, and developmental delay.11-13

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Acute pancreatitis is a rare, but one of the most severe VPA-related toxic effects. It makes a total of around 100 cases reported in the literature from 1979 to the time of writing.12,14-18 Necrotising pancreatitis is an extreme complication of acute pancreatitis.19 Most of the cases are children with epilepsy; however, only one bipolar disorder patient with renal failure, who underwent haemodialysis and subsequently developed acute pancreatitis due to VPA treatment, has been reported.20 Herein, the authors present a rare case of adult valproate-induced acute pancreatitis, presenting 12 years after starting the drug, and who had a good outcome due to early diagnosis and resuscitation.

CASE REPORT The patient was a 61-year-old male who was known to have diabetes and hypertension. They were diagnosed with bipolar disorder 12 years ago. The patient presented to the hospital with a chief complaint of 3 days of abdominal pain, which was diffuse in nature. The patient had no history of duodenal or gastric ulceration and no other digestive system disease; they did not have metabolic syndrome or food poisoning. The pain was associated with multiple episodes of vomiting, containing partially-digested gastric contents, and was non-bilious and non-bloody in nature. These complaints were accompanied by decreased urine output for 2 days. The patient also complained of bloating and obstipation. The patient’s medication history showed that they were taking a 500 mg tablet of valproate thrice a day for 12 years. A complete list of medications being taken by the patient includes valproate 500 mg thrice a day; lorazepam 2 mg at bedtime; quetiapine at bedtime; glimepiride 2 mg once daily; and telmisartan 40 mg once daily.

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The patient was a non-smoker and did not drink or partake in recreational drug use. The patient had their first dose of the COVID-19 vaccination in early 2021, and was found negative for COVID-19 on testing. Bedside screening echocardiography was done to rule out any cardiac event, and it was suggestive of left ventricle diastolic dysfunction, preserved left ventricle systolic function, and no regional wall motion abnormality. The patient was managed with intravenous fluids and analgesics. VPA was stopped, while lorazepam and quetiapine were continued in consultation with the psychiatrist. Abdominal distension was present. The patient was kept nil per oral. A Ryles tube was inserted, which yielded 300 mL aspirate. Patient was given a proctoclysis enema, after which the patient passed stool. An ultrasound of the abdomen was done, which showed bulky pancreas and mildly prominent common bile duct (CBD) at porta. Investigations revealed a raised serum amylase (861 U/L) and lipase (690 U/L). Serum calcium was 9.0 mg/dL and serum triglycerides were 175 mg/dL. The patient was managed conservatively and monitored to detect earliest signs of systemic inflammatory response syndrome or organ dysfunction or failure. MRI cholangiography was done on the third day to rule out CBD stones. MRI cholangiography was suggestive of acute pancreatitis with minimal ascites and bilateral minimal pleural effusion. The right and left hepatic ducts, along with the CBD, were normal. There was no intra-luminal filling defect. The patient had a form of mild acute pancreatitis, probably due to the prompt and early treatment (initiated by the intensive care doctors). There were no signs and symptoms of acute organ failure. There were no local or systemic complications. The patient was moved to the ward after 4 days.

DISCUSSION The present patient presented to the hospital with acute abdomen. Clinically, after laboratory and radiological investigations, a diagnosis of acute pancreatitis was made.

After making this diagnosis, a search for the probable aetiology was made. A list of the causes of acute pancreatitis was made to reach the probable cause, as enumerated below:21 gallstones, which were ruled out after an ultrasound; alcohol, but the patient did not drink alcohol; and hypertriglyceridemia, but serum triglycerides were borderline high and, at these levels, are highly unlikely to be cause the of acute pancreatitis. The authors also considered drugs. Drug‑induced pancreatitis (DIP) is a rare cause.22 Drugs are responsible for only 0.1–2.0% of acute pancreatitis incidents.23-25 According to a German retrospective study, the incidence of pancreatitis caused by drugs was 1.4%.26 A nationwide survey conducted in Japan showed that 1.2% of all cases of acute pancreatitis were caused by drugs.27 The list of drugs responsible for this complication has increased to about 500 agents.28 A substantial number of drugs commonly prescribed for gastrointestinal disorders are known to cause acute pancreatitis.22,27 Antiepileptics have been implicated as a cause of acute pancreatitis, though the incidence is rare.29 The diagnosis is essentially a diagnosis of exclusion. The patient was not subjected to endoscopy before presentation with features of acute pancreatitis, ruling out endoscopic retrograde cholangiopancreatography induced pancreatitis. Other causes were also considered, including accidental damage or injury to the pancreas, but there was no such history in this patient; viruses like mumps or measles, but no such disease history was given by the patient; hypercalcaemia, but serum calcium was within normal limits; and autoimmune pancreatitis, but the patient had no such history of any autoimmune disease, and no history was present in their family. Based on the above, and a thorough literature search, the treating physicians made the probable diagnosis of valproate-induced pancreatitis, which is essentially a diagnosis of exclusion. VPA is a broad-spectrum antiepileptic, which is commonly prescribed for seizures, epilepsy,

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and bipolar disorder. Commonly reported side effects of VPA include congenital anomalies, infection, abdominal pain, asthenia, drowsiness, nausea, tremor, vomiting, alopecia, diarrhoea, dizziness, flu-like symptoms, thrombocytopenia, and anorexia. Rare side effects include fulminant hepatitis, pancreatitis, encephalopathy, and pedal oedema.30 Acute pancreatitis is one of the rare complications of this drug. Santos et al.31 published about VPA-induced pancreatitis in an adult in less than 2 months of treatment.They discussed about the history of the entity. The first cases of acute pancreatitis associated to VPA were reported in 1979 by Bataladen et al.32 and Camfield et al.,33 with several sporadic cases being published subsequently. There are two cases described in Brazil, the first in 1986 by Barros et al.34 and the other in 1998 by Munhoz et al.35 Barbosa et al.36 published a case report of VPAinduced necrohaemorragic pancreatitis. The authors discussed the diagnostic approach in this uncommon pancreatitis, and mentioned that it is convenient to consider medication (such as VPA) induced acute pancreatitis in patients without a clear causative agent. Huang W et al.4 published a case report of VPAinduced acute pancreatitis in a patient with bipolar disorder presenting within 1 year of starting the drug. The authors mentioned acute pancreatitis to be considered as one of the idiosyncratic adverse reactions to antiepileptic drugs. Idiosyncratic reactions to drugs are adverse effects that are not directly related to pharmacodynamic mechanisms of the drug, and they take place by abnormal interaction between the drug and the organism in unpredictable manner, usually mediated by immunologic or cytotoxic effects triggered by the drug or its metabolites.37 The risk of idiosyncratic drug reactions is affected by several factors. The first factor is genetic determinants. If a patient has reacted to an immune-mediated aromatic antiepileptic drug, there is a 25% chance that their siblings will experience similar responses when exposed to the same class of drugs. The second factor is age. Many idiosyncratic reactions are typically age-dependent, in which age influences the metabolism of drugs.38 Specifically, the decreased glucuronide conjugation is commonly seen in

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young babies. The younger age group is at higher risk of idiosyncratic drug effects and increased reactive metabolite production.39 The third factor is initial dose and titration rate. Allergic reactions are generally regarded as not relative to drug dosage. However, allergic reactions may occur when the drug level reaches a certain threshold. Drugs with low effective doses (below 10 mg/ day) are less likely to trigger immune-mediated reactions.40 The titration speed is also a matter of great importance. Generally, the allergic reactions may not occur at low initial dose and slow titration rate, giving time for the body to become desensitised. Additionally, other factors include the similar responses to prior treatment, or disease-related factors such as patients with metabolic disorder and sodium valproate-related liver toxicity.37 Other valproate-related idiosyncratic reactions reported are alopecia, bone marrow aplasia, and immune-mediated hepatotoxicity.37 Werlin et al.41 published about the spectrum of VPA-associated pancreatitis in paediatric patients, and mentioned that VPA-associated pancreatitis does not depend on VPA serum level and may occur any time after the onset of therapy. In most cases reported in the literature, the serum VPA level was within the normal range, again pointing towards idiosyncratic nature of this side effect.13 Gerstner et al.12 published about VPA-induced pancreatitis in 16 patients, the average age of whom was 11.3 years (median age: 10.3 years). The authors estimated the incidence of VPAinduced pancreatitis to be 1:40,000. Similar data was presented by Genton et al.42 The authors mention that pancreatitis is an unusual reaction to VPA therapy, approximately seen in one out of 40,000 patients.42 Gerstner et al.12 mention the proposed mechanism of action of valproate-induced acute pancreatitis as a direct toxic effect of free radicals on the pancreatic tissue and a depletion of superoxide dismutase, catalase, and glutathione peroxidise. Similarly, Sanfey et al.43 and Pellock et al.44 theorised that the depletion of the free radical scavengers, superoxide dismutase, catalase, and glutathione peroxidase occurs in patients receiving VPA. It has also been suggested that the reduction of carnitine brought

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about by the use of VPA has an important role in the damage caused to the pancreas.45

quetiapine is combined with VPA to react against the pathogenesis of this disease.

Previous literature mentions that the incidence of valproate-induced acute pancreatitis is higher in paediatric and young patients.46 According to earlier reports (Hamad et al.),13 such adverse effects can occur after prescribing this medicine for 1 week to 8 years. Yazdani et al.47 have also mentioned pancreatitis to develop as an idiosyncratic reaction within 1 week to 8 years of exposure to VPA, with no association between dosage and serum levels of valproate.

During the follow-up period, the patient remained symptom-free under the treatment of quetiapine. Taken together, the authors assumed that the cause of acute pancreatitis in this patient was VPA treatment.

Despite appropriate treatment, DIP demonstrated severe complications and high mortality.48 The onset can be a slight asymptomatic hyperamylasaemia up to a fatal necrohaemorragic pancreatitis, with many complications in the non-lethal cases (pseudocysts, infections, septic shock, chronic pancreatitis, and endocrine pancreatic insufficiency).49 Some cases show rapid progression in bleeding from the initial symptoms until death; some are observed after the initial medication; and some appear after few years of medication.50 The mortality rates of acute pancreatitis in children and adult patients are 15.4% and 21.4%, respectively.12 A pharmacological research has recommended the maximum daily VPA dose of 2,500 mg, and the authors’ patient was taking 1,500 mg of VPA a day.51 Furthermore, there was no identifiable cause of acute pancreatitis, as the patient had no history of trauma, no overeating or drinking, no history of drug sensitivity, and was free of biliary system disorders, such as gallstones.52 In this study, the patient exhibited abdominal pain, accompanied by nausea and vomiting. Laboratory findings indicated that the levels of amylase elevated sharply to 861 U/L and lipase to 690 U/L, while other laboratory indices were within the normal range. In addition, ultrasound was used to confirm the abnormality of their pancreas. The patient presented with symptoms while receiving sodium VPA, a Class I medication associated with acute pancreatitis. Quetiapineinduced acute pancreatitis has been reported in few cases, which is difficult to distinguish from VPA-induced pancreatitis because they share similar symptoms.53-55 Moreover, there is limited existing evidence to support the assumption that

The novelty of the authors’ case is the age of the patient and the duration after which the entity developed. The patient was an adult and not from the most affected paediatric age group, the disease developed after 12 years of taking the drug, while commonly it is reported in up to 8 years of taking the drug. Valproate induced adult pancreatitis is rare; only a few cases have been reported and, hence, this case adds to the knowledge on the topic. Acute pancreatitis can be fatal. Although DIP is rare, physicians should bear in mind its possibility. To prevent DIP, the latest knowledge of medicines connecting their use to the occurrence of pancreatitis is required.23 Physicians should be aware of drug allergy history and a patient’s comorbid conditions, while maintaining vigilance against the signs of severe toxic reactions. To prescribe VPA as bipolar disorder treatment, patients need to be closely monitored in order to prevent severe adverse effects, including acute pancreatitis. Physicians must be vigilant about this diagnosis even after many years of starting the implicating drug.

