47 - World Journal of Gastroenterology

World Journal ofGastroenterology

ISSN 1007-9327 (print)ISSN 2219-2840 (online)

World J Gastroenterol 2020 December 21; 26(47): 7436-7592

Published by Baishideng Publishing Group Inc

WJG https://www.wjgnet.com I December 21, 2020 Volume 26 Issue 47

World Journal of

GastroenterologyW J GContents Weekly Volume 26 Number 47 December 21, 2020

OPINION REVIEW

Artificial intelligence-aided colonoscopy: Recent developments and future perspectives7436

Antonelli G, Gkolfakis P, Tziatzios G, Papanikolaou IS, Triantafyllou K, Hassan C

REVIEW

Therapeutic efficiency of bone marrow-derived mesenchymal stem cells for liver fibrosis: A systematic review of in vivo studies

7444

Al-Dhamin Z, Liu LD, Li DD, Zhang SY, Dong SM, Nan YM

MINIREVIEWS

Molecular overview of progressive familial intrahepatic cholestasis7470

Amirneni S, Haep N, Gad MA, Soto-Gutierrez A, Squires JE, Florentino RM

Invasive fungal infection before and after liver transplantation7485

Ferrarese A, Cattelan A, Cillo U, Gringeri E, Russo FP, Germani G, Gambato M, Burra P, Senzolo M

ORIGINAL ARTICLE

Basic Study

Blockage of ETS homologous factor inhibits the proliferation and invasion of gastric cancer cells through the c-Met pathway

7497

Gu ML, Zhou XX, Ren MT, Shi KD, Yu MS, Jiao WR, Wang YM, Zhong WX, Ji F

Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway

7513

Wang Z, Cheng ZX, Abrams ST, Lin ZQ, Yates E, Yu Q, Yu WP, Chen PS, Toh CH, Wang GZ

Case Control Study

Prevalence and associated factors of obesity in inflammatory bowel disease: A case-control study7528

Losurdo G, La Fortezza RF, Iannone A, Contaldo A, Barone M, Ierardi E, Di Leo A, Principi M

Retrospective Study

Towards an evaluation of alcoholic liver cirrhosis and nonalcoholic fatty liver disease patients with hematological scales

7538

Michalak A, Cichoż-Lach H, Guz M, Kozicka J, Cybulski M, Jeleniewicz W, Stepulak A

Clinical features of multiple gastrointestinal stromal tumors: A pooling analysis combined with evidence and gap map

7550

Li C, Yang KL, Wang Q, Tian JH, Li Y, Gao ZD, Yang XD, Ye YJ, Jiang KW

WJG https://www.wjgnet.com II December 21, 2020 Volume 26 Issue 47

World Journal of GastroenterologyContents

Weekly Volume 26 Number 47 December 21, 2020

Randomized Controlled Trial

Evaluation of an educational telephone intervention strategy to improve non-screening colonoscopy attendance: A randomized controlled trial

7568

Seoane A, Font X, Pérez JC, Pérez R, Enriquez CF, Parrilla M, Riu F, Dedeu JM, Barranco LE, Duran X, Ibáñez IA, Álvarez MA

CASE REPORT

Multiple cerebral lesions in a patient with refractory celiac disease: A case report7584

Horvath L, Oberhuber G, Chott A, Effenberger M, Tilg H, Gunsilius E, Wolf D, Iglseder S

WJG https://www.wjgnet.com III December 21, 2020 Volume 26 Issue 47

World Journal of GastroenterologyContents

Weekly Volume 26 Number 47 December 21, 2020

ABOUT COVER

Editorial Board Member of World Journal of Gastroenterology, Dr. Mohammad Rostami-Nejad is an Assistant Professor at the Shahid Beheshti University of Medical Sciences (SBMU), Tehran, Iran. He acquired his Bachelor’s degree from Azad University, Science and Research Branch (Tehran) and graduated with a Master’s degree in Medical Parasitology in 2009. Since 2005, he has worked as research fellow at the Research Institute for Gastroenterology and Liver Diseases (RIGLD), SBMU. His PhD thesis was on epidemiology, genetic and clinical behavior of celiac disease in the Middle East, under Dr. Kamran Rostami, Prof. Mohammad Reza Zali and Prof. Chris Mulder (The Netherlands). His ongoing research interest is the immunopathogenesis of celiac disease and other gluten-related disorders, and he has published more than 180 articles in peer-reviewed journals and 6 books. Currently, he serves as Head of the Celiac Disease Department in RIGLD, SBMU. (L-Editor: Filipodia)

AIMS AND SCOPE

The primary aim of World Journal of Gastroenterology (WJG, World J Gastroenterol) is to provide scholars and readers from various fields of gastroenterology and hepatology with a platform to publish high-quality basic and clinical research articles and communicate their research findings online. WJG mainly publishes articles reporting research results and findings obtained in the field of gastroenterology and hepatology and covering a wide range of topics including gastroenterology, hepatology, gastrointestinal endoscopy, gastrointestinal surgery, gastrointestinal oncology, and pediatric gastroenterology.

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The WJG is now indexed in Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®), Journal Citation Reports®, Index Medicus, MEDLINE, PubMed, PubMed Central, and Scopus. The 2020 edition of Journal Citation Report® cites the 2019 impact factor (IF) for WJG as 3.665; IF without journal self cites: 3.534; 5-year IF: 4.048; Ranking: 35 among 88 journals in gastroenterology and hepatology; and Quartile category: Q2.

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GastroenterologyW J GSubmit a Manuscript: https://www.f6publishing.com World J Gastroenterol 2020 December 21; 26(47): 7436-7443

DOI: 10.3748/wjg.v26.i47.7436 ISSN 1007-9327 (print) ISSN 2219-2840 (online)

OPINION REVIEW

Artificial intelligence-aided colonoscopy: Recent developments and future perspectives

Giulio Antonelli, Paraskevas Gkolfakis, Georgios Tziatzios, Ioannis S Papanikolaou, Konstantinos Triantafyllou, Cesare Hassan

ORCID number: Giulio Antonelli 0000-0003-1797-3864; Paraskevas Gkolfakis 0000-0002-9677-4013; Georgios Tziatzios 0000-0002-2945-6007; Ioannis S Papanikolaou 0000-0002-7368-6168; Konstantinos Triantafyllou 0000-0002-5183-9426; Cesare Hassan 0000-0001-7167-1459.

Author contributions: Antonelli G conceived the idea for the manuscript; Antonelli G and Gkolfakis P reviewed the literature and drafted the manuscript; Tziatzios G, Papanikolaou IS, Triantafyllou K and Hassan C drafted and finally approved the manuscript.

Conflict-of-interest statement: All authors declare no conflict of interest.

Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/License

Giulio Antonelli, Cesare Hassan, Gastroenterology Unit, Nuovo Regina Margherita Hospital, Rome 00153, Italy

Giulio Antonelli, Department of Translational and Precision Medicine, “Sapienza” University of Rome, Rome 00185, Italy

Paraskevas Gkolfakis, Department of Gastroenterology Hepatopancreatology and Digestive Oncology, Erasme University Hospital, Université Libre de Bruxelles, Brussels 1070, Belgium

Georgios Tziatzios, Ioannis S Papanikolaou, Konstantinos Triantafyllou, Hepatogastroenterology Unit, Second Department of Internal Medicine – Propaedeutic, Medical School, National and Kapodistrian University of Athens, ‘‘Attikon” University General Hospital, Athens 12462, Greece

Corresponding author: Paraskevas Gkolfakis, MD, Consultant Physician-Scientist, Department of Gastroenterology Hepatopancreatology and Digestive Oncology, Erasme University Hospital, Université Libre de Bruxelles, Rue de Lennik 808, Brussels 1070, Belgium. [email protected]

AbstractArtificial intelligence (AI) systems, especially after the successful application of Convolutional Neural Networks, are revolutionizing modern medicine. Gastrointestinal Endoscopy has shown to be a fertile terrain for the development of AI systems aiming to aid endoscopists in various aspects of their daily activity. Lesion detection can be one of the two main aspects in which AI can increase diagnostic yield and abilities of endoscopists. In colonoscopy, it is well known that a substantial rate of missed neoplasia is still present, representing the major cause of interval cancer. In addition, an extremely high variability in adenoma detection rate, the main key quality indicator in colonoscopy, has been extensively reported. The other domain in which AI is believed to have a considerable impact on everyday clinical practice is lesion characterization and aid in “optical diagnosis”. By predicting in vivo histology, such pathology costs may be averted by the implementation of two separate but synergistic strategies, namely the “leave-in-situ” strategy for < 5 mm hyperplastic lesions in the rectosigmoid tract, and “resect and discard” for the other diminutive lesions. In this opinion review we present current available evidence regarding the role of AI in improving lesions’ detection and characterization during colonoscopy.


https://dx.doi.org/10.3748/wjg.v26.i47.7436

http://orcid.org/0000-0003-1797-3864

http://orcid.org/0000-0003-1797-3864

http://orcid.org/0000-0002-9677-4013

http://orcid.org/0000-0002-9677-4013

http://orcid.org/0000-0002-2945-6007

http://orcid.org/0000-0002-2945-6007

http://orcid.org/0000-0002-7368-6168

http://orcid.org/0000-0002-7368-6168

http://orcid.org/0000-0002-7368-6168

http://orcid.org/0000-0002-5183-9426

http://orcid.org/0000-0002-5183-9426

http://orcid.org/0000-0001-7167-1459

http://orcid.org/0000-0001-7167-1459

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Antonelli G et al. AI for colonoscopy


s/by-nc/4.0/

Manuscript source: Invited manuscript

Specialty type: Gastroenterology and hepatology

Country/Territory of origin: Italy

Peer-review report’s scientific quality classificationGrade A (Excellent): A Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0

Received: October 13, 2020 Peer-review started: October 13, 2020 First decision: November 13, 2020 Revised: November 18, 2020 Accepted: November 29, 2020 Article in press: November 29, 2020 Published online: December 21, 2020

P-Reviewer: Krishna SG, Sharara AI S-Editor: Gao CC L-Editor: A P-Editor: Ma YJ

Key Words: Artificial intelligence; Colonoscopy; Polyp; Adenoma; Detection; Characterization

©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.

Core Tip: Artificial intelligence systems using deep learning techniques are constantly developing in all fields of medicine including diagnostic colonoscopy. They aim to become part of daily routine and eliminate inherent examination’s shortcomings and lead to a higher level of provided health services. In this opinion review we present the existing evidence regarding the impact of artificial intelligence systems on the improvement of colonoscopy’s outcomes, namely adenoma detection rate and adenoma miss rate, focusing mainly on clinical trials and meta-analyses evaluating real-time computer aided detection and characterization.

Citation: Antonelli G, Gkolfakis P, Tziatzios G, Papanikolaou IS, Triantafyllou K, Hassan C. Artificial intelligence-aided colonoscopy: Recent developments and future perspectives. World J Gastroenterol 2020; 26(47): 7436-7443URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7436.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7436

INTRODUCTIONColonoscopy and polypectomy are the mainstay in the prevention of colorectal cancer (CRC), and have been shown to reduce its incidence and mortality[1-3]. The development of quality improvement programs and performance measures, their measurement with audit and eventual retraining have led to an increase in adenoma detection rate (ADR), directly associated with a decrease in interval cancer (i.e., a cancer that is identified before the next recommended screening or surveillance examination)[4-6]. Notwithstanding the increasing awareness and the ever-improving quality, a substantial rate of colorectal neoplasia is still missed during colonoscopy, variably reported between 5% and 25%, leading to an interval colorectal cancer rate ranging between 0.5 and 1 per 1000 person-years[7]. The main reasons identified for colorectal neoplasia miss rate are both failure in recognising a lesion although fully visible on the endoscopy screen, due to attention or recognition issues, and failure to expose enough colorectal mucosa and incomplete resection. While mucosal exposure depends on the endoscopist’s examination technique and the quality of bowel preparation, failure to recognise a polyp when visible on the endoscopy screen can be addressed and improved by the application of artificial intelligence (AI), or “deep learning” systems[8,9]. Contrary to human-programmed computer systems, “deep learning” systems autonomously learn to distinguish the characteristics within the images provided using multiple levels of processing[10]. In this way, AI systems can recognize discriminatory characteristics between images that differ from those commonly used and elaborated by the human brain. In addition, AI systems developed with deep learning techniques can acquire fast image processing that can be used real time during an endoscopic examination. Consequently, AI systems can flag the suspect area during the endoscopic examination. These systems have shown a high accuracy when retrospectively applied to still images or stored videos, and more recently have been tested in trials during endoscopic examinations[10]. The other domain in which AI is believed to have a considerable impact on everyday clinical practice is lesion characterization and aid in “optical diagnosis”. When considering the magnitude of colonoscopies performed, covering between 1% and 6% of the target general population per year, the financial and economic burden is relevant[11]. A relevant contribution of such burden is represented by the post-polypectomy histology cost, mostly attributed to diminutive polyps that represent over 90% of all the resected lesions[12-14]. By predicting in vivo histology, such pathology costs may be averted by the implementation of two strategies, namely the “leave-in-situ” or the “resect and discard” strategy for < 5 mm hyperplastic lesions in the rectosigmoid[15,16]. Despite the acceptance by experts, the accuracy of optical diagnosis in the community setting has been suboptimal, preventing the implementation of these cost-saving inter-


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ventions[17,18]. In addition, the clinical relevance of these lesions has been debated, being mostly represented by either non-advanced adenomas or indolent hyperplastic polyps. In addition, the role of pathology as reference standard has been questioned because of possible high interobserver agreement, inadequate orientation or insufficient material[19]. By automatizing the perception phase, AI may overcome both of these pitfalls in detection and characterization. Based on deep learning Computer Aided Detection (CADe), can recognize in real-time lesions that are present on the screen and that may have been missed by the endoscopist. Similarly, Computer Aided Characterization (CADx) can predict the histology of the lesion providing the correct classification to the endoscopist[8]. We present here an overview of recent literature regarding the real-time clinical application of CADe and CADx for colorectal neoplasia.

BRIEF DESCRIPTION OF AI SYSTEM DEVELOPMENT PROCESSThe goal of AI system development is to build a mathematical model from a set of pre-marked data (e.g., images) that will allow interpretation of new, unknown data with a reasonable amount of accuracy[10]. Deep learning systems autonomously “learn” (i.e., build their own algorithms) starting from libraries of labelled data (images containing a polyp) and subsequently acquire parameters that recognize a polyp in an image they have never been presented before. The phases of AI system development can be summarised in the training phase, the validation phase and the testing phase. In the training phase, an exceptionally large number of images labelled for the regions/ features of interest are presented to the system, that learns to recognise the labelled features building its own algorithms. The system is then initially tested on another set of unknown images, the validation set, in which the performance is evaluated, and the system is fine-tuned by the use of “hyperparameters”, optional settings calibrated by the programmers to optimise the system’s performance. Lastly, a third, unseen set of data (the “test” set) is presented to the system, to evaluate its standalone performance. Ideally, the test set should be a library of unseen images completely different from those of the training and validation sets. The last step is to test it in a randomised controlled clinical trial to face the pitfalls of clinical practice, like suboptimal preparation, patient compliance, operator skills, etc.

In the near future it is conceivable that many different AI systems will be available. Taking into account that many endoscope manufacturers will probably include computer aided diagnosis (CAD) in their new hardware releases and that different systems will be applicable in different endoscopy systems, it will be at the discretion of the different centres to decide how to implement CAD systems in their endoscopy suites.

DETECTION OF COLORECTAL NEOPLASIA: EVIDENCE FROM CLINICAL TRIALS After a long experimental phase, in the last two years the results of the first clinical trials testing the performance of CADe systems in real-life clinical practice have been published, mostly from Chinese groups[20-25]. A summary of AI systems that are currently available is found in Table 1[20,23,26,27].

Interestingly, no clinical trial showed differences in colonoscopy withdrawal times between groups undergoing CADe examinations and controls. All published trials showed ADR increase in the CADe groups: Wang et al[20] reported a significant (P < 0.001) ADR increase from 20% in the control group to 29% in the CADe group. Su et al[22] and Liu et al[25] reported a significant ADR increase, 39% vs 24% (P < 0.001) and 29% vs 17% (P < 0.001), respectively. All three trials had the limitation of low ADR in the control group, raising concerns about whether AI might compensate, albeit partially, a poor operator technique. However, in a trial by Repici et al[23] high baseline ADR at 40.4% in the control group was outmatched by a 54.8% ADR in the CADe group. A recent meta-analysis of published randomised control trials[28], has shown that the increase in ADR was consistent across all trials. Among the included 4354 patients, the ADR in the control and the CADe groups was 25.2% and 36.6%, respectively, with risk ratio (RR) = 1.44 [95% confidence interval (CI), 1.27-1.62; P < 0.01]. Sub-analysis revealed that the increase in ADR was mainly due to detection of more diminutive adenomas in all studies included in the meta-analysis. No study



Table 1 Current standalone performance of approved and not approved computer aided diagnosis systems

Regulatory approved Company Sensitivity (%) False positives/specificity (%)

GI-Genius[23] Medtronic 99.70 0.9 (FP)

Discovery AI Pentax 90 80 (spec)

CAD-EYE detection1 Fujifilm 92.90 90.6 (spec)

CAD-EYE characterization1 Fujifilm 85 79.4 (spec)

Endobrain-EYE[27] Cybernet 95 89 (spec)

Not regulatory approved

Yamada et al[26] NEC 97.30 99 (spec)

Wang et al[20] Wision AI 94 96 (spec)

1Submitted data. CAD: Computer aided diagnosis; FP: False positive; spec: Specificity; AI: Artificial intelligence.

showed advanced adenoma (> 10mm) ADR increase, while only Repici et al[23] showed higher detection rates for adenomas measuring between 6 and 9 mm in the CADe group (12.7% vs 17.2%, P < 0.05)[28]. Only one study[24], not included in the aforementioned meta-analysis, has shown a role of CADe in increasing advanced adenoma detection rate, so far. This study showed an increase of advanced adenoma ADR from 1% in the control group to 3% in the CADe group, and this difference proved statistically significant. However, in this study participants had very low ADR (8% in the control group, 16% in the CADe group) overall; thus concerns are raised regarding the interpretation of the results.

CADe has shown interesting results also among the other colonoscopy quality indicators. In detail, the “doppelganger” of ADR, namely adenoma miss rate (AMR), was recently reported in a back-to-back randomised trial from Wang et al[29], with an impressive improvement from a worrying 40% in the control group to a quite low (still not negligible) 13% in the CADe group. In this study the authors also underwent an elegant analysis regarding the difference in the miss rate between “visible” (i.e., exposed, but not recognised by the operating endoscopist) and “invisible” (i.e., not exposed by the endoscopist) polyps. Interestingly, they confirmed that when mucosa containing a polyp is effectively exposed by the endoscopist, CADe almost never misses the polyp [AMR-visible in the CADe group: 1.59%; polyp miss rate (PMR)-visible in the CADe group: 2.36%]. This observation further confirms the growing awareness of the importance of effectively exposing all colonic mucosa to increase neoplasia detection. Reduction in AMR was significant for diminutive (39.6% vs 13.1%, P = 0.001) and small polyps (46.9% vs 13.7%, P < 0.0001), but not for adenomas larger than 10mm (15.3% vs 33.3%), confirming that the detection of advanced adenomas is independent of CAD use[29]. Regarding polyp detection rate, meta-analyses have shown[28,30] significantly improved colonoscopy performance regarding PDR in CAD groups: (50.3% vs 34.6%; RR 1.43; 95%CI, 1.34-1.53; P < 0.01), overall. CADe use was also associated with a higher adenoma per colonoscopy (APC) rate, irrespectively of polyp size: overall APC: 0.58 vs 0.36 [RR (95%CI): 1.70 (1.53-1.89), P < 0.01]; while for polyps < 5 mm, 6-9 mm and ≥ 10 mm RR (95%CI) was 1.69 (1.48-1.84), 1.44 (1.19-1.75) and 1.46 (1.04-2.06), respectively. Lastly, a meta-analysis showed improved serrated lesion detection rates by CADe (0.06 vs 0.04, RR: 1.52; 95%CI, 1.14-2.02; P < 0.01)[30]. However, serrated miss rate was found not to be significantly different between the two groups in the back to back study by Wang et al[29]. This discrepancy could be explained either by an inadequate sample size for this specific indicator, or by a CAD system that has still to be optimized (improved training) for serrated adenoma detection.

CHARACTERIZATION OF COLORECTAL NEOPLASIA: EVIDENCE FROM CLINICAL TRIALSCADx is the other promising field of clinical application of AI in colonoscopy. While the human operator depends on the application of virtual or physical chromo-endoscopy to improve visualisation of mucosal and vascular patterns in order to



predict lesion histology, the adequately trained on a wide library AI system should be able to predict histology regardless of the optical visualisation modality[30]. Currently, no randomised clinical trial is available evaluating performance of detection systems. However, many systems are under development and their standalone performance has been evaluated. A recent metanalysis has summarised existing literature, showing how among the 3 prospective studies on CADx[30], AI showed an impressive 92.3% (95%CI, 88.8%-94.9%) sensitivity on polyp histology prediction and a high specificity: 89.8% (95%CI, 85.3%-93.0%). Among the considerable number of retrospective studies, similar pooled results were found[30]. It is important to notice that the majority of these systems are shallow machine learning systems abandoned in favour of deep learning systems, and that solid data from randomised trials, using real-life images will be needed before a true estimate of CADx performance can be made. The performance during live colonoscopy is of course the main focus around this kind of system, where pitfalls such as inadequate bowel preparation or incomplete lesion visualisation are common.

It is well known that extensive training is needed for an endoscopist to achieve acceptable results in predicting in vivo histology of encountered lesions and that this knowledge must be regularly updated and retrained. Thus, measuring the advantage of CADx vs optical diagnosis performance of expert and non-expert endoscopists, is expected. According to the available limited evidence, AI performs similarly to experts but better than non-expert endoscopists in lesion characterisation[29]. Therefore, a significant improvement of non-experts’ performance through CADx could be of great interest, both for training and for quality assurance.

FUTURE PERSPECTIVES AND CONTROVERSIESA possible drawback of CADe is the potential large number of false positive results[31]. As previously discussed, CADe systems autonomously learn their own detection algorithms and therefore its outcomes incorporate some unpredictability in the clinical setting that must be interpreted cautiously. Indeed, the system may flag frames that the endoscopists may never have selected as suspicious areas and consequently reduce colonoscopy efficiency. The endoscopist might spend an excessive amount of time to discriminate between an actual false positive and a possible false negative result. Furthermore, although areas flagged by CADe must always be interpreted by trained endoscopists, it is still possible that a false positive area may result in unnecessary polypectomy with related avoidable adverse events. In a recent study[31], authors underwent a post-hoc analysis of a randomized controlled trial (RCT) on CADe performance, where they measured false positive burden and clinical relevance and classified false positives in two broad categories: artefacts from bowel wall and artefacts from bowel content. Overall, they found a mean 27.3% false positive activations per colonoscopy, with nearly 90% of them due to artefacts from the bowel wall (folds, ileocecal valve, diverticula, appendicular foramen, etc.). Interestingly, according to their measurements, less than 10% of the false positive activations resulted in additional time spent by the endoscopist in examining the flagged area, while the majority were instantly dismissed as not relevant. These results must be confirmed with other systems and other settings.

Another domain in which CADe performance has yet to be improved is the detection of non-polypoid lesions[32]. These colorectal lesions account for a large portion of missed colorectal neoplasia and may be associated with a more aggressive biological behaviour. A recent review[32] has shown that among the published RCTs on CADe systems, some of them did not report the number of flat lesions included in the training sets and others did not report sub-analysis on the performance of AI specifically for flat lesions. The authors concluded that in future CADe systems, development and refinement, additional training and validation for the recognition of the individual subtypes of non-polypoid lesions, especially for non-granular lateral spreading tumors (LST-NG), is urgently needed. The authors speculate that a joint partnership between Eastern and Western centres should be prioritized to create datasets with a large number of flat lesions.

In the era of colonoscopy quality measurement and improvement, CAD systems that can integrate quality measurement and reporting have been initially evaluated[4,24]. The indicators that have been measured with CAD are caecal intubation rate, withdrawal time, and even slipping of the scope that can leave areas of the colon uninspected.

The cost-effectiveness of CAD systems has yet to be fully analysed. Only one



preliminary study has been published so far by Mori et al[33] focusing on the implementation of AI alongside a “diagnose and leave behind” strategy, showing that this can lead to substantial cost reductions regarding the annual reimbursement for colonoscopies conducted under public health insurances in Japan, England, Norway, and the United States, respectively. Further cost-effectiveness models could further tailor this analysis, for example in the setting of organised screening programs, that in most of the Western world currently account for the greater part of the colonoscopy burden in public health systems. A considerable improvement in AI-aided colonoscopy should be the implementation of systems that integrate CADe and CADx in the same machine[34]. This could reduce costs and increase the practical considerations regarding clinical use. Randomised trials and cost-effectiveness models combining the additional detection provided by CADe to the optical diagnosis improvement provided by CADx could pave the way to a swift implementation of these systems in clinical practice.

CONCLUSIONArtificial Intelligence is a major breakthrough in the whole medical field, and endoscopy is a very fertile terrain for its development and refinement. However, it may not come without harm. The excessive reliance on AI systems may trigger a relaxation in endoscopic performance with the (un-)conscious thought that “the system is watching”. Moreover, implementation of AI may discourage endoscopists from improving optical diagnosis skills or update their knowledge. As already discussed, the presence of false positives may also push the novice or un-expert to perform unnecessary resections or biopsies, increasing cost and pathology burden.

In the case of CADe, this problem seems of a lesser grade, since regardless of the level of expertise we can affirm that the endoscopists will be able to confirm or discard the region flagged by the AI system with a reasonable level of confidence. The “one and done” issue of ADR might be taken into account, but this is true irrespective of the presence of a CAD system.

On the contrary, when dealing with CADx, only a trained endoscopist with a good confidence in optical diagnosis will be able to accept or refuse the AI characterization output and give the final diagnosis with its consequent actions. It is conceivable that non-experts might passively accept the CADx prediction without the competence to challenge it, raising also the legal issue of the final responsibility of an incorrect diagnosis: the operator, the AI system developer, or the health system?

This argues against using AI accuracy to bypass a suboptimal competence in optical diagnosis, and actually strengthens guideline recommendations that specifically affirm that optical diagnosis can be only performed by endoscopists who are proficient in the technique and are actively trained and audited.

We strongly believe that in every dominion in which we seek AI assistance, competence is the prerequisite and not the final outcome of AI implementation.

REFERENCESAtkin W, Wooldrage K, Brenner A, Martin J, Shah U, Perera S, Lucas F, Brown JP, Kralj-Hans I, Greliak P, Pack K, Wood J, Thomson A, Veitch A, Duffy SW, Cross AJ. Adenoma surveillance and colorectal cancer incidence: a retrospective, multicentre, cohort study. Lancet Oncol 2017; 18: 823-834 [PMID: 28457708 DOI: 10.1016/S1470-2045(17)30187-0]

1

Brenner H, Arndt V, Stürmer T, Stegmaier C, Ziegler H, Dhom G. Long-lasting reduction of risk of colorectal cancer following screening endoscopy. Br J Cancer 2001; 85: 972-976 [PMID: 11592768 DOI: 10.1054/bjoc.2001.2023]

2

Brenner H, Stock C, Hoffmeister M. Effect of screening sigmoidoscopy and screening colonoscopy on colorectal cancer incidence and mortality: systematic review and meta-analysis of randomised controlled trials and observational studies. BMJ 2014; 348: g2467 [PMID: 24922745 DOI: 10.1136/bmj.g2467]

3

Kaminski MF, Thomas-Gibson S, Bugajski M, Bretthauer M, Rees CJ, Dekker E, Hoff G, Jover R, Suchanek S, Ferlitsch M, Anderson J, Roesch T, Hultcranz R, Racz I, Kuipers EJ, Garborg K, East JE, Rupinski M, Seip B, Bennett C, Senore C, Minozzi S, Bisschops R, Domagk D, Valori R, Spada C, Hassan C, Dinis-Ribeiro M, Rutter MD. Performance measures for lower gastrointestinal endoscopy: a European Society of Gastrointestinal Endoscopy (ESGE) Quality Improvement Initiative. Endoscopy 2017; 49: 378-397 [PMID: 28268235 DOI: 10.1055/s-0043-103411]

4

Kaminski MF, Regula J, Kraszewska E, Polkowski M, Wojciechowska U, Didkowska J, Zwierko M, Rupinski M, Nowacki MP, Butruk E. Quality indicators for colonoscopy and the risk of interval

5

http://www.ncbi.nlm.nih.gov/pubmed/28457708

https://dx.doi.org/10.1016/S1470-2045(17)30187-0


https://dx.doi.org/10.1054/bjoc.2001.2023


https://dx.doi.org/10.1136/bmj.g2467


https://dx.doi.org/10.1055/s-0043-103411



cancer. N Engl J Med 2010; 362: 1795-1803 [PMID: 20463339 DOI: 10.1056/NEJMoa0907667]Corley DA, Jensen CD, Marks AR, Zhao WK, Lee JK, Doubeni CA, Zauber AG, de Boer J, Fireman BH, Schottinger JE, Quinn VP, Ghai NR, Levin TR, Quesenberry CP. Adenoma detection rate and risk of colorectal cancer and death. N Engl J Med 2014; 370: 1298-1306 [PMID: 24693890 DOI: 10.1056/NEJMoa1309086]

6

Zhao S, Wang S, Pan P, Xia T, Chang X, Yang X, Guo L, Meng Q, Yang F, Qian W, Xu Z, Wang Y, Wang Z, Gu L, Wang R, Jia F, Yao J, Li Z, Bai Y. Magnitude, Risk Factors, and Factors Associated With Adenoma Miss Rate of Tandem Colonoscopy: A Systematic Review and Meta-analysis. Gastroenterology 2019; 156: 1661-1674. e11 [PMID: 30738046 DOI: 10.1053/j.gastro.2019.01.260]

7

Bisschops R, East JE, Hassan C, Hazewinkel Y, Kamiński MF, Neumann H, Pellisé M, Antonelli G, Bustamante Balen M, Coron E, Cortas G, Iacucci M, Yuichi M, Longcroft-Wheaton G, Mouzyka S, Pilonis N, Puig I, van Hooft JE, Dekker E. Advanced imaging for detection and differentiation of colorectal neoplasia: European Society of Gastrointestinal Endoscopy (ESGE) Guideline - Update 2019. Endoscopy 2019; 51: 1155-1179 [PMID: 31711241 DOI: 10.1055/a-1031-7657]

8

de Groen PC. Using artificial intelligence to improve adequacy of inspection in gastrointestinal endoscopy. Tech Innov Gastrointest Endosc 2020; 22: 71-79 [DOI: 10.1016/j.tgie.2019.150640]

9

van der Sommen F, de Groof J, Struyvenberg M, van der Putten J, Boers T, Fockens K, Schoon EJ, Curvers W, de With P, Mori Y, Byrne M, Bergman JJGHM. Machine learning in GI endoscopy: practical guidance in how to interpret a novel field. Gut 2020; 69: 2035-2045 [PMID: 32393540 DOI: 10.1136/gutjnl-2019-320466]

10

Greuter MJE, de Klerk CM, Meijer GA, Dekker E, Coupé VMH. Screening for Colorectal Cancer With Fecal Immunochemical Testing With and Without Postpolypectomy Surveillance Colonoscopy: A Cost-Effectiveness Analysis. Ann Intern Med 2017; 167: 544-554 [PMID: 28973514 DOI: 10.7326/M16-2891]

11

Gupta N, Bansal A, Rao D, Early DS, Jonnalagadda S, Wani SB, Edmundowicz SA, Sharma P, Rastogi A. Prevalence of advanced histological features in diminutive and small colon polyps. Gastrointest Endosc 2012; 75: 1022-1030 [PMID: 22405698 DOI: 10.1016/j.gie.2012.01.020]

12

Laish I, Sergeev I, Stein A, Naftali T, Konikoff FM. Risk of metachronous advanced lesions after resection of diminutive and small, non-advanced adenomas. Clin Res Hepatol Gastroenterol 2019; 43: 201-207 [PMID: 30266580 DOI: 10.1016/j.clinre.2018.03.001]

13

Schachschal G, Sehner S, Choschzick M, Aust D, Brandl L, Vieth M, Wegscheider K, Baretton GB, Kirchner T, Sauter G, Rösch T. Impact of reassessment of colonic hyperplastic polyps by expert GI pathologists. Int J Colorectal Dis 2016; 31: 675-683 [PMID: 26847619 DOI: 10.1007/s00384-016-2523-8]

14

Hassan C, Pickhardt PJ, Rex DK. A resect and discard strategy would improve cost-effectiveness of colorectal cancer screening. Clin Gastroenterol Hepatol 2010; 8: 865-869, 869.e1-869. e3 [PMID: 20621680 DOI: 10.1016/j.cgh.2010.05.018]

15

Rex DK, Kahi C, O'Brien M, Levin TR, Pohl H, Rastogi A, Burgart L, Imperiale T, Ladabaum U, Cohen J, Lieberman DA. The American Society for Gastrointestinal Endoscopy PIVI (Preservation and Incorporation of Valuable Endoscopic Innovations) on real-time endoscopic assessment of the histology of diminutive colorectal polyps. Gastrointest Endosc 2011; 73: 419-422 [PMID: 21353837 DOI: 10.1016/j.gie.2011.01.023]

16

Rex DK. Can we do resect and discard with artificial intelligence-assisted colon polyp “optical biopsy? Tech Innov Gastrointest Endosc 2020; 22: 52-55 [DOI: 10.1016/j.tgie.2019.150638]

17

Vu HT, Sayuk GS, Hollander TG, Clebanoff J, Edmundowicz SA, Gyawali CP, Thyssen EP, Weinstock LB, Early DS. Resect and discard approach to colon polyps: real-world applicability among academic and community gastroenterologists. Dig Dis Sci 2015; 60: 502-508 [PMID: 25287002 DOI: 10.1007/s10620-014-3376-z]

18

Vennelaganti S, Cuatrecasas M, Vennalaganti P, Kennedy KF, Srinivasan S, Patil DT, Plesec T, Lanas A, Hörndler C, Andraws N, Cherian R, Mathur S, Hassan C, Repici A, Klotz D, Musulen E, Risio M, Castells A, Gupta N, Sharma P. Interobserver Agreement Among Pathologists in the Differentiation of Sessile Serrated From Hyperplastic Polyps. Gastroenterology 2020; Online ahead of print [PMID: 32950521 DOI: 10.1053/j.gastro.2020.09.015]

19

Wang P, Berzin TM, Glissen Brown JR, Bharadwaj S, Becq A, Xiao X, Liu P, Li L, Song Y, Zhang D, Li Y, Xu G, Tu M, Liu X. Real-time automatic detection system increases colonoscopic polyp and adenoma detection rates: a prospective randomised controlled study. Gut 2019; 68: 1813-1819 [PMID: 30814121 DOI: 10.1136/gutjnl-2018-317500]

20

Wang P, Liu X, Berzin TM, Glissen Brown JR, Liu P, Zhou C, Lei L, Li L, Guo Z, Lei S, Xiong F, Wang H, Song Y, Pan Y, Zhou G. Effect of a deep-learning computer-aided detection system on adenoma detection during colonoscopy (CADe-DB trial): a double-blind randomised study. Lancet Gastroenterol Hepatol 2020; 5: 343-351 [PMID: 31981517 DOI: 10.1016/S2468-1253(19)30411-X]

21

Su JR, Li Z, Shao XJ, Ji CR, Ji R, Zhou RC, Li GC, Liu GQ, He YS, Zuo XL, Li YQ. Impact of a real-time automatic quality control system on colorectal polyp and adenoma detection: a prospective randomized controlled study (with videos). Gastrointest Endosc 2020; 91: 415-424. e4 [PMID: 31454493 DOI: 10.1016/j.gie.2019.08.026]

22

Repici A, Badalamenti M, Maselli R, Correale L, Radaelli F, Rondonotti E, Ferrara E, Spadaccini M, Alkandari A, Fugazza A, Anderloni A, Galtieri PA, Pellegatta G, Carrara S, Di Leo M, Craviotto V, Lamonaca L, Lorenzetti R, Andrealli A, Antonelli G, Wallace M, Sharma P, Rosch T, Hassan C. Efficacy of Real-Time Computer-Aided Detection of Colorectal Neoplasia in a Randomized Trial.

23


https://dx.doi.org/10.1056/NEJMoa0907667


https://dx.doi.org/10.1056/NEJMoa1309086


https://dx.doi.org/10.1053/j.gastro.2019.01.260


https://dx.doi.org/10.1055/a-1031-7657

https://dx.doi.org/10.1016/j.tgie.2019.150640


https://dx.doi.org/10.1136/gutjnl-2019-320466


https://dx.doi.org/10.7326/M16-2891


https://dx.doi.org/10.1016/j.gie.2012.01.020


https://dx.doi.org/10.1016/j.clinre.2018.03.001


https://dx.doi.org/10.1007/s00384-016-2523-8


https://dx.doi.org/10.1016/j.cgh.2010.05.018



https://dx.doi.org/10.1016/j.tgie.2019.150638


https://dx.doi.org/10.1007/s10620-014-3376-z






https://dx.doi.org/10.1016/S2468-1253(19)30411-X





Gastroenterology 2020; 159: 512-520. e7 [PMID: 32371116 DOI: 10.1053/j.gastro.2020.04.062]Gong D, Wu L, Zhang J, Mu G, Shen L, Liu J, Wang Z, Zhou W, An P, Huang X, Jiang X, Li Y, Wan X, Hu S, Chen Y, Hu X, Xu Y, Zhu X, Li S, Yao L, He X, Chen D, Huang L, Wei X, Wang X, Yu H. Detection of colorectal adenomas with a real-time computer-aided system (ENDOANGEL): a randomised controlled study. Lancet Gastroenterol Hepatol 2020; 5: 352-361 [PMID: 31981518 DOI: 10.1016/S2468-1253(19)30413-3]

24

Liu WN, Zhang YY, Bian XQ, Wang LJ, Yang Q, Zhang XD, Huang J. Study on detection rate of polyps and adenomas in artificial-intelligence-aided colonoscopy. Saudi J Gastroenterol 2020; 26: 13-19 [PMID: 31898644 DOI: 10.4103/sjg.SJG_377_19]

25

Yamada M, Saito Y, Imaoka H, Saiko M, Yamada S, Kondo H, Takamaru H, Sakamoto T, Sese J, Kuchiba A, Shibata T, Hamamoto R. Development of a real-time endoscopic image diagnosis support system using deep learning technology in colonoscopy. Sci Rep 2019; 9: 14465 [PMID: 31594962 DOI: 10.1038/s41598-019-50567-5]

26

Kudo SE, Misawa M, Mori Y, Hotta K, Ohtsuka K, Ikematsu H, Saito Y, Takeda K, Nakamura H, Ichimasa K, Ishigaki T, Toyoshima N, Kudo T, Hayashi T, Wakamura K, Baba T, Ishida F, Inoue H, Itoh H, Oda M, Mori K. Artificial Intelligence-assisted System Improves Endoscopic Identification of Colorectal Neoplasms. Clin Gastroenterol Hepatol 2020; 18: 1874-1881. e2 [PMID: 31525512 DOI: 10.1016/j.cgh.2019.09.009]

27

Hassan C, Spadaccini M, Iannone A, Maselli R, Jovani M, Chandrasekar VT, Antonelli G, Yu H, Areia M, Dinis-Ribeiro M, Bhandari P, Sharma P, Rex DK, Rösch T, Wallace M, Repici A. Performance of artificial intelligence in colonoscopy for adenoma and polyp detection: a systematic review and meta-analysis. Gastrointest Endosc 2020 [PMID: 32598963 DOI: 10.1016/j.gie.2020.06.059]

28

Wang P, Liu P, Glissen Brown JR, Berzin TM, Zhou G, Lei S, Liu X, Li L, Xiao X. Lower Adenoma Miss Rate of Computer-Aided Detection-Assisted Colonoscopy vs Routine White-Light Colonoscopy in a Prospective Tandem Study. Gastroenterology 2020; 159: 1252-1261. e5 [PMID: 32562721 DOI: 10.1053/j.gastro.2020.06.023]

29

Lui TKL, Guo CG, Leung WK. Accuracy of artificial intelligence on histology prediction and detection of colorectal polyps: a systematic review and meta-analysis. Gastrointest Endosc 2020; 92: 11-22. e6 [PMID: 32119938 DOI: 10.1016/j.gie.2020.02.033]

30

Hassan C, Badalamenti M, Maselli R, Correale L, Iannone A, Radaelli F, Rondonotti E, Ferrara E, Spadaccini M, Alkandari A, Fugazza A, Anderloni A, Galtieri PA, Pellegatta G, Carrara S, Di Leo M, Craviotto V, Lamonaca L, Lorenzetti R, Andrealli A, Antonelli G, Wallace M, Sharma P, Rösch T, Repici A. Computer-aided detection-assisted colonoscopy: classification and relevance of false positives. Gastrointest Endosc 2020; 92: 900-904. e4 [PMID: 32561410 DOI: 10.1016/j.gie.2020.06.021]

31

Hassan C, Bhandari P, Antonelli G, Repici A. Artificial intelligence for non-polypoid colorectal neoplasms. Dig Endosc 2020; Online ahead of print [PMID: 32767704 DOI: 10.1111/den.13807]

32

Mori Y, Kudo SE, East JE, Rastogi A, Bretthauer M, Misawa M, Sekiguchi M, Matsuda T, Saito Y, Ikematsu H, Hotta K, Ohtsuka K, Kudo T, Mori K. Cost savings in colonoscopy with artificial intelligence-aided polyp diagnosis: an add-on analysis of a clinical trial (with video). Gastrointest Endosc 2020; 92: 905-911. e1 [PMID: 32240683 DOI: 10.1016/j.gie.2020.03.3759]

33

Kudo SE, Mori Y, Misawa M, Takeda K, Kudo T, Itoh H, Oda M, Mori K. Artificial intelligence and colonoscopy: Current status and future perspectives. Dig Endosc 2019; 31: 363-371 [PMID: 30624835 DOI: 10.1111/den.13340]

34




https://dx.doi.org/10.1016/S2468-1253(19)30413-3


https://dx.doi.org/10.4103/sjg.SJG_377_19


https://dx.doi.org/10.1038/s41598-019-50567-5












https://dx.doi.org/10.1111/den.13807




https://dx.doi.org/10.1111/den.13340


World Journal of



REVIEW

Therapeutic efficiency of bone marrow-derived mesenchymal stem cells for liver fibrosis: A systematic review of in vivo studies

Zaid Al-Dhamin, Ling-Di Liu, Dong-Dong Li, Si-Yu Zhang, Shi-Ming Dong, Yue-Min Nan

ORCID number: Zaid Al-Dhamin 0000-0002-0503-2592; Ling-Di Liu 0000-0003-4286-5603; Dong-Dong Li 0000-0002-4210-5551; Si-Yu Zhang 0000-0002-7979-4610; Shi-Ming Dong 0000-0003-33621-1443; Yue-Min Nan 0000-0003-4192-099X.

Author contributions: Al-Dhamin Z and Nan YM designed the work, performed the extensive literature search, and drafted the manuscript; Al-Dhamin Z and Liu LD analyzed and interpreted the data; Al-Dhamin Z, Zhang SY, and Dong SM provided important intellectual content by generating the figures and tables; Al-Dhamin Z and Li DD reviewed and edited the manuscript; Nan YM revised the work critically and approved the final version to be published.

Supported by Key Research and Development Program of Hebei Province, No. 19277779D; and The Program of Introduce International Intelligence of Hebei Province.

Conflict-of-interest statement: The authors declare that they have no conflicts of interest.

Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution

Zaid Al-Dhamin, Ling-Di Liu, Dong-Dong Li, Si-Yu Zhang, Shi-Ming Dong, Yue-Min Nan, Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University & Hebei Key Laboratory of Mechanism of Liver Fibrosis in Chronic Liver Disease, Shijiazhuang 050051, Hebei Province, China

Corresponding author: Yue-Min Nan, MD, PhD, Director, Doctor, Professor, Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University & Hebei Key Laboratory of Mechanism of Liver Fibrosis in Chronic Liver Disease, No. 139 Ziqiang Road, Shijiazhuang 050051, Hebei Province, China. [email protected]

AbstractAlthough multiple drugs are accessible for recovering liver function in patients, none are considered efficient. Liver transplantation is the mainstay therapy for end-stage liver fibrosis. However, the worldwide shortage of healthy liver donors, organ rejection, complex surgery, and high costs are prompting researchers to develop novel approaches to deal with the overwhelming liver fibrosis cases. Mesenchymal stem cell (MSC) therapy is an emerging alternative method for treating patients with liver fibrosis. However, many aspects of this therapy remain unclear, such as the efficiency compared to conventional treatment, the ideal MSC sources, and the most effective way to use it. Because bone marrow (BM) is the largest source for MSCs, this paper used a systematic review approach to study the therapeutic efficiency of MSCs against liver fibrosis and related factors. We systematically searched multiple published articles to identify studies involving liver fibrosis and BM-MSC-based therapy. Analyzing the selected studies showed that compared with conventional treatment BM-MSC therapy may be more efficient for liver fibrosis in some cases. In contrast, the cotreatment presented a more efficient way. Nevertheless, BM-MSCs are lacking as a therapy for liver fibrosis; thus, this paper also reviews factors that affect BM-MSC efficiency, such as the implementation routes and strategies employed to enhance the potential in alleviating liver fibrosis. Ultimately, our review summarizes the recent advances in the BM-MSC therapy for liver fibrosis. It is grounded in recent developments underlying the efficiency of BM-MSCs as therapy, focusing on the preclinical in vivo experiments, and comparing to other treatments or sources and the strategies used to enhance its potential while mentioning the research gaps.

Key Words: Bone marrow; Mesenchymal stem cells; Liver fibrosis; In vivo; Efficiency



http://orcid.org/0000-0002-0503-2592

http://orcid.org/0000-0002-0503-2592

http://orcid.org/0000-0003-4286-5603

http://orcid.org/0000-0003-4286-5603

http://orcid.org/0000-0002-4210-5551

http://orcid.org/0000-0002-4210-5551

http://orcid.org/0000-0002-7979-4610

http://orcid.org/0000-0002-7979-4610

http://orcid.org/0000-0003-33621-1443

http://orcid.org/0000-0003-33621-1443

http://orcid.org/0000-0003-4192-099X

http://orcid.org/0000-0003-4192-099X


Al-Dhamin Z et al. BM-MSCs in liver fibrosis


NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/

Manuscript source: Unsolicited manuscript


Country/Territory of origin: China

Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): B, B, B, B Grade C (Good): C Grade D (Fair): D Grade E (Poor): 0

Received: August 21, 2020 Peer-review started: August 21, 2020 First decision: October 18, 2020 Revised: October 31, 2020 Accepted: November 13, 2020 Article in press: November 13, 2020 Published online: December 21, 2020

P-Reviewer: Caboclo JF, Chiba T, Chiu KW, Corrales FJ, Sipos F S-Editor: Huang P L-Editor: Filipodia P-Editor: Ma YJ


Core Tip: Bone marrow (BM)- mesenchymal stem cells (MSCs) are a promising therapy for liver fibrosis. However, several aspects, such as efficiency, of this treatment are vague. This review summarizes the recent advances and effectiveness of BM-MSC therapy, its mechanisms, and related factors by focusing on preclinical in vivo experiments. While BM-MSCs appear to be effective in some cases, cotreatment appears to be a better option. Studies on strategies, implementation routes, and cotreatments are helping to strengthen the efficiency. While the potential of this therapy continues to advance, research is still needed to achieve full potential.

Citation: Al-Dhamin Z, Liu LD, Li DD, Zhang SY, Dong SM, Nan YM. Therapeutic efficiency of bone marrow-derived mesenchymal stem cells for liver fibrosis: A systematic review of in vivo studies. World J Gastroenterol 2020; 26(47): 7444-7469URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7444.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7444

INTRODUCTIONThe worldwide incidence of liver fibrosis has been increasing steadily in recent years; even though antiviral agents are widely used, the ultimate treatment for liver fibrosis is liver transplantation. Because transplants are not available in many countries and when they are, high costs and organ shortages cause this to be an unfeasible option for many patients. Development of curative therapies for end-stage liver disease is a necessity. Stem cell transplantation represents a promising solution because it involves recovery of the liver and production of hepatic stem cells (HSCs) in sufficient quantities to overcome the shortage of liver donors. Hepatocyte expansion from HSCs facilitates both physiological turnover and homeostatic regeneration[1].

Mesenchymal stem cells (MSCs) boast the benefits of being acquired relatively easily and stimulating low immunogenicity[2]. These cells are also characterized by a self-renewal ability and a capacity to differentiate into cells of various lineages, including osteoblasts, adipocytes, and chondrocytes[3]. MSCs also elicit less of an ethical concern because they do not originate from somatic cells. Their transplantation is considered safe and has been widely assessed in clinical settings and various diseases, yielding promising results[4].

Because bone marrow (BM) is the largest supplier of MSC sources, we focused this work on BM-MSC transplantation for liver fibrosis and its therapeutic efficiency. Many elements affect the therapeutic efficacy of BM-MSCs, such as culture method, strategies, and transplantation routes. Thus, understanding liver regeneration through BM-MSCs is crucial to offer new perspectives for treatment of liver diseases. This includes the underlying therapeutic mechanism that facilitates alleviation of liver fibrosis, its efficiency compared to other treatments, or dependence on the transplantation route and the strategies used for the procedure. This review outlines the recent advances of BM-MSCs for liver fibrosis, the main aspects of its utility steps, and their therapeutic effects on liver fibrosis to address questions regarding efficacy and gaps in the knowledge, opening a new path toward further studies (Figure 1).

LIVER FIBROSISLiver fibrosis is the extreme accumulation of extracellular matrix (ECM) proteins, including collagen, and appears in most chronic liver diseases. Distinct types of hepatotoxic agents produce mediators that induce inflammatory actions in hepatic cell types. Following chronic liver injury, symptoms associated with advanced hepatic fibrosis will appear. When advanced, liver fibrosis results in cirrhosis, liver failure, and portal hypertension, often requiring liver transplantation[5]. Alternatively, it can be resolved if the underlying cause is removed or through the use of an antifibrotic drug or cell therapy (Figure 2). It is possibly a reversible response that resulted from either hepatic insults generated by different chronic diseases, such as nonalcoholic fatty liver








Figure 1 Introduction of the main parts of the review. First, bone marrow-derived mesenchymal stem cells (BM-MSCs) are introduced as a therapy for liver fibrosis, then the steps before transplantation (culture, strategies, and the choice of transplantation route) are discussed. After that, the efficiency of BM-MSCs for liver fibrosis in vivo are explained through studies that research the efficiency, studies that compare the therapy to other medication sources, and the strategies to enhance the therapeutic efficiency.

disease[6] or repetitive chronic liver injury induced by hepatitis, fat deposition, and continued alcohol consumption[7,8]; for both, the liver may accumulate aberrant myofibroblasts and ECM thus generating liver fibrosis. Depending on the inducing liver disease, liver fibrosis pathogenesis differs; for example, schistosomiasis induces liver fibrosis by accumulating parasitic ova and periocular granulomas in portal veins[9]. Wilson’s disease (or hepatolenticular degeneration), caused by a mutation in the Wilson disease protein (ATP7B) gene, frequently induces liver fibrosis[10]. Furthermore, it has recently been found that metabolic syndromes, including obesity, insulin resistance, and diabetes, are closely related to end-stage liver fibrosis[11].

Physiologically speaking, liver fibrosis is a healing response to liver injury. It is characterized by excessive deposition of ECM proteins as an outcome of different chronic liver diseases, including viral hepatitis and alcoholic or nonalcoholic steatohepatitis[5,12]. Liver fibrosis is beneficial at first because it can encapsulate the injury and is considered a reversible process at this stage[13,14]; however, it ultimately develops into advanced fibrosis or cirrhosis, which might be irreversible and impairs liver function that leads to subsequent morbidity and mortality[15].

After a severe liver injury, parenchymal cells regenerate and substitute the necrotic or apoptotic cells associated with an inflammatory response and an incomplete ECM deposition. The liver regeneration fails if the hepatic injury persists, and hepatocytes are replaced with abundant ECM containing fibrillar collagen. Depending on the origin of the liver injury, the distribution of this fibrous material differs. In chronic cholestatic disorders and chronic viral hepatitis, the fibrotic tissue is first located around portal tracts. In alcohol-induced liver disease, it is instead situated in pericentral and perisinusoidal areas[16]. Liver fibrosis is related to significant alterations



Figure 2 The transition from a healthy liver to liver fibrosis. Different types of hepatotoxic agents produce mediators that induce inflammatory actions in hepatic cell types. Following chronic liver injury, symptoms associated with advanced hepatic fibrosis will appear. This can either lead to liver cirrhosis, liver failure, and portal hypertension or can be resolved under the conditions mentioned. HCV: Hepatitis C virus; LPS: Lipopolysaccharide.

in the quantity and composition of ECM[17]. In advanced stages, the liver holds about six times more ECM than ordinary, including collagens (I, III, and IV), fibronectin, undulin, elastin, laminin, hyaluronan, and proteoglycans[18]. Decreased activity of ECM-removing matrix metalloproteinases (MMPs) is mainly related to the overexpression of their inhibitors (i.e. TIMPs)[12].

Succeeding a chronic injury, HSCs, the primary ECM-producing cells[19], activate and transdifferentiate into myofibroblast-like cells, acquiring contractile, proinflammatory, and fibrogenic properties[20,21]. The activated HSCs accumulate at the spots of tissue repair, discharging significant ECM amounts and regulating ECM degradation. Kupffer cells (KCs) are the primary producer of PDGF, which is the main mitogen for activated HSCs. At the transcriptional and posttranscriptional levels, collagen synthesis in HSCs is regulated[22].

There is a complex interplay among different hepatic cell types the occurs during hepatic fibrogenesis. Many hepatotoxic agents can damage hepatocytes[23]; these damaged hepatocytes release reactive oxygen species (ROS) and fibrogenic mediators and induce white blood cell recruitment via inflammatory cells. Apoptosis of damaged hepatocytes stimulates the fibrogenic actions of liver myofibroblasts[23]. Inflammatory cells activate HSCs to secret collagen, emit inflammatory chemokines, and modulate lymphocyte activation[24,25]. Consequently, a vicious circle of inflammatory and fibrogenic cells stimulating each other occurs[26]. Fibrosis is affected by different T helper subsets, with the Th2 response being associated with more active fibrogenesis[27]. KCs play a main role in liver inflammation by releasing ROS and cytokines[28,29]. Also, changes in the composition of the ECM can directly promote fibrogenesis. Fibrinogen, type IV collagen, and urokinase-type plasminogen activator stimulate resident HSCs by activating latent cytokines, such as transforming growth factor (TGF)-β1[30]. Fibrillar collagens can attach and stimulate HSCs via the discoidin domain receptor and integrins. Furthermore, altered ECM can act as a reservoir for growth factors and MMPs[31].

BM-MSCSIdentification of MSCsModern science has witnessed an essential thrust in stem cell research[32], identifying their presence in limited amounts in adult tissues, such as adipose tissue (AD-MSCs)[33,34], umbil ical cord (UC) tissue[35], amniotic fluid[36,37], breast milk[38,39], synovium[40], BM-MSCs[41], placental cells[42], dental pulp[43], lung, and liver (both adult and fetal)[44]. They are multipotent cells capable of differentiating into distinct cell groups, such as hepatocytes[45].



The therapeutic eminence index represents the amount of research that has advanced into clinical trials in the last 10 years, based on the Macrin et al[46] study (Figure 3). The eminent sources of adult MSCs are ordered following their therapeutic eminence index and are presented as follows: UC is the most eminent, then comes the placenta, AD, endometrium, dental pulp, and dermis successively, and the least eminent sources are amniotic fluid, synovium, and breast milk. Moreover, the cell types into which the isolated MSCs can differentiate vary, ranging from neurons and enterocytes to osteocytes and chondrocytes[47], etc. Among the eminent MSCs, three primary sources are capable of treating liver disease, namely the BM-MSCs, UC-MSCs, and AD-MSCs. Usually, MSCs derived from these sources express no signicant differences concerning the morphology and immune phenotype[47].

According to the research by Liu et al[48], the choice of MSCs should be related to the function and repair potentiality of the liver. Therefore, BM was selected as the best source compared to the three most capable sources to treat liver diseases. Accordingly, we decided to concentrate on BM-derived MSCs; as promising as this therapy can be, there are still many aspects of this therapy that need to be investigated.

Identification of BM-MSCsAs presented in Figure 4, the BM consists of two different cell lineages: the hematopoietic tissue cells and the associated stromal cells[49]. BM contains more than one stem cell population, and these include: (1) Hematopoietic stem cells and endothelial progenitor cells (EPCs) obtained through flow cytometric cell sorting (known as FACS) according to cell surface markers; (2) Side population cells present in the subpopulation as side scatters on FACS plot, owing to their ability to efflux Hoechst 33342 dye; (3) MSCs; and (4) Multipotent adult progenitor cells, which are derived through the characterization of adherent cell populations.

Isolation and expansion of BM-MSCs involve aspiration of the iliac crest followed by separation of the mononuclear cell fraction by density-gradient centrifugation and plating for expansion. BM-MSCs can differentiate into ectodermal cell lineages that include neurons, endodermal cell lineages, such as hepatocytes[50], and mesodermal lineages, such as myocytes, chondrocytes, osteocytes, and adipocytes[51]. Considering this differentiation capacity, several possible applications of BM-MSCs have been suggested, tested, and studied[46].

There is a presence of pluripotency markers in BM-MSCs, suggesting that they can differentiate into cell lineages of all three germ layers. These surface marker expression levels and transcription factors play a significant role in distinguishing the stem cell populations[46]. Besides the regenerative and differentiation potentials of MSCs, the immunosuppressive and immunomodulatory properties are critical to their use in cellular therapy[52].

The potential contribution of BM-MSCs to liver fibrosis is presented in Figure 5. Each of the presented elements is a different mechanism that has a specific role that can alleviate liver fibrosis, such as the higher differentiation of AFP, CK18, and CK19, the activation of HSCs, and the higher mobility of KCs. The most dominant axis seems to be transdifferentiation to a collagen-producing myofibroblast cell population. However, other factors can also show a potential contribution to liver fibrosis.

BM-MSCs are known to express the MHC class I antigen but not the MHC class II antigen[53,54]. The coculture of BM-MSCs and HSCs inhibited the proliferation of HSCs and promoted cell apoptosis of HSCs through downregulating the E3 ligase S phase kinase associated protein 2 level, attenuating ubiquitination and increasing the stability of p27[55]. Moreover, BM-MSCs produce various growth factors and cytokines with anti-inflammatory effects in vitro and in vivo to inverse the fibrotic liver state. As transplantation of MSCs upsurges, the serum levels of vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF), IL-10, and MMP-9 increase in injured livers[56].

BM-MSCs attenuate hepatic fibrosis in vivo by decreased serum levels of collagen I, collagen IV, type III procollagen, hyaluronic acid, laminin, downregulated liver collagen proportionate area, hepatic hydroxyproline, and liver α-smooth muscle actin (SMA). This improvement is accompanied by reduced hepatic levels of TGF-β1, decreased expression of serum TGF-β1, Smad3, and Smad4 but increased Smad7 expression[57,58]. BM-MSCs significantly ameliorate liver fibrosis in mice via stimulation of interferon-γ and inhibition of lymphocyte proliferation; the BM-MSCs also significantly decreased the number of IL-17 producing Th17 cells and the serum level of inflammatory IL-17 while increasing the serum levels of kynurenine, immunosuppressive IL-10, indoleamine 2,3-dioxygenase, and a number of CD4+ IL-10+ T cells to attenuate liver fibrosis[59].

BM-MSCs are also confronting various challenges to reach clinical application



Figure 3 Eminent sources of adult mesenchymal stem cells and the cells into which they can differentiate. Eminent sources of adult mesenchymal stem cells are ordered through their therapeutic eminence index. Umbilical cord is the most eminent, followed by the placenta, adipose tissue, endometrium, dental pulp, and dermis. The least eminent sources are amniotic fluid, synovium, and breast milk. The cell types, such as neurons, enterocytes, osteocytes, and chondrocytes, into which the isolated mesenchymal stem cells can differentiate are variant.

requirements, such as the highly invasive donation procedure, the decline in MSC number and differentiation potential with increasing age, demands of a large number of cells for therapy, heterogeneic character of cell quality, low survival ability after transplantation, the weakening of MSC capacities in two-dimensional (2D) culture, and unclear mechanism of MSC function for disease therapy. An essential need for MSC therapy is to produce enough high-quality MSCs in vitro to meet clinical demand.

PRE-TRANSPLANTATION STEPSCultureAs explained in Figure 6, different methods are used to culture stem cells; the general way is to culture MSCs in 2D dishes as a monolayer for fast expansion. This method conjures changes in MSCs, including cellular senescence, immunogenicity, losses of their stemness properties and paracrine activity, genetic expression of cells, and altered inner structure of cells[60,61]. The second way is the three-dimensional (3D) culture, which artificially creates an environment in which cells can interact or grow with their surroundings in all three dimensions. Thus, 3D culture is regarded as a more suitable and closer physiological microenvironment for cell growth[62,63]. There are numerous 3D culture methods developed to form MSC spheroids, such as



Figure 4 Bone marrow extracted cells. Bone marrow contains a variety of stem cell populations that can be extracted, either through specific growth factor media, such as that for multipotent adult progenitor cells (MAPCs) and mesenchymal stem cells (MSCs) or through flow cytometric cell sorting (FACS) technology, such as for the endothelial progenitor cells (EPCs), hematopoietic stem cells (HPSCs), and side population cells (SPs).

hanging-drop, magnetic levitation, chitosan membrane culture, microgravity bioreactor, and rotating culture[1,64]. These methods provide cells with a suspension culture condition where the 3D spheroids were formed mainly relying on cell-cell adhesion and interaction that promoted the self-assembly tendency of MSCs.

There are two main types of spheroid used for the 3D culture. The first of these is initially formed and derived from the aggregation of many individual cells and is named multiple cells-derived spheroid (MCDS). Growing evidence has shown that, in comparison to 2D culture, 3D MCDS culture enhances the characteristics of MSCs on cell survival, factor secretion, stemness maintenance, migration, and antisenescence in vitro and improves the capacities of anti-inflammation, angiogenesis, tissue repair, and regeneration in vivo[65,66]. However, despite the many advantages reported, visible defects restrict the direct application of MCDS-cultured MSCs in the clinic. These include the heterogeneity of cell quality in the whole spheroid, the multitudinous presence of individual MSCs with distinct viabilities, and the large size of MCDS resulting in different distributions of nutrients, oxygen, and waste metabolism between the core and periphery of the spheroid; moreover, the cells in the core are subjected to hostile metabolic stresses and tend to undergo apoptosis[67]. The large size (diameter > 100 μm) makes MCDSs unable to be directly injected into the body, as it poses risk of blood vessel blockage. So, the MCDSs generally must be dissociated into single cells by an enzymatic process before vein injection, but this affects the cells by causing damage and impairing viability[68].

The second type of spheroid is formed through a single cell-derived sphere (SCDS), based on the report by Qiao et al[69]. This formation can enhance the effectiveness of UC-MSCs thereby optimizing the quality of MSCs to meet the demand of the clinical application. In vitro and in vivo results have indicated that compared to 2D and MCDS cultures SCDS culture possesses some advantages for MSCs optimization, such as in cell stemness properties, survival ability, and therapeutic potential. However, despite this, there are still some questions that need to be explored further in the future; in particular, these questions involve the effects of SCDS culture on immunomodulatory capacities, inflammatory response, paracrine capacities, and cellular metabolism. Whether SCDS culture could markedly optimize BM-MSCs for potentially meeting the demand for clinical application also remains an unanswered question. In general, after cell transplantation, only a small number of MSCs migrate to injured tissues, so various studies have investigated effective strategies for improving the survival rate



Figure 5 Potential contributions of bone marrow-derived mesenchymal stem cells to liver fibrosis. Each of the presented elements represent a distinct mechanism that has a specific role that can contribute to alleviating liver fibrosis. BM-MSCs: Bone marrow-derived mesenchymal stem cells; HSCs: Hepatic stellate cells; KC: Kupffer cells.

and activity of MSCs to treat liver fibrosis.

Strategies to improve MSCs efficiencyBM-MSCs have limited viability, with as low as < 1% of transplanted cells predicated to survive. Inflexibility of the microenvironment encountered upon transplantation may be the cause[70]. Various strategies have been developed and implemented to improve cell therapy. In this section, we will focus on: genetic engineering and the preconditioning used during the culture phase; tissue engineering used on a 3D matrix and involving signaling molecules; and cell-free therapy achieved through the use of exomes (Ex) and microvesicles (MVs) (Figure 7).

Genetic engineering: BM-MSCs have also been genetically engineered to overexpress the desired gene to improve their therapeutic efficacy further. They can be used for the targeted delivery of therapeutic gene products as gene therapy. The genes capable of manipulation could be genes encoding receptors, growth factors, and cytokines. Genetically-engineered BM-MSCs have been applied as treatment to a range of genetic and acquired diseases. Genetic modification of BM-MSCs improves their therapeutic potential by enhancing various cellular features, like endurance and survival of the transplanted BM-MSC, angiogenesis, differentiation, homing, and anti-inflammatory effects[71].

This strategy investigated approaches to promote the expression of proteins involved in the homing of donor cells[72]. MSCs express low levels of molecules, including the homing factor stromal cell-derived factor-1 (SDF-1) and chemokine receptors[73]. Genetic manipulation of prosurvival or antiapoptosis genes have been



Figure 6 Two main types of mesenchymal stem cell culture. The two-dimensional (2D) culture using 2D dishes as a monolayer for fast expansion, and the three-dimensional (3D) culture with two main types of spheroid use: multiple cell-derived spheroid and single cell-derived sphere. MSCs: Mesenchymal stem cells.

shown to increase BM-MSC survival in vivo[74]. Through modulation of cellular networks, microRNAs can regulate mRNAs, including those involved in cell survival. MicroRNA overexpression can enhance BM-MSC survival[75]. Nonetheless, this strategy presents many risks, including carcinogenesis, that should be carefully considered when applying genetic manipulations.

Tissue engineering: Strategies that allow for BM-MSC homing and adaptation in the liver before initiating their regeneration will help improve cell survival. Several approaches have been investigated, involving coculture and the development of 3D systems that can involve a scaffold-based or scaffold-free system[76,77]. Cells grown in 3D systems would behave more like cells in vivo and could be implanted directly. Numerous synthetic polymers as well as natural materials have been assessed for their ability to raise the expression of hepatocyte-specific genes in BM-MSCs through hepatic differentiation[78]. The most significant performance effect was observed when a 1:5 ratio of BM-MSCs to hepatocytes was used both in vitro and in vivo[79]. Decellularized tissue is another system in use for tissue engineering; the decellularized liver tissue forms an ECM scaffold, improving MSC engraftment by offering a more physiological environment[80].

Preconditioning: Priming methods avoid genetic and chemical modifications entirely by altering culture conditions to influence gene expression[81]. These methods have been used to improve the tethering, activation, and transmigration steps of systemic homing. Preconditioning improves the survival signals and resistance of MSCs against stress and insults in the pathological environment[82]. In the preconditioning process, BM-MSCs can be pretreated or exposed to a sublethal dose of various insults, such as apoptotic cascade activation, hypoxia, toxins, ROS, inflammatory response, autophagy, and many others. Furthermore, preconditioning can enhance cell survival following the transplantation because it considerably induces therapeutic benefits of BM-MSCs by increasing the potential of cell differentiation and its paracrine protective effect, improving migration and homing of BM-MSCs to the lesion site, increasing regenerative and repair potentials, and suppressing inflammatory and immune responses that occur after transplantation[83]. Many preconditioning strategies involve



Figure 7 Strategies to enhance bone marrow-derived mesenchymal stem cell therapeutic efficiency. Genetic engineering and preconditioning used during the culture phase; tissue engineering used on a three-dimensional (3D) matrix and involving signaling molecules; cell-free therapy through the use of exomes and microvesicles. MSCs: Mesenchymal stem cells.

exposing cells to a physical or an environmental shock and/or pharmacological modulators of targeted molecules[83,84]; the following three strategies exemplify such.

The first is a thermal preconditioning strategy carried out at 42 °C for 1-2 h before transplantation. It has been demonstrated to promote cell survival in vivo, and this outcome is related to the induction of heat shock protein expression, which inhibits apoptotic pathways[85,86].

The second is a hypoxic preconditioning strategy based upon the knowledge that hypoxia can promote defense mechanisms against oxidative stress. Hypoxia is a significant feature of MSCs; it plays a vital role in maintaining stem cell fate, self-renewal, and multipotency. Cultivating MSCs under hypoxia is an essential preconditioning step because it mimics the natural microenvironment of BM. The reaction of MSCs to hypoxic conditions is contradictory, however, indicating both damaging and ameliorating effects.

The third is a pharmacologic strategy to maintain cell viability after transplantation. This process includes the use of antioxidants and HIF-1α stabilizers to contribute to cell survival, as well as antimycin and mitochondrial electron transport inhibitors to promote cell survival by activating mitochondrial death pathways[87].

Extracellular vesicles as a cell-free therapyWorries regarding the use of MSCs as a cellular therapeutic approach for the liver include their potential for aberrant differentiation, the peril of tumor formation, and



the half-life of transplanted MSCs inadequate for tissue regeneration by differentiation[88]. To deal with these issues, the MSC secretome has been introduced as an acellular alternative therapy. Indeed, these soluble proteins or extracellular vesicles residing among the BM-MSCs and released by paracrine mechanisms could be a practical option and offer numerous advantages compared to the use of cellular therapies for liver diseases[89].

BM-MSCs can also release more elaborate structures, called extracellular vehicles (EVs)[90]. These EVs can be engineered to enhance anticipated activities or introduce specific effector molecules[91,92]. MSC-derived EVs were shown to improve hepatic injury and inflammation[93]. EVs from human MSCs preserve at least some of the immunomodulatory properties of the cells. MSC-derived induced pluripotent stem cell-EVs hold the EV characteristics that are usually obtained from tissue-derived MSCs, regardless of origin[94]. EVs could be a better therapeutic strategy because they characterize a physically different fraction and transport signals with more predictable effects. Although, the complex functions of EVs are still mostly undiscovered. Additional studies are needed to determine how long-circulating MSC-EVs survive after administration and what recognition pathways are used by the target cells.

Choice of transplantation routesMany BM-MSC transplantation routes can be used for liver disease, in general. Some of the routes are direct, such as the portal vein and the hepatic artery; others are indirect routes, such as the peripheral vein, intrasplenic, intraperitoneal, BM reconstitution, and extra-corporeal liver assist device (Figure 8).

BM-MSC transplantation routes can affect the therapy’s potential because they conceptually represent the simplest method to improve MSCs homing by administrating the cells at or near the target tissue instead of infusing them through standard intravenous routes. It may seem intuitive that direct delivery of MSCs to a target tissue could result in higher retention[95].

There are conflicting data about the engraftment of transplanted BM-MSCs, and some concerns around the fibrogenic potential have been raised. Unwanted effects may depend on the route and dose of BM-MSC infusion[96,97]. Different routes can be used to transplant BM-MSCs, such as intravenous, intraperitoneal, intrahepatic, intrasplenic, or portal vein injection, but the effectiveness varies depending on the injection route. The peripheral vein is the most common transplantation route followed by the hepatic artery, intrasplenic, intrahepatic and portal vein injection. It has been shown that BM-MSCs administered through the peripheral vein migrate well into the liver parenchyma in chronic injury in vivo. Simultaneously, limited BM-MSC engraftment has been observed in an acute injury environment[98].

Furthermore, BM-MSCs endured in liver tissues when injected through the intrahepatic artery, indicating that BM-MSCs were present without differentiating into hepatocytes. Moreover, the intraportal infusion was found to be more efficient than the peripheral route in clinical trials. However, direct approaches, such as via the portal vein or hepatic artery, carry a risk of portal hypertensive bleeding following cell injection[99]. Generally, the lines of evidence provided by most of these clinical studies have been lacking.

EFFICIENCY OF BM-MSCS FOR LIVER FIBROSIS IN VIVOSeveral in vivo studies have continued to prove the efficiency of BM-MSCs in attenuating liver fibrosis induced by tioacetamide or carbon tetrachloride. These studies have investigated the efficiency and role of MSCs in liver fibrosis to elucidate the mechanism underlying the mobilization and function of BM-MSCs.

Mehrabani et al[100] investigated the regenerative effect of BM-MSCs in a rat model of liver fibrosis induced by tioacetamide. The study demonstrated that BM-MSCs could open a new window and be a therapy of choice in the amelioration of liver fibrosis because it alleviated liver fibrosis through the antifibrotic potential of BM-MSCs. The paracrine and endocrine functions of BM-MSCs also underlay the efficacy of these cells in the amelioration of liver damage through reducing inflammatory cells in the hepatic tissue and decreasing the alanine aminotransferase level.

Another study investigated the link between natural killer cells and liver fibrosis and their link to regenerative medicine. It was found that BM-MSCs alleviated liver fibrosis through suppressing the inflammatory response and the local proinflammatory cytokines. A significant increase in intrahepatic natural killer cells was also noted upon BM-MSC treatment[101]. BM-MSCs attenuate liver brosis by



Figure 8 Bone marrow-derived mesenchymal stem cell transplantation routes for liver disease. The routes are either direct (portal vein and hepatic artery) or indirect (peripheral vein, intrasplenic, intraperitoneal, bone marrow reconstitution, and extra-corporeal liver assist). BM-MSCs: Bone marrow-derived mesenchymal stem cells.

decreasing serum levels of the inammatory cytokine IL-17, increasing the immunosuppressive cytokine IL-10 and the related factors indoleamine 2,3-dioxygenase and kynurenine, reducing the number of IL-17-producing Th17 cells, and expanding the percentage of CD4+ IL-10+ T cells[59].

The BM-MSCs have also been shown to improve thioacetamide-induced liver fibrosis in rats by remolding the collagen bers, which could be lysed by MMPs, namely MMP-2-mediated degradation of the ECM. PKH26-labeled BM-MSCs were seeded into liver tissue and found to differentiate into healthy cells replacing the damaged ones with either hepatocytes or cholangiocytes. The reduction in α-SMA expression that was observed reected a diminution in the number of activated HSCs[102].

BM-MSCs might play an immunomodulatory role in treating liver fibrosis through the down-regulation of IL-17A, affecting the IL-6/STAT3 signaling pathway. It was noted that after the treatment there was modulation of the cytokine milieu and among the signal transducers, including a significant downregulation of the genes encoding cytokines IL-17A, IL-17RA, IL-17F, and IL-17RC. In accordance with the BM-MSC administration was a decline in IL-17, IL-2, and IL-6 serum proteins and downregulation in the IL-17A and IL-17RA proteins in liver tissue. The BM-MSC administration also resulted in downregulation of both Stat3 mRNA expression and p-STAT3 protein as well as Stat5a gene expression and p-SMAD3 and TGF-βR2 proteins and elevated p-STAT5 protein[103].