CONCLUSION Valproate-induced acute pancreatitis is a rare and potentially fatal complication in patients on valproate. This is a diagnosis of exclusion, and is labelled only after the other common causes of pancreatitis have been ruled out. The authors’ patient had a good outcome because of early diagnosis and resuscitation. Though the complication is most commonly seen in paediatric patients up to 8 years after starting the drug, caregivers should be vigilant about the early signs and symptoms of pancreatitis, including acute abdomen, vomiting, and raised serum amylase and lipase, even in adult patients taking valproate for more than 8 years. Patients

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on long-term valproate should be counselled to make them aware, so that patients seek immediate medical attention if any of these symptoms develop. Prompt blood investigations for acute pancreatitis (serum amylase and lipase tests) and radiological imaging must be done

References 1.

14. Biour M et al. [Drug-induced pancreatitis: second edition of the bibliographic database of pancreatitis related to drugs.] Gastroenterol Clin Biol. 2005;29(4):353-9. (In French).

Browne TR, Koch-Weser J. Drug therapy: valproic acid. N Engl J Med. 1980;302(12):661-6.

Ray S et al. Valproic acid-induced severe acute pancreatitis with pseudocyst formation: report of a case. Cureus. 2015;7(8):e297.

A case series that combines the published cases until the time of writing, would improve understanding of the association.

Neurosciences (Riyadh). 2000;5(3):156-8.

Tripathi KD, “Antiepileptic drugs,” Essentials of Medical Pharmacology (2018) 8th edition, New Delhi: Jaypee Brothers, pp.438-51.

in patients on VPA who present at a healthcare setting with features of acute abdomen.

15. Salem CB et al. Valproic acid-induced pancreatitis. J Gastroenterol. 2007;42(7):598-9. 16. Hurdle AC, Moss RD. Unrecognized valproic acid intoxication. Am J Emerg Med. 2009;27(2):250.e1-2.

Huang W et al. Sodium valproate induced acute pancreatitis in a bipolar disorder patient: a case report. BMC Pharmacol Toxicol. 2019;20(1):71.

17. Sikma MA et al. Massive valproic acid overdose, a misleading case. Am J Emerg Med. 2008;26(1):110. e3-6.

Chapman SA et al. Pancreatitis associated with valproic acid: a review of the literature. Pharmacotherapy. 2001;21(12):1549-60.

18. Atam V et al. A case report of valproate-induced acute pancreatitis. J Med Soc. 2017;31(1):48-9.

Bialer M. Why are antiepileptic drugs used for nonepileptic conditions? Epilepsia. 2012;53(Suppl 7):26-33.

19. Goud SS, Indla V. Sodium valproate-induced necrotizing pancreatitis in a patient with bipolar mania. Telangana J Psychiatry. 2021;7(1):70-1.

Johannessen CU, Johannessen SI. Valproate: past, present, and future. CNS Drug Rev. 2003;9(2):199-216.

20. Okayasu H et al. Development of acute pancreatitis caused by sodium valproate in a patient with bipolar disorder on hemodialysis for chronic renal failure: a case report. BMC Psychiatry. 2014;14:93.

Chateauvieux S et al. Molecular and therapeutic potential and toxicity of valproic acid. J Biomed Biotechnol. 2010;2010:479365.

21. Szatmary P et al. Acute pancreatitis: diagnosis and treatment. Drugs. 2022;82(12):1251-76.

Phiel CJ et al. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001;276(39):36734-41.

22. Quan W et al. Acute pancreatitis associated with valproate treatment. Chin Med J (Engl.). 2018;131(15):1889-90.

10. Rosenberg HK, Ortega W. Hemorrhagic pancreatitis in a young child following valproic acid therapy. Clinical and ultrasonic assessment. Clin Pediatr (Phila). 1987;26(2):98-101.

23. Jones MR et al. Drug-induced acute pancreatitis: a review. Ochsner J. 2015;15(1):45-51.

11. Ali MF, Loh KY. Sodium valproate induced necrotising pancreatitis: a case report. Malays Fam Physician. 2013;8(3):28-30.

24. Bologa C et al. An unusual etiology of acute necrotic pancreatitis in a comatose patient. J Emerg Med Case Rep. 2019;10(2):43-6.

12. Gerstner T et al. Valproic-acid induced pancreatitis: 16 new cases and a review of the literature. J Gastroenterol. 2007;42(1):39-48.

25. Urbanek K et al., “Acute pancreatitis induced by drugs,” Rodrigo L (ed.), Acute Pancreatitis (2012), London: InTech Open, pp.17-34.

13. Hamad AE, Fawzi ME. Valproate associated acute pancreatitis.

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26. Lankisch PG et al. Drug induced acute pancreatitis: incidence and severity. Gut. 1995;37(4):565-7. 27. Ksiądzyna D. Drug induced acute pancreatitis related to medications commonly used in gastroenterology. Eur J Intern Med. 2011;22(1):20-5. 28. Cofini M et al. Valproic acidinduced acute pancreatitis in pediatric age: case series and review of literature. G Chir. 2015;36(4):158-60. 29. Trivedi CD, Pitchumoni CS. Drug-induced pancreatitis: an update. J Clin Gastroenterol. 2005;39(8):709-16. 30. Cho S, Atwood JE. Peripheral edema. Am J Med. 2002;113(7):580-6. 31. Santos BL et al. Valproic acidinduced pancreatitis in an adult. Arq Neuropsiquiatr. 2010;68(1):135-6. 32. Bataladen PB et al. Pancreatitis associated with valproic acid therapy. Pediatrics. 1979;64(4):520-2. 33. Camfield PR et al. Pancreatitis due to valproate acid. Lancet. 1979;1(8127):1198-9. 34. Barros HMT, Barros EJG. [Pancreatite aguda associada ao uso de valproato de sódio.] Rev Assoc Med Bras. 1986;32(1):33-4. (In Portuguese). 35. Munhoz RP et al. [Fatal necrohemorrhagic pancreatitis related to sodium valproate: case report.] Arq Neuropsiquiatr. 2001;59(3-B):821-3. (In Portuguese). 36. Barbosa SC et al. Valproic acid induced necrohemorragic pancreatitis: case report and diagnos-tic approach in uncommon pancreatitis. Int J Surg Case Rep. 2019;60:126-9. 37. Zaccara G et al. Idiosyncratic adverse reactions to antiepileptic drugs. Epilepsia. 2007;48(7):122344. 38. Perucca E. Clinical pharmacokinetics of new generation antiepileptic drugs

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at the extremes of age. Clin Pharmacokinet. 2006;45(4):35163. 39. Johnson TN. The development of drug metabolising enzymes and their influence on the susceptibility to adverse drug reactions in children. Toxicology. 2003;192(1):37-48. 40. Seguin B, Uetrecht J. The danger hypothesis applied to idiosyncratic drug reactions. Curr Opin Allergy Clin Immunol. 2003;3(4):235-42. 41. Werlin SL, Fish DL. The spectrum of valproic acid-associated pancreatitis. Pediatrics. 2006;118(4):1660-3. 42. Genton P et al., “Adverse effects,” Levy RH et al. (eds.), Antipepileptic drugs (2002) 5th edition, Philadelphia: Lippincott William & Wilkins, pp.838-51. 43. Sanfey H et al. The role of oxygen-derived free radicals in the pathogenesis of acute pancreatitis. Ann Surg. 1984;200(4):405-13.

44. Pellock JM et al. Acute pancreatitis coincident with valproate use: a critical review. Epilepsia. 2002;43(11):1421-4. 45. Moreno FA, Macey H, Schreiber B. Carnitine levels in valproic acid-treated psychiatric patients: a cross-sectional study. J Clin Psychiatry. 2005;66(5):555-8. 46. Jain A et al. Valproic acid-induced acute pancreatitis. Indian J Psychiatry. 2019;61(4):421-2. 47. Yazdani K et al. Fatal pancreatitis associated with valproic acid: review of the literature. Medicine (Baltimore). 2002;81(4):305-10. 48. Kaurich T. Drug induced acute pancreatitis. Proc (Bayl Univ Med Cent). 2008;21(1):77-81. 49. Binek J et al. Valproic-acidinduced pancreatitis: case report and review of the literature. J Clin Gastroenterol. 1991;13(6):690-3. 50. Guevara Campos J et al. Acute pancreatitis associated to the use of valproic acid. Arq Neurop-

siquiatr. 2009;67(2):513-5. 51. Perrott J et al. L-carnitine for acute valproic acid overdose: a systematic review of published cases. Ann Pharmacother. 2010;44(7-8):1287-93. 52. Sinclair DB et al. Valproic acidinduced pancreatitis in childhood epilepsy: case series and review. J Child Neurol. 2004;19(7):498-502. 53. Sharma R et al. Quetiapine induced acute pancreatitis: a case report from Eastern Nepal. Tropical Gastroenterology. 2008;DOI:10.7869/tg.450. 54. Franco JM et al. Quetiapineinduced hypertriglyceridaemia causing acute pancreatitis. BMJ Case Rep. 2015;2015:bcr2015209571. 55. Liou L et al. Aggravation of hypertriglyceridemia and acute pancreatitis in a bipolar patient treated with quetiapine. Yonsei Med J. 2014;55(3):831-3.

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SAPHO Syndrome in Crohn's Disease Successfully Treated with Ustekinumab: Case Report and Review of the Literature Authors:

Sara Caporuscio,1 *Daniela Maggi,2 Annalisa Aratari,2 Claudio Papi,2 Stefano Festa2 1. Internal Medicine, San Filippo Neri Hospital, Rome, Italy 2. IBD Unit, San Filippo Neri Hospital, Rome, Italy *Correspondence to daniela.maggi@aslroma1.it

Disclosure:

This case report was written according to CARE guidelines. Informed patient consent was obtained for publication of the case details. The authors contributed equally to the manuscript. The authors declare that there is no conflict of interest that would prejudice the impartiality of the report.

Received:

24.08.23

Accepted:

27.09.23

Keywords:

Crohn’s disease, inflammatory bowel disease (IBD), spondyloarthropathy, synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO).

Citation:

EMJ Gastroenterol. 2023; DOI/10.33590/emjgastroenterol/10303080. https://doi.org/10.33590/emjgastroenterol/10303080.

Abstract The authors report a case of synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome in a patient with Crohn’s disease (CD). SAPHO syndrome is a rare disease characterised by the association, even if not simultaneous, of joint and skin manifestations. A young patient with CD was admitted to the authors’ hospital for the onset of bloody diarrhoea, persistent chest pain, headache, and fever while on maintenance therapy with vedolizumab. At visit, sternocostoclavicular and temporomandibular joints were tender and painful. Magnetic resonance was performed, and showed bone oedema of involved joints, while ileocolonoscopy revealed ulcers in the transverse colon. At laboratory, tests marked phlogosis and Campylobacter jejuni infection was observed. A challenge in differential diagnosis arose: atypical drug-induced extraintestinal manifestations, reactive arthritis, or extraintestinal manifestation directly associated with intestinal flare? In relation to the patient's age, the involved joints, and magnetic resonance findings, SAPHO syndrome was diagnosed. Systemic steroids were used with a rapid clinical improvement; vedolizumab was withdrawn and ustekinumab was started with sustained clinical response.

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Key Points 1. The present case report describes the occurrence of an acute osteochondritis in a young male affected by Crohn’s disease (CD) that the authors diagnosed as synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome. This challenging clinical picture raised interest on the problem of differential diagnosis in those patients in which the same presentation can hide several, and even rare, clinical entities. 2. This case highlights the great importance of joint management between gastroenterologists and rheumatologists, to optimise clinical outcomes and therapeutic approaches. 3. In this case, ustekinumab allowed control of both intestinal manifestations and sternoclavicular osteitis, proving to be an effective therapeutic approach in patients with Crohn's disease and SAPHO syndrome.

INTRODUCTION Inflammatory bowel diseases (IBD) are chronic diseases characterised by an unpredictable course that can be complicated by extraintestinal manifestations (EIM). EIMs affect approximately 50% of patients with IBD at any time during the disease course, and most commonly involve musculoskeletal (causing arthralgia due to arthritis, either axial or peripheral), cutaneous, and ocular systems.1-3 Synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome has been considered for a long time a rare form of seronegative spondyloarthropathy. The association between SAPHO and IBD has been known since 1992;4,5 however, it is often underdiagnosed because of its rarity, and similarity with other more common IBDassociated EIMs. The authors report a case of SAPHO syndrome in Crohn’s disease (CD). The aim is to highlight the challenges in differential diagnosis of EIMs in the context of IBD; and to point out the importance of joint management between gastroenterologists and rheumatologists for a correct diagnosis, an effective therapeutic approach to achieve an optimal outcome, and improved quality of life in patients with IBD.

CASE REPORT In August 2022, a 24-year-old White male affected by ileocolonic CD was hospitalised in the authors’ IBD unit for the appearance of

persistent chest and temporomandibular pain, with remitting fever up to 39 °C. The patient was diagnosed with CD in 2018 (A2, L3, B1, according to Montreal classification), and regularly followed up in the authors’ outpatient IBD clinic. An early treatment with adalimumab was started at diagnosis due to the presence of multiple unfavourable prognostic factors (extensive colonic involvement, severe endoscopic lesions, young age, anaemia, and inflammatory systemic impact). However, after experiencing primary failure to adalimumab, and subsequently infusion reaction to infliximab, a swap to vedolizumab (300 mg at 0, 4, and 12 weeks, and subsequently every 8 weeks) was initiated in February 2021. Sustained steroid-free clinical remission and endoscopic improvement were achieved after 18 months of vedolizumab treatment. It must be specified that no EIMs were present at diagnosis. At the last outpatient visit, the patient was in intestinal clinical remission, but complained of dull pain, progressively increasing, and exacerbated by exertion, located in the middle of the chest, and radiating to the right shoulder and neck. The physical examination revealed a warm and erythematous swelling of approximately 4 cm in diameter in the left sternoclavicular joint region, fluctuating, and tender to palpation. Ultrasonography of superficial lymph nodes was negative. In suspicion of synovitis, the initial treatment proposed was ibuprofen 1,200 mg/ day, with slight improvement in chest pain, but worsening of headache and temporomandibular joint paint. Furthermore, due to the appearance of a fever up to 39 °C and bloody diarrhoea (4–6

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bowel movements/day), the patient was admitted to hospital. At admission, laboratory tests revealed elevated C-reactive protein levels (26.2 mg/dL) and faecal calprotectin (1,865 mg/kg), together with inflammatory anaemia (haemoglobin: 10.4 g/dL; ferritin: 198 ng/mL). Autoimmunity panel showed slight positivity for c-ANCA (1:40) and anti-PR3 (94 chemiluminescent units; normal value <20 chemiluminescent units), and absence of the allele HLA-B27. Blood and urinary culture, urethral swab for sexually transmitted disease, cytomegalovirus, Epstein–Barr virus, HIV serology, and interferon gamma release assay test were performed with negative results, whereas stool pathogens test revealed a positivity for Campylobacter jejuni. Ileocolonoscopy showed severe activity

with ulcers in the transverse colon (Figure 1), in absence of ileal lesions, and histologic examination confirmed colic moderate-severe activity with neutrophilic infiltrate. T1 and T2-weighted fat-suppressed gadolinium-enhanced MRI of temporomandibular and chest joints showed alteration of signal characterised by hyperintensity in the long tandem repeat sequences of the distal epiphysis, and the third distal of the diaphysis of the right clavicle. Areas with similar appearance were found in the proximal and distal portion of the sternal body. After administration of gadolinium, impregnation of the areas described was detected, and the finding was to be referred to areas of bone oedema (Figure 2).

Figure 1: Colonoscopy imaging showing ulcers of transverse colon.

Figure 2: MRI at onset of symptoms, showing hyperintensity in the long tandem repeat sequences of the right clavicle and in the sternal body.

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Figure 3: MRI after 10 months, showing resolution of inflammatory findings.

In relation to the patient’s age, the insidious onset initially, with the involvement of the costosternal, and then of the temporomandibular joint, and the presence of fever and MRI findings, SAPHO syndrome was suspected. Since initial treatment with non-steroidal anti-inflammatory drugs was ineffective on articular manifestations, and no improvement was observed after a course of azithromycin 500 mg once daily given for Campylobacter infection, systemic steroids (methylprednisolone 0.75 mg/kg) were started, leading to rapid improvements of joints pain, resolution of fever, and intestinal clinical remission. In light of severe endoscopic lesions, vedolizumab was withdrawn, and the patient was swapped to ustekinumab. After 1 year of ustekinumab therapy, the patient was still in complete clinical remission for both gastrointestinal and extraintestinal manifestations. MRI of chest joints showed complete resolution of bone oedema and other inflammatory findings (Figure 3). Faecal calprotectin after 1 year was 259 mg/kg.

DISCUSSION The present case report describes the occurrence of an acute osteocondritis in a young male affected by CD, which the authors diagnosed as SAPHO syndrome. This challenging

clinical picture raised the interest on the problem of differential diagnosis. In this patient, the authors could, in fact, formulate three main diagnostic hypotheses: atypical drug-induced extraintestinal manifestations, reactive arthritis, or EIMs directly associated with intestinal flare. The first option was excluded because there is no agreement in the literature regarding the role of vedolizumab in triggering de novo extraintestinal manifestations.6,7 It is theoretically possible that its gastrointestinal selectivity, useful to reduce systemic side effects and adverse reactions, may lead α4β7-expressing lymphocytes to other organs, predisposing patients to develop EIMs with a parallel course to IBD. Some studies, as post hoc analyses of GEMINI-2,8 have shown that the onset or worsening of joint manifestations is less likely with vedolizumab compared to placebo. Other studies have reported the drug as a cause of arthritis, with patients being 28% more likely to develop such EIMs than those treated with antiTNF-α antagonists.9 In the treatment of those extraintestinal manifestations, both stopping vedolizumab and adding a new drug (e.g. systemic steroids, azathioprine, methotrexate) appear to be effective, and even better results have been reported in patients who switched to ustekinumab.10 As far as concerns the second hypothesis of reactive arthritis, the authors performed an

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extensive screening for infectious disease, and only a concomitant enteric infection with C. jejuni was observed; up to 3% of cases may be followed by Reiter’s syndrome, or by a reactive arthritis. However, this hypothesis was excluded because of the lack of compatible clinical features (reactive arthritis is usually oligoarticular, asymmetric, and most frequently involves the knees, ankles, or wrists), and lack of improvement after antibiotic therapy for C. jejuni infection.11,12

and subsequently in several case reports and case series.16-19 The prevalence of IBD among patients with SAPHO has been estimated to be from 8–13% in two large studies, with higher prevalence in ileocolic CD compared to ulcerative colitis. Typically, SAPHO occurs in young patients with CD who have colonic involvement; however, prevalence of this association might be underestimated among patients with IBD because of clinical similarities with other EIMs, and drug-related side effects.

The last hypothesis the authors have taken into consideration was EIM associated with intestinal flare. A wide range of EIMs can occur in up to 50% of patients with IBD, and their onset may precede the diagnosis of IBD or arise later, as single or multiple manifestations. The most frequent are spondyloarthritis, pauciarticular arthritis (directly associated with intestinal flare), and ankylosing spondylitis, independent of intestinal disease activity. Nevertheless, the patient’s symptoms did not completely fit diagnostic criteria for any of the above-mentioned manifestations. In light of sternoclavicular and temporomandibular joint involvement, the presence of fever and headache, and the absence of axial involvement, SAPHO syndrome was hypothesised.

The literature on SAPHO syndrome consists mainly of case reports, case series, or observational cohorts.20 Treatment, as for seronegative spondyloarthropathies, consists primarily of non-steroidal anti-inflammatory drugs, which, however, are ineffective in some cases. The second step involves diseasemodifying antirheumatic drugs, such as methotrexate, sulfasalazine, and cyclosporin A, but even with these, a significant proportion of patients are unable to achieve remission. In the biologic era, anti-TNFα are considered the first choice treatment in patients with IBD and EIMs; however, more and more evidence is emerging on the efficacy and safety of alternative therapeutic options, such as ustekinumab, vedolizumab, and secukinumab, especially in patients with arthralgia.21,22 This may be of particular importance in SAPHO syndrome, since the IL-23/Th17 axis appears to be implicated in its pathogenesis, and an increase in Th17 cells in peripheral blood has been demonstrated.23 In the authors’ patient, who had a previous drug history of adalimumab failure, infliximab intolerance, and evidence of moderate-to-severe flare, together with severe endoscopic activity while on vedolizumab as a maintenance treatment, the swap to ustekinumab was proposed with sustained clinical remission, both on intestinal and articular manifestations.

Although SAPHO syndrome was initially considered to be a seronegative spondyloarthropathy, recent evidence suggests that it may be better described as primary inflammatory osteitis, within the spectrum of autoinflammatory diseases.13-15 The onset of the disease varies from infancy to late adulthood, with an average age between 30–40 years. The main clinical features are osteitis and hyperostosis, which typically involve the anterior chest wall (in particular sternocostal joints, sternoclavicular joints, and the costoclavicular ligament), followed by the axial skeleton, the long bones of the extremities, the irregular bones (such as mandible), and the peripheral joints. Skin involvement usually appears 1–2 years after skeletal changes, but can also be simultaneous, or arise after more than 20 years, and may or may not be present in patients with these osteoarticular features, especially in association with IBD. The association of SAPHO syndrome with IBD was first reported by Kahn et al.4 in 1992,

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CONCLUS ION This case underlines the importance of joint management between gastroenterologists and rheumatologists for patients with IBD with articular manifestation. In particular, the authors want to highlight the exciting challenge of differential diagnosis in those patients in which the same presentation can hide several

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Case Report

clinical entities, even those as rare as SAPHO syndrome. A correct differential diagnosis guarantees the best clinical outcome, and to follow a therapeutic approach that should always consider the coexistence of intestinal disease

References

Hepatol (N Y). 2020;16(2):75-81.

Felice C et al. Clinical management of rheumatologic conditions cooccurring with inflammatory bowel diseases. Expert Rev Clin Immunol. 2018;14(9):751-9.

Fragoulis GE et al. Inflammatory bowel diseases and spondyloarthropathies: from pathogenesis to treatment. World J Gastroenterol. 2019; 25(18):216276.

and extraintestinal manifestations. In this case, ustekinumab proved to be an effective therapy in patients with CD with SAPHO syndrome, allowing the control of both intestinal and articular manifestations.

Rogler G et al. Extraintestinal manifestations of inflammatory bowel disease: current concepts, treatment, and implications for disease management. Gastroenterology. 2021;161(4):1118-32. Kahn MF et al. [Chronic enterocolopathies and SAPHO syndrome. 8 cases]. Rev Rhum Mal Osteoartic. 1992;59(2):91-4. In French. Naves JE et al. A systematic review of SAPHO syndrome and inflammatory bowel disease association. Dig Dis Sci. 2013;58(8):2138-47. Dubash S et al. Emergence of severe spondyloarthropathyrelated entheseal pathology following successful vedolizumab therapy for inflammatory bowel disease. Rheumatology (Oxford). 2019;58(6):963-8. Diaz LI et al. Vedolizumabinduced de novo extraintestinal manifestations. Gastroenterol

2021;13(9):e18332.

Feagan BG et al. Incidence of arthritis/arthralgia in inflammatory bowel disease with long-term vedolizumab treatment: post hoc analyses of the GEMINI trials. J Crohns Colitis. 2019;13(1):50-7.

16. Siau K, Laversuch CJ. SAPHO syndrome in an adult with ulcerative colitis responsive to intravenous pamidronate: a case report and review of the literature. Rheumatol Int. 2010;30(8):1085-8

Dubinsky MC et al. Extraintestinal manifestations in vedolizumab and anti-TNF-treated patients with inflammatory bowel disease. Inflamm Bowel Dis. 2018;24(9):1876-82.

17. Marrani E et al. SAPHO syndrome in pediatric patients with inflammatory bowel disease treated with infliximab. Dig Liver Dis. 2018;50(11):1249-51.