It was also evidenced that the SDF-1α/CXCR4, which is essential among the chemotactic axis regulating MSC migration from BM to fibrotic liver, can attenuate



liver damage and thus alleviate liver fibrosis[104]. That same study identified VEGF as the key cytokine that contributes to MSC proliferation. These results provide further evidence for the role of MSCs in liver fibrosis and help to elucidate the mechanism underlying MSC mobilization under the condition of carbon tetrachloride-induced liver injury (the model system used in the study).

Transplantation of stem cells, including BM-MSCs, has proven to be competent for repairing fibrotic livers. The underlying mechanism promotes hepatocyte transdifferentiation and hepatocyte proliferation while inhibiting activated hepatic stellate cells, upregulating the activity of MMPs, and promoting neovascularization in liver tissues[105].

Considering that BM-MSCs have demonstrated a strong proliferative ability, multilineage potential, and no ethical considerations for widespread application to repair various organ injuries, they are currently transplanted in vivo to reduce hepatocyte apoptosis and promote hepatocyte regeneration[106]. Thus, in this review, we focused on the in vivo research in the literature starting from the year 2015 to address these two main issues: (1) The therapeutic efficiency of BM-MSCs compared to other types of treatments; and (2) The possibility to enhance the therapeutic efficiency of BM-MSCs through various strategies.

Therapeutic efficiency of BM-MSCs compared to other treatments and stem cell sourcesThe therapeutic efficiency of BM-MSCs depends on various factors, but there remains a need to compare this efficiency to that of other treatments and other MSC sources. In Table 1[107-116], we present those studies in the literature that have compared BM-MSC efficiency to other existing therapies and other sources up to the year 2015.

We noted that compared to such standard treatments as resveratrol and silybum marianum the regenerative capabilities and resolution of hepatic fibrosis was higher for the BM-MSCs. Treatment with BM-MSCs enhanced the liver state more effectively than either of the two drugs. It also significantly decreased levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, malondialdehyde, TNF-α, and CYP450 and increased levels of albumin, superoxide dismutase, glutathione, glutathione S-transferase, and catalase. BM-MSCs could also reestablish liver structure and function, ameliorating the toxicity of carbon tetrachloride and improving liver function tests[107].

In other cases, drug treatment with imatinib, simvastatin, and decorin was found to be more efficient when used concomitant to BM-MSCs. For example, with imatinib the single treatment and combination therapy significantly reduced serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase and downregulated α-SMA, procollagen I, procollagen III, collagen IV, and laminin. However, in pathological observations the highest therapeutic potential was achieved with a combination of BM-MSCs and imatinib[108]. Also, compared to simvastatin and decorin the cotreatment exhibited better histological improvement than was achieved with simvastatin or decorin alone. The combination treatment also lowered the hydroxyproline content, decreased hepatic collagen distribution, and rescued liver function impairment. The upregulation of α-SMA, collagen-1, TGF-b1, and p-Smad3 was prevented by the administration of the cotreatment, which exerted strong protective effects against hepatic fibrosis[109,110].

Depending on the source, the efficiency of MSCs differ; for instance, compared to human UC CD34+ cells, both the CD34+ cells and BM-MSCs have the same efficacy in significantly reducing TNF-α. Nevertheless, concerning liver function and gene expression, the UC CD34+ cells were more efficient in elevating albumin and reducing alanine aminotransferase concentrations and gene expression of collagen Iα, TGF-β1, α-SMA, albumin, and MMP-9. Thus, human UC CD34+ stem cells were deemed more efficient than the BM-MSCs[111].

While the UC-MSCs present similar effectiveness to BM-MSCs, levels were also compared to UC-EPCs and AD-MSCs. The UC-EPCs showed higher MMP-2 and VEGF gene expression than BM-MSCs. Moreover, the UC-EPCs were more effective than BM-MSCs in increasing gene expression of HGF, α-SMA, and Ki-67. The UC-EPCs also showed significantly higher TGF-β than BM-MSCs[112]. Comparison of BM-MSCs and AD-MSCs showed them to be similarly efficient at attenuating liver fibrosis, both using a mechanism that involves inhibiting the activation and proliferation of HSCs and boosting apoptosis of HSCs. The AD-MSCs may be a better candidate than BM-MSCs for cell-based therapy to treat liver fibrosis because they improved the anti-inflammatory and antifibrotic effects to a slightly greater extent than the BM-MSCs, they are easier to prepare, and they are more effective at inhibiting HSC proliferation



Table 1 Bone marrow-derived mesenchymal stem cell therapeutic efficiency compared to other treatments and other stem cell sources

Ref. Year Pathogenesis Model Route BM-MSCs compared to Effect on liver fibrosis Efficiency

comparison[107] 2020 CCl4 Rats Penile vein Standard therapy:

resveratrol and silybum marianum

Decreased AST, ALT, MDA, ALP, TNF-α, and CYP450 and increased albumin, SOD, GSH, GST, and CAT

BM-MSCs were more efficient

Restored liver structure and function and markedly decreased the induced liver fibrosis

[108] 2020 CCl4 Rats Intravenous Imatinib High therapeutic potential of utilizing BM-MSCs and imatinib, either individually or combined

Combined treatment was the most efficient

Reduced serum levels of ALT, AST, and ALP concomitantly

Downregulated α-SMA, procollagen I, procollagen III, collagen IV, and laminin

[109] 2018 TAA Rats Right lobe of the liver

Simvastatin Reduced TGF-β1, α-SMA, and collagen-1 expression

Combined treatment was more efficient

Inhibited TGF-β/Smad signaling

Sim-MSCs strongly inhibited the progression of TAA-induced hepatic fibrosis

[110] 2016 TAA Rats Intrahepatic Decorin DCN and BM-MSCs alleviated liver fibrosis through: (1) decreased proliferation of HSCs; (2) suppressed TGF-β/Smad signaling; and (3) antifibrotic effect

Combined treatment was more efficient

[111] 2016 CCl4 Rats Intravenous Endothelial progenitor cells

Elevated albumin and reduced ALT concentrations

No statistically significant difference

UC-EPCs were more valuable in increasing gene expression of HGF and immunohistochemistry of α-SMA and Ki-67; BM-MSCs had significantly lower TGF-β compared to UC-EPCs

[112] 2020 CCl4 Rats Tail vein Human UC CD34+

Expressing liver-specific genes BM-MSCs were less efficient

Decreased gene expression of profibrotic genes (collagen Iα, TGFβ1, α-SMA) and of albumin

Increased antifibrotic gene (MMP-9) expression and decreased proinflammatory gene (TNF-α) expression

Reduced ALT concentration

[113] 2017 CCl4 Rats Intravenous WJ-MSCs Decreased hepatic hydroxyproline content and the percentage of collagen proportionately

BM-MSCs were more efficient

Reduced α-SMA and myofibroblasts

Increased number of EpCAM+ hepatic progenitor cells along with Ki-67+ and human matrix metalloprotease-1+ (MMP-1+) cells

[114] 2017 CCI4 Rats Portal vein AD-MSCs Prevented activation and proliferation of HSCs, and promoted apoptosis of HSCs

Similar efficiency



Implantation of AD-MSCs exhibited slightly improved anti-inflammatory and antiliver fibrotic activities compared to BM-MSCs

[115] 2018 CCl4 Rats Intravenous and intrasplenic

Intravenous and intrasplenic route

Elevated serum albumin levels and reduced serum ALT levels

Intravenous route was more efficient

Decreased inflammation by reducing the gene expression of proinflammatory cytokines (IL-1β, IL-6, and INF-γ)

An antifibrotic effect via reduced profibrogenic factors (TGF-β1, α-SMA, CTGF) and increased antifibrogenic factors (CK18, HGF)

Increased VEGF protein levels

[116] 2016 CCl4 Mice Portal and tail vein

Tail and portal vein route

Reduced AST/ALT levels There were no efficiency differences

Stimulated positive changes in serum bilirubin and albumin

Downregulated expression of integrins (600-7000-fold), TGF, and procollagen-α1

α-SMA: Alpha-smooth muscle actin; a-SMA: Anti-alpha-smooth muscle actin; AD-MSCs: Adipose-derived mesenchymal stem cells; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; BM-MSCs: Bone marrow-mesenchymal stem cells; CAT: Catalase; CCl4: Carbon tetrachloride; CK18: Cytokeratin 18; CTGF: Connective tissue growth factor; CYP450: Cytochrome P450; DCN: Decorin; GSH: Glutathione reductase; GST: Glutathione S-transferase; HGF: Hepatocyte growth factor; HSCs: Hepatic stellate cells; IL-1β: Interleukin-1β; IL-6: Interleukin 6; MDA: Malondialdehyde; MMP: Matrix metalloproteinase; MSCs: Mesenchymal stem cells; Sim-MSCs: Simvastatin-mesenchymal stem cells; SOD: Superoxide dismutase; TAA: Thioacetamide; TGF-β: Transforming growth factor-beta; TNF-α: Tumor necrosis factor-alpha; UC CD34+: Umbilical cord blood CD34+; UC-EPCs: Umbilical cord-endothelial progenitor cells; WJ-MSCs: Wharton’s jelly-derived mesenchymal stem cells; VEGF: Vascular endothelial growth factor.

and apoptosis in the coculture system. However, the slight improvement in anti-inflammatory and antifibrotic effects did not reach the threshold of statistically significant difference[113,114].

The transplantation route also impacts BM-MSC competence. This is not the case for all though, as there are no notable differences in the impact from portal and tail vein injections. The former involves different procollagen gene expression than the latter; nevertheless, the liver serum markers and liver histology classification show no differences postinjection. Remarkably, there are also no differences in treatment effects for those two administrations. When considering safety, though, BM-MSC transfusion via a peripheral vein is a safer potential method. In other cases, the impact results differ; for example, the intravenous route is remarkably more efficient than the intrasplenic one. Although both routes achieve a similar enhancement of liver function, the intravenous route provides greater reduction in cytokine gene expression levels (IL-1β, IL-6, and INF-γ)[115,116].

As a result, we note that although BM-MSCs are more efficient than other treatments, combination treatment with other therapies can itself become a strategy to enhance the therapeutic potential of BM-MSCs. We also want to point out that even though BM-MSCs are considered the most significant source of stem cells and are more efficient in some cases, there should be more studies to consider the potential of other sources potential. Lastly, many possible transplantation routes can affect the therapeutic impact of BM-MSCs on liver fibrosis, but we have observed a lack of studies that research the advantages and disadvantages of these routes.

Possibility to enhance the therapeutic efficiency of BM-MSCsThe therapeutic efficiency of BM-MSCs as a treatment for liver fibrosis is influenced by numerous factors including culture conditions, delivery route, number of infused cells, gene modification of MSCs, and other potential factors. Hence, we herein arrange and analyze the current evidence related to BM-MSC transplantation in liver fibrosis and summarize the strategies for promoting the therapeutic efficiency of BM-MSC transplantation. We expect to develop other strategies to improve BM-MSC activities in vivo to restore liver function and alleviate liver fibrosis. Based on Table 2[117-129], the



Table 2 Strategies to enhance bone marrow-derived mesenchymal stem cell therapeutic efficiency

Ref. Year Pathogenesis Model Route Strategy Strategy efficiency[117] 2020 CCl4 Mice Tail vein Preconditioning: Autophagy regulation

in BM-MSCsBoosted antifibrotic potential primed by autophagy inhibition in BM-MSCs may be attributed to their suppressive effect on CD4+ and CD8+ lymphocytes infiltration and HSC proliferation, which were regulated by elevated PTGS2/PGE2 via a paracrine pathway

BM-MSC-based remedy in liver fibrosis and other inflammatory disorders

[118] 2019 CCL4 Rats Tail vein Preconditioning: Conditioned media Increasing antioxidant enzyme activity

Increased gene expression levels attenuated by CCl4 up to basal levels

Normalized the organization apart from some dilated sinusoids and vacuolated cells

Improved morphological, immunohistochemical, and biochemical measures

[119] 2016 CCl4 Rats Tail vein Preconditioning: With melatonin Enhanced homing ability of MSCs

Enhanced liver function

Enhanced the interaction of melatonin receptors and matrix enzymes

Expressed a high level of CD44

Ability to differentiate into adipocytes and Schwann cells

[120] 2017 CCI4 Rats Tail vein Preconditioning: With melatonin High ability of homing into the injured liver (P ≤ 0.05 vs BM-MSCs)

Higher percentage of glycogen storage but a lower percentage of collagen and lipid accumulation (P ≤ 0.05 vs CCl4 + BM-MSCs)

Low expression of TGF-β1 and Bax and lower content of serum ALT but higher expressions of MMPs and Bcl2

The effectiveness of MT preconditioning in cell therapy

[121] 2019 CCL4 Rats Tail vein Cell-free therapy: MSC-derived macrovesicles BM- MSC-MVs

Increased serum albumin levels and VEGF quantitative gene expression (P < 0.05)

Decreased serum ALT enzyme levels, quantitative gene expression of TGF-β, collagen-1α, and IL-1β

Decreased the collagen deposition and improvement of the histopathological picture

Antifibrotic, anti-inflammatory, and proangiogenic effects

[122] 2019 CCl4 Rats Tail vein Cell free therapy: hBM-MSCs-Ex Inhibition of Wnt/β-catenin signaling (PPARγ, Wnt10b, Wnt3a, β-catenin)

Downregulation of downstream gene expression (cyclin D1, WISP1)



[123] 2015 CCl4 Rats Intravenous Genetically modified BM-MSCs expressing TIMP-1-shRNA

Decreased TIMP-1 expression thereby regulating HSC survival

Decreased serum levels of ALT and AST, fibrotic areas, and collagens

Reduction of the fibrotic area

Restoration of the liver function

[124] 2020 CCl4 Mice Intraperitoneal injection MSCs expressing EPO Promoted cell viability and migration of BM-MSCs

Enhanced antibrotic efcacy with higher cell viability and stronger migration ability

Alleviated liver brosis

[125] 2015 BDL or CCl4 Mice Underneath the kidney capsule

Microencapsulated BM-MSCs Activated HSCs

Released antiapoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines

Decreased mRNA levels of collagen type I

Increased levels of MMPs

[126] 2018 CCl4 Rats Tail vein Genetically modified BM-MSCs with human MMP-1

Biochemical parameters and hepatic architecture improved

Decreased collagen content

Suppressed activation of HSCs

Improvement of both liver injury and fibrosis

[127] 2016 CCl4 Rats Tail vein Human urokinase-type plasminogen activator gene-modified BM-MSCs

Decreased serum levels of ALT, AST, total bilirubin, hyaluronic acid, laminin, and procollagen type III

Genetically modified BM-MSCs with human urokinase-type plasminogen activator

Increased levels of serum albumin

Downregulated both protein and mRNA expression of β-catenin, Wnt4, and Wnt5a

Decreased the Wnt signaling pathway

Decreased mRNA and protein expression of molecules involved in Wnt signaling thus working as an antifibrotic

[128] 2015 TAA Mice Tail vein Genetically modified BM-MSCs, MSCs engineered to produce IGF-I

Enhanced the effects of MSC transplantation

Decreased inflammatory responses



Decreased collagen deposition

Increased growth factor like-I, IGF-I, and HGF

Reduced fibrogenesis and the stimulation of hepatocellular proliferation

[129] 2017 CCl4, BDL Mice Intraperitoneal BM-MSCs triggered by sphingosine 1-phosphate

Increased HuR expression and cytoplasmic localization

S1P-induced migration of HBM-MSCs via S1PR3 and HuR

HuR regulated S1PR3 mRNA expression by binding with S1PR3 mRNA 3’ UTR

S1P-induced HuR phosphorylation and cytoplasmic translocation via S1PR3

HuR regulated S1PR3 expression by competing with miR-30e

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BDL: Bile duct ligation; BM-MSCs: Bone marrow-mesenchymal stem cells; CCl4: Carbon tetrachloride; EPO: Erythropoietin; hBM-MSCs-Ex: Human BM-MSCs-exomes; HBM: Human bone marrow; HGF: Hepatocyte growth factor; HSCs: Hepatic stellate cells; IGF-I: Insulin growth factor like-I; IL: Interleukin; MMPs: Matrix metalloproteinases; MSCs: Mesenchymal stem cells; MT: Melatonin; MVs: Microvesicles; PGE2: Prostaglandin E2; PPARγ: Peroxisome proliferator-activated receptor-gamma; PTGS2: Prostaglandin-endoperoxide synthase-2; S1P: Sphingosine 1-phosphate; S1PR3: Sphingosine-1-phosphate receptor 3; TGF: Transforming growth factor; TIMP-1: Tissue inhibitor of metalloproteases 1; UTR: Untranslated region; VEGF: Vascular endothelial growth factor; WISP1: Wnt-1-induced secreted protein 1.

overall strategies used have a positive therapeutic impact on liver fibrosis. Notable among the various strategies are preconditioning using autophagy regulation, microencapsulation, and preconditioned media.

BM-MSCs conditioned medium preparation provided a predominant therapeutic role in experimentally-induced chronic liver fibrosis as demonstrated by improved morphological, immunohistochemical, and biochemical measures[118]. Still, future studies should be carried out to further delineate the mechanisms underlying their action.

Cell-free therapy with BM-MSC-MVs and human BM-MSCs-Ex is another method by which the healing effect is enhanced as demonstrated by human BM-MSCs-Ex providing a meaningfully greater therapeutic effect than the human BM-MSCs. In fact, the human BM-MSCs-Ex effectively alleviated liver fibrosis as evidenced by reduced collagen accumulation, inhibition of inflammation, enhanced liver functionality, and increased hepatocyte regeneration. Besides, the administration of hBM-MSCs-Ex reduced liver fibrosis via inhibition of Wnt/β-catenin signaling to prevent HSC activation. Therefore, the use of hBM-MSCs-Ex presents a new and promising therapeutic strategy for hepatic disease in the clinical setting[122].

Adding to that, genetically-modified BM-MSCs expressing TIMP-1-short hairpin RNA or with human MMP-1 boosted the original effects of BM-MSCs through a mechanism that involved enhancing the antifibrotic potential as well as the anti-inflammatory and proangiogenic effects. Although BM-MSC administration reduced liver fibrosis, transplantation of the BM-MSCs/MMP-1 enhanced the reduction in liver fibrosis to a greater extent. Therapy with BMSCs/MMP-1 also reduced collagen



content and suppressed activation of HSCs in the fibrotic liver, leading to the subsequent improvement of liver injury and fibrosis[126].

In other cases, such strategies as preconditioning with melatonin enhanced the homing and transplantation of the BM-MSCs. Remarkably, melatonin-BM-MSCs showed better therapeutic outcomes, likely facilitated through a mechanism involving the improvement of cell homing and better maintenance of the balance between matrix degradation and accumulating factors that had a high ability to home into the injured liver. This could be related to a higher percentage of glycogen storage but a lower percentage of collagen and lipid accumulation. On the other hand, this could be due to lower expressions of TGF-β1 and Bax, and lower content of serum alanine aminotransferase but higher expressions of MMPs and Bcl2[120].

Simultaneously, other strategies, such as MSCs expressing EPO promoted cell viability and strengthened their migration ability to damaged cells. As an example, compared to BM-MSCs EPO-MSC treatment was found to promote cell viability and migration of the BM-MSCs and to enhance the antibrotic efcacy without inducing apoptosis[124]. This finding supported improving the efciency of MSC transplantation as a potential therapeutic strategy for liver brosis.

Overall, each strategy tackles specific aspects that can enhance the therapeutic potential of BM-MSCs, but there are no strategies that tackle several aspects at the same time. Therefore, there is a need to create strategies that can affect various aspects and mechanisms related to improving the potential of BM-MSCs as a therapy for liver fibrosis. Moreover, these strategies need to have sufficient safety, proficiency, and productivity features. It is also important to mention a deficiency in all types of strategies to enhance the therapy’s efficiency. Because there are many undiscovered and unclear aspects of the therapeutic mechanism of BM-MSCs for liver fibrosis, there is also room for new strategies to be discovered and researched.

Limitations and further studyThis review successfully studied the recent advances in therapeutic efficiency of BM-MSCs for liver fibrosis, focusing on the preclinical in vivo experiments while mentioning the gaps that still exist within the field. However, this review remains limited because it focused mostly on preclinical studies performed on animals; although these studies targeted many aspects of the topic, different targets still need to be addressed. This review did not address clinical studies, but it is crucial to address all the concerns regarding long-term follow-up exams for humans. Only then may we draw solid conclusions on the therapeutic effects of BM-MSC transplantation on liver fibrosis. Ultimately, such additional work will also help to further improve the therapeutic effects of BM-MSC transplantation for liver fibrosis, enhancing the quality of life and prolonging patient survival time with liver fibrosis.

CONCLUSIONVarious therapeutic methods are used to alleviate liver fibrosis; BM-MSCs are a promising therapy being investigated in vivo. This type of therapy has diverse advantages, including anti-inflammatory, self-renewal, and multipotency abilities. Thus, more and more studies are investigating the anti-inflammatory and immunomodulatory effects of BM-MSCs and focusing on comparing them with other stem cell sources and treatments to identify and develop an optimal treatment for the regression of liver fibrosis. Ongoing studies are focused on determining the different factors affecting the therapeutic efficiency of BM-MSCs, such as the transplantation route, where the portal vein route may be the optimum choice for restoring liver function in liver fibrosis, and the strategies used, including BM-MSC-based cell-free therapy and preconditioned, tissue-engineered, and genetic-engineered BM-MSC transplantation. These strategies have thus far presented promising results, but more research and experiments need to be done to find the optimum and most efficient strategy (or strategies) to enhance the therapeutic effect and be elevated to clinical trials.

REFERENCESJiang B, Yan L, Miao Z, Li E, Wong KH, Xu RH. Spheroidal formation preserves human stem cells for prolonged time under ambient conditions for facile storage and transportation. Biomaterials 2017; 133: 275-286 [PMID: 28460350 DOI: 10.1016/j.biomaterials.2017.03.050]

1


https://dx.doi.org/10.1016/j.biomaterials.2017.03.050



Kim G, Eom YW, Baik SK, Shin Y, Lim YL, Kim MY, Kwon SO, Chang SJ. Therapeutic Effects of Mesenchymal Stem Cells for Patients with Chronic Liver Diseases: Systematic Review and Meta-analysis. J Korean Med Sci 2015; 30: 1405-1415 [PMID: 26425036 DOI: 10.3346/jkms.2015.30.10.1405]

2

Secunda R, Vennila R, Mohanashankar AM, Rajasundari M, Jeswanth S, Surendran R. Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study. Cytotechnology 2015; 67: 793-807 [PMID: 24798808 DOI: 10.1007/s10616-014-9718-z]

3

Ji ST, Kim H, Yun J, Chung JS, Kwon SM. Promising Therapeutic Strategies for Mesenchymal Stem Cell-Based Cardiovascular Regeneration: From Cell Priming to Tissue Engineering. Stem Cells Int 2017; 2017: 3945403 [PMID: 28303152 DOI: 10.1155/2017/3945403]

4

Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115: 209-218 [PMID: 15690074 DOI: 10.1172/JCI24282]

5

Ebrahimi H, Naderian M, Sohrabpour AA. New Concepts on Pathogenesis and Diagnosis of Liver Fibrosis; A Review Article. Middle East J Dig Dis 2016; 8: 166-178 [PMID: 27698966 DOI: 10.15171/mejdd.2016.29]

6

Xu L, Gong Y, Wang B, Shi K, Hou Y, Wang L, Lin Z, Han Y, Lu L, Chen D, Lin X, Zeng Q, Feng W, Chen Y. Randomized trial of autologous bone marrow mesenchymal stem cells transplantation for hepatitis B virus cirrhosis: regulation of Treg/Th17 cells. J Gastroenterol Hepatol 2014; 29: 1620-1628 [PMID: 24942592 DOI: 10.1111/jgh.12653]

7

Sourianarayanane A, Arikapudi S, McCullough AJ, Humar A. Nonalcoholic steatohepatitis recurrence and rate of fibrosis progression following liver transplantation. Eur J Gastroenterol Hepatol 2017; 29: 481-487 [PMID: 28253211 DOI: 10.1097/MEG.0000000000000820]

8

Sombetzki M, Fuchs CD, Fickert P, Österreicher CH, Mueller M, Claudel T, Loebermann M, Engelmann R, Langner C, Sahin E, Schwinge D, Guenther ND, Schramm C, Mueller-Hilke B, Reisinger EC, Trauner M. 24-nor-ursodeoxycholic acid ameliorates inflammatory response and liver fibrosis in a murine model of hepatic schistosomiasis. J Hepatol 2015; 62: 871-878 [PMID: 25463533 DOI: 10.1016/j.jhep.2014.11.020]

9

Todorov T, Balakrishnan P, Savov A, Socha P, Schmidt HH. Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson’s Disease Pathogenesis. PLoS One 2016; 11: e0168372 [PMID: 27992490 DOI: 10.1371/journal.pone.0168372]

10

Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol 2013; 58: 593-608 [PMID: 23419824 DOI: 10.1016/j.jhep.2012.12.005]

11

Schuppan D, Ruehl M, Somasundaram R, Hahn EG. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 2001; 21: 351-372 [PMID: 11586465 DOI: 10.1055/s-2001-17556]

12

Bonis PA, Friedman SL, Kaplan MM. Is liver fibrosis reversible? N Engl J Med 2001; 344: 452-454 [PMID: 11172184 DOI: 10.1056/NEJM200102083440610]

13

Shen X, Cheng S, Peng Y, Song H, Li H. Attenuation of early liver fibrosis by herbal compound “Diwu Yanggan” through modulating the balance between epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition. BMC Complement Altern Med 2014; 14: 418 [PMID: 25345787 DOI: 10.1186/1472-6882-14-418]

14

Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008; 134: 1655-1669 [PMID: 18471545 DOI: 10.1053/j.gastro.2008.03.003]

15

Pinzani M. Liver fibrosis. Springer Semin Immunopathol 1999; 21: 475-490 [PMID: 10945037 DOI: 10.1007/s002810000037]

16

Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000; 46: 443-446 [PMID: 10716665 DOI: 10.1136/gut.46.4.443]

17

Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2000; 279: G245-G249 [PMID: 10915630 DOI: 10.1152/ajpgi.2000.279.2.G245]

18

Gäbele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci 2003; 8: d69-d77 [PMID: 12456323 DOI: 10.2741/887]

19

Milani S, Herbst H, Schuppan D, Kim KY, Riecken EO, Stein H. Procollagen expression by nonparenchymal rat liver cells in experimental biliary fibrosis. Gastroenterology 1990; 98: 175-184 [PMID: 2293576 DOI: 10.1016/0016-5085(90)91307-r]

20

Marra F. Hepatic stellate cells and the regulation of liver inflammation. J Hepatol 1999; 31: 1120-1130 [PMID: 10604588 DOI: 10.1016/s0168-8278(99)80327-4]

21

Lindquist JN, Marzluff WF, Stefanovic B. Fibrogenesis. III. Posttranscriptional regulation of type I collagen. Am J Physiol Gastrointest Liver Physiol 2000; 279: G471-G476 [PMID: 10960344 DOI: 10.1152/ajpgi.2000.279.3.G471]

22

Higuchi H, Gores GJ. Mechanisms of liver injury: an overview. Curr Mol Med 2003; 3: 483-490 [PMID: 14527080 DOI: 10.2174/1566524033479528]

23

Canbay A, Friedman S, Gores GJ. Apoptosis: the nexus of liver injury and fibrosis. Hepatology 2004; 39: 273-278 [PMID: 14767974 DOI: 10.1002/hep.20051]

24

Viñas O, Bataller R, Sancho-Bru P, Ginès P, Berenguer C, Enrich C, Nicolás JM, Ercilla G, Gallart T, Vives J, Arroyo V, Rodés J. Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation. Hepatology 2003; 38: 919-929 [PMID: 14512879 DOI: 10.1053/jhep.2003.50392]

25


https://dx.doi.org/10.3346/jkms.2015.30.10.1405




https://dx.doi.org/10.1155/2017/3945403


https://dx.doi.org/10.1172/JCI24282


https://dx.doi.org/10.15171/mejdd.2016.29


https://dx.doi.org/10.1111/jgh.12653


https://dx.doi.org/10.1097/MEG.0000000000000820


https://dx.doi.org/10.1016/j.jhep.2014.11.020


https://dx.doi.org/10.1371/journal.pone.0168372




https://dx.doi.org/10.1055/s-2001-17556


https://dx.doi.org/10.1056/NEJM200102083440610


https://dx.doi.org/10.1186/1472-6882-14-418




https://dx.doi.org/10.1007/s002810000037


https://dx.doi.org/10.1136/gut.46.4.443


https://dx.doi.org/10.1152/ajpgi.2000.279.2.G245


https://dx.doi.org/10.2741/887


https://dx.doi.org/10.1016/0016-5085(90)91307-r


https://dx.doi.org/10.1016/s0168-8278(99)80327-4




https://dx.doi.org/10.2174/1566524033479528


https://dx.doi.org/10.1002/hep.20051


https://dx.doi.org/10.1053/jhep.2003.50392



Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 2001; 21: 417-426 [PMID: 11586469 DOI: 10.1055/s-2001-17555]

26

Shi Z, Wakil AE, Rockey DC. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci USA 1997; 94: 10663-10668 [PMID: 9380692 DOI: 10.1073/pnas.94.20.10663]

27

Naito M, Hasegawa G, Ebe Y, Yamamoto T. Differentiation and function of Kupffer cells. Med Electron Microsc 2004; 37: 16-28 [PMID: 15057601 DOI: 10.1007/s00795-003-0228-x]

28

Thurman RG. II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin. Am J Physiol 1998; 275: G605-G611 [PMID: 9756487 DOI: 10.1152/ajpgi.1998.275.4.G605]

29

Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci 2002; 7: d793-d807 [PMID: 11897555]

30

Olaso E, Ikeda K, Eng FJ, Xu L, Wang LH, Lin HC, Friedman SL. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells. J Clin Invest 2001; 108: 1369-1378 [PMID: 11696582 DOI: 10.1172/JCI12373]

31

Hipp J, Atala A. Sources of stem cells for regenerative medicine. Stem Cell Rev 2008; 4: 3-11 [PMID: 18286392 DOI: 10.1007/s12015-008-9010-8]

32

Gruber HE, Deepe R, Hoelscher GL, Ingram JA, Norton HJ, Scannell B, Loeffler BJ, Zinchenko N, Hanley EN, Tapp H. Human adipose-derived mesenchymal stem cells: direction to a phenotype sharing similarities with the disc, gene expression profiling, and coculture with human annulus cells. Tissue Eng Part A 2010; 16: 2843-2860 [PMID: 20408770 DOI: 10.1089/ten.TEA.2009.0709]

33

Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, Okochi H, Ochiya T. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology 2007; 46: 219-228 [PMID: 17596885 DOI: 10.1002/hep.21704]

34

McElreavey KD, Irvine AI, Ennis KT, McLean WH. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton’s jelly portion of human umbilical cord. Biochem Soc Trans 1991; 19: 29S [PMID: 1709890 DOI: 10.1042/bst019029s]

35

Tsai MS, Hwang SM, Tsai YL, Cheng FC, Lee JL, Chang YJ. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod 2006; 74: 545-551 [PMID: 16306422 DOI: 10.1095/biolreprod.105.046029]

36

Yu X, Wang N, Qiang R, Wan Q, Qin M, Chen S, Wang H. Human amniotic fluid stem cells possess the potential to differentiate into primordial follicle oocytes in vitro. Biol Reprod 2014; 90: 73 [PMID: 24571984 DOI: 10.1095/biolreprod.113.112920]

37

Hosseini SM, Talaei-Khozani T, Sani M, Owrangi B. Differentiation of human breast-milk stem cells to neural stem cells and neurons. Neurol Res Int 2014; 2014: 807896 [PMID: 25506428 DOI: 10.1155/2014/807896]

38

Fan Y, Chong YS, Choolani MA, Cregan MD, Chan JK. Unravelling the mystery of stem/progenitor cells in human breast milk. PLoS One 2010; 5: e14421 [PMID: 21203434 DOI: 10.1371/journal.pone.0014421]

39

Ogata Y, Mabuchi Y, Yoshida M, Suto EG, Suzuki N, Muneta T, Sekiya I, Akazawa C. Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration. PLoS One 2015; 10: e0129096 [PMID: 26053045 DOI: 10.1371/journal.pone.0129096]

40

Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells: isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol 2009; 482: 281-294 [PMID: 19089363 DOI: 10.1007/978-1-59745-060-7_18]

41

In ‘t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE, Kanhai HH. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004; 22: 1338-1345 [PMID: 15579651 DOI: 10.1634/stemcells.2004-0058]

42

Ponnaiyan D, Bhat KM, Bhat GS. Comparison of immuno-phenotypes of stem cells from human dental pulp and periodontal ligament. Int J Immunopathol Pharmacol 2012; 25: 127-134 [PMID: 22507325 DOI: 10.1177/039463201202500115]

43

In ‘t Anker PS, Noort WA, Scherjon SA, Kleijburg-van der Keur C, Kruisselbrink AB, van Bezooijen RL, Beekhuizen W, Willemze R, Kanhai HH, Fibbe WE. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica 2003; 88: 845-852 [PMID: 12935972]

44

Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, Chen JR, Chen YP, Lee OK. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 2004; 40: 1275-1284 [PMID: 15562440 DOI: 10.1002/hep.20469]

45

Macrin D, Joseph JP, Pillai AA, Devi A. Eminent Sources of Adult Mesenchymal Stem Cells and Their Therapeutic Imminence. Stem Cell Rev Rep 2017; 13: 741-756 [PMID: 28812219 DOI: 10.1007/s12015-017-9759-8]

46

Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006; 24: 1294-1301 [PMID: 16410387 DOI: 10.1634/stemcells.2005-0342]

47

Liu WH, Song FQ, Ren LN, Guo WQ, Wang T, Feng YX, Tang LJ, Li K. The multiple functional roles of mesenchymal stem cells in participating in treating liver diseases. J Cell Mol Med 2015; 19: 511-520 [PMID: 25534251 DOI: 10.1111/jcmm.12482]

48

Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001; 19: 180-192 [PMID: 11359943 DOI:

49


https://dx.doi.org/10.1055/s-2001-17555


https://dx.doi.org/10.1073/pnas.94.20.10663


https://dx.doi.org/10.1007/s00795-003-0228-x







https://dx.doi.org/10.1007/s12015-008-9010-8


https://dx.doi.org/10.1089/ten.TEA.2009.0709




https://dx.doi.org/10.1042/bst019029s


https://dx.doi.org/10.1095/biolreprod.105.046029


https://dx.doi.org/10.1095/biolreprod.113.112920


https://dx.doi.org/10.1155/2014/807896






https://dx.doi.org/10.1007/978-1-59745-060-7_18


https://dx.doi.org/10.1634/stemcells.2004-0058


https://dx.doi.org/10.1177/039463201202500115





https://dx.doi.org/10.1007/s12015-017-9759-8


https://dx.doi.org/10.1634/stemcells.2005-0342


https://dx.doi.org/10.1111/jcmm.12482




10.1634/stemcells.19-3-180]Dueñas F, Becerra V, Cortes Y, Vidal S, Sáenz L, Palomino J, De Los Reyes M, Peralta OA. Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses. BMC Vet Res 2014; 10: 154 [PMID: 25011474 DOI: 10.1186/1746-6148-10-154]

50

Wagner W, Feldmann RE Jr, Seckinger A, Maurer MH, Wein F, Blake J, Krause U, Kalenka A, Bürgers HF, Saffrich R, Wuchter P, Kuschinsky W, Ho AD. The heterogeneity of human mesenchymal stem cell preparations--evidence from simultaneous analysis of proteomes and transcriptomes. Exp Hematol 2006; 34: 536-548 [PMID: 16569600 DOI: 10.1016/j.exphem.2006.01.002]

51

Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringdén O; Developmental Committee of the European Group for Blood and Marrow Transplantation. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008; 371: 1579-1586 [PMID: 18468541 DOI: 10.1016/S0140-6736(08)60690-X]

52

In ‘t Anker PS, Scherjon SA, Kleijburg-van der Keur C, Noort WA, Claas FH, Willemze R, Fibbe WE, Kanhai HH. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003; 102: 1548-1549 [PMID: 12900350 DOI: 10.1182/blood-2003-04-1291]

53

Deuse T, Stubbendorff M, Tang-Quan K, Phillips N, Kay MA, Eiermann T, Phan TT, Volk HD, Reichenspurner H, Robbins RC, Schrepfer S. Immunogenicity and immunomodulatory properties of umbilical cord lining mesenchymal stem cells. Cell Transplant 2011; 20: 655-667 [PMID: 21054940 DOI: 10.3727/096368910X536473]

54

Wang L, Bai G, Chen F. Human bone marrow mesenchymal stem cells suppress the proliferation of hepatic stellate cells by inhibiting the ubiquitination of p27. Biochem Cell Biol 2017; 95: 628-633 [PMID: 28746817 DOI: 10.1139/bcb-2017-0127]

55

Li T, Zhu J, Ma K, Liu N, Feng K, Li X, Wang S, Bie P. Autologous bone marrow-derived mesenchymal stem cell transplantation promotes liver regeneration after portal vein embolization in cirrhotic rats. J Surg Res 2013; 184: 1161-1173 [PMID: 23809154 DOI: 10.1016/j.jss.2013.04.054]

56

Jang YO, Kim MY, Cho MY, Baik SK, Cho YZ, Kwon SO. Effect of bone marrow-derived mesenchymal stem cells on hepatic fibrosis in a thioacetamide-induced cirrhotic rat model. BMC Gastroenterol 2014; 14: 198 [PMID: 25425284 DOI: 10.1186/s12876-014-0198-6]

57

Wang ZC, Yang S, Huang JJ, Chen SL, Li QQ, Li Y. Effect of bone marrow mesenchymal stem cells on the Smad expression of hepatic fibrosis rats. Asian Pac J Trop Med 2014; 7: 321-324 [PMID: 24507685 DOI: 10.1016/S1995-7645(14)60048-1]

58

Milosavljevic N, Gazdic M, Simovic Markovic B, Arsenijevic A, Nurkovic J, Dolicanin Z, Jovicic N, Jeftic I, Djonov V, Arsenijevic N, Lukic ML, Volarevic V. Mesenchymal stem cells attenuate liver fibrosis by suppressing Th17 cells - an experimental study. Transpl Int 2018; 31: 102-115 [PMID: 28805262 DOI: 10.1111/tri.13023]

59

Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E, Suzuki S, Miyauchi-Hara C, Nagoshi N, Sunabori T, Shimmura S, Miyawaki A, Nakagawa T, Suda T, Okano H, Matsuzaki Y. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med 2009; 206: 2483-2496 [PMID: 19841085 DOI: 10.1084/jem.20091046]

60

Toma C, Wagner WR, Bowry S, Schwartz A, Villanueva F. Fate of culture-expanded mesenchymal stem cells in the microvasculature: in vivo observations of cell kinetics. Circ Res 2009; 104: 398-402 [PMID: 19096027 DOI: 10.1161/CIRCRESAHA.108.187724]

61

Antoni D, Burckel H, Josset E, Noel G. Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci 2015; 16: 5517-5527 [PMID: 25768338 DOI: 10.3390/ijms16035517]

62

Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev 2008; 14: 61-86 [PMID: 18454635 DOI: 10.1089/teb.2007.0150]

63

Saleh FA, Frith JE, Lee JA, Genever PG. Three-dimensional in vitro culture techniques for mesenchymal stem cells. Methods Mol Biol 2012; 916: 31-45 [PMID: 22914931 DOI: 10.1007/978-1-61779-980-8_4]

64

Domnina A, Novikova P, Obidina J, Fridlyanskaya I, Alekseenko L, Kozhukharova I, Lyublinskaya O, Zenin V, Nikolsky N. Human mesenchymal stem cells in spheroids improve fertility in model animals with damaged endometrium. Stem Cell Res Ther 2018; 9: 50 [PMID: 29482664 DOI: 10.1186/s13287-018-0801-9]

65

Lin RZ, Chang HY. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J 2008; 3: 1172-1184 [PMID: 18566957 DOI: 10.1002/biot.200700228]

66

Tsai AC, Liu Y, Yuan X, Ma T. Compaction, fusion, and functional activation of three-dimensional human mesenchymal stem cell aggregate. Tissue Eng Part A 2015; 21: 1705-1719 [PMID: 25661745 DOI: 10.1089/ten.TEA.2014.0314]

67

Seiberg M, Siock P, Wisniewski S, Cauwenbergh G, Shapiro SS. The effects of trypsin on apoptosis, utriculi size, and skin elasticity in the Rhino mouse. J Invest Dermatol 1997; 109: 370-376 [PMID: 9284107 DOI: 10.1111/1523-1747.ep12336244]

68

Qiao Y, Xu Z, Yu Y, Hou S, Geng J, Xiao T, Liang Y, Dong Q, Mei Y, Wang B, Qiao H, Dai J, Suo G. Single cell derived spheres of umbilical cord mesenchymal stem cells enhance cell stemness properties, survival ability and therapeutic potential on liver failure. Biomaterials 2020; 227: 119573

69

https://dx.doi.org/10.1634/stemcells.19-3-180


https://dx.doi.org/10.1186/1746-6148-10-154


https://dx.doi.org/10.1016/j.exphem.2006.01.002


https://dx.doi.org/10.1016/S0140-6736(08)60690-X


https://dx.doi.org/10.1182/blood-2003-04-1291


https://dx.doi.org/10.3727/096368910X536473


https://dx.doi.org/10.1139/bcb-2017-0127


https://dx.doi.org/10.1016/j.jss.2013.04.054


https://dx.doi.org/10.1186/s12876-014-0198-6


https://dx.doi.org/10.1016/S1995-7645(14)60048-1


https://dx.doi.org/10.1111/tri.13023


https://dx.doi.org/10.1084/jem.20091046


https://dx.doi.org/10.1161/CIRCRESAHA.108.187724


https://dx.doi.org/10.3390/ijms16035517


https://dx.doi.org/10.1089/teb.2007.0150


https://dx.doi.org/10.1007/978-1-61779-980-8_4


https://dx.doi.org/10.1186/s13287-018-0801-9


https://dx.doi.org/10.1002/biot.200700228


https://dx.doi.org/10.1089/ten.TEA.2014.0314


https://dx.doi.org/10.1111/1523-1747.ep12336244



[PMID: 31670080 DOI: 10.1016/j.biomaterials.2019.119573]Dhawan A, Puppi J, Hughes RD, Mitry RR. Human hepatocyte transplantation: current experience and future challenges. Nat Rev Gastroenterol Hepatol 2010; 7: 288-298 [PMID: 20368738 DOI: 10.1038/nrgastro.2010.44]

70

Hodgkinson CP, Gomez JA, Mirotsou M, Dzau VJ. Genetic engineering of mesenchymal stem cells and its application in human disease therapy. Hum Gene Ther 2010; 21: 1513-1526 [PMID: 20825283 DOI: 10.1089/hum.2010.165]

71

Alfaifi M, Eom YW, Newsome PN, Baik SK. Mesenchymal stromal cell therapy for liver diseases. J Hepatol 2018; 68: 1272-1285 [PMID: 29425678 DOI: 10.1016/j.jhep.2018.01.030]

72

Di Rocco G, Gentile A, Antonini A, Ceradini F, Wu JC, Capogrossi MC, Toietta G. Enhanced healing of diabetic wounds by topical administration of adipose tissue-derived stromal cells overexpressing stromal-derived factor-1: biodistribution and engraftment analysis by bioluminescent imaging. Stem Cells Int 2010; 2011: 304562 [PMID: 21234108 DOI: 10.4061/2011/304562]

73

Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, Dzau VJ. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003; 9: 1195-1201 [PMID: 12910262 DOI: 10.1038/nm912]

74

Xu J, Huang Z, Lin L, Fu M, Gao Y, Shen Y, Zou Y, Sun A, Qian J, Ge J. miR-210 over-expression enhances mesenchymal stem cell survival in an oxidative stress environment through antioxidation and c-Met pathway activation. Sci China Life Sci 2014; 57: 989-997 [PMID: 25168379 DOI: 10.1007/s11427-014-4725-z]

75

Zeilinger K, Freyer N, Damm G, Seehofer D, Knöspel F. Cell sources for in vitro human liver cell culture models. Exp Biol Med (Maywood) 2016; 241: 1684-1698 [PMID: 27385595 DOI: 10.1177/1535370216657448]

76

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 2013; 87: 1315-1530 [PMID: 23974980 DOI: 10.1007/s00204-013-1078-5]

77

Li J, Tao R, Wu W, Cao H, Xin J, Li J, Guo J, Jiang L, Gao C, Demetriou AA, Farkas DL, Li L. 3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes. Stem Cells Dev 2010; 19: 1427-1436 [PMID: 20055663 DOI: 10.1089/scd.2009.0415]

78

Liu M, Yang J, Hu W, Zhang S, Wang Y. Superior performance of co-cultured mesenchymal stem cells and hepatocytes in poly(lactic acid-glycolic acid) scaffolds for the treatment of acute liver failure. Biomed Mater 2016; 11: 015008 [PMID: 26836957 DOI: 10.1088/1748-6041/11/1/015008]

79

Mazza G, Rombouts K, Rennie Hall A, Urbani L, Vinh Luong T, Al-Akkad W, Longato L, Brown D, Maghsoudlou P, Dhillon AP, Fuller B, Davidson B, Moore K, Dhar D, De Coppi P, Malago M, Pinzani M. Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Sci Rep 2015; 5: 13079 [PMID: 26248878 DOI: 10.1038/srep13079]

80

Saeedi P, Halabian R, Imani Fooladi AA. A revealing review of mesenchymal stem cells therapy, clinical perspectives and Modification strategies. Stem Cell Investig 2019; 6: 34 [PMID: 31620481 DOI: 10.21037/sci.2019.08.11]

81

Augustin M. Preconditioning Methods in Cell Therapy of the Heart. Conference Proceedings, 2012 Oct 12; Helsingin yliopisto. Available from: http://hdl.handle.net/10138/36906

82

Yu SP, Wei Z, Wei L. Preconditioning strategy in stem cell transplantation therapy. Transl Stroke Res 2013; 4: 76-88 [PMID: 23914259 DOI: 10.1007/s12975-012-0251-0]

83

Haider HKh, Ashraf M. Preconditioning and stem cell survival. J Cardiovasc Transl Res 2010; 3: 89-102 [PMID: 20560023 DOI: 10.1007/s12265-009-9161-2]

84

Feng Y, Huang W, Meng W, Jegga AG, Wang Y, Cai W, Kim HW, Pasha Z, Wen Z, Rao F, Modi RM, Yu X, Ashraf M. Heat shock improves Sca-1+ stem cell survival and directs ischemic cardiomyocytes toward a prosurvival phenotype via exosomal transfer: a critical role for HSF1/miR-34a/HSP70 pathway. Stem Cells 2014; 32: 462-472 [PMID: 24123326 DOI: 10.1002/stem.1571]

85

Qiao PF, Yao L, Zhang XC, Li GD, Wu DQ. Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy. World J Gastroenterol 2015; 21: 12822-12834 [PMID: 26668506 DOI: 10.3748/wjg.v21.i45.12822]

86

Carrière A, Ebrahimian TG, Dehez S, Augé N, Joffre C, André M, Arnal S, Duriez M, Barreau C, Arnaud E, Fernandez Y, Planat-Benard V, Lévy B, Pénicaud L, Silvestre JS, Casteilla L. Preconditioning by mitochondrial reactive oxygen species improves the proangiogenic potential of adipose-derived cells-based therapy. Arterioscler Thromb Vasc Biol 2009; 29: 1093-1099 [PMID:

87


https://dx.doi.org/10.1016/j.biomaterials.2019.119573


https://dx.doi.org/10.1038/nrgastro.2010.44


https://dx.doi.org/10.1089/hum.2010.165




https://dx.doi.org/10.4061/2011/304562


https://dx.doi.org/10.1038/nm912




https://dx.doi.org/10.1177/1535370216657448


https://dx.doi.org/10.1007/s00204-013-1078-5


https://dx.doi.org/10.1089/scd.2009.0415


https://dx.doi.org/10.1088/1748-6041/11/1/015008


https://dx.doi.org/10.1038/srep13079


https://dx.doi.org/10.21037/sci.2019.08.11

http://hdl.handle.net/10138/36906


https://dx.doi.org/10.1007/s12975-012-0251-0


https://dx.doi.org/10.1007/s12265-009-9161-2


https://dx.doi.org/10.1002/stem.1571





19423864 DOI: 10.1161/ATVBAHA.109.188318]Volarevic V, Markovic BS, Gazdic M, Volarevic A, Jovicic N, Arsenijevic N, Armstrong L, Djonov V, Lako M, Stojkovic M. Ethical and Safety Issues of Stem Cell-Based Therapy. Int J Med Sci 2018; 15: 36-45 [PMID: 29333086 DOI: 10.7150/ijms.21666]

88

Eggenhofer E, Luk F, Dahlke MH, Hoogduijn MJ. The life and fate of mesenchymal stem cells. Front Immunol 2014; 5: 148 [PMID: 24904568 DOI: 10.3389/fimmu.2014.00148]

89

Camussi G, Deregibus MC, Cantaluppi V. Role of stem-cell-derived microvesicles in the paracrine action of stem cells. Biochem Soc Trans 2013; 41: 283-287 [PMID: 23356298 DOI: 10.1042/BST20120192]

90

Koppers-Lalic D, Hogenboom MM, Middeldorp JM, Pegtel DM. Virus-modified exosomes for targeted RNA delivery; a new approach in nanomedicine. Adv Drug Deliv Rev 2013; 65: 348-356 [PMID: 22820525 DOI: 10.1016/j.addr.2012.07.006]

91

Kooijmans SAA, Schiffelers RM, Zarovni N, Vago R. Modulation of tissue tropism and biological activity of exosomes and other extracellular vesicles: New nanotools for cancer treatment. Pharmacol Res 2016; 111: 487-500 [PMID: 27394168 DOI: 10.1016/j.phrs.2016.07.006]

92

Li T, Yan Y, Wang B, Qian H, Zhang X, Shen L, Wang M, Zhou Y, Zhu W, Li W, Xu W. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis. Stem Cells Dev 2013; 22: 845-854 [PMID: 23002959 DOI: 10.1089/scd.2012.0395]

93

Nong K, Wang W, Niu X, Hu B, Ma C, Bai Y, Wu B, Wang Y, Ai K. Hepatoprotective effect of exosomes from human-induced pluripotent stem cell-derived mesenchymal stromal cells against hepatic ischemia-reperfusion injury in rats. Cytotherapy 2016; 18: 1548-1559 [PMID: 27592404 DOI: 10.1016/j.jcyt.2016.08.002]

94

Ullah M, Liu DD, Thakor AS. Mesenchymal Stromal Cell Homing: Mechanisms and Strategies for Improvement. iScience 2019; 15: 421-438 [PMID: 31121468 DOI: 10.1016/j.isci.2019.05.004]

95

Meier RP, Müller YD, Morel P, Gonelle-Gispert C, Bühler LH. Transplantation of mesenchymal stem cells for the treatment of liver diseases, is there enough evidence? Stem Cell Res 2013; 11: 1348-1364 [PMID: 24090934 DOI: 10.1016/j.scr.2013.08.011]

96

di Bonzo LV, Ferrero I, Cravanzola C, Mareschi K, Rustichell D, Novo E, Sanavio F, Cannito S, Zamara E, Bertero M, Davit A, Francica S, Novelli F, Colombatto S, Fagioli F, Parola M. Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential. Gut 2008; 57: 223-231 [PMID: 17639088 DOI: 10.1136/gut.2006.111617]

97

Amer ME, El-Sayed SZ, El-Kheir WA, Gabr H, Gomaa AA, El-Noomani N, Hegazy M. Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells. Eur J Gastroenterol Hepatol 2011; 23: 936-941 [PMID: 21900788 DOI: 10.1097/MEG.0b013e3283488b00]

98

Salama H, Zekri AR, Zern M, Bahnassy A, Loutfy S, Shalaby S, Vigen C, Burke W, Mostafa M, Medhat E, Alfi O, Huttinger E. Autologous hematopoietic stem cell transplantation in 48 patients with end-stage chronic liver diseases. Cell Transplant 2010; 19: 1475-1486 [PMID: 20587151 DOI: 10.3727/096368910X514314]

99

Mehrabani D, Khajehahmadi Z, Tajik P, Tamadon A, Rahmanifar F, Ashraf M, Tanideh N, Zare S. Regenerative Effect of Bone Marrow-derived Mesenchymal Stem Cells in Thioacetamide-induced Liver Fibrosis of Rats. Arch Razi Inst 2019; 74: 279-286 [PMID: 31592593 DOI: 10.22092/ari.2018.110029.1120]

100

Duman DG, Zibandeh N, Ugurlu MU, Celikel C, Akkoc T, Banzragch M, Genc D, Ozdogan O, Akkoc T. Mesenchymal stem cells suppress hepatic fibrosis accompanied by expanded intrahepatic natural killer cells in rat fibrosis model. Mol Biol Rep 2019; 46: 2997-3008 [PMID: 30850965 DOI: 10.1007/s11033-019-04736-4]

101

102 Mansour FAA, Shaheed I, Hassan NRA. Use of Bone Marrow-Derived Mesenchymal Stem Cells in Improving Thioacetamide Induced Liver Fibrosis in Rats. In: Polychroniadis E, Oral A, Ozer M, 2nd ed. International Multidisciplinary Microscopy and Microanalysis Congress. Springer Proceedings in Physics, 2015; 164. Springer, Cham [DOI: 10.1007/978-3-319-16919-4_29]

102

Farouk S, Sabet S, Abu Zahra FA, El-Ghor AA. Bone marrow derived-mesenchymal stem cells downregulate IL17A dependent IL6/STAT3 signaling pathway in CCl4-induced rat liver fibrosis. PLoS One 2018; 13: e0206130 [PMID: 30346985 DOI: 10.1371/journal.pone.0206130]

103

Liu Y, Yang X, Jing Y, Zhang S, Zong C, Jiang J, Sun K, Li R, Gao L, Zhao X, Wu D, Shi Y, Han Z, Wei L. Contribution and Mobilization of Mesenchymal Stem Cells in a mouse model of carbon tetrachloride-induced liver fibrosis. Sci Rep 2015; 5: 17762 [PMID: 26643997 DOI: 10.1038/srep17762]

104

Vainshtein JM, Kabarriti R, Mehta KJ, Roy-Chowdhury J, Guha C. Bone marrow-derived stromal cell therapy in cirrhosis: clinical evidence, cellular mechanisms, and implications for the treatment of hepatocellular carcinoma. Int J Radiat Oncol Biol Phys 2014; 89: 786-803 [PMID: 24969793 DOI: 10.1016/j.ijrobp.2014.02.017]

105

Hu C, Li L. In Vitro and In Vivo Hepatic Differentiation of Adult Somatic Stem Cells and Extraembryonic Stem Cells for Treating End Stage Liver Diseases. Stem Cells Int 2015; 2015: 871972 [PMID: 26347063 DOI: 10.1155/2015/871972]

106

Khalil MR, El-Demerdash RS, Elminshawy HH, Mehanna ET, Mesbah NM, Abo-Elmatty DM. Therapeutic effect of bone marrow mesenchymal stem cells in a rat model of carbon tetrachloride induced liver fibrosis. Biomed J2020 [PMID: 32389821 DOI: 10.1016/j.bj.2020.04.011]

107


https://dx.doi.org/10.1161/ATVBAHA.109.188318


https://dx.doi.org/10.7150/ijms.21666


https://dx.doi.org/10.3389/fimmu.2014.00148


https://dx.doi.org/10.1042/BST20120192


https://dx.doi.org/10.1016/j.addr.2012.07.006


https://dx.doi.org/10.1016/j.phrs.2016.07.006




https://dx.doi.org/10.1016/j.jcyt.2016.08.002


https://dx.doi.org/10.1016/j.isci.2019.05.004


https://dx.doi.org/10.1016/j.scr.2013.08.011


https://dx.doi.org/10.1136/gut.2006.111617


https://dx.doi.org/10.1097/MEG.0b013e3283488b00


https://dx.doi.org/10.3727/096368910X514314


https://dx.doi.org/10.22092/ari.2018.110029.1120


https://dx.doi.org/10.1007/s11033-019-04736-4

https://dx.doi.org/10.1007/978-3-319-16919-4_29






https://dx.doi.org/10.1016/j.ijrobp.2014.02.017


https://dx.doi.org/10.1155/2015/871972


https://dx.doi.org/10.1016/j.bj.2020.04.011



Mazhari S, Gitiara A, Baghaei K, Hatami B, Rad RE, Asadirad A, Joharchi K, Tokhanbigli S, Hashemi SM, Łos MJ, Aghdaei HA, Zali MR, Ghavami S. Therapeutic potential of bone marrow-derived mesenchymal stem cells and imatinib in a rat model of liver fibrosis. Eur J Pharmacol 2020; 882: 173263 [PMID: 32535098 DOI: 10.1016/j.ejphar.2020.173263]

108

Jang YO, Kim SH, Cho MY, Kim KS, Park KS, Cha SK, Kim MY, Chang SJ, Baik SK. Synergistic effects of simvastatin and bone marrow-derived mesenchymal stem cells on hepatic fibrosis. Biochem Biophys Res Commun 2018; 497: 264-271 [PMID: 29428718 DOI: 10.1016/j.bbrc.2018.02.067]

109

Jang YO, Cho MY, Yun CO, Baik SK, Park KS, Cha SK, Chang SJ, Kim MY, Lim YL, Kwon SO. Effect of Function-Enhanced Mesenchymal Stem Cells Infected With Decorin-Expressing Adenovirus on Hepatic Fibrosis. Stem Cells Transl Med 2016; 5: 1247-1256 [PMID: 27365486 DOI: 10.5966/sctm.2015-0323]

110

Sayyed HG, Osama A, Idriss NK, Sabry D, Abdelrhim AS, Bakry R. Comparison of the therapeutic effectiveness of human CD34+ and rat bone marrow mesenchymal stem cells on improvement of experimental liver fibrosis in Wistar rats. Int J Physiol Pathophysiol Pharmacol 2016; 8: 128-139 [PMID: 27785340]

111

Abdelgwad M, Ewaiss M, Sabry D, Khalifa WA, Altaib ZM, Alhelf M. Comparative study on effect of mesenchymal stem cells and endothelial progenitor cells on treatment of experimental CCL4-induced liver fibrosis. Arch Physiol Biochem 2020; 1-10 [PMID: 32374186 DOI: 10.1080/13813455.2020.1752256]

112

Rengasamy M, Singh G, Fakharuzi NA, Siddikuzzaman, Balasubramanian S, Swamynathan P, Thej C, Sasidharan G, Gupta PK, Das AK, Rahman AZA, Fakiruddin KS, Nian LM, Zakaria Z, Majumdar AS. Transplantation of human bone marrow mesenchymal stromal cells reduces liver fibrosis more effectively than Wharton’s jelly mesenchymal stromal cells. Stem Cell Res Ther 2017; 8: 143 [PMID: 28610623 DOI: 10.1186/s13287-017-0595-1]

113

Hao T, Chen J, Zhi S, Zhang Q, Chen G, Yu F. Comparison of bone marrow-vs. adipose tissue-derived mesenchymal stem cells for attenuating liver fibrosis. Exp Ther Med 2017; 14: 5956-5964 [PMID: 29285145 DOI: 10.3892/etm.2017.5333]

114

Idriss NK, Sayyed HG, Osama A, Sabry D. Treatment Efficiency of Different Routes of Bone Marrow-Derived Mesenchymal Stem Cell Injection in Rat Liver Fibrosis Model. Cell Physiol Biochem 2018; 48: 2161-2171 [PMID: 30114694 DOI: 10.1159/000492558]

115

Truong NH, Nguyen NH, Le TV, Vu NB, Huynh N, Nguyen TV, Le HM, Phan NK, Pham PV. Comparison of the Treatment Efficiency of Bone Marrow-Derived Mesenchymal Stem Cell Transplantation via Tail and Portal Veins in CCl4-Induced Mouse Liver Fibrosis. Stem Cells Int 2016; 2016: 5720413 [PMID: 26839564 DOI: 10.1155/2016/5720413]

116

Wang HY, Li C, Liu WH, Deng FM, Ma Y, Guo LN, Kong H, Hu KA, Liu Q, Wu J, Sun J, Liu YL. Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis. J Cell Physiol 2020; 235: 2722-2737 [PMID: 31508820 DOI: 10.1002/jcp.29176]

117

Abdel Aal S, Abdelrahman S, Raafat N. Comparative therapeutic effects of mesenchymal stem cells vs their conditioned media in alleviation of CCL4-induced liver fibrosis in rats: Histological and biochemical study. J Med Histol 2019; 3: 1-20 [DOI: 10.21608/jmh.2019.10310.1051]

118

Mortezaee K, Pasbakhsh P, Ragerdi Kashani I, Sabbaghziarani F, Omidi A, Zendedel A, Ghasemi S, Dehpour AR. Melatonin Pretreatment Enhances the Homing of Bone Marrow-derived Mesenchymal Stem Cells Following Transplantation in a Rat Model of Liver Fibrosis. Iran Biomed J 2016; 20: 207-216 [PMID: 27130910 DOI: 10.7508/ibj.2016.04.004]

119

Mortezaee K, Khanlarkhani N, Sabbaghziarani F, Nekoonam S, Majidpoor J, Hosseini A, Pasbakhsh P, Kashani IR, Zendedel A. Preconditioning with melatonin improves therapeutic outcomes of bone marrow-derived mesenchymal stem cells in targeting liver fibrosis induced by CCl4. Cell Tissue Res 2017; 369: 303-312 [PMID: 28413861 DOI: 10.1007/s00441-017-2604-1]

120

Sabry D, Mohamed A, Monir M, Ibrahim HA. The Effect of Mesenchymal Stem Cells Derived Microvesicles on the Treatment of Experimental CCL4 Induced Liver Fibrosis in Rats. Int J Stem Cells 2019; 12: 400-409 [PMID: 31474025 DOI: 10.15283/ijsc18143]

121

Rong X, Liu J, Yao X, Jiang T, Wang Y, Xie F. Human bone marrow mesenchymal stem cells-derived exosomes alleviate liver fibrosis through the Wnt/β-catenin pathway. Stem Cell Res Ther 2019; 10: 98 [PMID: 30885249 DOI: 10.1186/s13287-019-1204-2]

122

Zhu Y, Miao Z, Gong L, Chen W. Transplantation of mesenchymal stem cells expressing TIMP-1-shRNA improves hepatic fibrosis in CCl₄-treated rats. Int J Clin Exp Pathol 2015; 8: 8912-8920 [PMID: 26464632]

123

Wang X, Wang H, Lu J, Feng Z, Liu Z, Song H, Wang H, Zhou Y, Xu J. Erythropoietin-Modified Mesenchymal Stem Cells Enhance Anti-fibrosis Efficacy in Mouse Liver Fibrosis Model. Tissue Eng Regen Med 2020; 17: 683-693 [PMID: 32621283 DOI: 10.1007/s13770-020-00276-2]

124

Meier RP, Mahou R, Morel P, Meyer J, Montanari E, Muller YD, Christofilopoulos P, Wandrey C, Gonelle-Gispert C, Bühler LH. Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice. J Hepatol 2015; 62: 634-641 [PMID: 25450712 DOI: 10.1016/j.jhep.2014.10.030]

125

Du C, Jiang M, Wei X, Qin J, Xu H, Wang Y, Zhang Y, Zhou D, Xue H, Zheng S, Zeng W. Transplantation of human matrix metalloproteinase-1 gene-modified bone marrow-derived mesenchymal stem cell attenuates CCL4-induced liver fibrosis in rats. Int J Mol Med 2018; 41: 3175-3184 [PMID: 29512750 DOI: 10.3892/ijmm.2018.3516]

126


https://dx.doi.org/10.1016/j.ejphar.2020.173263


https://dx.doi.org/10.1016/j.bbrc.2018.02.067


https://dx.doi.org/10.5966/sctm.2015-0323



https://dx.doi.org/10.1080/13813455.2020.1752256


https://dx.doi.org/10.1186/s13287-017-0595-1


https://dx.doi.org/10.3892/etm.2017.5333


https://dx.doi.org/10.1159/000492558


https://dx.doi.org/10.1155/2016/5720413


https://dx.doi.org/10.1002/jcp.29176

https://dx.doi.org/10.21608/jmh.2019.10310.1051


https://dx.doi.org/10.7508/ibj.2016.04.004


https://dx.doi.org/10.1007/s00441-017-2604-1


https://dx.doi.org/10.15283/ijsc18143


https://dx.doi.org/10.1186/s13287-019-1204-2



https://dx.doi.org/10.1007/s13770-020-00276-2




https://dx.doi.org/10.3892/ijmm.2018.3516



Ma ZG, Lv XD, Zhan LL, Chen L, Zou QY, Xiang JQ, Qin JL, Zhang WW, Zeng ZJ, Jin H, Jiang HX, Lv XP. Human urokinase-type plasminogen activator gene-modified bone marrow-derived mesenchymal stem cells attenuate liver fibrosis in rats by down-regulating the Wnt signaling pathway. World J Gastroenterol 2016; 22: 2092-2103 [PMID: 26877613 DOI: 10.3748/wjg.v22.i6.2092]

127

Fiore EJ, Bayo JM, Garcia MG, Malvicini M, Lloyd R, Piccioni F, Rizzo M, Peixoto E, Sola MB, Atorrasagasti C, Alaniz L, Camilletti MA, Enguita M, Prieto J, Aquino JB, Mazzolini G. Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice. Stem Cells Dev 2015; 24: 791-801 [PMID: 25315017 DOI: 10.1089/scd.2014.0174]

128

Chang N, Ge J, Xiu L, Zhao Z, Duan X, Tian L, Xie J, Yang L, Li L. HuR mediates motility of human bone marrow-derived mesenchymal stem cells triggered by sphingosine 1-phosphate in liver fibrosis. J Mol Med (Berl) 2017; 95: 69-82 [PMID: 27543493 DOI: 10.1007/s00109-016-1460-x]

129








World Journal of



MINIREVIEWS

Molecular overview of progressive familial intrahepatic cholestasis

Sriram Amirneni, Nils Haep, Mohammad A Gad, Alejandro Soto-Gutierrez, James E Squires, Rodrigo M Florentino

ORCID number: Sriram Amirneni 0000-0002-5434-8745; Nils Haep 0000-0001-8149-4011; Mohammad A Gad 0000-0002-8318-2844; Alejandro Soto-Gutierrez 0000-0002-9838-1862; James E Squires 0000-0001-6979-8987; Rodrigo M Florentino 0000-0001-7033-176X.

Author contributions: All authors contributed to this paper with literature review and analysis and approval of the final version.

Supported by NIH, No. UG3TR003289 to Soto-Gutierrez A.

Conflict-of-interest statement: A.S.-G., is co-founder and have a financial interest in Von Baer Wolff, Inc. a company focused on biofabrication of autologous human hepatocytes from stem cells technology and programming liver failure and their interests are managed by the Conflict of Interest Office at the University of Pittsburgh in accordance with their policies

Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially,

Sriram Amirneni, Nils Haep, Mohammad A Gad, Alejandro Soto-Gutierrez, Rodrigo M Florentino, Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, United States

Alejandro Soto-Gutierrez, James E Squires, Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15213, United States

James E Squires, Division of Gastroenterology, Hepatology, and Nutrition, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, United States

Corresponding author: Rodrigo M Florentino, PhD, Research Fellow, Department of Pathology, University of Pittsburgh, 200 Lothrop Street S420 Biomedical Science Tower, Pittsburgh, PA 15213, United States. [email protected]

AbstractCholestasis is a clinical condition resulting from the imapairment of bile flow. This condition could be caused by defects of the hepatocytes, which are responsible for the complex process of bile formation and secretion, and/or caused by defects in the secretory machinery of cholangiocytes. Several mutations and pathways that lead to cholestasis have been described. Progressive familial intrahepatic cholestasis (PFIC) is a group of rare diseases caused by autosomal recessive mutations in the genes that encode proteins expressed mainly in the apical membrane of the hepatocytes. PFIC 1, also known as Byler’s disease, is caused by mutations of the ATP8B1 gene, which encodes the familial intrahepatic cholestasis 1 protein. PFIC 2 is characterized by the downregulation or absence of functional bile salt export pump (BSEP) expression via variations in the ABCB11 gene. Mutations of the ABCB4 gene result in lower expression of the multidrug resistance class 3 glycoprotein, leading to the third type of PFIC. Newer variations of this disease have been described. Loss of function of the tight junction protein 2 protein results in PFIC 4, while mutations of the NR1H4 gene, which encodes farnesoid X receptor, an important transcription factor for bile formation, cause PFIC 5. A recently described type of PFIC is associated with a mutation in the MYO5B gene, important for the trafficking of BSEP and hepatocyte membrane polarization. In this review, we provide a brief overview of the molecular mechanisms and clinical features associated with each type of PFIC based on peer reviewed journals published between 1993 and 2020.

Key Words: Progressive familial intrahepatic cholestasis; ATP8B1/familial intrahepatic cholestasis 1; ABCB11/bile salt export pump; ABCB4/multidrug resistance class 3; Intrahepatic cholestasis; Bile



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Amirneni S et al. PFIC on a molecular overview


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Received: September 11, 2020 Peer-review started: September 11, 2020 First decision: October 27, 2020 Revised: November 5, 2020 Accepted: November 29, 2020 Article in press: November 29, 2020 Published online: December 21, 2020

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Core Tip: Progressive familial intrahepatic cholestasis (PFIC) is an expanding group of genetically-based diseases caused by various autosomal recessive mutations which result in the inability to appropriately form and excrete bile from hepatocytes. There are three classic types of PFIC. Type 1 is caused by mutations of the ATP8B1 gene, encoding familial intrahepatic cholestasis 1. PFIC 2 is the result of mutations in the ABCB11 gene, reducing the apical membrane expression of bile salt export pump. PFIC 3 is caused by variations in the gene that encodes the multidrug resistance class 3 glycoprotein. With advances in DNA sequencing, new forms of this disease have been documented. Alterations in the NR1H4, tight junction protein 2, and MYO5B genes have been linked with new phenotypes of PFIC. Here, we provide a molecular and clinical overview of PFIC.

Citation: Amirneni S, Haep N, Gad MA, Soto-Gutierrez A, Squires JE, Florentino RM. Molecular overview of progressive familial intrahepatic cholestasis. World J Gastroenterol 2020; 26(47): 7470-7484URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7470.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7470

INTRODUCTIONAmong the liver functions, bile production is one of the most important. Hepatocytes and cholangiocytes work together in order to produce a mixture of organic and inorganic compounds that aid in the digestion process and excrete metabolites[1]. The formation of the final product, bile, is divided into two main processes: The formation and conjugation of the bile acids by the hepatocytes and the alkalization and dilution by the cholangiocytes. The bile can be stored in the gallbladder and secreted into the duodenum after a meal by a cholecystokinin stimulus. The detergent properties of bile give it the ability to break lipids into small particles, helping digestion[1].

The impairment of bile flow is responsible for generating a clinical condition called cholestasis[2]. Alterations in the bile formation process, at the hepatocyte and/or cholangiocyte level, lead to some complications, potentially resulting in liver tumors or necessitating liver transplantation (LT)[2]. Several point mutations and pathways that contribute to this clinical condition have been elucidated. In hepatocytes, mutations and polymorphisms of the genes responsible for bile acid transporters, as well as farnesoid X receptor (FXR), an important transcription factor for the bile formation, were described[3,4]. In cholangiocytes, defects of the cystic fibrosis transmembrane conductance regulator (CFTR) and alterations in calcium (Ca2+) signaling contribute to cholestasis establishment and development[5,6].

Nowadays, several cholestasis-related diseases have been described. At the biliary level, there are two such diseases: Primary sclerosing cholangitis and primary biliary cirrhosis. These diseases both result in nonfunctional cholangiocytes, leading to viscous bile[7-9]. Problems in the hepatocyte machinery are also related to cholestatic diseases. Through an unclear genetic, hormonal, and environmental mechanism, some women in late term pregnancy develop intrahepatic cholestasis due to the downregulation of some bile transporters of the hepatocyte membrane[10]. Some drugs, such as antimicrobials or acetaminophen, may also induce the impairment of bile flow through hepatocellular damage, resulting in cholestasis[11,12].

Some autosomal recessive mutations of the bile acid membrane transporters that affect hepatocytes result in a group of diseases known as progressive familial intrahepatic cholestasis (PFIC). Historically, three types of PFIC were described: Type 1 due to mutations of the ATP8B1 gene, type 2 resulting from mutations of the ABCB11 gene, and type 3 caused by variations in the ABCB4 gene. Recently, new types of this disease have been reported. Here, we will discuss the recent molecular and clinical findings of not only PFIC 1, PFIC 2 and PFIC 3, but also of the new types that have recently been reported. This group of hereditary diseases causes severe cholestasis in infants and patients regularly have to undergo LT (Table 1).