10. Livne-Margolin M et al. Ustekinumab and vedolizumab for extraintestinal manifestations in inflammatory bowel disease - a retrospective study. Dig Liver Dis. 2023;55(2):223-9. 11. Peterson MC. Clinical aspects of Campylobacter jejuni infections in adults. West J Med. 1994;161(2):148-52. 12. Smith JL. Arthritis, Guillain-Barré syndrome, and other sequelae of campylobacter jejuni enteritis †. J Food Prot. 1995;58(10):1153-70.

18. Sayeed S et al. SAPHO syndrome in an adult male with ulcerative colitis. J Coll Physicians Surg Pak. 2019;29(7):671-3. 19. Amano H et al. Paradoxical SAPHO syndrome observed during antiTNFα therapy for Crohn’s disease. Biologics. 2017;11:65-9. 20. Li C et al. Synovitis, acne, pustulosis, hyperostosis and osteitis syndrome: a single centre study of a cohort of 164 patients. Rheumatology (Oxford). 2016;55(6):1023-30.

13. Rukavina I. SAPHO syndrome: a review. J Child Orthop. 2015;9(1):19-27.

21. Cheng W et al. New insights in the treatment of SAPHO syndrome and medication recommendations. J Inflamm Res. 2022;15:2365-80.

14. Liu S et al. Synovitis, acne, pustulosis, hyperostosis, and osteitis syndrome: review and update. Ther Adv Musculoskelet. 2020;12:1759720X20912865.

22. Wang G et al. Off-label use of secukinumab: a potential therapeutic option for sapho syndrome. J Rheumatol. 2022;49(6):656.

15. Condé K et al. Synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome: clinical and therapeutic aspects. Cureus.

23. Wendling D et al. IL-23/Th17 targeted therapies in SAPHO syndrome. A case series. Joint Bone Spine. 2017;84(6):733-5.

FOR REPRINT QUERIES PLEASE CONTACT: INFO@EMJREVIEWS.COM

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Is Intestinal Cell Death in Necrotising Enterocolitis Assorted and Multifarious? A Special Focus on Risk Factors and Their Pathogenic Mechanisms Authors:

*Sri Harsha Kanuri,1 Newly Bagang,2 Ayse Sena Ulucay,1 Gamine Preet Singh1 1. Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, USA 2. Kasturba Medical College (KMC), Manipal, Karnataka, India *Correspondence to harsha9009@gmail.com

Disclosure:

The authors have disclosed no conflicts of interest.

Received:

29.06.23

Accepted:

24.10.23

Keywords:

Apoptosis, autophagy, inflammation, intestinal cell death, ischaemia, necroptosis, necrosis, necrotising enterocolitis (NEC), pyroptosis.

Citation:

EMJ Gastroenterol. 2023;12[1]:66-85. DOI/10.33590/emjgastroenterol/10303125. https://doi.org/10.33590/emjgastroenterol/10303125.

Abstract Necrotising enterocolitis (NEC) is a serious gastrointestinal clinical disorder primarily affecting preterm newborns. It is characterised by a wider histological spectrum, ranging from mild mucosal injury, microvascular thrombosis, and localised necrosis, to transmural intestinal necrosis. In the most severe form, it can be fatal, with complications such as intestinal perforation, peritonitis, and sepsis. Medical management is not clinically efficacious except in mild and self-limiting cases, as it is only focused on symptomatic treatment. Intestinal cell death is regarded as a crucial nascent cellular event in the pathogenesis of NEC that leads to a leaky intestinal barrier, as well as local and systemic inflammation. Therefore, cellular events that besiege intestinal cell death in NEC should be understood in a meticulous and precise manner. This review provides an extensive overview of the different types of cell death in NEC, including apoptosis, necrosis, autophagy, necroptosis, and pyroptosis. This is critically important as it helps us to comprehend the downstream signalling events that play a vital role in the initiation and progression of disease in NEC. Pertinent research studies performed in this regard would unravel novel molecular targets that could form the basis for drafting innovative therapeutic agents for optimising clinical outcomes in NEC.

Key Points 1. This article focuses on necrotising enterocolitis (NEC); a gastrointestinal disorder primarily affecting preterm newborns. Non-specific clinical presentation and rapid disease progression predispose them to develop local and systemic complications. Although most cases are managed with medical management, approximately 20–40% of cases are referred to surgical therapy. Unfortunately, surgery is associated with 50% mortality and risk of complications (strictures, abscesses, short bowel syndrome, and recurrent NEC).

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2. Mostly localised to the distal small intestine or proximal colon, the pathological findings range from intestinal inflammation to transmural intestinal necrosis. Intestinal cell death is regarded as a crucial nascent cellular event in the pathogenesis of NEC. The different types of cell death in NEC are reviewed in this paper, including apoptosis, necrosis, autophagy, necroptosis, and pyroptosis. It is regarded as a harbinger for a leaky intestinal barrier, intestinal complications, systemic inflammatory response syndrome, and multiorgan failure. 3. Comprehending the underlying critical pathogenic mechanisms that provoke intestinal injury, perturb intestinal homeostasis, as well as augment intestinal permeability in NEC, is a current pressing need, which this article aims to rectify. Necessary research studies implemented in this regard might unravel novel molecular targets that play a crucial role in provoking intestinal cell death in the NEC. This can form the foundation for crafting disease-specific cell-based novel therapeutic interventions for counteracting cell death, halting disease progression, and decreasing clinical mortality in NEC.

INTRODUCTION Necrotising enterocolitis (NEC) is a gastrointestinal disease affecting preterm and very low birth weight infants (birth weight of <1,500 g, and born at <32 weeks gestation), as well as extremely low birth weight infants (birth weight of <1,000 g, and born at <28 weeks gestation).1,2 In the neonatal intensive care unit (NICU), NEC is one of the most common, costly, and critical gastrointestinal emergencies associated with increased morbidity and mortality in preterm infants. A retrospective study evaluating NEC cases from January 2014– January 2019 revealed that the overall survival of NEC infants is around 44.2%, and it is strongly associated with sex (hazard ratio: 3.10; 95% confidence interval [CI]: 1.21–7.93; p=0.018) and NEC staging (hazard ratio: 0.44; 95% CI: 0.22–0.87; p=0.019).3 Upon analysis of live births from the years 1999–2020, infant mortality due to NEC in Black, White, and mixed race infants is approximately 16.1 per 100,000 live births (95% CI: 13.1–19.2), 6.4 per 100,000 live births (95% CI: 5.5–7.4), and 10.2 per 100,000 live births (95% CI: 10.0–10.4), respectively.4 The mortality of NEC is estimated to approximately range from 10–50%.5 In a multicentre cohort study performed between 2005–2017, the mortality rate of NEC was 36.7% in 2005–2008 compared with 26.6% in 2016–2017.6 Nevertheless, in preterm infants presenting with severe complications, such as full-blown tissue necrosis, perforation, and peritonitis, the mortality is almost 100%.5 According to a 2020 report, the overall mortality of NEC is around 23.5%, with lower birth weight

infants (<1,000 g) with surgical NEC experiencing relatively higher mortality (50.9%) compared with non-surgical patients.7 A 15-year outcome study from the UK that examined the NEC cases from years 2000–2015 revealed that the 30-day mortality of NEC is approximately 18.9%, and it was significantly correlated with the gestational age, area of bowel involvement, and presence of pneumoperitoneum.8 According to a recent systematic review and meta-analysis, global incidence of NEC was estimated to be around 6–7%.9 According to a retrospective follow-up study, 6.8% of the premature infants with gestational age 28–32 weeks develop NEC within 1–7 days of newborn life.10 In a Spanish study, which included 25,821 preterm infants (<32 weeks gestation) between January 2005–December 2017, the incidence of non-surgical NEC increased from 4.8% in 2005 to 6% in 2016. On the contrary, the incidence of surgical NEC demonstrated a decreasing trend (3.9% in 2005 versus 2.8% in 2016).6 In another study including low birth weight infants admitted to NICU in Pediatrix Medical Group (Sunrise, Florida, USA) from 1997–2015, non-Hispanic Black and Hispanic infants developed NEC at a higher rate (adjusted odds ratio [aOR]: 1.31; 95% CI: 1.24–1.39; p<0.001) compared with nonHispanic White infants (aOR: 1.30; 95% CI: 1.21– 1.39; p< 0.001).11 In the same study, non-Hispanic Black and Hispanic infants who developed NEC experienced higher mortality (aOR: 1.35; 95% CI: 1.15–1.58; p<0.001), compared with non-Hispanic White infants (aOR: 1.31; 95% CI: 1.09–1.56; p=0.003). The prevalence of NEC is around 1–5 in low birth weight infants.12 Analysis of 24,731 infants (<34 weeks gestational age) from May

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2015–May 2018 in 25 tertiary care hospitals in China indicate incidence of NEC in very low birth weight (<1,500 g) and extremely low birth weight (<1,000 g) infants is 4.8% and 7.6%, respectively.13 Furthermore, the overall casefatality rate and case-fatality of surgical NEC in the previous Chinese study is 9.5% and 13.6%, respectively.13 A retrospective cross-sectional study performed in Ethiopian public hospitals from 25th March– 10th May 2020 indicated that prevalence of NEC was 25.4% (n=89; 95% CI: 21.1–30.0), which is much higher compared with the USA (7.0%).2,11 Higher prevalence of NEC in Ethiopia compared with the USA can be attributed to differences in population demographics, and healthcare access, between them.2 Regardless of recent developments in the obstetric care of high-risk pregnancies, the incidence and prevalence of NEC have gradually increased globally at a steady pace over the past 25 years.2 There are no disease-specific therapeutic interventions that can reverse the clinical progression of NEC after the onset of intestinal inflammation with the pathogen insult. Even after surgical referral, the clinical prognosis of moderate to severe NEC is not very encouraging, as it is associated with many endorgan and systemic complications. NEC accounts for 2.0–7.5% of NICU admissions, and there is some variation between high-income and lowincome countries.1,9,14,15 The average incidence of NEC in the USA is approximately 1.1–3.0 per 1,000 live births, and its prevalence is around 1–5% of the total number of births in the NICU.12,16,17 Racebased analysis performed in upstate New York, USA, revealed that non-Hispanic Black infants had a higher incidence of NEC compared with non-Hispanic White infants (2.2 versus 0.5 per 1,000 live births).18 Multicentre cohort studies reported a higher incidence of mortality in nonHispanic Black and Hispanic infants compared with non-Hispanic White infants (aOR: 1.35; 95% CI: 1.15–1.58; p<0.001 and aOR: 1.31; 95% CI: 1.09–1.56; p=0.003, respectively).11 Additionally, it was reported that non-Hispanic Black infants had higher incidence of first week (aOR: 2.29; 95% CI: 2.21–2.37), neonatal (aOR: 2.23; 95% CI: 2.17-2.30), post-neonatal (aOR: 1.74; 95% CI: 1.68–1.81), and infant mortality (aOR: 2.05; 95% CI: 2.00–2.15) compared with non-Hispanic White infants.19 In the USA, the average cost

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of surgical procedures in NEC, which usually entails peritoneal drainage with or without laparotomy, can range from 300,000–660,000 USD.20 Despite these high costs, the clinical outcomes of surgically corrected NEC are poor, and are associated with many end-organ sequelae. The incidence of complications in advanced NEC includes short bowel syndrome (20–35%), intestinal strictures (12–35%), stoma complications (50%), neurodevelopmental impairment (30–50%), and growth delay (10%).21 It is important to note that most NEC cases occur in infants who are born at 32 weeks gestation, and with a body weight of <1,500 g.1 In clinical cohort studies, the incidence rates of NEC in infants weighing 401–750 g, 751–1,000 g, 1,001–1,250 g, and 1,251–1,500 g is approximately 11.5%, 9.0%, 6.0%, and 4.0%, respectively.16

CLINICAL PRESENTATION RISK FACTORS, PATHOLOGY, AND CURRENT TREATMENT MODALITIES The clinical presentation of NEC usually starts with non-specific symptoms, such as rapid respiration, periodic breathing, lethargy, and irritability.21,22 Later, the preterm infant starts developing abdominal swelling; diarrhoea with bloody stool; constipation; vomiting; bradycardia; hypotension; difficulty in feeding; apnoea; lethargy; and fluctuating heart rate, blood pressure, and body temperature.21,22 In some cases, spillage of intestinal inflammation can cause pathological sequelae in the brain, resulting in neuro-inflammation, serious neurodevelopmental delay, and microcephaly.23,24 X-ray examination of the abdomen might reveal non-specific signs, such as widespread bowel distension, abdominal wall thickening, and fixation of intestinal loops until disease progression leads to intestinal perforation with peritonitis.25 Specific radiological signs diagnosing NEC can include pneumatosis, portal venous gas, pneumoperitoneum, and gasless abdomen.26 A recent study demonstrated that ultrasonography is far better and more advanced than X-ray in managing NEC, because it is more reliable in detecting bowel thickness, vascular perfusion, peristalsis, and intestinal perforation, and is associated with optimal clinical outcomes.27,28 Ultrasonography of the abdomen provides little more information, ranging from

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wall thickness >2.6 mm, abnormal bowel wall echoic pattern, as well as increased vascularity of bowel tissues and mesenteric peri-visceral tissues in earlier stages of NEC.25 In the later stages of NEC, ultrasonography might reveal the presence of microbubbles in a circumferential pattern within one or more loops of the intestine, the presence of air in the main portal vein or intra-hepatic portal venous branches or liver parenchyma, microbubbles appearing between the front surface of the liver and abdominal wall, and a reduction of intestinal wall thickness due to bowel ischaemia.25,29 Some of the alarming signs demonstrated by ultrasonography that necessitate immediate surgical attention include focal fluid collections, complex echogenic ascites, bowel wall thickness less than 1 mm, loss of intestinal peristalsis, increased intestinal wall echogenicity, and loss of intestinal wall perfusion.29 Other clinical signs in NEC include signs of peritonitis, abdominal tenderness, hypotension, hypovolaemic shock, metabolic acidosis, and thrombocytopenia.21 Although no specific aetiological cause has been identified so far, previous studies identified a few risk factors that can potentially make preterm infants susceptible and vulnerable to the disease. Some of the risk factors hypothesised for developing NEC include sepsis, chorioamnionitis, patent ductus arteriosus, indomethacin therapy, glucocorticoid therapy, mechanical ventilation, antenatal cocaine use, perinatal asphyxia, Black race, prolonged course of antibiotic therapy, intrahepatic cholestatic syndrome of pregnancy, meconium aspiration syndrome, congenital heart disease, prolonged rupture of membranes, anaemia, blood transfusions, pregnancy-induced hypertension, hypoalbuminemia, formula feeding, prolonged parenteral feeding, immature intestinal motility, immature intestinal barrier, immature intestinal immunity, and abnormal bacterial colonisation.16,30-34 The underlying hallmark histological features of NEC include intestinal inflammation, enterocyte apoptosis, decreased enterocyte proliferation, mucosal oedema, haemorrhage, transmural necrosis, necroptosis, and distortion of villus and cryptic architecture. These changes are mostly localised to the distal small intestine and proximal colon.16,35,36 In a clinical research study entailing the inspection of the resected intestinal

specimens of infants with NEC who underwent bowel resection over a 10-year period, it was revealed that the depth of bacterial invasion, transmural necrosis, and presence of bacteria in tissue specimens was directly proportional to increased morbidity and mortality in these preterm babies.37 In the most severe form, it has been shown to be associated with various end-organ and systemic complications, such as peritonitis, intestinal rupture, sepsis, and multi-organ failure, resulting in higher mortality in premature infants.16,38 Most of the preterm infants with NEC are managed with medical management, and approximately 20–40% of clinically severe cases are referred for surgical management. The case fatality rate of these surgically managed NEC cases is very high (50%).39 All infants with NEC are initially managed with medical therapy. The main aim of medical management is to restore intestinal homeostasis and facilitate intestinal tissue healing. Medical therapy usually entails administrating interventions, including bowel rest and tissue regeneration, gastric decompression, systemic antibiotics, and parenteral nutrition.40 Initially, mechanical ventilation and tracheal intubation might be necessary to secure the airway.41 Peripheral arterial access should be established so that arterial blood gases and systemic blood pressure can be frequently monitored.41 Signs of hypovolaemia and low blood pressure should be actively looked for so that intravenous fluids can be administered in a timely manner.41 In rare scenarios, hypoalbuminaemia due to capillary leak might necessitate the administration of colloids to combat fluid losses.41 If the infant does not respond to above-mentioned fluid resuscitation, then administrating pressors, such as epinephrine and dopamine, might be required.41 Treatment of bacterial infections associated with NEC usually includes initial delivery of a cocktail of broad-spectrum antibiotics including vancomycin, gentamycin, and clindamycin.40,41 However, due to the recent rise in the development of resistant bacterial species secondary to unrestrained antibiotics usage, their choice should be guided by incidence and prevalence of resistant bacterial cultures in the respective NICU unit.41 As NEC has been shown to be associated with medical complications, such as electrolyte disturbances, acid-base imbalance (metabolic

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acidosis), and coagulopathy, these should be adequately addressed by providing appropriate therapies.40,41 In addition to these therapies, frequent measurements of abdominal girth, X-ray, and abdominal sonographs should be performed regularly to monitor for any onset of local intestinal complications that warrant immediate referral to surgical therapy.41 In a recent retrospective study, it was demonstrated that medical therapy afforded lower mortality rate compared with surgical intervention (15.6% versus 50.0%).42 The infants with NEC who deteriorate in spite of medical therapy, due to rapid disease progression, should be referred for surgical management. The indications for referral to surgical management in NEC include pneumoperitoneum, portal venous gas, ascites, fixed persistent intestinal loop, and rapid clinical deterioration.35 Surgically corrected NEC cases frequently have associated post-operative complications, such as wound dehiscence, wound infections, intra-abdominal abscesses, intestinal strictures, adhesions, cholestasis, short bowel syndrome, and recurrent NEC.16,35,43-45 Therefore, earlier detection, prompt diagnosis, and supportive management is the key to preventing morbidity and mortality in infants with NEC.

CAUSES OF EPITHELIAL CELL DEATH IN NECROTISING ENTEROCOLITIS One of the most important causes of intestinal epithelial cell death is hypoxia.46 Xue et al.47 demonstrated increased levels of hypoxia in the intestinal tissues of ulcerative colitis and Crohn’s disease by immunohistochemistry. Some of the important reasons speculated for occurrence of hypoxia during intestinal inflammation include oedema, vasoconstriction, vasculitis, and increased O2 consumption.46 Due to the abovementioned reasons, intestinal epithelial cells were found to be extremely hypoxic in the mouse models of intestinal inflammation.48 Moreover, bacterial infection of the intestinal epithelial cells also causes hypoxia via inciting vasculitis, as well as by increasing the O2 demand in the surrounding intestinal milieu.49 Fingerprints of hypoxia were widely demonstrated in the colitis intestine by measuring the levels of

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hypoxia-inducible factor, which is regarded as an important hypoxic marker.49 Intestinal inflammation tends to promote overexpression of hypoxia-inducible factor, whose main function is to preserve the integrity of intestinal epithelial barrier, and safeguard against epithelial apoptosis through nuclear transcription of protective genes.46 Hypoxia-inducible factor-1 is directly involved in regulating the ratio of T helper 17 cell/regulatory T cells in the intestinal milieu, and this dysregulation might result in increased production of proinflammatory cytokines, leading to intestinal epithelial cell death.50 Intestinal dysbiosis is defined by the downregulation of commensal bacteria and the upregulation of pathogenic bacteria resulting in the intestinal hyper-inflammatory storm.51 In a few cases, pathogenic bacterial infection might result in the destabilisation of the intestinal epithelial barrier, resulting in increased intestinal permeability, bacterial translocation, and intestinal cell death.51 Lipopolysaccharide (LPS) released from the pathogenic bacteria can act through Toll-like receptor-4 (TLR4) in the intestinal epithelial cells and neighbouring immune cells.52 LPS induced activation of TLR4 results in the secretion of cytokines (IL-6, IL-8, IL-10, and TNF-α), chemokines, and immune cell recruitment, leading to exacerbated inflammatory response and enterocyte cell death.51,52 After enterocyte cell death occurs, disrupted TLR4 signalling is hypothesised to limit tissue healing process by impairing the migration of intestinal stem cells from base of the intestinal crypt to the villus tips, thereby delaying the replacement of dead epithelial cells with fresh ones.53 Previous studies also demonstrated that increased TLR4 activation in the endothelial cells of the small intestine can impair intestinal microcirculation by downregulating nitric oxide synthase production, resulting in intestinal ischaemia and enterocyte death.54 Although neutrophil recruitment is regarded as an important first-line defence mechanism following intestinal epithelial injury in NEC, excessive neutrophil accumulation as well as their defective removal can be detrimental by contributing to intestinal epithelial cell death via release of reactive oxygen species (ROS), oxidative

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stress, and further release of cytokines.55 Furthermore, alteration of balance between cytotoxic (T helper 17 cell) and regulatory T cells in the intestinal milieu is postulated to be one of the most important predisposing factors for hyper-inflammatory state in the NEC intestinal tissues.56 Proinflammatory cytokines secreted by cytotoxic T cells, mainly TNF-α and interferon (IFN)-γ are the most common factors implicated in the disruption of tight junctions between the epithelial cells by altering the lipid composition, as well as by swapping the fatty acyl moieties in the phospholipids of tight junctions.57 In few instances, IL-17 secreted by cytotoxic T cells is also capable to instigate disrupted epithelial tight junctions, excess epithelial cell death, and attenuated epithelial proliferation.58 Using colon biopsy samples from patients with inflammatory bowel disease, it was recently demonstrated that IFN-γ and TNF-α were implicated in tight junction openings, resulting in intraepithelial seepage of pathogenic bacteria.59 Some previous research studies tried to investigate and identify the pathological mechanisms that underlie intestinal epithelial cell death in preterm infants with NEC. Excess ROS production, oxidative stress, and inflammation are some of the well documented mechanisms for intestinal cell death in premature infants with NEC.60,61 TNF-α, which is primarily produced by LPS stimulation, has been implicated in the intestinal epithelial cell death via the downregulation of tight junction proteins, increasing autophagy and suppression of proliferation.51 Platelet-activating factor (PAF), hypoxia-reoxygenation, ischaemia-reperfusion, peroxynitrite, bacterial invasion, and endotoxin are some other additional mechanisms proposed for the execution of intestinal cell apoptosis.3 Interestingly, the downregulation of epithelial growth factor in the serum and saliva in infants also predisposes to intestinal epithelial cell death in NEC through compromised mucosal barrier function in the intestine.62,63 Overexpression of β-arrestin-2 (cytosolic protein) can cause apoptosis of intestinal epithelial cells by pathological endoplasmic reticulum stress, which promotes the release of pro-apoptotic molecule BCL-2 interacting killer (BIK) and the activation of caspase-mediated signalling mechanism.64 The various mechanisms of intestinal cell death that occur in NEC are summarised in the next section, where each type of cell death and their relevance

to the pathogenesis of the disease process are discussed.

TYPES OF CELL DEATH IN NECROTISING ENTEROCOLITIS The different types of cell death that can occur in NEC can be broadly classified into five categories: apoptosis, necrosis, necroptosis, autophagy, and pyroptosis (Figure 1).