Table 1 Overview of progressive familial intrahepatic cholestasis subtypes

PFIC 1 PFIC 2 PFIC 3 PFIC 4 PFIC 5 PFICAssociated with MYO5B defects

Gene ATP8B1 ABCB11 ABCB4 TJP2 NR1H4 MYO5B

Molecular findings

Impaired hepatocyte bile salt secretion; canalicular cholestasis; giant cell transformation; portal and lobular fibrosis; lack of ductular proliferation

Increased intracellular bile salt concentrations; canalicular cholestasis; lobular and portal fibrosis; hepatocellular necrosis

Absence of PC from the biliary canaliculi; increased free bile salts in canaliculi; cholangiocyte damage; increased cholesterol crystallization nonspecific portal inflammation; portal fibrosis; giant cell hepatitis

Abnormal CLDN1 localization; normal CLDN2 localization; compromised tight junctions; bile salt leakage into the paracellular space; hepatocyte and cholangiocyte damage; giant cell transformation

Undetectable expression of BSEP in the bile canaliculi; intralobular cholestasis with ductular reaction; hepatocellular ballooning; giant cell transformation

Possibly mislocalized BSEP and MDR3 upon staining; portal and lobular fibrosis; giant cell transformation. Intestinal MVID findings (mislocalized apical brush border proteins, villus atrophy, presence of microvillus inclusion bodies)

Clinical findings

Low GGT cholestasis; jaundice caused by hyperbilirubinemia; pruritus; hepatosplenomegaly; diarrhea; stunted growth; exocrine pancreatic insufficiency; progressive sensorineural hearing loss; fat-soluble vitamin deficiencies; can lead to cirrhosis and end stage liver disease

Low GGT cholestasis; elevated transaminases; elevated serum bilirubin; elevated AFP; jaundice; pruritus; scleral icterus; hepatomegaly; chronic skin picking; reduced growth

Elevated GGT cholestasis; pruritus; hepatosplenomegaly; variceal bleeding; portal hypertension; jaundice; acholic stools; stunted growth; reduced bone density; learning disabilities; elevated transaminases; elevated bilirubin; elevated AP

Low GGT cholestasis; neurological and respiratory symptoms

Neonatal onset of normal GGT associated cholestasis; elevated serum bilirubin; elevated serum AFP; vitamin K independent coagulopathy; fibrosis progressing into micronodular cirrhosis

Normal GGT cholestasis; jaundice; pruritus; mildly elevated ALT and AST; elevated serum BS; hepatomegaly

Clinical outcomes

Medical management is the first line of defense, but if it is insufficient, surgical is next. Bilary diversion has been effective in around 80% of patients. The last resort is LT in patients who develop cirrhosis and liver failure. Extrahepatic symptoms can persist (or worsen) after LT

There is a 15% chance of cirrhosis developing into HCC or cholangiocarcinoma. Genetic defect(s) can aid in determination of success of biliary diversion. LT in PFIC 2 patients might lead to development of BSEP specific allo-reactive antibodies (in approximately 8% of PFIC 2 patients who undergo LT). In these cases, a second LT may be needed if BSEP deficiency develops in the new liver

HCC and cholangiocarcinoma have been associated with PFIC 3. The severity varies, with those retaining MDR3 expression responding better to medical treatment. Biliary diversion procedures aren’t as effective due to severity upon presentation, but LT is curative

There have been some reports of HCC occurrence in PFIC 4 patients, though not much is known about the mechanism. LT has been successful, with no reported recurrence of the PFIC 4 phenotype after LT

PFIC 5 is very rare (only 8 cases reported in the literature so far). LT has been used, but steatosis in the transplanted liver has been reported in some cases

MVID has been associated with MYO5B defects. Lifelong TPN is required but is associated with increased risk of sepsis and small bowel transplant. Combined bowel-liver transplants may reduce the risk of post transplant onset of cholestasis

Treatments Medical: Vitamin supplementation; UDCA; Rifampin; Cholestyramine; CFTR folding correctors

Medical: 4PBA. Surgical: Biliary diversion procedures; LT (might need increased immunosuppression)

Medical: UDCA; Rifampin. Surgical: Biliary diversion; LT

Surgical: LT Medical: UDCA; Rifampin; ASBT inhibitors; OCA. Surgical: LT

Medical: UDCA; rifampin; Cholestyramine. Surgical: PEBD; isolated or Combined liver-bowel transplant

PC: Phosphatidylcholine; CLDN1: Claudin-1; CLDN2: Claudin-2; BSEP: Bile salt export pump; MDR3: Multidrug resistance class 3 glycoprotein; MVID: Microvillus inclusion disease; GGT: Gamma-glutamyl transferase; AFP: Alpha-fetoprotein; AP: Alkaline phosphatase; ALT: Alanine transaminase; AST: Aspartate transaminase; BS: Bile salt; HCC: Hepatocellular carcinoma; MYO5B: Myosin VB; TPN: Total parenteral nutrition; UDCA: Ursodeoxycholic acid; CFTR: Cystic fibrosis transmembrane conductance regulator; 4PBA: 4-phenylbutyrate; ASBT: Apical sodium-dependent bile acid transporter; OCA: Obeticholic acid; PEBD: Partial external biliary diversion; PIBD: Partial internal biliary diversion; IE: Ileal exclusion; LT: Liver transplant; PFIC: Progressive Familial Intrahepatic Cholestasis; TJP2: Tight junction protein-2.



BILE FORMATIONPrior to describing the molecular and clinical features of PFIC, it is important to comprehend the bile formation process in order to understand how complex it is and how alterations could lead not only to hepatic disease, but also to dermatologic and neurologic symptoms.

The main primary bile acids produced by the human liver are cholic acid (CA) and chenodeoxycholic acid (CDCA), generated by cholesterol metabolism[13]. In the hepatocytes, these compounds are conjugated with taurine and glycine in order to form bile salts, increasing their solubility. After the bile is secreted into the duodenum, the bile salts are reabsorbed by the brush border membrane of the terminal ileum. These reabsorbed bile salts return to the liver through portal blood circulation. A small amount of bile salts are lost in the feces but are replaced by de novo synthesis in the liver. Daily, the human liver produces around 200 to 600 mg of bile acids, replenishing the bile acid pool in the enterohepatic circulation[13].

The hepatocellular part of bile formation can be divided into four stages. Stage 0 is characterized by the uptake of the compounds that will be processed to form the bile. This occurs at the basolateral hepatocyte membrane. Stage I is characterized by the activity of cytochrome P450 which turns the lipid compounds into soluble substrates for the next stages. Stage II is marked by the reactions that conjugate the bile acids. Finally, stage III is when the bile salts are secreted into the biliary canaliculus, the virtual space between two hepatocytes[1,13]. There are several different proteins and enzymes related to each stage, as shown in Figure 1.

The two main pathways to produce bile acids involve 17 different enzymes located in the cytoplasm, endoplasmic reticulum, mitochondria, and peroxisomes[13]. In the classic pathway, also known as the neutral pathway, cholesterol is first metabolized by CYP7A1, generating CDCA. In the presence of CYP8B1, CDCA is converted into CA. The alternative pathway, known as the acid pathway, begins with cholesterol side chain metabolism by CYP27A1, which is expressed in the mitochondrial membrane[14]. The alternative pathway is important for bile acid production in neonates as well as in diseased livers.

After bile acid synthesis and conjugation with the amino acids taurine and glycine, the bile salts are secreted into the canalicular region, a major step for bile flow. Most of the transporters belong to the ATP-binding cassette transporters (ABC transporters) superfamily[1]. As shown in Figure 1, there are several proteins involved in these steps and each one is responsible for transporting one compound in order to form the final product, the bile. Bile salt export pump (BSEP), multidrug resistance class 1 (MDR1), MDR3, MRP2, Breast Cancer Resistance Protein, sterolins 1 and 2, familial intrahepatic cholestasis 1 (FIC1) and others are examples of these transporters.

After the bile salts are secreted into the canalicular space, the cholangiocytes are responsible for alkalinizing and diluting the bile by secreting bicarbonate (HCO3

-) and water[1,15,16]. This process is initiated by the stimulation of secretin, a hormone produced in the S cells of the duodenum in response to the change in pH and presence of peptides after a meal. When this hormone binds to its receptor at the cholangiocyte basolateral membrane, it leads to a cytosolic increase in cAMP which activates CFTR, thereby releasing chloride (Cl-) into the bile duct lumen. This Cl- is exchanged with HCO3

-, mediated by the chloride-bicarbonate exchanger which is expressed on the apical membrane of cholangiocytes[15]. The release of HCO3

- is enhanced by local increases in Ca2+ signaling that activate the M3 muscarinic receptor. When acetylcholine binds to the M3 muscarinic receptor, InsP3, a second intracellular messenger, is produced. This InsP3 mediates the release of a Ca2+ signal via the type 3 inositol 1,4,5-trisphosphate receptor. The Ca2+ release near the apical membrane activates the Ca2+-dependent chloride channel, which releases more Cl- into the luminal space to be exchanged by the chloride-bicarbonate exchanger[6]. The activation of purinergic receptors also increases bicarbonate secretion, resulting in a pH increase. The presence of bicarbonate in the luminal space creates an osmotic gradient that is important for the flux of water from the cholangiocyte cytosol to the extracellular space. This transport is mediated by small integral membrane proteins known as aquaporins. The water makes the bile less viscous and leads the bile to the gallbladder, where it will be stored until a cholecystokinin stimulus occurs after a meal[1,15,16].

Mutations in genes that encode proteins expressed in the hepatocyte canalicular membrane and involved in Phase III of bile formation are responsible for the development of a cholestatic disease known as PFIC. Now, we will provide a molecular and clinical overview of the three classic types of PFIC and discuss the newer types of this disease.



Figure 1 Bile acid production. In phase 0, bile acids and other organic products are transported through the basolateral membrane of the hepatocyte. Phase I and II take place in the cytoplasm through activation and metabolization of differents CYPs. In phase III the bile acids are secreted into the biliary canalicus by the transmebrane transporters. NTCP: Na+-taurocholate cotransporting polypeptide; OATPs: Organic anion transporting polypeptides; OATs: Organic anion transporter; OCT1: Organic cation transporter 1; CYP7A1: Cytochrome P450 Family 7 Subfamily A Member 1; CYP8B1: Cytochrome P450 Family 8 Subfamily b Member 1; CYP27A1: Cytochrome P450 Family 27 Subfamily A Member 1; BSEP: Bile salt export pump; BRCP: Breast cancer resistance protein; ABCG5/8: ATP-binding cassette sub-family G member 5/8; MATE-1: Multidrug and toxin extrusion 1; MRP2: Multidrug resistance-associated protein 2; MDR1: Multidrug resistance class 1 glycoprotein; FIC1: Familial intrahepatic cholestasis 1; MDR3: Multidrug resistance class 3 glycoprotein; BC: Biliary canaliculus.

PFIC 1 PFIC type 1, also known as Byler’s disease, is caused by homozygous or compound heterozygous mutations of the ATP8B1 gene on chromosome 18 (18q21), which encodes the FIC1 protein[17]. FIC1 is part of the type 4 subfamily of P-type adenosine triphosphatases, which are involved in translocating phospholipids in membranes[18]. This protein, present at the apical membrane of hepatocytes, is thought to function as an aminophospholipid translocase, carrying phospholipids, specifically phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the outside, ectoplasmic leaflet of the canalicular membrane, to the inside, cytoplasmic leaflet in hepatocytes[17-19]. When functioning normally, FIC1 protects the hepatocyte from high bile salt concentrations[17]. By maintaining the asymmetry of the plasma membrane, FIC1 guards the canalicular membrane from the detergent nature of hydrophobic bile salts present in the canalicular lumen[17,18].

FIC1 was found to be successful at flopping phospholipids only when it was coexpressed with CDC50 proteins, specifically CDC50A[18]. This coexpression allows the FIC1-CDC50A heterodimer to leave the endoplasmic reticulum and localize to the apical membrane[18]. Mutations in CDC50A may also lead to the PFIC 1 phenotype if FIC1 fails to localize as a result[18]. In CDC50A-depleted intestinal cells, not only the apical membrane expression of FIC1 is reduced, but also the expression of the Solute Carrier Family 10 Member 2 (SLC10A2) also known as apical sodium-dependent bile acid transporter (ASBT)[20]. This transporter is responsible for the reuptake of bile acids in the intestines, so mutations in either the ATP8B1 gene or the CDC50A gene will impair localization of ASBT, reducing bile uptake[20]. This suggests that the FIC1-CDC50A heterodimer is involved in the localization of SLC10A2 to the apical membrane, resulting in bile salt malabsorption, which may be the cause of the diarrhea that PFIC 1 patients experience[20].

There is also evidence that FIC1 is involved in the organization of the apical membrane of polarized cells[21]. In ATP8B1-deficient intestinal Caco-2 cells, the apical actin cytoskeleton was found to be disorganized, there was a loss of microvilli, and the translation of apical membrane protein mRNA was disrupted[21]. The reduced



microvilli in the ATP8B1-deficient intestinal cells mirrors the loss of stereocilia on the apical membrane of inner hair cells in mice with an ATP8B1 mutation, which is linked to hearing loss, an event that is also observed in humans[21,22]. These examples support the case for the function of FIC1 in forming or stabilizing microvillar structures throughout the body[21].

In the disease phenotype, the lipid flipping process does not occur, interfering with the secretion of bile salt from the hepatocytes[23]. Lower expression of FIC1 downregulates FXR expression, encoded by the NR1H4 gene[17]. The function of FXR is two-fold. In hepatocytes, elevated bile salts concentrations activate FXR, which induces the expression of the BSEP[17,19]. BSEP allows for the pumping of bile salts out of the hepatocyte into the canalicular lumen[17,19]. Additionally, when expressed in the intestines, FXR represses the expression of ASBT. By doing so, bile salt reabsorption is reduced in the intestines. The bile salt transport defect is secondary in the case of the ATP8B1 mutation. The accumulation of bile salts inside the hepatocytes, coupled with the absence of the protective lipid flipping by FIC1 has a cytotoxic effect, harming the cells and leading to the cholestatic phenotype[17,19].

With the lack of lipid flipping, PS is freely present on the outer leaflet of the canalicular membrane[23]. This gives hydrophobic bile salts that exist in the canalicular space the opportunity to extract the PS from the membrane[23]. In normal hepatocytes, FIC1’s lipid flipping activity is necessary in maintaining the asymmetric and liquid ordered state of the outer leaflet of the canalicular membrane[23]. In diseased hepatocytes with an ATP8B1 mutation, the liquid ordered state becomes disordered because FIC1 is not able to flip the excess aminophospholipids from the outer to the inner leaflet[23]. This makes the membrane more susceptible to phospholipid, cholesterol, and ectoenzyme extraction[23]. Reduced cholesterol presence in the outer leaflet is vital in the functioning of certain integral membrane proteins such as BSEP (implicated in PFIC 2) and MRP1[23].

Each specific mutation comes with its own presentation of cholestatic disease. The G308V mutation was first identified in the Amish population and in homozygotes with this mutation, the FIC1 protein is undetectable in the liver[24]. The D554N mutation was found in Greenland familial cholestasis and also resulted in reduced expression of FIC1[24]. The G1040R mutation, first found in two Saudi families, did not result in a significantly decreased FIC1 expression when compared to the wild-type[24]. As expected, the interaction between FIC1 and CDC50A was undetectable in both the G308V and D554N mutations[24]. It was, however, still present in the G1040R mutation, although at a much lower level than the wild-type[24]. This is likely the reason why these three FIC1 mutant proteins are not localized to the canalicular membrane in WIF-B9 cell models[24].

Clinically, PFIC 1 presents with low gamma-glutamyl transferase (GGT) cholestasis[19]. Recurrent episodes of jaundice caused by hyperbilirubinemia, along with uncontrollable itching, or pruritus also occur[19]. Transaminases, as well as serum bile acids are elevated[19]. In infants with this disease, jaundice with pruritus, as well as hepatosplenomegaly develop early in life[19]. In severe cases, a progressive cholestasis presents along with portal hypertension[19]. Histologically, canalicular cholestasis, giant cell transformation, portal and lobular fibrosis, as well as lack of ductular proliferation are most common[17,19]. Because FIC1 is also expressed in cholangiocytes, enterocytes, and in the pancreas, there are extrahepatic manifestations of PFIC 1, setting it apart from the other types of intrahepatic cholestasis[17,19]. Some of these symptoms include diarrhea, stunted growth, exocrine pancreatic insufficiency, and progressive sensorineural hearing loss[17,19]. Nutritional deficiencies are also common, especially in fat-soluble vitamins A, D, E, and K[19].

Children with PFIC 1 often require vitamin supplementation, as well as other dietary aids in order to manage malnutrition[19]. Ursodeoxycholic acid (UDCA) is the first-line of medication used to treat pruritus[19]. It is a hydrophilic bile acid that is hypothesized to induce the expression of BSEP and MDR3, improving cholestatic symptoms[19]. Rifampin and cholestyramine have also been used in order to reduce pruritus, but have not been as effective as UDCA[19]. In addition, CFTR folding correctors may be a promising future therapy to enhance the trafficking of FIC1 in the hepatocytes[19].

In cases where medical management is inadequate, surgery is the next option. The purpose of these surgeries is to bypass the enterohepatic circulation, thereby lowering the amount of bile salts that are reabsorbed by the digestive system. Partial external biliary diversion (PEBD), partial internal biliary diversion (PIBD), and ileal exclusion (IE) have led to improvement in some cases[19]. PEBD has been shown to improve liver function, reduce serum bile acid levels, and slow progression of liver fibrosis in 80% of patients with either PFIC 1 or PFIC 2[25]. In this procedure, an external stomal conduit



is created to divert bile flow outside of the body[19]. PIBD diverts bile flow from the gall bladder to the colon in order to bypass the terminal ileum, where bile salt reabsorption occurs[19]. This procedure has been shown to improve pruritus, as well as decrease bilirubin and plasma bile acid levels[19]. IE bypasses the distal 15% of the ileum to prevent bile acid reabsorption[19]. It provides relief from pruritus and may be used as a bridge to transplant[26]. Ileal adaptation may occur, reducing the effectiveness of this surgery[25].

When no other treatment is successful, liver transplant is the final option. Lobular fibrosis may develop into cirrhosis and end stage liver disease[19]. There is no known association between FIC1 deficiency and tumorigenesis[19]. At this point, LT is indicated, which will improve cholestatic symptoms[19]. However, since FIC1 is expressed outside of the liver, extrahepatic symptoms may persist, or worsen[19]. Refractory diarrhea, coupled with the development of fatty liver changes in the allograft and fibrosis, may lead to re-transplant[27]. Ileal diversion during transplant may be used to potentially prevent these developments[19].

PFIC 2Homozygous or compound heterozygous mutations in the ABCB11 gene (chromosome 2q31), which encodes the BSEP, results in PFIC 2[17]. BSEP is an adenosine triphosphate-binding cassette transporter involved in actively transporting bile salts out of hepatocytes into biliary canaliculi[17]. It is crucial in maintaining enterohepatic circulation of bile salts[17]. Mutations in BSEP interrupt the process of pumping bile out of hepatocytes, leading to increased intracellular bile salt concentrations which consequently damage the hepatocytes[17].

FXR, activated in a heterodimer with retinoid X receptor α (RXRα), has been linked to upregulating enzymes and bile salt transporters when bound to bile salts in the cell[28]. BSEP is one of these bile salt transporters that FXR activates[28]. CDCA and the FXR/RXRα heterodimer collectively work together to transactivate the ABCB11 promoter[28]. The level of activation of the ABCB11 gene is dependent on the concentration of CDCA[28]. When more bile acids are present in the hepatocyte, more BSEP is made to maintain the equilibrium.

There are over 200 mutations that have been linked with causing PFIC 2[19]. Some of these mutations cause splicing defects while most cause protein processing defects[29]. The majority of missense mutations resulted in a reduction of BSEP levels in vitro[29]. Some common mutations — G238V, D482G, G982R, R1153C, R1286Q, and ΔGly — cause the mutant protein to be retained in the endoplasmic reticulum[30]. Proteosomes are the main cellular component that breaks down these mutant proteins, but the D482G mutant protein can also be broken down by lysosomes[30]. The degradation occurs through Endoplasmic Reticulum Associated Degradation, part of the quality control system of the endoplasmic reticulum[30]. This process involves ubiquitination, retro-translocation out of the endoplasmic reticulum, and degradation by the proteasome[30].

PFIC 2 presents with low GGT cholestasis as well as highly elevated transaminases, serum bilirubin, and alpha-fetoprotein (AFP) levels[17,19]. Jaundice, pruritus, scleral icterus, hepatomegaly, chronic skin picking, and reduced growth are also common[17,19]. Liver histology shows canalicular cholestasis, lobular and portal fibrosis, and hepatocellular necrosis[17]. Unlike PFIC 1, there is up to a 15% chance of cirrhosis developing into either hepatocellular carcinoma (HCC) or cholangiocarcinoma in patients with PFIC 2[19].

Treatment of PFIC 2 is similar to PFIC 1. A promising therapy includes treatment with cell surface BSEP-enhancer molecules such as 4-phenylbutyrate (4PBA)[19]. 4PBA has been shown to improve liver function and pruritus in patients with certain mutations of BSEP[19]. Bile salt excretion was improved, although not completely[31]. 4PBA helps increase cell surface expression of the mutant BSEPs, likely altering a post-transcriptional mechanism[32]. This therapy works best when the mutation results in normal transport ability, but impaired membrane trafficking[32,33]. Biliary diversion procedures have a better response in cases where there is still some BSEP function than in cases where function is nonexistent[19].

LT is the ultimate therapeutic option, but there is an additional risk in patients with PFIC 2. In severe cases, patients might develop allo-reactive antibodies specific to BSEP in the allograft, causing BSEP deficiency in the transplanted liver[19]. This happens in up to 8% of PFIC 2 patients who undergo LT[27]. Increased immuno-suppression may prevent these antibodies from developing, but if the disease recurs,



plasmapheresis to remove anti-BSEP antibodies, B-cell depleting antibody therapy, or allogeneic hematopoietic stem cell transplant may help[19,27]. If none of these therapies succeed, a second LT might be required[27].

There have been many scientific advances offering hope for potential treatments for BSEP deficiency. One group led by Jillian Ellis developed a zebrafish model of BSEP deficiency[34]. Treating these zebrafish with rapamycin partially restored bile excretion as well as lengthened their lifespans[34]. The rapamycin likely localizes other transporters, such as MDR1, to the canaliculus in order to compensate for the lack of BSEP[34]. Rapamycin is an autophagy inducer, so the increased trafficking of MDR1 in rapamycin treated zebrafish might be the result of increased autophagy[34]. Another group led by Imagawa et al[33] generated a BSEP-deficient cellular model using patient cells[33]. They generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells[33]. Then, they were able to differentiate these iPSCs into hepatocyte-like cells that mimicked the BSEP deficiency of the patient from whom they were derived[33]. These cells had abnormal BSEP expression, as well as reduced biliary excretion[33]. When the cells were treated with 4PBA, there was an increase in membrane expression of BSEP[33]. In vitro models such as this one provide a practical way to test the effect of many different therapeutic agents on cells derived from individual patients themselves. Advances in stem cell research might make it possible to determine what drugs a patient will respond best to, based on their specific mutation, without having to test them all on the patient.

PFIC 3Mutations in the ABCB4 gene, located on chromosome 7q21, which encodes the MDR3 glycoprotein, cause not only PFIC 3, but also other cholestasis related diseases such as gallbladder disease 1/low phospholipid-associated cholelithiasis (GBD1/LPAC), parenteral nutrition-associated liver disease, transient neonatal cholestasis and intrahepatic cholestasis of pregnancy[17]. This phosphatidylcholine (PC) flippase is localized only to the canalicular membrane of hepatocytes and consists of two cytoplasmic nucleotide binding domains and two transmembrane domains, each with six transmembrane segments. This structure facilitates the transport of PC out of the hepatocyte and into the biliary canaliculi. This process is supported by the over-expression of MDR3 in polarized cells, increasing the rate of transport of fluorescently labeled PC, but not other phospholipids. MDR2, the mouse homolog of human MDR3, has been used in knockout (KO) mice in order to model MDR3 deficiency[35]. The absence of the PC flippase leads to hepatocyte necrosis, dilated biliary canaliculus, periportal inflammation and bile duct proliferation[36]. These MDR2 KO animals develop HCC earlier, within 4-6 mo, due to an accumulation of carcinogenic factors[37-39]. The expression of human MDR3 in the MDR2 KO animals restores the function and histological features of the hepatocytes[38,40].

Several variations in the ABCB4 gene have been described. Delaunay and colleagues used site-directed mutagenesis in a cell line model to show that the mutations I541F, L556R, and Q855L result in protein retention in the endoplasmic reticulum[41]. Another four mutations have no effect on MDR3 function: T424A and N510S had a normal pattern of canalicular expression with lower protein stability, while T175A and R652G had no detectable defect. Degiorgio et al[42] analyzed ABCB4 gene mutations in 68 PFIC 3 patients. The authors found 29 mutations in the coding region, including 23 missense, four nonsense, and two short insertion mutations in the gene. 10 of these mutations were present in the transmembrane domains (TM), specifically in TM7, involved in PC translocation[42]. Delaunay et al[41] devised a classification system for the various forms of these mutations[41]. Class I includes mutations that result in defective synthesis, class II covers variations that prevent protein maturation, class III mutants result in mature but defective proteins, class IV variations are unstable, and class V mutants have unknown pathogenicity[41]. These classifications are useful in determining potential therapies for patients, depending on their genotype.

When MDR3 does not function properly, PC is absent from the biliary canaliculi. The free hydrophobic bile acids in this space will damage cholangiocytes, leading to cholestasis[19]. In addition, cholesterol is more likely to crystallize into stones, damaging liver structures by obstructing small bile ducts[25]. PFIC 3 often presents later on in life than both PFIC 1 and PFIC 2, as late as adulthood in some cases[19]. In early onset, pruritus, hepatosplenomegaly, variceal bleeding caused by portal hypertension, alcoholic stools, stunted growth, jaundice, reduced bone density, and learning disabilities have all been associated with MDR3 deficiency[17,19]. Adult onset can present



anywhere from cholelithiasis to benign recurrent intrahepatic cholestasis (BRIC)[19]. Unlike PFIC 1 and 2, PFIC 3 presents with elevated GGT[19]. Transaminases, bilirubin, and alkaline phosphatase are elevated as well[17,19]. Although serum bile salt and cholesterol can be in the normal range, biliary phospholipids will be reduced[17]. Histological findings include nonspecific portal inflammation, portal fibrosis, cholestasis with bile duct proliferation, and slight giant cell hepatitis[17,19]. MDR3 expression upon staining will depend on the type of protein defect, with functional protein defects staining normally and truncated protein defects not staining[17,19]. Additionally, hepatocellular carcinoma and cholangiocarcinoma have been associated with PFIC 3[19].

UDCA treatment has been effective in 70% of cases where the patient retains MDR3 expression and residual PC secretion[17]. Rifampin could also improve pruritus[19]. Chemical chaperones could improve MDR3 trafficking, depending on the specific mutation[43]. Biliary diversion procedures are not as successful with MDR3 deficiency because of the severity upon presentation[19]. LT is currently the only known curative treatment[19]. Gene therapy via adeno-associated virus has shown promise in restoring phospholipid excretion and preventing liver injury in murine models of PFIC 3[44]. This may be an alternative therapy for MRD3 deficiency in the near future.

NEWER TYPES OF PFICPFIC 4PFIC 4 is caused by a loss of function mutation in the tight junction protein 2 (TJP2), also called zona occludens 2, present on chromosome 9q21[17]. In the liver, this protein is involved in forming tight junctions by interacting with transmembrane tight junction proteins and the actin cytoskeleton[45]. Tight junctions are essential in the liver because they help prevent the leakage of biliary components into the liver parenchyma[46]. Claudin-1 (CLDN1) and claudin-2 (CLDN2) are two integral tight junction proteins that are expressed in the liver, aiding in the formation of tight junctions[45]. Normally, these two proteins are localized to the canalicular membrane, but in TJP2 mutation, CLDN1 fails to localize, especially in the parenchyma of the hepatic lobule[45,47]. These compromised tight junctions then allow cytotoxic bile salts to leak into the paracellular space, causing damage to the surrounding hepatocytes and cholangiocytes[19,45].

This disease presents with severe cholestasis and low GGT levels[17]. Patients lack mutations in ATP8B1 and ABCB11 genes, excluding the diagnoses of PFIC 1 and PFIC 2. Extrahepatic symptoms have been reported in some patients, mainly in the neurological and respiratory systems[45]. This is likely related to the expression of TJP2 in every epithelial cell in the body[45]. The reason the liver is consistently affected by TJP2 deficiency is because of the particularly damaging nature of the bile acids present in the canaliculi[47]. Pathological findings include intracellular cholestasis, giant cell transformation, lack of TJP2 with staining, abnormal CLDN1 localization, and normal CLDN2 localization[19,47]. The lack of TJP2 could be tied to the nonsense mediated mRNA decay (NMD) process[45]. This process degrades mutant transcripts, such as TJP2 in the case of a mutation[45]. Even though the protein could have residual function, it is still degraded[45]. TJP2 was found to have low mRNA expression in PFIC 4 patients[45]. Western blotting revealed no TJP2 expression, indicating a possible link between the mutation in TJP2 and NMD[45].

There have been several reports of HCC occurrence in patients suffering with TJP2 deficiency[48-50]. The exact mechanism of tumorigenesis has not yet been elucidated. In terms of treatment, there have been no reported cases of recurrence after LT[17].

PFIC 5Loss of function mutations in the NR1H4 gene (chromosome 12q23), encoding FXR, result in PFIC 5[4,17]. FXR is a nuclear receptor activated by bile acids and directly involved in the expression of both BSEP and MDR3, proteins affected in PFIC 2 and PFIC 3 respectively[4]. This protein is activated by elevated bile acids levels in the ileum, inducing expression of fibroblast growth factor 19 (FGF19)[51]. In the liver, FGF19 binds to the fibroblast growth factor receptor 4/β-Klotho complex, which in turn represses cytochrome P450 7A1 (CYP7A1)[51]. Repression of this enzyme reduces de novo bile acid synthesis[51].

Features of this disease include neonatal onset of normal GGT associated cholestasis, elevated serum bilirubin, elevated serum AFP levels, undetectable expression of BSEP in the bile canaliculi, and vitamin K independent coagulopathy[4].



This vitamin K independent coagulopathy is unique to PFIC 5 and has been shown to be a direct result of FXR mutation[4]. Three fibrinogen genes, as well as some coagulation factors have been linked with FXR-dependent induction, which does not occur in PFIC 5 patients[4]. Histological findings are intralobular cholestasis with ductular reaction, hepatocellular ballooning, fibrosis progressing into micronodular cirrhosis, and giant cell transformation[19]. NR1H4/FXR associated PFIC is very rare as only eight cases have been reported in the literature[4,52,53]. LT has been used, but steatosis in the transplanted liver has been reported in some of these cases[4,53]. UDCA and rifampicin may be used in order to improve pruritus, but ASBT inhibitors, as well as FXR agonists such as obeticholic acid, both show promise as future medical therapies[51,54].

PFIC associated with MYO5B defectsThe MYO5B gene (chromosome 18q21.1) encodes an actin-associated molecular motor known as MYO5B[17]. The interaction between MYO5B and RAS-related GTP-binding protein 11A (RAB11A) is essential for the polarization of epithelial cells, as well as localizing BSEP to the canalicular membrane[55]. Diminished activity of the MYO5B/RAB11A recycling endosome pathway is related to disrupting the localization of BSEP[56]. Mutations of this gene are associated with microvillus inclusion disease (MVID) which affects the enterocytes and leads to diarrhea, as well as malabsorption[57]. Mislocalized apical brush border proteins, villus atrophy, and the presence of microvillus inclusion bodies are all associated with MVID[57-60]. Total parenteral nutrition (TPN) is required throughout life, but it has been associated with a high risk of sepsis and small bowel transplant[61].

MVID has been associated with cholestatic liver disease, which possibly occurs as a result of TPN[57]. In fact, MYO5B gene mutations may account for 20% of the idiopathic low-GGT associated cholestasis in pediatric patients[60]. This cholestasis presents with low to normal GGT levels, jaundice, pruritus, mildly elevated Alanine transaminase and Aspartate transaminase, elevated serum BS levels, hepatomegaly, portal and lobular fibrosis, and giant cell transformation[17,19,60]. Staining of BSEP and MDR3 may show that they are present, but not localized to the canalicular membrane, although this may not occur in every patient[19,60].

There is evidence for different phenotypes resulting from the type of MYO5B gene mutation[55,60]. Mutations with limited effects on MYO5B functionality are linked with canalicular transport defects in hepatocytes but are not known to have as significant of an effect on enterocytes[60]. In more damaging mutations, the hepatocyte defects may be greater, but there will also be enterocyte disruption[60]. Enterocytes will not uptake bile acids as efficiently, reducing the amount of bile product taken back to the liver via enterohepatic circulation, potentially alleviating or protecting against cholestatic symptoms[60]. There seems to be an inverse relationship between cholestasis and intestinal function. UDCA, rifampin, and cholestyramine have been used to treat pruritus in these patients[19]. If medical treatment is not sufficient, PEBD may be successful[19]. When symptoms necessitate transplant, combined bowel-liver transplant should be considered in order to avoid post-transplant onset of cholestasis[60].

HOW TO STUDY PFIC?After the identification of mutations in patients, several models have been created to better understand not only the pathophysiology of PFIC, but also to investigate drugs and treatments. The first model is the use of a cell line with mutations in PFIC related genes to study the mechanisms of these variants on both the molecular and clinical level. van der Velden et al[62], using different cell lines, studied some FIC1 mutations[62]. Delaunay et al[41], studied MDR3 gene mutations using HepG2 as a model. A second option to study PFIC are knockout mice[63]. Some studies have reported the use of ABCB11-/-mice as a model for PFIC 2, however the clinical findings are less severe than those of humans[63]. MDR2 KO mice are used to model complications resulting from MDR3 deficiency in humans and are also used to study treatments for all MDR3-related diseases[35,40].

In 2012, when Shinya Yamanaka and John B. Gurdon were awarded The Nobel Prize in Physiology or Medicine for their discovery in pluripotency, a new method to study PFIC became available. The possibility of transforming any somatic cell into an iPSCs was revolutionary. Okita and colleagues used plasmids to express c-Myc, SRY-box transcription factor 2 (Sox2), Kruppel-like factor 4, and octamer-binding transcription factor 4 (Oct4) in reprogrammed mature cells[64]. This last set of genes is



known as Yamanaka’s Factors. However, there are many different protocols to generate iPSCs. Si-Tayeb et al[65] used the expression of Oct4, Sox2, Nanog, and Lin-28 homolog A in a lentivirus delivery system to generate iPS cells[65]. In this way, Imagawa et al[33] generated a BSEP-deficient cellular model using patient cells and tested the effects of 4PBA treatment on protein expression and localization[33]. With this system of reprogramming cells, and access to patient cells with different mutations, a new model became available to better understand the various types of PFIC. The possibility of differentiating these cells into liver cells, such as hepatocytes and cholangiocytes, gives researchers the opportunity to test potential treatments in vitro. This can be extremely useful since very few patients have these rare diseases and are available to enroll in clinical trials. Large scale drug trials may not be feasible.

CONCLUSIONPFIC is a rare disease that affects the bile secretion process of the hepatocytes due to alterations in the proteins involved, as shown in Figure 2. Better understanding of the molecular alterations in this context is necessary to elucidate new targets and pathways that could be the focus of new treatments in the future. With advances in inducing pluripotent cells, a new model that is more precise and closer to humans has become available. Also, the progress in gene editing will help in the development of new models and potentially be used as a treatment method in the future.



Figure 2 The molecular mechanisms behind progressive familial intrahepatic cholestasis. A: The classic types of Progressive Familial Intrahepatic Cholestasis (PFIC). PFIC 1 is related to mutations in the genes that encode the flippase familial intrahepatic cholestasis 1, which flips phospholipids in the plasma membrane. Mutations in the bile salt export pump (BSEP) protein, a bile salt transporter, results in PFIC 2. The third type of PFIC is caused by mutations in the gene that encodes the Multidrug resistance class 3 glycoprotein (MDR3) protein, another lipid flippase; B: The newer types of PFIC. Mutations in the tight junction protein-2 protein, which prevents the mixing of blood and bile acids, are responsible for PFIC 4. PFIC 5 is a result of mutations in the farnesoid X receptor protein, a transcription factor important for BSEP and MDR3 ecpression. Mutations in Myosin VB result in a PFIC phenotype because the trafficking of the BSEP protein from the endoplasmic reticulum to the plasma membrane is disrupted. PFIC: Progressive Familial Intrahepatic Cholestasis; FIC1: Familial intrahepatic cholestasis 1; BSEP: Bile salt export pump; MDR3: Multidrug resistance class 3 glycoprotein; TJP2: Tight junction protein-2; FXR: Farnesoid X receptor.

REFERENCESBoyer JL. Bile formation and secretion. Compr Physiol 2013; 3: 1035-1078 [PMID: 23897680 DOI: 10.1002/cphy.c120027]

1

Hirschfield GM, Heathcote EJ, Gershwin ME. Pathogenesis of cholestatic liver disease and therapeutic approaches. Gastroenterology 2010; 139: 1481-1496 [PMID: 20849855 DOI: 10.1053/j.gastro.2010.09.004]

2

Alvarez L, Jara P, Sánchez-Sabaté E, Hierro L, Larrauri J, Díaz MC, Camarena C, De la Vega A, Frauca E, López-Collazo E, Lapunzina P. Reduced hepatic expression of farnesoid X receptor in hereditary cholestasis associated to mutation in ATP8B1. Hum Mol Genet 2004; 13: 2451-2460 [PMID: 15317749 DOI: 10.1093/hmg/ddh261]

3

Gomez-Ospina N, Potter CJ, Xiao R, Manickam K, Kim MS, Kim KH, Shneider BL, Picarsic JL, Jacobson TA, Zhang J, He W, Liu P, Knisely AS, Finegold MJ, Muzny DM, Boerwinkle E, Lupski JR, Plon SE, Gibbs RA, Eng CM, Yang Y, Washington GC, Porteus MH, Berquist WE, Kambham N, Singh RJ, Xia F, Enns GM, Moore DD. Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis. Nat Commun 2016; 7: 10713 [PMID: 26888176 DOI: 10.1038/ncomms10713]

4

Marson FAL, Bertuzzo CS, Ribeiro JD. Classification of CFTR mutation classes. Lancet Respir Med 2016; 4: e37-e38 [PMID: 27377414 DOI: 10.1016/S2213-2600(16)30188-6]

5

Trampert DC, Nathanson MH. Regulation of bile secretion by calcium signaling in health and disease. Biochim Biophys Acta Mol Cell Res 2018; 1865: 1761-1770 [PMID: 29787781 DOI: 10.1016/j.bbamcr.2018.05.010]

6

Dyson JK, Hirschfield GM, Adams DH, Beuers U, Mann DA, Lindor KD, Jones DE. Novel therapeutic targets in primary biliary cirrhosis. Nat Rev Gastroenterol Hepatol 2015; 12: 147-158 [PMID: 25645973 DOI: 10.1038/nrgastro.2015.12]

7

Arsenijevic A, Stojanovic B, Milovanovic J, Arsenijevic D, Arsenijevic N, Milovanovic M. Galectin-8


https://dx.doi.org/10.1002/cphy.c120027




https://dx.doi.org/10.1093/hmg/ddh261


https://dx.doi.org/10.1038/ncomms10713


https://dx.doi.org/10.1016/S2213-2600(16)30188-6


https://dx.doi.org/10.1016/j.bbamcr.2018.05.010





3 in Inflammasome Activation and Primary Biliary Cholangitis Development. Int J Mol Sci 2020; 21: 5097 [PMID: 32707678 DOI: 10.1038/s41575-019-0226-7]Jiang X, Karlsen TH. Genetics of primary sclerosing cholangitis and pathophysiological implications. Nat Rev Gastroenterol Hepatol 2017; 14: 279-295 [PMID: 28293027 DOI: 10.1038/nrgastro.2016.154]

9

Palmer KR, Xiaohua L, Mol BW. Management of intrahepatic cholestasis in pregnancy. Lancet 2019; 393: 853-854 [PMID: 30773279 DOI: 10.1016/S0140-6736(18)32323-7]

10

Sundaram V, Björnsson ES. Drug-induced cholestasis. Hepatol Commun 2017; 1: 726-735 [PMID: 29404489 DOI: 10.1002/hep4.1088]

11

Hoofnagle JH, Björnsson ES. Drug-Induced Liver Injury - Types and Phenotypes. N Engl J Med 2019; 381: 264-273 [PMID: 31314970 DOI: 10.1056/NEJMra1816149]

12

Chiang JY. Bile acid metabolism and signaling. Compr Physiol 2013; 3: 1191-1212 [PMID: 23897684 DOI: 10.1002/cphy.c120023]

13

Chiang JY. Bile acids: regulation of synthesis. J Lipid Res 2009; 50: 1955-1966 [PMID: 19346330 DOI: 10.1194/jlr.R900010-JLR200]

14

Banales JM, Huebert RC, Karlsen T, Strazzabosco M, LaRusso NF, Gores GJ. Cholangiocyte pathobiology. Nat Rev Gastroenterol Hepatol 2019; 16: 269-281 [PMID: 30850822 DOI: 10.1038/s41575-019-0125-y]

15

Tabibian JH, Masyuk AI, Masyuk TV, O'Hara SP, LaRusso NF. Physiology of cholangiocytes. Compr Physiol 2013; 3: 541-565 [PMID: 23720296 DOI: 10.1002/cphy.c120019]

16

Vitale G, Gitto S, Vukotic R, Raimondi F, Andreone P. Familial intrahepatic cholestasis: New and wide perspectives. Dig Liver Dis 2019; 51: 922-933 [PMID: 31105019 DOI: 10.1016/j.dld.2019.04.013]

17

Paulusma CC, Folmer DE, Ho-Mok KS, de Waart DR, Hilarius PM, Verhoeven AJ, Oude Elferink RP. ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity. Hepatology 2008; 47: 268-278 [PMID: 17948906 DOI: 10.1002/hep.21950]

18

Henkel SA, Squires JH, Ayers M, Ganoza A, Mckiernan P, Squires JE. Expanding etiology of progressive familial intrahepatic cholestasis. World J Hepatol 2019; 11: 450-463 [PMID: 31183005 DOI: 10.4254/wjh.v11.i5.450]

19

van der Mark VA, de Waart DR, Ho-Mok KS, Tabbers MM, Voogt HW, Oude Elferink RP, Knisely AS, Paulusma CC. The lipid flippase heterodimer ATP8B1-CDC50A is essential for surface expression of the apical sodium-dependent bile acid transporter (SLC10A2/ASBT) in intestinal Caco-2 cells. Biochim Biophys Acta 2014; 1842: 2378-2386 [PMID: 25239307 DOI: 10.1016/j.bbadis.2014.09.003]

20

Verhulst PM, van der Velden LM, Oorschot V, van Faassen EE, Klumperman J, Houwen RH, Pomorski TG, Holthuis JC, Klomp LW. A flippase-independent function of ATP8B1, the protein affected in familial intrahepatic cholestasis type 1, is required for apical protein expression and microvillus formation in polarized epithelial cells. Hepatology 2010; 51: 2049-2060 [PMID: 20512993 DOI: 10.1002/hep.23586]

21

Stapelbroek JM, Peters TA, van Beurden DH, Curfs JH, Joosten A, Beynon AJ, van Leeuwen BM, van der Velden LM, Bull L, Oude Elferink RP, van Zanten BA, Klomp LW, Houwen RH. ATP8B1 is essential for maintaining normal hearing. Proc Natl Acad Sci USA 2009; 106: 9709-9714 [PMID: 19478059 DOI: 10.1073/pnas.0807919106]

22

Paulusma CC, Groen A, Kunne C, Ho-Mok KS, Spijkerboer AL, Rudi de Waart D, Hoek FJ, Vreeling H, Hoeben KA, van Marle J, Pawlikowska L, Bull LN, Hofmann AF, Knisely AS, Oude Elferink RP. Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport. Hepatology 2006; 44: 195-204 [PMID: 16799980 DOI: 10.1002/hep.21212]

23

Folmer DE, van der Mark VA, Ho-Mok KS, Oude Elferink RP, Paulusma CC. Differential effects of progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1 mutations on canalicular localization of ATP8B1. Hepatology 2009; 50: 1597-1605 [PMID: 19731236 DOI: 10.1002/hep.23158]

24

Srivastava A. Progressive familial intrahepatic cholestasis. J Clin Exp Hepatol 2014; 4: 25-36 [PMID: 25755532 DOI: 10.1016/j.jceh.2013.10.005]

25

Van Vaisberg V, Tannuri ACA, Lima FR, Tannuri U. Ileal exclusion for pruritus treatment in children with progressive familial intrahepatic cholestasis and other cholestatic diseases. J Pediatr Surg 2020; 55: 1385-1391 [PMID: 31708211 DOI: 10.1016/j.jpedsurg.2019.09.018]

26

van der Woerd WL, van Mil SW, Stapelbroek JM, Klomp LW, van de Graaf SF, Houwen RH. Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy. Best Pract Res Clin Gastroenterol 2010; 24: 541-553 [PMID: 20955958 DOI: 10.1016/j.bpg.2010.07.010]

27

Plass JR, Mol O, Heegsma J, Geuken M, Faber KN, Jansen PL, Müller M. Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 2002; 35: 589-596 [PMID: 11870371 DOI: 10.1053/jhep.2002.31724]

28

Byrne JA, Strautnieks SS, Ihrke G, Pagani F, Knisely AS, Linton KJ, Mieli-Vergani G, Thompson RJ. Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing. Hepatology 2009; 49: 553-567 [PMID: 19101985 DOI: 10.1002/hep.22683]

29

Wang L, Dong H, Soroka CJ, Wei N, Boyer JL, Hochstrasser M. Degradation of the bile salt export 30


https://dx.doi.org/10.1038/s41575-019-0226-7




https://dx.doi.org/10.1016/S0140-6736(18)32323-7


https://dx.doi.org/10.1002/hep4.1088


https://dx.doi.org/10.1056/NEJMra1816149




https://dx.doi.org/10.1194/jlr.R900010-JLR200


https://dx.doi.org/10.1038/s41575-019-0125-y




https://dx.doi.org/10.1016/j.dld.2019.04.013




https://dx.doi.org/10.4254/wjh.v11.i5.450


https://dx.doi.org/10.1016/j.bbadis.2014.09.003




https://dx.doi.org/10.1073/pnas.0807919106






https://dx.doi.org/10.1016/j.jceh.2013.10.005


https://dx.doi.org/10.1016/j.jpedsurg.2019.09.018


https://dx.doi.org/10.1016/j.bpg.2010.07.010







pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II. Hepatology 2008; 48: 1558-1569 [PMID: 18798335 DOI: 10.1002/hep.22499]Naoi S, Hayashi H, Inoue T, Tanikawa K, Igarashi K, Nagasaka H, Kage M, Takikawa H, Sugiyama Y, Inui A, Nagai T, Kusuhara H. Improved liver function and relieved pruritus after 4-phenylbutyrate therapy in a patient with progressive familial intrahepatic cholestasis type 2. J Pediatr 2014; 164: 1219-1227. e3 [PMID: 24530123 DOI: 10.1016/j.jpeds.2013.12.032]

31

Hayashi H, Sugiyama Y. 4-phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps. Hepatology 2007; 45: 1506-1516 [PMID: 17538928 DOI: 10.1002/hep.21630]

32

Imagawa K, Takayama K, Isoyama S, Tanikawa K, Shinkai M, Harada K, Tachibana M, Sakurai F, Noguchi E, Hirata K, Kage M, Kawabata K, Sumazaki R, Mizuguchi H. Generation of a bile salt export pump deficiency model using patient-specific induced pluripotent stem cell-derived hepatocyte-like cells. Sci Rep 2017; 7: 41806 [PMID: 28150711 DOI: 10.1038/srep41806]

33

Ellis JL, Bove KE, Schuetz EG, Leino D, Valencia CA, Schuetz JD, Miethke A, Yin C. Zebrafish abcb11b mutant reveals strategies to restore bile excretion impaired by bile salt export pump deficiency. Hepatology 2018; 67: 1531-1545 [PMID: 29091294 DOI: 10.1002/hep.29632]

34

Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993; 75: 451-462 [PMID: 8106172 DOI: 10.1016/0092-8674(93)90380-9]

35

Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes. J Hepatol 2005; 43: 1045-1054 [PMID: 16223543 DOI: 10.1016/j.jhep.2005.06.025]

36

Jacquemin E. Role of multidrug resistance 3 deficiency in pediatric and adult liver disease: one gene for three diseases. Semin Liver Dis 2001; 21: 551-562 [PMID: 11745043 DOI: 10.1055/s-2001-19033]

37

Oude Elferink RP, Paulusma CC. Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein). Pflugers Arch 2007; 453: 601-610 [PMID: 16622704 DOI: 10.1007/s00424-006-0062-9]

38

Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes. Semin Liver Dis 2007; 27: 77-98 [PMID: 17295178 DOI: 10.1055/s-2006-960172]

39

Smith AJ, de Vree JM, Ottenhoff R, Oude Elferink RP, Schinkel AH, Borst P. Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (-/-) mice. Hepatology 1998; 28: 530-536 [PMID: 9696021 DOI: 10.1002/hep.510280234]

40

Delaunay JL, Durand-Schneider AM, Dossier C, Falguières T, Gautherot J, Davit-Spraul A, Aït-Slimane T, Housset C, Jacquemin E, Maurice M. A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3. Hepatology 2016; 63: 1620-1631 [PMID: 26474921 DOI: 10.1002/hep.28300]

41

Degiorgio D, Colombo C, Seia M, Porcaro L, Costantino L, Zazzeron L, Bordo D, Coviello DA. Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3). Eur J Hum Genet 2007; 15: 1230-1238 [PMID: 17726488 DOI: 10.1038/sj.ejhg.5201908]

42

Gordo-Gilart R, Andueza S, Hierro L, Jara P, Alvarez L. Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones. PLoS One 2016; 11: e0150098 [PMID: 26900700 DOI: 10.1371/journal.pone.0150098]

43

Aronson SJ, Bakker RS, Shi X, Duijst S, Ten Bloemendaal L, de Waart DR, Verheij J, Ronzitti G, Oude Elferink RP, Beuers U, Paulusma CC, Bosma PJ. Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice. J Hepatol 2019; 71: 153-162 [PMID: 30935993 DOI: 10.1016/j.jhep.2019.03.021]

44

Sambrotta M, Thompson RJ. Mutations in TJP2, encoding zona occludens 2, and liver disease. Tissue Barriers 2015; 3: e1026537 [PMID: 26451340 DOI: 10.1080/21688370.2015.1026537]

45

González-Mariscal L, Gallego-Gutiérrez H, González-González L, Hernández-Guzmán C. ZO-2 Is a Master Regulator of Gene Expression, Cell Proliferation, Cytoarchitecture, and Cell Size. Int J Mol Sci 2019; 20: 4128 [PMID: 31450555 DOI: 10.3390/ijms20174128]

46

Sambrotta M, Strautnieks S, Papouli E, Rushton P, Clark BE, Parry DA, Logan CV, Newbury LJ, Kamath BM, Ling S, Grammatikopoulos T, Wagner BE, Magee JC, Sokol RJ, Mieli-Vergani G; University of Washington Center for Mendelian Genomics; Smith JD; Johnson CA; McClean P; Simpson MA; Knisely AS; Bull LN; Thompson RJ. Mutations in TJP2 cause progressive cholestatic liver disease. Nat Genet 2014; 46: 326-328 [PMID: 24614073 DOI: 10.1038/ng.2918]

47

Vij M, Shanmugam NP, Reddy MS, Sankaranarayanan S, Rela M. Paediatric hepatocellular carcinoma in tight junction protein 2 (TJP2) deficiency. Virchows Arch 2017; 471: 679-683 [PMID: 28733884 DOI: 10.1007/s00428-017-2204-1]

48

Wei CS, Becher N, Friis JB, Ott P, Vogel I, Grønbæk H. New tight junction protein 2 variant causing progressive familial intrahepatic cholestasis type 4 in adults: A case report. World J Gastroenterol 2020; 26: 550-561 [PMID: 32089630 DOI: 10.3748/wjg.v26.i5.550]

49

Zhou S, Hertel PM, Finegold MJ, Wang L, Kerkar N, Wang J, Wong LJ, Plon SE, Sambrotta M, Foskett P, Niu Z, Thompson RJ, Knisely AS. Hepatocellular carcinoma associated with tight-junction

50




https://dx.doi.org/10.1016/j.jpeds.2013.12.032








https://dx.doi.org/10.1016/0092-8674(93)90380-9




https://dx.doi.org/10.1055/s-2001-19033


https://dx.doi.org/10.1007/s00424-006-0062-9


https://dx.doi.org/10.1055/s-2006-960172






https://dx.doi.org/10.1038/sj.ejhg.5201908






https://dx.doi.org/10.1080/21688370.2015.1026537


https://dx.doi.org/10.3390/ijms20174128


https://dx.doi.org/10.1038/ng.2918


https://dx.doi.org/10.1007/s00428-017-2204-1





protein 2 deficiency. Hepatology 2015; 62: 1914-1916 [PMID: 25921221 DOI: 10.1002/hep.27872]Cariello M, Piccinin E, Garcia-Irigoyen O, Sabbà C, Moschetta A. Nuclear receptor FXR, bile acids and liver damage: Introducing the progressive familial intrahepatic cholestasis with FXR mutations. Biochim Biophys Acta Mol Basis Dis 2018; 1864: 1308-1318 [PMID: 28965883 DOI: 10.1016/j.bbadis.2017.09.019]

51

Chen HL, Li HY, Wu JF, Wu SH, Chen HL, Yang YH, Hsu YH, Liou BY, Chang MH, Ni YH. Panel-Based Next-Generation Sequencing for the Diagnosis of Cholestatic Genetic Liver Diseases: Clinical Utility and Challenges. J Pediatr 2019; 205: 153-159. e6 [PMID: 30366773 DOI: 10.1016/j.jpeds.2018.09.028]

52

Himes RW, Mojarrad M, Eslahi A, Finegold MJ, Maroofian R, Moore DD. NR1H4-related Progressive Familial Intrahepatic Cholestasis 5: Further Evidence for Rapidly Progressive Liver Failure. J Pediatr Gastroenterol Nutr 2020; 70: e111-e113 [PMID: 32443034 DOI: 10.1097/MPG.0000000000002670]

53

Carino A, Biagioli M, Marchianò S, Fiorucci C, Bordoni M, Roselli R, Di Giorgio C, Baldoni M, Ricci P, Monti MC, Morretta E, Zampella A, Distrutti E, Fiorucci S. Opposite effects of the FXR agonist obeticholic acid on Mafg and Nrf2 mediate the development of acute liver injury in rodent models of cholestasis. Biochim Biophys Acta Mol Cell Biol Lipids 2020; 1865: 158733 [PMID: 32371093 DOI: 10.1016/j.bbalip.2020.158733]

54

Qiu YL, Gong JY, Feng JY, Wang RX, Han J, Liu T, Lu Y, Li LT, Zhang MH, Sheps JA, Wang NL, Yan YY, Li JQ, Chen L, Borchers CH, Sipos B, Knisely AS, Ling V, Xing QH, Wang JS. Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis. Hepatology 2017; 65: 1655-1669 [PMID: 28027573 DOI: 10.1002/hep.29020]

55

Girard M, Lacaille F, Verkarre V, Mategot R, Feldmann G, Grodet A, Sauvat F, Irtan S, Davit-Spraul A, Jacquemin E, Ruemmele F, Rainteau D, Goulet O, Colomb V, Chardot C, Henrion-Caude A, Debray D. MYO5B and bile salt export pump contribute to cholestatic liver disorder in microvillous inclusion disease. Hepatology 2014; 60: 301-310 [PMID: 24375397 DOI: 10.1002/hep.26974]

56

Overeem AW, Li Q, Qiu YL, Cartón-García F, Leng C, Klappe K, Dronkers J, Hsiao NH, Wang JS, Arango D, van Ijzendoorn SCD. A Molecular Mechanism Underlying Genotype-Specific Intrahepatic Cholestasis Resulting From MYO5B Mutations. Hepatology 2020; 72: 213-229 [PMID: 31750554 DOI: 10.1002/hep.31002]

57

Gonzales E, Taylor SA, Davit-Spraul A, Thébaut A, Thomassin N, Guettier C, Whitington PF, Jacquemin E. MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease. Hepatology 2017; 65: 164-173 [PMID: 27532546 DOI: 10.1002/hep.28779]

58

Jarasvaraparn C, He M, Granadillo JL, Kulkarni S, Stoll J, Liss K. MYO5B Pathogenic Variants Found to Cause Intestinal Symptoms Without Microvillus Inclusion Disease In A Child Who Previously Underwent Liver Transplantation For PFIC-like Cholestasis. J Pediatr Gastroenterol Nutr2020 [PMID: 32459745 DOI: 10.1097/MPG.0000000000002792]

59

van IJzendoorn SCD, Li Q, Qiu YL, Wang JS, Overeem AW. Unequal effects of MYO5B mutations in liver and intestine determine the clinical presentation of low-GGT cholestasis. Hepatology2020 [PMID: 32583448 DOI: 10.1002/hep.31430]

60

Engevik AC, Kaji I, Engevik MA, Meyer AR, Weis VG, Goldstein A, Hess MW, Müller T, Koepsell H, Dudeja PK, Tyska M, Huber LA, Shub MD, Ameen N, Goldenring JR. Loss of MYO5B Leads to Reductions in Na+ Absorption With Maintenance of CFTR-Dependent Cl- Secretion in Enterocytes. Gastroenterology 2018; 155: 1883-1897. e10 [PMID: 30144427 DOI: 10.1053/j.gastro.2018.08.025]

61

van der Velden LM, Stapelbroek JM, Krieger E, van den Berghe PV, Berger R, Verhulst PM, Holthuis JC, Houwen RH, Klomp LW, van de Graaf SF. Folding defects in P-type ATP 8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate. Hepatology 2010; 51: 286-296 [PMID: 19918981 DOI: 10.1002/hep.23268]

62

Mariotti V, Strazzabosco M, Fabris L, Calvisi DF. Animal models of biliary injury and altered bile acid metabolism. Biochim Biophys Acta Mol Basis Dis 2018; 1864: 1254-1261 [PMID: 28709963 DOI: 10.1016/j.bbadis.2017.06.027]

63

Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S. A more efficient method to generate integration-free human iPS cells. Nat Methods 2011; 8: 409-412 [PMID: 21460823 DOI: 10.1038/nmeth.1591]

64

Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010; 51: 297-305 [PMID: 19998274 DOI: 10.1002/hep.23354]

65






https://dx.doi.org/10.1016/j.jpeds.2018.09.028


https://dx.doi.org/10.1097/MPG.0000000000002670


https://dx.doi.org/10.1016/j.bbalip.2020.158733










https://dx.doi.org/10.1097/MPG.0000000000002792










https://dx.doi.org/10.1038/nmeth.1591




World Journal of



MINIREVIEWS

Invasive fungal infection before and after liver transplantation

Alberto Ferrarese, Annamaria Cattelan, Umberto Cillo, Enrico Gringeri, Francesco Paolo Russo, Giacomo Germani, Martina Gambato, Patrizia Burra, Marco Senzolo

ORCID number: Alberto Ferrarese 0000-0002-3248-2038; Annamaria Cattelan 0000-0003-4678-9070; Umberto Cillo 0000-0002-2310-0245; Enrico Gringeri 0000-0002-3459-7306; Francesco Paolo Russo 0000-0003-4127-8941; Giacomo Germani 0000-0002-4332-2072; Martina Gambato 0000-0002-0101-1938; Patrizia Burra 0000-0002-8791-191X; Marco Senzolo 0000-0002-7261-6520.

Author contributions: Ferrarese A, Cattelan A and Senzolo M participated in research design, data analysis, and writing of the manuscript; Cillo U, Gringeri E, Russo FP, Germani G, Gambato M and Burra P participated in research design and preparation of the manuscript; all authors have contributed to, read, and approved the manuscript.

Conflict-of-interest statement: The Authors have nothing to disclose regarding this manuscript.

Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the

Alberto Ferrarese, Francesco Paolo Russo, Giacomo Germani, Martina Gambato, Patrizia Burra, Marco Senzolo, Multivisceral Transplant Unit, Padua University Hospital, Padua 35128, Italy

Annamaria Cattelan, Tropical and Infectious Disease Unit, Padua University Hospital, Padua 35128, Italy

Umberto Cillo, Enrico Gringeri, Padua University Hospital, Hepatobiliary Surgery and Liver Transplant Center, Padua 35128, Italy

Corresponding author: Alberto Ferrarese, MD, Multivisceral Transplant Unit, Padua University Hospital, via Giustiniani 2, Padua 35128, Italy. [email protected]

AbstractInvasive infections are a major complication before liver transplantation (LT) and in the early phase after surgery. There has been an increasing prevalence of invasive fungal disease (IFD), especially among the sickest patients with decompensated cirrhosis and acute-on-chronic liver failure, who suffer from a profound state of immune dysfunction and receive intensive care management. In such patients, who are listed for LT, development of an IFD often worsens hepatic and extra-hepatic organ dysfunction, requiring a careful evaluation before surgery. In the post-transplant setting, the burden of IFD has been reduced after the clinical advent of antifungal prophylaxis, even if several major issues still remain, such as duration, target population and drug type(s). Nevertheless, the development of IFD in the early phase after surgery significantly impairs graft and patient survival. This review outlines presentation, prophylactic and therapeutic strategies, and outcomes of IFD in LT candidates and recipients, providing specific considerations for clinical practice.

Key Words: Acute-on-chronic liver failure; Sepsis; Cirrhosis; Candidemia; Acute liver failure; Invasive fungal infection


Core Tip: Invasive fungal infection significantly influences the outcome of patients with acute liver failure or cirrhosis awaiting liver transplantation, as well as their post-



http://orcid.org/0000-0002-3248-2038

http://orcid.org/0000-0002-3248-2038

http://orcid.org/0000-0003-4678-9070

http://orcid.org/0000-0003-4678-9070

http://orcid.org/0000-0002-2310-0245

http://orcid.org/0000-0002-2310-0245

http://orcid.org/0000-0002-3459-7306

http://orcid.org/0000-0002-3459-7306

http://orcid.org/0000-0003-4127-8941

http://orcid.org/0000-0003-4127-8941

http://orcid.org/0000-0003-4127-8941

http://orcid.org/0000-0002-4332-2072

http://orcid.org/0000-0002-4332-2072

http://orcid.org/0000-0002-0101-1938

http://orcid.org/0000-0002-0101-1938

http://orcid.org/0000-0002-8791-191X

http://orcid.org/0000-0002-8791-191X

http://orcid.org/0000-0002-7261-6520

http://orcid.org/0000-0002-7261-6520


Ferrarese A et al. Fungal infection in liver transplant recipients


original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/




Peer-review report’s scientific quality classificationGrade A (Excellent): A Grade B (Very good): 0 Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0

Received: October 18, 2020 Peer-review started: October 18, 2020 First decision: November 13, 2020 Revised: November 15, 2020 Accepted: November 29, 2020 Article in press: November 29, 2020 Published online: December 21, 2020

P-Reviewer: Kang KJ S-Editor: Fan JR L-Editor: A P-Editor: Liu JH

operative course. This state-of-the-art review comprehensively describes the epidemiology and the therapeutic options on this field.

Citation: Ferrarese A, Cattelan A, Cillo U, Gringeri E, Russo FP, Germani G, Gambato M, Burra P, Senzolo M. Invasive fungal infection before and after liver transplantation. World J Gastroenterol 2020; 26(47): 7485-7496URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7485.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7485

INTRODUCTIONLiver transplantation (LT) represents the best therapeutic option for end-stage liver diseases and hepatocellular carcinoma. The LT landscape has changed rapidly in the last decades, with a widespread diffusion of this practice, a significant expansion of indications, and an evolution in medical and surgical care. Therefore, although more patients than in the past are offered a graft and can survive after surgery, this changing scenario has determined a huge modification of characteristics of LT candidates and recipients, who are older, sicker and often display many extra-hepatic comorbidities[1].

In this setting, the burden of invasive infection, both before LT [especially in those with advanced cirrhosis or acute-on-chronic liver failure (ACLF)] and in the early post-operative course is still a major issue. Cirrhosis is a predisposing condition to such infections, because of a profound immune dysfunction, due to both an exhaustion of response to pathogens and persistent systemic inflammation[2]. Bacteria are responsible for the majority of invasive infections, determining a further impairment of hepatic and extra-hepatic organ disfunction in the pre-operative phase, and significantly affecting graft and patient’s survival in the early phase after surgery[3-5].

Nevertheless, considered rare in the past, invasive fungal infection occurs with an increasing prevalence in LT candidates, mostly due to the refinement of diagnostic criteria and the increasing burden of predisposing conditions. In the post-LT phase, the institution of antifungal prophylactic strategies has significantly improved patient outcome.

INVASIVE FUNGAL DISEASE IN PATIENTS AWAITING LTEpidemiology, risk factors, therapeutic options, outcomesBy definition, an invasive fungal disease (IFD) is a disease process caused by invasive fungal infection. Current diagnostic criteria rely on three different levels of probability (proven, probable and possible IFD), mixing together host factors, clinical manifestations, and mycological evidence[6].

The epidemiology of IFD in cirrhotic patients has been heterogeneously reported, mainly in retrospective, single-center series, which included patients with different disease stages, prognosis (i.e., waitlisted for a transplant) and hospital settings [i.e., intensive care unit (ICU) vs regular ward]. Moreover, heterogeneous prevalence, diagnostic criteria and treatment protocols applied throughout the literature may have further influenced the actual epidemiology of such infections.

According to multicenter studies on hospitalized patients with cirrhosis, the prevalence of IFD is nearly 4%[7,8], although only proven IFD are usually considered. Most infections are caused by Candida; according to recent evidence, albicans and non-albicans strains have roughly similar prevalence[9].

The institution of surveillance protocols appears mandatory for an early diagnosis. These protocols should focus on patients at highest risk of IFD development, such as those with ACLF. Indeed, they encompass several risk factors, such as a profound immune-dysfunction, prolonged hospitalization, hepatic and extra-hepatic failure(s), indwelling (vascular) catheters, and long-term antibiotic therapies[3,10]. According to available studies on this specific population[11-16], the prevalence of IFD ranges between 1% and 47% (depending on diagnostic criteria and surveillance policies), significantly affecting short-term survival. Nevertheless, heterogeneous selection criteria have not








allowed a refinement of risk stratification to date (Table 1). Patients with severe alcoholic hepatitis are another high-risk group for IFD, especially for invasive aspergillosis (IA). Gustot et al[17] reported a high incidence of such infection in a prospective cohort of 94 patients with biopsy-proven severe alcoholic hepatitis, after a median time of 25 d from steroids introduction, and with a 100% transplant-free mortality. This report raised the question about the potential role of steroids for IA development in such a population; a meta-analysis in this field[18] partly confirmed this hypothesis, suggesting that opportunistic infections, especially fungal, seemed to be more frequent in this high-risk group, and may deserve special attention. IFD is a less frequent, but highly relevant complication also in patients with acute liver failure (ALF), carrying a high mortality risk, especially in case of a delayed diagnosis or institution of inappropriate treatment[19,20].

The occurrence of IFD often represents a detrimental event in patients with cirrhosis, leading to a significant increase in short-term mortality (35% to 50%), at a similar rate to that experienced after a multidrug-resistant organism bloodstream infection, especially when an appropriate antifungal treatment is not promptly initiated[7,9,21].

A detailed treatment algorithm for IFD in patients with cirrhosis is beyond the scope of this manuscript. The clinical keys of a successful treatment are early diagnosis, early administration of appropriate antifungal treatment, in close cooperation with Infectious Disease specialists. Considering Candida related IFD, ophthalmologic evaluation and removal of vascular/peritoneal catheters, as well as a shift towards non-albicans strains should be considered before starting antifungal therapy. Echinocandins are now considered the drugs of choice, to be continued for 2 weeks after clearance of Candida from the bloodstream or symptoms resolution[22]. Considering IA, voriconazole represents the first therapeutic option, whereas echinocandins and liposomal amphotericin B (L-AmB) are other, albeit less effective, available drugs[23]. It is worth mentioning that voriconazole has been associated with hepatic and renal dysfunction, therefore therapeutic drug monitoring is recommended[24].

Specific issues in the liver transplant settingIFD are a major issue in patients waiting for LT. As discussed above, occurrence of an IFD highlights the already impaired patient’s general condition, with an unpredictable evolution of hepatic and extra-hepatic organ(s) failure. This may potentially increase the need for a transplant, especially in a urgency-based system of organ allocation[25]. Nevertheless, according to the available data, several points should be considered; first, the effectiveness and treatment length of an appropriate antifungal therapy are very different from antibiotic therapies. Second, an IFD seems to develop in sicker patients than in the case of a bacterial infection, often as a superimposed infection[7,8]. Therefore, an active IFD should be viewed as a temporary contraindication for LT[26] (Figure 1). For the sickest patients who are waiting for a graft, surveillance protocols are mandatory, and antifungal prophylaxis has been advocated in selected cases. For instance, Gustot et al[27] suggested ICU admission and a baseline MELD score > 24 as factors for considering a prophylaxis against IA in patients with acute alcoholic hepatitis[16,27], but more data are needed before considering it as a standard practice. After diagnosis of IFD, consultation by expert Infectious Disease specialists should be always considered, in order to establish the best targeted antifungal treatment and its length. Moreover, antifungal stewardship aiming to avoid both adverse events and increasing resistance should always be pursued in the transplant setting.

The assessment of short-term outcome for each waitlisted patient should be individually discussed by the LT team, in order to consider the best timing for a waiting-list readmission (and a possible prioritization after infection recovery[4]). Conversely, other therapeutic options should be taken into account, to avoid futile transplantation[28,29].

FUNGAL INFECTIONS EARLY AFTER LTEpidemiology, risk factors, and outcomeAlthough better outcomes have been reported after the introduction of novel antifungal agents and significant progress has been obtained after antifungal prophylaxis, IFD remains an important cause of early morbidity and mortality after solid organ transplantation (SOT). Recent large cohort studies on SOT recipients showed a 1-year post-transplant IFD rate of 4%-8%[30-32], with a changing epidemiology



Table 1 Studies assessing the prevalence of invasive fungal disease in patients with acute-on-chronic liver failure

Ref. Study design Diagnostic criteria for IFD Prevalence of IFD Outcome Risk factors for IFD

Verma et al[11], 2019 Single-center, retrospective study on ICU patients from India

EORTC/MSG diagnostic criteria 39/264 (14.7%). 11 (28%) proven. 25 (64%) IC and 14 (36%) IA In-hospital mortality 77% Hemodialysis. Prior antibiotic use

Fernández et al[12], 2018

Multi-center, prospective study on non-ICU ACLF patients across Europe

EORTC/MSG diagnostic criteria 8/407 (1.9%). 7 (87%) IC. 1 (13%) IA 28-d and 90-d mortality 57% and 71%, respectively

NR

Theocharidou et al[13]1, 2016

Analysis from prospectively collected database on ICU patients across the United Kingdom

EORTC/MSG diagnostic criteria (only proven IFD considered for the analysis)

8/782 (1%) In-ICU and in-hospital mortality 0%

NR

Chen et al[14]2, 2013 Retrospective single center study from China on IA

EORTC/MSG diagnostic criteria 39/787 (4.9%) Cumulative mortality 61% Age. Hepatic encephalopathy. Steroid use

Lin et al[15]2, 2013 Single center retrospective study from non-ICU hepatitis B cirrhotic patients from China

EORTC/MSG diagnostic criteria 60/126 (47.6%). Proven IFD: 14 (23%). 9 (64%) C. Albicans 2 (14%) Criptococcus neoformans: 1 (7%) C. Tropicalis; 1 (7%) C. Glabrata; 1 (7%) IA

Cumulative mortality 40% Hepatitis B viral load

Levesque et al[16], 2019

Single center retrospective study on ICU patients with cirrhosis and IA in France

EORTC/MSG diagnostic criteria 60/362 (16.6%). 43/60 (71.7%) fulfilled ACLF criteria. 17/60 (28%) had IA

IA associated cumulative in-hospital mortality 71%

NR

1The manuscript did not extensively classify patients according to acute-on-chronic liver failure (ACLF) criteria.2This study used the APASL criteria for ACLF diagnosis. Colonizations are not reported. ACLF: Acute-on-chronic liver failure; IA: Invasive aspergillosis; IC: Invasive candidiasis; IFD: Invasive fungal disease; NR: Not reported; ICU: Intensive care unit; EORTC: European Organization for Research and Treatment of Cancer; MSG: Mycoses Study Group.

over time. Indeed, if Candida spp. and Aspergillus spp. are still the most common molds, there has been a rise of non-albicans Candida species, carrying a higher mortality[33].

Broad-spectrum antibiotic therapy, parenteral nutrition, prolonged neutropenia, ICU stay, diabetes, pre-LT colonization, renal replacement therapy, cytomegalovirus (CMV) infection, re-interventions and choledochojejunostomy are established risk factors for post-LT IC[34,35], whereas pre-LT steroid administration, ALF, and renal replacement therapy seem to be more frequently associated with IA[36-38]. Recently, pre-LT Aspergillus colonization has been considered not a contraindication to LT in a single-center cohort of 27 patients; although they received appropriate post-operative prophylaxis (voriconazole +/- echinocandin), post-LT IA occurrence was 11%[39]. Most of the abovementioned risk factors are associated with patient’s severity at time of transplantation. This concept has been well demonstrated in ACLF patients, who experienced a significantly increasing post-LT IFD incidence, according to disease stage (ACLF grade 3 vs -2 vs -1: 15% vs 6.2% vs 3.4%)[40].

Although active IFD in the donor is a contraindication to donation, several cases of donor-derived IFD have been reported in the literature, mostly due to a undiagnosed infection at time of surgery[41]. Contamination of the organ during procurement



Figure 1 CT-scan. A: Chest CT-scan of a young male patient with hepatitis B virus related cirrhosis and acute-on-chronic liver failure, waitlisted for liver transplantation, who developed invasive aspergillosis; B: He was temporarily withdrawn from the waiting list, and received antifungal treatment for a total of 13 d, with a clinical and radiological improvement. He subsequently died of bacterial super-infection before liver transplantation.

appears as another important issue. For instance, a large retrospective multicenter study from France showed a 1.33% Candida spp. prevalence in preservation fluid, being associated with a high rate of post-operative IFD and impaired survival[42].

Despite the adoption of preventive measures and antifungal stewardship, IFD still significantly affect the overall graft and patient survival. For instance, the TRANSNET study[43] reported 90 d cumulative mortality of 26% after IC occurrence, and 1-year survival of 59% after development of IA.

Post-LT antifungal prophylaxisAntifungal prophylaxis is now being considered a cornerstone after LT, due to its safety and effectiveness[44,45]. A systematic review and metanalysis by Evans et al[46] showed a significant reduction in the odds for proven IFD and for IFD-related mortality among LT patients who received prophylaxis, even if overall mortality did not change significantly. Notably, this study provided robust data about fluconazole and L-AmB, whereas echinocandins were not investigated. That said, several issues in the field of antifungal prophylaxis, such as the type (universal vs targeted approach), length, and preferred molecule(s) to use, are currently debated.

The rationale of a targeted prophylaxis is to capture only high-risk patients (based on pre- and early post-LT characteristics), in order to avoid antifungal over-use, and to administer highly effective molecules. Indeed, several studies have clearly demonstrated the cost-ineffectiveness of antifungal prophylaxis in low-risk patients.

Considering the optimal prophylaxis duration, current guidelines suggest that targeted prophylaxis against IC and IA should be administered for 14-21 d[34,36], but heterogeneous lengths have been adopted in the post-transplant setting, also in view of the dynamic, poorly predictable post-operative course. Further, many attempts at regimen simplification or stratification according to patients’ risk factors have been proposed. Table 2 summarizes the current evidence on antifungal prophylaxis after LT[35,37,47-60]. Notably, heterogeneous inclusion criteria, treatment algorithms, and endpoints adopted, do not allow a robust comparison between studies, but it is worth mentioning that a large amount of data has been available in the last years.

A randomized, double-blind clinical trial including 200 high-risk LT recipients, compared prophylaxis with fluconazole 400 mg/d with anidulafungin 100 mg/d to be continued for 3 wk or until hospital discharge. The study showed a similar IFD occurrence between cohorts (5.1% vs 8%, P = 0.4), with no post-LT IFD related deaths in either. Furthermore, only one patient had to stop anidulafungin prophylaxis due to adverse drug-related events, strengthening the safety of this molecule in the post-LT setting. Another multicenter, randomized, controlled trial including 347 LT recipients recruited across 37 European Centers[51] demonstrated that micafungin prophylaxis (100 mg/d for 21 d or until hospital discharge) was equally effective and safe as standard of care (i.e., fluconazole, caspofungin, or L-AmB), according to composite primary and secondary endpoints. The effectiveness of caspofungin (50 mg/d) has been also demonstrated in a large retrospective study from Spain, after comparison with standard fluconazole prophylaxis[56].