Apoptosis

Apoptosis is one of the most important types of intestinal cell death seen in NEC tissues. Apoptotic cell death in NEC can occur in three pathways, namely the death receptor pathway (extrinsic), mitochondrial pathway (intrinsic), and perforin/granzyme pathway (T cell-mediated) pathways. All these pathways converge over the final execution pathway (caspase-3 cleavage), ultimately leading to apoptotic type of intestinal cell death in NEC. The sequelae of intestinal apoptosis in NEC can range from DNA fragmentation, degradation of cytoskeletal proteins, and cross-linking of proteins, to formation of apoptotic bodies.65 One of the most common sites for occurrence of apoptotic type cell death in NEC is the small intestine (ileum).66 Bacterial invasion and proliferation is the cardinal reason for apoptotic type of cell death in NEC.5 The intestinal micro-environment gets exposed to other pathological stimuli once bacterial infection of intestinal epithelial cells ensues. Some of these inflammatory stimuli that are implicated in the apoptotic cell death in NEC include TNF-α, LPS, ischaemia, and ischaemia-reperfusion injury.67 Accumulation of TNF-α in the intestinal cells provokes apoptotic type of cell death via Jun N-terminal kinases/p38 (mitogen-activated protein kinase [MAPK] pathway), as well as through production of ROS.68 On top of that, poor blood flow to the intestines with resultant ischaemia and immature intestinal epithelial barrier in preterm babies also increases the propensity of intestinal epithelium to undergo apoptotic cell death.69 Under the influence of these pathological stimuli, recovery and regeneration following intestinal desecration is derailed. As a result, there is excessive pathological shedding of intestinal epithelial cells

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Figure 1: Illustration of different types of intestinal cell death. Hypoxia inflammation free radicals

LPS TWF TLR4

TNFR

Cell membrane

Necroptosis

RIPK1 Necrosome

Autophasic cell death

Necrosis

Apoptosis

Autophasic lysosome

Death receptor pathway

Granzyme pathway

Pyroptosis

Caspase-1 ASC NLRP3

Death Innate/adaptive immunity

MLKL

Apoptotic bodies DNA cleavage Cytoskeletal cleavage Protein crosslinking

Cytokinesis Chemokinesis DAMPs Na+/K+channels

IL-1B IL-18

Activation of TLR4 and TNFR could produce necroptosis by formation of necrosome by involving the RIPK1 and MLKL. Intestinal cells can also undergo autophagic cell death due to formation of lysosome. The necrosis and apoptotic cell death is followed by death receptor pathway and granzyme formation inducing DNA cleavage. In another pathway, caspase-1 release and NLRP3 activation produces pyroptosis. ASC: apoptosis-associated speck-like protein containing a CARD domain; DAMP: damage-associated molecular pattern; K⁺: potassium ions; LPS: lipopolysaccharide; MLKL: mixed lineage kinase domain-like protein; Na+: sodium ions; NLRP3: nucleotide binding oligomerisation domain-like receptor family pyrin domain containing 3; RIPK1: receptor-interacting protein kinase 1; TLR4: Toll-like receptor 4; TNFR: TNF receptor.

leading to mechanical defect in the intestinal epithelial barrier. This will, ultimately, pave the way for superfluous bacterial invasion, massive ingress of inflammatory cytokines, extensive gut inflammation, systemic complications, and sepsis.67,70,71 In a rat model of NEC, unhindered and rapid progressive apoptosis in the intestinal epithelial cells can be a harbinger for kickstarting ensuing gross bowel necrosis of the intestinal tissues.3 In NEC, apoptosis can be considered as a defence mechanism to curb the local bacterial spread as well as to eliminate the infection.

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This is accomplished by sequestering and destroying the infected intestinal epithelial cells through activation of host defence mechanisms during apoptosis.72 This is important as earlier bacterial eradication will speed up intestinal repair, regeneration, and tissue homeostasis. The process of apoptosis usually occurs in acute and chronic stages. That being said, apoptosis in acute stage is protective by limiting tissue spread, whereas in the chronic stage it metamorphoses into a counterproductive process by amplifying the proinflammatory environment. So, chronic apoptosis can be considered as a harbinger for accelerated

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disease progression and tissue complications, thus leading to an increase in mortality and morbidity in NEC.

cytokines such as TNF-α. TNF-α usually acts through TNF receptor 1 and results in formation of complex 1 (TNF receptor type 1-associated death domain) and receptor-interacting protein kinase 1 (RIPK1).74,75 This ultimately leads to increased activation of nuclear factor NF-κ-B p105-mediated survival pathways, including proinflammatory cytokine secretion, resulting in the elimination of infected IECs from the body.72,76 Increased secretion of proinflammatory cytokines contributes to accumulation of peroxy-nitrate, thus increasing the risk of apoptotic cell death in NEC.77 LPS-TLR4 interaction within the intestinal epithelium and crypts results in increased apoptosis through endoplasmic reticulum (ER) stress-mediated by protein kinase-related extracellular signal-regulated kinases [PKR]like ER kinase, C/EBP homologous protein, and myeloid differentiation primary response gene 88 signalling molecules.78

The process of acute apoptosis in NEC commences with bacterial proteins interacting with receptors expressed on the epithelial cell membrane (Figure 2). Bacterial LPS acts on the TLR4 receptors on the intestinal epithelium, leading to decreased proliferation and increased apoptosis of crypts and villi through involvement of p53 upregulated mediator of apoptosis.73 Upon bacterial infection, intestinal epithelial cells (IEC) recognise the pathogen-associated molecular patterns (PAMP) and damageassociated molecular patterns (DAMP) through Toll-like receptors (TLR), nucleotide binding oligomerisation domain (NOD)-like receptors (NLR), and upregulated proinflammatory

Figure 2: Representation of acute and chronic necrotising enterocolitis.

Bacterial invasion

Bacterial DAMP NLR

Pathological epithelial shedding

Steam cells

PAMP

TLR

LPS TNF

TNF

Gut inflammation sepsis FasL, TRAD

Steam cells

TNFR

TRAD–RIPK1

Acute

NF-Kβ

Complex II Initiator caspase-8

Executioner caspase-3, 7, 8, 9 NF-Kβ

ProInflammatory cytokines Apoptosis

Chronic

Apoptotic dying cells

Decreased proliferation

Impaired epithelial barrier

Susceptibility to Infections

IEC elimination NEC

In the acute phase TLR, NLR activation enhances NF-κ-B transcription that damage IEC. Whereas in the chronic phase gut inflammation activates TNFR Complex II, which results in release of initiation and executioner caspases leading to apoptosis, decreased proliferation, intestinal epithelial barrier damage, and NEC. DAMP: damage-associated molecular pattern; FasL: Fas ligand; IEC: intestinal epithelial cells; LPS: lipopolysaccharide; PAMP: pathogen-associated molecular pattern; NEC: necrotising enterocolitis; NLR: nucleotide binding oligomerisation domain-like receptor; RIPK1: receptor-interacting protein kinase 1; TLR: Toll-like receptor; TNFR: TNF receptor; TRAD: TNFR1-associated death domain protein.

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Failure to eliminate infection due to the persistence of microbial insults leads to progression to a chronic stage. In the chronic stage, apoptosis is no longer protective but rather counterproductive, and leads to pathological consequences and disease progression (Figure 2). In the chronic stage, IECs upregulate ligands, such as Fas ligand and TNFrelated apoptosis-inducing ligand, which can act via TNF receptor 1, and leads to complex II formation (Figure 2).72,79 This leads to stimulation of initiator caspases (caspase-8) and executioner caspases (caspase-3, 6, 7, and 9), ultimately resulting in the activation of nuclear factor NFκ-B p100 pro-apoptotic pathways and inhibition of survival pathways (Figure 2).72,79-81 The end target effects of these downstream signalling events in the IECs include increased apoptosis; decreased proliferation of intestinal epithelial cells; shattered intestinal epithelial barrier; and susceptibility to several inflammatory conditions, including necrotising enterocolitis (Figure 2).72 Few studies were previously performed demonstrating the presence of intestinal apoptosis in the animal models of NEC, and by blocking apoptosis they were able to show improved clinical outcomes in these disease models. Intestinal tissues of neonatal rats exposed to formula feeding and cold/hypoxia stress had imprints of apoptosis, namely increased expression of caspase-3 and DNA fragmentation in the initial stages before progressing to transmural epithelial necrosis in the rat model of NEC.3 The LPS/TLR4/NF-κ-B signalling pathway-mediated proinflammatory cytokine secretion and mitochondrialcaspase pathway are primarily responsible for intestinal epithelial apoptosis in the rat model of necrotising enterocolitis.82 ER stressinduced activation of β-arrestin-2 causes polyubiquitination of binding Ig protein, and release of pro-apoptotic molecule Bcl-2 interacting killer, into the cytosol, resulting in caspase-mediated intestinal epithelial apoptosis in the animal, cell culture, and human models of NEC.83 Administering milk polar lipids decreased the intestinal apoptosis in NEC through downregulation of Bax, caspase-3 and 9 expression, as well as upregulation of antiapoptotic Bcl-2 expression.82 Administering porcine milk exosome microRNAs resulted in decreased intestinal apoptosis and inflammation

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via downregulation of TLR4 pathway as well as p53 pathway in the NEC model.84 Similarly, microRNA-23a and miR-339 have been shown to decrease LPS-induced intestinal epithelial apoptosis due to their influence on TLR4 and Rho kinase inhibitor 1– sirtuin 1–NF-κ-B signalling pathways, respectively.85,86 Probiotic bacteria Lactobacillus acidophilus has been shown to attenuate intestinal epithelial apoptosis as well as cytokine production (IL-6) in the animal model and (LPS and TNF) cell culture model of NEC.87 Furthermore, another probiotic bacteria (Bifidobacterium bifidum) has been shown to attenuate intestinal epithelial apoptosis through a COX-dependent mechanism, thereby preserving intestinal mucosal integrity in the animal and cell culture models of NEC.88 Erythropoetin and epidermal growth factor have been known to have therapeutic efficacy in the animal model of NEC through reduction of intestinal epithelial apoptosis via alteration of MAPK/ERK pathway and Bax-to-Bcl-2 ratio respectively.66,89

Necrosis

Once the intestinal epithelial cells undergo apoptotic cell death, they should be cleared in a timely fashion by macrophages by a physiological process known as efferocytosis, otherwise they proceed to undergo a more devastating necrotic type of cell death.90,91 Some of the predisposing factors implicated in necrotic cell death include infectious agents, ischaemia, inflammation, and toxins.92,93 Infections are usually associated with liquefactive necrosis, which is characterised by slimy and liquefactive appearance due to the digestion of intracellular debris in dying intestinal epithelial cells by bacterial enzymes (proteases, DNases, and lysosomal enzymes).92 The morphological features of necrosis can range from cellular swelling, extensive vacuole formation, rupture of plasma membrane, and cell lysis, to nuclear and chromatin condensation in the later stages.94,95 The initial trigger for necrosis is disruption of the intestinal barrier, which recruits mucosal T-lymphocytes, and is responsible for causing upregulation of inflammatory mediators, such as TNF-α at the site of mucosal injury.94 Recruited TNF-α acts on the intestinal epithelial cells via TNF receptors to induce the expression of ROS production, which acts as a secondary messenger to incite various cellular derangements ranging from DNA damage and proinflammatory signalling

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pathways to membrane damage in the presence of pathological stimuli and stressors.94 Since necrosis is associated with cellular rupture, it can cause the dumping of cellular debris into the extracellular microenvironment with the inherent risk of developing secondary systemic inflammatory response syndrome.93 NEC is the most common cause of intestinal necrosis in premature neonates, and is associated with infection-related intestinal ischaemia.96 It is important to understand that bacterial invasion of the intestinal epithelium is one of the most important primary events for the occurrence of necrotic type of cell death in NEC. Hypoxia, anaerobic metabolism, swelling, rupture of IECs, and breaching the intestinal epithelial barrier are the initial events that can accentuate bacterial infiltration into the intestinal mucosa in NEC.97 Bacterial invasion of the intestinal wall can result in proinflammatory mediator secretion, as well as activation of TLR4 receptors in the mucosal microvasculature through nitrate-nitrite signalling, resulting in intestinal ischaemia and transmural necrosis.54,97 One of the most promising screening tools for monitoring the extent of transmural necrosis in the small intestine in patients with NEC is Doppler ultrasonography.98 Efforts need to be made to identify the NEC in earlier stages, before the occurrence of intestinal transmural coagulative necrosis, since its occurrence heralds the onset of severe complications, such as perforation, bacterial overgrowth, pneumatosis intestinalis, inflammation, and peritonitis.99 The prognosis of patients with above-mentioned complications is very poor and mostly necessitates surgical interventions with associated morbidity and mortality. Intestinal epithelial apoptosis is the initial cellular event that slowly progresses to morphological transmural necrosis of the small intestine in neonatal rats subjected to formula feeding and asphyxia stress in the experimental model of NEC.3 Previous studies have demonstrated that there is no positive relationship between mortality of surgical NEC cases and severity of histological necrosis evident in the small intestine, although upregulation of immunological markers, such as cleaved caspase-3 and vascular cell adhesion protein, has been associated with poor survival.100 In

another multicentre study, Heida et al.101 had demonstrated that intestinal fatty acid binding protein levels in the plasma and urine directly correlate with the extent of surgical resection in the NEC, and it has the potential to become a reliable serum biomarker for monitoring transmural necrosis of the small intestine in later stages of NEC. Apart from bacteria and inflammatory cytokines, another important mediator that can cause bowel necrosis in NEC is platelet activating factor (PAF).102-104 A previous study reported that a combination of LPS with PAF administered intra-aortically is sufficient to induce ischaemic intestinal necrosis in rats with lesions exhibiting morphological similarity to the tell-tale signs of NEC.102 Furthermore, the Sun et al.104 study revealed that TNF-α-induced bowel necrosis is mediated by PAF as it was attenuated by administration of PAF inhibitor. In the same study, it was also demonstrated that additive synergism between LPS, TNF-α, and PAF is necessary for the occurrence of intestinal necrosis, and this was associated with hypovolaemic shock in rats treated with these toxins.104 The underlying mechanism through which PAF can induce bowel necrosis in NEC is via activation of the lipoxygenase pathway of arachnoid metabolism and upregulation of leukotrienes.103

Necroptosis

Necroptosis is a programmed type of cell death that has the features of apoptosis and necrosis, and occurs with inflammatory conditions.105 Receptor interacting protein kinase (RIPK) is a protein kinase that also fulfils a physiological role of protecting IECs from TNF induced caspase-8 apoptosis, and thereby maintaining intestinal homeostasis.106 Caspase-8 inhibits the RIPK1 and RIPK3 in the physiological conditions and prevents their interaction (Figure 3). Classical necroptosis is usually initiated by formation of complex of RIPK1 and RIPK3 when caspase-8 is inhibited, resulting in the formation of necrosome complex (Figure 3).107,108 Once the necrosome complex forms, it activates mixed lineage kinase domain-like protein (MLKL), resulting in its phosphorylation and oligomerisation. MLKL is regarded as a chief executioner of necroptosis type of cell death (Figure 3).107,108 Activated MLKL migrates to the plasma membrane and executes necroptosis by pore formation, thereby secreting

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cytokines, chemokines, and DAMPs, as well as engaging sodium and potassium channels (Figure 3).107

The morphological features of necroptosis type of cell death include cellular swelling, membrane rupture, the release of DAMPs, and tissue inflammation.108 During bacterial infections, LPS induced activation of TLR4 receptors in the mouse macrophages initiates the formation of a complex between RIPK3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-β (TRIF), resulting in the occurrence of non-classical necroptosis.109,110 Salmonella enterica typhimurium-induced interferon type I expression in the macrophages leads to RIPK1-RIPK3 induced necroptosis, resulting in macrophage depletion and evading of innate immune response.111 Alternatively, the LPS/ TLR4/TRIF/receptor-interacting protein kinase-3 (RIPK3) axis in the macrophages can potentially induce the formation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and caspase-1 activation through alternate pathways, resulting in the production of proinflammatory cytokines, such as IL-1β and IL-18 for the amplification of inflammatory response.112 According to Sinclair et al.,113 the development dependent enterocyte metabolism might predispose to the development of necroptosis induced intestinal injury in the NEC.113 Upregulation of necroptosis proteins (receptorinteracting protein kinase-1 [RIPK1], RIPK3, and MLKL) and activation of necroptosis occurred in Toll-like receptor dependent manner in the differentiated intestinal epithelium of immature ileum with LPS treatment of wild-type mice, as well as in a novel NEC-in-a-dish cell culture system.114 Breast milk and pharmacological inhibition of necroptosis resulted in decreased epithelial cell death and mucosal injury in the mouse model of NEC.114 In consensus with this study, Liu et al.115 demonstrated that necroptosis was induced in the patients with NEC and cell lines through the TLR4/TRIF/RIKI/MLKL axis, as well as TNF-α/TNFR/RIKI/MLKL axis. With necroptosis proven to be involved in the pathogenesis of NEC models, efforts to inhibit necroptosis yielded encouraging results in the cell culture and animal models of NEC.

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Necrostatin-1 (receptor-interacting protein kinase 1 antagonist) treatment resulted in decreased necroptosis protein expression, decreased intestinal cell death, and preserved intestinal barrier in the mice model of NEC.115 In another study, LPS challenged weaning piglets treated with medium chain triglyceride diet displayed decreased expression of necroptosis proteins (receptor-interacting protein kinase 1 and MLKL) as well as TLR4 and nucleotide binding oligomerisation domain proteins, leading to enhanced intestinal barrier function, disaccharidase activities, and better intestinal morphology.116 MiR-143-3p was downregulated in the serum of patients with NEC compared with healthy controls, and its depletion promoted RIKI induced necroptosis in the LPS treated caco-2 cells.117 In the in vitro studies, miR-1433p supplementation resulted in the decreased expression of necroptosis proteins (RIKI and MLKL), attenuated the necroptosis, and associated inflammation in the LPS-treated caco2 intestinal epithelial cells.117

Autophagy

Autophagy (eating of self) is a self-destructive process of removal of damaged organelles, misfolded proteins, and intracellular organisms.118 There are three main types of autophagy, namely, macro-autophagy, micro-autophagy, and chaperone-mediated autophagy.118,119 The stages in autophagy include initiation, nucleation, elongation, maturation, lysosomal fusion, and degration.119 The core machinery required for the autophagic process is summarised as Initiation (ULK1 complex [FIP200, ATGB, ATG10]), nucleation (Class III phosphatidylinositol 3-kinase complex [BECWI, AMBRA, ATG14L, UPS15, UPS34]), elongation (WIPI proteins), maturation (E3 complex [ATG5-ATG12-ATG164]), lysosomal fusion (phosphatidylethanolamine [PE]), and degradation (LC3).119,120 In normal homeostatic conditions, the autophagic process is involved in the regulation of numerous physiological functions in the human intestine, including preserving barrier integrity, prevention of bacterial dissemination, hostmicrobial interaction, goblet cells mediated mucin secretion, macrophage-induced pathogen clearance, dendritic cell-mediated antigen presentation, effective and memory T cell development, and secondary antibody

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response.121 IECs infected with vibrio cholera secreting pore-forming toxin (cytolysin) responded initially with increased Microtubuleassociated proteins 1A/1B light chain 3B (LC3B-II) autophagic vacuole formation to neutralise the pathogen spread.122 Next, when autophagy is inhibited in the IECs infected with vibrio cholera, they demonstrated decreased survival, which supports the presumption that autophagy is a cellular defence mechanism to localise, subdue, and destroy the intracellular bacteria.122 In another research study, as bacteria such as S. typhimurium enters the cells through entry vacuoles, they are immediately ubiquitinated, and coated with p62 so that destruction of bacteria can proceed by cellular autophagy apparatus to restrict their rapid replication and limit cell-tocell spread.123 It is important to keep in mind that the process of autophagy not only serves to destroy the pathogen but also promotes the initiation of immune response for its destabilisation and impairment of its multiplication, and resultant cellular spread. Degradation of bacterial/viral components by autophagic process drives the activation of innate and adaptive immunity, resulting in the IFN-γ production and major histocompatibility complex Class II-mediated CD4+ T cell response, respectively.124 But, some microbial agents cleverly have intrinsic mechanisms set in place that tend to cripple the process of autophagy, and thereby escape the host immunity by impaired trafficking into the autophagolysosome and destabilising the autophagy proteins.124 For example, Shigella flexneri secretes IcsB protein due to which it escapes from binding to the autophagy apparatus (autophagy related [Atg] 5), leading to successful evading of autophagy process, and this results in its eventual multiplication and enhanced cellular spread.125 Understanding the shifts in autophagy regulation in the different stages of pregnancy is essential as it can have direct influence on the occurrence of infection in the maternal-fetal unit. It has been demonstrated that expression of autophagy marker LC3B-II in the placenta decreases during first trimester and increases during term pregnancy with TNF-α treatment.126 This fluctuation of LC3B-II during different stages of pregnancy mediated by inflammatory cytokines indicates that dysregulation of autophagy can

have potential implications on the regulation of infection and apoptosis in the maternal-fetal unit.126 Autophagy is deemed to be a protective mechanism against intestinal inflammation; however, it can be counterproductive in some instances depending upon on the presence of toxins and cytokines in the intestinal milieu. In preterm infants, increased autophagic protein gene expression and TLR4 signalling can kickstart the autophagy process that can secondarily impair intestinal epithelial migration through inactivation of Rho-GTPase, a predisposing factor that can ultimately lead to NEC.127 It is quite plausible that an increased autophagic process tends to be protective at least in the earlier phases of NEC intestinal injury, by limiting the progression of the disease.128 Electron microscopy evaluation of intestinal epithelial cells revealed that there is upregulated expression of autophagy proteins (light chain 3 and beclin), increased formation of autophagic vacuoles, as well as degradation of p62 in the ileum of neonatal rats with NEC.128 During pathogen-mediated intestinal inflammation, signalling pathways that tend to inactivate the autophagic process favour disease progression in NEC (Figure 3). Studies have shown that LPS mediates an increase in the ROS, including peroxy-nitrate production, which can lead to epithelial injury and intestinal damage in the NEC disease models.129 The increase in ROS production in the intestinal tissues also evokes inactivation of autophagy marker Atg4 and lipidation of Atg8, which in combination cripples the autophagy process in the intestinal epithelial cells. It is important to keep in mind that the decreased autophagy process in the IECs is a liability factor for development of NEC, as disease progression moves forward unchecked without this important protective mechanism.130 But in the later stages of the inflammatory disease process, the presence of continuous stressors and chronic tissue injury becomes detrimental as they convert the protective autophagic process into a destructive mechanism that is incapable of protecting against disease progression. In other words, autophagy tends to be dysregulated, counterproductive, and becomes less beneficial in controlling the infection and therefore accentuates the mucosal tissue injury in the NEC.128

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Figure 3: Various pathological events in necroptosis process. Necroptosis

Blocked

Caspase-8 (–) RIP K1

(–) RIP K3 Necrosome

TRIF Oligomerisation Phosphorylation

TLR4 LPS Dependent

MLKL Migration to plasma membrane

• Cytokines • Chemokines • DAMPs

• Na+ channels • K+ channels

Perforation

Membrance rupture

Cellular swelling DAMP release

IEC

Activation of TLR4 induces necrosome formation due to increase in the RIPK1/3 and TRIF signalling, leading to the caspase-8 inhibition. The necrosome formation causes MLKL phosphorylation and migration to the plasma membrane. These pathogenic responses induce cytokines, chemokines, and DAMPs release, which can activate Na+ and K+ channels and increase membrane permeability, rupturing, and cell swelling. DAMP: damage-associated molecular pattern; IEC: intestinal epithelial cell; K+: potassium ions; LPS: lipopolysaccharide; MLKL: mixed lineage kinase domain-like protein; Na+: sodium ions; RIPK1: receptor-interacting protein kinase 1; RIPK3: receptor-interacting protein kinase 3; TLR4: Toll-like receptor 4; TRIF: IR-domain-containing adapter-inducing interferon-β.