Specific treatment issues in the liver transplant settingA detailed therapeutic algorithm for the treatment of each IFD is beyond the scope of this manuscript. Nevertheless, some treatment principles could be of help for clinical



Table 2 Studies published in the last 10 years on fungal prophylaxis in the liver transplantation setting

Ref. Study design Prophylaxis regimen Patient selection criteria Outcomes

Saliba et al[37], 2013

Single-center study. LTs between 1999-2005. Effectiveness of targeted prophylaxis

Group 1: L-AmB (1 mg/kg/d for 1 wk, then 2.5 mg/kg/ twice a week for 3 wk) OR fluconazole (200-400 mg/d for 3 wk for those with pre-LT Candida colonization). Group 2: No prophylaxis

High risk group (≥ 1 RF): ALF; ICU prior to LT; re-LT; re-operation Group 1: 198 LT recipients (n.146 L-Amb, n. 50 fluconazole, n. 2 amphotericinB). Group 2: 467 LT recipients. Lower 1 yr IFD occurrence in Group 1 (17.7% vs 32.4%; P < 0.001). IA occurrence not significantly different between groups. 1 yr graft and patient survival impaired after IFD occurrence

Sun et al[47], 2013

Single-center study. LTs between 1997-2009. Comparative study for targeted prophylaxis in at-risk patients

Group 1: Amphotericin B lipid complex (5 mg/kg/d for 21 d). Group 2: Micafungin (100 mg/d for 21 d)

High risk group (≥ 1 RF): Post-LT RRT; re-LT; re-operation Group 1 vs 2: 24 vs 18 LT recipients. Similar 90d IFD occurrence (11% vs 8.3%) and 90d mortality (29.2% and 22.2%) between groups

Trudeau et al[48], 2013

Single-center study. LTs between 2005-2008. Effectiveness of universal prophylaxis

Fluconazole (200 mg i.v./p.o. once weekly for 3 mo)

High risk group (≥ 2 RF): Re-LT; sCr > 2 mg/dL or RRT within 48 h prior to LT; choledochojejunostomy; transfusion of > 40 BP; operation time > 11 h; peri-operative fungal colonization

221 LTs (18 fulfilled high risk criteria). 6 mo overall IFD occurrence equal to 4.9%. Higher IFD occurrence in high-risk patients (16.7% vs 3.4%, P = 0.03)

Antunes et al[49], 2014


Group 1 (high risk): L-AmB 100 mg/d for 2 wk OR nystatin alone. Group 2 (low-risk): Nystatin

High risk (≥ 1 RF): Urgent LT; sCr > 2 mg/Dl; AKI after LT; re-LT; re-operation; transfusion of > 40 BP

Group 1 vs Group 2: 104 vs 357 LT recipients. 66 (63%) patients belonging to group 1 received L-AmB prophylaxis. Cumulative 3-mo IFD occurrence 2.5%. Higher IFD occurrence in high-risk patients who didn’t receive L-AmB prophylaxis (4.5% vs 13%, P = 0.01)

Winston et al[50], 2014

Randomized, double-blind trial. LTs between 2010-2011. Comparative trial for targeted prophylaxis

Group 1: Anidulafungin (200 mg/d loading those, then 100 mg/d) for 3 wk or until discharge. Group 2: fluconazole (400 mg/d, adjusted according renal function) for 3 wk or until discharge

High risk group (≥ 1 RF): Re-LT; ALF; Steroids for at least 2 wk before LT; ICU stay > 48 h. Colonization with Candida (> 2 sites) within 4 wk before LT; transfusion of ≥ 15 BP; operative time > 6 h; RRT at the time or within 7 d of LT; re-operation

200 patients 1:1 randomized. Similar cumulative IFD occurrence between cohorts (5.1% vs 8%, P = 0.4). Equal 3 mo post-LT mortality (12% each arm). 0% IFD related deaths

Saliba et al[51], 2015

Randomized, open-label study. LTs between 2009-2012. Comparative trial for targeted prophylaxis

Group 1: Micafungin (100 mg/d for 21 d or until discharge) in high risk patients. Group 2: Center-specific standard care (fluconazole 200–400 mg/d OR L-AmB 1–3 mg/kg/d OR caspofungin 70 mg loading dose followed by 50 mg/d) in high risk patients

High risk patients (≥ 1 RF): Re-LT; ALF; Pre- or post-operative sCr clearance ≤ 40 mL/min) or RRT; ICU 48 h prior to LT; re-operation within 5d of LT; choledochojejunostomy; peri-operative Candida colonization (≥ 2 positive cultures); prolonged mechanical ventilation > 48 h after LT; transfusion of ≥ 20 BP

Group 1 vs Group 2: 174 vs 173 LT recipients (140 and 137 LT completed the study in each arm). Micafungin was non inferior to standard of care (composite primary and secondary efficacy endpoints)

Giannella et al[52], 2015

Prospective non-randomized trial. LTs between 2009-2013. Safety of high dose L-AmB for targeted prophylaxis

L-AmB 10 mg/Kg once a week until hospital discharge for a minimum of 2 wk

High risk for IC (≥ 2 RF): ICU in 90d prior LT; perioperative Candida colonization; Choledochojejunostomy; transfusion of > 40 BP; AKI; rejection within 2 wk after LT; CMV DNA > 100.000 copies/mL; prolonged or repeated operation. High risk for IA (≥ 1 RF): ALF; steroid treatment before LT; multivisceral transplant; RRT; rejection; re-LT; re-operation

76 patients enrolled (39 having ≥ 2 RF for IC; 37 having ≥ 1 RF for IA). 10 patients discontinued therapy (6 for L-AmB related adverse events; 4 for IFD). 2 episodes of proven IC occurred

Eschenauer et al[53], 2015


Universal prophylaxis (LTs between 2008-2010): Voriconazole 200 mg BID. Targeted prophylaxis (LTs between 2010-2012): Group 1: Voriconazole 200 mg BID for 30 d. Group 2: Fluconazole 400 mg/d during post-LT ICU stay. Group 3: No prophylaxis

Inclusion criteria for Group 1 (≥ 1 RF): re-LT; ALF; RRT; re-operation within 30 d after LT. Inclusion criteria for Group 2 (≥ 1 RF): Choledochojejunostomy; transfusion of > 40 BP and operation time ≥ 11 h; candida colonization or infection within 3 mo before LT

Universal prophylaxis: 236 LTs. Targeted prophylaxis: 145 LTs (group 1 vs 2 vs 3: 78 vs 11 vs 55). Cumulative IFD occurrence 5.2% (targeted vs universal group: 6.9% vs 4.2%; P = 0.34). 40% breakthrough IFD. Similar 100-d mortality between targeted and universal prophylaxis group



Balogh et al[54], 2016

Single-center study. LTs between 2008-2014. Targeted prophylaxis against IA

Group 1: Voriconazole 200 mg BID for 90 d. Group 2: Oral nystatin OR fluconazole

High risk group: MELD score > 25. OR ≥ 2 RF: Pre-LT ICU stay > 24h; inotropic support; RRT; re-LT; Combined transplant; pre-LT mechanical ventilation; ALF

Group 1 vs Group 2: 174 vs 140 LT recipients; no episodes of IA occurred; no difference in graft and patient survival curves between cohorts

Perrella et al[55], 2016

Single-center study. LTs between 2006-2012. Comparative observational study for targeted prophylaxis

Group 1: L-AmB (3 mg/kg/d). Group 2: Caspofungin (70 mg/d loading dose, then 50 mg/d)

High risk patients (≥ 3 RF): sCr clearance < 30 mL/min and/or sCr > 4 mg/mL. Pre-LT Candida colonization. Pre-LT antibiotic use > 10 d. Pre-LT hospitalization > 7 d. Operation time ≥ 9 h. Warm ischemia ≥ 45’. Re-LT. Transfusion of > 14 BP. Choledochojejunostomy

Group 1 vs Group 2: 28 vs 26 LTs. No episodes of IFD occurred in both groups

Fortún et al[56], 2016

Multicenter study. LTs between 2005-2012. Comparative observational study for targeted prophylaxis

Group 1: Caspofugin (50 mg/d). Group 2: Fluconazole 100-400 mg/d (median 200 mg/d)

High risk group (≥ 1 RF): Re-LT; RRT within 30 d; LT for ALF. OR ≥ 2 of the following RF: Transfusion of ≥ 20 BP; Choledochojejunostomy; Peri-operative Candida colonization (≥ 2 sites); re-operation within 7 d

Group 1 vs Group 2: 97 vs 98 LT recipients. Median prophylaxis duration: 22 and 24 d, respectively. Similar 6-mo IFD occurrence (5.2% vs 12.2%). Reduced risk of IA in LT recipients receiving caspofungin. Similar overall mortality and IFD-related mortality between groups

Chen et al[57], 2016


Group 1: Anidulafungin (100 mg/d) OR micafungin (100 mg/d)1. Group 2: No prophylaxis

High risk patients: MELD ≥ 20 Group 1 vs 2: 201 vs 201 LT recipients (propensity score matching). Similar IFD occurrence (11.2% vs 18.9%, P = 0.052). Lower cumulative mortality in Group 1 (23.4% vs 40.8%, P = 0.001)

Giannella et al[35], 2016

Retrospective, single-center study. LTs between 2010-2014. Evaluation of risk factors for a targeted antifungal prophylaxis

Group 1 (no RF): No prophylaxis. Group 2 (1 RF IC): Fluconazole. Group 3 (high risk patients): Anti-mold agent

High-risk patients for IC (≥ 2 RF): Prolonged operation; choledochojejunostomy; Pre-LT Candida colonization; re-LT; AKI. High-risk patients for IA (≥ 1 RF): ALF; RRT after LT; re-operation; re-LT

303 patients evaluated (Groups 1 vs 2 vs 3: 91 vs 61 vs 151). Antifungal prophylaxis administered to 45.9% patients (80 L-AmB; 18 caspofungin; 41 fluconazole). Cumulative IFD prevalence 6.3%. Fluconazole prophylaxis independently associated with IFD development.

Lavezzo et al[58], 2018


Group 1 (high risk): Amphotericin B lipid complex (3 mg/kg/d) OR L-AmB (2 mg/kg/d), for 5 to 10 d after LT. Group 2 (low risk): No prophylaxis

High-risk group (≥ 1 RF): Hospitalization at LT or in the 30 d prior LT for infection; ALF; Primary-non-function; Steroid treatment at LT; sCr > 2 mg/dl before LT; RRT before or after LT; MELD > 30 at LT; re-LT, split liver, combined transplantation; Transfusion of ≥ 20 BP; choledochojejunostomy; re-operation; thymoglobulin therapy; positive fungal culture of donor preservation fluid

Overall IFD prevalence 2.8% (all in the targeted prophylaxis group). 1 yr mortality higher in prophylaxis group (12.5% vs 1.8%, P = 0.001). 1-yr mortality higher in IFD patients (33.3% vs 6.4%; P < 0.001)

Jorgenson et al[59], 2019

Single-center study. LTs between 2009-2016. Effectiveness of fixed dose prophylaxis

Group 1: Fluconazole fixed dose (400 mg/d for 14d) in at-risk patients. Group 2: Unsupervised antifungal protocols

High risk group (≥ 1 RF): Operation time > 10 h; re-operation within 30 d; re-LT; Pre LT dialysis; pre-LT Candida colonization; pre-LT hospitalization > 7 d; Choledochojejunostomy; MELD ≥ 35; transfusion ≥ 40 BP

High-risk patients: Group 1 vs Group 2: 50 vs 139. Reduction of 1-yr IFD among high-risk cohorts (12.5% vs 26.6%). Similar 1 yr patient and graft survival

Kang et al[60], 2020

Multicenter, randomized, open-label trial. Living donor LTs 2012-2015. Comparative study for universal prophylaxis

Group 1: Micafungin (100 mg/d for 3 wk or until hospital discharge). Group 2: Fluconazole (100-200 mg/d for 3 wk or until hospital discharge)

Universal prophylaxis Group 1 vs Group 2: 69 vs 75 LT recipients. IFD occurrence within 3 wk: 1/69 vs 0/75. Micafungin was non-inferior to fluconazole

1Duration of prophylaxis not reported. BP: Blood products; IA: Invasive aspergillosis; IC: Invasive candidiasis; IFD: Invasive fungal disease; L-AmB: Liposomal amphotericin B; LT: Liver transplantation; RF: Risk factor; RRT: Renal replacement therapy; sCr: Serum creatinine; ICU: Intensive care unit.

practice. As in the pre-LT setting, echinocandins and fluconazole are the most effective molecules for the treatment of IC, whereas L-AmB should be used as first-line therapy only in selected cases. A thorough knowledge of local epidemiology, as well as pre-



operative colonization(s) represent crucial information before starting a therapeutic regimen. Source control, obtained by removal of indwelling vascular/abdominal catheters, is another important option to be considered. Regarding echinocandins, both micafungin and anidulafungin have been demonstrated to be safe and effective at therapeutic dose[51,61]. Notably, micafungin does influence through levels of m-TOR inhibitors, but not of tacrolimus and cyclosporine[62].

Current guidelines recommend voriconazole as the drug of choice for IA, whereas isavuconazole and L-AmB can be considered as alternatives[36]. Isavuconazole seems to have similar effectiveness to voriconazole, but with fewer side effects–also liver-related –, being a promising option especially in the early post-operative phase[63]. During the course of therapy (usually 12 wk regimen), a careful assessment of IS, liver and renal function are mandatory, as well as therapeutic drug monitoring. Moreover, daily dose of calcineurin inhibitors should be carefully reduced (about by 50%), whereas co-administration of voriconazole and mTORs should be avoided due to a high increase of serum concentration[64]. Other molecules could be of help for the treatment of rarer species, or as rescue therapies[65,66].

CONCLUSIONThe occurrence of an invasive fungal disease significantly affects the natural history of LT candidates and recipients. In the peri-operative setting, it usually develops in the sickest patients, impairing hepatic and extra-hepatic organ function and being associated with high short-term mortality. An active IFD is still considered a contraindication to LT. Therefore, response to appropriate antifungal therapy and patient’s global outcome should be strictly evaluated by the LT team in accordance with Infectious Disease Specialists, in order to re-consider transplantation as a cost-effective therapeutic option. In the post-operative setting, IFD occurrence has been significantly reduced since the institution of prophylaxis, but it is still a serious complication, affecting graft and patient survival. Prophylactic regimens in patients deemed at high-risk may take into account the local epidemiology, risk of resistance, and potential adverse drug-related effects or interactions.

REFERENCESToniutto P, Zanetto A, Ferrarese A, Burra P. Current challenges and future directions for liver transplantation. Liver Int 2017; 37: 317-327 [PMID: 27634369 DOI: 10.1111/liv.13255]

1

Albillos A, Lario M, Álvarez-Mon M. Cirrhosis-associated immune dysfunction: distinctive features and clinical relevance. J Hepatol 2014; 61: 1385-1396 [PMID: 25135860 DOI: 10.1016/j.jhep.2014.08.010]

2

Ferrarese A, Zanetto A, Becchetti C, Sciarrone SS, Shalaby S, Germani G, Gambato M, Russo FP, Burra P, Senzolo M. Management of bacterial infection in the liver transplant candidate. World J Hepatol 2018; 10: 222-230 [PMID: 29527258 DOI: 10.4254/wjh.v10.i2.222]

3

Ferrarese A, Vitale A, Sgarabotto D, Russo FP, Germani G, Gambato M, Cattelan AM, Angeli P, Cillo U, Burra P, Senzolo M. Outcome of a First Episode of Bacterial Infection in Candidates for Liver Transplantation. Liver Transpl 2019; 25: 1187-1197 [PMID: 31021050 DOI: 10.1002/lt.25479]

4

Dionigi E, Garcovich M, Borzio M, Leandro G, Majumdar A, Tsami A, Arvaniti V, Roccarina D, Pinzani M, Burroughs AK, O'Beirne J, Tsochatzis EA. Bacterial Infections Change Natural History of Cirrhosis Irrespective of Liver Disease Severity. Am J Gastroenterol 2017; 112: 588-596 [PMID: 28220780 DOI: 10.1038/ajg.2017.19]

5

De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, Denning DW, Patterson TF, Maschmeyer G, Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC, Kullberg BJ, Marr KA, Muñoz P, Odds FC, Perfect JR, Restrepo A, Ruhnke M, Segal BH, Sobel JD, Sorrell TC, Viscoli C, Wingard JR, Zaoutis T, Bennett JE; European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 2008; 46: 1813-1821 [PMID: 18462102 DOI: 10.1086/588660]

6

Bajaj JS, Reddy RK, Tandon P, Wong F, Kamath PS, Biggins SW, Garcia-Tsao G, Fallon M, Maliakkal B, Lai J, Vargas HE, Subramanian RM, Thuluvath P, Thacker LR, OʼLeary JG. Prediction of Fungal Infection Development and Their Impact on Survival Using the NACSELD Cohort. Am J Gastroenterol 2018; 113: 556-563 [PMID: 29257141 DOI: 10.1038/ajg.2017.471]

7

Piano S, Singh V, Caraceni P, Maiwall R, Alessandria C, Fernandez J, Soares EC, Kim DJ, Kim SE, 8


https://dx.doi.org/10.1111/liv.13255






https://dx.doi.org/10.1002/lt.25479


https://dx.doi.org/10.1038/ajg.2017.19


https://dx.doi.org/10.1086/588660


https://dx.doi.org/10.1038/ajg.2017.471



Marino M, Vorobioff J, Barea RCR, Merli M, Elkrief L, Vargas V, Krag A, Singh SP, Lesmana LA, Toledo C, Marciano S, Verhelst X, Wong F, Intagliata N, Rabinowich L, Colombato L, Kim SG, Gerbes A, Durand F, Roblero JP, Bhamidimarri KR, Boyer TD, Maevskaya M, Fassio E, Kim HS, Hwang JS, Gines P, Gadano A, Sarin SK, Angeli P; International Club of Ascites Global Study Group. Epidemiology and Effects of Bacterial Infections in Patients With Cirrhosis Worldwide. Gastroenterology 2019; 156: 1368-1380. e10 [PMID: 30552895 DOI: 10.1053/j.gastro.2018.12.005]Bassetti M, Peghin M, Carnelutti A, Righi E, Merelli M, Ansaldi F, Trucchi C, Alicino C, Sartor A, Toniutto P, Wauters J, Laleman W, Tascini C, Menichetti F, Luzzati R, Brugnaro P, Mesini A, Raviolo S, De Rosa FG, Lagunes L, Rello J, Dimopoulos G, Colombo AL, Nucci M, Vena A, Bouza E, Muñoz P, Tumbarello M, Losito R, Martin-Loeches I, Viscoli C. Clinical characteristics and predictors of mortality in cirrhotic patients with candidemia and intra-abdominal candidiasis: a multicenter study. Intensive Care Med 2017; 43: 509-518 [PMID: 28271321 DOI: 10.1007/s00134-017-4717-0]

9

Clària J, Stauber RE, Coenraad MJ, Moreau R, Jalan R, Pavesi M, Amorós À, Titos E, Alcaraz-Quiles J, Oettl K, Morales-Ruiz M, Angeli P, Domenicali M, Alessandria C, Gerbes A, Wendon J, Nevens F, Trebicka J, Laleman W, Saliba F, Welzel TM, Albillos A, Gustot T, Benten D, Durand F, Ginès P, Bernardi M, Arroyo V; CANONIC Study Investigators of the EASL-CLIF Consortium and the European Foundation for the Study of Chronic Liver Failure (EF-CLIF). Systemic inflammation in decompensated cirrhosis: Characterization and role in acute-on-chronic liver failure. Hepatology 2016; 64: 1249-1264 [PMID: 27483394 DOI: 10.1002/hep.28740]

10

Verma N, Singh S, Taneja S, Duseja A, Singh V, Dhiman RK, Chakrabarti A, Chawla YK. Invasive fungal infections amongst patients with acute-on-chronic liver failure at high risk for fungal infections. Liver Int 2019; 39: 503-513 [PMID: 30276951 DOI: 10.1111/liv.13981]

11

Fernández J, Acevedo J, Wiest R, Gustot T, Amoros A, Deulofeu C, Reverter E, Martínez J, Saliba F, Jalan R, Welzel T, Pavesi M, Hernández-Tejero M, Ginès P, Arroyo V; European Foundation for the Study of Chronic Liver Failure. Bacterial and fungal infections in acute-on-chronic liver failure: prevalence, characteristics and impact on prognosis. Gut 2018; 67: 1870-1880 [PMID: 28847867 DOI: 10.1136/gutjnl-2017-314240]

12

Theocharidou E, Agarwal B, Jeffrey G, Jalan R, Harrison D, Burroughs AK, Kibbler CC. Early invasive fungal infections and colonization in patients with cirrhosis admitted to the intensive care unit. Clin Microbiol Infect 2016; 22: 189.e1-189. e7 [PMID: 26551838 DOI: 10.1016/j.cmi.2015.10.020]

13

Chen J, Yang Q, Huang J, Li L. Risk Factors for Invasive Pulmonary Aspergillosis and Hospital Mortality in Acute-On-Chronic Liver Failure Patients: A Retrospective-Cohort Study. Int J Med Sci 2013; 10: 1625-1631 [PMID: 24151434 DOI: 10.7150/ijms.6824]

14

Lin LN, Zhu Y, Che FB, Gu JL, Chen JH. Invasive fungal infections secondary to acute-on-chronic liver failure: a retrospective study. Mycoses 2013; 56: 429-433 [PMID: 23368965 DOI: 10.1111/myc.12044]

15

Levesque E, Ait-Ammar N, Dudau D, Clavieras N, Feray C, Foulet F, Botterel F. Invasive pulmonary aspergillosis in cirrhotic patients: analysis of a 10-year clinical experience. Ann Intensive Care 2019; 9: 31 [PMID: 30778699 DOI: 10.1186/s13613-019-0502-2]

16

Gustot T, Maillart E, Bocci M, Surin R, Trépo E, Degré D, Lucidi V, Taccone FS, Delforge ML, Vincent JL, Donckier V, Jacobs F, Moreno C. Invasive aspergillosis in patients with severe alcoholic hepatitis. J Hepatol 2014; 60: 267-274 [PMID: 24055548 DOI: 10.1016/j.jhep.2013.09.011]

17

Hmoud BS, Patel K, Bataller R, Singal AK. Corticosteroids and occurrence of and mortality from infections in severe alcoholic hepatitis: a meta-analysis of randomized trials. Liver Int 2016; 36: 721-728 [PMID: 26279269 DOI: 10.1111/liv.12939]

18

Rolando N, Philpott-Howard J, Williams R. Bacterial and fungal infection in acute liver failure. Semin Liver Dis 1996; 16: 389-402 [PMID: 9027952 DOI: 10.1055/s-2007-1007252]

19

Antoniades CG, Berry PA, Wendon JA, Vergani D. The importance of immune dysfunction in determining outcome in acute liver failure. J Hepatol 2008; 49: 845-861 [PMID: 18801592 DOI: 10.1016/j.jhep.2008.08.009]

20

Bartoletti M, Giannella M, Lewis R, Caraceni P, Tedeschi S, Paul M, Schramm C, Bruns T, Merli M, Cobos-Trigueros N, Seminari E, Retamar P, Muñoz P, Tumbarello M, Burra P, Torrani Cerenzia M, Barsic B, Calbo E, Maraolo AE, Petrosillo N, Galan-Ladero MA, D'Offizi G, Bar Sinai N, Rodríguez-Baño J, Verucchi G, Bernardi M, Viale P; ESGBIS/BICHROME Study Group. A prospective multicentre study of the epidemiology and outcomes of bloodstream infection in cirrhotic patients. Clin Microbiol Infect 2018; 24: 546.e1-546. e8 [PMID: 28818628 DOI: 10.1016/j.cmi.2017.08.001]

21

Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, Ostrosky-Zeichner L, Reboli AC, Schuster MG, Vazquez JA, Walsh TJ, Zaoutis TE, Sobel JD. Executive Summary: Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis 2016; 62: 409-417 [PMID: 26810419 DOI: 10.1093/cid/civ1194]

22

Patterson TF, Thompson GR 3rd, Denning DW, Fishman JA, Hadley S, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Nguyen MH, Segal BH, Steinbach WJ, Stevens DA, Walsh TJ, Wingard JR, Young JA, Bennett JE. Executive Summary: Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis 2016; 63: 433-442 [PMID: 27481947 DOI: 10.1093/cid/ciw444]

23

Wang T, Yan M, Tang D, Xue L, Zhang T, Dong Y, Zhu L, Wang X, Dong Y. Therapeutic drug monitoring and safety of voriconazole therapy in patients with Child-Pugh class B and C cirrhosis: A

24




https://dx.doi.org/10.1007/s00134-017-4717-0








https://dx.doi.org/10.1016/j.cmi.2015.10.020


https://dx.doi.org/10.7150/ijms.6824


https://dx.doi.org/10.1111/myc.12044


https://dx.doi.org/10.1186/s13613-019-0502-2






https://dx.doi.org/10.1055/s-2007-1007252




https://dx.doi.org/10.1016/j.cmi.2017.08.001


https://dx.doi.org/10.1093/cid/civ1194


https://dx.doi.org/10.1093/cid/ciw444



multicenter study. Int J Infect Dis 2018; 72: 49-54 [PMID: 29793038 DOI: 10.1016/j.ijid.2018.05.009]Tschuor C, Ferrarese A, Kuemmerli C, Dutkowski P, Burra P, Clavien PA; Liver Allocation Study Group. Allocation of liver grafts worldwide - Is there a best system? J Hepatol 2019; 71: 707-718 [PMID: 31199941 DOI: 10.1016/j.jhep.2019.05.025]

25

Trebicka J, Sundaram V, Moreau R, Jalan R, Arroyo V. Liver Transplantation for Acute-on-Chronic Liver Failure: Science or Fiction? Liver Transpl 2020; 26: 906-915 [PMID: 32365422 DOI: 10.1002/lt.25788]

26

Gustot T, Fernandez J, Szabo G, Albillos A, Louvet A, Jalan R, Moreau R, Moreno C. Sepsis in alcohol-related liver disease. J Hepatol 2017; 67: 1031-1050 [PMID: 28647569 DOI: 10.1016/j.jhep.2017.06.013]

27

Linecker M, Krones T, Berg T, Niemann CU, Steadman RH, Dutkowski P, Clavien PA, Busuttil RW, Truog RD, Petrowsky H. Potentially inappropriate liver transplantation in the era of the "sickest first" policy - A search for the upper limits. J Hepatol 2018; 68: 798-813 [PMID: 29133246 DOI: 10.1016/j.jhep.2017.11.008]

28

Karvellas CJ, Garcia-Lopez E, Fernandez J, Saliba F, Sy E, Jalan R, Pavesi M, Gustot T, Ronco JJ, Arroyo V; Chronic Liver Failure Consortium and European Foundation for the Study of Chronic Liver Failure. Dynamic Prognostication in Critically Ill Cirrhotic Patients With Multiorgan Failure in ICUs in Europe and North America: A Multicenter Analysis. Crit Care Med 2018; 46: 1783-1791 [PMID: 30106759 DOI: 10.1097/CCM.0000000000003369]

29

Hosseini-Moghaddam SM, Ouédraogo A, Naylor KL, Bota SE, Husain S, Nash DM, Paterson JM. Incidence and outcomes of invasive fungal infection among solid organ transplant recipients: A population-based cohort study. Transpl Infect Dis 2020; 22: e13250 [PMID: 31981389 DOI: 10.1111/tid.13250]

30

van Delden C, Stampf S, Hirsch HH, Manuel O, Meylan P, Cusini A, Hirzel C, Khanna N, Weisser M, Garzoni C, Boggian K, Berger C, Nadal D, Koller M, Saccilotto R, Mueller NJ; Swiss Transplant Cohort Study. Burden and Timeline of Infectious Diseases in the First Year After Solid Organ Transplantation in the Swiss Transplant Cohort Study. Clin Infect Dis 2020; 71: e159-e169 [PMID: 31915816 DOI: 10.1093/cid/ciz1113]

31

Andes DR, Safdar N, Baddley JW, Alexander B, Brumble L, Freifeld A, Hadley S, Herwaldt L, Kauffman C, Lyon GM, Morrison V, Patterson T, Perl T, Walker R, Hess T, Chiller T, Pappas PG; TRANSNET Investigators. The epidemiology and outcomes of invasive Candida infections among organ transplant recipients in the United States: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Transpl Infect Dis 2016; 18: 921-931 [PMID: 27643395 DOI: 10.1111/tid.12613]

32

Fernández-Ruiz M, Cardozo C, Salavert M, Aguilar-Guisado M, Escolà-Vergé L, Muñoz P, Gioia F, Montejo M, Merino P, Cuervo G, García-Vidal C, Aguado JM; CANDIPOP Project; the CANDI-Bundle Group; GEIRAS-GEMICOMED (SEIMC)REIPI. Candidemia in solid organ transplant recipients in Spain: Epidemiological trends and determinants of outcome. Transpl Infect Dis 2019; 21: e13195 [PMID: 31610077 DOI: 10.1111/tid.13195]

33

Aslam S, Rotstein C; AST Infectious Disease Community of Practice. Candida infections in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant 2019; 33: e13623 [PMID: 31155770 DOI: 10.1111/ctr.13623]

34

Giannella M, Bartoletti M, Morelli M, Cristini F, Tedeschi S, Campoli C, Tumietto F, Bertuzzo V, Ercolani G, Faenza S, Pinna AD, Lewis RE, Viale P. Antifungal prophylaxis in liver transplant recipients: one size does not fit all. Transpl Infect Dis 2016; 18: 538-544 [PMID: 27237076 DOI: 10.1111/tid.12560]

35

Husain S, Camargo JF. Invasive Aspergillosis in solid-organ transplant recipients: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant 2019; 33: e13544 [PMID: 30900296 DOI: 10.1111/ctr.13544]

36

Saliba F, Delvart V, Ichaï P, Kassis N, Botterel F, Mihaila L, Azoulay D, Adam R, Castaing D, Bretagne S, Samuel D. Fungal infections after liver transplantation: outcomes and risk factors revisited in the MELD era. Clin Transplant 2013; 27: E454-E461 [PMID: 23656358 DOI: 10.1111/ctr.12129]

37

Neofytos D, Chatzis O, Nasioudis D, Boely Janke E, Doco Lecompte T, Garzoni C, Berger C, Cussini A, Boggian K, Khanna N, Manuel O, Mueller NJ, van Delden C; Swiss Transplant Cohort Study. Epidemiology, risk factors and outcomes of invasive aspergillosis in solid organ transplant recipients in the Swiss Transplant Cohort Study. Transpl Infect Dis 2018; 20: e12898 [PMID: 29668068 DOI: 10.1111/tid.12898]

38

Amin A, Molina A, Quach L, Ito T, McMillan R, DiNorcia J, Agopian VG, Kaldas FM, Farmer DG, Busuttil RW, Winston DJ. Liver Transplantation in Patients with Pretransplant Aspergillus Colonization: Is It Safe To Proceed? Transplantation 2020 [PMID: 32301905 DOI: 10.1097/TP.0000000000003276]

39

Artru F, Louvet A, Ruiz I, Levesque E, Labreuche J, Ursic-Bedoya J, Lassailly G, Dharancy S, Boleslawski E, Lebuffe G, Kipnis E, Ichai P, Coilly A, De Martin E, Antonini TM, Vibert E, Jaber S, Herrerro A, Samuel D, Duhamel A, Pageaux GP, Mathurin P, Saliba F. Liver transplantation in the most severely ill cirrhotic patients: A multicenter study in acute-on-chronic liver failure grade 3. J Hepatol 2017; 67: 708-715 [PMID: 28645736 DOI: 10.1016/j.jhep.2017.06.009]

40

Echenique IA, Ison MG. Update on donor-derived infections in liver transplantation. Liver Transpl 41


https://dx.doi.org/10.1016/j.ijid.2018.05.009










https://dx.doi.org/10.1097/CCM.0000000000003369


https://dx.doi.org/10.1111/tid.13250


https://dx.doi.org/10.1093/cid/ciz1113






https://dx.doi.org/10.1111/ctr.13623










https://dx.doi.org/10.1097/TP.0000000000003276





2013; 19: 575-585 [PMID: 23526639 DOI: 10.1002/lt.23640]Levesque E, Paugam-Burtz C, Saliba F, Khoy-Ear L, Merle JC, Jung B, Stecken L, Ferrandiere M, Mihaila L, Botterel F. Fungal complications after Candida preservation fluid contamination in liver transplant recipients. Transpl Int 2015; 28: 1308-1316 [PMID: 26147662 DOI: 10.1111/tri.12633]

42

Pappas PG, Alexander BD, Andes DR, Hadley S, Kauffman CA, Freifeld A, Anaissie EJ, Brumble LM, Herwaldt L, Ito J, Kontoyiannis DP, Lyon GM, Marr KA, Morrison VA, Park BJ, Patterson TF, Perl TM, Oster RA, Schuster MG, Walker R, Walsh TJ, Wannemuehler KA, Chiller TM. Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis 2010; 50: 1101-1111 [PMID: 20218876 DOI: 10.1086/651262]

43

European Association for the Study of the Liver. EASL Clinical Practice Guidelines: Liver transplantation. J Hepatol 2016; 64: 433-485 [PMID: 26597456 DOI: 10.1016/j.jhep.2015.10.006]

44

Lucey MR, Terrault N, Ojo L, Hay JE, Neuberger J, Blumberg E, Teperman LW. Long-term management of the successful adult liver transplant: 2012 practice guideline by the American Association for the Study of Liver Diseases and the American Society of Transplantation. Liver Transpl 2013; 19: 3-26 [PMID: 23281277 DOI: 10.1002/lt.23566]

45

Evans JD, Morris PJ, Knight SR. Antifungal prophylaxis in liver transplantation: a systematic review and network meta-analysis. Am J Transplant 2014; 14: 2765-2776 [PMID: 25395336 DOI: 10.1111/ajt.12925]

46

Sun HY, Cacciarelli TV, Singh N. Micafungin versus amphotericin B lipid complex for the prevention of invasive fungal infections in high-risk liver transplant recipients. Transplantation 2013; 96: 573-578 [PMID: 23842191 DOI: 10.1097/TP.0b013e31829d674f]

47

Trudeau RE, Bowman LJ, Wills AR, Crippin JS, Chapman WC, Anderson C. Once weekly fluconazole for antifungal prophylaxis post-liver transplantation. HPB (Oxford) 2013; 15: 541-547 [PMID: 23458063 DOI: 10.1111/hpb.12006]

48

Antunes AM, Teixeira C, Corvo ML, Perdigoto R, Barroso E, Marcelino P. Prophylactic use of liposomal amphotericin B in preventing fungal infections early after liver transplantation: a retrospective, single-center study. Transplant Proc 2014; 46: 3554-3559 [PMID: 25498088 DOI: 10.1016/j.transproceed.2014.06.065]

49

Winston DJ, Limaye AP, Pelletier S, Safdar N, Morris MI, Meneses K, Busuttil RW, Singh N. Randomized, double-blind trial of anidulafungin versus fluconazole for prophylaxis of invasive fungal infections in high-risk liver transplant recipients. Am J Transplant 2014; 14: 2758-2764 [PMID: 25376267 DOI: 10.1111/ajt.12963]

50

Saliba F, Pascher A, Cointault O, Laterre PF, Cervera C, De Waele JJ, Cillo U, Langer RM, Lugano M, Göran-Ericzon B, Phillips S, Tweddle L, Karas A, Brown M, Fischer L; TENPIN (Liver Transplant European Study Into the Prevention of Fungal Infection) Investigators; TENPIN Liver Transplant European Study Into the Prevention of Fungal Infection Investigators. Randomized trial of micafungin for the prevention of invasive fungal infection in high-risk liver transplant recipients. Clin Infect Dis 2015; 60: 997-1006 [PMID: 25520332 DOI: 10.1093/cid/ciu1128]

51

Giannella M, Ercolani G, Cristini F, Morelli M, Bartoletti M, Bertuzzo V, Tedeschi S, Faenza S, Puggioli C, Lewis RE, Pinna AD, Viale P. High-dose weekly liposomal amphotericin b antifungal prophylaxis in patients undergoing liver transplantation: a prospective phase II trial. Transplantation 2015; 99: 848-854 [PMID: 25531982 DOI: 10.1097/TP.0000000000000393]

52

Eschenauer GA, Kwak EJ, Humar A, Potoski BA, Clarke LG, Shields RK, Abdel-Massih R, Silveira FP, Vergidis P, Clancy CJ, Nguyen MH. Targeted versus universal antifungal prophylaxis among liver transplant recipients. Am J Transplant 2015; 15: 180-189 [PMID: 25359455 DOI: 10.1111/ajt.12993]

53

Balogh J, Gordon Burroughs S, Boktour M, Patel S, Saharia A, Ochoa RA, McFadden R, Victor DW, Ankoma-Sey V, Galati J, Monsour HP Jr, Fainstein V, Li XC, Grimes KA, Gaber AO, Aloia T, Ghobrial RM. Efficacy and cost-effectiveness of voriconazole prophylaxis for prevention of invasive aspergillosis in high-risk liver transplant recipients. Liver Transpl 2016; 22: 163-170 [PMID: 26515643 DOI: 10.1002/lt.24365]

54

Perrella A, Esposito C, Amato G, Perrella O, Migliaccio C, Pisaniello D, Calise F, Cuomo O, Santaniello W. Antifungal prophylaxis with liposomal amphotericin B and caspofungin in high-risk patients after liver transplantation: impact on fungal infections and immune system. Infect Dis (Lond) 2016; 48: 161-166 [PMID: 26513601 DOI: 10.3109/23744235.2015.1100322]

55

Fortún J, Muriel A, Martín-Dávila P, Montejo M, Len O, Torre-Cisneros J, Carratalá J, Muñoz P, Fariñas C, Moreno A, Fresco G, Goikoetxea J, Gavaldá J, Pozo JC, Bodro M, Vena A, Casafont F, Cervera C, Silva JT, Aguado JM; Grupo de Estudio de Infección en Pacientes Trasplantados-Grupo de Estudio de Micología Médica (Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica); and Red Española de Investigación en Patología Infecciosa. Caspofungin versus fluconazole as prophylaxis of invasive fungal infection in high-risk liver transplantation recipients: A propensity score analysis. Liver Transpl 2016; 22: 427-435 [PMID: 26709146 DOI: 10.1002/lt.24391]

56

Chen YC, Huang TS, Wang YC, Cheng CH, Lee CF, Wu TJ, Chou HS, Chan KM, Lee WC, Soong RS. Effect of Prophylactic Antifungal Protocols on the Prognosis of Liver Transplantation: A Propensity Score Matching and Multistate Model Approach. Biomed Res Int 2016; 2016: 6212503 [PMID: 27747235 DOI: 10.1155/2016/6212503]

57

Lavezzo B, Patrono D, Tandoi F, Martini S, Fop F, Ballerini V, Stratta C, Skurzak S, Lupo F, Strignano P, Donadio PP, Salizzoni M, Romagnoli R, De Rosa FG. A simplified regimen of targeted

58




https://dx.doi.org/10.1111/tri.12633


https://dx.doi.org/10.1086/651262






https://dx.doi.org/10.1111/ajt.12925


https://dx.doi.org/10.1097/TP.0b013e31829d674f


https://dx.doi.org/10.1111/hpb.12006


https://dx.doi.org/10.1016/j.transproceed.2014.06.065




https://dx.doi.org/10.1093/cid/ciu1128


https://dx.doi.org/10.1097/TP.0000000000000393






https://dx.doi.org/10.3109/23744235.2015.1100322




https://dx.doi.org/10.1155/2016/6212503



antifungal prophylaxis in liver transplant recipients: A single-center experience. Transpl Infect Dis 2018; 20: e12859 [PMID: 29427394 DOI: 10.1111/tid.12859]Jorgenson MR, Descourouez JL, Marka NA, Leverson GE, Smith JA, Andes DR, Fernandez LA, Foley DP. A targeted fungal prophylaxis protocol with static dosed fluconazole significantly reduces invasive fungal infection after liver transplantation. Transpl Infect Dis 2019; 21: e13156 [PMID: 31390109 DOI: 10.1111/tid.13156]

59

Kang WH, Song GW, Lee SG, Suh KS, Lee KW, Yi NJ, Joh JW, Kwon CHD, Kim JM, Choi DL, Kim JD, Kim MS. A Multicenter, Randomized, Open-Label Study to Compare Micafungin with Fluconazole in the Prophylaxis of Invasive Fungal Infections in Living-Donor Liver Transplant Recipients. J Gastrointest Surg 2020; 24: 832-840 [PMID: 31066013 DOI: 10.1007/s11605-019-04241-w]

60

Aguado JM, Varo E, Usetti P, Pozo JC, Moreno A, Catalán M, Len O, Blanes M, Solé A, Muñoz P, Montejo M; TOSCANA Study Group. Safety of anidulafungin in solid organ transplant recipients. Liver Transpl 2012; 18: 680-685 [PMID: 22328277 DOI: 10.1002/lt.23410]

61

Muilwijk EW, Lempers VJ, Burger DM, Warris A, Pickkers P, Aarnoutse RE, Brüggemann RJ. Impact of special patient populations on the pharmacokinetics of echinocandins. Expert Rev Anti Infect Ther 2015; 13: 799-815 [PMID: 25947367 DOI: 10.1586/14787210.2015.1028366]

62

Maertens JA, Raad II, Marr KA, Patterson TF, Kontoyiannis DP, Cornely OA, Bow EJ, Rahav G, Neofytos D, Aoun M, Baddley JW, Giladi M, Heinz WJ, Herbrecht R, Hope W, Karthaus M, Lee DG, Lortholary O, Morrison VA, Oren I, Selleslag D, Shoham S, Thompson GR 3rd, Lee M, Maher RM, Schmitt-Hoffmann AH, Zeiher B, Ullmann AJ. Isavuconazole versus voriconazole for primary treatment of invasive mould disease caused by Aspergillus and other filamentous fungi (SECURE): a phase 3, randomised-controlled, non-inferiority trial. Lancet 2016; 387: 760-769 [PMID: 26684607 DOI: 10.1016/S0140-6736(15)01159-9]

63

Groll AH, Townsend R, Desai A, Azie N, Jones M, Engelhardt M, Schmitt-Hoffman AH, Brüggemann RJM. Drug-drug interactions between triazole antifungal agents used to treat invasive aspergillosis and immunosuppressants metabolized by cytochrome P450 3A4. Transpl Infect Dis 2017; 19 [PMID: 28722255 DOI: 10.1111/tid.12751]

64

Becchetti C, Ferrarese A, Cattelan A, Barbieri S, Feltracco P, Saluzzo F, Cillo U, Senzolo M, Germani G, Burra P. Geotrichum capitatum Invasive Infection Early After Liver Transplant. Exp Clin Transplant 2020; 18: 737-740 [PMID: 31801448 DOI: 10.6002/ect.2019.0170]

65

Shoham S, Dominguez EA; AST Infectious Diseases Community of Practice. Emerging fungal infections in solid organ transplant recipients: Guidelines of the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant 2019; 33: e13525 [PMID: 30859651 DOI: 10.1111/ctr.13525]

66






https://dx.doi.org/10.1007/s11605-019-04241-w




https://dx.doi.org/10.1586/14787210.2015.1028366


https://dx.doi.org/10.1016/S0140-6736(15)01159-9




https://dx.doi.org/10.6002/ect.2019.0170




World Journal of



ORIGINAL ARTICLE

Basic Study

Blockage of ETS homologous factor inhibits the proliferation and invasion of gastric cancer cells through the c-Met pathway

Meng-Li Gu, Xin-Xin Zhou, Meng-Ting Ren, Ke-Da Shi, Mo-Sang Yu, Wen-Rui Jiao, Ya-Mei Wang, Wei-Xiang Zhong, Feng Ji

ORCID number: Meng-Li Gu 0000-0001-5851-3456; Xin-Xin Zhou 0000-0003-0183-6400; Meng-Ting Ren 0000-0002-3807-4596; Ke-Da Shi 0000-0001-9215-328X; Mo-Sang Yu 0000-0002-5614-0227; Wen-Rui Jiao 0000-0002-0247-4694; Ya-Mei Wang 0000-0001-7092-694X; Wei-Xiang Zhong 0000-0002-5493-893X; Feng Ji 0000-0003-4252-8850.

Author contributions: Gu ML and Zhou XX contributed equally to this work; Gu ML designed the research and wrote the manuscript; Gu ML, Zhou XX, Ren MT, and Jiao WR performed the experiments and acquired the data; Shi KD and Yu MS analyzed and interpreted the data; Wang YM performed some of the experiments; Zhong WX provided valuable suggestions for this study; Ji F contributed to study supervision; All authors have read and approved the final manuscript.

Supported by The Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province, No. 2017ZZ010; and Zhejiang Medical Science and Technology Program, No. 2018266817.

Institutional review board statement: The study was reviewed and approved by the

Meng-Li Gu, Xin-Xin Zhou, Meng-Ting Ren, Ke-Da Shi, Mo-Sang Yu, Wen-Rui Jiao, Feng Ji, Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China

Ya-Mei Wang, Department of Gastroenterology, The Fourth Affiliated Hospital, College of Medicine, Zhejiang University, Yiwu 322000, Zhejiang Province, China

Wei-Xiang Zhong, Department of Pathology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China

Corresponding author: Feng Ji, MD, PhD, Chief Doctor, Professor, Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou 310003, Zhejiang Province, China. [email protected]

AbstractBACKGROUND Gastric cancer (GC) is one of the most common and deadliest types of cancer worldwide due to its delayed diagnosis and high metastatic frequency, but its exact pathogenesis has not been fully elucidated. ETS homologous factor (EHF) is an important member of the ETS family and contributes to the pathogenesis of multiple malignant tumors. To date, whether EHF participates in the development of GC via the c-Met signaling pathway remains unclear.

AIM To investigate the role and mechanism of EHF in the occurrence and development of GC.

METHODS The expression of EHF mRNA in GC tissues and cell lines was measured by quantitative PCR. Western blotting was performed to determine the protein expression of EHF, c-Met, and its downstream signal molecules. The EHF expression in GC tissues was further detected by immunohistochemical staining. To investigate the role of EHF in GC oncogenesis, small interfering RNA (siRNA) against EHF was transfected into GC cells. The cell proliferation of GC cells was determined by Cell Counting Kit-8 and colony formation assays. Flow cytometry was performed following Annexin V/propidium iodide (PI) to identify apoptotic cells and PI staining to analyze the cell cycle. Cell migration and invasion were



http://orcid.org/0000-0001-5851-3456

http://orcid.org/0000-0001-5851-3456

http://orcid.org/0000-0001-5851-3456

http://orcid.org/0000-0003-0183-6400

http://orcid.org/0000-0003-0183-6400

http://orcid.org/0000-0003-0183-6400

http://orcid.org/0000-0002-3807-4596

http://orcid.org/0000-0002-3807-4596

http://orcid.org/0000-0001-9215-328X

http://orcid.org/0000-0001-9215-328X

http://orcid.org/0000-0002-5614-0227

http://orcid.org/0000-0002-5614-0227

http://orcid.org/0000-0002-0247-4694

http://orcid.org/0000-0002-0247-4694

http://orcid.org/0000-0001-7092-694X

http://orcid.org/0000-0001-7092-694X

http://orcid.org/0000-0002-5493-893X

http://orcid.org/0000-0002-5493-893X

http://orcid.org/0000-0003-4252-8850

http://orcid.org/0000-0003-4252-8850


Gu ML et al. EHF blockage inhibits proliferation and invasion


Institutional Review Board of The First Affiliated Hospital, College of Medicine, Zhejiang University, Approval No. 2019-1218.

Conflict-of-interest statement: The authors have no conflicts of interest to declare.

Data sharing statement: No additional data are available.

Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/




Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0

Received: July 21, 2020 Peer-review started: July 21, 2020 First decision: September 30, 2020 Revised: October 13, 2020 Accepted: November 2, 2020 Article in press: November 2, 2020 Published online: December 21, 2020

P-Reviewer: Li Y, Neninger E S-Editor: Huang P L-Editor: Filipodia P-Editor: Wang LL

assessed by transwell assays.

RESULTS The data showed that EHF was upregulated in GC tissues and cell lines in which increased expression of c-Met was also observed. Silencing of EHF by siRNA reduced the proliferation of GC cells. Inhibition of EHF induced significant apoptosis and cell cycle arrest in GC cells. Cell migration and invasion were significantly inhibited. EHF silencing led to c-Met downregulation and further blocked the Ras/c-Raf/extracellular signal-related kinase 1/2 (Erk1/2) pathway. Additionally, phosphatase and tensin homolog was upregulated and glycogen synthase kinase 3 beta was deactivated. Moreover, inactivation of signal transducer and activator of transcription 3 was detected following EHF inhibition, leading to inhibition of the epithelial-to-mesenchymal transition (EMT).

CONCLUSION These results suggest that EHF plays a key role in cell proliferation, invasion, apoptosis, the cell cycle and EMT via the c-Met pathway. Therefore, EHF may serve as an antineoplastic target for the diagnosis and treatment of GC.

Key Words: Gastric cancer; ETS homologous factor; c-Met; Antineoplastic target; Signaling pathway


Core Tip: The purpose of this study was to investigate the role and mechanism of ETS homologous factor (EHF) in the occurrence and development of gastric cancer (GC). The results showed that EHF plays a key role in cell proliferation, invasion, apoptosis, the cell cycle and epithelial-to-mesenchymal transition. EHF may contribute to the tumorigenesis and progression of GC via the c-Met pathway. Therefore, EHF is a promising target for the diagnosis and treatment of GC.

Citation: Gu ML, Zhou XX, Ren MT, Shi KD, Yu MS, Jiao WR, Wang YM, Zhong WX, Ji F. Blockage of ETS homologous factor inhibits the proliferation and invasion of gastric cancer cells through the c-Met pathway. World J Gastroenterol 2020; 26(47): 7497-7512URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7497.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7497

INTRODUCTIONGastric cancer (GC) is a common malignant tumor in the gastrointestinal tract and the third leading cause of cancer-related mortality worldwide[1]. Risk factors such as Helicobacter pylori infection, atrophic gastritis, a high-salt diet and heavy alcohol drinking contribute to the tumorigenesis and progression of GC, but its exact pathogenesis has not been fully elucidated[2,3]. Currently, surgical resection represents the best curative option for GC. On the other hand, advanced novel targets for GC diagnosis and treatment have resulted in decreased incidence and mortality rates in recent years[4]. However, the 5-year overall survival rate of patients with advanced GC is less than 30% due to delayed diagnosis and recurrence[5]. Therefore, revealing the oncogenic mechanisms of GC and further exploring the strategies for GC diagnosis and treatment are urgent.

The ETS family consists of various transcriptional factors that contain a conservative ETS structure domain, which specifically identifies and binds to the GGAA/T sequence within the promoter or enhancer region of target genes to regulate their transcription[6]. ETS homologous factor (EHF) is a member of the ETS family of transcription factors expressed in multiple epithelial cell types, which participates in the oncogenesis of various malignant tumors via regulating cell proliferation and differentiation[7,8]. The aberrant expression of EHF is involved in the epithelial-to-mesenchymal transition (EMT) and is a risk factor for the increased recurrence and reduced overall survival of prostate cancer[9]. A recent study demonstrated that








upregulated EHF expression is correlated with the poor prognosis of ovarian cancer patients and that the inhibition of EHF exerts an antineoplastic effect on ovarian cancer cells[10]. Additionally, EHF is highly expressed in colon cancers, and the down-regulation of EHF leads to apoptosis of colon cancer cells[11,12]. However, the roles of EHF in the tumorigenesis and progression of GC need further investigation.

A previous study indicated that EHF regulates the expression of c-Met by directly binding to the promoter region of the c-Met coding sequence[7]. c-Met is a receptor tyrosine kinase (RTK), and its dysregulation is closely related to the pathogenesis and development of GC[13]. In advanced GC patients, c-Met-positive status is an independent prognostic factor for poor outcomes[14]. To date, whether EHF participates in the development of GC through the c-Met pathway remains unclear. Herein, we investigated EHF expression in GC and elucidated its potential function and regulatory mechanisms involving the oncogenesis of GC, providing important evidence for the exploration of novel therapeutic targets for GC.

MATERIALS AND METHODSHuman samplesA total of 10 paired GC tissues and adjacent normal tissues were obtained from The First Affiliated Hospital of Zhejiang University (Zhejiang, China). The patients did not receive radiotherapy or chemotherapy prior to collection. The tissues were immediately stored in liquid nitrogen postoperatively. This study was performed according to the Declaration of Helsinki and approved by The Research Ethics Committee of The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 20191218. Informed consent was obtained from all subjects involved in the study.

Cell cultureThe human normal gastric epithelial cell line (GES-1) and human GC cell lines (BGC-823, KATO III and SGC-7901) were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

Transfection of small interfering RNAsSmall interfering RNA (siRNA) transfections were performed using Lipofectamine™ 3000 reagent (Invitrogen, San Diego, CA, United States) in antibiotic-free RPMI 1640 according to the manufacturer’s instructions. The GC cell lines were transfected with 10 nmol/L siRNA. The control siRNA and siRNA targeting EHF were purchased from Invitrogen.

Quantitative PCRThe total RNA of each cell line was extracted using RNAiso Plus reagent (Takara, Dalian, China) following the manufacturer's protocol. The quantitative polymerase chain reaction (qPCR) assay was performed as previously described[15]. The primers were obtained from Sangon Biotech (Shanghai, China). Primer sequences were listed as follows: EHF, forward 5'-GAAGAACAACAGCAGCATGACC-3' and reverse 5'-TCAGGTTTCGGTGTATGAGTTG-3' ; GAPDH, forward 5 ' -AGAAGGCT GGGGCTCATTTG-3' and reverse 5'-AGGGGCCATCCACAGTCTT-3'. The relative gene expression levels of the tested genes were calculated using the 2-ΔΔCt method[16].

Cell proliferationCell proliferation was determined using the Cell Counting Kit-8 assay according to our previous study[15].

Colony formation assayAfter transfection for 48 h, BGC-823, KATO III, and SGC-7901 cells were seeded into 6-well plates at a density of 2 × 103 cells per well and incubated for 10 d at 37 °C. Then, the culture medium was removed, and cells were washed with phosphate-buffered saline (referred to as PBS). The colonies were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The colony numbers were ultimately counted.



Cell cycle assay and cell apoptosis assayCells were inoculated into 6-well plates at a density of 4 × 105 cells per well and incubated for 24 h. Then the cells were transfected with negative control and EHF-targeted siRNA for 72 h. Finally, cell cycle assays and cell apoptosis assays were performed as previously described[15].

Transwell assayFollowing the transfection of EHF-targeted siRNA for 24 h, 2.5 × 104 cells were plated into 24-well chambers (Corning, NY, United States) with medium containing 1% FBS. Medium containing 10% FBS was added into the lower wells. For the invasion assays, Matrigel was added to each transwell chamber and incubated for 5 h before the cells were seeded. After 48 h of incubation, the cells remaining in the upper chamber were carefully removed with cotton swabs. The membrane-penetrating cells were stained with crystal violet for 15 min. The cells that migrated and invaded were counted from at least five randomly selected fields.

Immunohistochemical analysisThe expression of EHF was evaluated by immunohistochemical staining. Paraffin blocks that contained formalin-fixed GC and adjacent normal tissues were sectioned at 4 μm. The sections were deparaffinized in 100% xylene and re-hydrated at graded ethanol concentrations. Endogenous peroxidase activity was blocked using fresh 3% hydrogen peroxide for 10 min at room temperature. Antigen retrieval was done by boiling the slides in sodium citrate buffer (pH 6.0). After blocking in 3% bovine serum albumin, sections were incubated with anti-EHF antibody (1:100 dilution in PBS; Abcam, Cambridge, MA, United States) overnight at 4 °C. Subsequently, the sections were incubated for 30 min at room temperature with anti-rabbit horseradish peroxidase-conjugated secondary antibody. The immunostaining was performed using diaminobenzidine solution. The sections were then counterstained with hematoxylin. dehydrated and mounted. Blinded evaluations of EHF immunostaining were carried out independently by two experienced pathologists.

Western blot analysisAccording to the manufacturer's instructions, the total protein was extracted from cells by using cell lysis buffer (Cell Signaling, Beverly, MA, United States). Western blotting analysis was performed as previously described[15]. Rabbit antibodies corresponding to EHF were purchased from Abcam. Antibodies against c-Met, Ras, total c-Raf, phospho-c-Raf (p-c-Raf), total Erk1/2, p-Erk1/2, phosphatase and tensin homolog (PTEN), total glycogen synthase kinase 3 beta (GSK-3β), phospho-GSK-3β, Bcl-2-associated X protein (Bax), survivin, total signal transducer and activator of transcription 3 (STAT3), p-STAT3, Twist, Snail, E-cadherin, and GAPDH were purchased from Cell Signaling Technology, Inc.

Statistical analysesStatistical analyses were performed using SPSS version 20 (IBM Corp., Armonk, NY, United States). Results are presented as the mean ± standard deviation. The statistical differences between two groups was assessed by the Student’s t-test. One-way analysis of variance followed by Tukey's multiple comparison test was used to determine the statistical difference among multiple groups. The significance level was set at P < 0.05. All experiments were performed in triplicate.

RESULTSEHF is overexpressed in GC specimens and cell lines in which c-Met expression is also upregulatedThe expression of EHF mRNA in paired adjacent normal tissues and GC tissues were first measured via qPCR and Western blotting. The mRNA expression level of EHF was significantly upregulated in GC tissues compared with adjacent control tissues (Figure 1A). The protein expression of EHF was increased in GC tissues overexpressing c-Met (Figure 1B). As shown in Figure 1C, increased expression of EHF was also detected in GC tissues by immunohistochemistry. Second, the gene and protein expression of EHF in human GES-1 and GC cell lines (BGC-823, KATO III and SGC-7901) was detected. The expression of EHF mRNA was increased in these GC cell lines compared with GES-1 (Figure 1D). In GC cell lines with upregulated EHF, the



Figure 1 ETS homologous factor and c-Met expression in gastric cancer tissues and cell lines. A: Quantitative PCR analysis of mRNAs revealed the upregulation of ETS homologous factor (EHF) mRNA in gastric cancer (GC) tissues compared with their matched adjacent tissues; B: Western blotting results showed that the expression of EHF and c-Met were both upregulated in GC tissues; C: Immunohistochemical staining of EHF indicated that increased expression of EHF was higher in GC tissues than that in matched adjacent tissues (400 ×); D: The mRNA expression level of EHF was enhanced in GC cell lines compared with that in normal gastric epithelial cells; E: EHF protein expression was increased in GC cell lines in which c-Met was also upregulated. aP < 0.05 vs matched adjacent tissues; eP < 0.001 vs normal gastric epithelial cells. T: GC tissues; N: Matched adjacent tissues.



protein expression of c-Met was also increased in a similar pattern (Figure 1E).

Silencing EHF by siRNA leads to c-Met downregulationTo clarify the potential function of EHF in the tumorigenesis of GC, EHF silencing was performed by transfecting EHF-targeted siRNA into human GC cell lines (BGC-823, KATO III and SGC-7901). As shown in Figure 2, the mRNA and protein expression levels of EHF significantly decreased in the GC cell lines compared with the negative control group after transfection for 72 h (P < 0.05). In addition, the inhibition of EHF also led to the downregulation of c-Met in these GC cell lines.

EHF silencing suppresses GC cell viability and colony formationThe aforementioned results demonstrated that EHF expression was higher in GC cell lines and GC tissues. The potential functional roles of EHF in GC cells were further explored. Knockdown of EHF significantly reduced the cell viability of GC cell lines after transfection for 5 d (Figure 3A). In addition, EHF silencing also significantly inhibited the colony formation of GC cell lines compared with the negative control group (Figure 3B and C). Thus, EHF plays a crucial role in the regulation of proliferation in GC.

Depletion of EHF promotes apoptosis and induces cell cycle arrestTo investigate whether EHF can affect apoptotic cell death in GC, Annexin V-FITC/propidium iodide (PI) staining and flow cytometry were performed. As shown in Figure 4A and B, EHF downregulation induced significant apoptosis in the GC cell lines (P < 0.05), suggesting that EHF exerts an antiapoptotic effect in these GC cell lines. To further assess the cell cycle status following EHF silencing, PI staining, and flow cytometry were performed. As shown in Figure 4C and D, the inhibition of EHF caused an increase in GC cells in the G1 phase, but a significant decrease in the S phase (P < 0.05). These data indicate that EHF participates in regulating cell cycle progression in GC cell lines by promoting the transition from the G1 phase to the S phase.

Downregulation of EHF inhibits the migration and invasion of GC cellsTranswell assays were used to detect cell migration after transfection for 24 h. As shown in Figure 5A and B, the migration of GC cell lines significantly decreased following EHF silencing (P < 0.01). The effect of EHF on GC cell invasion ability was further investigated. As shown in Figure 5C and D, the number of cells that invaded through the filters significantly decreased in the EHF silencing group compared with the negative control group in these GC cell lines (P < 0.01). These results suggested that EHF promotes the migration and invasion of BGC-823, KATO III and SGC-7901 cells, contributing to the high metastatic potential of GC.

EHF may regulate GC cell malignant behaviors through the c-Met pathwayPrevious findings have demonstrated that c-Met has a pivotal role in the development and progression of GC[17]. Aberrantly activated c-Met triggers tumor growth, survival, metastasis, and EMT via mitogen-activated protein kinases (MAPK), PTEN, and STAT3 signaling pathways[18-20]. Consequently, the expression of c-Met protein and its downstream molecules in the GC cell lines following EHF downregulation were detected. As shown in Figure 6A, the results suggested that EHF silencing led to the considerable inhibition of c-Met and Ras expression in BGC-823, KATO III and SGC-7901 cells. Moreover, the inactivation of c-Raf and Erk1/2 were both observed after EHF downregulation. In addition to the MAPK pathway, expression of the tumor suppressor molecule PTEN in these GC cells was significantly upregulated (Figure 6B). The decreased phosphorylation of GSK-3β, the downstream target of PTEN, was observed in BGC-823 and KATO III cells. Moreover, the expression of antiapoptotic survivin was remarkably inhibited in these GC cells, whereas the expression of proapoptotic Bax was significantly upregulated in BGC-823 and KATO III cells. Furthermore, the data indicated that STAT3, a signal transducer of the c-Met pathway, was inactivated in BGC-823, KATO III, and SGC-7901 cells following the downregulation of EHF (Figure 6C). The protein expression of the downstream transcriptional factors of STAT3 was also examined. Interestingly, the expression of Twist decreased in BGC-823 and SGC-7901 cells, while the inhibitory effect of Snail expression was considerable in KATO III and SGC-7901 cells. E-cadherin is a target of Twist and Snail, while it is also known as a biomarker for the EMT. Herein, we found that E-cadherin was significantly increased in the EHF silencing group, indicating an inhibitory effect on EMT in GC cells.



Figure 2 Transfection of ETS homologous factor-targeted small interfering RNA reduces c-Met expression in gastric cancer cell lines. A: Quantitative PCR was performed to evaluate the mRNA levels of ETS homologous factor (EHF) in gastric cancer (GC) cells following transfection for 72 h; B: Western blotting was used to detect the protein expression of EHF and c-Met after small interfering RNA transfection. bP < 0.01 vs negative control (NC) group; eP < 0.001 vs NC group.

DISCUSSIONThe ETS family is a transcription factor family consisting of various members, which can be divided into 11 subfamilies according to their structural characteristics[21]. EHF belongs to the epithelium-specific ETS (ESE) subfamily, and consequently is also known as ESE-3. According to previous studies, EHF is mainly expressed in multiple types of epithelial cells; its dysregulation is often detected in multiple malignant tumors, and it is associated with poor clinical outcomes[9,22-25]. However, the biological roles and potential regulatory mechanisms of EHF in GC is not completely clear.

In this study, qPCR was performed to detect the expression of EHF mRNA in GC cell lines and tissues compared with that in the gastric mucosal epithelial cell line and the adjacent tissues. The data demonstrated that EHF is overexpressed in GC tissues and three GC cell lines (BGC-823, KATO III, and SGC-7901), which is consistent with the alterations in protein expression. As a potential downstream target of EHF, the expression of c-Met was also increased in GC tissues and cell lines with high EHF expression. c-Met is an RTK encoded by the proto-oncogene MET, and hepatocyte growth factor is its only known ligand, whose aberrant expression and functional changes are closely related to the occurrence and development of GC[26]. The expression of c-Met in GC tissues is positively associated with distant metastasis and poor patient prognosis[14]. Several in vitro experiments have indicated that activated c-Met triggers its downstream MAPK, Akt/mTOR and other signaling pathways, thereby inhibiting the apoptosis of tumor cells and promoting their proliferation and migration[27]. Additionally, the inhibition of c-Met leads to the apoptosis of GC cells and promotes their sensitivity to chemotherapeutic agents[28].

The results demonstrated that EHF silencing can significantly reduce cell proliferation, promote apoptosis and induce cell cycle arrest in GC cell lines (BGC-823, KATO III and SGC-7901). On the other hand, both the migration and invasion abilities of these GC cell lines were significantly inhibited following the interference of EHF. These data indicate that EHF positively regulates the malignant biological behaviors of





Figure 3 The effects of ETS homologous factor silencing on gastric cancer cell proliferation. A: Cell viability was evaluated by the Cell Counting Kit-8 after transfection for 1, 3, 5, and 7 d; B and C: Cell colony formation was measured to verify the changes in gastric cancer (GC) cell proliferation following ETS homologous factor downregulation. eP < 0.001 vs negative control (NC) group.

GC. Moreover, knockdown of EHF by specific siRNA led to the downregulation of c-Met in both GC tissues and cell lines. Currently, whether EHF participates in the tumorigenesis and progression of GC via the c-Met pathway remains unknown. Previous investigations have shown that the inhibition of c-Met in GC results in the suppression of cell proliferation both in vitro and in vivo[29]. Selective c-Met inhibitors block cell DNA synthesis and induce cell cycle arrest at the G1-S phase transition in GC cells[30]. The inactivation of c-Met can also reduce migration and invasion in vitro and ultimately block the malignant progression of GC[31]. EHF directly regulates c-Met expression via binding to its promoter region[7]. A recent study showed that EHF contributes to the metastasis and chemoresistance of oral squamous cell carcinoma through the positive regulation of the c-Met pathway[32]. Thus, EHF may be involved in the regulation of the biological behaviors of GC via promoting c-Met expression.

Recently, it has been verified that the abnormally upregulated expression of at least one RTK (human epidermal growth factor receptor 2, epidermal growth factor receptor, c-Met or fibroblast growth factor receptor 2) was found in approximately two-thirds of GC patients, indicating that the detection of RTK expression levels or their gene mutations can provide important evidence for the diagnosis and treatment of GC[33]. However, the identification of one single type of RTK expression or mutation is inadequate to accurately predict chemotherapy sensitivity or overall prognosis[34]. In recent years, the detection of multiple RTKs has improved the efficiency of predicting the prognosis to some extent, whereas the accuracy of RTK pathway analysis is still limited by the variable mutation sites and complex mutation types of these coding genes. It is possible to determine the abnormal expression or activation of RTKs by examining the expression level of the upstream regulators that affect RTK transcription regardless of their mutation status. Consequently, the combined detection of EHF and c-Met may serve as potential markers for the diagnosis and prognosis of GC.



Figure 4 Downregulation of ETS homologous factor induces apoptosis and cell cycle arrest in gastric cancer cells. A and B: The analysis of apoptosis was performed by flow cytometry to detect the induction of apoptosis caused by ETS homologous factor (EHF) silencing following V-FITC/propidium iodide (PI) staining; C and D: The cell cycle arrest induced by EHF downregulation was assessed by PI staining and further analyzed by flow cytometry. aP < 0.05 vs negative control (NC) group; bP < 0.01 vs NC group; eP < 0.001 vs NC group. GC: Gastric cancer.



Activation of c-Met and its key downstream MAPK cascades are responsible for maintaining the abnormal biological function of tumor cells[35]. Erk1/2 is a subtype of MAPK that is closely associated with cell proliferation, differentiation and migration[36]. The current study demonstrated that the inhibition of EHF leads to the impaired biological function of GC cell lines via the blockage of Ras-Erk1/2 cascades.

PTEN is a tumor suppressor, and its expression level is negatively correlated with the tumor-node-metastasis stage of GC. In vivo experiments have confirmed that the absence of PTEN promotes the tumorigenesis of GC[37]. It has been reported that PTEN interacts with the c-Met signaling pathway[38], and a high expression level of c-Met is associated with the downregulation of PTEN[39]. The inhibition of PTEN expression leads to the increased proliferation activity of GC cells via GSK-3β inactivation[40]. The present study demonstrated that EHF may affect the apoptosis and cell cycle progression of GC via the c-Met/PTEN/GSK-3β cascade. Interestingly, the upregulation of PTEN was detected in these GC cell lines, whereas considerable inactivation of GSK-3β was not observed in SGC-7901 cells. Previous findings have suggested that survivin is a direct target of GSK-3β, which inhibits survivin expression, leading to apoptosis in GC cells[41]. Herein, the expression of survivin increased in all the GC cells, indicating that survivin may not be completely controlled by GSK-3β in SGC-7901 cells.

In this study, STAT3 was deactivated following EHF silencing. In addition, as a biomarker for EMT, E-cadherin expression was upregulated in the GC cell lines. The activation of STAT3 promotes the expression of Twist and Snail that reduces E-cadherin expression, resulting in the EMT of malignant tumor cells[42-44]. EMT causes significant epithelial phenotypic changes and is associated with the progression, metastasis, and drug resistance of GC[45]. A previous study indicated that abnormally activated STAT3 plays an important role in c-Met-mediated EMT in GC[46]. Accordingly, the results suggested that EHF may regulate EMT in GC cell lines via the c-Met/STAT3 pathway. However, the downregulation of Twist was not observed in KATO III cells, and Snail expression was not significantly affected in BGC-823 cells, indicating that STAT3 may affect the EMT of BGC-823 and KATO III cells through Twist and Snail, respectively.

This study had some limitations. Further investigations with large sample sizes are needed to clarify the relationship between EHF and c-Met expression in GC. In the present study, the potential role of EHF in GC tumorigenesis was demonstrated and the possible mechanisms underlying the antineoplastic effects induced by EHF downregulation were further sought. Nevertheless, the advantage of EHF as an antineoplastic target for GC diagnosis and treatment has not yet been fully elucidated and remains to be validated both in vitro and in vivo.

CONCLUSIONIn summary, EHF may promote cell proliferation and cell cycle progression and inhibit the apoptosis of GC cells via regulating the c-Met pathway. Furthermore, EHF may contribute to the migration and invasion of GC cells by inducing EMT via the STAT3 pathway. Therefore, by targeting the c-Met pathway, EHF can be regarded as a promising candidate target for the development of a novel antineoplastic strategy for GC treatment.



Figure 5 ETS homologous factor regulates the cell migration and invasion abilities of gastric cancer cells. A and B: The transwell assay was performed to assess the cell migration ability of gastric cancer (GC) cells following ETS homologous factor (EHF) downregulation; C and D: Invasion assays were performed by adding Matrigel into the Transwell chamber to determine the effects of EHF silencing on the invasion ability of GC cells. eP < 0.001 vs negative control (NC) group.



Figure 6 ETS homologous factor promotes the malignant biological behaviors of gastric cancer cells through the c-Met pathway. Western blotting was conducted to investigate the effects of ETS homologous factor (EHF) silencing on the expression and activities of signaling molecules in the c-Met pathway. A: Alterations in the Ras- extracellular signal-related kinase 1/2 (Erk1/2) cascade after EHF inhibition; B: The molecular changes in phosphatase and tensin homolog (PTEN) and glycogen synthase kinase-3β (GSK3β) and their potential targets following EHF silencing; C: The altered activities of signal transducer and activator of transcription 3 (STAT3) and the changed expression of its downstream effectors after EHF downregulation. GC: Gastric cancer; NC: Negative control group.

ARTICLE HIGHLIGHTSResearch backgroundGastric cancer (GC) is one of the most common and deadliest types of cancer worldwide due to its delayed diagnosis and high metastatic frequency, and its exact pathogenesis has not been fully elucidated. ETS homologous factor (EHF) is an important member of the ETS family and contributes to the pathogenesis of multiple malignant tumors. To date, whether EHF participates in the development of GC via the c-Met signaling pathway remains unclear.

Research motivationCurrently, surgical resection represents the best curative option for GC. On the other hand, advanced novel targets for GC diagnosis and treatment have resulted in decreased incidence and mortality rates in recent years. However, the 5-year overall survival rate of patients with advanced GC is less than 30% due to delayed diagnosis and recurrence. Therefore, revealing the oncogenic mechanisms of GC and further exploring the strategies for GC diagnosis and treatment are urgent.

Research objectivesTo investigate the role and mechanism of EHF in the occurrence and development of GC.



Research methodsThe expression of EHF mRNA in GC tissues and cell lines was measured by qPCR. Western blotting was performed to determine the protein expression of EHF, c-Met and its downstream signal molecules. The EHF expression in GC tissues was further checked by immunohistochemical staining. To investigate the role of EHF in GC oncogenesis, small interfering RNAs (siRNAs) against EHF were transfected into GC cells. The cell proliferation of GC cells was determined by cell counting kit-8 and colony formation assays. Flow cytometry was performed following annexin V/PI and PI staining to determine apoptosis and the cell cycle, respectively. Cell migration and invasion were assessed by transwell assays.

Research resultsThe data indicated that EHF was upregulated in GC tissues and cell lines in which increased expression of c-Met was also observed. Silencing of EHF by siRNA reduced cell proliferation in GC cells. Inhibition of EHF induced significant apoptosis and cell cycle arrest in GC cells. Cell migration and invasion were significantly inhibited. EHF silencing led to the c-Met downregulation and further blocked Ras/c-Raf/Erk1/2. Additionally, PTEN was upregulated and GSK-3β was deactivated. Moreover, inactivation of STAT3 was detected following EHF inhibition, leading to the blockage of the epithelial-to-mesenchymal transition (EMT).

Research conclusionsThese results suggested that EHF plays a key role in cell proliferation, invasion, apoptosis, the cell cycle and EMT via the c-Met pathway.

Research perspectivesEHF may serve as an antineoplastic target for the diagnosis and treatment of GC.

ACKNOWLEDGEMENTSWe are very grateful to The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases of The First Affiliated Hospital of Zhejiang University for providing excellent technical assistance.

REFERENCESBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394-424 [PMID: 30207593 DOI: 10.3322/caac.21492]

1

Doorakkers E, Lagergren J, Engstrand L, Brusselaers N. Helicobacter pylori eradication treatment and the risk of gastric adenocarcinoma in a Western population. Gut 2018; 67: 2092-2096 [PMID: 29382776 DOI: 10.1136/gutjnl-2017-315363]

2

Fang X, Wei J, He X, An P, Wang H, Jiang L, Shao D, Liang H, Li Y, Wang F, Min J. Landscape of dietary factors associated with risk of gastric cancer: A systematic review and dose-response meta-analysis of prospective cohort studies. Eur J Cancer 2015; 51: 2820-2832 [PMID: 26589974 DOI: 10.1016/j.ejca.2015.09.010]

3

Lordick F, Allum W, Carneiro F, Mitry E, Tabernero J, Tan P, Van Cutsem E, van de Velde C, Cervantes A. Unmet needs and challenges in gastric cancer: the way forward. Cancer Treat Rev 2014; 40: 692-700 [PMID: 24656602 DOI: 10.1016/j.ctrv.2014.03.002]

4

Allemani C, Weir HK, Carreira H, Harewood R, Spika D, Wang XS, Bannon F, Ahn JV, Johnson CJ, Bonaventure A, Marcos-Gragera R, Stiller C, Azevedo e Silva G, Chen WQ, Ogunbiyi OJ, Rachet B, Soeberg MJ, You H, Matsuda T, Bielska-Lasota M, Storm H, Tucker TC, Coleman MP; CONCORD Working Group. Global surveillance of cancer survival 1995-2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet 2015; 385: 977-1010 [PMID: 25467588 DOI: 10.1016/S0140-6736(14)62038-9]

5

Feng FY, Brenner JC, Hussain M, Chinnaiyan AM. Molecular pathways: targeting ETS gene fusions in cancer. Clin Cancer Res 2014; 20: 4442-4448 [PMID: 24958807 DOI: 10.1158/1078-0432.CCR-13-0275]

6

Kas K, Finger E, Grall F, Gu X, Akbarali Y, Boltax J, Weiss A, Oettgen P, Kapeller R, Libermann TA. ESE-3, a novel member of an epithelium-specific ets transcription factor subfamily, demonstrates different target gene specificity from ESE-1. J Biol Chem 2000; 275: 2986-2998 [PMID: 10644770 DOI: 10.1074/jbc.275.4.2986]

7

Tugores A, Le J, Sorokina I, Snijders AJ, Duyao M, Reddy PS, Carlee L, Ronshaugen M, Mushegian 8


https://dx.doi.org/10.3322/caac.21492




https://dx.doi.org/10.1016/j.ejca.2015.09.010


https://dx.doi.org/10.1016/j.ctrv.2014.03.002


https://dx.doi.org/10.1016/S0140-6736(14)62038-9


https://dx.doi.org/10.1158/1078-0432.CCR-13-0275


https://dx.doi.org/10.1074/jbc.275.4.2986



A, Watanaskul T, Chu S, Buckler A, Emtage S, McCormick MK. The epithelium-specific ETS protein EHF/ESE-3 is a context-dependent transcriptional repressor downstream of MAPK signaling cascades. J Biol Chem 2001; 276: 20397-20406 [PMID: 11259407 DOI: 10.1074/jbc.M010930200]Albino D, Longoni N, Curti L, Mello-Grand M, Pinton S, Civenni G, Thalmann G, D'Ambrosio G, Sarti M, Sessa F, Chiorino G, Catapano CV, Carbone GM. ESE3/EHF controls epithelial cell differentiation and its loss leads to prostate tumors with mesenchymal and stem-like features. Cancer Res 2012; 72: 2889-2900 [PMID: 22505649 DOI: 10.1158/0008-5472.CAN-12-0212]

9

Cheng Z, Guo J, Chen L, Luo N, Yang W, Qu X. Knockdown of EHF inhibited the proliferation, invasion and tumorigenesis of ovarian cancer cells. Mol Carcinog 2016; 55: 1048-1059 [PMID: 26258986 DOI: 10.1002/mc.22349]

10

Hufton SE, Moerkerk PT, Brandwijk R, de Bruïne AP, Arends JW, Hoogenboom HR. A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization. FEBS Lett 1999; 463: 77-82 [PMID: 10601642 DOI: 10.1016/s0014-5793(99)01578-1]

11

Taniue K, Oda T, Hayashi T, Okuno M, Akiyama T. A member of the ETS family, EHF, and the ATPase RUVBL1 inhibit p53-mediated apoptosis. EMBO Rep 2011; 12: 682-689 [PMID: 21617703 DOI: 10.1038/embor.2011.81]

12

Kawakami H, Okamoto I. MET-targeted therapy for gastric cancer: the importance of a biomarker-based strategy. Gastric Cancer 2016; 19: 687-695 [PMID: 26690587 DOI: 10.1007/s10120-015-0585-x]

13

Fuse N, Kuboki Y, Kuwata T, Nishina T, Kadowaki S, Shinozaki E, Machida N, Yuki S, Ooki A, Kajiura S, Kimura T, Yamanaka T, Shitara K, Nagatsuma AK, Yoshino T, Ochiai A, Ohtsu A. Prognostic impact of HER2, EGFR, and c-MET status on overall survival of advanced gastric cancer patients. Gastric Cancer 2016; 19: 183-191 [PMID: 25682441 DOI: 10.1007/s10120-015-0471-6]

14

Wang YM, Gu ML, Meng FS, Jiao WR, Zhou XX, Yao HP, Ji F. Histone acetyltransferase p300/CBP inhibitor C646 blocks the survival and invasion pathways of gastric cancer cell lines. Int J Oncol 2017; 51: 1860-1868 [PMID: 29075795 DOI: 10.3892/ijo.2017.4176]

15

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408 [PMID: 11846609 DOI: 10.1006/meth.2001.1262]

16

Matsumoto K, Umitsu M, De Silva DM, Roy A, Bottaro DP. Hepatocyte growth factor/MET in cancer progression and biomarker discovery. Cancer Sci 2017; 108: 296-307 [PMID: 28064454 DOI: 10.1111/cas.13156]

17

Choi KM, Cho E, Kim E, Shin JH, Kang M, Kim B, Han EH, Chung YH, Kim JY. Prolonged MEK inhibition leads to acquired resistance and increased invasiveness in KRAS mutant gastric cancer. Biochem Biophys Res Commun 2018; 507: 311-318 [PMID: 30466782 DOI: 10.1016/j.bbrc.2018.11.030]

18

Wadhwa R, Song S, Lee JS, Yao Y, Wei Q, Ajani JA. Gastric cancer-molecular and clinical dimensions. Nat Rev Clin Oncol 2013; 10: 643-655 [PMID: 24061039 DOI: 10.1038/nrclinonc.2013.170]

19

Lai AZ, Cory S, Zhao H, Gigoux M, Monast A, Guiot MC, Huang S, Tofigh A, Thompson C, Naujokas M, Marcus VA, Bertos N, Sehat B, Perera RM, Bell ES, Page BD, Gunning PT, Ferri LE, Hallett M, Park M. Dynamic reprogramming of signaling upon met inhibition reveals a mechanism of drug resistance in gastric cancer. Sci Signal 2014; 7: ra38 [PMID: 24757178 DOI: 10.1126/scisignal.2004839]

20

Kar A, Gutierrez-Hartmann A. Molecular mechanisms of ETS transcription factor-mediated tumorigenesis. Crit Rev Biochem Mol Biol 2013; 48: 522-543 [PMID: 24066765 DOI: 10.3109/10409238.2013.838202]

21

Gutierrez-Hartmann A, Duval DL, Bradford AP. ETS transcription factors in endocrine systems. Trends Endocrinol Metab 2007; 18: 150-158 [PMID: 17387021 DOI: 10.1016/j.tem.2007.03.002]

22

Brenne K, Nymoen DA, Hetland TE, Trope' CG, Davidson B. Expression of the Ets transcription factor EHF in serous ovarian carcinoma effusions is a marker of poor survival. Hum Pathol 2012; 43: 496-505 [PMID: 21855111 DOI: 10.1016/j.humpath.2011.05.023]

23

Zhao T, Jiang W, Wang X, Wang H, Zheng C, Li Y, Sun Y, Huang C, Han ZB, Yang S, Jia Z, Xie K, Ren H, Hao J. ESE3 Inhibits Pancreatic Cancer Metastasis by Upregulating E-Cadherin. Cancer Res 2017; 77: 874-885 [PMID: 27923832 DOI: 10.1158/0008-5472.CAN-16-2170]

24

He J, Pan Y, Hu J, Albarracin C, Wu Y, Dai JL. Profile of Ets gene expression in human breast carcinoma. Cancer Biol Ther 2007; 6: 76-82 [PMID: 17172821 DOI: 10.4161/cbt.6.1.3551]

25

Ahn SY, Kim J, Kim MA, Choi J, Kim WH. Increased HGF Expression Induces Resistance to c-MET Tyrosine Kinase Inhibitors in Gastric Cancer. Anticancer Res 2017; 37: 1127-1138 [PMID: 28314274 DOI: 10.21873/anticanres.11426]

26

Wu Y, Yao X, Zhu M, Qian H, Jiang L, Lan T, Wu M, Pang J, Chen Y. PKG II reverses HGF-triggered cellular activities by phosphorylating serine 985 of c-Met in gastric cancer cells. Oncotarget 2016; 7: 34190-34200 [PMID: 27147579 DOI: 10.18632/oncotarget.9074]

27

Zhang Z, Kong Y, Yang W, Ma F, Zhang Y, Ji S, Ma EM, Liu H, Chen Y, Hua Y. Upregulation of microRNA-34a enhances the DDP sensitivity of gastric cancer cells by modulating proliferation and apoptosis via targeting MET. Oncol Rep 2016; 36: 2391-2397 [PMID: 27513895 DOI: 10.3892/or.2016.5016]

28

Ohashi Y, Okamura M, Hirosawa A, Tamaki N, Akatsuka A, Wu KM, Choi HW, Yoshimatsu K, Shiina I, Yamori T, Dan S. M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET

29


https://dx.doi.org/10.1074/jbc.M010930200


https://dx.doi.org/10.1158/0008-5472.CAN-12-0212


https://dx.doi.org/10.1002/mc.22349


https://dx.doi.org/10.1016/s0014-5793(99)01578-1


https://dx.doi.org/10.1038/embor.2011.81




https://dx.doi.org/10.1007/s10120-015-0471-6


https://dx.doi.org/10.3892/ijo.2017.4176


https://dx.doi.org/10.1006/meth.2001.1262


https://dx.doi.org/10.1111/cas.13156


https://dx.doi.org/10.1016/j.bbrc.2018.11.030


https://dx.doi.org/10.1038/nrclinonc.2013.170


https://dx.doi.org/10.1126/scisignal.2004839


https://dx.doi.org/10.3109/10409238.2013.838202


https://dx.doi.org/10.1016/j.tem.2007.03.002


https://dx.doi.org/10.1016/j.humpath.2011.05.023




https://dx.doi.org/10.4161/cbt.6.1.3551


https://dx.doi.org/10.21873/anticanres.11426


https://dx.doi.org/10.18632/oncotarget.9074


https://dx.doi.org/10.3892/or.2016.5016



Protein and Exhibits Antitumor Activity against MET-Addicted Gastric Cancers. Cancer Res 2016; 76: 3895-3903 [PMID: 27197184 DOI: 10.1158/0008-5472.CAN-15-2220]Park CH, Cho SY, Ha JD, Jung H, Kim HR, Lee CO, Jang IY, Chae CH, Lee HK, Choi SU. Novel c-Met inhibitor suppresses the growth of c-Met-addicted gastric cancer cells. BMC Cancer 2016; 16: 35 [PMID: 26801760 DOI: 10.1186/s12885-016-2058-y]

30

Toiyama Y, Yasuda H, Saigusa S, Matushita K, Fujikawa H, Tanaka K, Mohri Y, Inoue Y, Goel A, Kusunoki M. Co-expression of hepatocyte growth factor and c-Met predicts peritoneal dissemination established by autocrine hepatocyte growth factor/c-Met signaling in gastric cancer. Int J Cancer 2012; 130: 2912-2921 [PMID: 21796631 DOI: 10.1002/ijc.26330]

31

Huang WC, Jang TH, Tung SL, Yen TC, Chan SH, Wang LH. A novel miR-365-3p/EHF/keratin 16 axis promotes oral squamous cell carcinoma metastasis, cancer stemness and drug resistance via enhancing β5-integrin/c-met signaling pathway. J Exp Clin Cancer Res 2019; 38: 89 [PMID: 30782177 DOI: 10.1186/s13046-019-1091-5]

32

Nagatsuma AK, Aizawa M, Kuwata T, Doi T, Ohtsu A, Fujii H, Ochiai A. Expression profiles of HER2, EGFR, MET and FGFR2 in a large cohort of patients with gastric adenocarcinoma. Gastric Cancer 2015; 18: 227-238 [PMID: 24626858 DOI: 10.1007/s10120-014-0360-4]

33

Satoh T, Xu RH, Chung HC, Sun GP, Doi T, Xu JM, Tsuji A, Omuro Y, Li J, Wang JW, Miwa H, Qin SK, Chung IJ, Yeh KH, Feng JF, Mukaiyama A, Kobayashi M, Ohtsu A, Bang YJ. Lapatinib plus paclitaxel versus paclitaxel alone in the second-line treatment of HER2-amplified advanced gastric cancer in Asian populations: TyTAN--a randomized, phase III study. J Clin Oncol 2014; 32: 2039-2049 [PMID: 24868024 DOI: 10.1200/JCO.2013.53.6136]

34

Asati V, Mahapatra DK, Bharti SK. PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents: Structural and pharmacological perspectives. Eur J Med Chem 2016; 109: 314-341 [PMID: 26807863 DOI: 10.1016/j.ejmech.2016.01.012]

35

Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene 2007; 26: 3279-3290 [PMID: 17496922 DOI: 10.1038/sj.onc.1210421]

36

Kim SJ, Lee HW, Baek JH, Cho YH, Kang HG, Jeong JS, Song J, Park HS, Chun KH. Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer. Oncogene 2016; 35: 251-260 [PMID: 25823029 DOI: 10.1038/onc.2015.80]

37

Abounader R, Reznik T, Colantuoni C, Martinez-Murillo F, Rosen EM, Laterra J. Regulation of c-Met-dependent gene expression by PTEN. Oncogene 2004; 23: 9173-9182 [PMID: 15516982 DOI: 10.1038/sj.onc.1208146]

38

Ach T, Zeitler K, Schwarz-Furlan S, Baader K, Agaimy A, Rohrmeier C, Zenk J, Gosau M, Reichert TE, Brockhoff G, Ettl T. Aberrations of MET are associated with copy number gain of EGFR and loss of PTEN and predict poor outcome in patients with salivary gland cancer. Virchows Arch 2013; 462: 65-72 [PMID: 23242174 DOI: 10.1007/s00428-012-1358-0]

39

Tseng PC, Huang WC, Chen CL, Sheu BS, Shan YS, Tsai CC, Wang CY, Chen SO, Hsieh CY, Lin CF. Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer. Immunobiology 2012; 217: 926-934 [PMID: 22325465 DOI: 10.1016/j.imbio.2012.01.001]

40

Li HL, Liang S, Cui JH, Han GY. Targeting of GSK-3β by miR-214 to facilitate gastric cancer cell proliferation and decrease of cell apoptosis. Eur Rev Med Pharmacol Sci 2018; 22: 127-134 [PMID: 29364479 DOI: 10.26355/eurrev_201801_14109]

41

Wang BJ, Zhang ZQ, Ke Y. Conversion of cadherin isoforms in cultured human gastric carcinoma cells. World J Gastroenterol 2006; 12: 966-970 [PMID: 16521229 DOI: 10.3748/wjg.v12.i6.966]

42

Hsu KW, Hsieh RH, Huang KH, Fen-Yau Li A, Chi CW, Wang TY, Tseng MJ, Wu KJ, Yeh TS. Activation of the Notch1/STAT3/Twist signaling axis promotes gastric cancer progression. Carcinogenesis 2012; 33: 1459-1467 [PMID: 22581828 DOI: 10.1093/carcin/bgs165]

43

Berx G, van Roy F. Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb Perspect Biol 2009; 1: a003129 [PMID: 20457567 DOI: 10.1101/cshperspect.a003129]

44

Peng Z, Wang CX, Fang EH, Wang GB, Tong Q. Role of epithelial-mesenchymal transition in gastric cancer initiation and progression. World J Gastroenterol 2014; 20: 5403-5410 [PMID: 24833870 DOI: 10.3748/wjg.v20.i18.5403]

45

Okamoto W, Okamoto I, Arao T, Yanagihara K, Nishio K, Nakagawa K. Differential roles of STAT3 depending on the mechanism of STAT3 activation in gastric cancer cells. Br J Cancer 2011; 105: 407-412 [PMID: 21730976 DOI: 10.1038/bjc.2011.246]

46




https://dx.doi.org/10.1186/s12885-016-2058-y


https://dx.doi.org/10.1002/ijc.26330


https://dx.doi.org/10.1186/s13046-019-1091-5


https://dx.doi.org/10.1007/s10120-014-0360-4


https://dx.doi.org/10.1200/JCO.2013.53.6136


https://dx.doi.org/10.1016/j.ejmech.2016.01.012


https://dx.doi.org/10.1038/sj.onc.1210421


https://dx.doi.org/10.1038/onc.2015.80


https://dx.doi.org/10.1038/sj.onc.1208146


https://dx.doi.org/10.1007/s00428-012-1358-0


https://dx.doi.org/10.1016/j.imbio.2012.01.001


https://dx.doi.org/10.26355/eurrev_201801_14109




https://dx.doi.org/10.1093/carcin/bgs165


https://dx.doi.org/10.1101/cshperspect.a003129




https://dx.doi.org/10.1038/bjc.2011.246


World Journal of



ORIGINAL ARTICLE

Basic Study

Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway

Zhi Wang, Zhen-Xing Cheng, Simon T Abrams, Zi-Qi Lin, ED Yates, Qian Yu, Wei-Ping Yu, Ping-Sheng Chen, Cheng-Hock Toh, Guo-Zheng Wang

ORCID number: Zhi Wang 0000-0002-1270-8087; Zhen-Xing Cheng 0000-0003-0847-2582; Simon T Abrams 0000-0003-3949-2455; Zi-Qi Lin 0000-0002-0445-9607; ED Yates 0000-0001-8004-4801; Qian Yu 0000-0003-0808-6550; Wei-Ping Yu 0000-0003-3968-3104; Ping-Sheng Chen 0000-0002-3876-1994; Cheng-Hock Toh 0000-0002-9708-8883; Guo-Zheng Wang 0000-0001-5525-3548.

Author contributions: Wang Z conceived the study; Cheng ZX assisted animal experiments and hydroxyproline measurements; Lin ZQ and Abrams ST assisted in performing in vitro experiments; Yates E synthesized and characterised non-anticoagulant heparin; Abrams ST helped edit figures; Yu Q, Yu WP, Chen PS, Toh CH and Wang GZ supervised the work and were involved in data analysis and manuscript writing; and all authors have read and agreed to the published version of the manuscript.

Supported by Key R & D Program of Jiangsu Province, No. BE2019712; British Heart Foundation, No. PG/14/19/30751 and No. PG/16/65/32313.

Institutional review board

Zhi Wang, Zhen-Xing Cheng, Wei-Ping Yu, Ping-Sheng Chen, Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing 210009, Jiangsu Province, China

Zhi Wang, Qian Yu, Department of Gastroenterology, Zhongda Hospital, Nanjing 210009, Jiangsu Province, China

Zhen-Xing Cheng, Simon T Abrams, Cheng-Hock Toh, Guo-Zheng Wang, Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool L69 7BE, United Kingdom

Zhen-Xing Cheng, Department of Gastroenterology, The First Affiliated Hospital, Anhui Medical University, Hefei 230032, Anhui Province, China

Zi-Qi Lin, Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China

ED Yates, Department of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom

Cheng-Hock Toh, Roald Dahl Haemostasis & Thrombosis Ctr, Royal Liverpool University Hospital, Liverpool L69 7BE, United Kingdom

Corresponding author: Ping-Sheng Chen, MBChB, MD, PhD, Professor, Research Dean, Department of Pathology and Pathophysiology, Medical School, Southeast University, No. 87 Dingjiaqiao, Nanjing 210009, Jiangsu Province, China. [email protected]

AbstractBACKGROUND Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis



http://orcid.org/0000-0002-1270-8087

http://orcid.org/0000-0002-1270-8087

http://orcid.org/0000-0002-1270-8087

http://orcid.org/0000-0003-0847-2582

http://orcid.org/0000-0003-0847-2582

http://orcid.org/0000-0003-3949-2455

http://orcid.org/0000-0003-3949-2455

http://orcid.org/0000-0002-0445-9607

http://orcid.org/0000-0002-0445-9607

http://orcid.org/0000-0001-8004-4801

http://orcid.org/0000-0001-8004-4801

http://orcid.org/0000-0003-0808-6550

http://orcid.org/0000-0003-0808-6550

http://orcid.org/0000-0003-0808-6550

http://orcid.org/0000-0003-3968-3104

http://orcid.org/0000-0003-3968-3104

http://orcid.org/0000-0003-3968-3104

http://orcid.org/0000-0002-3876-1994

http://orcid.org/0000-0002-3876-1994

http://orcid.org/0000-0002-9708-8883

http://orcid.org/0000-0002-9708-8883

http://orcid.org/0000-0001-5525-3548

http://orcid.org/0000-0001-5525-3548


Wang Z et al. Extracellular histones promote liver fibrosis


statement: The study was reviewed and approved by Medical School of Southeast University.

Institutional animal care and use committee statement: All procedures were performed according to State laws and monitored by local inspectors, and approved by the Animal Research Ethics Committee at the Medical School of the Southeast University.

Conflict-of-interest statement: No conflict interested has been claimed by any author.


ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.




Country/Territory of origin: United Kingdom

Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C, C, C Grade D (Fair): D Grade E (Poor): 0

have not been investigated.

AIM To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis.

METHODS In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated.

RESULTS Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice.

CONCLUSION Extracellular histones potentially enhance fibrogenesis via the TLR4–MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

Key Words: Liver fibrosis; Extracellular histones; Non-anticoagulant heparin; TLR4; MyD88; CCl4


Core Tip: This study fills the gap between recurrent liver injury and liver fibrosis. When liver cells die, histones are released. High levels of extracellular histones not only cause secondary liver injury, but also activate the TLR4-MyD88 signaling pathway to enhance collagen I production and liver fibrosis. Binding of non-anticoagulant heparin (NAHP) to extracellular histones reduces histone toxicity, alleviates liver injury, and prevents histones from activating the TLR4-MyD88 signaling pathway. These results may explain why NAHP reduces liver fibrosis in this animal model, although further investigations are required.

Citation: Wang Z, Cheng ZX, Abrams ST, Lin ZQ, Yates E, Yu Q, Yu WP, Chen PS, Toh CH, Wang GZ. Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway. World J Gastroenterol 2020; 26(47): 7513-7527URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7513.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7513

INTRODUCTIONLiver fibrosis progression to liver cirrhosis and hepatocellular carcinoma is common, in China due to high incidence of hepatitis B and in western countries due to alcohol consumption and hepatitis C[1-3]. In 2017, liver cirrhosis caused more than 1.3 million








Received: August 27, 2020 Peer-review started: August 27, 2020 First decision: October 17, 2020 Revised: November 8, 2020 Accepted: December 6, 2020 Article in press: December 6, 2020 Published online: December 21, 2020

P-Reviewer: Cuevas-Covarrubias SA, Cui J, Ilangumaran S, Tanabe S, Zhu Y S-Editor: Huang P L-Editor: A P-Editor: Ma YJ

deaths globally, and constituted 2.4% of total deaths[4]. However, elucidating the underlying molecular mechanism and the development of specific therapies for fibrosis and cirrhosis have progressed slowly and significant discoveries in this area are urgently needed.

Liver fibrosis is a natural wound healing response to chronic liver injury, which has many common causes, including viral infection, alcohol abuse, autoimmune diseases, drug toxicity, schistosomiasis, and metabolic diseases[5]. This process can be easily reconstituted in animals by administering chemicals toxic to hepatic cells, including CCl4, thioacetamide, and dimethyl or diethyl nitrosamine[6-8]. Bile duct ligation (BDL) and animal models mimicking specific chronic liver diseases are also used[6]. CCl4 is most commonly used to study the underlying molecular mechanisms and evaluate therapeutic reagents for inhibiting liver fibrosis in animals[6]. CCl4 directly damages hepatocytes by ligation of CCl3 to plasma, lysosomal and mitochondrial membranes, as well as to highly reactive free radical metabolites[9]. A single dose of CCl4 causes centrizonal necrosis and steatosis, with recurrent administration leading to liver fibrosis[10].

The pathological feature of liver fibrosis is the extensive deposition of extracellular collagen produced by myofibroblasts[1]. In the healthy liver, no myofibroblasts are present but they accumulate in the damaged liver and play a pivotal role in fibrogenesis[11]. Myofibroblasts are derived from different types of cells. Hepatic stellate cells (HSCs) in the perisinusoidal Disse space are recognized as the most important source[12]. Bone marrow also contributes to both macrophages and to HSCs within the injured liver[13]. Recurrent injury, chronic inflammation and other chronic stimuli can cause HSCs to evolve into a myofibroblast-like phenotype and produce an extracellular matrix[14]. The myofibroblast-like phenotype also develops and expresses α-smooth muscle actin (α-SMA) in vitro when quiescent HSCs were isolated and cultured over a period of 5-10 d[15].

HSCs activated both in vivo and in vitro express TLR4, CD14 and MD2, therefore, lipopolysaccharide (LPS) is able to assemble a complex to activate TLR4 on HSCs[16]. TLR4 signals via MyD88-dependent and TRIF-dependent pathways that can activate fibroblasts, to promote fibrogenesis in numerous organs, including the liver, lungs, kidneys, heart and skin[17-20]. Since circulating LPS is produced primarily in Gram-negative bacterial infections[21], ligands that activate TLR4 in chronic liver injury without concomitant bacterial infection are yet to be identified. Increased intestinal permeability allows LPS to enter the circulation in the later stages of liver cirrhosis, but this leakage has not been demonstrated in the early stages of fibrogenesis[22].

Liver cell injury and death also release damage-associated molecular patterns (DAMPs) that may initiate the wound-repair process[23]. Extracellular histones, the most abundant DAMPs, have gained much attention in recent years, as they play key roles in many pathological processes and human diseases[24-35]. Histones are well-conserved proteins that are essential for DNA packaging and gene regulation[36]. During tissue damage and cell death, nuclear chromatin is cleaved into nucleosomes that are released extracellularly[37] and further degraded into individual histones[38]. Although histones are rapidly cleared by the liver[39] and rarely detected in blood[25,26,31,32], liver cell death is likely to release histones locally[40-43], which stimulate adjacent cells, including HSCs. Histones can activate TLR2, TLR4 and TLR9[24,40,43-46] in the early stage of chronic liver injury and fibrogenesis. Therefore, TLR4 activation is more likely to be maintained by extracellular histones rather than by LPS.

In this study, we investigated the roles and mechanisms of extracellular histones in liver fibrogenesis. Circulating histone levels were measured in a CCl4-induced mouse liver fibrosis model. Extracellular histones were used to stimulate human HSC cell lines to demonstrate their direct effect on collagen I production. In addition, we used non-anticoagulant heparin (NAHP) to neutralize circulating histones in both the cell culture system and mouse liver fibrosis model to explore the role of anti-histone therapy in reducing collagen I production and fibrosis. To clarify the potential molecular mechanisms, we used a TLR4-neutralizing antibody to block histone-enhanced collagen production by an HSC cell line and compared the fibrogenesis in TLR4 and MyD88 knockout mice with their wild-type (WT) parental mice in response to CCl4 treatment.

MATERIALS AND METHODSCell cultureLX2 cells, a human HSC cell line, were purchased from Shanghai Meixuan Biological



Sciences and Technology Ltd. and routinely cultured.

Histone treatmentAfter optimization of the doses and times, 0-20 μg/mL calf thymus histones (Merck, Armstadt, Germany) were added to cell culture medium to treat the LX2 cells. Medium with different concentrations of histones was changed every 48 h. On day 6, cell supernatant and lysates were collected for western blotting.

TLR4-neutralizing antibody and NAHP treatmentThe neutralizing antibody, PAb-hTLR4, was purchased from Invitrogen (Carlsbad, CA, United States). LX2 cells were treated with 5 μg/mL histones with and without 5 μg/mL antibody. NAHP was synthesized and characterized by Dr. Yates at the University of Liverpool. NAHP (25 and 50 μg/mL) was used to treat LX2 cells together with 5 μg/mL histones. The cell culture medium was changed every 48 h. The cell lysates of the treated LX2 cells were collected on day 6.

Western blottingOne milliliter of cell supernatant was collected and proteins were precipitated using ice-cold acetone and suspended in 50 μL clear lysis buffer [1% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 120 mmol/L Tris–HCl, 25 mmol/L EDTA, pH 6.8]. Cells were washed with phosphate-buffered saline (PBS) three times and lysed using clear lysis buffer. Protein concentrations were determined using Bio-Rad protein assay kit II (Bio-Rad, Watford, United Kingdom). Both supernatant and cell lysates (20 μg protein) were subjected to SDS-PAGE and electrically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Watford, United Kingdom). After blocking with 5% (w/v) dried milk in TBST buffer [50 mmol/L Tris-HCl, pH 7.6; 150 mmol/L NaCl, 0.1% (v/v) Tween-20], the membranes were incubated with the sheep anti-human collagen I α1 antibody (1:2000, R&D Systems, Abingdon, United Kingdom) overnight at 4 °C. After extensive washings with TBST, the membranes were incubated with rabbit anti-sheep IgG-horseradish peroxidase (HRP, 1:10000, Santa Cruz Biotechnology, Dallas, TX, United States) for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (ECL, Millipore). For β-actin, primary antibody was purchased from Abcam (Abcam, Cambridge, United Kingdom) and anti-rabbit IgG-HRP was purchased from Santa Cruz Biotechnology. The band density was analyzed using Image J software and then the ratios of collagen I/β-actin were calculated.

Immunofluorescent stainingLX2 cells were seeded in eight-well chamber slides and treated with or without 5 μg/mL histones for 6 d. The cells were fixed with 4% (w/v) paraformaldehyde for 10 min and permeabilized with PBS + 1% (v/v) Triton X-100 for 10 min. After blocking with 5% (w/v) dried milk + 2% (w/v) bovine serum albumin in PBS, the cells were incubated with sheep anti-human collagen I α1 antibody (1:200) or rabbit-anti-α-SMA antibody (1:200, Proteintech, Rosemont, IL, United States) at 4 °C overnight. After extensive washing, the cells were incubated with anti-sheep-fluorescein isothiocyanate (FITC, Abcam, 1:2000) or anti-rabbit-FITC (Abcam, 1:2000) at room temperature for 45 min. Following extensive washing, the slides were mounted on coverslips using mounting medium and antifade (Thermo Fisher Scientific, Renfrew, United Kingdom). The fluorescent images were visualized using an LSM-10 confocal microscope (excitation: 488 nm and emission: 512 nm).

AnimalsMice were housed and used in sterile conditions at the Research Centre of Genetically Modified Mice, Southeast University, Nanjing, China. The mice were kept in a temperature and humidity controlled laminar flow room with an artificial 10-14 h light/dark cycle. All mice had free access to water. Sample sizes were calculated using power calculation based on our previous data and experience. All procedures were performed according to State laws and monitored by local inspectors, and approved by the Animal Research Ethics Committee at the Medical School of the Southeast University. Wang Z and Cheng ZX hold a full animal license for use of mice (Jiangsu Province, No. 2151981 and No. 2131272).

CCl4 model in ICR miceHealthy male, 6-week-old ICR mice were purchased from Yangzhou University Animal Experiment Centre and divided randomly into three groups after blood



sampling: (1) 5 μL/g body weight of 25% (w/v) CCl4 in olive oil injected intraperitoneally (i.p.), twice per week; (2) CCl4 + NAHP: On the day of CCl4 injection, 8 μL/g body weight NAHP (4 mg/mL in saline, filtered for sterility) were injected subcutaneously (s.c.) every 8 h. On the remaining days, 8 μL/g body weight NAHP was injected s.c. every 12 h. Both groups of mice were monitored every day and euthanized by neck dislocation at 4, 8 and 24 h (3 mice per group at each time point) after the first dose of CCl4 each week to collect the blood and organs for further analysis (Supplementary Figure 1); and (3) Saline was injected i.p. and NAHP injected s.c. as a control; nine mice in total were euthanized by the end of 4 wk. No mice died before euthanization.

CCl4 model using genetically modified miceC57BL/6JGpt (WT), TLR-4-KO (B6/JGpt-Tlr4em1Cd/Gpt) and MyD88-KO(B6/JGpt-Myd88em1Cd/Gpt) mice were purchased from Gempharmatech (Nanjing, China). Each type of mouse was divided into three groups as above after blood sampling. Eight mice in each group were treated with 5 μL/g body weight of 25% (w/v) CCl4 in olive oil i.p., twice per week for 4 wk. All mice were euthanized by neck dislocation and blood and organs were collected for further analysis (Supplementary Figure 2). No mice died before euthanization.

Tissue processing and stainingIn our pathology laboratory, organ tissues were routinely processed and 4-μm sections were prepared. Staining with hematoxylin and eosin (H&E) and Sirius red was routinely performed and α-SMA was detected by immunohistochemical staining using anti-α-SMA (Proteintech), as described previously[47]. Liver histological grading was as previously reported[48] and as performed by two investigators in a blinded manner. The staining areas of α-SMA and fibrosis were quantified using Image J software.

Measurement of blood histones and alanine aminotransferaseVenous blood was collected with citrate anticoagulant and immediately centrifuged at 1500 × g for 25 min at room temperature. Plasma was collected and alanine aminotransferase (ALT) levels were measured by an automatic blood biochemical analyzer in the clinical laboratory and histone levels were measured by western blotting using anti-histone H3 (Abcam), as described previously[32]. Plasma and histone H3 protein (New England Biolabs, Herts, United Kingdom) as standard were subjected to SDS-PAGE and electrically transferred onto PVDF membranes (Millipore). After blocking, the blot was probed by anti-histone H3 (Abcam, 1:2000) at 4 °C overnight. After extensive washings, HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:10000) was incubated at room temperature for 1 h. After extensive washing in TBST, ECL (Millipore) was used to visualize the protein bands, which were quantified against histone H3 protein standard to obtain the concentration of histone H3 in plasma using Image J software. The total histones were estimated based on the molar ratio of H3 in the cell nuclei.

Measurement of hydroxyproline in liver tissuesPart of the liver from mice was washed with saline and frozen at -80 °C. Two hundred and fifty milligrams of frozen liver tissues were ground using a low temperature Tissuelyser (Shanghai Jingxin Industrial Development Co. Ltd.) and hydroxyproline was measured using a colorimetric kit from Beijing Solarbio Science & Technology Co. Ltd.

Statistical analysisContinuous variables are presented as mean ± standard deviation (SD) or standard error (SE). Differences in means between any two groups were compared using unpaired Student’s t test. Differences in means between more than two groups were compared using analysis of variance followed by Student-Newman-Keuls test. P < 0.05 was considered significant. Statistical analysis was performed using SPSS version 25.

RESULTSHigh levels of circulating histones are detected in mice treated with CCl4

CCl4 is a hepatotoxin that directly damages hepatocytes. CCl4 administration in mice is the most commonly used animal model to generate liver fibrosis, but it is not clear

https://f6publishing.blob.core.windows.net/acc76f97-dcf6-4d6f-a87d-e1a6d0bc6de1/WJG-26-7513-supplementary-material.pdf




whether high levels of histones actually exist under these conditions in the circulation. Circulating histone H3 was detected by western blotting and the total circulating histone levels were calculated based on these levels of H3, as described previously[32]. Histone H3 was detectable at 4 h after the first administration of CCl4 alone or CCl4 + NAHP (Figure 1A and B) at week 1. At week 4, before or 24 h after the last dose of CCl4, H3 was also detectable (Figure 1C). Total circulating histones reached peak values (around 200 μg/mL) between 8 and 24 h after CCl4 was administered and then gradually reduced to low levels (10-60 μg/mL) prior to the subsequent dose of CCl4 (Figure 1D). The mean ± SD of total histones during each week from 8 and 24 h after CCl4 injection is shown in Figure 1E. In contrast, histone H3 was barely detectable in the control group treated with saline + NAHP (data not shown). To monitor liver injury, blood ALT level was measured using a clinical biochemical analyzer. The mean ± SD from nine mice on day 28 was compared, and NAHP alone did not increase ALT levels, while CCl4 caused a significant elevation in circulating ALT (approximately 10-fold), but this elevation was significantly reduced by NAHP (approximately 40% reduction) (Figure 1F). Liver injury scores of H&E-stained liver sections were also significantly reduced by NAHP treatment (Figure 1G). These observations indicated that NAHP treatment reduced liver injury.

Extracellular histones stimulate HSCs to increase production of collagen I and α-SMAExtracellular histones are TLR ligands, including TLR2 and TLR4[40,45]; both of which signal via the downstream MyD88 pathway. During liver fibrogenesis, HSCs express TLR4 and its signaling enhances activation of fibroblasts[17]. We directly treated LX2 cells with calf thymus histones and found that histone concentrations between 2 and 10 μg/mL significantly increased collagen I production. Histones (5 μg/mL) incubated for 6 d were the optimal condition. This increased collagen I released into the culture media by nearly 2.5-fold and increased collagen I expression by 2.7-fold in cell lysates (Figure 2A and B). Concentrations of ≥ 20 μg/mL histones caused cell death (data not shown). Using immunofluorescent staining, we found that LX2 cells treated with 5 μg/mL histones were positively stained with anti-collagen I and anti-α-SMA compared to LX2 cells treated without histones (Figure 2C), supporting that LX2 cells were activated by histone treatment and induced collagen production.

NAHP reduces histone-enhanced collagen production in LX2 cells and liver fibrosis induced by CCl4

NAHP has been demonstrated to bind histones and block their cytotoxicity[26,32,49]. We added NAHP (25 and 50 μg/mL) to culture medium to neutralize 5 μg/mL calf thymus histones (Histones: NAHP approximately 1:2 and 1:4 molar ratio), and found that NAHP inhibited histone-enhanced collagen I synthesis in LX2 cells (Figure 3A and B). In H&E-stained liver sections from the CCl4-induced mouse liver fibrosis model, CCl4 caused extensive hepatocytic damage compared to controls (Figure 3C), including hepatocyte swelling, vacuole formation, necrosis, Kupffer cell proliferation and immune cell infiltration (Figure 3C). NAHP intervention significantly reduced CCl4-induced hepatocyte vacuolization, necrosis and immune cell infiltration (Figure 3C). Immunohistochemical staining with anti-α-SMA showed a larger area of positive staining in CCl4-treated mice compared to controls (Figure 3C). In NAHP-treated mice, the CCl4-induced α-SMA staining was reduced (Figure 3C). Using Sirius red staining to visualize collagen, only the vascular walls were stained positively in controls (Figure 3C). In contrast, large areas of positive staining appeared in CCl4-treated mice (Figure 3C), which were reduced by NAHP intervention (Figure 3C). Using Image J software to quantify the areas of positive staining for α-SMA (Figure 3D) and collagen (Figure 3E), we demonstrated that NAHP significantly reduced both α-SMA and collagen production by 50%-70%. Using a colorimetric assay, hydroxyproline in liver tissues was detected and NAHP was shown to significantly reduce CCl4-induced hydroxyproline elevation by about 40% (Figure 3F).

TLR4 is involved in histone-enhanced collagen production in LX2 cells and in liver fibrosis induced by CCl4

To understand how histones are involved in liver fibrosis, we specifically tested if histone-activated TLR/MyD88 signaling played a major role in this process. This hypothesis was based on previous reports that TLR4 and MyD88 were involved in fibrogenesis[17,19]. We used human TLR4-neutralizing antibodies to treat LX2 cells and found that histone-enhanced collagen I production was significantly reduced



Figure 1 Circulating histones are elevated in ICR mice treated with CCl4. Typical western blots of histone H3 standard and histone H3 in plasma from mice treated with the first dose of CCl4 (A) and CCl4 + non-anticoagulant heparin (NAHP) (B), and a typical H3 blot before and 24 h after the last dose of CCl4 (C); D: The mean ± SD of circulating histones at different time points, including 0 h (before the experiment), and 4, 8, 24, 48 and 84 h after first dose of CCl4 (orange) and CCl4 + NAHP (blue) of each week are presented. ANOVA test, aP < 0.05 compared to time 0; E: The mean ± SD of peak circulating histones (8 and 24 h after first CCl4 injection of each week). ANOVA test, aP < 0.05 compared to time 0 (before first injection). The mean ± SE of blood alanine aminotransferase levels (F) and liver injury scores (G) from nine mice in each group following 4 wk of treatment. ANOVA test, aP < 0.05 compared to mice without treatment (before). bP < 0.05 compared to CCl4 group. ANOVA: Analysis of variance; NAHP: Non-anticoagulant heparin; ALT: Alanine aminotransferase.

(Figure 4A). To confirm that the TLR4-MyD88 signaling pathway was involved in CCl4

-induced liver fibrosis, TLR4 and MyD88 gene knockout mice were used. The extent of fibrosis was significantly less in both TLR4-/- and MyD88-/- mice than in WT mice treated with CCl4 (Figure 4B and C). These results are consistent with a previous study that demonstrated TLR4 and MyD88 deficiency reduced liver fibrosis in a mouse BDL model[50]. These findings strongly suggest that extracellular histones activate the TLR4-MyD88 signaling pathway to enhance collagen I production and liver fibrosis. However, further investigation is required to substantiate this point and the downstream pathways.

DISCUSSIONWe proposed a novel concept that extracellular histones act as pathogenic factors in liver fibrosis. High levels of circulating histones in CCl4-treated mice were detected from 4 h after the first administration of CCl4 and remained high during fibrosis induction. In vitro, extracellular histones directly enhanced collagen I production in LX2 (HSC) cells by up to 2.7-fold. When NAHP was used to neutralize histones, it



Figure 2 Extracellular histones induced collagen I and α-smooth muscle actin production in LX2 cells. A: Typical western blots of collagen I in medium (upper panel) and lysates (middle panel) of LX2 cells treated with different concentrations of histones at day 6. Lower panel: β-actin in the cell lysates; B: The mean ± SD of the relative percentages of collagen/β-actin ratios with untreated LX2 cells set at 100% from five independent experiments. Analysis of variance test, aP < 0.05 compared to untreated cells; C: Immunofluorescent staining of LX2 cells with anti-collagen I and anti-α- smooth muscle actin (SMA) antibodies. Control: Cells were treated with culture medium without histones for 6 d. Histones: Cells were treated with culture medium + 5 μg/mL histones. Typical images are shown. White arrows indicate staining for collagen I, yellow arrows indicate staining for α-SMA. Bar = 50 m. α-SMA: α-smooth muscle actin.

significantly reduced histone-enhanced collagen I production in LX2 cells and significantly reduced fibrosis in CCl4-treated mice. When TLR4 neutralizing antibody was used, histone-enhanced collagen I production in LX2 cells was also reduced. When CCl4 was injected into TLR4 or MyD88 knockout mice, they showed significantly less fibrosis compared to WT mice, suggesting that both TLR4 and MyD88 were involved in histone-enhanced fibrosis.

TLRs not only recognize pathogen-associated molecular patterns from various microbial infections, but also respond to DAMPs from host cell damage[51]. TLR4 recognizes LPS from Gram-negative bacteria as well as histones released following cell death. TLR4 can activate nuclear factor-κB via MyD88[40,44,52,53]. The TLR4-MyD88 pathway has been demonstrated to promote liver fibrosis in a mouse BDL model by enhancing transforming growth factor (TGF)-β signaling[50]. LPS as a TLR4 ligand was elevated 3-6-fold in this model likely due to obstruction of bile ducts and subsequent bacterial infection[50]. However, circulating LPS concentration did not increase in the CCl4 mouse model[54]. Without infection, LPS from digestive tracts enters the circulation in the late stage of fibrosis or cirrhosis, while the wound-healing process is initiated immediately after injury. Therefore, LPS is unlikely to be an initiating factor in fibrosis. Extracellular histones are newly identified TLR4 ligands and are most likely to mediate the initial stage of fibrogenesis by activating the TLR4-MyD88 signaling pathway[40,44]. However, the downstream signaling pathways requires further experimental clarification. In addition, fibrogenesis not only involves collagen production but also many molecular and cellular mechanisms[55]. The co-operation of the TLR4-MyD88 pathway with other signaling pathways, such as TGF-β signaling and inflammatory response, also requires further investigation to determine their relative contributions to liver fibrosis.

The level of extracellular histones required for stimulating LX2 (HSC) cells is 2-10 μg/mL, which is lower than that detected in the circulation of CCl4-treated mice. All groups, including ICR mice treated with CCl4 and CCl4 + NAHP, and C57BL/6,



Figure 3 Effect of anti-histone reagent, non-anticoagulant heparin, on histone-enhanced collagen I production in LX2 cells and CCl4-induced fibrosis in mice. A: Typical western blots of collagen I in LX2 cells treated with histones or histones + non-anticoagulant heparin (NAHP). Beta-actin is shown as a loading reference; B: The mean ± SD of relative percentage of collagen/actin ratios in untreated LX-2 cells set at 100% from three independent experiments. ANOVA test, aP < 0.05 compared to untreated cells. bP < 0.05 compared to cells treated with 5 μg/mL histones; C: Typical images of stained liver sections (hematoxylin and eosin staining: a-c; immunohistochemical staining with anti-α-SMA: d-f; and Sirius red staining: g-i) from normal mice (a, d, g); mice treated with CCl4 (b, e, h) and mice treated with CCl4 + NAHP (CCl4 + H) (c, f, i). Arrows in b and c: Black indicate hepatocyte swelling and white indicate necrosis and immune cell infiltration. Arrows in e and f: Indicate staining for smooth muscle actin. Arrows in h and i: Indicate collagen deposition. The mean ± SD of percentage of areas of staining for α-SMA (D) and Sirius red staining for collagen (E) (nine mice per group, and six randomly selected sections per mouse). ANOVA test, aP < 0.05



compared to controls. bP < 0.05 compared to CCl4 alone; F: The mean ± SD of hydroxyproline levels in liver tissues from nine mice per group. ANOVA test, aP < 0.05 compared to controls. bP < 0.05 compared to CCl4 alone. α-SMA: α-smooth muscle actin; ANOVA: Analysis of variance; NAHP: Non-anticoagulant heparin.

Figure 4 TLR4 is involved in histone-enhanced collagen I production and CCl4-induced mouse liver fibrosis. A: LX2 cells were treated with 5 μg/mL histones (Hist) in the presence or absence of TLR4 neutralizing antibody (TLR4i). The mean ± SD of the relative percentage of collagen Ι/β-actin ratios are presented with control (UT) set at 100% from three independent experiments. ANOVA test, aP < 0.05 compared to UT. bP < 0.05 compared to histone alone; B: Typical images of Sirius red staining of liver section from untreated wt C57BL/j mice (a), CCl4-treated wt mice (b), and TLR4 and MyD88 knockout mice; C: The mean ± SD of stained areas of liver sections from untreated wt mice (UT), CCl4-treated wt mice (WT), CCl4-treated TLR4-/- and MyD88-/- mice. Eight mice were in each group and six sections from each mouse were analyses. ANOVA test, aP < 0.05 compared to untreated wt mice (UT). bP < 0.05 compared to CCl4-treated wt mice. ANOVA: Analysis of variance; wt: Wild type; UT: Control.

TLR4-/- and MyD88-/- mice treated with CCl4, had comparable levels of peak circulating histones (Supplementary Figure 3). This is due to the severe and extensive damage to hepatocytes caused by the radical CCl3, a metabolite of CCl4. The highest levels of histones detected after CCl4 administration are comparable to those we have detected in critical illnesses[26,31,32] and are most likely able to cause further liver injury[56]. However, plasma can neutralize histones at a concentration of up to 50 μg/mL in vivo or ex vivo, making cells more resistant than those in culture medium[32]. Additionally,




in our mouse model, the interval between two doses of CCl4 was 84 h; therefore, more than half of this period had histone levels around 50 μg/mL, which may be sufficient in maintaining TLR4 activation. Elevation of circulating histones was also found in many types of liver disease and the high levels of circulating histones could cause secondary liver injury[26,40,57]. Some human chronic liver diseases, such as chronic hepatitis B and C, and alcoholic and autoimmune liver diseases may have low but constant levels of circulating histones to serve as alarmins, which signal tissue injury and initiate repair processes. However, this needs further clinical investigation.

NAHP can bind to histones, but does not lower the levels of circulating histones. In the first week, NAHP seems to increase the levels of circulating histones in the CCl4-treated mice and this result could be explained by its ability to reduce histone clearance, as we have observed previously[26]. NAHP binding to extracellular histones prevents their binding to the cell plasma membrane and other targets, such as prothrombin[32-34] and thereby detoxifies histones. In this study, we found that ALT and liver injury scores were significantly reduced by NAHP in CCl4-treated mice, strongly indicating that high levels of circulating histones are toxic to liver cells, which can be neutralized by NAHP. This protective effect may also contribute to the reduction of liver fibrosis.

Heparin was reported to reduce collagen fiber formation and liver fibrosis two decades ago[58-60]. Enzymatically depolymerized low-molecular-weight heparins were shown to inhibit CCl4-induced liver fibrosis by reducing tumor necrosis factor (TNF) α and interleukin (IL)-1β[61]. Although extracellular histones could enhance cytokine release, including IL-6, IL-1β, and TNFα[33,40], we suggest that the major mechanism for heparin-inhibited liver fibrosis is most likely due to its blocking the ability of extracellular histones to activate the TLR4-MyD88 signaling pathway. Heparin has been used as an anticoagulant for many decades and overdoses may cause bleeding, which can be avoided by using NAHP[26]. Additionally, NAHP alone appears to have no significant liver toxicity, but it significantly reduced histone-enhanced collagen I expression in LX2 cells and reduced CCl4-induced liver fibrosis. Therefore, this novel finding may help to advance translating this old discovery into clinical application.

CONCLUSIONThis study has demonstrated that high levels of circulating histones exist in mice treated with CCl4 during induction of liver fibrosis. Treatment of LX2 cells with extracellular histones enhanced production of collagen I and α-SMA. Neutralizing histones using NAHP significantly reduced the production of collagen I and the extent of liver fibrosis. These data demonstrate that extracellular histones released after liver injury promote liver fibrosis. Blocking TLR4 receptor on LX2 cells reduced histone-enhanced collagen I production and CCl4 induced less fibrosis in TLR4 and MyD88 knockout mice than in WT parental mice, strongly suggesting that histone-enhanced collagen production is via the TLR4-MyD88 signaling pathway. However, there were a few limitations to this study. Firstly, in our in vivo study, no direct link was established between histones and their binding to TLR4. The relative contribution of the histone-TLR4-MyD88 axis and downstream signaling pathways is still not clear. Secondly, only one type of animal model for liver fibrosis was used and it may not truly represent the fibrosis caused by different diseases. However, the results are sufficient to demonstrate the novel concept that extracellular histones are involved in liver fibrosis. Thirdly, liver fibrosis is a complicated process involving many types of cells and signaling pathways. Besides collagen overproduction, many other biological processes are also involved in liver fibrosis. High levels of extracellular histones may also affect many other types of cells besides HSCs and collagen production. The detailed roles of extracellular histones in the complex processes of fibrogenesis require extensive investigation. Similarly, NAHP may have other effects besides blocking histones to activate TLR4 and to injury hepatocytes. Therefore, further in vivo mechanistic studies and development of better reagents to target histones and potential signaling pathways may offer a better understanding of fibrogenesis and more effective therapies in the future.



ARTICLE HIGHLIGHTSResearch backgroundCurrently, the molecular mechanisms of liver fibrosis are not fully understood. Recurrent liver injury or inflammation initiates wound healing along with fibrogenesis. However, what initiates this process is not clear. When cells die, damage-associated molecular patterns (DAMPs) are released. Histones are the most abundant DAMPs and are also ligands for TLR4, which in turn has been demonstrated to be involved in bile-duct-ligation-induced liver fibrosis. Lipopolysaccharide (LPS) is proposed to be a ligand for TLR4. Since recurrent liver injury does not naturally produce LPS but abundant extracellular histones, this study sought to investigate the potential roles of extracellular histones as TLR4 ligands in liver fibrosis.

Research motivationSince our laboratory has been involved in studying the roles of DAMPs in critical illnesses, extracellular histones in liver fibrosis are of interest in terms of biological and clinical significance.

Research objectivesOur study aimed to clarify the roles of extracellular histones in fibrogenesis in vitro and in vivo.

Research methodsIn our study, a hepatic stellate (HSC) cell line and animal models of liver fibrosis were used. Intervention studies with non-anticoagulant heparin (NAHP) to detoxify histones and TLR4-blocking antibodies to inhibit TLR4 were performed. In addition, TLR4 and MyD88 knockout mice were used to support the theory that the TLR4-MyD88 signaling pathway is involved in liver fibrosis in the CCl4 mouse model.

Research resultsHigh levels of circulating histones were present when fibrosis was induced by CCl4 in the mouse model. Extracellular histones stimulated HSC cells in vitro to increase production of collagen I and alpha-smooth muscle actin. NAHP inhibited histone-enhanced collagen production in vitro, and reduce liver injury and fibrosis in vivo. TLR4 was involved in histone-enhanced collagen I production by HSC cells. In vivo, the TLR4-MyD88 signaling pathway mediated liver fibrosis, but whether circulating histones were the major activators of the pathway was not clear.

Research conclusionsRecurrent liver injury releases extracellular histones that potentially activate TLR4-MyD88 signaling to promote liver fibrosis. The ability of NAHP to detoxify circulating histones has the potential for treatment of liver injury and prevention of liver fibrosis.

Research perspectivesFuture studies demonstrating the contribution of circulating histones to activation of the TLR4-MyD88 signaling and downstream pathways will validate their role in liver fibrosis. Development of effective anti-histone therapies to reduce liver injury and prevent liver fibrosis have potential in the management of diseases with recurrent liver injury.

ACKNOWLEDGEMENTSThanks to all the staff in the animal house for their support and to the technicians in the pathology laboratory for technical support and clinical biochemistry for ALT measurement in Zhongda Hospital, Nanjing, China. Thanks to Professor Rudland P in the University of Liverpool for his critical reading and correction of the manuscript. Thanks to Dr. Lane S in The Department of Statistics, University of Liverpool for his assistance with statistical analysis.



REFERENCESBataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115: 209-218 [PMID: 15690074 DOI: 10.1172/JCI24282]

1

Schuppan D, Afdhal NH. Liver cirrhosis. Lancet 2008; 371: 838-851 [PMID: 18328931 DOI: 10.1016/S0140-6736(08)60383-9]

2

Friedman SL. Liver fibrosis -- from bench to bedside. J Hepatol 2003; 38 Suppl 1: S38-S53 [PMID: 12591185 DOI: 10.1016/s0168-8278(02)00429-4]

3

GBD 2017 Cirrhosis Collaborators. The global, regional, and national burden of cirrhosis by cause in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol Hepatol 2020; 5: 245-266 [PMID: 31981519 DOI: 10.1016/S2468-1253(19)30349-8]

4

Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011; 6: 425-456 [PMID: 21073339 DOI: 10.1146/annurev-pathol-011110-130246]

5

Liu Y, Meyer C, Xu C, Weng H, Hellerbrand C, ten Dijke P, Dooley S. Animal models of chronic liver diseases. Am J Physiol Gastrointest Liver Physiol 2013; 304: G449-G468 [PMID: 23275613 DOI: 10.1152/ajpgi.00199.2012]

6

Liedtke C, Luedde T, Sauerbruch T, Scholten D, Streetz K, Tacke F, Tolba R, Trautwein C, Trebicka J, Weiskirchen R. Experimental liver fibrosis research: update on animal models, legal issues and translational aspects. Fibrogenesis Tissue Repair 2013; 6: 19 [PMID: 24274743 DOI: 10.1186/1755-1536-6-19]

7

Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest 2007; 117: 539-548 [PMID: 17332881 DOI: 10.1172/JCI30542]

8

Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003; 33: 105-136 [PMID: 12708612 DOI: 10.1080/713611034]

9

Fujii T, Fuchs BC, Yamada S, Lauwers GY, Kulu Y, Goodwin JM, Lanuti M, Tanabe KK. Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor. BMC Gastroenterol 2010; 10: 79 [PMID: 20618941 DOI: 10.1186/1471-230X-10-79]

10

Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology 2017; 65: 1039-1043 [PMID: 27859502 DOI: 10.1002/hep.28948]

11

Gäbele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci 2003; 8: d69-d77 [PMID: 12456323 DOI: 10.2741/887]

12

Russo FP, Alison MR, Bigger BW, Amofah E, Florou A, Amin F, Bou-Gharios G, Jeffery R, Iredale JP, Forbes SJ. The bone marrow functionally contributes to liver fibrosis. Gastroenterology 2006; 130: 1807-1821 [PMID: 16697743 DOI: 10.1053/j.gastro.2006.01.036]

13

Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest 1990; 86: 1641-1648 [PMID: 2243137 DOI: 10.1172/JCI114886]

14

Friedman SL, Rockey DC, McGuire RF, Maher JJ, Boyles JK, Yamasaki G. Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture. Hepatology 1992; 15: 234-243 [PMID: 1735526 DOI: 10.1002/hep.1840150211]

15

Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA. Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells. Hepatology 2003; 37: 1043-1055 [PMID: 12717385 DOI: 10.1053/jhep.2003.50182]

16

Bhattacharyya S, Wang W, Qin W, Cheng K, Coulup S, Chavez S, Jiang S, Raparia K, De Almeida LMV, Stehlik C, Tamaki Z, Yin H, Varga J. TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung. JCI Insight 2018; 3: e98850 [PMID: 29997297 DOI: 10.1172/jci.insight.98850]

17

Huebener P, Schwabe RF. Regulation of wound healing and organ fibrosis by toll-like receptors. Biochim Biophys Acta 2013; 1832: 1005-1017 [PMID: 23220258 DOI: 10.1016/j.bbadis.2012.11.017]

18

Yang L, Seki E. Toll-like receptors in liver fibrosis: cellular crosstalk and mechanisms. Front Physiol 2012; 3: 138 [PMID: 22661952 DOI: 10.3389/fphys.2012.00138]

19

Cengiz M, Ozenirler S, Elbeg S. Role of serum toll-like receptors 2 and 4 in non-alcoholic steatohepatitis and liver fibrosis. J Gastroenterol Hepatol 2015; 30: 1190-1196 [PMID: 25684563 DOI: 10.1111/jgh.12924]

20

Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zähringer U, Seydel U, Di Padova F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 1994; 8: 217-225 [PMID: 8119492 DOI: 10.1096/fasebj.8.2.8119492]

21

Pascual S, Such J, Esteban A, Zapater P, Casellas JA, Aparicio JR, Girona E, Gutiérrez A, Carnices F, Palazón JM, Sola-Vera J, Pérez-Mateo M. Intestinal permeability is increased in patients with advanced cirrhosis. Hepatogastroenterology 2003; 50: 1482-1486 [PMID: 14571769]

22

Vénéreau E, Ceriotti C, Bianchi ME. DAMPs from Cell Death to New Life. Front Immunol 2015; 6: 422 [PMID: 26347745 DOI: 10.3389/fimmu.2015.00422]

23

Silk E, Zhao H, Weng H, Ma D. The role of extracellular histone in organ injury. Cell Death Dis 2017; 8: e2812 [PMID: 28542146 DOI: 10.1038/cddis.2017.52]

24

Moxon CA, Alhamdi Y, Storm J, Toh JMH, McGuinness D, Ko JY, Murphy G, Lane S, Taylor TE, Seydel KB, Kampondeni S, Potchen M, O'Donnell JS, O'Regan N, Wang G, García-Cardeña G,

25




https://dx.doi.org/10.1016/S0140-6736(08)60383-9


https://dx.doi.org/10.1016/s0168-8278(02)00429-4


https://dx.doi.org/10.1016/S2468-1253(19)30349-8


https://dx.doi.org/10.1146/annurev-pathol-011110-130246


https://dx.doi.org/10.1152/ajpgi.00199.2012


https://dx.doi.org/10.1186/1755-1536-6-19




https://dx.doi.org/10.1080/713611034


https://dx.doi.org/10.1186/1471-230X-10-79














https://dx.doi.org/10.1172/jci.insight.98850




https://dx.doi.org/10.3389/fphys.2012.00138


https://dx.doi.org/10.1111/jgh.12924


https://dx.doi.org/10.1096/fasebj.8.2.8119492





https://dx.doi.org/10.1038/cddis.2017.52



Molyneux M, Craig AG, Abrams ST, Toh CH. Parasite histones are toxic to brain endothelium and link blood barrier breakdown and thrombosis in cerebral malaria. Blood Adv 2020; 4: 2851-2864 [PMID: 32579667 DOI: 10.1182/bloodadvances.2019001258]Cheng Z, Abrams ST, Alhamdi Y, Toh J, Yu W, Wang G, Toh CH. Circulating Histones Are Major Mediators of Multiple Organ Dysfunction Syndrome in Acute Critical Illnesses. Crit Care Med 2019; 47: e677-e684 [PMID: 31162199 DOI: 10.1097/CCM.0000000000003839]

26

Qaddoori Y, Abrams ST, Mould P, Alhamdi Y, Christmas SE, Wang G, Toh CH. Extracellular Histones Inhibit Complement Activation through Interacting with Complement Component 4. J Immunol 2018; 200: 4125-4133 [PMID: 29752310 DOI: 10.4049/jimmunol.1700779]

27

Alhamdi Y, Toh CH. Recent advances in pathophysiology of disseminated intravascular coagulation: the role of circulating histones and neutrophil extracellular traps. F1000Res 2017; 6: 2143 [PMID: 29399324 DOI: 10.12688/f1000research.12498.1]

28

Liu T, Huang W, Szatmary P, Abrams ST, Alhamdi Y, Lin Z, Greenhalf W, Wang G, Sutton R, Toh CH. Accuracy of circulating histones in predicting persistent organ failure and mortality in patients with acute pancreatitis. Br J Surg 2017; 104: 1215-1225 [PMID: 28436602 DOI: 10.1002/bjs.10538]

29

Alhamdi Y, Toh CH. The role of extracellular histones in haematological disorders. Br J Haematol 2016; 173: 805-811 [PMID: 27062156 DOI: 10.1111/bjh.14077]

30

Alhamdi Y, Abrams ST, Cheng Z, Jing S, Su D, Liu Z, Lane S, Welters I, Wang G, Toh CH. Circulating Histones Are Major Mediators of Cardiac Injury in Patients With Sepsis. Crit Care Med 2015; 43: 2094-2103 [PMID: 26121070 DOI: 10.1097/CCM.0000000000001162]

31

Abrams ST, Zhang N, Manson J, Liu T, Dart C, Baluwa F, Wang SS, Brohi K, Kipar A, Yu W, Wang G, Toh CH. Circulating histones are mediators of trauma-associated lung injury. Am J Respir Crit Care Med 2013; 187: 160-169 [PMID: 23220920 DOI: 10.1164/rccm.201206-1037OC]

32

Abrams ST, Zhang N, Dart C, Wang SS, Thachil J, Guan Y, Wang G, Toh CH. Human CRP defends against the toxicity of circulating histones. J Immunol 2013; 191: 2495-2502 [PMID: 23894199 DOI: 10.4049/jimmunol.1203181]

33

Abrams ST, Su D, Sahraoui Y, Lin Z, Cheng Z, Nesbitt K, Alhamdi Y, Harrasser M, Du M, Foley J, Lillicrap D, Wang G, Toh CH. Assembly of alternative prothrombinase by extracellular histones initiate and disseminate intravascular coagulation. Blood2020 [PMID: 32722805 DOI: 10.1182/blood.2019002973]

34

Alhamdi Y, Zi M, Abrams ST, Liu T, Su D, Welters I, Dutt T, Cartwright EJ, Wang G, Toh CH. Circulating Histone Concentrations Differentially Affect the Predominance of Left or Right Ventricular Dysfunction in Critical Illness. Crit Care Med 2016; 44: e278-e288 [PMID: 26588828 DOI: 10.1097/CCM.0000000000001413]

35

Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997; 389: 251-260 [PMID: 9305837 DOI: 10.1038/38444]

36

Holdenrieder S, Stieber P, Bodenmüller H, Fertig G, Fürst H, Schmeller N, Untch M, Seidel D. Nucleosomes in serum as a marker for cell death. Clin Chem Lab Med 2001; 39: 596-605 [PMID: 11522104 DOI: 10.1515/CCLM.2001.095]

37

Wu D, Ingram A, Lahti JH, Mazza B, Grenet J, Kapoor A, Liu L, Kidd VJ, Tang D. Apoptotic release of histones from nucleosomes. J Biol Chem 2002; 277: 12001-12008 [PMID: 11812781 DOI: 10.1074/jbc.M109219200]

38

Gauthier VJ, Tyler LN, Mannik M. Blood clearance kinetics and liver uptake of mononucleosomes in mice. J Immunol 1996; 156: 1151-1156 [PMID: 8557992]

39

Xu J, Zhang X, Monestier M, Esmon NL, Esmon CT. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol 2011; 187: 2626-2631 [PMID: 21784973 DOI: 10.4049/jimmunol.1003930]

40

Li X, Gou C, Yao L, Lei Z, Gu T, Ren F, Wen T. Patients with HBV-related acute-on-chronic liver failure have increased concentrations of extracellular histones aggravating cellular damage and systemic inflammation. J Viral Hepat 2017; 24: 59-67 [PMID: 27660136 DOI: 10.1111/jvh.12612]

41

Yang R, Zou X, Tenhunen J, Tønnessen TI. HMGB1 and Extracellular Histones Significantly Contribute to Systemic Inflammation and Multiple Organ Failure in Acute Liver Failure. Mediators Inflamm 2017; 2017: 5928078 [PMID: 28694564 DOI: 10.1155/2017/5928078]

42

Huang H, Evankovich J, Yan W, Nace G, Zhang L, Ross M, Liao X, Billiar T, Xu J, Esmon CT, Tsung A. Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice. Hepatology 2011; 54: 999-1008 [PMID: 21721026 DOI: 10.1002/hep.24501]

43

Allam R, Scherbaum CR, Darisipudi MN, Mulay SR, Hägele H, Lichtnekert J, Hagemann JH, Rupanagudi KV, Ryu M, Schwarzenberger C, Hohenstein B, Hugo C, Uhl B, Reichel CA, Krombach F, Monestier M, Liapis H, Moreth K, Schaefer L, Anders HJ. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4. J Am Soc Nephrol 2012; 23: 1375-1388 [PMID: 22677551 DOI: 10.1681/ASN.2011111077]

44

Semeraro F, Ammollo CT, Morrissey JH, Dale GL, Friese P, Esmon NL, Esmon CT. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 2011; 118: 1952-1961 [PMID: 21673343 DOI: 10.1182/blood-2011-03-343061]

45

Guo HY, Cui ZJ. Extracellular Histones Activate Plasma Membrane Toll-Like Receptor 9 to Trigger Calcium Oscillations in Rat Pancreatic Acinar Tumor Cell AR4-2J. Cells 2018; 8: 3 [PMID: 30577532 DOI: 10.3390/cells8010003]

46

Liu J, Li W, Limbu MH, Li Y, Wang Z, Cheng Z, Zhang X, Chen P. Effects of Simultaneous 47


https://dx.doi.org/10.1182/bloodadvances.2019001258




https://dx.doi.org/10.4049/jimmunol.1700779


https://dx.doi.org/10.12688/f1000research.12498.1


https://dx.doi.org/10.1002/bjs.10538


https://dx.doi.org/10.1111/bjh.14077




https://dx.doi.org/10.1164/rccm.201206-1037OC




https://dx.doi.org/10.1182/blood.2019002973






https://dx.doi.org/10.1515/CCLM.2001.095


https://dx.doi.org/10.1074/jbc.M109219200





https://dx.doi.org/10.1111/jvh.12612


https://dx.doi.org/10.1155/2017/5928078




https://dx.doi.org/10.1681/ASN.2011111077




https://dx.doi.org/10.3390/cells8010003



Downregulation of PHD1 and Keap1 on Prevention and Reversal of Liver Fibrosis in Mice. Front Pharmacol 2018; 9: 555 [PMID: 29899699 DOI: 10.3389/fphar.2018.00555]Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, Desmet V, Korb G, MacSween RN. Histological grading and staging of chronic hepatitis. J Hepatol 1995; 22: 696-699 [PMID: 7560864 DOI: 10.1016/0168-8278(95)80226-6]

48

Abrams ST, Morton B, Alhamdi Y, Alsabani M, Lane S, Welters ID, Wang G, Toh CH. A Novel Assay for Neutrophil Extracellular Trap Formation Independently Predicts Disseminated Intravascular Coagulation and Mortality in Critically Ill Patients. Am J Respir Crit Care Med 2019; 200: 869-880 [PMID: 31162936 DOI: 10.1164/rccm.201811-2111OC]

49

Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 2007; 13: 1324-1332 [PMID: 17952090 DOI: 10.1038/nm1663]

50

Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol 2014; 5: 461 [PMID: 25309543 DOI: 10.3389/fimmu.2014.00461]

51

Yang X, Li L, Liu J, Lv B, Chen F. Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1. Thromb Res 2016; 137: 211-218 [PMID: 26476743 DOI: 10.1016/j.thromres.2015.10.012]

52

Wang K, Yang X, Wu Z, Wang H, Li Q, Mei H, You R, Zhang Y. Dendrobium officinale Polysaccharide Protected CCl4-Induced Liver Fibrosis Through Intestinal Homeostasis and the LPS-TLR4-NF-κB Signaling Pathway. Front Pharmacol 2020; 11: 240 [PMID: 32226380 DOI: 10.3389/fphar.2020.00240]

53

Kumar S, Wang J, Shanmukhappa SK, Gandhi CR. Toll-Like Receptor 4-Independent Carbon Tetrachloride-Induced Fibrosis and Lipopolysaccharide-Induced Acute Liver Injury in Mice: Role of Hepatic Stellate Cells. Am J Pathol 2017; 187: 1356-1367 [PMID: 28412299 DOI: 10.1016/j.ajpath.2017.01.021]

54

Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol2020 [PMID: 33128017 DOI: 10.1038/s41575-020-00372-7]

55

Wen Z, Lei Z, Yao L, Jiang P, Gu T, Ren F, Liu Y, Gou C, Li X, Wen T. Circulating histones are major mediators of systemic inflammation and cellular injury in patients with acute liver failure. Cell Death Dis 2016; 7: e2391 [PMID: 27685635 DOI: 10.1038/cddis.2016.303]

56

Wen Z, Liu Y, Li F, Ren F, Chen D, Li X, Wen T. Circulating histones exacerbate inflammation in mice with acute liver failure. J Cell Biochem 2013; 114: 2384-2391 [PMID: 23696007 DOI: 10.1002/jcb.24588]

57

Akimoto K, Ikeda M, Sorimachi K. Inhibitory effect of heparin on collagen fiber formation in hepatic cells in culture. Cell Struct Funct 1997; 22: 533-538 [PMID: 9431458 DOI: 10.1247/csf.22.533]

58

Shi J, Hao JH, Ren WH, Zhu JR. Effects of heparin on liver fibrosis in patients with chronic hepatitis B. World J Gastroenterol 2003; 9: 1611-1614 [PMID: 12854176 DOI: 10.3748/wjg.v9.i7.1611]

59

Sorimachi K. Inhibition of collagen fiber formation by dextran sulfate in hepatic cells. Biomed Res 1992; 13: 75-79

60

Yan Y, Guan C, Du S, Zhu W, Ji Y, Su N, Mei X, He D, Lu Y, Zhang C, Xing XH. Effects of Enzymatically Depolymerized Low Molecular Weight Heparins on CCl4-Induced Liver Fibrosis. Front Pharmacol 2017; 8: 514 [PMID: 28871223 DOI: 10.3389/fphar.2017.00514]

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https://dx.doi.org/10.3389/fphar.2018.00555


https://dx.doi.org/10.1016/0168-8278(95)80226-6


https://dx.doi.org/10.1164/rccm.201811-2111OC


https://dx.doi.org/10.1038/nm1663




https://dx.doi.org/10.1016/j.thromres.2015.10.012




https://dx.doi.org/10.1016/j.ajpath.2017.01.021


https://dx.doi.org/10.1038/s41575-020-00372-7


https://dx.doi.org/10.1038/cddis.2016.303


https://dx.doi.org/10.1002/jcb.24588


https://dx.doi.org/10.1247/csf.22.533






World Journal of



ORIGINAL ARTICLE

Case Control Study

Prevalence and associated factors of obesity in inflammatory bowel disease: A case-control study

Giuseppe Losurdo, Rosa Federica La Fortezza, Andrea Iannone, Antonella Contaldo, Michele Barone, Enzo Ierardi, Alfredo Di Leo, Mariabeatrice Principi

ORCID number: Giuseppe Losurdo 0000-0001-7038-3287; Rosa Federica La Fortezza 0000-0003-2587-097X; Andrea Iannone 0000-0002-5468-9515; Antonella Contaldo 0000-0002-5901-1340; Michele Barone 0000-0001-8284-5127; Enzo Ierardi 0000-0001-7275-5080; Alfredo Di Leo 0000-0003-2026-1200; Mariabeatrice Principi 0000-0003-0545-5656.

Author contributions: Iannone A, Di Leo A and Principi M planned the study; Losurdo G, La Fortezza RF, Iannone A, Contaldo A and Barone M collected data; Ierardi, Barone M and Principi M supervised the study; Losurdo G performed statistical analysis; Losurdo G and Ierardi E wrote the manuscript; all authors read and approved the final version.

Institutional review board statement: The study was approved by the independent Ethics Committee of the Policlinico di Bari (protocol No. 4862) and was performed according to the Helsinki declaration 1975 statements.

Informed consent statement: All patients gave informed consent.

Conflict-of-interest statement: No benefits in any form have been received or will be received from a

Giuseppe Losurdo, Rosa Federica La Fortezza, Andrea Iannone, Antonella Contaldo, Michele Barone, Enzo Ierardi, Alfredo Di Leo, Mariabeatrice Principi, Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Bari 70124, Italy

Corresponding author: Giuseppe Losurdo, MD, Academic Fellow, Doctor, Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Piazza Giulio Cesare 11, Bari 70124, Italy. [email protected]

AbstractBACKGROUND In recent years, an increasing prevalence of obesity in inflammatory bowel disease (IBD) has been observed. Obesity, moreover, has been directly correlated with a more severe clinical course and loss of response to treatment.

AIM To assess the prevalence and associated factors of obesity in IBD.

METHODS We collected data about IBD disease pattern and activity, drugs and laboratory investigations in our center. Anthropometric measures were retrieved and obesity defined as a body mass index (BMI) > 30. Then, we compared characteristics of obese vs non obese patients, and Chi-squared test and Student’s t test were used for discrete and continuous variables, respectively, at univariate analysis. For multivariate analysis, we used binomial logistic regression and estimated odd ratios (OR) and 95% confidence intervals (CI) to ascertain factors associated with obesity.

RESULTS We enrolled 807 patients with IBD, either ulcerative colitis (UC) or Crohn’s disease (CD). Four hundred seventy-four patients were male (58.7%); the average age was 46.2 ± 13.2 years; 438 (54.2%) patients had CD and 369 (45.8%) UC. We enrolled 378 controls, who were comparable to IBD group for age, sex, BMI, obesity, diabetes and abdominal circumference, while more smokers and more subjects with hypertension were observed among controls. The prevalence of obesity was 6.9% in IBD and 7.9% in controls (not statistically different; P = 0.38). In the comparison of obese IBD patients and obese controls, we did not find any



http://orcid.org/0000-0001-7038-3287

http://orcid.org/0000-0001-7038-3287

http://orcid.org/0000-0003-2587-097X

http://orcid.org/0000-0003-2587-097X

http://orcid.org/0000-0002-5468-9515

http://orcid.org/0000-0002-5468-9515

http://orcid.org/0000-0002-5901-1340

http://orcid.org/0000-0002-5901-1340

http://orcid.org/0000-0002-5901-1340

http://orcid.org/0000-0001-8284-5127

http://orcid.org/0000-0001-8284-5127

http://orcid.org/0000-0001-8284-5127

http://orcid.org/0000-0001-7275-5080

http://orcid.org/0000-0001-7275-5080

http://orcid.org/0000-0001-7275-5080

http://orcid.org/0000-0003-2026-1200

http://orcid.org/0000-0003-2026-1200

http://orcid.org/0000-0003-0545-5656

http://orcid.org/0000-0003-0545-5656


Losurdo G et al. IBD and obesity


commercial party related directly or indirectly to the subject of this article.


STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.





Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): B, B, B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0

Received: September 30, 2020 Peer-review started: September 30, 2020 First decision: November 13, 2020 Revised: November 18, 2020 Accepted: November 29, 2020 Article in press: November 29, 2020 Published online: December 21, 2020

P-Reviewer: Li C, Szilagyi A S-Editor: Gao CC L-Editor: A P-Editor: Liu JH

difference regarding diabetes and hypertension prevalence, nor in sex or smoking habits. Obese IBD patients were younger than obese controls (51.2 ± 14.9 years vs 60.7 ± 12.1 years, P = 0.03). At univariate analysis, obese IBD were older than normal weight ones (51.2 ± 14.9 vs 44.5 ± 15.8, P = 0.002). IBD onset age was earlier in obese population (44.8 ± 13.6 vs 35.6 ± 15.6, P = 0.004). We did not detect any difference in disease extension. Obese subjects had consumed more frequently long course of systemic steroids (66.6% vs 12.5%, P = 0.02) as well as antibiotics such as metronidazole or ciprofloxacin (71.4% vs 54.7%, P = 0.05). No difference about other drugs (biologics, mesalazine or thiopurines) was observed. Disease activity was similar between obese and non obese subjects both for UC and CD. Obese IBD patients suffered more frequently from arterial hypertension, type 2 diabetes, non-alcoholic fatty liver disease. Regarding laboratory investigations, obese IBD patients had higher levels of triglyceridemia, fasting blood glucose, gamma-glutamyl-transpeptidase. On multivariate analysis, however, the only factor that appeared to be independently linked to obesity in IBD was the high abdominal circumference (OR = 16.3, 95%CI: 1.03-250, P = 0.04).

CONCLUSION Obese IBD patients seem to have features similar to general obese population, and there is no disease-specific factor (disease activity, extension or therapy) that may foster obesity in IBD.

Key Words: Inflammatory bowel disease; Obesity; Body mass index; Antibiotics; Risk factor; Corticosteroids


Core Tip: Obesity in inflammatory bowel disease (IBD) may be correlated with a more severe clinical course and loss of response to treatment. We did not find any peculiar difference between obese IBD patients and controls. On the other hand, it is possible that some drugs, such as steroids or antibiotics may contribute to the development of obesity in IBD, despite our results suggest that a more complex interaction of several factors could be more likely.

Citation: Losurdo G, La Fortezza RF, Iannone A, Contaldo A, Barone M, Ierardi E, Di Leo A, Principi M. Prevalence and associated factors of obesity in inflammatory bowel disease: A case-control study. World J Gastroenterol 2020; 26(47): 7528-7537URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7528.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7528

INTRODUCTIONObesity is a growing problem in developed countries, since it is going to become the leading cause for mortality due to cardiovascular events[1]. In Italy, it is estimated that about the 18% of population is suffering from obesity[2]. The World Health Organization (WHO) defines obesity by a value of body mass index (BMI) above 30 kg/m2, but obesity underlies as well an excessive visceral fat distribution, with several alterations at hormonal, inflammatory and endothelial level[3]. Inflammatory bowel disease (IBD) is a group of chronic inflammatory autoimmune disorders of gastrointestinal tract, mainly represented by Crohn’s disease (CD) and ulcerative colitis (UC). The problem of obesity is spreading in the context of IBD, since, in the past, it has been rarely recognized for the frequent association between IBD and malnutrition. However, nowadays, novel and more effective drugs are able to stop the progression of the disease, thus preventing malnutrition[4]. Indeed, a study performed in 2002 found a prevalence of obesity in CD of about 3%[5]; one decade later, however, in another study a prevalence of 31.5% was recorded[6]. Co-occurrence of obesity and IBD is not just a casual phenomenon and it has been emphasized that obesity may lead to a higher risk of perianal complications, higher hospitalization rates and greater risk of disease flares[5]. Moreover, obese patients with IBD on azathioprine were more likely








to need courses of systemic corticosteroids and had higher recurrence rates after stopping the drug[7]. Furthermore, it has been estimated that an increase of one unit of BMI may increase the risk of therapeutic failure of 4%[8] and, in particular, a high BMI was an independent predictor of adalimumab therapy failure[9]. Considering that IBD per se could increase the risk of endothelial dysfunction and cardiovascular risk[10,11], the association between obesity and IBD may represent a very important issue.

Therefore, we aimed, in a case-control study, to investigate the prevalence of obesity in IBD patients and detect possible factors associated to this condition.

MATERIALS AND METHODSPatientsWe consecutively recruited IBD patients referred to our outpatient tertiary Gastroenterology Unit (University Hospital Policlinico, Bari, Italy) in the period October 2016-October 2017. We only excluded patients aging < 18, doubtful IBD diagnosis and those who refused to participate in the study. Outpatients with functional gastrointestinal disorders constituted the control group. The study was approved by the independent Ethics Committee of the Policlinico di Bari (protocol No. 4862) and was performed according to the Helsinki declaration 1975 statements.

For each patient, age, sex, abdominal circumference, weight and BMI, smoking habits and relevant comorbidities were collected. Obesity was diagnosed when BMI > 30[3]. For IBD patients, the diagnosis was achieved by a combination of endoscopy, histology (in all cases) and, for all CD patients, a transmural evaluation by magnetic resonance enterography. Then, we collected data about IBD staging (according to Montreal classification), clinical disease activity [partial Mayo for UC and Harvey-Bradshaw index (HBI) for CD] and specific therapies. For IBD patients we recorded data about laboratory investigations, in particular full blood count, erythrocyte sedimentation rate (ESR), C reactive protein (CRP), parameters of liver function, glucose and fat homeostasis. Liver steatosis was diagnosed by abdominal ultrasound, according to known criteria and already described in a previous experience[12,13].

Controls underwent only anthropometric and clinical history assessment, because all examinations that have been performed for IBD are not indicated nor refunded by Italian Health Service.

Statistical analysisAt univariate analysis, we compared IBD patients with and without obesity. Student's t test was used for continuous variables, while the chi-square test was used for discrete variables. Correlation was analyzed by Pearson’s r. Significant factors in univariate analysis were analyzed at multivariate analysis by binary logistic regression, considering obesity as an independent variable. Odds ratios (ORs) and respective 95% confidence intervals (CI) were calculated. All analyses were two-tailed; P values < 0.05 were considered statistically significant. The analysis was carried out using SPSS.21 software for Windows.

RESULTSPatients characteristicsWe enrolled 807 patients with IBD. The process of patients selection is reported in Figure 1. Four hundred seventy-four patients were male (58.7%); the average age was 46.2 ± 13.2 years. The average age of onset of IBD was 20 ± 9.4 years and we did not find any difference of age onset between UC and CD (19.8 ± 6.8 vs 20.4 ± 6.2; P = 0.19). Of these, 438 (54.2%) patients had CD and 369 (45.8%) UC.

The majority of CD patients had an inflammatory behavior (54.1%) and an ileal localization (45.2%); perianal involvement was reported only in 3 patients (0.68%). The 24.9% of patients with CD had undergone previous surgical treatment. The average clinical disease activity detected at enrollment and assessed by Harvey Bradshaw Index was 1.3 ± 2.4.

Regarding UC, 164 (44.4%) patients had proctitis. 30 patients (8.1%) had undergone colectomy. The mean clinical activity of disease detected at the time of enrollment, assessed by Mayo partial Score, was 1.7 ± 1. Further details about baseline features of IBD population are reported in Table 1.

We enrolled 378 controls, who were comparable to IBD group for age and sex, as



Table 1 Baseline demographic and disease characteristics of inflammatory bowel disease population

Parameter mean ± SD or n (%)

Age (yr) 46.2 ± 13.2

Sex

Male 474 (58.7)

Female 333 (41.3)

Smokers 132 (16.4)

BMI (kg/m2) 24.5 ± 5.0

CD 438 (54.2)

UC 369 (45.8)

IBD age onset 20.0 ± 9.4

HBI 1.3 ± 2.4

CD behavior

Inflammatory B1 237 (54.1)

Stricturing B2 128 (29.2)

Penetrating B3 60 (16.7)

CD extension

L1 Ileal 198 (45.2)

L2 colic 44 (10.0)

L3 ileocolic 164 (37.4)

Perianal 3 (0.68)

L4 upper GI 16 (3.6)

UC extension

Proctitis E1 164 (44.4)

Left colitis E2 55 (14.9)

Pancolitis E3 123 (40.7)

Mayo Score 1.7 ± 1

CD previous surgery 109 (24.9)

Colectomy for UC 30 (8.1)

Azathioprine 300 (37.2)

Systemic corticosteroids

< 3 courses/yr 471 (58.4)

> 3 courses/yr 101 (12.5)

Topical corticosteroids

In course 94 (11.6)

Previously taken 190 (23.5)

Infliximab 147 (18.2)

Adalimumab 131 (16.2)

Golimumab 28 (3.5)

Vedolizumab 35 (4.3)

Antibiotics 451 (55.9)

Diabetes 39 (4.8)

Hypertension 79 (9.8)



Waist circumference (> 102 cm males, > 88 females) 138 (17.1)

Liver steatosis 232 (28.7)

BMI: Body mass index; UC: Ulcerative colitis; CD: Crohn’s disease; IBD: Inflammatory bowel disease; HBI: Harvey-Bradshaw index; SD: Standard deviation; GI: Gastrointestinal.

Figure 1 Flowchart reporting the process of patients selection. IBD: Inflammatory bowel disease.

shown in Table 2, reporting the main characteristics of IBD patients and controls.The prevalence of obesity was 6.9% in IBD and 7.9% in controls (not statistically

different; P = 0.38). Values of BMI were comparable between the two groups (24.5 ± 5.0 vs 24.4 ± 4.4, P = 0.74). More patients with hypertension and more smokers were observed in control group. Obesity rate did not differ between CD and UC (respectively 7.3% and 6.5%, P = 0.65).

Obesity in IBD vs controlsIn the comparison of obese IBD patients and obese controls, we did not find any difference in main comorbidities (diabetes and hypertension). No differences in sex or smoking habits were observed. Obese IBD patients were younger than obese controls (51.2 ± 14.9 years vs 60.7 ± 12.1 years, P = 0.03). Additionally, an abdominal circumference > 102 cm in males and > 88 cm in females was observed more frequently in obese IBD group (83.9% vs 46.7%, P < 0.001). The results of such analyses are graphically represented in Figure 2.

Factors associated to obesity in IBDAt univariate analysis, we compared IBD obese vs IBD non obese patients. We observed that obese ones were older than normal weight subjects (51.2 ± 14.9 vs 44.5 ± 15.8, P = 0.002). IBD onset age was earlier in obese population (44.8 ± 13.6 vs 35.6 ± 15.6, P = 0.004). We did not detect any difference regarding other considered characteristics of IBD, such as disease location according to Montreal classification. When the drugs used for IBD therapy were taken into account, obese subjects had consumed more frequently long courses of systemic steroids (66.6% vs 12.5%, P = 0.02) as well as antibiotics such as metronidazole or ciprofloxacin (71.4% vs 54.7%, P = 0.05). No difference about other drugs (biologics, mesalazine or thiopurines) was observed.

Disease activity was similar between obese and non obese subjects both for UC and CD. Indeed, among UC patients, we did not find any correlation between BMI and partial Mayo subscore (r = -0.06; P = 0.25), as illustrated in Figure 3A. Clinical remission phase was observed respectively in the 41.6% and 49.3% of obese and non obese UC patients (P = 0.53; Figure 3B). Among CD patients, we similarly did not detect any correlation between BMI and HBI (r = -0.09; P = 0.08), as reported in Figure 4A. Clinical remission phase was observed respectively in the 93.7% and 87.7% of obese and non obese CD patients (P = 0.41; Figure 4B).

Obese IBD patients suffered more frequently from arterial hypertension (42.8% vs 15.3%, P < 0.001), type 2 diabetes (21.4% vs 3.7%, P < 0.001), liver steatosis (76.8% vs 25.2%, P < 0.001), and had a significantly higher value of abdominal circumference equal to or greater than the cut-offs of central obesity (83.9% vs 17.3%, P < 0.001). Regarding laboratory investigations, obese IBD patients had higher levels of triglyceridemia (161 ± 71 vs 107 ± 55, P < 0.001), fasting blood glucose (113 ± 46 vs 89 ± 18, P < 0.001) , gamma-glutamyl-transpeptidase (0.89 ± 0.99 vs 0.55 ± 0.91, P = 0.04), and low blood levels of HDL Cholesterol (47 ± 12 vs 56 ± 19, P = 0.001).



Table 2 Main characteristics of inflammatory bowel disease patients and controls

Variable IBD (n = 807) Controls (n = 378) P value

Age 46.2 ± 13.2 45.9 ± 17.7 0.66

Male sex 474 (58.7%) 210 (55.5%) 0.30

BMI 24.5 ± 5.0 24.4 ± 4.4 0.74

Obesity 56 (6.9%) 30 (7.9%) 0.38

Diabetes 39 (4.8%) 14 (3.7%) 0.77

Smokers 132 (16.4%) 90 (23.8%) 0.002

Hypertension 79 (9.8%) 82 (21.7%) < 0.001

Abdominal circumference > 102 in males, > 88 in females 138 (17.1%) 76 (20.1%) 0.21

BMI: Body mass index; IBD: Inflammatory bowel disease.

Figure 2 Comparison between obese inflammatory bowel disease and controls. A: Age; B: Body mass index; C: Sex; D: Smoking habits; E: Diabetes; F: Hypertension; G: Abdominal circumference > 102 in males and > 88 in females; H: Liver steatosis. BMI: Body mass index.

On multivariate analysis, however, the only factor that appeared to be independently linked to obesity in IBD was the high abdominal circumference (OR = 16.3, 95%CI: 1.03-250, P = 0.04).

DISCUSSIONThe prevalence of obesity in IBD, a hot topic at the moment, has been investigated in several studies, showing highly variable values ranging from 5% to 30%[5,6]. A Scottish study based on a population of 489 IBD patients showed that 18% of patients had the features of obesity (compared to 23% of the general population); obese patients with CD were 18%, while obese patients with UC were 17.5%[14]. In the present study, we found a similar prevalence between controls and IBD which seems to confirm data from literature. The obesity rate in our cohort (6.9%) was only slightly lower than the 10.8% in the general population reported according to the European Eurostat survey[15]. Our results, additionally, underline that obesity in IBD patients has some peculiar features in comparison with obese controls. In detail, obese IBD subjects are younger than control counterpart, and this could be explained by the fact that, during



Figure 3 Correlation between ulcerative colitis clinical activity and body mass index. A: Scatterplot of body mass index and partial Mayo scores; B: Distribution of activity phase according to obesity status. BMI: Body mass index.

Figure 4 Correlation between Crohn’s disease clinical activity and body mass index. A: Scatterplot of body mass index and Harvey-Bradshaw index scores; B: Distribution of activity phase according to obesity status. BMI: Body mass index; HBI: Harvey-Bradshaw index.

the history of the disease, some factors such as steroid consumption could have favored weight gain. However, only at univariate analysis, steroid use was a predictor of obesity in IBD even if we were able to evaluate simply the exposure, and not its amount, and this could be considered as a limitation in our study. Additionally, the number of relapses occurred during the clinical history of patients was not collected from medical records, and this could be another limitation. Another feature of obese IBD patients is that they tend to have less frequently a large abdominal circumference than obese controls (Figure 2G), thus suggesting the possibility that fat distribution could be also localized in areas different than waist, such as hip or limbs. Indeed, some studies evidenced that in IBD the ratio between visceral and subcutaneous fat is altered[16] compared to healthy population, and this may explain our finding. Unfortunately, we did not take other anthropometric measurements, therefore we were unable to confirm this hypothesis.

Furthermore, we did not find any correlation between disease activity and BMI. This could be in disagreement with some literature data, showing that visceral fat[17,18] and high BMI[19] are associated with a dismal prognosis. However, our study was cross-sectional, therefore we could not evaluate the evolution of the disease during a follow up period. This could be acknowledged as another limitation. Nevertheless, some other studies did not find a strong association between BMI and disease activity and prognosis[20-23], and this underlines how this topic is still debated and with conflicting evidences.

Another important finding was that only the abdominal circumference was independently associated with obesity in IBD. We are aware that this could be an obvious result, but if we consider that it was not very common in our cohort (less than



50%), it is noteworthy to be underlined. Systemic steroids were associated with obesity only at univariate analysis, and this is an easily comprehensible link that has been already elucidated in literature[24]. Another interesting result was the most extensive use of antibiotics in obese IBD subjects. It is presumable that they could promote a dysbiosis, which in turn might facilitate the development of obesity, as already suggested by many clinical and basic science studies[25]. Finally, the high risk of diabetes, hypertension and liver steatosis is a well know phenomenon that seems to be related to obesity rather than to IBD itself[26].

CONCLUSIONIn conclusion, our study may lay the foundation for some additional speculations. Since epidemiologically IBD are increasing in developed countries, the pathogenetic role and influence on the outcome of disease played by the diet should not be underestimated and must be further investigated. In this regard, it has been already demonstrated that IBD patients, even in remission phase, tend to have a high lipid and low fiber intake[27]. Furthermore, It may be useful to plan new clinical studies aimed at evaluating clinical, laboratory and endoscopic parameters at the baseline and following BMI changes induced by dietary regimens, since this topic is still very poorly investigated.

ARTICLE HIGHLIGHTSResearch backgroundIn recent years, an increasing prevalence of obesity in inflammatory bowel disease (IBD) has been observed.

Research motivationTo investigate the relationship between obesity and IBD.

Research objectivesTo evaluate the prevalence of obesity in IBD and associated factors.

Research methodsWe collected data about IBD disease pattern and activity, drugs and laboratory investigations in our center. Anthropometric measures were retrieved and obesity defined as a body mass index (BMI) > 30. Then, we compared characteristics of obese vs non obese patients, and Chi-squared test and Student’s t test were used for discrete and continuous variables, respectively, at univariate analysis. For multivariate analysis, we used binomial logistic regression and estimated odd ratios and 95% confidence intervals to ascertain factors associated with obesity.

Research resultsThe prevalence of obesity was 6.9% in IBD and 7.9% in controls (not statistically different; P = 0.38). Obese IBD were older than normal weight ones. IBD onset age was earlier in obese population. Obese subjects had consumed more frequently long course of systemic steroids as well as antibiotics such as metronidazole or ciprofloxacin. Obese IBD patients suffered more frequently from arterial hypertension, type 2 diabetes, non-alcoholic fatty liver disease. On multivariate analysis, however, the only factor that appeared to be independently linked to obesity in IBD was the high abdominal circumference.

Research conclusionsObese IBD patients seem to have features similar to general obese population, and there is no disease-specific factor (disease activity, extension or therapy) that may foster obesity in IBD.

Research perspectivesDietary interventions to explore whether BMI variation may have some benefit on IBD course are warranted.



REFERENCESYoon KH, Lee JH, Kim JW, Cho JH, Choi YH, Ko SH, Zimmet P, Son HY. Epidemic obesity and type 2 diabetes in Asia. Lancet 2006; 368: 1681-1688 [PMID: 17098087 DOI: 10.1016/S0140-6736(06)69703-1]

1

Seravalle G, Grassi G. Obesity and hypertension. Pharmacol Res 2017; 122: 1-7 [PMID: 28532816 DOI: 10.1016/j.phrs.2017.05.013]

2

World Health Organization. Obesity. [cited April 13, 2020]. Available from: https://www.who.int/topics/obesity/en/

3

Vadan R, Gheorghe LS, Constantinescu A, Gheorghe C. The prevalence of malnutrition and the evolution of nutritional status in patients with moderate to severe forms of Crohn's disease treated with Infliximab. Clin Nutr 2011; 30: 86-91 [PMID: 20719413 DOI: 10.1016/j.clnu.2010.07.019]

4

Blain A, Cattan S, Beaugerie L, Carbonnel F, Gendre JP, Cosnes J. Crohn's disease clinical course and severity in obese patients. Clin Nutr 2002; 21: 51-57 [PMID: 11884013 DOI: 10.1054/clnu.2001.0503]

5

Seminerio JL, Koutroubakis IE, Ramos-Rivers C, Hashash JG, Dudekula A, Regueiro M, Baidoo L, Barrie A, Swoger J, Schwartz M, Weyant K, Dunn MA, Binion DG. Impact of Obesity on the Management and Clinical Course of Patients with Inflammatory Bowel Disease. Inflamm Bowel Dis 2015; 21: 2857-2863 [PMID: 26241001 DOI: 10.1097/MIB.0000000000000560]

6

Holtmann MH, Krummenauer F, Claas C, Kremeyer K, Lorenz D, Rainer O, Vogel I, Böcker U, Böhm S, Büning C, Duchmann R, Gerken G, Herfarth H, Lügering N, Kruis W, Reinshagen M, Schmidt J, Stallmach A, Stein J, Sturm A, Galle PR, Hommes DW, D'Haens G, Rutgeerts P, Neurath MF. Significant differences between Crohn's disease and ulcerative colitis regarding the impact of body mass index and initial disease activity on responsiveness to azathioprine: results from a European multicenter study in 1,176 patients. Dig Dis Sci 2010; 55: 1066-1078 [PMID: 19513841 DOI: 10.1007/s10620-009-0846-9]

7

Kurnool S, Nguyen NH, Proudfoot J, Dulai PS, Boland BS, Vande Casteele N, Evans E, Grunvald EL, Zarrinpar A, Sandborn WJ, Singh S. High body mass index is associated with increased risk of treatment failure and surgery in biologic-treated patients with ulcerative colitis. Aliment Pharmacol Ther 2018; 47: 1472-1479 [PMID: 29665045 DOI: 10.1111/apt.14665]

8

Chiu YL, Rubin DT, Vermeire S, Louis E, Robinson AM, Lomax KG, Pollack PF, Paulson SK. Serum adalimumab concentration and clinical remission in patients with Crohn's disease. Inflamm Bowel Dis 2013; 19: 1112-1122 [PMID: 23584130 DOI: 10.1097/MIB.0b013e3182813242]

9

Principi M, Mastrolonardo M, Scicchitano P, Gesualdo M, Sassara M, Guida P, Bucci A, Zito A, Caputo P, Albano F, Ierardi E, Di Leo A, Ciccone MM. Endothelial function and cardiovascular risk in active inflammatory bowel diseases. J Crohns Colitis 2013; 7: e427-e433 [PMID: 23473915 DOI: 10.1016/j.crohns.2013.02.001]

10

Principi M, Montenegro L, Losurdo G, Zito A, Devito F, Bulzis G, Carbonara R, Ierardi E, Di Leo A, Ciccone MM. Endothelial function and cardiovascular risk in patients with inflammatory bowel disease in remission phase. Scand J Gastroenterol 2016; 51: 253-255 [PMID: 26212627 DOI: 10.3109/00365521.2015.1070901]

11

Losurdo G, Iannone A, Contaldo A, Barone M, Ierardi E, Di Leo A, Principi M. Chronic Viral Hepatitis in a Cohort of Inflammatory Bowel Disease Patients from Southern Italy: A Case-Control Study. Pathogens 2020; 9 [PMID: 33113974 DOI: 10.3390/pathogens9110870]

12

European Association for the Study of the Liver (EASL). EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol 2016; 64: 1388-1402 [PMID: 27062661 DOI: 10.1016/j.jhep.2015.11.004]

13

Steed H, Walsh S, Reynolds N. A brief report of the epidemiology of obesity in the inflammatory bowel disease population of Tayside, Scotland. Obes Facts 2009; 2: 370-372 [PMID: 20090388 DOI: 10.1159/000262276]

14

European Commission. European health interview survey (EHIS). [cited April 21, 2020]. Available from: https://ec.europa.eu/eurostat/web/microdata/european-health-interview-survey

15

Bryant RV, Schultz CG, Ooi S, Goess C, Costello SP, Vincent AD, Schoeman S, Lim A, Bartholomeusz FD, Travis SPL, Andrews JM. Visceral Adipose Tissue Is Associated With Stricturing Crohn's Disease Behavior, Fecal Calprotectin, and Quality of Life. Inflamm Bowel Dis 2019; 25: 592-600 [PMID: 30215805 DOI: 10.1093/ibd/izy278]

16

Wei Y, Zhu F, Gong J, Yang J, Zhang T, Gu L, Zhu W, Guo Z, Li Y, Li N, Li J. High Visceral to Subcutaneous Fat Ratio Is Associated with Increased Postoperative Inflammatory Response after Colorectal Resection in Inflammatory Bowel Disease. Gastroenterol Res Pract 2018; 2018: 6270514 [PMID: 29849595 DOI: 10.1155/2018/6270514]

17

Stidham RW, Waljee AK, Day NM, Bergmans CL, Zahn KM, Higgins PD, Wang SC, Su GL. Body fat composition assessment using analytic morphomics predicts infectious complications after bowel resection in Crohn's disease. Inflamm Bowel Dis 2015; 21: 1306-1313 [PMID: 25822011 DOI: 10.1097/MIB.0000000000000360]

18

Yerushalmy-Feler A, Ben-Tov A, Weintraub Y, Amir A, Galai T, Moran-Lev H, Cohen S. High and low body mass index may predict severe disease course in children with inflammatory bowel disease. Scand J Gastroenterol 2018; 53: 708-713 [PMID: 29688090 DOI: 10.1080/00365521.2018.1464595]

19

Mentella MC, Scaldaferri F, Pizzoferrato M, Gasbarrini A, Miggiano GAD. The Association of Disease Activity, BMI and Phase Angle with Vitamin D Deficiency in Patients with IBD. Nutrients

20


https://dx.doi.org/10.1016/S0140-6736(06)69703-1


https://dx.doi.org/10.1016/j.phrs.2017.05.013

https://www.who.int/topics/obesity/en/


https://dx.doi.org/10.1016/j.clnu.2010.07.019


https://dx.doi.org/10.1054/clnu.2001.0503


https://dx.doi.org/10.1097/MIB.0000000000000560


https://dx.doi.org/10.1007/s10620-009-0846-9


https://dx.doi.org/10.1111/apt.14665


https://dx.doi.org/10.1097/MIB.0b013e3182813242


https://dx.doi.org/10.1016/j.crohns.2013.02.001


https://dx.doi.org/10.3109/00365521.2015.1070901


https://dx.doi.org/10.3390/pathogens9110870




https://dx.doi.org/10.1159/000262276

https://ec.europa.eu/eurostat/web/microdata/european-health-interview-survey


https://dx.doi.org/10.1093/ibd/izy278


https://dx.doi.org/10.1155/2018/6270514


https://dx.doi.org/10.1097/MIB.0000000000000360


https://dx.doi.org/10.1080/00365521.2018.1464595



2019; 11 [PMID: 31717788 DOI: 10.3390/nu11112583]Connelly TM, Juza RM, Sangster W, Sehgal R, Tappouni RF, Messaris E. Volumetric fat ratio and not body mass index is predictive of ileocolectomy outcomes in Crohn's disease patients. Dig Surg 2014; 31: 219-224 [PMID: 25277149 DOI: 10.1159/000365359]

21

Singh S, Proudfoot J, Xu R, Sandborn WJ. Obesity and Response to Infliximab in Patients with Inflammatory Bowel Diseases: Pooled Analysis of Individual Participant Data from Clinical Trials. Am J Gastroenterol 2018; 113: 883-889 [PMID: 29867171 DOI: 10.1038/s41395-018-0104-x]

22

Singh S, Proudfoot J, Xu R, Sandborn WJ. Impact of Obesity on Short- and Intermediate-Term Outcomes in Inflammatory Bowel Diseases: Pooled Analysis of Placebo Arms of Infliximab Clinical Trials. Inflamm Bowel Dis 2018; 24: 2278-2284 [PMID: 29788260 DOI: 10.1093/ibd/izy135]

23

Jahnsen J, Falch JA, Mowinckel P, Aadland E. Body composition in patients with inflammatory bowel disease: a population-based study. Am J Gastroenterol 2003; 98: 1556-1562 [PMID: 12873577 DOI: 10.1111/j.1572-0241.2003.07520.x]

24

Cammarota G, Ianiro G, Cianci R, Bibbò S, Gasbarrini A, Currò D. The involvement of gut microbiota in inflammatory bowel disease pathogenesis: potential for therapy. Pharmacol Ther 2015; 149: 191-212 [PMID: 25561343 DOI: 10.1016/j.pharmthera.2014.12.006]

25

Principi M, Iannone A, Losurdo G, Mangia M, Shahini E, Albano F, Rizzi SF, La Fortezza RF, Lovero R, Contaldo A, Barone M, Leandro G, Ierardi E, Di Leo A. Nonalcoholic Fatty Liver Disease in Inflammatory Bowel Disease: Prevalence and Risk Factors. Inflamm Bowel Dis 2018; 24: 1589-1596 [PMID: 29688336 DOI: 10.1093/ibd/izy051]

26

Principi M, Losurdo G, Iannone A, Contaldo A, Deflorio V, Ranaldo N, Pisani A, Ierardi E, Di Leo A, Barone M. Differences in dietary habits between patients with inflammatory bowel disease in clinical remission and a healthy population. Ann Gastroenterol 2018; 31: 469-473 [PMID: 29991892 DOI: 10.20524/aog.2018.0273]

27


https://dx.doi.org/10.3390/nu11112583


https://dx.doi.org/10.1159/000365359






https://dx.doi.org/10.1111/j.1572-0241.2003.07520.x


https://dx.doi.org/10.1016/j.pharmthera.2014.12.006




https://dx.doi.org/10.20524/aog.2018.0273


World Journal of



ORIGINAL ARTICLE

Retrospective Study

Towards an evaluation of alcoholic liver cirrhosis and nonalcoholic fatty liver disease patients with hematological scales

Agata Michalak, Halina Cichoż-Lach, Małgorzata Guz, Joanna Kozicka, Marek Cybulski, Witold Jeleniewicz, Andrzej Stepulak

ORCID number: Agata Michalak 0000-0003-4426-6321; Halina Cichoż-Lach 0000-0002-7337-835X; Małgorzata Guz 0000-0001-6783-8017; Joanna Kozicka 0000-0002-3094-8789; Marek Cybulski 0000-0003-0540-1199; Witold Jeleniewicz 0000-0003-1423-0504; Andrzej Stepulak 0000-0002-1872-394X.

Author contributions: Michalak A and Cichoż-Lach H designed and coordinated the study; Guz M, Kozicka A and Jeleniewicz W performed the experiments, acquired and analyzed data; Cybulski M and Stepulak A interpreted the data; Michalak A and Cichoż-Lach H wrote the manuscript; all authors approved the final version of the article.

Institutional review board statement: The local ethics committee of the Medical University of Lublin approved the study (Approval No. KE-0254/86/2016).

Informed consent statement: All patients signed an informed written consent in accordance with the Helsinki Declaration for the procedures they underwent.

Conflict-of-interest statement: Nothing to disclose.

Agata Michalak, Halina Cichoż-Lach, Joanna Kozicka, Department of Gastroenterology, Medical University of Lublin, Lublin 20-954, Jaczewskiego 8, Poland

Małgorzata Guz, Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin 20-093, Chodźki 3, Poland

Marek Cybulski, Witold Jeleniewicz, Andrzej Stepulak, Department of Biochemistry and Molecular Biology, Medical Univeristy of Lublin, Lublin 20-093, Chodźki 3, Poland

Corresponding author: Halina Cichoż-Lach, MD, PhD, Professor, Department of Gastroenterology, Medical University of Lublin, Jaczewskiego 8, Lublin 20-954, Jaczewskiego 8, Poland. [email protected]

AbstractBACKGROUND Seeking potentially novel blood markers of liver fibrosis and steatosis is constantly of crucial importance. Despite a growing number of studies in this field of hepatology, a certain role of hematological indices in the course of liver disorders has not been fully elucidated, yet.

AIM To evaluate a diagnostic accuracy of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and mean platelet volume-to-platelet-ratio (MPR) in the course of alcoholic liver cirrhosis (ALC) and nonalcoholic fatty liver disease (NAFLD).

METHODS One hundred forty-two patients with ALC, 92 with NAFLD and 68 persons in control group were enrolled in the study. Hematological indices (NLR, PLR and MPR), indirect and direct markers of liver fibrosis (aspartate transaminase to alkaline transaminase ratio, aspartate transaminase to platelet ratio index, fibrosis-4, gamma-glutamyl transpeptidase to platelet ratio, procollagen I carboxyterminal propeptide, procollagen III aminoterminal propeptide, transforming growth factor-α, platelet-derived growth factor AB, laminin) were measured in each person. Model for end-stage liver disease (MELD) score in ALC group and NAFLD fibrosis score together with BARD score were calculated in NAFLD patients. Receiver operating characteristic (ROC) curves and area under the curve



http://orcid.org/0000-0003-4426-6321

http://orcid.org/0000-0003-4426-6321

http://orcid.org/0000-0002-7337-835X

http://orcid.org/0000-0002-7337-835X

http://orcid.org/0000-0001-6783-8017

http://orcid.org/0000-0001-6783-8017

http://orcid.org/0000-0002-3094-8789

http://orcid.org/0000-0002-3094-8789

http://orcid.org/0000-0002-3094-8789

http://orcid.org/0000-0003-0540-1199

http://orcid.org/0000-0003-0540-1199

http://orcid.org/0000-0003-0540-1199

http://orcid.org/0000-0003-1423-0504

http://orcid.org/0000-0003-1423-0504

http://orcid.org/0000-0002-1872-394X

http://orcid.org/0000-0002-1872-394X


Michalak A et al. Hematological scales in ALC and NAFLD


Data sharing statement: Dataset available from the corresponding author at [email protected].




Country/Territory of origin: Poland

Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0

Received: July 27, 2020 Peer-review started: July 27, 2020 First decision: September 30, 2020 Revised: October 12, 2020 Accepted: November 29, 2020 Article in press: November 29, 2020 Published online: December 21, 2020

P-Reviewer: Dumitrascu DL, Lan C, Que J S-Editor: Gao CC L-Editor: A P-Editor: Liu JH

(AUC) values were applied to assess the sensitivity and specificity of examined markers and to evaluate proposed cut-offs of measured indices in the course of ALC and NAFLD.

RESULTS MPR and NLR values in ALC patients were significantly higher in comparison to control group; PLR level was significantly lower. MPR and PLR correlated with assessed indirect and direct markers of liver fibrosis. MPR, NLR and PLR correlated with MELD score. NLR level in NAFLD patients was significantly higher in comparison to controls. MPR correlated with indirect markers of liver fibrosis and NAFLD fibrosis score. AUC values and proposed cut-offs for NLR, PLR and MPR in ALC patients were: 0.821 (> 2.227), 0.675 (< 70.445) and 0.929 (> 0.048), respectively. AUC values and proposed cut-offs for NLR, PLR and MPR in NAFLD group were: 0.725 (> 2.034), 0.528 (> 97.101) and 0.547 (> 0.038), respectively.

CONCLUSION Hematological markers are inseparably connected with serological indices of liver fibrosis in ALC and NAFLD patients. MPR and NLR turned out to be the most powerful parameters in ALC patients.

Key Words: Hematological markers; Alcoholic liver cirrhosis; Nonalcoholic fatty liver disease; Neutrophil-to-lymphocyte ratio; Platelet-to-lymphocyte ratio; Mean platelet volume-to-platelet-ratio


Core Tip: Neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and mean platelet volume-to-platelet-ratio (MPR) seem to be unexplored in Polish population of patients with alcoholic liver cirrhosis (ALC) and non-alcoholic fatty liver disease (NAFLD). What is more, according to available literature, relationships between NLR, MPR, PLR and serological (indirect and indirect) markers of liver fibrosis have never been investigated in a single study, yet. We found MPR to be a parameter with high diagnostic accuracy in the course ALC, correlating with model for end-stage liver disease score and serological markers of liver fibrosis. Hematological indices should be considered as potential tools in the noninvasive diagnostics in hepatology.

Citation: Michalak A, Cichoż-Lach H, Guz M, Kozicka J, Cybulski M, Jeleniewicz W, Stepulak A. Towards an evaluation of alcoholic liver cirrhosis and nonalcoholic fatty liver disease patients with hematological scales. World J Gastroenterol 2020; 26(47): 7538-7549URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7538.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7538

INTRODUCTIONA reliable noninvasive assessment of liver fibrosis remains a key goal in the field of hepatology. Liver biopsy is still perceived as a gold standard, however elastography in ultrasound or magnetic resonance mode have gained importance. Despite a great advance in the development of imaging techniques, simple blood surrogates in liver fibrosis would be the most appreciated diagnostic tools. A new potential player has been arising among direct and indirect markers of liver fibrosis for several years—hematological parameters. The utility of hematological indices definitely exceeded differential diagnosis of anemia or inflammatory process. It came out several years ago that routinely used parameters, like neutrophil (NEU)-to-lymphocyte (LYM) ratio (NLR), platelet (PLT)-to-LYM ratio (PLR) and mean PLT volume (MPV)-to-PLT-ratio (MPR) can be applied as markers of the prognosis in cancer, inflammatory bowel disease and cardiovascular patients. Some reports proved their involvement in the course of liver disorders, too[1-4]. Nevertheless, they are present in subsequent surveys









rather than in everyday clinical practice. A vast majority of studies explored a role of NLR and PLR in the decompensation of liver fibrosis or the development of hepatocellular carcinoma (HCC) due to a tight linkage between liver pathologies and inflammation. Moreover, MPR was described in a single study as a predictor of liver fibrosis[5-9]. But available data on their role in the course of liver disorders are still scanty and unclear. Subsequently, a potential role of hematological indices has been poorly explored in the course of liver steatosis.

For these reasons we decided to explore NLR, PLR and MPR role in alcohol-related liver cirrhosis (ALC) and nonalcoholic fatty liver disease (NAFLD) patients and to find out if there are any dependences between these hematological indices and serological (indirect and direct) markers of liver fibrosis. To the best of our knowledge, correlations between aforementioned hematological indices and serological markers of liver fibrosis have not been explored in a single study, yet and PLR has not been explored in NAFLD population, either. Because of a great worldwide clinical significance of ALC and NAFLD we decided to explore this group of patients. According to already collected data, a potential value of hematological indices in the populations of patients with ALC and NAFLD is poorly explored. Moreover, it appears to be the first study on Polish patients, assessing the relationships between hematological markers and serological indices of liver fibrosis.

MATERIALS AND METHODSThe local ethics committee of the Medical University of Lublin approved the study (No. KE-0254/86/2016) and all patients signed an informed written consent in accordance with the Helsinki Declaration for the procedures they underwent.

Study population and research design This study assessed 302 persons: 142 patients with ALC, 92 with NAFLD and 68 healthy volunteers in control group. Table 1 presents clinical features of study population. The diagnosis of liver cirrhosis was based on commonly used criteria. The presence of portal hypertension was proved in the doppler mode abdominal ultrasound examination (diameter of portal vein ≥ 13 mm) and other potential reasons of existing portal hypertension were excluded. All ALC patients underwent panendoscopy of the gastrointestinal tract — in 126 persons varices of the esophagus/stomach in the different stage were found. Ninety-two people were diagnosed with ascites and 84 of them underwent paracentesis. The presence of hepatic encephalopathy and spontaneous bacterial peritonitis were excluded in the whole group. All participants included to the survey gained 0/9 points in clinical hepatic encephalopathy staging scale (CHESS) scale. Alcoholic background of liver cirrhosis (LC) was diagnosed according to the proved daily intake of pure ethanol exceeding 30 g. A history of alcohol abuse was obtained directly from the patients or their family members. Moreover, all enrolled in the study ALC patients presented positive result of CAGE test. A diagnosis of NAFLD was established due to the history, physical examination, laboratory testing, and ultrasound imaging. A daily alcohol consumption did not exceed 20 g in men and 10 g in women. Certain diseases that can lead to steatosis (hepatobiliary infections, celiac disease, Wilson's disease, and alpha-1-antitrypsin deficiency) have been excluded. Twenty-two persons were diagnosed with diabetes mellitus type 2. People with diabetes mellitus type 1 were excluded from the study. None of the patients presented impaired fasting glucose. Forty-six NAFLD patients were found to have arterial hypertension and metabolic syndrome was diagnosed in 84 persons. Viral, cholestatic and autoimmune liver disorders together with the presence of clinically significant inflammatory process were excluded in all participants. Antinuclear antibody (ANA), antimitochondrial antibody (AMA), anti-smooth muscle antibodies (ASMA), liver-kidney microsome type 1 (anti-LKM-1) antibodies, hepatitis B virus (HBV) and hepatitis C virus (HCV) tests were negative. Hepatobiliary infections, celiac disease, Wilson’s disease, and alpha-1-antitrypsin deficiency were excluded as well. We aimed to exclude potential factors influencing the level of hematological parameters evaluated in our survey. None of the persons included to the study was on steroid therapy.

ProceduresVenous blood samples (peripheral blood) were collected from the studied patients and controls (S-Monovette, SARSTEDT, Aktiengesellschaft and Co., Nubrecht, Germany). Ethylenediamine tetraacetic acid was used to obtain hematological parameters and



Table 1 Clinical characteristics of study participants

Parameter ALC (n = 142) NAFLD (n = 92) Controls (n = 68) Together (n = 302)

Sex (F/M) 36/106 33/59 36/32 105/197

Age (yr), (mean ± SD; median; min-max) 54 ± 12; 55; 31-84 60 ± 15; 61; 22-90 46 ± 16; 45; 20-85 54 ± 15; 55; 20-90

BMI (kg/m2) (mean ± SD; median; min-max)

25.89 ± 9.31; 25.91; 16.7-36.71

29.49 ± 4.9; 28.7; 16.26-43.01

21.95 ± 2.62; 22.45; 16.18-24.86

-

DM type 2 0/142 22/92 - -

AH 32/142 46/92 - -

F: Female; M: Male; SD: Standard deviation; min: Minimum; max: Maximum; BMI: Body mass index; DM: Diabetes mellitus; AH: Arterial hypertension.

citrate to assess clotting indices. Biochemical markers were measured from the remaining blood sample without anticoagulant. The blood was obtained after at least 12 h of fasting. Hematological and biochemical parameters were obtained 4 h after blood samples collection. All the tests were performed in the laboratory of Clinical Hospital Number 4, Lublin, Poland. The analysis of morphotic blood indices was done with automatic ADVIA 2120i analyzer, Siemens and biochemical markers with ADVIA 1800 analyzer, Siemens. Prothrombin time (PT) and its International Normalized Ratio (INR) were measured with ACL TOP 500 analyzer, Instrumentation Laboratory. The part of blood samples without an anticoagulant was centrifuged at speed 2000 g for 10 min within 15 min from blood collection. Obtained serum was stored in 1 mL Eppendorf test tubes in the temperature of -80° Celsius until the measurement of direct markers of liver fibrosis with enzyme-linked immunosorbent assay (ELISA). Among morphotic parameters of the blood NLR, PLR and MPR were obtained. The assessment of indirect indices of liver fibrosis included: AAR — AST (aspartate transaminase)/ALT (alkaline transaminase) (AST to ALT Ratio), APRI — [(AST/*ULN)/PLT × (109/L)] × 100; *ULN — upper limit of normal (AST to PLT Ratio Index), FIB-4 — [age × AST/PLT × (109/L)] × ALT1/2 (fibrosis-4), GPR — [GGT (γ-glutamyl transpeptidase)/ULN/PLT × (109/L)] × 100 (GGT to PLT Ratio). Model for end-stage liver disease (MELD) score was used in ALC patients and NAFLD fibrosis score and BARD score were used in NAFLD group: MELD - 3.8 [*Ln bilirubin (mg/dL)] + 11.2 [Ln INR] + 9.6 [Ln creatinine (mg/dL)] + 6.4. *Ln — natural logarithm, NAFLD fibrosis score - (-1.675) + 0.037 × age (years) + 0.094 × BMI (body mass index) (kg/m2) + 1.13 × impaired fasting glucose/diabetes (YES — 1 point, NO — 0 points) + 0.99 × AST/ALT - 0.013 × PLT (× 109/L) - 0.66 × albumin (mg/dL), BARD score — AST/ALT ≥ 0.8, 2 points, BMI ≥ 28, 1 point; IFG/diabetes, 1 point; together 0-4 points. Among direct indices of liver fibrosis, procollagen I carboxyterminal propeptide (PICP), procollagen III aminoterminal propeptide (PIIINP), platelet-derived growth factor AB (PDGF-AB), transforming growth factor-α (TGF-α) and laminin were obtained. Laboratory test were done in the Department of Biochemistry and Molecular Biology, Medical University of Lublin according to the manufacturer's instructions. The measurement of PICP and PIIINP was performed with quantitative ELISA tests (Wuhan EIAab Science, Wuhan China). The measurement of PDGF-AB and TGFα was done with R&D Systems Quantikine ELISA Kits (Minneapolis, MN, United States). Finally, the measurement of laminin was performed with Takara Laminin EIA Kit without Sulphuric Acid (Kusatsu, Shiga, Japonia).

Statistical analyses Statistical analysis of the results was conducted using Statistica 13.0 (StatSoft Polska Sp. z o.o., Kraków, Poland) for Windows system. The demographic data and results of laboratory tests were presented as the mean value ± standard deviation and Student’s t test was used to compare these results. Deviation from normality was evaluated by Kolmogorov–Smirnov test. Data were expressed as the median and range (minimum- maximum). The Mann-Whitney U test was used for between-group comparisons because of non-normal distribution. Spearman correlation analyses were used to verify the correlations. All probability values were two-tailed, and a value of P less than 0.05 was considered statistically significant. Receiver operating characteristic (ROC) curves and area under the curve (AUC) values were applied to assess the sensitivity and specificity of examined markers and to evaluate proposed cut-offs of measured indices



in the course of ALC and NAFLD.

RESULTSTable 2 shows results of used scores in research group. Table 3 presents results of hematological indices and serological (indirect and direct) markers of liver fibrosis in examined patients. MPR and NLR medians in ALC groups were significantly higher in comparison to controls (P < 0.0001); PLR level was significantly lower (P < 0.0001). NLR level in NAFLD patients was significantly higher compared to control group (P < 0.0001). MPR and PLR values did not differ significantly. The analysis of AAR, APRI, FIB-4 and GPR revealed their significantly higher medians in ALC patients compared to controls (P < 0.0001). Except for AAR, patients with NAFLD were found to have significantly higher values of all above-mentioned indices in comparison to controls (P < 0.0001). Among direct markers of liver fibrosis, laminin median in ALC group was significantly higher than in controls (P < 0.05). Beside of PICP, medians of PIIINP, PDGF-AB and TGF-α were significantly lower (P < 0.01, P < 0.001, P < 0.0001, respectively). Medians of TGF-α and laminin in NAFLD patients compared to controls turned out to be significantly lower (P < 0.0001). PICP, PIIINP and PDGF-AB medians did not differ significantly. Table 4 shows observed correlations between assessed markers in ALC and NAFLD patients. MPR and PLR correlated positively with indirect markers of liver fibrosis (APRI, FIB-4; P < 0.001) in examined ALC patients. Positive (but weaker) relationships were found between NLR and both: AAR and GPR (P < 0.05). PLR correlated positively with PDGF-AB and MPR-negatively (P < 0.001 and P < 0.01, respectively); a negative relationship was observed between NLR and PIIINP (P < 0.05). MELD score correlated positively with both: NLR and MPR (P < 0.0001) and negatively with PLR (P < 0.001). MPR correlated positively with indirect markers of liver fibrosis—APRI (P < 0.0001), FIB-4 (P < 0.0001) and GPR (P < 0.01) in NAFLD group. A strong positive relationship between MPR and NAFLD fibrosis score was noted, too (P < 0.0001). Diagnostic accuracy of examined hematological indices is shown in Table 5. ROCs presenting examined parameters in ALC and NAFLD patients are presented below in Figures 1-3. AUC values and proposed cut-offs for NLR, PLR and MPR in ALC patients were: 0.821 (> 2.227), 0.675 (< 70.445) and 0.929 (> 0.048), respectively. AUC values and proposed cut-offs for NLR, PLR and MPR in NAFLD patients were: 0.725 (> 2.034), 0.528 (< 97.101) and 0.547 (> 0.038), respectively.

DISCUSSIONMonitoring of liver fibrosis and clinical decompensation of liver failure with reliable and simple noninvasive markers obtained from the blood are two of the most essential research pathways in hepatology. On the other hand, the detection and careful monitoring of liver steatosis is also of great importance because of a significant prevalence of NAFLD all over the world and its possible severe complications. Looking for meaningful dependences between hematological parameters and the phenomenon of liver disorders has been intriguing scientists for several years. Despite their proved involvement in the course of liver fibrosis, there is still no clear answer whether to include them into the panel of diagnostic tests assessing cirrhotic patients. There were numerous attempts to evaluate a potential role of NLR in this area. Its increased level is explained to be the result of the release of interleukin-6 and tumor necrosis factor α together with coexisting bacterial translocation, followed by elevated NEUs count. Simultaneously, activated immune cells releasing cytokines and reactive oxygen species may inhibit lymphocytic immune response[10]. Of note, high level of NLR has been already proposed in several observations as a predictor of mortality in cirrhotic patients (independently from MELD score)[11-20]. Recently, Abu Omar et al[21] found NLR to be the marker of poor survival in alcoholic hepatitis patients, too. A coexisting inflammatory process (independent from liver cirrhosis) is an essential limitation connected with the utility of NLR and influencing its reliability. Thus, we excluded from our study all the participants suspected of the inflammation. NLR in studied ALC and NAFLD groups was characterized by quite high diagnostic accuracy (AUC = 0.821 and AUC = 0.725, respectively). It correlated significantly with MELD score and serological (AAR, GPR, PIIINP) markers of liver fibrosis in ALC patients. The role of NLR in the course of NAFLD remains ambiguous, however there are evidences suggesting that an increase in NLR might accompany the transformation from simple steatosis to steatohepatitis, highlighting the role of inflammatory process



Table 2 Results of used scores in research group

ALC NAFLDScore

Mean SD Median Min Max Mean SD Median Min Max

MELD 17 8 16 6 45 - - - - -

BARD - - - - - 2 1 2 0 4

NAFLD fibrosis score - - - - - -1.36 1.5 -1.16 -5.83 1.74

SD: Standard deviation; Min: Minimum; Max: Maximum; MELD: Model for end-stage liver disease; NAFLD: Nonalcoholic fatty liver disease.

in the elevation of NLR[22,23]. PLR seems to be mostly explored among chronic HBV/HCV patients — recent investigations were performed by Lu et al[24] and Alsebaey et al[25]. Lower values of this parameter accompanied more advanced liver fibrosis, but the number of existing surveys is definitely small. On the other hand, high levels of PLR (together with NLR) were noted in patients with more advanced HCC and greater recurrence risk; similar observations concerned patients with pancreatic cancer and cholangiocarcinoma[26-29]. To the best of our knowledge, this is the first study figuring out the role of PLR in ALC and NAFLD population. PLR had relatively moderate diagnostic value in the research group, but it was significantly lower compared to controls and correlated with MELD score and both APRI and FIB-4 in ALC patients. It was carried out in former studies that higher levels of MPR correspond with histopathologically diagnosed liver cirrhosis; however available data on this issue are strictly limited and do not concern ALC and NAFLD patients. Cho et al[30] even found MPR as a potential marker of the development of HCC. In our studied ALC patients MPR obtained high diagnostic accuracy (AUC = 0.928); a cut-off value of 0.048 had a sensitivity of 85% and a specificity of 94%. It also correlated significantly with MELD score, serum concentration of PDGF-AB, APRI and FIB-4. According to available literature, it seems to be the first report concerning dependences between PLR and serological markers of liver fibrosis. In NAFLD group PLR level did not differ significantly from controls.

The goal of our survey was not to compare a diagnostic accuracy of selected hematological indices between ALC and NAFLD patients. We tried to figure out whether an isolated liver steatosis might be affected by certain deviations in hematological indices. Our survey evaluated the population of patients with NAFLD without the assessment of coexisting hepatitis in liver biopsy. A general division of the research group into only two subgroups (ALC and NAFLD) can be perceived as a limitation, however it was the beginning of our exploration in this field of hepatology and our further direction will be the evaluation of the markers presented in this study among patients with different stages of ALC and NAFLD, including simple steatosis and steatohepatitis. A clinical stage of ALC was evaluated with MELD score and we did not find any significant differences according to the severity of the disease. The idea of the current study was caused by our clinical practice and a common presence of hematological parameters disturbances in the patients with liver disorder, especially ALC and NAFLD. These pathologies have an unquestionable global impact and there is still a great demand on finding new markers in their monitoring. PLR and MPR have been poorly explored in ALC and NAFLD patients, so far and the current study fills this important gap. Hematological markers are inseparably connected with serological markers of liver fibrosis in ALC and NAFLD patients. MPR and NLR turned out to be the most powerful markers in ALC patients.

CONCLUSIONIn conclusion, we demonstrated that NLR, MPR and PLR belong to hematological parameters with a relatively high diagnostic accuracy especially in the course of ALC. They are closely related to indirect and direct markers of liver fibrosis. Moreover, NLR, MPR and PLR seem to correlate with a clinical progression of liver cirrhosis (MELD score). These relationships propose evaluated hematological indices to be explored as potential parameters of liver disorders, especially liver cirrhosis.



Table 3 Results of hematological indices and serological (indirect and indirect) markers of liver fibrosis in examined patients

ALC NAFLD ControlsParameter (reference range) Mean SD Median Min Max Mean SD Median Min Max Mean SD Median Min Max

NLR 6.3 6.99 4.09d 0.53 49.84 3.4 2.84 2.63d 0.17 17.33 1.97 1.09 1.77 0.81 6.2

PLR 120.85 87.18 99.49d 0.7 435.82 182.78 128.93 139.55 8.94 742.86 154.88 64.92 141.59 56.9 327.27

MPR 0.15 0.29 0.09d 0.02 3.28 0.04 0.02 0.03 0.01 0.1 0.03 0.01 0.03 0.02 0.06

AAR 2.19 1.16 1.89d 0.18 7.57 1.03 0.55 0.91a 0.23 3.1 1.15 0.43 1.1 0.43 2.86

APRI 4.35 7.02 2.43d 0.15 68.38 0.81 1.04 0.48d 0.13 7.67 0.25 0.13 0.23 0.11 0.86

FIB-4 11.67 25.46 6.34d 0.69 287.59 1.92 1.63 1.57d 0.23 11.58 0.85 0.54 0.71 0.28 3.27

GPR 15.73 28.54 6.65d 0.18 188.71 2.76 5.57 0.54d 0.13 35.41 0.25 0.1 0.24 0.06 0.63

PICP (ng/mL) 63.32 31.53 60.53 6.15 161.12 52.14 27.56 46.08 10.10 147.27 58.26 37.39 44.18 0 202.89

PIIINP (ng/mL) 9.28 4.33 8.4b 2.43 28.65 11.41 3.99 11.00 2.18 25.35 11.07 5.61 10.25 4.35 43.63

PDGF-AB (pg/mL) 18280.47 8061.06 17343.71c 1925.68 42823.84 26858.68 7335.09 26682.83 10821.02 49808.07 23579.28 10068.8 25623.2 1638.2 47758.7

TGF-α (pg/mL) 24 45.33 13.77d 0.872 507.09 17.89 19.18 12.09d 1.39 142.63 28.44 17.21 24.59 1.31 93.55

Laminin (ng/mL) 976.34 705.29 832.06a 101.933 3301.00 48 230.24 375.23d 72.87 1335.92 718.24 386.1 663.27 140.88 1813.88

aP < 0.05.bP < 0.01.cP < 0.001.dP < 0.0001. ALC: Alcoholic liver cirrhosis; NAFLD: Nonalcoholic fatty liver disease; SD: Standard deviation; Min: Minimum; Max: Maximum; NLR: Neutrophil-to-lymphocyte ratio; PLR: Platelet-to-lymphocyte ratio; MPR: Mean platelet volume-to-platelet-ratio; AAR: Aspartate transaminase to alkaline transaminase ratio; APRI: Aspartate transaminase to platelet ratio index; FIB-4: Fibrosis-4; GPR: Gamma-glutamyl transpeptidase to platelet ratio; PICP: Procollagen I carboxyterminal propeptide; PIIINP: Procollagen III aminoterminal propeptide; PDGF-AB: Platelet-derived growth factor AB; TGF-α: Transforming growth factor-α.



Table 4 Correlations between examined parameters in examined alcoholic liver cirrhosis and nonalcoholic fatty liver disease patients

Pair R Spearman P value

ALC

MPR and APRI 0.691 c

MPR and FIB-4 0.776 c

NLR and AAR 0.173 a

NLR and GPR 0.183 a

PLR and APRI -0.535 c

PLR and FIB-4 -0.557 c

MPR and MELD 0.343 d

NLR and MELD 0.379 d

PLR and MELD -0.235 b

NLR and PIIINP -0.183 a

MPR and PDGF-AB -0.366 c

PLR and PDGF-AB 0.272 b

NAFLD

MPR and APRI 0.557 d

MPR and FIB-4 0.603 d

MPR and GPR 0.303 b

MPR and NFS 0.587 d

aP < 0.05.bP < 0.01.cP < 0.001.dP < 0.0001. ALC: Alcoholic liver cirrhosis; NAFLD: Nonalcoholic fatty liver disease; NLR: Neutrophil-to-lymphocyte ratio; PLR: Platelet-to-lymphocyte ratio; MPR: Mean platelet volume-to-platelet-ratio; AAR: Aspartate transaminase to alkaline transaminase ratio; APRI: Aspartate transaminase to platelet ratio index; FIB-4: Fibrosis-4; GPR: Gamma-glutamyl transpeptidase to platelet ratio; PICP: Procollagen I carboxyterminal propeptide; PIIINP: Procollagen III aminoterminal propeptide; PDGF-AB: Platelet-derived growth factor AB.

Table 5 Diagnostic accuracy of hematological indices in examined alcoholic liver cirrhosis and nonalcoholic fatty liver disease patients

ALC NAFLD

Diagnostic accuracy Diagnostic accuracyParameter

AUC Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

P value AUC Sensitivity

(%)Specificity (%)

PPV (%)

NPV (%)

P value

NLR 0.821 78 80 89 63 d 0.725 72 71 77 65 d

PLR 0.675 35 97 96 42 d 0.528 88 18 59 52 -

MPR 0.929 85 94 97 75 d 0.547 39 78 71 49 -

dP < 0.0001. ALC: Alcoholic liver cirrhosis; NAFLD: Nonalcoholic fatty liver disease; AUC: Area under the curve; PPV: Positive predictive value; NPV: Negative predictive value.



Figure 1 Receiver operating characteristics for neutrophil-to-lymphocyte ratio in alcoholic liver cirrhosis and nonalcoholic fatty liver disease groups. Area under the curve value (AUC) = 0.821 (cut-off > 2.227) and AUC = 0.725 (cut-off > 2.034), respectively. A: Alcoholic liver cirrhosis; B: Nonalcoholic fatty liver disease.

Figure 2 Receiver operating characteristics for platelet-to-lymphocyte ratio in alcoholic liver cirrhosis and nonalcoholic fatty liver disease groups. Area under the curve value (AUC) = 0.675 (cut off < 70.445) and AUC = 0.528 (cut-off < 97.101), respectively. A: Alcoholic liver cirrhosis; B: Nonalcoholic fatty liver disease.

Figure 3 Receiver operating characteristics for mean platelet volume-to-platelet-ratio in alcoholic liver cirrhosis (A) and nonalcoholic fatty liver disease (B) groups. Area under the curve value (AUC) = 0.929 (cut-off > 0.048) and AUC = 0.547 (cut-off > 0.038), respectively. A: Alcoholic liver cirrhosis; B: Nonalcoholic fatty liver disease.



ARTICLE HIGHLIGHTSResearch backgroundA noninvasive evaluation of liver fibrosis remains still an unexplored field of hepatology. Seeking potentially new parameters of liver disease progression is constantly a key task among hepatologists. Recently several new hematological markers have been proposed as potential indices in the monitoring of alcoholic liver cirrhosis (ALC) and non-alcoholic fatty liver disease (NAFLD) patients, however the number of available studies on them is strictly limited.

Research motivationSo far there is little evidence about the potential relationships between hematological indices [neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and mean platelet volume-to-platelet-ratio (MPR)] and serological markers of liver fibrosis in the course of ALC and NAFLD. Available data suggest their potential role in the monitoring and prediction of outcome in liver diseases.

Research objectivesWe performed a retrospective study to evaluate the clinical utility of selected hematological indices and their potential relationships with serological markers of liver fibrosis among patients with ALC and NAFLD.

Research methodsOne hundred forty two patients with ALC, 92 with NAFLD and 68 persons in control group were enrolled in the study. Hematological indices (NLR, PLR and MPR), indirect and direct markers of liver fibrosis [AST and ALT ratio (AAR), AST to platelet ratio index (APRI), fibrosis-4 (FIB-4), gamma-glutamyl transpeptidase to platelet ratio (GPR), procollagen I carboxyterminal propeptide (PICP), procollagen III aminoterminal propeptide (PIIINP), platelet-derived growth factor AB (PDGF-AB), transforming growth factor-α (TGF-α) and laminin] were measured in each person. Model for end-stage liver disease (MELD) score in ALC group and NAFLD fibrosis score together with BARD score were calculated in NAFLD patients. Receiver operating characteristic (ROC) curves and area under the curve (AUC) values were applied to assess the sensitivity and specificity of examined markers and to evaluate proposed cut-offs of measured indices in the course of ALC and NAFLD.

Research resultsMPR and NLR values in ALC patients were significantly higher compared to control group; PLR level was significantly lower. MPR and PLR correlated with assessed indirect and direct markers of liver fibrosis. MPR, NLR and PLR correlated with MELD score as well. NLR level in NAFLD patients was significantly higher in comparison to controls. MPR correlated with indirect markers of liver fibrosis and NAFLD fibrosis score. AUC values and proposed cut-offs for NLR, PLR and MPR in ALC patients were: 0.821 (> 2.227), 0.675 (< 70.445) and 0.929 (> 0.048), respectively. AUC values and proposed cut-offs for NLR, PLR and MPR in NAFLD group were: 0.725 (> 2.034), 0.528 (> 97.101) and 0.547 (> 0.038), respectively.

Research conclusionsWe demonstrated that NLR, MPR and PLR belong to hematological parameters with a relatively high diagnostic accuracy especially in the course of ALC. They are closely related to indirect and direct markers of liver fibrosis. Moreover, NLR, MPR and PLR seem to correlate with a clinical progression of liver cirrhosis (MELD score). These relationships propose evaluated hematological indices to be explored as potential parameters of liver disorders, especially liver cirrhosis.

Research perspectivesWe consider that further studies on NLR, MPR and PLR might broaden the range of noninvasive diagnostic tools in the evaluation of liver fibrosis and the decompensation of liver cirrhosis.



REFERENCESZhao Z, Liu J, Wang J, Xie T, Zhang Q, Feng S, Deng H, Zhong B. Platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR) are associated with chronic hepatitis B virus (HBV) infection. Int Immunopharmacol 2017; 51: 1-8 [PMID: 28759808 DOI: 10.1016/j.intimp.2017.07.007]

1

Meng X, Wei G, Chang Q, Peng R, Shi G, Zheng P, He F, Wang W, Ming L. The platelet-to-lymphocyte ratio, superior to the neutrophil-to-lymphocyte ratio, correlates with hepatitis C virus infection. Int J Infect Dis 2016; 45: 72-77 [PMID: 26948479 DOI: 10.1016/j.ijid.2016.02.025]

2

Biyik M, Ucar R, Solak Y, Gungor G, Polat I, Gaipov A, Cakir OO, Ataseven H, Demir A, Turk S, Polat H. Blood neutrophil-to-lymphocyte ratio independently predicts survival in patients with liver cirrhosis. Eur J Gastroenterol Hepatol 2013; 25: 435-441 [PMID: 23249602 DOI: 10.1097/MEG.0b013e32835c2af3]

3

Kara M, Dogru T, Genc H, Sertoglu E, Celebi G, Gurel H, Kayadibi H, Cicek AF, Ercin CN, Sonmez A. Neutrophil-to-lymphocyte ratio is not a predictor of liver histology in patients with nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 2015; 27: 1144-1148 [PMID: 26062078 DOI: 10.1097/MEG.0000000000000405]

4

Lai Q, Melandro F, Larghi Laureiro Z, Giovanardi F, Ginanni Corradini S, Ferri F, Hassan R, Rossi M, Mennini G. Platelet-to-lymphocyte ratio in the setting of liver transplantation for hepatocellular cancer: A systematic review and meta-analysis. World J Gastroenterol 2018; 24: 1658-1665 [PMID: 29686473 DOI: 10.3748/wjg.v24.i15.1658]

5

Chen K, Zhan MX, Hu BS, Li Y, He X, Fu SR, Xin YJ, Lu LG. Combination of the neutrophil to lymphocyte ratio and the platelet to lymphocyte ratio as a useful predictor for recurrence following radiofrequency ablation of hepatocellular carcinoma. Oncol Lett 2018; 15: 315-323 [PMID: 29285194 DOI: 10.3892/ol.2017.7291]

6

Yang HJ, Jiang JH, Liu QA, Zhou CM, Du YF, Wu T, Chen NZ, Xiang BD. Preoperative platelet-to-lymphocyte ratio is a valuable prognostic biomarker in patients with hepatocellular carcinoma undergoing curative liver resection. Tumour Biol 2017; 39: 1010428317707375 [PMID: 28639906 DOI: 10.1177/1010428317707375]

7

Min GT, Li YM, Yao N, Wang J, Wang HP, Chen W. The pretreatment neutrophil-lymphocyte ratio may predict prognosis of patients with liver cancer: A systematic review and meta-analysis. Clin Transplant 2018; 32 [PMID: 29112283 DOI: 10.1111/ctr.13151]

8

Iida H, Kaibori M, Matsui K, Ishizaki M, Kon M. Ratio of mean platelet volume to platelet count is a potential surrogate marker predicting liver cirrhosis. World J Hepatol 2018; 10: 82-87 [PMID: 29399281 DOI: 10.4254/wjh.v10.i1.82]

9

Kwon JH, Jang JW, Kim YW, Lee SW, Nam SW, Jaegal D, Lee S, Bae SH. The usefulness of C-reactive protein and neutrophil-to-lymphocyte ratio for predicting the outcome in hospitalized patients with liver cirrhosis. BMC Gastroenterol 2015; 15: 146 [PMID: 26498833 DOI: 10.1186/s12876-015-0378-z]

10

Lin L, Yang F, Wang Y, Su S, Su Z, Jiang X, Zheng Y, Deng Y, Lv H, Zhao J, Lin R, Wang B, Sun C. Prognostic nomogram incorporating neutrophil-to-lymphocyte ratio for early mortality in decompensated liver cirrhosis. Int Immunopharmacol 2018; 56: 58-64 [PMID: 29353688 DOI: 10.1016/j.intimp.2018.01.007]

11

Cervoni JP, Thévenot T, Weil D, Muel E, Barbot O, Sheppard F, Monnet E, Di Martino V. C-reactive protein predicts short-term mortality in patients with cirrhosis. J Hepatol 2012; 56: 1299-1304 [PMID: 22314431 DOI: 10.1016/j.jhep.2011.12.030]

12

Cai YJ, Dong JJ, Dong JZ, Chen Y, Lin Z, Song M, Wang YQ, Chen YP, Shi KQ, Zhou MT. A nomogram for predicting prognostic value of inflammatory response biomarkers in decompensated cirrhotic patients without acute-on-chronic liver failure. Aliment Pharmacol Ther 2017; 45: 1413-1426 [PMID: 28345155 DOI: 10.1111/apt.14046]

13

Kalra A, Wedd JP, Bambha KM, Gralla J, Golden-Mason L, Collins C, Rosen HR, Biggins SW. Neutrophil-to-lymphocyte ratio correlates with proinflammatory neutrophils and predicts death in low model for end-stage liver disease patients with cirrhosis. Liver Transpl 2017; 23: 155-165 [PMID: 28006875 DOI: 10.1002/lt.24702]

14

Cai YJ, Dong JJ, Dong JZ, Yang NB, Song M, Wang YQ, Chen YP, Lin Z, Shi KQ. Neutrophil-lymphocyte ratio predicts hospital-acquired bacterial infections in decompensated cirrhosis. Clin Chim Acta 2017; 469: 201-207 [PMID: 28412195 DOI: 10.1016/j.cca.2017.04.011]

15

Lin B, Geng L, Zheng Z, Jia J, Shen T, Zhang J, Zhou L, Zheng S. The predictive value of blood neutrophil-lymphocyte ratio in patients with end-stage liver cirrhosis following ABO-incompatible liver transplantation. J Res Med Sci 2016; 21: 69 [PMID: 27904614 DOI: 10.4103/1735-1995.189653]

16

Leithead JA, Rajoriya N, Gunson BK, Ferguson JW. Neutrophil-to-lymphocyte ratio predicts mortality in patients listed for liver transplantation. Liver Int 2015; 35: 502-509 [PMID: 25234369 DOI: 10.1111/liv.12688]

17

Zhang H, Sun Q, Mao W, Fan J, Ye B. Neutrophil-to-Lymphocyte Ratio Predicts Early Mortality in Patients with HBV-Related Decompensated Cirrhosis. Gastroenterol Res Pract 2016; 2016: 4394650 [PMID: 26949385 DOI: 10.1155/2016/4394650]

18

Liu H, Zhang H, Wan G, Sang Y, Chang Y, Wang X, Zeng H. Neutrophil-lymphocyte ratio: a novel predictor for short-term prognosis in acute-on-chronic hepatitis B liver failure. J Viral Hepat 2014;

19


https://dx.doi.org/10.1016/j.intimp.2017.07.007


https://dx.doi.org/10.1016/j.ijid.2016.02.025


https://dx.doi.org/10.1097/MEG.0b013e32835c2af3






https://dx.doi.org/10.3892/ol.2017.7291


https://dx.doi.org/10.1177/1010428317707375








https://dx.doi.org/10.1016/j.intimp.2018.01.007




https://dx.doi.org/10.1111/apt.14046




https://dx.doi.org/10.1016/j.cca.2017.04.011


https://dx.doi.org/10.4103/1735-1995.189653




https://dx.doi.org/10.1155/2016/4394650



21: 499-507 [PMID: 24750274 DOI: 10.1111/jvh.12160]Rice J, Dodge JL, Bambha KM, Bajaj JS, Reddy KR, Gralla J, Ganapathy D, Mitrani R, Reuter B, Palecki J, Acharya C, Shaw J, Burton JR, Biggins SW. Neutrophil-to-Lymphocyte Ratio Associates Independently With Mortality in Hospitalized Patients With Cirrhosis. Clin Gastroenterol Hepatol 2018; 16: 1786-1791. e1 [PMID: 29705264 DOI: 10.1016/j.cgh.2018.04.045]

20

Abu Omar Y, Randhawa T, Attar B, Agrawal R, Wang Y, Pichardo R, Majeed MB, Patel SA. Prognostic Value of Neutrophil-lymphocyte Ratio in Patients with Severe Alcoholic Hepatitis. Cureus 2019; 11: e6141 [PMID: 31886076 DOI: 10.7759/cureus.6141]

21

Yilmaz H, Yalcin KS, Namuslu M, Celik HT, Sozen M, Inan O, Nadir I, Turkay C, Akcay A, Kosar A. Neutrophil-Lymphocyte Ratio (NLR) Could Be Better Predictor than C-reactive Protein (CRP) for Liver Fibrosis in Non-alcoholic Steatohepatitis(NASH). Ann Clin Lab Sci 2015; 45: 278-286 [PMID: 26116591]

22

Alkhouri N, Morris-Stiff G, Campbell C, Lopez R, Tamimi TA, Yerian L, Zein NN, Feldstein AE. Neutrophil to lymphocyte ratio: a new marker for predicting steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease. Liver Int 2012; 32: 297-302 [PMID: 22097893 DOI: 10.1111/j.1478-3231.2011.02639.x]

23

Lu W, Zhang YP, Zhu HG, Zhang T, Zhang L, Gao N, Chang DY, Yin J, Zhou XY, Li MY, Li YT, Li ZZ, He Q, Geng Y. Evaluation and comparison of the diagnostic performance of routine blood tests in predicting liver fibrosis in chronic hepatitis B infection. Br J Biomed Sci 2019; 76: 137-142 [PMID: 31062646 DOI: 10.1080/09674845.2019.1615717]

24

Alsebaey A, Elhelbawy M, Waked I. Platelets-to-lymphocyte ratio is a good predictor of liver fibrosis and insulin resistance in hepatitis C virus-related liver disease. Eur J Gastroenterol Hepatol 2018; 30: 207-211 [PMID: 29240565 DOI: 10.1097/MEG.0000000000001013]

25

Wang D, Bai N, Hu X, OuYang XW, Yao L, Tao Y, Wang Z. Preoperative inflammatory markers of NLR and PLR as indicators of poor prognosis in resectable HCC. PeerJ 2019; 7: e7132 [PMID: 31632844 DOI: 10.7717/peerj.7132]

26

Ismael MN, Forde J, Milla E, Khan W, Cabrera R. Utility of Inflammatory Markers in Predicting Hepatocellular Carcinoma Survival after Liver Transplantation. Biomed Res Int 2019; 2019: 7284040 [PMID: 31737675 DOI: 10.1155/2019/7284040]

27

Dogan M, Algin E, Guven ZT, Baykara M, Kos TF, Bal O, Zengin N. Neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, neutrophil-platelet score and prognostic nutritional index: do they have prognostic significance in metastatic pancreas cancer? Curr Med Res Opin 2018; 34: 857-863 [PMID: 29161926 DOI: 10.1080/03007995.2017.1408579]

28

Kitano Y, Yamashita YI, Yamamura K, Arima K, Kaida T, Miyata T, Nakagawa S, Mima K, Imai K, Hashimoto D, Chikamoto A, Baba H. Effects of Preoperative Neutrophil-to-Lymphocyte and Platelet-to-Lymphocyte Ratios on Survival in Patients with Extrahepatic Cholangiocarcinoma. Anticancer Res 2017; 37: 3229-3237 [PMID: 28551669 DOI: 10.21873/anticanres.11685]

29

Cho SY, Yang JJ, You E, Kim BH, Shim J, Lee HJ, Lee WI, Suh JT, Park TS. Mean platelet volume/platelet count ratio in hepatocellular carcinoma. Platelets 2013; 24: 375-377 [PMID: 22835043 DOI: 10.3109/09537104.2012.701028]

30


https://dx.doi.org/10.1111/jvh.12160




https://dx.doi.org/10.7759/cureus.6141





https://dx.doi.org/10.1080/09674845.2019.1615717




https://dx.doi.org/10.7717/peerj.7132


https://dx.doi.org/10.1155/2019/7284040


https://dx.doi.org/10.1080/03007995.2017.1408579


https://dx.doi.org/10.21873/anticanres.11685


https://dx.doi.org/10.3109/09537104.2012.701028


World Journal of



ORIGINAL ARTICLE

Retrospective Study

Clinical features of multiple gastrointestinal stromal tumors: A pooling analysis combined with evidence and gap map

Chen Li, Ke-Lu Yang, Quan Wang, Jin-Hui Tian, Yang Li, Zhi-Dong Gao, Xiao-Dong Yang, Ying-Jiang Ye, Ke-Wei Jiang

ORCID number: Chen Li 0000-0003-3556-2719; Ke-Lu Yang 0000-0003-0240-9750; Quan Wang 0000-0002-2821-5017; Jin-Hui Tian 0000-0002-3859-9587; Yang Li 0000-0002-1473-0296; Zhi-Dong Gao 0000-0002-8415-7587; Xiao-Dong Yang 0000-0002-4326-0262; Ying-Jiang Ye 0000-0002-7904-3163; Ke-Wei Jiang 0000-0002-6706-4741.

Author contributions: Li C and Yang KL contributed equally to design the research and wrote the paper; Tian JH and Wang Q supervised the research and contributed to analysis; Li Y, Gao ZD, Yang XD, and Ye YJ provided the clinical advice and supervised the manuscript.

Institutional review board statement: This study was reviewed and approved by the Ethics Committee of the Peking University People’s Hospital.

Informed consent statement: Patients were not required to give informed consent to the study because the analysis used anonymous clinical data that were obtained after each patient agreed to treatment by written consent. The sample of informed consent had already uploaded.

Conflict-of-interest statement: We

Chen Li, Quan Wang, Yang Li, Zhi-Dong Gao, Xiao-Dong Yang, Ying-Jiang Ye, Ke-Wei Jiang, Department of Gastroenterological Surgery, Peking University People's Hospital, Beijing 100044, China

Ke-Lu Yang, Evidence-Based Nursing Center, School of Nursing, Lanzhou University, Lanzhou 730000, Gansu Province, China

Jin-Hui Tian, Evidence Based Medicine Center, School of Basic Medical Science of Lanzhou University, Lanzhou 730000, Gansu Province, China

Corresponding author: Ke-Wei Jiang, MD, PhD, Adjunct Professor, Chairman, Chief Doctor, Director, Surgeon, Surgical Oncologist, Department of Gastroenterological Surgery, Peking University People's Hospital, No. 11 Xizhimen South Street, Xicheng District, Beijing 100044, China. [email protected]

AbstractBACKGROUND Multiple gastrointestinal stromal tumors (MGISTs) are a very rare type of gastrointestinal stromal tumor (GIST) and are usually observed in syndrome.

AIM The paper aimed to describe the clinical and oncological features of MGISTs and to offer evidence for the diagnosis and treatment.

METHODS Data of consecutive patients with MGISTs who were diagnosed at Peking University People’s Hospital (PKUPH) from 2008 to 2019 were retrospectively evaluated. Further, a literature search was conducted by retrieving data from PubMed, EMBASE, and the Cochrane library databases from inception up to November 30, 2019.

RESULTS In all, 12 patients were diagnosed with MGISTs at PKUPH, and 43 published records were ultimately included following the literature review. Combined analysis of the whole individual patient data showed that female (59.30%), young (14.45%), and syndromic GIST (63.95%) patients comprised a large proportion of the total patient population. Tumors were mainly located in the small intestine



http://orcid.org/0000-0003-3556-2719

http://orcid.org/0000-0003-3556-2719

http://orcid.org/0000-0003-3556-2719

http://orcid.org/0000-0003-0240-9750

http://orcid.org/0000-0003-0240-9750

http://orcid.org/0000-0003-0240-9750

http://orcid.org/0000-0002-2821-5017

http://orcid.org/0000-0002-2821-5017

http://orcid.org/0000-0002-2821-5017

http://orcid.org/0000-0002-3859-9587

http://orcid.org/0000-0002-3859-9587

http://orcid.org/0000-0002-3859-9587

http://orcid.org/0000-0002-1473-0296

http://orcid.org/0000-0002-1473-0296

http://orcid.org/0000-0002-8415-7587

http://orcid.org/0000-0002-8415-7587

http://orcid.org/0000-0002-4326-0262

http://orcid.org/0000-0002-4326-0262

http://orcid.org/0000-0002-4326-0262

http://orcid.org/0000-0002-7904-3163

http://orcid.org/0000-0002-7904-3163

http://orcid.org/0000-0002-7904-3163

http://orcid.org/0000-0002-6706-4741

http://orcid.org/0000-0002-6706-4741

http://orcid.org/0000-0002-6706-4741


Li C et al. Clinical features of MGISTs


have no financial relationships to disclose.

Data sharing statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.





Peer-review report’s scientific quality classificationGrade A (Excellent): A Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0

Received: September 12, 2020 Peer-review started: September 12, 2020 First decision: October 27, 2020 Revised: November 9, 2020 Accepted: November 21, 2020 Article in press: November 21, 2020 Published online: December 21, 2020

P-Reviewer: Bhat YR, Hann HW S-Editor: Gao CC L-Editor: Filipodia P-Editor: Li JH

(58.92%), and both CD117 and CD34 were generally positive. After a mean 78.32-mo follow-up, the estimated median overall survival duration (11.5 years) was similar to single GISTs, but recurrence-free survival was relatively poorer.

CONCLUSION The clinical and oncological features are potentially different between MGISTs and single GIST. Further studies are needed to explore appropriate surgical approach and adjuvant therapy.

Key Words: Gastrointestinal stromal tumor; Multiple; Pooling analysis; Cross sectional study; Evidence and gap map


Core Tip: The study retrospectively collected 12 patients of Peking University People’s Hospital and 161 patients of literature research to illustrate the demographic, oncological, and surgical features of patients with multiple gastrointestinal stromal tumors (MGISTs). After analysis, MGISTs might have unique characteristics, such as lower morbidity, female predominance, young age, multiple organ involvement, and more likely to occur in syndrome. Although overall survival was similar to single gastrointestinal stromal tumor, the high rate of metastasis resulted in a poor recurrence free survival in MGISTs. Based upon evidence and gap map, gene detection and molecular biological analysis are necessary to explore the mechanism and provide appropriate therapy.

Citation: Li C, Yang KL, Wang Q, Tian JH, Li Y, Gao ZD, Yang XD, Ye YJ, Jiang KW. Clinical features of multiple gastrointestinal stromal tumors: A pooling analysis combined with evidence and gap map. World J Gastroenterol 2020; 26(47): 7550-7567URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7550.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7550

INTRODUCTIONGastrointestinal (GI) stromal tumor (GIST) is the most common mesenchymal tumor of the GI tract, with an estimated unadjusted yearly incidence of 1-1.5 per 100000 individuals[1]. GISTs, with variable biological behavior ranging from benign to malignant types, usually occur in elderly individuals (age, 55-65 years; median age, 63 years) and are seldom observed in young individuals aged below 20 years (0.4%)[2,3]. The tumors generally occur in the stomach (55%-60%) and small intestine (30%-35%) and rarely in the esophagus (< 1%) and colon/rectum (5%)[1,2,4,5]. Particularly, GIST found elsewhere within the abdominal cavity, usually in the omentum, mesentery, or the retroperitoneum (accounting for < 5% of all GISTs), are referred to as extra-GI tract tumors (E-GISTs)[2,6]; these are considered to have metastasized from the stomach and/or small intestine during their development[2,7]. These tumors are derived from (or share a common stem cell with) intestinal Cajal cells[8,9] and have histological features including spindle, epithelioid, and mixed. Several immunohistochemical (IHC) markers such as CD117 (95%), CD34 (70%), DOG-1 (96%), SMA (25%), desmin (< 5%), and S100 (rare) are observed in GISTs[7,10]. Most GISTs show an oncogenic mutation in the KIT gene (80%-85%) or platelet-derived growth factor receptor alpha (PDGFRA, 5%-7%) gene[11].

Multiple GISTs (MGISTs) are very rare and are commonly observed in cases of syndromic GISTs, such as type 1 neurofibromatosis (NF1)-associated GISTs[12], familial GIST[13], pediatric GIST[14], Carney triad[15], and Carney-Stratakis syndrome[16]. Further, MGISTs are often misinterpreted as metastasis or recurrence using conventional diagnosis techniques[12,13,17-20]. During the past decades, few studies have been conducted on MGISTs, and the guidelines from National Comprehensive Cancer Network[21], European Society for Medical Oncology[22], United Kingdom[23], and France[24] in addition to Asian[25] and Chinese[26] consensus fail to describe specific diagnosis, treatment, and follow-up strategies for MGISTs. Therefore, it is urgently








required to understand deeply these serious tumors and to determine whether single GIST diagnosis, treatment, and follow-up strategies are appropriate for diagnosing and treating MGISTs and whether they offer a worthwhile reference for precise and individualized medical measures in the future.

The present study was performed in accordance with Surgical Case Report (SCARE) Guidelines[27] and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement[28], which provide the standard reporting guidelines for case reports and literature reviews, respectively.

MATERIALS AND METHODSDefinitionMultiple GISTs, also called multicentric or multifocal GISTs, do not have a formal definition at present. In our study, we defined MGISTs as no less than two GISTs located in the GI tract without any evidence of recurrence or metastasis despite one or more organs being involved[17,29]. Especially, GISTs located in the extra-GI tract are usually considered to have metastasized, although a small portion of them are primary. Because patients with MGISTs comprise only a small proportion of patients with GISTs, we excluded multiple EGIST patients (or those with only one tumor located in the GI tract and the others in the extra-GI tract) to prevent the interference of metastatic EGIST.

Peking University People’s Hospital patientsAs shown in Figure 1, there were two inclusion criteria for patients at Peking University People’s Hospital: (1) Diagnosis of GIST based on pathological results; and (2) Existence of multiple neoplasms (≥ 2 tumors). Patients were excluded if they: (1) Had only one tumor or none located in the GI tract and others were located in extra-GI tract sites, which are usually considered to have metastasized including mesentery, omentum, peritoneum, or abdomen; and (2) Substantial patient information such as baseline information or tumor features among others was missing.

Literature searchWe searched the following electronic databases from inception up to November 30, 2019: PubMed, EMBASE, and the Cochrane library. All published studies were searched without any language restriction. Search items including gastrointestinal stromal tumor, multiple, multicentric, and multifocal were searched using Medical Subject Headings terms combined with free text terms. We also performed a supplemental literature search through Google Scholar and identified two studies by manual search.

Study selectionEndnote software (version X9.2, Thomson Reuters, Philadelphia, PA, United States) was used for removing duplicates and facilitating the screening process. After two reviewers independently screened the titles and abstracts, unsuitable studies were excluded; further, observational studies were excluded after reading the full text, and the eligible trials were finally identified. Disagreements between reviewers were resolved through discussions. In some cases, case reports may be used as a part of patients group in same author’s or other author’s studies, and we excluded these patients’ data from the latter and reserved the case reports.

Data extractionTwo reviewers independently extracted the following data after literature search: Titles, years of publication, demographics and baseline characteristics, perioperative information, tumor features, pathological results, and follow-up duration. Meanwhile, methodological and reporting qualities were assessed by two reviewers as well.

Literature-based patientsAs shown in Figure 2, we retrieved studies focusing on patients with MGISTs from PubMed, EMBASE, and the Cochrane library using keywords mentioned before. After screening titles and abstracts, studies such as case reports, case series, or retrospective studies with detailed patient information were included. Following this, full-text articles were assessed for eligibility: Studies with either metastatic GIST or incomplete patient information (such as lack of tumor location data) were excluded. Finally, combined with two articles shortlisted by manual search, the evaluation of studies for



Figure 1 Flowchart of patient inclusion and exclusion criteria at Peking University People’s Hospital. GI: Gastrointestinal; GIST: Gastrointestinal stromal tumor; MGIST: Multiple gastrointestinal stromal tumor; PKUPH: Peking University People’s Hospital.

Figure 2 Flowchart of literature selection.

inclusion was completed.During full-text article assessment, data of literature review-based patients were

included if they were diagnosed with GIST based on pathological results and if they had ≥ 2 tumors. Accordingly, studies were excluded if patients had only one tumor in the GI tract and others were located in sites that are usually considered as metastasis sites and if explicit patient and tumor information was missing.

Assessment of reporting and methodological quality of including studiesThe SCARE guideline is a consensus-based surgical case report guideline[27]. Another tool, Joanna Briggs Institute (JBI) model, was used to enable the assessment of evidence-based healthcare and its role in improving global health[30]. We assessed the methodological and reporting quality of the included studies on the basis of the SCARE guideline and JBI model for quality evaluation. We recorded the issues, and each of the criteria was assigned different scores including “1 = Yes,” “0 = No,” and “0.5 = Unclear” to estimate the quality of the included studies. Particularly, some items were not applicable to certain articles; these were marked as “NA”. Subsequently, we classified the JBI model (case report/case series) and SCARE guideline points as follows: JBI-I (high, 6-8/8-10), JBI-II (intermediate, 3-5/5-7), JBI-III (low, 0-2/0-4); SCARE-A (very high, 28-30), SCARE-B (high, 21-27), SCARE-C (intermediate, 14-20), SCARE-D (low, 7-13), and SCARE-E (very low, 0-6). A detailed rating scale for the JBI model and SCARE guidelines is available in Supplementary Tables 1 and 2. Each

https://f6publishing.blob.core.windows.net/e6b557ed-f756-42b4-a473-67f82eb62ea1/WJG-26-7550-supplementary-material.pdf



study was subjected to quality assessment by two reviewers, and discrepancies were resolved by a discussion.

Evidence and gap mapEvidence and gap map (EGM) is a method of systematically identifying, reporting, and visualizing a body of evidence on a specific topic, which may show high-quality studies and the emphasis of studies. The scope of the EGM in our study was to cover the different types of MGISTs and their clinical and pathological information. The EGM adopted in our study was classified into five categories: Baseline characteristics, tumor features, pathological characteristics, perioperative information, and follow-up results. Further, the population was classified into six categories: Sporadic MGISTs, NF1-associated MGISTs, familial MGISTs, pediatric MGISTs, Carney triad syndrome, and Carney-Stratakis syndrome. A bubble diagram was used to visualize the EGM via Excel (Microsoft, 2016, Redmond, WA, United States). The size of a bubble represented the sample, and the color indicated whether the clinical characteristic was fully or partly reported in each study.

Statistical analysisThe statistical methods of this study were reviewed by Tian JH from Evidence-Based Medicine Center of Lanzhou University. Continuous variables are expressed as means, and categorical variables are expressed as numbers (%). Kaplan–Meier survival function and statistical analyses were performed using the SPSS software (version 25.0, SPSS Inc., Armonk, NY, United States), unless indicated otherwise.

RESULTSPeking University People’s Hospital patientsDuring the period between January 2008 and November 2019, 12 identified individual patients (males, six; females, six) aged 53 to 88 years (mean age, 65.33 years) were admitted to the Peking University People’s Hospital. All detailed information is available in Supplementary Table 3. Based on the patient age groups, we could determine that patients aged between 61 and 80 years comprised the major proportion of patients (9/12). With regard to the common symptoms, incidental finding without any subjective discomfort occurred in seven patients, and GI bleeding and abdominal pain were the most common symptoms (both 2/12). Sporadic MGISTs (10/12) were predominant and the others were NF1-associated type; no patient had familial history of GIST. Only one in 12 patients had a secondary malignant tumor (breast cancer). All patients at the Peking University People’s Hospital underwent computed tomography (CT), and half of them underwent an endoscopy; only two patients underwent magnetic resonance imaging. During the surgery, laparoscopy was performed in seven patients, and nine patients received en bloc (R0) resection. Among the 40 tumors of 12 patients, 22 tumors were located in the stomach and 17 in the small intestine. Three quarters of patients showed the involvement of only a single organ. With regard to the pathological results, minimal and maximum sizes were 0.10 cm and 8.00 cm, respectively. All tumors showed spindle morphology, and > 90% had a low mitosis rate (≤ 5), with a mean tumor size of 3.86 cm. Interestingly, more than half of the tumors were micro-GIST (sized < 1 cm). Because of the small size of tumors, the risks were predominantly low or very low (more than 85%). On immunohistochemical analysis, CD117 was extensively positive in 12 patients, and only one-third of the tumors were positive for CD34. Desmin, smooth muscle actin (SMA), and S-100 were almost negative, and Ki-67 level ranged from 0% to 20% with a mean value of 3.83%. After a mean 33.75-mo follow-up in the form of telephonic conversations and outpatient visits, imatinib was administered as an adjuvant therapy in seven patients, and all patients were alive without any evidence of metastasis or recurrence.

Literature-based patientsAfter literature retrieval, 43 published records were included in the study (Supplementary Table 4). Of these, 21 showed high-quality (level I) methodology, and the other 22 were of the intermediate level based on the JBI model. Meanwhile, level C (23/43) and level D (15/43) comprised the main proportion of the identified records with regard to reporting quality (according to SCARE guidelines). Finally, 161 patients with more than 798 tumors were recorded in total. Accordingly, SCARE guidelines (Supplementary Table 5) and PRISMA statement (Supplementary Table 6) were







adopted in the present study for improving the reporting quality.On observing the bubble diagram (Figure 3), we could see that the baseline

characteristics and tumor features showed high-level evidence, and perioperative information showed a lower level. In contrast, sporadic and NF1-associated MGISTs were reported more frequently than other types. In correspondence, partially reported articles were more frequently observed. Compared with the abovementioned types, familial type MGISTs showed a lack of evidence with regard to pathological characteristics and perioperative information. Further, pediatric and Carney triad syndromic MGISTs showed a weak evidence level, and evidence gaps were observed with regard to perioperative information in Carney triad syndromic and Carney-Stratakis syndromic MGISTs.

As shown in Supplementary Tables 7 and 8, there was a female predominance in literature-based patients (96/160, one patient’s gender is unknown), with a mean age of female patients being 48.70 years. The highest number of patients were admitted to the hospital after GI bleeding as the first symptom, followed by abdominal pain and incidental findings. Apart from sporadic GISTs, NF1-associated MGISTs were the most common in the 161 patients. Unlike patients from Peking University People’s Hospital, literature-based patients underwent endoscopy more frequently than CT and predominantly underwent laparotomy. Referring to tumor features, 201 tumors were located in the small intestine and 121 in the stomach. Of these, most tumors were spindle type and had a low mitosis rate (< 5/50 high-power fields). Accordingly, low and very low risk tumors comprised the highest proportion among all tumors. Both CD117 and CD34 were extensively positive in tumors; further, the mean value of Ki-67 was similar between literature review patients and Peking University People’s Hospital patients, and desmin, SMA, and S-100 were almost negative, although 29.63% tumors were S-100 positive. After a mean 83.01-mo follow-up, 62.60% patients were alive without any evidence of recurrence, and seven patients died of MGISTs. The frequency of recurrence was 2.75% and in all, 16 patients showed evidence of metastasis. Peritoneum, liver, and lymph nodes were the common sites of metastasis.

Individual patient dataAmong the 173 patients shown in Table 1, 102 (59.30%) were females and 70 (40.70%) were males, with a mean age of 49.85 years. Patients between 41 to 60 years (n = 53; 30.81%) and 61 to 80 years (n = 60; 34.88%) comprised the majority among patients with MGISTs.

The dominant symptom was GI bleeding (33/67), followed by incidental finding and abdominal pain. NF1-associated (42.77%) and sporadic type (36.42%) MGISTs were the predominant types among patients; pediatric-type MGISTs comprised 16.18% of all cases, followed by familial MGISTs (4.62%). With regard to secondary malignant tumors, the breast (4.62%), genitourinary tract (2.89%), and gastrointestinal tract (1.73%) were the three common sites.

Among 218 tumors located in the small intestine (58.92%), the jejunum (19.46%) was the most common site compared with the ileum (7.57%) and duodenum (7.57%). The stomach was the second most common site, comprising 38.65% of all tumors. In addition to the tumors in the stomach and small intestine, tumors at rare sites such as the colon (case 5, 6, 9,14 and 124) and rectum (case 5, 145) were also observed (2.43%). Approximately 69.64% patients had only a single organ involved. Tumors sized ≤ 1 cm and 2-5 cm were the most common, accounting for 32.68% and 30.70% of all tumors, respectively; the mean tumor size was 3.12 cm.

As shown in Table 2, most tumors had a spindle morphology (80.46%), low mitosis rate (< 5/50 high power fields, 87.34%), and low or very low risk classification (69.36%). Among the resected specimens, 179 (97.28%) and 126 (72.00%) were positive for CD117 and CD34, respectively. Further, Ki-67 value ranged from 0% to 33.8% with a mean value of 3.91%; simultaneously, positivity for desmin, S-100, and SMA was rare, accounting for 1.50%, 23.08%, and 15.48% of all specimens.

On considering the Peking University People’s Hospital and literature review-based patient data (Table 3), CT (31/41) was the most commonly used detection method for patients with MGISTs, followed by endoscopy (27/41). A traditional open surgery was conducted in 23 (23/37) patients, and 31 (31/42) patients underwent a radical operation. After surgery, imatinib was administered to 32 (32/49) patients as adjuvant therapy.

After a mean 78.32-mo follow-up, 65.93% (89/135) patients were alive without any evidence of recurrence or metastasis. Unfortunately, 5.19% patients died of MGISTs. Of 121 patients with clear follow-up results, 2.48% patients had a relapse, and 13.22% patients were metastatic. Common sites of metastasis were the peritoneum (7.44%), liver (6.61%), and lymph nodes (4.13%). As shown in Figure 4 and Figure 5, all patients




Table 1 Baseline characteristics of patients with multiple gastrointestinal stromal tumors, n (%)

PKUPH patients, n = 121 Literature-based patients, n = 1611 Total patients, n = 1731

Sex n = 12 n = 160 n = 172

Female 6 (50.00) 96 (60.00) 102 (59.30)

F/M 1.00 1.48 1.46

Age in yr n = 12 n = 161 n = 173

Range 53-88 8-84 8-88

≤ 20 0 (0.00) 25 (15.63) 25 (14.45)

21-40 0 (0.00) 29 (18.12) 29 (16.86)

41-60 2 (16.67) 51 (31.88) 53 (30.81)

61-80 9 (75.00) 51 (31.88) 60 (34.88)

> 80 1 (8.33) 5 (3.13) 6 (3.49)

Mean (SD) 65.33 (9.48) 48.70 (21.17) 49.85 (20.99)

Symptoms n = 12 n = 55 n = 67

GI bleeding 2 (16.67) 31 (56.36) 33 (49.25)

Hematochezia 1 (8.33) 13 (24.07) 14 (21.21)

Anemia 0 (0.00) 11 (20.37) 11 (16.67)

Hematemesis 1 (8.33) 2 (3.70) 3 (4.55)

Incidental finding 7 (58.33) 15 (27.27) 22 (32.83)

Abdominal pain 2 (16.67) 14 (25.45) 16 (23.88)

Palpable mass 0 (0.00) 2 (3.64) 2 (2.99)

Others 1 (8.33) 11 (20.00) 12 (17.91)

Classification n = 12 n = 161 n = 173

Sporadic multiple GIST 10 (83.33) 53 (32.92) 63 (36.42)

NF-1 associated GIST 2 (16.67) 72 (44.72) 74 (42.77)

Primary familial GIST 0 (0.00) 8 (4.97) 8 (4.62)

Pediatric GIST 0 (0.00) 25 (15.53) 25 (14.45)

Carney-Stratakis syndrome 0 (0.00) 0 (0.00) 0 (0.00)

Carney triads 0 (0.00) 3 (1.86) 3 (1.73)

Combined diseases n = 12 n = 161 n = 173

GI tumors 0 (0.00) 3 (1.86) 3 (1.73)

GU tumors 0 (0.00) 5 (3.11) 5 (2.89)

Breast tumors 1 (8.33) 7 (4.35) 8 (4.62)

Other tumors 0 (0.00) 5 (3.11) 5 (2.89)

1n For total number of patients, other n for number of patients with relevant data. GI: Gastrointestinal; GIST: Gastrointestinal stromal tumor; GU: Genitourinary tract; PKUPH: Peking University People’s Hospital; SD: Standard deviation.

at the Peking University People’s Hospital showed an estimated median overall survival (OS) duration of 11.5 years (138 mo, 95% confidence interval: 8.7-14.3) and estimated 5-year, 10-year, and 15-year recurrence-free (RF) survival rates of 89.4%, 76.3%, and 50.8%, respectively.



Table 2 Tumor and pathological features of patients with multiple gastrointestinal stromal tumors, n (%)


Site n = 40 n = 330 n = 370

Stomach 22 (55.00) 121 (36.67) 143 (38.65)

Small intestine 17 (42.50) 201 (60.91) 218 (58.92)

Duodenum 1 (2.50) 27 (8.18) 28 (7.57)

Jejunum 4 (10.00) 68 (20.61) 72 (19.46)

Ileum 3 (7.50) 25 (7.58) 28 (7.57)

Other sites 1 (2.50) 8 (2.42) 9 (2.43)

Involving organ n = 12 n = 156 n = 168

Single 9 (75.00) 108 (69.23) 117 (69.64)

Two or more 3 (25.00) 48 (30.77) 51 (30.36)

Size in cm n = 40 n = 315 n = 355

Range 0.10-8.00 0.05-27.00 0.05-27.00

≤ 1 22 (55.00) 94 (29.84) 116 (32.68)

1-2 4 (10.00) 65 (20.64) 69 (19.44)

2-5 11 (27.75) 98 (31.11) 109 (30.70)

5-10 3 (7.50) 45 (14.29) 48 (13.52)

> 10 0 (0.00) 13 (4.13) 13 (3.56)

Mean (SD) 2.00 (2.16) 3.26 (3.50) 3.12 (3.40)

Cellular type n = 40 n = 180 n = 220

Spindle 40 (100.00) 137 (76.11) 177 (80.46)

Epithelial 0 (0.00) 23 (12.78) 23 (10.46)

Mixed 0 (0.00) 20 (11.11) 20 (9.09)

Mitosis, /50 HPFs n = 35 n = 202 n = 237

Range 0-9 0-48 0-48

≤ 5 33 (94.29) 174 (86.14) 207 (87.34)

5-10 2 (5.71) 15 (7.43) 17 (7.17)

> 10 0 (0.00) 13 (6.44) 13 (5.49)

Mean (SD) 3.86 (1.87) 3.32 (5.91) 3.40 (5.50)

Risk classification n = 35 n = 249 n = 284

Very low risk 20 (57.14) 54 (21.69) 74 (26.05)

Low risk 10 (28.57) 113 (45.38) 123 (43.31)

Median risk 3 (8.57) 26 (10.44) 29 (10.21)

High risk 2 (5.71) 56 (22.49) 58 (20.42)

CD117 n = 35 n = 149 n = 184

Positive 35 (100.00) 144 (96.64) 179 (97.28)

CD34 n = 35 n = 140 n = 175

Positive 12 (34.29) 114 (81.43) 126 (72.00)

Ki-67 n = 35 n = 47 n = 82

Range 0-20 1-33.8 0-33.8

Mean (SD) 3.83 (4.85) 3.96 (5.11) 3.91 (4.97)



Desmin n = 35 n = 98 n = 133

Positive 1 (2.86) 1 (1.02) 2 (1.50)

S-100 n = 35 n = 108 n = 143

Positive 1 (2.86) 32 (29.63) 33 (23.08)

SMA n = 35 n = 120 n = 155

Positive 5 (14.29) 19 (15.83) 24 (15.48)

1n For total number of patients, other n for number of patients with relevant data. HPFs: High-power fields; PKUPH: Peking University People’s Hospital; SD: Standard deviation.

DISCUSSIONThe majority of GISTs occur as sporadic solitary neoplasms resulting from somatic mutations in KIT or PDGFRA genes[8]. MGISTs are rare and were often misinterpreted previously as a recurrent or metastatic disease[20], leading to inappropriate treatment. Until now, there are no established criteria for confirming the diagnosis and treatment of MGISTs. Given the lack of clinical trials, single GIST therapy has conflicts in MGIST patients with regard to factors such as surgical excision and perioperative adjuvant therapy. KIT or PDGFRA mutation analysis and pattern of muscularis involvement can contribute to differential diagnosis[13,17,20]. As molecular analysis is generally not available in routine practices, basic clinical characteristics, distinctive syndromic manifestation, and pathological features of MGISTs are required to be known in routine examination.

In this study, unlike the similar prevalence of men and women in GISTs[31], female prevalence (F/M = 1.46) was higher in MGISTs, which may because of female predominance in syndromic GIST. GIST can develop at any age, but no less than 80% of these were reported in middle-aged and elderly patients (mean age, 64-69 years)[5,32,33]. Compared with single GIST, MGISTs were usually observed in younger individuals (mean, 49.85 years) and showed a variant age predominance in different types. Sporadic MGISTs comprise the highest proportion of cases among all types and have similar demographics with single GIST[5,12,19,29,34,35]. According to published studies, NF1-associated GIST patients were younger (49 years) than single GIST patients without obvious sex predominance[12]. Similarly, familial GISTs equally appear in men and in women and are observed in younger patients, with a mean age of 46 years[13]. Moreover, pediatric, Carney triad, and Carney-Stratakis syndromic GIST often occur in young patients (approximately 80% being women) who are < 20-years-old[36-38]. Because of the lack of department of pediatric surgery and Grade 3A classification of the Peking University People’s Hospital, fewer young female patients and more elderly patients may have been included in the study.

On MGISTs classification, NF1-associated GIST and sporadic GIST were found to be the main types. Although multiplicity is very rare (1.1%-1.6%) in sporadic GIST[20, 29], it was the second frequent type due to the large cardinal number. In contrast, multiple growth patterns are a characteristic feature of NF1-associated GIST and familial GISTs[13] (up to 70% NF1-associated GIST patients have multiple lesions[36,39,40]). Pediatric, Carney triad, and Carney-Stratakis syndromic GISTs showed multiplicity in approximately 23%-81%, 40%, and 80% cases, respectively, and GIST may be the first sign in the latter two syndromes[41-43]. In particular, Armed Forces Institute of Pathology (Washington, DC, United States) revealed that GIST in young patients who lack other features of the Carney triad syndrome are clinically, phenotypically, and molecularly similar to those in patients with Carney triad syndromic GIST and might represent an attenuated manifestation of the triad[19]. We could infer that pediatric MGIST patients may be heterogeneous and may include Carney triad syndrome, Carney-Stratakis syndrome, or an attenuated manifestation of them. Hereinafter, we use “pediatric-type MGISTs” to represent pediatric, Carney triad syndromic, and Carney-Stratakis syndromic MGISTs. Interestingly, some previous studies[38,44] indicated that some adult GIST patients also have clinical and pathological characteristics similar to those of pediatric GIST patients, and these special groups are also included under pediatric-type GISTs. However, these may have been classified under sporadic GISTs in our study because of the ambiguous diagnosis criteria, and this may have led to an increase in the number of sporadic MGIST cases.

With regard to clinical symptoms, some infrequent and specific symptoms require to be paid more attention. Patients with NF1 often present with specific subcutaneous



Table 3 Perioperative information and follow-up results of patients with multiple gastrointestinal stromal tumors, n (%)


Preoperative I.E. n = 12 n = 29 n = 41

CT 12 (100.00) 19 (65.52) 31 (75.61)

MRI 2 (16.67) 2 (6.90) 4 (9.76)

Endoscopy 6 (50.00) 21 (72.41) 27 (65.85)

Approach n = 12 n = 25 n = 37

Laparotomy 5 (41.67) 18 (72.00) 23 (62.16)

Laparoscopy 7 (58.33) 7 (28.00) 14 (37.84)

Excision Extension n = 12 n = 30 n = 42

Radical resection 9 (75.00) 22 (73.33) 31 (73.81)

Palliative resection 3 (25.00) 8 (26.67) 11 (26.19)

Imatinib n = 12 n = 37 n = 49

Apply 7 (58.33) 25 (67.57) 32 (65.31)

Not apply 5 (41.67) 12 (32.43) 17 (34.70)

Follow-up time in mo n = 12 n = 114 n = 126

Range 3-86 3-396 3-396

Mean (SD) 33.75 (27.28) 83.01 (82.26) 78.32 (79.96)

Outcome n = 12 n = 123 n = 135

ANED 12 (100.00) 77 (62.60) 89 (65.93)

AWD 0 (0.00) 21 (17.07) 21 (15.56)

ATSU 0 (0.00) 2 (1.63) 2 (1.48)

DOD 0 (0.00) 7 (5.69) 7 (5.19)

DUC 0 (0.00) 9 (7.32) 9 (6.67)

DOPC 0 (0.00) 2 (1.63) 2 (1.48)

DUNC 0 (0.00) 5 (4.07) 5 (3.70)

Recurrence 0/12 (0.00) 3/123 (2.44) 3/135 (2.22)

Metastasis n = 12 n = 123 n = 135

Lymph node 0 (0.00) 5 (4.07) 5 (3.70)

Liver 0 (0.00) 8 (6.50) 8 (5.93)

Peritoneum 0 (0.00) 9 (7.32) 9 (6.67)

Lung 0 (0.00) 1 (0.76) 1 (0.74)

Abdomen 0 (0.00) 1 (0.76) 1 (0.74)

Omentum 0 (0.00) 1 (0.76) 1 (0.74)

1n For total number of patients, other n for number of patients with relevant data. CT: Computed tomography; I.E.: Imaging examinations; MRI: Magnetic resonance imaging; PKUPH: Peking University People’s Hospital; SD: Standard deviation.

nodules and Cafe-au-Lait Spots; Carney triad patients manifest pulmonary chondroma and paraganglioma; and Carney-Stratakis syndrome patients present with only paraganglioma. Further, familial GIST patients normally suffer from skin pigmentation and dysphagia[45]. Although most patients had symptoms or syndromes, approximately 30% patients were diagnosed incidentally during imaging or surgery for other disorders, and quite a few were diagnosed during autopsy. It is worth noting that specific symptoms and family history are vital information for our surgeons to make a correct clinical diagnosis.

CT is currently the preferred imaging examination[46-49] because it can clearly show



Figure 3 Evidence and gap map of multiple gastrointestinal stromal tumors (Bubble diagram). MGISTs: Multiple gastrointestinal stromal tumors; NF1: Type 1 neurofibromatosis.

Figure 4 Overall survival of patients with multiple gastrointestinal stromal tumors.

GISTs in the small intestine. Small-sized GISTs in the small intestine usually show higher enhancement than those in the stomach[50]. More remarkable, micro-GISTs that comprise the main parts of MGISTs are, however, difficult to detect by CT. Therefore, to avoid a misdiagnosis, preoperative endoscopy is necessary. However, we should factor in that small GISTs may have a large extra-extension that is not visible during endoscopy. Thus, CT and endoscopy are complementary to each other.

A previous systemic review showed that 49% of single GIST were measured to be 5-10 cm in size[1,2]. In this study, groups of ≤ 1 cm (called as micro-GISTs) and 2-5 cm tumors were the main components of MGISTs, which may be because of satellite tumors. Roughly 30% of middle-aged and elderly general population may be detected with micro-GISTs, which have a high frequency of KIT mutation and almost no malignant potential, although they are considered to be a precursor lesion or the origin



Figure 5 Recurrence-free survival of patients with multiple gastrointestinal stromal tumors.

of GIST[31,34,51]. According to previous studies, sporadic, NF1-associated, and pediatric-type GISTs are mainly located in the stomach[12,36,52,53]. Interestingly, we found that MGISTs in our study usually affected the small intestine, followed by stomach and other sites. On considering both Peking University People’s Hospital and literature review, approximately 30% tumors were shown to affect two or more organs.

En bloc (R0) resection and minimally invasive surgery are the first choice of treatment. For some local MGISTs (≥ 2 cm), a segmental or wedge resection instead of an extended anatomic resection is appropriate to obtain negative margins. Especially, unlike single GISTs, MGISTs may affect one or more long segments of the GI tract; therefore, segmental resection is performed more frequently. A multidisciplinary team (MDT, including experienced oncologists, gastroenterologists, and radiologists among others) are needed in all MGIST patients, especially in patients with multiple organ involvement, to assess surgical excision and perioperative adjuvant therapy. All 12 patients form the Peking University People’s Hospital were assessed by MDT and underwent the most current appropriate individual-based treatment. Lympha-denectomy might not be required in most MGIST patients. A laparoscopic approach by experienced surgeons could be considered for select MGISTs located at favorable anatomic locations because of the fragile texture of tumors. Either laparotomy or laparoscopy must follow the basic oncological principles of GIST resection, and generally, multi-visceral resection and re-resection are not indicated. Imatinib was used as an auxiliary therapy in KIT/PDGFRA mutation MGISTs[29]. Gene detection is vital in precision therapy, as cases without KIT or PDGFRA mutation, such as syndromic MGISTs cases, may not respond to Imatinib[38,54,55], although some patients[56] have reported contrasting outcomes.

The most important independent prognostic factor for GIST recurrence after surgery is a high tumor mitotic rate[2,35,57,58]. Of note, IHC staining for Ki-67 antigen has been suggested as an alternative to mitosis rate counting, which is affected by subjective factors to some extent[59,60]. As approximately 70% of overall tumors in this study were low or very low risk tumors compared with 30%-45%[5,57,58] reported in previous studies, small satellite tumors may interfere with the results. Interestingly, pediatric-type GISTs are slightly unpredictable and have an indolent clinical progression; further, they may be more prone to be metastatic irrespective of the prognostic criteria used in adults, such as tumor site, size, and mitotic rate[7,37,61,62].

With regard to IHC results, a number of previous studies have documented only one tumor’s results, even though multiple lesions were observed; and this phenomenon was also observed in patients of the Peking University People’s Hospital. Accordingly, it was difficult to determine the accurate IHC manifestation of each tumor and summarize the different marker expressions of the main large tumor and small satellite tumors in each MGIST patient. Therefore, we recommend that, if possible, appropriately detailed pathological examination should be conducted for



each tumor.In an analysis including 10 population-based series and 2459 patients[2], the

estimated median OS duration was 12.4 years (95% confidence interval: 10.8-14.0), and the estimated 5-year, 10-year, and 15-year RF survival rates of patients with GISTs treated via surgery alone were 70.5%, 62.9%, and 59.9%, respectively. Only a few tumors relapsed after the first 10 years of follow-up, suggesting that most patients (approximately 60%) with operable GIST were probably cured by surgery[31]. Tumor size, site, and rupture before or during surgery were independent prognostic factors recurrence[2,57,58,63-66]. Despite the high tendency for metastasis and recurrence in syndromic MGISTs (particularly in pediatric-type MGISTs after up to 3-5 years, predominantly via a hematogenous pathway)[38,67], the 5-year and 10-year clinical course is usually indolent with a favorable prognosis similar to that of (or even better than that of) single GISTs but 15-year RF survival rate is poorer.

As per the evidence gap map (Figure 3), most of the current studies mainly focus on the demographic and oncological characteristics, but few pay attention to perioperative and operative information. In other words, patients benefit from treatment strategies such as neoadjuvant chemotherapy, selection of operative extensions, or genetic detection. Furthermore, it is necessary to form a standard medical diagnosis and surgical procedures of the MGISTs

There were some limitations to the current study. First, inclusion bias existed among Peking University People’s Hospital patients because of the hospital category and the lack of pediatric surgery at the hospital; this may have led to the recruitment of few young female patients and more elderly patients. Further, all 12 patients were unwilling to undergo gene detection because of the high cost and limited medical insurance coverage. Second, in the present study, we only included articles published in English; this may cause a language bias. Moreover, only case reports and series, as the current best evidence, were included, and SCARE and JBI data were not completely available for all included studies.

CONCLUSIONIn conclusion, MGISTs may have unique characteristics such as lower morbidity, female predominance, young age, multiple organ involvement, and possible syndromic GIST. Although OS was similar between single GISTs and MGISTs, a high rate of metastasis in MGIST patients resulted in a poor long-time RF survival rate. Based on the current EGM, focusing on gene detection and molecular biological analysis can contribute to the determination of the mechanism underlying this special type of GIST in future studies. Furthermore, an appropriate surgical approach and auxiliary therapy are urgently need to be determined by prospective, multicenter, and large-scale studies.

ARTICLE HIGHLIGHTSResearch backgroundMultiple gastrointestinal stromal tumors (MGISTs) is a very rare type of gastrointestinal stromal tumor (GIST) and is usually misdiagnosed as metastatic tumors.

Research motivationAs physicians become more aware of MGISTs, researchers believed that it was imperative to describe MGISTs to help surgeons make appropriate diagnosis and treatment.

Research objectivesThe study aimed to describe the clinical and oncological features of MGISTs and to offer evidence for MGISTs diagnosis and treatment.

Research methodsData of consecutive patients with MGISTs who were diagnosed at Peking University People’s Hospital (PKUPH) from 2008 to 2019 were retrospectively evaluated. Further, a literature search was conducted by retrieving data from PubMed, EMBASE, and the



Cochrane library databases from inception up to November 30, 2019.

Research resultsIn all, 12 patients were diagnosed with MGISTs at PKUPH, and 43 published records were ultimately included following literature review. Combined analysis of all the individual patient data showed that female (59.30%), young (14.45%), and syndromic GIST (63.95%) patients comprised a large proportion of the total patient population. Tumors were mainly located in the small intestine (58.92%), and both CD117 and CD34 were generally positive. After a mean 78.32-mo follow-up, the estimated median overall survival duration (11.5 years) was similar to single GISTs, but recurrence-free survival was relatively poorer.

Research conclusionsThe clinical and oncological features are potentially different between MGISTs and single GIST, such as lower morbidity, female predominance, young age, multiple organ involvement, and possible syndromic GIST. Although overall survival was similar between single GISTs and MGISTs, a high rate of metastasis in MGIST patients resulted in a poorer long-time RF survival rate.

Research perspectivesIn further studies, focusing on gene detection and molecular biological analysis can contribute to the understanding of the mechanism underlying this special type of GIST in future studies. Moreover, an appropriate surgical approach and auxiliary therapy are urgently need to be determined by prospective, multicenter, and large-scale studies.

ACKNOWLEDGEMENTSWe would like to thank the library of Peking University for database accessing and acquiring full texts and Dai JL from the Liver Transplantation Center, West China Hospital of Sichuan University for providing help with the diagram. Finally, we give our respect to all patients involved in the study.

REFERENCESSøreide K, Sandvik OM, Søreide JA, Giljaca V, Jureckova A, Bulusu VR. Global epidemiology of gastrointestinal stromal tumours (GIST): A systematic review of population-based cohort studies. Cancer Epidemiol 2016; 40: 39-46 [PMID: 26618334 DOI: 10.1016/j.canep.2015.10.031]

1

Joensuu H, Vehtari A, Riihimäki J, Nishida T, Steigen SE, Brabec P, Plank L, Nilsson B, Cirilli C, Braconi C, Bordoni A, Magnusson MK, Linke Z, Sufliarsky J, Federico M, Jonasson JG, Dei Tos AP, Rutkowski P. Risk of recurrence of gastrointestinal stromal tumour after surgery: an analysis of pooled population-based cohorts. Lancet Oncol 2012; 13: 265-274 [PMID: 22153892 DOI: 10.1016/S1470-2045(11)70299-6]

2

Ducimetière F, Lurkin A, Ranchère-Vince D, Decouvelaere AV, Péoc'h M, Istier L, Chalabreysse P, Muller C, Alberti L, Bringuier PP, Scoazec JY, Schott AM, Bergeron C, Cellier D, Blay JY, Ray-Coquard I. Incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing. PLoS One 2011; 6: e20294 [PMID: 21826194 DOI: 10.1371/journal.pone.0020294]

3

Gatta G, van der Zwan JM, Casali PG, Siesling S, Dei Tos AP, Kunkler I, Otter R, Licitra L, Mallone S, Tavilla A, Trama A, Capocaccia R; RARECARE working group. Rare cancers are not so rare: the rare cancer burden in Europe. Eur J Cancer 2011; 47: 2493-2511 [PMID: 22033323 DOI: 10.1016/j.ejca.2011.08.008]

4

Nilsson B, Bümming P, Meis-Kindblom JM, Odén A, Dortok A, Gustavsson B, Sablinska K, Kindblom LG. Gastrointestinal stromal tumors: the incidence, prevalence, clinical course, and prognostication in the preimatinib mesylate era--a population-based study in western Sweden. Cancer 2005; 103: 821-829 [PMID: 15648083 DOI: 10.1002/cncr.20862]

5

Kim KH, Nelson SD, Kim DH, Choi KU, Kim SJ, Min KW, Jang KS, Paik SS, Oh YH, Chae SW, Sohn JH, Kim HJ, Cho YK, Kim BI, Park DI, Sohn CI, Oh S, Choi SH, Choi YJ, Woo HY, Park YL, Park SJ, Lee SH, Ryu S, DO SI, Kang G, Kim K, Cho YH, Pyo JS. Diagnostic relevance of overexpressions of PKC-θ and DOG-1 and KIT/PDGFRA gene mutations in extragastrointestinal stromal tumors: a Korean six-centers study of 28 cases. Anticancer Res 2012; 32: 923-937 [PMID: 22399613 DOI: 10.1111/j.1442-2042.1997.tb00317.x]

6

Charville GW, Longacre TA. Surgical Pathology of Gastrointestinal Stromal Tumors: Practical 7


https://dx.doi.org/10.1016/j.canep.2015.10.031


https://dx.doi.org/10.1016/S1470-2045(11)70299-6




https://dx.doi.org/10.1016/j.ejca.2011.08.008


https://dx.doi.org/10.1002/cncr.20862


https://dx.doi.org/10.1111/j.1442-2042.1997.tb00317.x



Implications of Morphologic and Molecular Heterogeneity for Precision Medicine. Adv Anat Pathol 2017; 24: 336-353 [PMID: 28820749 DOI: 10.1097/PAP.0000000000000166]Corless CL, Barnett CM, Heinrich MC. Gastrointestinal stromal tumours: origin and molecular oncology. Nat Rev Cancer 2011; 11: 865-878 [PMID: 22089421 DOI: 10.1038/nrc3143]

8

Min KW. Gastrointestinal stromal tumor: an ultrastructural investigation on regional differences with considerations on their histogenesis. Ultrastruct Pathol 2010; 34: 174-188 [PMID: 20455665 DOI: 10.3109/01913121003689075]

9

Wang C, Jin MS, Zou YB, Gao JN, Li XB, Peng F, Wang HY, Wu ZD, Wang YP, Duan XM. Diagnostic significance of DOG-1 and PKC-θ expression and c-Kit/PDGFRA mutations in gastrointestinal stromal tumours. Scand J Gastroenterol 2013; 48: 1055-1065 [PMID: 23862765 DOI: 10.3109/00365521.2013.816770]

10

Heinrich MC, Corless CL, Demetri GD, Blanke CD, von Mehren M, Joensuu H, McGreevey LS, Chen CJ, Van den Abbeele AD, Druker BJ, Kiese B, Eisenberg B, Roberts PJ, Singer S, Fletcher CD, Silberman S, Dimitrijevic S, Fletcher JA. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 2003; 21: 4342-4349 [PMID: 14645423 DOI: 10.1200/JCO.2003.04.190]

11

Miettinen M, Fetsch JF, Sobin LH, Lasota J. Gastrointestinal stromal tumors in patients with neurofibromatosis 1: a clinicopathologic and molecular genetic study of 45 cases. Am J Surg Pathol 2006; 30: 90-96 [PMID: 16330947 DOI: 10.1097/01.pas.0000176433.81079.bd]

12

Haller F, Schulten HJ, Armbrust T, Langer C, Gunawan B, Füzesi L. Multicentric sporadic gastrointestinal stromal tumors (GISTs) of the stomach with distinct clonal origin: differential diagnosis to familial and syndromal GIST variants and peritoneal metastasis. Am J Surg Pathol 2007; 31: 933-937 [PMID: 17527083 DOI: 10.1097/01.pas.0000213440.78407.27]

13

Kang G, Park YS, Jung ES, Joo M, Kang MS, Ahn S, Kang GH, Kim KM. Gastrointestinal stromal tumors in children and young adults: a clinicopathologic and molecular genetic study of 22 Korean cases. APMIS 2013; 121: 938-944 [PMID: 23755839 DOI: 10.1111/apm.12085]

14

Zhang L, Smyrk TC, Young WF Jr, Stratakis CA, Carney JA. Gastric stromal tumors in Carney triad are different clinically, pathologically, and behaviorally from sporadic gastric gastrointestinal stromal tumors: findings in 104 cases. Am J Surg Pathol 2010; 34: 53-64 [PMID: 19935059 DOI: 10.1097/PAS.0b013e3181c20f4f]

15

Pasini B, McWhinney SR, Bei T, Matyakhina L, Stergiopoulos S, Muchow M, Boikos SA, Ferrando B, Pacak K, Assie G, Baudin E, Chompret A, Ellison JW, Briere JJ, Rustin P, Gimenez-Roqueplo AP, Eng C, Carney JA, Stratakis CA. Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD. Eur J Hum Genet 2008; 16: 79-88 [PMID: 17667967 DOI: 10.1038/sj.ejhg.5201904]

16

Kang DY, Park CK, Choi JS, Jin SY, Kim HJ, Joo M, Kang MS, Moon WS, Yun KJ, Yu ES, Kang H, Kim KM. Multiple gastrointestinal stromal tumors: Clinicopathologic and genetic analysis of 12 patients. Am J Surg Pathol 2007; 31: 224-232 [PMID: 17255767 DOI: 10.1097/01.pas.0000213318.66800.94]

17

Gasparotto D, Rossi S, Bearzi I, Doglioni C, Marzotto A, Hornick JL, Grizzo A, Sartor C, Mandolesi A, Sciot R, Debiec-Rychter M, Dei Tos AP, Maestro R. Multiple primary sporadic gastrointestinal stromal tumors in the adult: an underestimated entity. Clin Cancer Res 2008; 14: 5715-5721 [PMID: 18779314 DOI: 10.1158/1078-0432.CCR-08-0622]

18

Agaimy A, Dirnhofer S, Wünsch PH, Terracciano LM, Tornillo L, Bihl MP. Multiple sporadic gastrointestinal stromal tumors (GISTs) of the proximal stomach are caused by different somatic KIT mutations suggesting a field effect. Am J Surg Pathol 2008; 32: 1553-1559 [PMID: 18724245 DOI: 10.1097/PAS.0b013e31817587ea]

19

Agaimy A, Märkl B, Arnholdt H, Wünsch PH, Terracciano LM, Dirnhofer S, Hartmann A, Tornillo L, Bihl MP. Multiple sporadic gastrointestinal stromal tumours arising at different gastrointestinal sites: pattern of involvement of the muscularis propria as a clue to independent primary GISTs. Virchows Arch 2009; 455: 101-108 [PMID: 19572146 DOI: 10.1007/s00428-009-0803-1]

20

von Mehren M, Randall RL, Benjamin RS, Boles S, Bui MM, Ganjoo KN, George S, Gonzalez RJ, Heslin MJ, Kane JM, Keedy V, Kim E, Koon H, Mayerson J, McCarter M, McGarry SV, Meyer C, Morris ZS, O'Donnell RJ, Pappo AS, Paz IB, Petersen IA, Pfeifer JD, Riedel RF, Ruo B, Schuetze S, Tap WD, Wayne JD, Bergman MA, Scavone JL. Soft Tissue Sarcoma, Version 2.2018, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2018; 16: 536-563 [PMID: 29752328 DOI: 10.6004/jnccn.2018.0025]

21

Casali PG, Abecassis N, Aro HT, Bauer S, Biagini R, Bielack S, Bonvalot S, Boukovinas I, Bovee JVMG, Brodowicz T, Broto JM, Buonadonna A, De Álava E, Dei Tos AP, Del Muro XG, Dileo P, Eriksson M, Fedenko A, Ferraresi V, Ferrari A, Ferrari S, Frezza AM, Gasperoni S, Gelderblom H, Gil T, Grignani G, Gronchi A, Haas RL, Hassan B, Hohenberger P, Issels R, Joensuu H, Jones RL, Judson I, Jutte P, Kaal S, Kasper B, Kopeckova K, Krákorová DA, Le Cesne A, Lugowska I, Merimsky O, Montemurro M, Pantaleo MA, Piana R, Picci P, Piperno-Neumann S, Pousa AL, Reichardt P, Robinson MH, Rutkowski P, Safwat AA, Schöffski P, Sleijfer S, Stacchiotti S, Sundby Hall K, Unk M, Van Coevorden F, van der Graaf WTA, Whelan J, Wardelmann E, Zaikova O, Blay JY; ESMO Guidelines Committee and EURACAN. Gastrointestinal stromal tumours: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2018; 29: iv267 [PMID: 30188977 DOI: 10.1093/annonc/mdy320]

22


https://dx.doi.org/10.1097/PAP.0000000000000166


https://dx.doi.org/10.1038/nrc3143


https://dx.doi.org/10.3109/01913121003689075


https://dx.doi.org/10.3109/00365521.2013.816770




https://dx.doi.org/10.1097/01.pas.0000176433.81079.bd


https://dx.doi.org/10.1097/01.pas.0000213440.78407.27


https://dx.doi.org/10.1111/apm.12085


https://dx.doi.org/10.1097/PAS.0b013e3181c20f4f


https://dx.doi.org/10.1038/sj.ejhg.5201904






https://dx.doi.org/10.1097/PAS.0b013e31817587ea


https://dx.doi.org/10.1007/s00428-009-0803-1


https://dx.doi.org/10.6004/jnccn.2018.0025


https://dx.doi.org/10.1093/annonc/mdy320



Judson I, Bulusu R, Seddon B, Dangoor A, Wong N, Mudan S. UK clinical practice guidelines for the management of gastrointestinal stromal tumours (GIST). Clin Sarcoma Res 2017; 7: 6 [PMID: 28465823 DOI: 10.1186/s13569-017-0072-8]

23

Landi B, Blay JY, Bonvalot S, Brasseur M, Coindre JM, Emile JF, Hautefeuille V, Honore C, Lartigau E, Mantion G, Pracht M, Le Cesne A, Ducreux M, Bouche O; «Thésaurus National de Cancérologie Digestive (TNCD)» (Fédération Francophone de Cancérologie Digestive (FFCD); Fédération Nationale de Centres de Lutte Contre les Cancers (UNICANCER); Groupe Coopérateur Multidisciplinaire en Oncologie (GERCOR); Société Française de Chirurgie Digestive (SFCD); Société Française de Radiothérapie Oncologique (SFRO); Société Française d’Endoscopie Digestive (SFED); Société Nationale Française de Gastroentérologie (SNFGE). Gastrointestinal stromal tumours (GISTs): French Intergroup Clinical Practice Guidelines for diagnosis, treatments and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO). Dig Liver Dis 2019; 51: 1223-1231 [PMID: 31387778 DOI: 10.1016/j.dld.2019.07.006]

24

Koo DH, Ryu MH, Kim KM, Yang HK, Sawaki A, Hirota S, Zheng J, Zhang B, Tzen CY, Yeh CN, Nishida T, Shen L, Chen LT, Kang YK. Asian Consensus Guidelines for the Diagnosis and Management of Gastrointestinal Stromal Tumor. Cancer Res Treat 2016; 48: 1155-1166 [PMID: 27384163 DOI: 10.4143/crt.2016.187]

25

Li J, Ye Y, Wang J, Zhang B, Qin S, Shi Y, He Y, Liang X, Liu X, Zhou Y, Wu X, Zhang X, Wang M, Gao Z, Lin T, Cao H, Shen L, Chinese Society Of Clinical Oncology Csco Expert Committee On Gastrointestinal Stromal Tumor. Chinese consensus guidelines for diagnosis and management of gastrointestinal stromal tumor. Chin J Cancer Res 2017; 29: 281-293 [PMID: 28947860 DOI: 10.21147/j.issn.1000-9604.2017.04.01]

26

Agha RA, Fowler AJ, Saeta A, Barai I, Rajmohan S, Orgill DP; SCARE Group. The SCARE Statement: Consensus-based surgical case report guidelines. Int J Surg 2016; 34: 180-186 [PMID: 27613565 DOI: 10.1016/j.ijsu.2016.08.014]

27

Moher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. BMJ 2009; 339: b2535 [PMID: 19622551 DOI: 10.1136/bmj.b2535]

28

Li K, Tjhoi W, Shou C, Yang W, Zhang Q, Liu X, Yu J. Multiple gastrointestinal stromal tumors: analysis of clinicopathologic characteristics and prognosis of 20 patients. Cancer Manag Res 2019; 11: 7031-7038 [PMID: 31413638 DOI: 10.2147/CMAR.S197560]

29

Pearson A, Wiechula R, Court A, Lockwood C. The JBI model of evidence-based healthcare. Int J Evid Based Healthc 2005; 3: 207-215 [PMID: 21631749 DOI: 10.1111/j.1479-6988.2005.00026.x]

30

Rubin BP, Heinrich MC, Corless CL. Gastrointestinal stromal tumour. Lancet 2007; 369: 1731-1741 [PMID: 17512858 DOI: 10.1016/S0140-6736(07)60780-6]

31

Ma GL, Murphy JD, Martinez ME, Sicklick JK. Epidemiology of gastrointestinal stromal tumors in the era of histology codes: results of a population-based study. Cancer Epidemiol Biomarkers Prev 2015; 24: 298-302 [PMID: 25277795 DOI: 10.1158/1055-9965.EPI-14-1002]

32

Tryggvason G, Gíslason HG, Magnússon MK, Jónasson JG. Gastrointestinal stromal tumors in Iceland, 1990-2003: the icelandic GIST study, a population-based incidence and pathologic risk stratification study. Int J Cancer 2005; 117: 289-293 [PMID: 15900576 DOI: 10.1002/ijc.21167]

33

Corless CL, McGreevey L, Haley A, Town A, Heinrich MC. KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size. Am J Pathol 2002; 160: 1567-1572 [PMID: 12000708 DOI: 10.1016/S0002-9440(10)61103-0]

34

DeMatteo RP, Lewis JJ, Leung D, Mudan SS, Woodruff JM, Brennan MF. Two hundred gastrointestinal stromal tumors: recurrence patterns and prognostic factors for survival. Ann Surg 2000; 231: 51-58 [PMID: 10636102 DOI: 10.1097/00000658-200001000-00008]

35

Miettinen M, Lasota J, Sobin LH. Gastrointestinal stromal tumors of the stomach in children and young adults: a clinicopathologic, immunohistochemical, and molecular genetic study of 44 cases with long-term follow-up and review of the literature. Am J Surg Pathol 2005; 29: 1373-1381 [PMID: 16160481 DOI: 10.1097/01.pas.0000172190.79552.8b]

36

Agaram NP, Laquaglia MP, Ustun B, Guo T, Wong GC, Socci ND, Maki RG, DeMatteo RP, Besmer P, Antonescu CR. Molecular characterization of pediatric gastrointestinal stromal tumors. Clin Cancer Res 2008; 14: 3204-3215 [PMID: 18483389 DOI: 10.1158/1078-0432.CCR-07-1984]

37

Rege TA, Wagner AJ, Corless CL, Heinrich MC, Hornick JL. "Pediatric-type" gastrointestinal stromal tumors in adults: distinctive histology predicts genotype and clinical behavior. Am J Surg Pathol 2011; 35: 495-504 [PMID: 21358303 DOI: 10.1097/PAS.0b013e31820e5f7d]

38

Andersson J, Sihto H, Meis-Kindblom JM, Joensuu H, Nupponen N, Kindblom LG. NF1-associated gastrointestinal stromal tumors have unique clinical, phenotypic, and genotypic characteristics. Am J Surg Pathol 2005; 29: 1170-1176 [PMID: 16096406 DOI: 10.1097/01.pas.0000159775.77912.15]

39

Maertens O, Prenen H, Debiec-Rychter M, Wozniak A, Sciot R, Pauwels P, De Wever I, Vermeesch JR, de Raedt T, De Paepe A, Speleman F, van Oosterom A, Messiaen L, Legius E. Molecular pathogenesis of multiple gastrointestinal stromal tumors in NF1 patients. Hum Mol Genet 2006; 15: 1015-1023 [PMID: 16461335 DOI: 10.1093/hmg/ddl016]

40

Carney JA. Gastric stromal sarcoma, pulmonary chondroma, and extra-adrenal paraganglioma (Carney Triad): natural history, adrenocortical component, and possible familial occurrence. Mayo Clin Proc 1999; 74: 543-552 [PMID: 10377927 DOI: 10.4065/74.6.543]

41

Carney JA, Stratakis CA. Familial paraganglioma and gastric stromal sarcoma: a new syndrome distinct from the Carney triad. Am J Med Genet 2002; 108: 132-139 [PMID: 11857563 DOI:

42


https://dx.doi.org/10.1186/s13569-017-0072-8




https://dx.doi.org/10.4143/crt.2016.187


https://dx.doi.org/10.21147/j.issn.1000-9604.2017.04.01


https://dx.doi.org/10.1016/j.ijsu.2016.08.014


https://dx.doi.org/10.1136/bmj.b2535


https://dx.doi.org/10.2147/CMAR.S197560




https://dx.doi.org/10.1016/S0140-6736(07)60780-6


https://dx.doi.org/10.1158/1055-9965.EPI-14-1002


https://dx.doi.org/10.1002/ijc.21167


https://dx.doi.org/10.1016/S0002-9440(10)61103-0


https://dx.doi.org/10.1097/00000658-200001000-00008


https://dx.doi.org/10.1097/01.pas.0000172190.79552.8b




https://dx.doi.org/10.1097/PAS.0b013e31820e5f7d




https://dx.doi.org/10.1093/hmg/ddl016


https://dx.doi.org/10.4065/74.6.543




10.1002/ajmg.10235]Diment J, Tamborini E, Casali P, Gronchi A, Carney JA, Colecchia M. Carney triad: case report and molecular analysis of gastric tumor. Hum Pathol 2005; 36: 112-116 [PMID: 15712189 DOI: 10.1016/j.humpath.2004.10.003]

43

Pappo AS, Janeway KA. Pediatric gastrointestinal stromal tumors. Hematol Oncol Clin North Am 2009; 23: 15-34, vii [PMID: 19248968 DOI: 10.1016/j.hoc.2008.11.005]

44

Maeyama H, Hidaka E, Ota H, Minami S, Kajiyama M, Kuraishi A, Mori H, Matsuda Y, Wada S, Sodeyama H, Nakata S, Kawamura N, Hata S, Watanabe M, Iijima Y, Katsuyama T. Familial gastrointestinal stromal tumor with hyperpigmentation: association with a germline mutation of the c-kit gene. Gastroenterology 2001; 120: 210-215 [PMID: 11208730 DOI: 10.1053/gast.2001.20880]

45

Vernuccio F, Taibbi A, Picone D, LA Grutta L, Midiri M, Lagalla R, Lo Re G, Bartolotta TV. Imaging of Gastrointestinal Stromal Tumors: From Diagnosis to Evaluation of Therapeutic Response. Anticancer Res 2016; 36: 2639-2648 [PMID: 27272772 DOI: 10.1097/MD.0000000000019275]

46

Parab TM, DeRogatis MJ, Boaz AM, Grasso SA, Issack PS, Duarte DA, Urayeneza O, Vahdat S, Qiao JH, Hinika GS. Gastrointestinal stromal tumors: a comprehensive review. J Gastrointest Oncol 2019; 10: 144-154 [PMID: 30788170 DOI: 10.21037/jgo.2018.08.20]

47

Werewka-Maczuga A, Osiński T, Chrzan R, Buczek M, Urbanik A. Characteristics of computed tomography imaging of gastrointestinal stromal tumor (GIST) and related diagnostic problems. Pol J Radiol 2011; 76: 38-48 [PMID: 22802840]

48

King DM. The radiology of gastrointestinal stromal tumours (GIST). Cancer Imaging 2005; 5: 150-156 [PMID: 16361144 DOI: 10.1102/1470-7330.2005.0109]

49

Inoue A, Ota S, Nitta N, Murata K, Shimizu T, Sonoda H, Tani M, Ban H, Inatomi O, Ando A, Kushima R, Watanabe Y. Difference of computed tomographic characteristic findings between gastric and intestinal gastrointestinal stromal tumors. Jpn J Radiol 2020; 38: 771-781 [PMID: 32246352 DOI: 10.1007/s11604-020-00962-0]

50

Duensing A, Heinrich MC, Fletcher CD, Fletcher JA. Biology of gastrointestinal stromal tumors: KIT mutations and beyond. Cancer Invest 2004; 22: 106-116 [PMID: 15069768 DOI: 10.1081/cnv-120027585]

51

Prakash S, Sarran L, Socci N, DeMatteo RP, Eisenstat J, Greco AM, Maki RG, Wexler LH, LaQuaglia MP, Besmer P, Antonescu CR. Gastrointestinal stromal tumors in children and young adults: a clinicopathologic, molecular, and genomic study of 15 cases and review of the literature. J Pediatr Hematol Oncol 2005; 27: 179-187 [PMID: 15838387 DOI: 10.1097/01.mph.0000157790.81329.47]

52

Miettinen M, Lasota J. Gastrointestinal stromal tumors--definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch 2001; 438: 1-12 [PMID: 11213830 DOI: 10.1007/s004280000338]

53

Mussi C, Schildhaus HU, Gronchi A, Wardelmann E, Hohenberger P. Therapeutic consequences from molecular biology for gastrointestinal stromal tumor patients affected by neurofibromatosis type 1. Clin Cancer Res 2008; 14: 4550-4555 [PMID: 18628470 DOI: 10.1158/1078-0432.CCR-08-0086]

54

Neppala P, Banerjee S, Fanta PT, Yerba M, Porras KA, Burgoyne AM, Sicklick JK. Current management of succinate dehydrogenase-deficient gastrointestinal stromal tumors. Cancer Metastasis Rev 2019; 38: 525-535 [PMID: 31773431 DOI: 10.1007/s10555-019-09818-0]

55

Bamba S, Hirota S, Inatomi O, Ban H, Nishimura T, Shioya M, Imaeda H, Nishida A, Sasaki M, Murata S, Andoh A. Familial and multiple gastrointestinal stromal tumors with fair response to a half-dose of imatinib. Intern Med 2015; 54: 759-764 [PMID: 25832938 DOI: 10.2169/internalmedicine.54.3585]

56

Miettinen M, Sobin LH, Lasota J. Gastrointestinal stromal tumors of the stomach: a clinicopathologic, immunohistochemical, and molecular genetic study of 1765 cases with long-term follow-up. Am J Surg Pathol 2005; 29: 52-68 [PMID: 15613856 DOI: 10.1097/01.pas.0000146010.92933.de]

57

Miettinen M, Makhlouf H, Sobin LH, Lasota J. Gastrointestinal stromal tumors of the jejunum and ileum: a clinicopathologic, immunohistochemical, and molecular genetic study of 906 cases before imatinib with long-term follow-up. Am J Surg Pathol 2006; 30: 477-489 [PMID: 16625094 DOI: 10.1097/00000478-200604000-00008]

58

Jiang J, Jin MS, Suo J, Wang YP, He L, Cao XY. Evaluation of malignancy using Ki-67, p53, EGFR and COX-2 expressions in gastrointestinal stromal tumors. World J Gastroenterol 2012; 18: 2569-2575 [PMID: 22654456 DOI: 10.3748/wjg.v18.i20.2569]

59

Liang YM, Li XH, Li WM, Lu YY. Prognostic significance of PTEN, Ki-67 and CD44s expression patterns in gastrointestinal stromal tumors. World J Gastroenterol 2012; 18: 1664-1671 [PMID: 22529697 DOI: 10.3748/wjg.v18.i14.1664]

60

Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ, Miettinen M, O'Leary TJ, Remotti H, Rubin BP, Shmookler B, Sobin LH, Weiss SW. Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol 2002; 33: 459-465 [PMID: 12094370 DOI: 10.1053/hupa.2002.123545]

61

Price VE, Zielenska M, Chilton-MacNeill S, Smith CR, Pappo AS. Clinical and molecular characteristics of pediatric gastrointestinal stromal tumors (GISTs). Pediatr Blood Cancer 2005; 45: 20-24 [PMID: 15795882 DOI: 10.1002/pbc.20377]

62

Hohenberger P, Ronellenfitsch U, Oladeji O, Pink D, Ströbel P, Wardelmann E, Reichardt P. Pattern of recurrence in patients with ruptured primary gastrointestinal stromal tumour. Br J Surg 2010; 97:

63

https://dx.doi.org/10.1002/ajmg.10235


https://dx.doi.org/10.1016/j.humpath.2004.10.003


https://dx.doi.org/10.1016/j.hoc.2008.11.005


https://dx.doi.org/10.1053/gast.2001.20880


https://dx.doi.org/10.1097/MD.0000000000019275


https://dx.doi.org/10.21037/jgo.2018.08.20



https://dx.doi.org/10.1102/1470-7330.2005.0109


https://dx.doi.org/10.1007/s11604-020-00962-0


https://dx.doi.org/10.1081/cnv-120027585


https://dx.doi.org/10.1097/01.mph.0000157790.81329.47


https://dx.doi.org/10.1007/s004280000338




https://dx.doi.org/10.1007/s10555-019-09818-0


https://dx.doi.org/10.2169/internalmedicine.54.3585


https://dx.doi.org/10.1097/01.pas.0000146010.92933.de


https://dx.doi.org/10.1097/00000478-200604000-00008






https://dx.doi.org/10.1053/hupa.2002.123545


https://dx.doi.org/10.1002/pbc.20377



1854-1859 [PMID: 20730857 DOI: 10.1002/bjs.7222]Dematteo RP, Gold JS, Saran L, Gönen M, Liau KH, Maki RG, Singer S, Besmer P, Brennan MF, Antonescu CR. Tumor mitotic rate, size, and location independently predict recurrence after resection of primary gastrointestinal stromal tumor (GIST). Cancer 2008; 112: 608-615 [PMID: 18076015 DOI: 10.1002/cncr.23199]

64

Rutkowski P, Bylina E, Wozniak A, Nowecki ZI, Osuch C, Matlok M, Switaj T, Michej W, Wroński M, Głuszek S, Kroc J, Nasierowska-Guttmejer A, Joensuu H. Validation of the Joensuu risk criteria for primary resectable gastrointestinal stromal tumour - the impact of tumour rupture on patient outcomes. Eur J Surg Oncol 2011; 37: 890-896 [PMID: 21737227 DOI: 10.1016/j.ejso.2011.06.005]

65

Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin Diagn Pathol 2006; 23: 70-83 [PMID: 17193820 DOI: 10.1053/j.semdp.2006.09.001]

66

Iwasaki M, Morimoto T, Sano K, Fukumitsu K, Takeda R, Iwata S, Nakamura Y, Yasui H, Urata Y, Mukaihara S. A case of pediatric gastrointestinal stromal tumor of the stomach. Pediatr Int 2005; 47: 102-104 [PMID: 15693878 DOI: 10.1111/j.1442-200x.2004.02016.x]

67


https://dx.doi.org/10.1002/bjs.7222




https://dx.doi.org/10.1016/j.ejso.2011.06.005


https://dx.doi.org/10.1053/j.semdp.2006.09.001


https://dx.doi.org/10.1111/j.1442-200x.2004.02016.x


World Journal of



ORIGINAL ARTICLE

Randomized Controlled Trial

Evaluation of an educational telephone intervention strategy to improve non-screening colonoscopy attendance: A randomized controlled trial

Agustín Seoane, Xènia Font, Juan C Pérez, Rocío Pérez, Carlos F Enriquez, Miriam Parrilla, Faust Riu, Josep M Dedeu, Luis E Barranco, Xavier Duran, Inés A Ibáñez, Marco A Álvarez

ORCID number: Agustín Seoane 0000-0001-8023-4445; Xènia Font 0000-0001-7605-2622; Juan C Pérez 0000-0002-4927-9546; Rocío Pérez 0000-0003-2092-0616; Carlos F Enriquez 0000-0001-9077-2618; Miriam Parrilla 0000-0003-1740-0396; Faust Riu 0000-0002-6576-4226; Josep M Dedeu 0000-0003-0522-1032; Luis E Barranco 0000-0002-7352-7415; Xavier Duran 0000-0001-8517-9254; Inés A Ibáñez 0000-0002-9642-6545; Marco A Álvarez 0000-0002-9312-0268.

Author contributions: Seoane A contributed to conception and designed of the study, analysis and interpretation of data; Font X, Pérez JC, Pérez R, Enriquez CF and Parrilla M contributed to acquisition of data; Duran X contributed to the statistical analysis; Riu F, Dedeu JM, Barranco LE, Ibáñez IA and Álvarez MA contributed making critical revisions and related to important intellectual content of the manuscript.

Supported by Hospital del Mar, Parc de Salut Mar.

Institutional review board statement: The study was approved by the ethics committee

Agustín Seoane, Xènia Font, Juan C Pérez, Rocío Pérez, Carlos F Enriquez, Miriam Parrilla, Faust Riu, Josep M Dedeu, Luis E Barranco, Inés A Ibáñez, Marco A Álvarez, Digestive Department, Endoscopy Unit, Hospital del Mar, Parc de Salut Mar, Barcelona 08003, Spain

Agustín Seoane, Faust Riu, Josep M Dedeu, Luis E Barranco, Marco A Álvarez, Colorectal Cancer Research Group, IMIM (Hospital del Mar Medical Research Institute), Barcelona 08003, Spain

Josep M Dedeu, Marco A Álvarez, Department of Medicine, Autonomous University of Barcelona, Barcelona 08003, Spain

Xavier Duran, Consulting Service on Methodology for Biomedical Research, IMIM (Hospital del Mar Medical Research Institute), Barcelona 08003, Spain

Corresponding author: Agustín Seoane, MD, Attending Doctor, Research Scientist, Digestive Department, Endoscopy Unit, Hospital del Mar, Parc de Salut Mar, Passeig Marítim, 25-29, Barcelona 08003, Spain. [email protected]

AbstractBACKGROUND Colonoscopy attendance is a key quality parameter in colorectal cancer population screening programmes. Within these programmes, educative interventions with bidirectional contact carried out by trained personnel have been proved to be an important tool for colonoscopy attendance improvement, and because of its huge clinical and economic impact, they have been widely implemented. However, outside of this population programmes, educative measures to improve colonoscopy attendance have been poorly studied and no navigation interventions are usually performed.

AIM To investigate the clinical and economic impacts of an educational telephone intervention on colonoscopy attendance outside colorectal cancer screening programmes.

METHODS This randomized controlled trial included consecutive patients referred to



http://orcid.org/0000-0001-8023-4445

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http://orcid.org/0000-0002-4927-9546

http://orcid.org/0000-0002-4927-9546

http://orcid.org/0000-0003-2092-0616

http://orcid.org/0000-0003-2092-0616

http://orcid.org/0000-0001-9077-2618

http://orcid.org/0000-0001-9077-2618

http://orcid.org/0000-0003-1740-0396

http://orcid.org/0000-0003-1740-0396

http://orcid.org/0000-0002-6576-4226

http://orcid.org/0000-0002-6576-4226

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http://orcid.org/0000-0003-0522-1032

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http://orcid.org/0000-0002-7352-7415

http://orcid.org/0000-0002-7352-7415

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http://orcid.org/0000-0001-8517-9254

http://orcid.org/0000-0001-8517-9254

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http://orcid.org/0000-0002-9642-6545

http://orcid.org/0000-0002-9642-6545

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http://orcid.org/0000-0002-9312-0268

http://orcid.org/0000-0002-9312-0268


Seoane A et al. Educational intervention to improve colonoscopy attendance


of IMIM, Hospital del Mar Medical Research Institute (7739/I).

Clinical trial registration statement: This study is registered at https://clinicaltrials.gov/ct2/show/NCT03458377. The registration identification number is NCT03458377.

Informed consent statement: All involved persons (subjects and legally authorized representatives) gave their written informed consent.

Conflict-of-interest statement: There are no conflicts of interest for any of the authors.


CONSORT 2010 statement: The authors have read the CONSORT 2010 Statement, and the manuscript was prepared and revised according to the CONSORT 2010 Statement.