Accordingly, controlling the dysregulated autophagy was used as a therapeutic strategy in regulating apoptosis and reducing tissue injury in the NEC models. In a recent study, TNF-α treatment resulted in decreased proliferation and increased apoptosis in the IECs (IEC-6 cells) through autophagy induction (increased LC3II/I ratio, beclin induction, and increased autophagy vacuoles), and use of autophagy inhibitor (wortmannin) reversed these effects, emphasising the importance of autophagy in regulation of apoptosis in the in vitro NEC models.131 In the rat model of NEC, it was revealed that autophagy preceded apoptosis and administration of erythropoietin resulted in suppressing autophagy markers (beclin 1 and LC3II), as well of apoptosis through

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phosphoinositide 3 kinase (PI3K)/Akt/ mammalian (or mechanistic) target of rapamycin signalling pathway and the MAPK/ERK pathway, respectively.89 By controlling excessive autophagic activity in enterocytes, human β-defensin-3 (cationic host defence peptide) is able to promote epithelial migration, attenuate mucosal inflammation, maintain mucosal integrity, and thereby offer protection against mucosal injury in the neonatal rat model of NEC.132 Likewise, supplementation of epidermal growth factor was beneficial in attenuating the intestinal injury in NEC via downregulating autophagy in the in vitro and in vivo models of NEC.128

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BACTERIAL INFECTION AND INNATE IMMUNE RECEPTOR RECOGNITION Bacterial infection of IECs leads to eliciting of immune response via downstream signalling of pathogen recognition receptors (PRR).133 There different kinds of PRRs include TLRs, NLRs, retinoic acid-inducible gene-I-like receptors , C-type lectin receptors, and AIM2like receptors.133 Lipoproteins and proteins of bacteria are recognised by TLR receptors (1, 2, 4, 5, 6, and 10), whereas nucleic acid of bacteria was identified by TLR receptors (3, 7, 8, and 9), expressed on the surface of IECs.134 These will lead to stimulation of TRIF3 (Toll/IL-1R domain containing adaptor protein inducing IFN-β) and myeloid differentiation primary response gene 88 pathways leading to activation of NF-κ-B, IFN regulatory factor 3, and MAPK (mitogenactivated protein kinase) mediated downstream signalling events.133,135 This will eventually result in nuclear transcription of proinflammatory pathways, and elicit a robust immune response against the pathogen.133,135 Sometimes, bacteria craft mechanisms to escape binding to these surface PRRs and undergo phagocytosis to enter IECs. Once inside, they will be able to bind to intracellular PRRs, such as NLRs. These intracellular NLRs have ligand recognition receptors at the C-terminal domain, central NACHT domain (a domain coined due to the presence of four NLR members, namely, NAIP, CIITA, HETE, and TP1) and caspase activation and recruitment domain or pyrin domain at the C-terminal domain.133,136-138 In physiological conditions, these NLRs are in inactive state due to their inherent U-shaped configuration, where the NACHT domain is masked and prevented from undergoing oligomerisation. As soon as bacteria are broken down by intracellular lysosomal proteases, PAMPs are released and bind to LRR. Binding of PAMPs to LRR results in the unmasking of the NACHT domain, and this leads to its subsequent oligomerisation and activation. Gram-positive and gram-negative peptidoglycans can activate NOD-containing protein 1 (and NODcontaining protein 2 receptors, leading to the stimulation of NF-κB pathways, which can be a starting point for activation of NOD leucine rich repeat pyrin domain containing protein 3 (NLRP3) inflammasome .135 Activation of NLRP3 inflammasome intracellularly promotes

innate immune response by secretion of proinflammatory cytokines, and provokes a specific type of cell death called pyroptosis, which enables clearance of pathogen infected intestinal epithelial cell.139,140 It is interesting to know that some pathogens can prevent NLRP3 activation and occurrence of pyroptosis by devising various mechanisms, such as masking of ligand expression, structural alteration of ligands limiting the detection of NLRs, and direct inhibition of inflammasome.140

Pyroptosis

Bacterial infection of IECs can release DAMPs and PAMPs, which can act on NLRs.93,141 The activation of NLRP3 inflammasome occurs in a two-phased manner: NF-κ-B meditated transcription of inflammasome components in Phase I, as well as assembly and activation of NLRP3 inflammasome in Phase II.142 Activation of NLR on IECs can induce the formation of inflammasome complex comprising of apoptosisassociated speck-like protein containing a caspase activation and recruitment domain, NLRP3, and capase-1 resulting in secretion of proinflammatory cytokines, such as IL-1β, and IL-18 in the classic canonical pathway.141,143 It is important to note that IECs secrete relatively less IL-1β secretion in contrast to lamina propria macrophages, which are the main source of IL-1β during intestinal inflammation.144 Alternatively, LPS released from gram-negative bacteria can cause activation of caspase-11, resulting in cytoplasmic swelling and release of DAMPs in the non-canonical pathway.93,142 Both canonical and non-canonical pathway result in pyroptosis, a special from of cell death specific to NLRP3 inflammasome activation.93,142,143 An important distinguishing feature of the non-canonical pathway is that it can directly induce pyroptosis by stimulating pore forming protein gasdermin D, and reinforce the activation of the classical canonical pathway, thereby potentiating the secretion of pro-inflammatory cytokines.145 The main physiological function of pyroptosis is to disrupt microbial replication and make them susceptible for phagocytosis by immune cells.140 The important morphological features of pyroptosis cell death include cellular swelling, osmotic lysis, exocytosis of inflammatory intracellular contents, DNA cleavage,

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nuclear condensation, and disruption of actin cytoskeleton.143 The benefits of inflammasome activation in the IECs can range from proinflammatory cytokine secretion, enhanced lipid production (membrane repair), preventing autophagic cell death, recruitment of macrophages, activation of B and T lymphocytes, stimulation of dendritic cells, and wound healing, to enhanced mucosal integrity.143,144 NLRP3 inflammasome activation can be regarded as an important intestinal immune defence mechanism as it inhibits bacterial growth, promotes cellular repair, and enhances cellular survival in the earlier stages of infection.143 However, in the later stages of infection (14–21 days post infection), cytosolic NLR Type 4 (NLRC4) has been implicated in the execution of an innate immune defence mechanism against Citrobacter rodentium by attenuating bacterial colonisation and associated intestinal inflammation.146 NLRC4 inflammasome is activated by ROS, lysosomal disruption, and calcium signalling, whereas the bacterial flagellins and Type III secretion system are capable of activating NLRP4 inflammasome.147 During physiological conditions, NLRP4 complexes with beclin-1 to negatively regulate the autophagy process.148 On the contrary, during bacterial infections, it dissociates from beclin-1 to migrate towards the phagosomes containing Streptococcus A, thereby promoting beclin-1-mediated autophagic degradation of bacterium.148 In the different phases of bacterial infection, IECs activate different types of inflammasomes as a part of an innate defence mechanism to neutralise the microbial threat.146 IECs initially sample the level of bacterial load and decide to move forward with inflammasome-mediated pyroptosis, if they sense that bacterial threat is high enough to warrant the activation of the immune system for limiting the intestinal damage.142 Suppression of NLRP3 inflammasome had offered clinical benefit in ulcerative colitis and mucosal colitis, underscoring its importance in the pathogenesis of inflammatory bowel diseases.149,150 Cronobacter sakazakii causes TLR4/NF-κ-B-mediated NLRP3 inflammasome activation, leading to IL-1β secretion and pyroptosis in the in vitro and rat models of NEC.151 According to Hu et al.152 there is increased expression of IL-1β, IL-18 (mRNA and protein),

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and NLRP3 (mRNA) in the NEC group of rats treated with hypoxia and cold stimulation, compared with the control rats, emphasising the importance of NLRP3 inflammasome and pyroptosis in the disease pathogenesis.152 NLRP3 inflammasome has been shown to be activated in the intestine, and brain NEC models, and its blockade with inhibitor MCC950 resulted in the substantial reduction in intestinal and brain inflammation, pathological severity, as well as long term neurological sequelae.153 In the LPStreated IECs, there was increased expression of high-mobility group protein 1 (HMGB1), NLRP3, TLR4, NF-κ-B, caspase-1, IL-1β, and TNF-α, as well as knockdown of HMGB1, resulting in the attenuation of these markers, highlighting the role of HMGB1 in inducing TLR4-induced activation of NLRP3 inflammasome.154 In line with these findings, increased expression of HMGB1, NLRP3, TLR4, NF-κ-B, caspase-1, IL1β, and TNF-α noted in the rat model of NEC compared with the control group was reversed with treatment of HMGB1 inhibitor.154 Fork 03A protein (FOXO3a), a member of the fork protein family and a transcription factor that functions to limit the translocation of NF-κ-B into the nucleus, derailed the proinflammatory cytokine production and associated inflammasome-induced inflammation in the animal models of NEC.155 In parallel to the above findings, Yin et al.155 demonstrated lower expression of FOXO3a and higher expression of NLRP3, TLR4, caspase-1, IL-1β, and IL-18 in the intestinal tissues of NEC rats and LPS-treated IECs. Overexpression of FOXO3a resulted in downregulation of NLRP3 inflammasome cytokines and reduction of pyroptosis, thereby highlighting its role as a potential therapeutic strategy in regulating inflammasome mediated intestinal injury in the in vitro and animal models of NEC.155 MiRNA-146a-5p was shown to downregulate NLRP3 related downstream signalling molecules including caspase-1, and its upstream chloride intracellular channel 4, in the THP-1 cells treated with low/moderate doses of LPS (0.5 μg/mL and 1.0 μg/mL) and its overexpression resulted in improved survival in the animal models of NEC.156 Increased expression of suppressor of cytokine signalling 3 protein resulted in the decreased expression of NLRP3, caspase-1, IL-1β, IL-18, and TNF-α in the LPS treated caco-2 and fetal human cells, as well as intestinal tissues of NEC,

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highlighting its therapeutic efficacy in suppressing NLRP3 inflammasome-induced inflammation in the NEC models.157

cellular signalling events surrounding intestinal cell death in the NEC need to be delved into and understood in a systematic and methodical manner.

MANY UNANSWERED QUESTIONS AND UNMET NEEDS: CONCLUSIONS AND FUTURE PERSPECTIVES

In this review, the authors provided a detailed overview of different types of cell death in NEC with a special focus on their individual pathogenic mechanisms and signalling pathways. Discussing the pathophysiological mechanisms for various types of cell death in the NEC will be helpful for understanding the downstream signalling events based on the type of risk factors present depending on the bacterial/ viral insults encountered in the intestinal milieu. Probing into the systematic downstream signalling events and mechanisms that underlie various types of IEC death can be very useful in comprehending the pathogenetic mechanisms that initiate and perpetuate disease progression in NEC. Appropriate basic science research studies implemented in this regard might unravel novel molecular targets, including membrane receptors, tyrosine kinases, and intermediate signalling factors that play a crucial role in provoking intestinal cell death, and inciting leaky intestinal barrier in NEC. The authors surmise that blocking these earlier signalling events that occur after bacterial insult with the help of these deciphered molecular targets might offer an opportunity to reverse the disease progression from the initial stages of NEC. Therefore, these molecular targets can form the structural basis for crafting disease-specific cell-based novel therapeutic interventions for counteracting cell death, halting the disease progression, and decreasing clinical severity and mortality in infants with NEC. Finally, clinical research studies to test the efficacies of these novel therapies on disease severity and systemic complications in NEC would be the logical next step and the need of the hour so that they can be implemented in the clinic for optimising clinical outcomes, as well as decreasing morbidity and mortality in NEC.

The different types of intestinal cell death that can possibly occur in NEC include apoptosis, necrosis, necroptosis, pyroptosis, and autophagy. The type and severity of cell death is mostly dependent on the various microbial insults such as type of bacteria, amount, and type of toxins released and immune responses that are present in the intestinal milieu. The current treatment strategy utilised in the treatment of NEC includes bowel rest, intravenous antibiotics, and regular radiographic monitoring.127 Despite the best efforts of clinicians in managing these NEC cases, therapeutic options currently employed have limited efficacy, and afforded just symptomatic relief while awaiting natural disease recovery with no effect in reversing the disease progression after its onset. Therefore, it is important to comprehend and analyse the underlying critical pathogenic mechanisms that provoke the intestinal injury, perturb intestinal homeostasis, as well as augment intestinal permeability in NEC. In this regard, pathogens that invade and precipitate IEC apoptosis can compromise the intestinal epithelial barrier, and trigger a wide variety of untoward pathological consequences, such as increased intestinal permeability, intestinal inflammation, systemic inflammatory response, translocation of bacteria into the systemic circulation, systemic inflammatory response syndrome, and multiorgan failure.27 Therefore, cell death of IECs is regarded as a critical nascent cellular event in the pathogenesis of intestinal inflammatory diseases such as NEC. So, underlying pathophysiology and associated

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Mekonnen SM et al. The prevalence of necrotizing enterocolitis and associated factors among enteral fed preterm and low birth weight neonates admitted in selected public hospitals in Addis Ababa, Ethiopia: a cross-sectional study. Global Pediatr Health.

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