Country/Territory of origin: Spain

Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C, C Grade D (Fair): 0 Grade E (Poor): 0

colonoscopy from primary care centres from November 2017 to May 2018. The intervention group (IG) received a telephone intervention, while the control group (CG) did not. Patients assigned to the IG received an educational telephone call 7 d before the colonoscopy appointment. The intervention was carried out by two nurses with deep endoscopic knowledge who were previously trained for a telephone educational intervention for colonoscopy. The impact on patient compliance with preparedness protocols related to bowel cleansing, anti-thrombotic management, and sedation scheduling was also evaluated. A second call was conducted to assess patient satisfaction. Intention-to-treat (ITT) and per-protocol (PP) analyses were performed.

RESULTS A total of 738 and 746 patients were finally included in the IG and CG respectively. Six hundred thirteen (83%) patients were contacted in the IG. The non-attendance rate was lower in the IG, both in the ITT analysis (IG 8.4% vs CG 14.3%, P < 0.001) and in the PP analysis (4.4% vs 14.3%, P < 0.001). In a multivariable analysis, belonging to the control group increased the risk of non-attendance in both, the ITT analysis (OR 1.81, 95%CI: 1.27 to 2.58, P = 0.001) and the PP analysis (OR 3.56, 95%CI: 2.25 to 5.64, P < 0.001). There was also a significant difference in compliance with preparedness protocols [bowel cleansing: IG 61.7% vs CG 52.6% (P = 0.001), antithrombotic management: IG 92.5% vs CG 62.8% (P = 0.001), and sedation scheduling: IG 78.8% vs CG 0% (P ≤ 0.001)]. We observed a net benefit of €55600/year after the intervention. The information given before the procedure was rated as excellent by 26% (CG) and 51% (IG) of patients, P ≤ 0.001.

CONCLUSION Educational telephone nurse intervention improves attendance, protocol compliance and patient satisfaction in the non-screening colonoscopy setting and has a large economic impact, which supports its imple-mentation and maintenance over time.

Key Words: Colonoscopy; Quality improvement; No-show patients; Nursing education; Patient compliance; Telephone intervention


Core Tip: This is the first randomized controlled trial that demonstrates that an educational intervention improves colonoscopy attendance in the non-screening colonoscopy setting. We found that bidirectional communication is the crucial point in reducing the non-attendance rate and telephone contact is a valid educational option. An endoscopy nurse is also a valid person to conduct the educational intervention and could be the ideal person to eliminate barriers that negatively influence the patient’s attendance. This educational intervention also improves protocol compliance and patient satisfaction and has a large economic impact, which supports its imple-mentation and maintenance over time.

Citation: Seoane A, Font X, Pérez JC, Pérez R, Enriquez CF, Parrilla M, Riu F, Dedeu JM, Barranco LE, Duran X, Ibáñez IA, Álvarez MA. Evaluation of an educational telephone intervention strategy to improve non-screening colonoscopy attendance: A randomized controlled trial. World J Gastroenterol 2020; 26(47): 7568-7583URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7568.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7568

INTRODUCTIONThe substantial advance in colonoscopy quality in recent years has been achieved at the expense of population screening colonoscopy programmes, launched due to their

https://clinicaltrials.gov/ct2/show/NCT03458377










Received: September 15, 2020 Peer-review started: September 15, 2020 First decision: November 3, 2020 Revised: November 16, 2020 Accepted: November 29, 2020 Article in press: November 29, 2020 Published online: December 21, 2020

P-Reviewer: Alsayid M, Ribeiro IB S-Editor: Huang P L-Editor: A P-Editor: Li JH

very large clinical and economic impact on the decrease in colorectal cancer mortality. At the European level, these programmes have drawn on multiple performance measures, subsequently used to improve non-screening colonoscopy quality[1].

Colonoscopy appointment attendance is a key quality parameter in screening programmes, and different improvement strategies have been developed using educational material (mainly web-based approaches and mailed print communication) and administrative reminder systems[2-6]. Another approach is patient navigation throughout the screening process. This intervention allows bidirectional contact to help address common barriers to attending colonoscopy by eliminating misconceptions, changing negative attitudes and helping patients gain control over factors that concern them with regard to their ability to comply with preparedness protocols[7,8]. This patient navigation strategy in screening programmes has also been reported to be cost-effective and to have a positive influence on patient satisfaction[9-11].

However, little effort has been made to improve colonoscopy attendance outside screening programmes in daily clinical practice, and educational measures are not usually performed. There are also very few published studies on educational measures aimed at increasing attendance. Some studies have identified several factors related to the no-show rate of endoscopic procedures in general, but specific information on the attendance rate[12-16] in the context of non-screening colonoscopy is not well known. Non-educational reminder systems have been shown to result in little improvement in colonoscopy attendance[17-19]. Nevertheless, an educational telephone nurse intervention has been shown to have better efficacy in reducing the no-show rate, and the intervention was also cost-effective[20].

Effective educational patient interventions have also been applied in other endoscopy quality areas, such as patient compliance with achieving complete examinations[21] and bowel cleansing for improving adequacy, a successful strategy to improve the adenoma detection rate[22-24]. All these data suggest the suitability of implementing educational patient interventions in endoscopy units to improve non-screening colonoscopy quality.

We designed a randomized controlled trial (RCT) to conduct an educational telephone nurse intervention with bidirectional contact directed to increase colonoscopy attendance, similar to the patient navigation carried out in the population screening setting. Second, we assessed the economic impact and the potential benefit of the intervention in regard to compliance with patient preparedness protocols, cleansing adequacy, and patient satisfaction.

MATERIALS AND METHODSStudy designThis is a prospective, RCT designed to assess the impact of an educational telephone intervention performed by digestive endoscopy nurses on colonoscopy attendance outside colorectal cancer screening programmes. This study was conducted in a tertiary centre at Hospital del Mar of Barcelona. The study was approved by the ethics committee of our centre (7739/I) in accordance with the Declaration of Helsinki, and the trial was registered at ClinicalTrials.gov (ID: NCT03458377).

Study population and treatment allocationAll consecutive outpatients referred for colonoscopy from November 2017 to May 2018 from the primary care centres in our health area were included. The exclusion criteria were unwillingness to participate, inability to obtain the signed informed consent form, hospital admission during the study period, simultaneous participation in another clinical trial and/or impossibility to carry out the educational intervention (colonoscopy cancellation or not having a telephone). Patients routinely excluded in endoscopic studies for presenting pathologies that condition the colon preparation regimen or the sedation plan (history of subtotal colectomy, active inflammatory bowel disease, or high comorbidity), were not excluded in our study according to the main objective of evaluating the attendance rate.

An intervention group (IG) and a control group (CG) were designed. We performed a prerandomization method (Zelen’s method) with a complete-double-consent-design to eliminate selection bias created by contact with the patient prior to randomization in a study where assessing the applicability of contact is crucial, and to avoid the risk of non-participation and drop out of the study, given the attractiveness of the designed intervention.

Patients were randomized to the CG and the IG 10 d before the colonoscopy



appointment using a computer-generated randomization list with a 1:1 allocation rate. In the IG, verbal consent was obtained in all patients after receiving a detailed explanation of the nature, aims, and consequences of the study, coinciding with the telephone call for intervention 7 d before the colonoscopy appointment. We obtained the signed informed consent form for both groups on the day of the colonoscopy. For those who did not attend the colonoscopy appointment, the signed informed consent form was obtained after later contact.

Patient preparedness protocols, colonoscopy procedure and patient informationPatient preparedness protocols included bowel cleansing, antithrombotic management and sedation scheduling. We recommended a low fibre diet 48 h before the colonoscopy and laxative intake in a split dose regimen, with an interval of less than 4 h between the last laxative dose and the colonoscopy, according to the European Society of Gastrointestinal Endoscopy (ESGE) Guidelines[25]. In the case of antithrombotic drug intake, we followed the withdrawal ESGE guideline algorithm[26]. Sedation was administered by an anaesthesiologist if the patient had a high comorbidity burden; otherwise, a nurse directed by a gastroenterologist administered the sedation[27]. A high comorbidity burden was defined in cases of serious COPD, tracheostomy, severe ventricular function, Mobitz II-III atrioventricular block, implantable cardioverter defibrillator, severe mitral or aortic valvular stenosis, severe sleep apnoea or body mass index > 40, which were verified by the nurse after reviewing medical history and interviewing the patient.

All of the patients received information about patient preparedness protocols for colonoscopy in the primary care centres at the time of the colonoscopy referral. Additionally, the patients received a certified letter including the appointment time and a copy of bowel cleansing instructions.

Educational interventionPatients assigned to the IG received an educational telephone call 7 d before the colonoscopy appointment. The intervention was carried out by two nurses with deep endoscopic knowledge who were previously trained for a telephone educational intervention for colonoscopy[22,28,29] and instructed to evaluate the need for anaesthesia.

Special emphasis was placed on the attempt to eliminate socioeconomic, psychological and clinical barriers that could affect attendance. Rescheduling was performed in case of work conflict, and language translation help was requested when needed. After reviewing the clinical history, the nurses focused on explaining the importance of getting tested and the possibility of important conditions not being diagnosed in case of non-attendance. Efforts were made to eliminate fears and misconceptions related to first colonoscopies and previous negative experiences. Clinical aspects were approached by giving the patients instructions and tricks to help them successfully complete the preparedness protocols. Finally, other possible questions were addressed, and the intervention ended after confirmation of a complete understanding and willingness of the patient to attend the appointment. In the case of a disabled patient, the intervention was performed with the patient’s caretaker. Three attempts were made to locate the patient. Both mobile and landline telephones were considered for the intervention.

Outcome measuresThe primary outcome was the rate of non-attendance. Non-attendance was defined in case the patient did not show up to the colonoscopy appointment or in case of rescheduling or cancellation in the 3 d prior to the appointment, given the impossibility of preparing another patient in the remaining time and being able to fill the gap with another colonoscopy.

The secondary outcomes included compliance with patient preparedness protocols, bowel cleansing adequacy, patient satisfaction, and cost analysis derived from the nurse educational intervention.

To evaluate compliance with the cleansing protocols we asked the patient about the correct diet, taking the last dose of the laxative between 2 and 4 h, total intake of the cleansing agent, and split-dose regimen. According to the predominant social-economic low class of our patients, we preferred to measure the compliance with the bowel preparation protocol with a short face-to-face interview conducted by an endoscopy nurse before the colonoscopy, instead of a survey. Cleansing compliance was defined as an aggregate of the 4 previous cleansing variables.

Skilled endoscopists (> 1000 colonoscopies each) blinded to the randomization evaluated the adequacy according to the Boston Bowel Preparation Scale (BBPS).



Cleansing adequacy was defined as all the colon segments scoring 2 or 3 points.Compliance with the management of antithrombotic drugs was assessed according

to ESGE guidelines. In the IG, patients with high comorbidity were referred to an endoscopy programme under anaesthesia. The appropriate scheduling according to the sedation administration was evaluated according to the presence of a high comorbidity burden. Disabled patients included any patient with physical or intellectual disability who needed help with colonoscopy preparedness.

Cost analysis was performed from the supplier’s perspective. We calculated the direct costs of the intervention compared with the cost of a colonoscopy that could not be billed. A colonoscopy service is invoiced based on its performance, which means it will not be charged if the patient does not show or there is a rescheduling need because of non-compliance with patient preparedness protocols. The cost of the colonoscopy was based on our hospital charges. The cost of the intervention was based on the nurse salary, according to our hospital’s collective agreement.

Patient satisfaction was quantified with the GHAA 9-me questionnaire from the ASGE[30] previously translated and validated into Spanish[31], including 7 items (waiting time until the appointment, waiting time on the day of examination, personal manner of staff, personal manner of the physician, adequacy of explanations given after the procedure, discomfort during examination and an overall rating of the visit). A specific item related to information given before the procedure was added (item 8). Each item was rated in an ordinal way (0-4), with 4 being the maximum satisfaction score, and satisfaction was assessed according to the score of the new item (0-4), the global score (0-32), and the percentage of patients who rated the information given before colonoscopy as excellent.

Data collectionDemographic, clinical and endoscopic variables known to potentially impact attendance were recorded from the hospital electronic database and the telephone interview. The study nurses registered all relevant information regarding non-compliance with preparedness protocols in the endoscopy room before the colonoscopy. Endoscopists registered the BBPS in the endoscopy report. Patient satisfaction was assessed with a nurse-led telephone interview 30 d after the colonoscopy.

Statistical analysisThe statistical methods of the study were reviewed by Duran X from Consulting service on methodology for Biomedical Research. Hospital del Mar Medical Research Institute (IMIM). Barcelona. Spain.

The sample size was estimated to demonstrate the superiority of the educational intervention. We retrospectively obtained a non-attendance rate of 13.9% from November 2016 to May 2017, the same period of the study the year before. We use this figure to calculate the sample size because scarce published data on non-attendance in non-screening colonoscopies are available. Expecting a non-attendance rate of 13.9% in the CG and 8.9% in the IG to detect a 5% reduction as clinically significant and applying a significance level of 5%, a power of 80% for the comparison of two independent proportions and a 20% loss ratio, a sample size of 764 patients per group was calculated.

The intention-to-treat (ITT) analysis included all randomized patients. The per-protocol (PP) analysis included the participants who were finally contacted by telephone. Categorical variables were described through frequencies table (number and percentage). Quantitative variables were described through mean and standard deviation. The categorical variables were compared between groups by the Pearson chi-squared test or Fisher’s exact test if applicable and continuous variables by using Student’s t-test. Multivariate logistic regression was used to identify factors associated with non-attendance. The backward stepwise criterion was used for variable selection. A P value of > 0.1 as removal criteria and < 0.05 for inclusion criteria. The results are expressed as odds ratios (ORs). Stata software version 15.1 was used by our research statistician to perform the analysis.

RESULTSPatient characteristicsA total of 1485 patients (738 in the IG and 746 in the CG) were finally enrolled (Figure 1). Telephone contact was achieved in 613 (83%) patients in the IG. At baseline,



Figure 1 Patient flow chart. ITT: Intention-to-treat; PP: Per-protocol.

the two groups did not differ in any of the variables analysed. Demographic, clinical and endoscopy data are shown in Table 1. The mean telephone time for the educational intervention was 12 min.

Primary outcomeAccording to the ITT analysis, the non-attendance rate was higher in the CG than in the IG (14.3% vs 8.4%, P ≤ 0.001). The absolute risk reduction (ARR) was 5.9% (95%CI: 2.7% to 9.2%). In the PP analysis, there was a greater reduction in non-attendance (14.3% vs 4.4%, P ≤ 0.001), with an ARR of 9.9% (95%CI: 6.9% to 13%).

The study of variables related to non-attendance was assessed with a bivariable (Table 2) and a multivariable analysis (Table 3), in which 12 variables were found to be independent predictors, with the treatment group being an independent factor for non-attendance, both in the ITT analysis (OR 1.81, 95%CI: 1.27 to 2.58, P = 0.001) and the PP analysis (OR 3.56, 95%CI: 2.25 to 5.64, P ≤ 0.001).

ComplianceIn the IG, there was an improvement in all compliance-related variables (Table 4). It is important to highlight that there was a modest benefit in compliance with cleansing items. The benefit was much greater in regard to the management of antithrombotics and sedation scheduling.

Cost analysisOur hospital invoices €240.71 for a performed colonoscopy. The calculated nurse cost of the telephone intervention was €5.12 per patient. With a non-attendance rate of 14.3% and a non-compliance rescheduling rate of 2.3% (Table 4) the net income in the CG per 100 patients was €20075 (the result of subtracting €3442 for non-attendance and €554 for non-compliance to the total amount of €24071). According to the non-attendance and non-compliance figures in the IG of 8.4% and 0.3% respectively (Table 4), the net income in the IG per 100 patients was €21465 (the result of subtracting €2022 for non-attendance, €72 for non-compliance, and €512 for the nurse intervention to the total amount of €24071).

Therefore, the intervention was a cost-saving measure, as the benefit was greater in the IG, resulting in a net balance of €1390 (€21465-€20075) per 100 patients, €13.9 per



Table 1 Baseline characteristics of the patients

Intervention (n = 738) Control (n = 746) P valueDemographic variables

Age (yr), mean (SD) 59.1 (16.2) 59.9 (16.0) 0.307

Male sex, n (%) 347 (47) 363 (48.7) 0.527

Foreign nationality, n (%) 94 (12.7) 102 (13.7) 0.594

Advanced studies, n (%) 148 (20.1) 149 (20.0) 0.834

Language barrier, n (%) 67 (9.1) 53 (7.1) 0.163

Clinical variables

BMI, mean (SD) 26.7 (4.7) 26.9 (4.6) 0.657

Diabetes mellitus, n (%) 126 (17.1) 125 (16.8) 0.871

Abdominal/pelvic surgery, n (%) 287 (38.9) 292 (39.1) 0.920

Constipation, n (%) 148 (20.1) 161 (21.6) 0.469

Anxiety-depression syndrome, n (%) 253 (34.3) 253 (33.9) 0.881

Disabled condition, n (%) 29 (3.9) 33 (4.4) 0.634

Charlson index, mean (SD) 0.5 (0.9) 0.5 (0.9) 0.322

ASA classification III-IV, n (%) 84 (11.4) 100 (13.4) 0.237

High comorbidity burden, n (%) 33 (4.5) 42 (5.6) 0.308

Antiaggregants, n (%)

Aspirin 73 (9.9) 72(9.7) 0.798

Clopidogrel 6 (0.8) 9 (1.2)

Dual APA 1 (0.1) 2 (0.3)

VKA, n (%) 24 (3.3) 28 (3.8) 0.600

DOAC, n (%)

Rivaroxaban 4 (0.5) 4 (0.5) 0.735

Apixaban 8 (1.1) (1.2)

Dabigatran 3 (0.4) 5 (0.7)

Endoscopic variables

Previous endoscopy, n (%) 335 (45.4) 323 (49.1) 0.417

Previous non-attendance, n (%) 77 (10.4) 86 (11.5) 0.500

Referring physician, n (%)

Primary care 668 (90.5) 684 (91.7) 0.128

Gastroenterologist 70 (9.5) 62 (8.3)

Indication, n (%)

Surveillance 211 (28.6) 197 (26.4) 0.620

Diagnostic 456 (61.8) (63.3)

Family history of CRC 71 (9.6) 77 (10.3)

Waiting time (d), mean (SD) 60.7 (56.6) 59.5 (60.9) 0.701

Afternoon timetable, n (%) 467 (63.3) 746 (67.3) 0.104

Laxative, n (%)

MCSP 102 (15.1) 80 (12.5) 0.372

PEG + ascorbate, 2 L 158 (23.4) 3.2)

PEG, 4 L 416 (61.5) 411 (64.3)



Previous cleansing problems, n (%) 37 (5) 44 (5.9) 0.453

BMI: Body mass index; ASA classification: American Society of Anaesthesiologists classification; APA: Antiplatelet agent; VKA: Vitamin K antagonist; DOAC: Direct oral anticoagulant; CRC: Colorectal cancer; MCPS: Magnesium citrate plus sodium picosulfate; PEG: Polyethylene glycol.

patient. Since our centre performs approximately 4000 colonoscopies in a year, the implementation of the intervention would represent a benefit of €55600.

Cleansing adequacyA total of 627 and 673 colonoscopies (584 in patients contacted) were finally performed in the CG and IG, respectively. The difference in cleansing adequacy did not reach statistical significance (CG 90.4% and IG 92.4%, P = 0.1); it also did not reach significance in the PP analysis (CG 90.4% and IG 93.2%, P = 0.08).

SatisfactionA total of 755 patients were contacted (50.8%). According to the total score and specific information item score, patient satisfaction improved with the intervention. The information was rated as excellent in 49.4% (IG) and 26% (CG) of patients, P ≤ 0.001 (Table 4).

DISCUSSIONThis is the first RCT that demonstrates that an educational measure reduces non-attendance in non-screening colonoscopies with a significant clinical and economic impact, suggesting the suitability of its implementation in endoscopy units. In addition, the educational intervention also improved the need to reschedule the colonoscopy for non-compliance with patient preparedness protocols, such as antithrombotic management, sedation scheduling or bowel cleansing instructions.

The educational intervention was an important factor for non-attendance reduction. We believe that this is probably related to the fact that the educational measure is capable of dealing with medical aspects related to care that are impossible to address from an administrative perspective and that the figure of an educator truly impacts the patient, especially if it is a health-care provider in the endoscopy unit. The drastic reduction observed in the PP analysis (9.9%) suggests that the bidirectional conversation with the patient, dedicating the necessary time to carry out the educational intervention in a personal way, is the key point. This agrees with published results of educational patient interventions in which bidirectional communication with the patient was effective in improving colon cleansing adequacy[21-23]. On the other hand, other educational measures that do not allow a “human” interaction (booklets, mailed print resources, web pages, telephone applications, etc.) have failed to achieve optimal results[8].

Bidirectional communication may be achieved through other means, such as face-to-face interviews and video conferences. A personal interview allows for conversation and facilitates the transmission of concepts through verbal and non-verbal communication. However, there are no published comparisons between a telephone and a face-to-face interview to improve patient education. In any case, a personal interview has some logistical issues, such as the need for an interview room, a shorter agenda capability, the transport and access of the patients to hospitals and a higher cost than a telephone intervention. These limitations are even more important during the present COVID-19 pandemic. Furthermore, a video conference is a new and promising communication method that needs to be evaluated. However, its applicability is more limited than a telephone call because some patients do not have the technology or the knowledge to use it.

Regarding the person responsible for providing medical education, there are no published comparisons between the prescribing doctor, administrative personnel, a primary care nurse or an endoscopy nurse. Nevertheless, our study demonstrates that endoscopy nurses are capable of conducting telephone educational interventions, and because of their involvement, knowledge and immediate access to endoscopy experts, they could be the ideal person to eliminate the barriers that negatively influence the patient's attendance, with the additional benefit of improving compliance with patient preparedness protocols.

The multivariate analysis showed several independent risk factors for non-



Table 2 Bivariable analysis of risk factors for non-attendance

ITT PP

A NA P value A NA P valueDemographic risk factors

Group, n (%) < 0.001 < 0.001

Intervention 676 (51.4) 62 (36.7) 586 (47.8) 27 (20.1)

Control 639 (48.6) 107 (63.3) 639 (52.2) 107 (79.9)

Age (yr), mean (SD) 60.2 (15.7) 54.2 (18.0) < 0.001 60.62 (15.6) 53.45 (18.2) < 0.001

Sex, n (%) 0.640 0.527

Female 683 (51.9) 91 (53.8) 626 (51.1) 75 (56)

Male 632 (48.1) 78 (46.2) 599 (48.9) 59 (44)

Nationality, n (%) < 0.001 < 0.001

Spanish 1169 (88.9) 119 (70.4) 1092 (89.1) 96 (71.6)

Foreign 146 (11.1) 50 (29.6) 133 (10.9) 38 (28.4)

Studies, n (%) < 0.001 < 0.001

Non-advanced 1033 (78.6) 154 (91.1) 947 (77.3) 121 (90.3)

Advanced 282 (21.4) 15 (8.9) 278 (22.7) 13 (9.7)

Language barrier, n (%) 0.318 0.276

No 1212 (92.2) 152 (89.9) 1130 (92.2) 120 (89.6)

Yes 103 (7.8) 17(10.1) 95 (7.8) 14 (10.4)

Clinical risk factors

BMI, mean (SD) 26.73 (4.6) 27.76 (4.9) 0.007 26.75 (4.5) 27.84 (4.9) 0.010

Diabetes mellitus, n (%) 0.758 0.528

No 1094 (83.2) 139 (82.2) 1014 (82.8) 108 (80.6)

Yes 221 (16.8) 30 (17.8) 211 (17.2) 26 (19.4)

Abdominal/pelvic surgery, n (%) 0.046 0.465

No 790 (60.1) 115 (60.8) 728 (59.4) 84 (62.7)

Yes 525 (39.9) 54 (32.0) 497 (40.6) 50 (37.3)

Constipation, n (%) 0.148 0.214

No 1034 (78.6) 141 (83.4) 958 (78.2) 11 (82.8)

Yes 429 (32.6) 77 (45.6) 276 (21.8) 23 (17.2)

Anxiety-depression syndrome, n (%) 0.001 0.002

No 886 (67.4) 92 (54.4) 832 (67.9) 73 (54.5)

Yes 429 (32.6) 77 (45.6) 393 (32.1) 61 (45.5)

Disabled condition, n (%) < 0.001 0.002

No 1269 (96.5) 153 (90.5) 1182 (96.5) 122 (91.0)

Yes 46 (3.5) 16 (9.5) 43 (3.5) 12 (9.0)

Charlson index, mean (SD) 0.57 (0.9) 0.54 (0.9) 0.771 0.57 (0.9) 0.50 (0.8) 0.403

ASA classification, n (%) 0.813 0.834

I/II 1151 (87.5) 149 (88.2) 1071 (87.4) 118 (88.1)

III/IV 164 (12.5) 20 (11.8) 154 (12.6) 16 (11.9)

High comorbidity burden, n (%) 0.840 0.769

No 1248 (94.9) 161 (95.3) 1163 (94.9) 128 (95.5)



Yes 67 (5.1) 8 (4.7) 62 (5.1) 6 (4.5)

Endoscopic risk factors

Previous endoscopy, n (%) < 0.001 < 0.001

No 697 (53) 129 (76.3) 646 (52.7) 98 (73.1)

Yes 618 (47) 40 (23.7) 579 (47.3) 36 (26.9)

Previous non-attendance, n (%) < 0.001 < 0.001

No 1192 (90.6) 129 (76.3) 1111 (90.7) 101 (75.4)

Yes 123 (9.4) 40 (23.7) 114 (9.3) 33 (24.6)

Referring physician, n (%) 0.125 0.293

Primary care 1178 (89.6) 159 (94.1) 1101 (89.9) 125 (93.3)

Gastroenterologist 122 (9.3) 10 (5.9) 109 (8.9) 9 (6.7)

Other specialities 15 (1.1) 0 (0.0) 15 (1.2) 0 (0)

Indication, n (%) < 0.001 < 0.001

Surveillance 338 (29.5) 20 (11.8) 357 (29.1) 15 (11.2)

Diagnostic 793 (60.3) 135 (79.9) 744 (60.7) 107 (79.9)

Family history of CRC 134 (10.2) 14 (8.3) 124 (10.1) 12 (9)

Waiting time (d), mean (SD) 59.3 (57.4) 66.2 (68.6) 0.149 58.3 (55.0) 66.1 (72.4) 0.134

Endoscopy timetable, n (%) 0.030 0.029

Morning 469 (35.7) 46 (27.2) 436 (35.6) 35 (26.1)

Afternoon 846 (64.3) 123 (72.8) 789 (64.4) 99 (73.9)

Previous cleansing problems, n (%) 0.318 0.180

No 1246 (94.8) 157 (92.9) 1159 (94.6) 123 (91.8)

Yes 69 (5.2) 12 (7.1) 66 (5.4) 11 (8.2)

ITT: Intention-to-treat analysis; PP: Per protocol analysis; A: Attendance; NA: Non-attendance; BMI: Body mass index; ASA classification: American Society of Anaesthesiologists classification; CRC: Colorectal cancer.

attendance. However, our intervention was the only modifiable factor. In this regard, when the patients were successfully contacted, the intervention was the most important predictor of attendance.

A quality improvement intervention is not implementable if despite generating a clinical improvement, it is not economically viable. It has been previously reported that the financial loss attributed to endoscopy non-attendance can be very high and can considerably decrease the expected net gain of outpatient procedure centres[32]. Similar to other educational studies[20,21], ours shows that non-attendance and rescheduling due to poor compliance are associated with a significant cost that nursing education intervention manages to reduce, providing data that support the conclusion that the educational intervention is not only cost-effective but also cost-saving.

Related to cleansing improvement, the intervention showed a numerically higher cleansing adequacy, but the difference was not statistically significant. However, this was not the main objective of our intervention. In any case, it must also be pointed out that both groups exceeded the 90% adequacy rate, which is over the quality recommendations[1], and it is possible that a ceiling effect is present.

We want to point out that our study has several strengths. First, this is the first RCT to evaluate an educational intervention to improve colonoscopy attendance outside colorectal screening programmes. Second, randomization was carried out with a large sample with broad inclusion criteria, which favours the extrapolation of the sample results from the study population. Third, we followed the most updated colonoscopy protocols recommended by the ESGE[26-28], allowing comparison with other studies. Finally, we performed a PP analysis and an economic study to measure the applicability of the intervention.

The main limitation of the study is the single centre design. There is room for improvement in the educational intervention since there are other independent factors



Table 3 Multivariable analysis of risk factors for non-attendance

ITT PP

OR 95%CI P value OR 95%CI P valueDemographic risk factors

Group

Intervention 1 1

Control 1.81 1.27-2.58 0.001 3.56 2.25-5.64 < 0.001

Age 0.98 0.97-0.99 < 0.001 0.97 0.96-0.98 < 0.001

Nationality

Spanish 1

Foreign 2.69 1.77-4.10 < 0.001 2.49 1.55-4.00 < 0.001

Studies level

Non-advanced 1 1

Advanced 0.47 0.27-0.83 0.010 0.51 0.27-0.94 0.031

Clinical risk factors

BMI 1.05 1.01-1.08 0.012 1.04 1.01-1.09 0.027

Anxiety-depression syndrome

No 1 1

Yes 2.00 1.40-2.86 < 0.001 2.02 1.35-3.02 0.001

High comorbidity burden

No 1 1

Yes 2.82 0.86-9.24 0.087 3.87 1.17-12.85 0.027

Disabled condition

No 1 1

Yes 3.39 1.68-6.84 0.001 3.37 1.50-7.58 0.003

Endoscopic risk factors

Previous endoscopy

No 1 1

Yes 0.35 0.22-0.56 < 0.001 0.46 0.28-0.77 0.003

Previous non-attendance

No 1 1

Yes 2.94 1.89-4.58 < 0.001 3.13 1.91-5.13 < 0.001

Previous cleansing problems

No 1 1

Yes 2.58 1.19-5.59 0.016 2.58 1.13-5.85 0.024

Endoscopy timetable

Morning 1 1

Afternoon 1.52 1.03-2.23 0.033 1.62 1.04-2.51 0.031

ITT: Intention-to-treat analysis; PP: Per protocol analysis; BMI: Body mass index; OR: Odds ratio; CI: Confidence interval.



that can help identify patients with high risk for non-attendance with whom to perform a more targeted intervention. It is also important to highlight that there are still several unknown factors to clarify regarding attendance outside population screening programmes. Studies are needed to determine the type and percentage of undiagnosed pathology and the prognostic and therapeutic implications of the diagnostic delay in the patients. We also do not know the effect that the intervention could have on attending future colonoscopies in this population although the satisfaction results observed in our study are promising in this regard.

Attendance is not currently considered a routine quality measurement parameter[1], but we believe this is arguable. Non-attendance, both in the diagnosis and in the surveillance of colonoscopies, has a significant clinical impact due to the diagnostic delay that it generates, which can be devastating in the case of colorectal cancer, which is mainly still diagnosed outside of colorectal screening programmes. Attendance is also a well-defined and reliable performance measure that is easy to measure and clearly susceptible to improvement. Its impact on the rest of the endoscopic procedures is also evident by increasing the waiting lists and making the endoscopic procedures more expensive. This means that attendance meets all the conditions that the European guideline considers as necessary to be able to include it as a key measure parameter for colonoscopy[1]. Colonoscopy quality parameters are not fixed and change over time based on data provided by new studies. We believe that our results support placing more importance on colonoscopy attendance as a routine quality measure parameter outside colorectal cancer screening programmes.

CONCLUSIONIn conclusion, we found that an educational telephone intervention carried out by endoscopy nurses improves attendance outside colorectal cancer screening programmes, facilitates compliance with colonoscopy preparedness protocols, increases patient satisfaction and results in a beneficial economic impact, all of which support its implementation and maintenance over time. We believe that due to its characteristics as a quality measure parameter and given the significant clinical and economic impact observed, attendance measures should be routinely incorporated into endoscopy units.



Table 4 Compliance, rescheduling and patient satisfaction questionnaire

Intervention Control P valueCompliance outcomes

Fasting 2-4 h, n/n (%)

ITT 464/702 (66.2) 427/720 (59.3) 0.007

PP 408/583 (70.1) 427/720 (59.3) < 0.001

Split regimen, n/n (%)

ITT 622/702 (88.6) 572/719 (79.6) < 0.001

PP 544/583 (93.3) 572/719 (79.6) < 0.001

Correct diet, n/n (%)

ITT 633/702 (90.2) 593/720 (82.4) < 0.001

PP 553/583 (94.9) 593/720 (82.4) < 0.001

Complete intake, n/n (%)

ITT 597/702 (85) 554/720 (76.9) < 0.001

PP 522/583 (89.5) 554/720 (76.9) < 0.001

Cleansing compliance, n/n (%)

ITT 433/702 (61.7) 379/720 (52.6) 0.001

PP 383/583 (65.7) 379/720 (52.6) < 0.001

Antithrombotic drugs, n/n (%)

ITT 37/40 (92.5) 27/43 (62.8) 0.001

PP 33/35 (94.3) 27/43 (62.8) 0.001

Endoscopy with anaesthesiologist, n/n (%)

ITT 26/33 (78.8) 0/42 (0) < 0.001

PP 26/26 (100) 0/42 (0) < 0.001

Rescheduling for non-compliance

Global rescheduling, n/n (%)

ITT 2/676 (0.3) 15/639 (2.3) 0.001

PP 1/586 (0.2) 15/639 (2.3) 0.001

Patient satisfaction questionnaire

Percentage of excellent (item 8), n/n (%)

ITT 195/382 (51) 97/373 (26) < 0.001

PP 171/346 (49.4) 97/373 (26) < 0.001

Information score (item 8)

ITT, mean (SD) 3.29 (0.88) 2.80 (1) < 0.001

PP, mean (SD) 3.31 (0.89) 2.80 (1) < 0.001

Total score of the questionnaire

ITT, mean (SD) 26.12 (3.76) 25.43 (3.82) 0.01

PP, mean (SD) 26.18 (3.87) 25.43 (3.82) 0.008

ITT: Intention-to-treat analysis; PP: Per protocol analysis.



ARTICLE HIGHLIGHTSResearch backgroundEducative interventions with bidirectional contact to the patient have shown to improve colonoscopy attendance in colorectal cancer population screening programmes and because of its huge clinical and economic impact, they have been widely implemented. However, outside of this population programmes, educative measures to improve colonoscopy attendance have been poorly studied and no navigation interventions are usually performed.

Research motivationWe thought this lack of research needed attention, so we designed a randomized controlled trial to conduct an educational telephone nurse intervention with bidirectional contact directed to increase colonoscopy attendance, similar to the patient navigation carried out in the population screening setting.

Research objectivesThe aim of the study was to determine the clinical and economic impact of this educational intervention.

Research methodsWe included all consecutive outpatients referred for colonoscopy from primary care centres in our health area. Patients randomized to the intervention group received a telephone call 7 d before colonoscopy appointment to eliminate socioeconomic, psychological and clinical barriers that could affect attendance. Baseline characteristics including demographics, clinical and endoscopic factors previously reported to be related to non-attendance were collected. The primary outcome was the attendance rate. The secondary outcomes included the economic impact and the potential benefit of the intervention in regard to compliance with patient preparedness protocols, cleansing adequacy, and patient satisfaction. We performed an intention-to-treat (ITT) and per-protocol (PP) analysis to measure the applicability of the telephone intervention.

Research resultsA total of 738 and 746 patients were finally included in the intervention and control group (CG) respectively. Six hundred thirteen (83%) patients were contacted in the intervention group (IG). The non-attendance rate was lower in the IG, both in the ITT analysis (IG 8.4% vs CG 14.3%, P < 0.001) and in the PP analysis (4.4% vs 14.3%, P < 0.001). In a multivariable analysis, belonging to the CG increased the risk of non-attendance in both, the ITT analysis (OR 1.81, 95%CI: 1.27 to 2.58, P = 0.001) and the PP analysis (OR 3.56, 95%CI: 2.25 to 5.64, P < 0.001). There was also a significant difference in compliance with preparedness protocols [bowel cleansing: IG 61.7% vs CG 52.6% (P = 0.001), antithrombotic management: IG 92.5% vs CG 62.8% (P = 0.001), and sedation scheduling: IG 78.8% vs CG 0% (P ≤ 0.001)]. We observed a net benefit of €55600/year after the intervention. The information given before the procedure was rated as excellent by 26% (CG) and 51% (IG) of patients, P ≤ 0.001.

Research conclusionsAccording to our results, non-attendance has a significant clinical and economic impact outside the population screening setting. This study proposes the necessity to routinely incorporate attendance measures into endoscopy units, not only in the population screening programmes but also in all colonoscopies. A telephone educative intervention by an endoscopy nurse seems to be a valid method.

Research perspectivesFurther multicentric studies on attendance outside colorectal cancer population screening programmes are needed. The type and percentage of undiagnosed pathology and the prognostic and therapeutic implications of the diagnostic delay in these patients have to be studied. We also do not know the effect that the intervention could have on attending future colonoscopies although the satisfaction results observed in our study are promising in this regard.



ACKNOWLEDGEMENTSThe authors would like to thank Serrano MC from The Epidemiology Department who assisted with the study design and Reguant FC for his help with the economic analysis.

REFERENCESKaminski MF, Thomas-Gibson S, Bugajski M, Bretthauer M, Rees CJ, Dekker E, Hoff G, Jover R, Suchanek S, Ferlitsch M, Anderson J, Roesch T, Hultcranz R, Racz I, Kuipers EJ, Garborg K, East JE, Rupinski M, Seip B, Bennett C, Senore C, Minozzi S, Bisschops R, Domagk D, Valori R, Spada C, Hassan C, Dinis-Ribeiro M, Rutter MD. Performance measures for lower gastrointestinal endoscopy: a European Society of Gastrointestinal Endoscopy (ESGE) Quality Improvement Initiative. Endoscopy 2017; 49: 378-397 [PMID: 28268235 DOI: 10.1055/s-0043-103411]

1

Weinberg DS, Keenan E, Ruth K, Devarajan K, Rodoletz M, Bieber EJ. A randomized comparison of print and web communication on colorectal cancer screening. JAMA Intern Med 2013; 173: 122-129 [PMID: 23128366 DOI: 10.1001/2013.jamainternmed.1017]

2

Helander S, Hakama M, Malila N. Effect of a pre-screening survey on attendance in colorectal cancer screening: a double-randomized study in Finland. J Med Screen 2014; 21: 82-88 [PMID: 24803481 DOI: 10.1177/0969141314534229]

3

Pisera M, Kaminski MF, Kraszewska E, Rupinski M, Regula J. Reinvitation to screening colonoscopy: a randomized-controlled trial of reminding letter and invitation to educational meeting on attendance in nonresponders to initial invitation to screening colonoscopy (REINVITE). Eur J Gastroenterol Hepatol 2016; 28: 538-542 [PMID: 26967693 DOI: 10.1097/MEG.0000000000000578]

4

Stratmann K, Bock H, Filmann N, Fister P, Weber C, Tacke W, Simonis B, Höftmann M, Schröder O, Hausmann J, Zeuzem S, Blumenstein I. Individual invitation letters lead to significant increase in attendance for screening colonoscopies: Results of a pilot study in Northern Hesse, Germany. United European Gastroenterol J 2018; 6: 1082-1088 [PMID: 30228897 DOI: 10.1177/2050640618769713]

5

Griffin JM, Hulbert EM, Vernon SW, Nelson D, Hagel EM, Nugent S, Baines Simon A, Bangerter A, van Ryn M. Improving endoscopy completion: effectiveness of an interactive voice response system. Am J Manag Care 2011; 17: 199-208 [PMID: 21504256]

6

Dougherty MK, Brenner AT, Crockett SD, Gupta S, Wheeler SB, Coker-Schwimmer M, Cubillos L, Malo T, Reuland DS. Evaluation of Interventions Intended to Increase Colorectal Cancer Screening Rates in the United States: A Systematic Review and Meta-analysis. JAMA Intern Med 2018; 178: 1645-1658 [PMID: 30326005 DOI: 10.1001/jamainternmed.2018.4637]

7

Calderwood AH, Roy HK. Increasing colorectal cancer screening adherence: comment on "A randomized comparison of print and web communication on colorectal cancer screening". JAMA Intern Med 2013; 173: 129-131 [PMID: 23247843 DOI: 10.1001/jamainternmed.2013.2527]

8

Bernardo BM, Zhang X, Beverly Hery CM, Meadows RJ, Paskett ED. The efficacy and cost-effectiveness of patient navigation programs across the cancer continuum: A systematic review. Cancer 2019; 125: 2747-2761 [PMID: 31034604 DOI: 10.1002/cncr.32147]

9

Hermann EA, Ashburner JM, Atlas SJ, Chang Y, Percac-Lima S. Satisfaction With Health Care Among Patients Navigated for Preventive Cancer Screening. J Patient Exp 2018; 5: 225-230 [PMID: 30214930 DOI: 10.1177/2374373517750413]

10

Post DM, McAlearney AS, Young GS, Krok-Schoen JL, Plascak JJ, Paskett ED. Effects of Patient Navigation on Patient Satisfaction Outcomes. J Cancer Educ 2015; 30: 728-735 [PMID: 25510369 DOI: 10.1007/s13187-014-0772-1]

11

Shuja A, Harris C, Aldridge P, Malespin M, de Melo SW Jr. Predictors of No-show Rate in the GI Endoscopy Suite at a Safety Net Academic Medical Center. J Clin Gastroenterol 2019; 53: 29-33 [PMID: 28961577 DOI: 10.1097/MCG.0000000000000928]

12

Laiyemo AO, Williams CD, Burnside C, Moghadam S, Sanasi-Bhola KD, Kwagyan J, Brim H, Ashktorab H, Scott VF, Smoot DT. Factors associated with attendance to scheduled outpatient endoscopy. Postgrad Med J 2014; 90: 571-575 [PMID: 25180285 DOI: 10.1136/postgradmedj-2012-131650]

13

Sola-vera J, Sáez J, Laveda R, Girona E, García-Sepulcre MF, Cuesta A, Vázquez N, Uceda F, Pérez E, Sillero C. Factors associated with non-attendance at outpatient endoscopy. Scand J Gastroenterol 2008; 43: 202-206 [PMID: 17852875 DOI: 10.1080/00365520701562056]

14

Chopra D, Hookey LC. Comorbid Illness, Bowel Preparation, and Logistical Constraints Are Key Reasons for Outpatient Colonoscopy Nonattendance. Can J Gastroenterol Hepatol 2016; 2016: 2179354 [PMID: 27478818 DOI: 10.1155/2016/2179354]

15

Chang JT, Sewell JL, Day LW. Prevalence and predictors of patient no-shows to outpatient endoscopic procedures scheduled with anesthesia. BMC Gastroenterol 2015; 15: 123 [PMID: 26423366 DOI: 10.1186/s12876-015-0358-3]

16

Lee CS, McCormick PA. Telephone reminders to reduce non-attendance rate for endoscopy. J R Soc Med 2003; 96: 547-548 [PMID: 14594962 DOI: 10.1258/jrsm.96.11.547]

17

Adams LA, Pawlik J, Forbes GM. Nonattendance at outpatient endoscopy. Endoscopy 2004; 36: 402-18


https://dx.doi.org/10.1055/s-0043-103411


https://dx.doi.org/10.1001/2013.jamainternmed.1017


https://dx.doi.org/10.1177/0969141314534229




https://dx.doi.org/10.1177/2050640618769713



https://dx.doi.org/10.1001/jamainternmed.2018.4637


https://dx.doi.org/10.1001/jamainternmed.2013.2527




https://dx.doi.org/10.1177/2374373517750413


https://dx.doi.org/10.1007/s13187-014-0772-1


https://dx.doi.org/10.1097/MCG.0000000000000928


https://dx.doi.org/10.1136/postgradmedj-2012-131650


https://dx.doi.org/10.1080/00365520701562056


https://dx.doi.org/10.1155/2016/2179354


https://dx.doi.org/10.1186/s12876-015-0358-3


https://dx.doi.org/10.1258/jrsm.96.11.547



404 [PMID: 15100947 DOI: 10.1055/s-2004-814329]Downer SR, Meara JG, Da Costa AC. Use of SMS text messaging to improve outpatient attendance. Med J Aust 2005; 183: 366-368 [PMID: 16201955]

19

Childers RE, Laird A, Newman L, Keyashian K. The role of a nurse telephone call to prevent no-shows in endoscopy. Gastrointest Endosc 2016; 84: 1010-1017. e1 [PMID: 27327847 DOI: 10.1016/j.gie.2016.05.052]

20

Abuksis G, Mor M, Segal N, Shemesh I, Morad I, Plaut S, Weiss E, Sulkes J, Fraser G, Niv Y. A patient education program is cost-effective for preventing failure of endoscopic procedures in a gastroenterology department. Am J Gastroenterol 2001; 96: 1786-1790 [PMID: 11419830 DOI: 10.1111/j.1572-0241.2001.03872.x]

21

Alvarez-Gonzalez MA, Flores-Le Roux JA, Seoane A, Pedro-Botet J, Carot L, Fernandez-Clotet A, Raga A, Pantaleon MA, Barranco L, Bory F, Lorenzo-Zuñiga V. Efficacy of a multifactorial strategy for bowel preparation in diabetic patients undergoing colonoscopy: a randomized trial. Endoscopy 2016; 48: 1003-1009 [PMID: 27490086 DOI: 10.1055/s-0042-111320]

22

Liu X, Luo H, Zhang L, Leung FW, Liu Z, Wang X, Huang R, Hui N, Wu K, Fan D, Pan Y, Guo X. Telephone-based re-education on the day before colonoscopy improves the quality of bowel preparation and the polyp detection rate: a prospective, colonoscopist-blinded, randomised, controlled study. Gut 2014; 63: 125-130 [PMID: 23503044 DOI: 10.1136/gutjnl-2012-304292]

23

Kurlander JE, Sondhi AR, Waljee AK, Menees SB, Connell CM, Schoenfeld PS, Saini SD. How Efficacious Are Patient Education Interventions to Improve Bowel Preparation for Colonoscopy? PLoS One 2016; 11: e0164442 [PMID: 27741260 DOI: 10.1371/journal.pone.0164442]

24

Hassan C, Bretthauer M, Kaminski MF, Polkowski M, Rembacken B, Saunders B, Benamouzig R, Holme O, Green S, Kuiper T, Marmo R, Omar M, Petruzziello L, Spada C, Zullo A, Dumonceau JM; European Society of Gastrointestinal Endoscopy. Bowel preparation for colonoscopy: European Society of Gastrointestinal Endoscopy (ESGE) guideline. Endoscopy 2013; 45: 142-150 [PMID: 23335011 DOI: 10.1055/s-0032-1326186]

25

Veitch AM, Vanbiervliet G, Gershlick AH, Boustiere C, Baglin TP, Smith LA, Radaelli F, Knight E, Gralnek IM, Hassan C, Dumonceau JM. Endoscopy in patients on antiplatelet or anticoagulant therapy, including direct oral anticoagulants: British Society of Gastroenterology (BSG) and European Society of Gastrointestinal Endoscopy (ESGE) guidelines. Endoscopy 2016; 48: 385-402 [PMID: 26890676 DOI: 10.1055/s-0042-102652]

26

Dumonceau JM, Riphaus A, Schreiber F, Vilmann P, Beilenhoff U, Aparicio JR, Vargo JJ, Manolaraki M, Wientjes C, Rácz I, Hassan C, Paspatis G. Non-anesthesiologist administration of propofol for gastrointestinal endoscopy: European Society of Gastrointestinal Endoscopy, European Society of Gastroenterology and Endoscopy Nurses and Associates Guideline--Updated June 2015. Endoscopy 2015; 47: 1175-1189 [PMID: 26561915 DOI: 10.1055/s-0034-1393414]

27

Alvarez-Gonzalez MA, Pantaleon MA, Flores-Le Roux JA, Zaffalon D, Amorós J, Bessa X, Seoane A, Pedro-Botet J; PROCOLON group. Randomized Clinical Trial: A Normocaloric Low-Fiber Diet the Day Before Colonoscopy Is the Most Effective Approach to Bowel Preparation in Colorectal Cancer Screening Colonoscopy. Dis Colon Rectum 2019; 62: 491-497 [PMID: 30844973 DOI: 10.1097/DCR.0000000000001305]

28

Alvarez-Gonzalez MA, Pantaleón Sánchez MÁ, Bernad Cabredo B, García-Rodríguez A, Frago Larramona S, Nogales O, Díez Redondo P, Puig Del Castillo I, Romero Mascarell C, Caballero N, Romero Sánchez-Miguel I, Pérez Berbegal R, Hernández Negrín D, Bujedo Sadornill G, Pérez Oltra A, Casals Urquiza G, Amorós Martínez J, Seoane Urgorri A, Ibáñez Zafón IA, Gimeno-García AZ. Educational nurse-led telephone intervention shortly before colonoscopy as a salvage strategy after previous bowel preparation failure: a multicenter randomized trial. Endoscopy 2020; 52: 1026-1035 [PMID: 32557475 DOI: 10.1055/a-1178-9844]

29

Johanson JF, Schmitt CM, Deas TM Jr, Eisen GM, Freeman M, Goldstein JL, Jensen DM, Lieberman DA, Lo SK, Sahai A, Schoenfeld P, Morgan M. Quality and outcomes assessment in Gastrointestinal Endoscopy. Gastrointest Endosc 2000; 52: 827-830 [PMID: 11182685]

30

Sánchez del Río A, Alarcón Fernández O, Baudet JS, Sainz Menéndez Z, Socas Méndez M. Reliability of the Spanish version of a brief questionnaire on patient satisfaction with gastrointestinal endoscopy. Rev Esp Enferm Dig 2005; 97: 554-561 [PMID: 16266222 DOI: 10.4321/s1130-01082005000800003]

31

Berg BP, Murr M, Chermak D, Woodall J, Pignone M, Sandler RS, Denton BT. Estimating the cost of no-shows and evaluating the effects of mitigation strategies. Med Decis Making 2013; 33: 976-985 [PMID: 23515215 DOI: 10.1177/0272989X13478194]

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https://dx.doi.org/10.1055/s-2004-814329







https://dx.doi.org/10.1055/s-0042-111320






https://dx.doi.org/10.1055/s-0032-1326186


https://dx.doi.org/10.1055/s-0042-102652


https://dx.doi.org/10.1055/s-0034-1393414


https://dx.doi.org/10.1097/DCR.0000000000001305


https://dx.doi.org/10.1055/a-1178-9844



https://dx.doi.org/10.4321/s1130-01082005000800003


https://dx.doi.org/10.1177/0272989X13478194


World Journal of



CASE REPORT

Multiple cerebral lesions in a patient with refractory celiac disease: A case report

Lena Horvath, Georg Oberhuber, Andreas Chott, Maria Effenberger, Herbert Tilg, Eberhard Gunsilius, Dominik Wolf, Sarah Iglseder

ORCID number: Lena Horvath 0000-0002-1509-4324; Georg Oberhuber 0000-0002-9701-821X; Andreas Chott 0000-0002-0179-838X; Maria Effenberger 0000-0002-0499-9953; Herbert Tilg 0000-0002-4235-2579; Eberhard Gunsilius 0000-0003-1327-2921; Dominik Wolf 0000-0002-4761-075X; Sarah Iglseder 0000-0002-0912-7257.

Author contributions: Horvath L and Iglseder S wrote and corrected the manuscript; Oberhuber G, Gunsilius E, Wolf D, Effenberger M, Tilg H and Chott A reviewed and corrected the manuscript; Oberhuber G and Iglseder S provided the figures; Iglseder S was the patient´s neurologist; Effenberger M and Tilg H were the patient´s gastroenterologists; Horvath L, Gunsilius E and Wolf D were the patient´s haematologists; Oberhuber G and Chott A were the pathologists involved; all authors issued final approval for the version to be submitted.

Informed consent statement: Any and all details that might disclose the identity of the patient described in this report are anonymized. The patient has deceased at the time point of enrollment, writing and submission of this case report and therefore not informed written

Lena Horvath, Eberhard Gunsilius, Dominik Wolf, Department of Internal Medicine V (Hematology and Medical Oncology), Medical University Innsbruck, Innsbruck 6020, Austria

Georg Oberhuber, InnPath GmbH, Institute of Pathology, Innsbruck 6020, Austria

Andreas Chott, Ottakring Clinic, Institute of Pathology and Microbiology, Vienna 1160, Austria

Maria Effenberger, Herbert Tilg, Department of Internal Medicine I (Gastroenterology, Hepatology, Endocrinology and Metabolism), Medical University Innsbruck, Innsbruck 6020, Austria

Sarah Iglseder, Department of Neurology, Medical University Innsbruck, Innsbruck 6020, Austria

Corresponding author: Sarah Iglseder, MD, Academic Fellow, Department of Neurology, Medical University Innsbruck, Anichstrasse 35, Innsbruck 6020, Austria. [email protected]

AbstractBACKGROUND Enteropathy-associated T cell lymphoma (EATL) is an aggressive intestinal T cell lymphoma derived from intraepithelial lymphocytes, which occurs in individuals with celiac disease (CD). Cerebral involvement is an extremely rare condition and as described so far, lymphoma lesions may present as parenchymal predo-minantly supratentorial or leptomeningeal involvement. We describe a case of EATL with multifocal supra- and infratentorial brain involvement in a patient with refractory celiac disease (RCD).

CASE SUMMARY A 58-years old man with known CD developed ulcerative jejunitis and was diagnosed with RCD type II. Six months later he presented with subacute cerebellar symptoms (gait ataxia, double vision, dizziness). Cranial magnetic resonance imaging (MRI) revealed multifocal T2 hyperintense supra- and infratentorial lesions. Laboratory studies of blood and cerebrospinal fluid were inconspicuous for infectious, inflammatory or autoimmune diseases. 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18FDG-PET/CT) scan showed a suspect hypermetabolic lesion in the left upper abdomen and consequent surgical jejunal resection revealed the diagnosis of



http://orcid.org/0000-0002-1509-4324

http://orcid.org/0000-0002-1509-4324

http://orcid.org/0000-0002-1509-4324

http://orcid.org/0000-0002-9701-821X

http://orcid.org/0000-0002-9701-821X

http://orcid.org/0000-0002-0179-838X

http://orcid.org/0000-0002-0179-838X

http://orcid.org/0000-0002-0499-9953

http://orcid.org/0000-0002-0499-9953

http://orcid.org/0000-0002-4235-2579

http://orcid.org/0000-0002-4235-2579

http://orcid.org/0000-0003-1327-2921

http://orcid.org/0000-0003-1327-2921

http://orcid.org/0000-0002-4761-075X

http://orcid.org/0000-0002-4761-075X

http://orcid.org/0000-0002-0912-7257

http://orcid.org/0000-0002-0912-7257

http://orcid.org/0000-0002-0912-7257



Horvath L et al. Cerebral lesions in refractory celiac disease


consent was provided.

Conflict-of-interest statement: The authors declare that they have no conflict of interest.

CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016).




Country/Territory of origin: Austria

Peer-review report’s scientific quality classificationGrade A (Excellent): 0 Grade B (Very good): B, B, B, B Grade C (Good): C Grade D (Fair): D Grade E (Poor): 0

Received: August 24, 2020 Peer-review started: August 24, 2020 First decision: October 18, 2020 Revised: October 31, 2020 Accepted: November 12, 2020 Article in press: November 12, 2020 Published online: December 21, 2020

P-Reviewer: Guo K, Lan C, Shi BM, Zavras N, Zhu WF, Xiao E S-Editor: Gao CC L-Editor: A P-Editor: Wang LL

EATL. During the diagnostic work-up, neurological symptoms aggravated and evolved refractory to high-dosage cortisone. Recurrent MRI scans showed progressive cerebral lesions, highly suspicious for lymphoma and methotrexate chemotherapy was initiated. Unfortunately, clinically the patient responded only transiently. Finally, cerebral biopsy confirmed the diagnosis of cerebral involvement of EATL. Considering the poor prognosis and deterioration of the performance status, best supportive care was started. The patient passed away three weeks after diagnosis.

CONCLUSION EATL with cerebral involvement must be considered as a possible differential diagnosis in patients with known RCD presenting with neurological symptoms.

Key Words: Enteropathy-associated T cell lymphoma; Brain neoplasm; Celiac disease; Cerebellar syndrome; Case report


Core Tip: Enteropathy-associated T cell lymphoma (EATL) is a rare type of peripheral T cell lymphoma frequently evolving from refractory celiac disease (RCD) type II. The prognosis is often dismal due to its aggressive clinical behavior. Extraintestinal manifestation may occur, but concerning cerebral involvement, only very few reports have been described so far, showing parenchymal lesions with predominantly supratentorial or leptomeningeal involvement. This case shows multifocal supratentorial, brainstem and cerebellar involvement of EATL in a patient with RCD type II, with a rarely observed gamma/delta T cell receptor immunophenotype. This report underlines the importance to consider EATL with cerebral involvement in patients with RCD who develop neurological symptoms.

Citation: Horvath L, Oberhuber G, Chott A, Effenberger M, Tilg H, Gunsilius E, Wolf D, Iglseder S. Multiple cerebral lesions in a patient with refractory celiac disease: A case report. World J Gastroenterol 2020; 26(47): 7584-7592URL: https://www.wjgnet.com/1007-9327/full/v26/i47/7584.htmDOI: https://dx.doi.org/10.3748/wjg.v26.i47.7584

INTRODUCTIONEnteropathy-associated T cell lymphoma (EATL) is a rare and aggressive type of peripheral T cell lymphoma (PTCL), accounting for approximately 5% of PTCL[1]. It is a primary intestinal lymphoma, typically affecting the jejunum and ileum, and is often associated with celiac disease (CD)[2]. Extraintestinal involvement is a rare complication with poor prognosis. Concerning cerebral involvement, predominantly supratentorial parenchymal lesions or leptomeningeal involvement have been described in few case reports, either as primary cerebral lymphoma or secondary to intestinal EATL, whereas infratentorial lesions are extremely rare and were only reported once[3-8]. We herein present an extraordinary case of EATL with simultaneous multifocal supra- and infratentorial brain involvement in a patient with refractory celiac disease (RCD).

CASE PRESENTATIONChief complaintsA 58-years old man with the known history CD consulted his gastroenterologist due to upper stomachache, postprandial bloating and involuntary weight loss (15 kg within one month).








History of present illnessNine years earlier the previously healthy man was diagnosed with CD (histologically Marsh-Oberhuber type 1, genotype HLA-DQB1*02, elevated immunoglobulin Atissue transglutaminase (tTG-IgA) antibodies) after a period of severe diarrhea. The patient swiftly recovered upon starting a strict gluten-free diet and went into histological and clinical remission.

History of past illnessApart from CD, the patient had no relevant previous illnesses.

Personal and family historyFamily history was inconspicuous.

Physical examinationAt the actual consultation the patient presented in normal nutritional, but slightly reduced general condition. Physical examination revealed diffuse tenderness of the abdomen, but otherwise no abnormal findings. Clinical neurological examination was unsuspicious.

Laboratory examinationsLaboratory studies, including total blood count, kidney, liver as well as inflammatory parameters did not reveal any abnormalities. Fecal analysis showed a calprotectin above 1000 µg/g (normal count < 50 µg/g). Serologic tTG-IgA antibodies were normal and suggested dietary well controlled CD.

Imaging examinationsAs initial gastroscopic evaluation was inconspicuous, contrast MRI of the abdomen was performed exposing prominent mesenterial lymphadenopathy in the left upper abdominal quadrant. Capsule endoscopy revealed ulcerative lesions in the proximal jejunum and ileum, which were consequently biopsied via push-endoscopy.

Further diagnostics and clinical courseHistological examination of the jejunal biopsies revealed ulcerative jejunitis type II (Figure 1) with an increased population of immunophenotypically abnormal intraepithelial lymphocytes (IEL) (Figure 2). Diagnosis of RCD type II was made and an immunosuppressive therapy with methylprednisolone and azathioprine initiated. The patient´s condition slightly improved within weeks.

Six months later, the patient suffered from subacute gait ataxia, undirected dizziness, double vision and cognitive impairment with aggravation over the course of four days. A cranial MRI revealed multifocal T2 hyperintense partially ring-enhancing lesions cortical, subcortical, periventricular, in the basal ganglia, mesencephalon and cerebellum. The cell count in the cerebrospinal fluid (CSF) was normal. No malignant tumor cells, pathogens or oligoclonal bands were detected. Neurotropic pathogens were ruled out via polymerase chain reaction (PCR). Electroencephalography (EEG) showed moderate diffuse cerebral dysfunction but no epileptiform abnormalities. Onconeural antibodies and antibodies to neuronal surface antigens in serum and CSF remained negative.

Due to rapid worsening of the patient’s neurological condition, particularly progression of the cerebellar syndrome, an empiric therapy with intravenous high-dose methylprednisolone (1 g daily for five days) was initiated and clinical improvement followed promptly. Yet, repeated cranial MRIs showed the intracranial lesions stable in size and location.

For further evaluation, an 18FDG-PET/CT scan was performed revealing a hypermetabolic lesion in the left upper abdomen. Recurrent endoscopic biopsies of the respective jejunal ulcerations indicated the known diagnosis of RCD type II. Due to high clinical suspicion of lymphoma, the patient consequently underwent surgical resection of the affected jejunal part.

During the period of diagnostic testing, the patient´s neurological condition worsened with permanent dizziness and progressive right-sided hemiparesis. Recurrent cranial MRIs revealed progressive multifocal supra- and infratentorial lesions with diffusion restriction as well as inhomogeneous and circular contrast enhancement, morphologically compatible with central nervous system (CNS) lymphoma (Figure 3). Once more, CSF analysis did not reveal any remarkable findings, especially no malignant cells.



Figure 1 Biopsies from an area with ulcerative jejunitis. On the right there is an erosion without lymphomatous involvement. Adjacent mucosa shows villous flattening. Magnification 200 ×.

Figure 2 Histological presentation of refractory celiac disease type 2. A: Normal villous architecture with an increase in intraepithelial lymphocytes. Hematoxylin and eosin; B: Intraepithelial CD3 positive lymphocytes (brown color) are abundant; C: Intraepithelial lymphocytes contain TIA-1 positive granules (brown color, arrow); D: Intraepithelial lymphocytes are CD8 negative (arrow), positive reaction in brown coloration. Magnification 200 × (A, B and D) and 400 × (C).

FINAL DIAGNOSISHistological workup of the jejunal specimen confirmed the diagnosis of EATL with infiltration of the subserosa and covered perforation (Figure 4A and B). Resection lines were free of tumor. Consequently, the cerebral lesions were interpreted as CNS involvement of the EATL.

TREATMENTA chemotherapy with intravenous methotrexate (4 mg/m2, total dose 8160 mg) was



Figure 3 Cerebral magnetic resonance imaging images. Cerebral magnetic resonance imaging demonstrated multifocal lesions in right temporal lobe, periventricular third ventricle right and lateral ventricle left, basal ganglia left and mesencephalon left with diffusion restriction on diffusion-weighted images as well as inhomogeneous and circular contrast enhancement as well as hyperintensities on T2-weighted images. A: Diffusion-weighted images (A1 and 2); B: Inhomogeneous and circular contrast enhancement (B1 and 2); C: T2-weighted images (C1 and 2).

administered as single dose.

OUTCOME AND FOLLOW-UPUnfortunately, the patient showed only transient and little clinical improvement in response to methotrexate. Given this discrepancy and the hope to re-define diagnosis, a brain biopsy was performed. Histopathological examination by Prof. Hainfellner J (Medical University Vienna, Division of Neuropathology) could lastly confirm the suspicion of cerebral involvement of primary intestinal EATL (Figure 4C and D).

Due to poor prognosis and lack of therapeutic options, the patient was transferred to a hospice facility. He passed away three weeks after the definitive diagnosis had been made.

DISCUSSIONEATL is a rare and aggressive primary intestinal lymphoma, accounting for approximately 5% of PTCL cases, with an incidence of 0.05-0.14/100000 in Europe[1]. It may evolve de novo or progress from RCD (particularly RCD type II) via a multi-step process. RCD is defined as lack of clinical and histological response to at least twelve months of rigid gluten free diet (i.e., normalization of antibody titers) and after exclusion of other possible causes of villous atrophy. RCD may present in form of chronic ulcerative jejunitis[1]. While patients with de novo EATL show a 5-year overall survival (OS) of 59%, those cases evolving from RCD type II show a poor prognosis with 5-year OS of 0%-8%[1]. In these patients extraintestinal manifestations are seen more frequently.

Clinical presentationEATL most frequently occurs in the 5th to 6th decade and is usually diagnosed in advanced disease stages, as it was seen in our case. Typical symptoms include



Figure 4 Histological presentation of enteropathy-associated T cell lymphoma. A and B: Jejunal biopsy. Hematoxylin and eosin staining (A) showing large anaplastic tumor cells and immunohistochemical staining (B) showing granzyme B positive (brown) tumor cells; C and D: Cerebral biopsy. Infiltration of CD3 positive lymphoma cells (arrow) and non-malignant T lymphocytes (dotted arrow). Magnification 600 × (A, B and D) and 400 × (C).

abdominal pain, vomiting/nausea, B symptoms as well as signs of intra-abdominal sepsis or intestinal perforation. Time span between diagnosis of CD and EATL varies between a few months to several decades[2]. In our patient, it was eight years.

The small intestine is the most commonly affected site (jejunum, ileum, duodenum in descending order) with frequent involvement of mesenteric, para-aortic or iliac lymph nodes[2], as also observed in our case. Bone marrow involvement is uncommon[9] and was not seen in our patient. Furthermore, EATL may be observed in the stomach[2], colon[10,11], liver, lung and skin[12]. Cerebral manifestation is extremely rare[13] and up to now, six case reports, either as primary cerebral lymphoma or secondary to intestinal EATL, have been published[3-8]. When EATL involves the CNS, it may present as lymphomatous dissemination in the form of parenchymal lesions predominantly supratentorial or as leptomeningeal involvement[3,6,14]. The MRI scans of our patient showed not only multifocal supratentorial lesions, but also brainstem and cerebellar involvement, which has been described only once so far[5].

In our patient, cerebellar ataxia was the most apparent neurological symptom. Differential diagnosis of acute or subacute cerebellar ataxia associated with RCD include gluten ataxia and paraneoplastic cerebellar syndrome. Other neurological symptoms from supratentorial metastasis involve headache, changes in mental status, seizures and paresis[3-8]. The time period from intestinal manifestation to CNS involvement varies, with development of neurological symptoms within month or years after primary diagnosis[14].

Diagnostic confirmationEATL is usually diagnosed by histopathological examination of small bowel biopsies or resection specimens and is based upon characteristic histological and immunophenotypic findings as well as T cell clonality analysis. In our patient, EATL only scarcely affected the intestinal mucosa but showed predominant destruction of muscularis and subserosa, therefore endoscopic biopsies were repeatedly inconclusive. In the jejunal resection specimen diagnosis could lastly be made. In case of brain involvement, radiological diagnosis is best accomplished by contrast-enhanced T1-and T2 weighted MRI, demonstrating either a diffuse high intensity signal along the CSF space in case of leptomeningeal seeding, or a localized irregular, ill-defined enhancing lesion exemplifying parenchymatous penetration[3,5,6]. As demonstrated in our patient, biopsies or resection of extraintestinal manifestations including cerebral lesions can be



considered to confirm diagnosis in cases of inconclusive histological results or poor treatment response.

MorphologyHistologically, the jejunal resection specimen showed an infiltration of medium- to large-sized lymphoid cells with anaplastic features (Figure 4A and B). Immunophenotypically, the neoplastic cells were CD30+, CD103+, CD8+, CD7+, granzyme B+ (Figure 4 B) as well as TCRδ+, but negative for βF1, CD4, CD2, CD5, and CD56. The cerebral biopsy showed a loosely scattered infiltration of very similar large anaplastic CD3+ tumor cells (Figure 4C and D).

TreatmentIn EATL, the primary treatment involves surgical local debulking followed by systemic chemotherapy, preferably two to five weeks after surgery[15-17], as also performed in our patient. Up to now, there is no standard chemotherapy protocol for EATL. In PTCL, anthracycline-based chemotherapy is widely used such as cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) or CHOP-like chemotherapy[18,19]. The aggressive combination of ifosfamide, epirubicin, and etoposide (IVE) with intermediate-dose methotrexate (MTX) followed by autologous stem cell transplantation (ASCT) has shown potent anti-tumor effect and improved response rate in patients with EATL[20]. Unfortunately, a great number of patients are unable to complete chemotherapy due to poor performance status and severe malnutrition[17,20]. As for targeted therapy approaches, the CD30-directed antibody drug conjugate brentuximab-vedotin (BV) is of particular interest, having shown to improve outcomes in patients with CD30+ PTCL in combination with chemotherapy. Currently, data on the effect of BV in patients with EATL is limited, however few encouraging results of BV either in combination with intensive chemotherapy/ ASCT[21] or as monotherapy[22] have been reported. Further studies are ongoing (e.g., NCT03217643). Other targeted drugs, such as histone deacetylase (HDAC) inhibitors or immune-checkpoint inhibitors are under clinical investigation in PTCL including EATL, however most studies exclude cerebral involvement.

Neurosurgical indication for resection of symptomatic cerebral lesions is similar to that of other types of intracranial metastasis and may be followed by focal or whole brain radiotherapy. In case of leptomeningeal involvement, the best treatment options are chemotherapy, followed by craniospinal irradiation[3-6]. In our patient, after surgical resection of the affected jejunal part, a systemic chemotherapy with methotrexate was started. Unfortunately, the patient´s status deteriorated quickly after a short transient clinical improvement after the chemotherapy, therefore treatment was switched to best supportive care. Considering our case and previously reported findings, the prognosis and outcome of EATL with CNS involvement is poor and is not significantly improved by aggressive surgical and systemic treatments.

In summary, EATL may present with primary or secondary extraintestinal manifestations, with brain involvement being an extremely rare complication with poor prognosis despite aggressive surgical and medical treatment. To our knowledge, this is the first reported EATL case with simultaneous multifocal supra- and infratentorial brain involvement. As demonstrated in our patient, a biopsy of extraintestinal manifestations including biopsy or resection of cerebral lesions could be considered to confirm diagnosis in case of inconclusive histological results or poor treatment response.

CONCLUSIONEATL with cerebral involvement is a rare but severe complication and must be considered as a diagnostic possibility when a patient with known RCD presents with neurological deterioration. A multidisciplinary approach to the disease is required for both an early diagnosis and prompt treatment, increasing the likelihood of a positive outcome.

ACKNOWLEDGEMENTSWe thank Professor Hainfellner J (Medical University Vienna, Division of Neuropathology) for providing histopathological figures of the cerebral biopsy.



REFERENCESChander U, Leeman-Neill RJ, Bhagat G. Pathogenesis of Enteropathy-Associated T Cell Lymphoma. Curr Hematol Malig Rep 2018; 13: 308-317 [PMID: 29943210 DOI: 10.1007/s11899-018-0459-5]

1

Delabie J, Holte H, Vose JM, Ullrich F, Jaffe ES, Savage KJ, Connors JM, Rimsza L, Harris NL, Müller-Hermelink K, Rüdiger T, Coiffier B, Gascoyne RD, Berger F, Tobinai K, Au WY, Liang R, Montserrat E, Hochberg EP, Pileri S, Federico M, Nathwani B, Armitage JO, Weisenburger DD. Enteropathy-associated T-cell lymphoma: clinical and histological findings from the international peripheral T-cell lymphoma project. Blood 2011; 118: 148-155 [PMID: 21566094 DOI: 10.1182/blood-2011-02-335216]

2

Gobbi C, Buess M, Probst A, Rüegg S, Schraml P, Herrmann R, Steck AJ, Dirnhofer S. Enteropathy-associated T-cell lymphoma with initial manifestation in the CNS. Neurology 2003; 60: 1718-1719 [PMID: 12771280 DOI: 10.1212/01.wnl.0000066052.84194.6e]

3

Tutt AN, Brada M, Sampson SA. Enteropathy associated T cell lymphoma presenting as an isolated CNS lymphoma three years after diagnosis of coeliac disease: T cell receptor polymerase chain reaction studies failed to show the original enteropathy to be a clonal disorder. Gut 1997; 40: 801-803 [PMID: 9245937 DOI: 10.1136/gut.40.6.801]

4

Shams PN, Waldman A, Dogan A, MacKenzie JM, Plant GT. Ataxia in the setting of complicated enteropathy: double jeopardy. J Neurol Neurosurg Psychiatry 2002; 72: 527-529 [PMID: 11909917 DOI: 10.1136/jnnp.72.4.527]

5

Berman EL, Zauber NP, Rickert RR, Diss TC, Isaacson PG. Enteropathy-associated T cell lymphoma with brain involvement. J Clin Gastroenterol 1998; 26: 337-341 [PMID: 9649024 DOI: 10.1097/00004836-199806000-00026]

6

Defillo A, Zelensky A, Simmons BH, Nussbaum ES. Supratentorial metastatic enteropathy-associated T-cell lymphoma: A case report and literature review. Surg Neurol Int 2012; 3: 144 [PMID: 23230525 DOI: 10.4103/2152-7806.103887]

7

Chuah YY, Tashi T, Lee YY, Fu TY, Shih CA. Enteropathy-associated T-cell Lymphoma (EATL) with intracranial metastasis : a rare and dismal condition. Acta Gastroenterol Belg 2020; 83: 77-80 [PMID: 32233276]

8

Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, Advani R, Ghielmini M, Salles GA, Zelenetz AD, Jaffe ES. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 2016; 127: 2375-2390 [PMID: 26980727 DOI: 10.1182/blood-2016-01-643569]

9

Hong YS, Woo YS, Park G, Lee K, Kang SH, Lee HW, Kim ER, Hong SN, Chang DK, Kim YH, Rhee PL, Kim JJ. Endoscopic Findings of Enteropathy-Associated T-Cell Lymphoma Type II: A Case Series. Gut Liver 2016; 10: 147-151 [PMID: 26260757 DOI: 10.5009/gnl14457]

10

Kim JB, Kim SH, Cho YK, Ahn SB, Jo YJ, Park YS, Lee JH, Kim DH, Lee H, Jung YY. A case of colon perforation due to enteropathy-associated T-cell lymphoma. World J Gastroenterol 2013; 19: 1841-1844 [PMID: 23555174 DOI: 10.3748/wjg.v19.i11.1841]

11

Malamut G, Chandesris O, Verkarre V, Meresse B, Callens C, Macintyre E, Bouhnik Y, Gornet JM, Allez M, Jian R, Berger A, Châtellier G, Brousse N, Hermine O, Cerf-Bensussan N, Cellier C. Enteropathy associated T cell lymphoma in celiac disease: a large retrospective study. Dig Liver Dis 2013; 45: 377-384 [PMID: 23313469 DOI: 10.1016/j.dld.2012.12.001]

12

Meijer JW, Mulder CJ, Goerres MG, Boot H, Schweizer JJ. Coeliac disease and (extra)intestinal T-cell lymphomas: definition, diagnosis and treatment. Scand J Gastroenterol Suppl 2004; : 78-84 [PMID: 15696854 DOI: 10.1080/00855920410014605]

13

Ang LC, Zochodne DW, Ebers GC, Kaufmann JC, Vinters HV. Severe cerebellar degeneration in a patient with T-cell lymphoma. Acta Neuropathol 1986; 69: 171-175 [PMID: 3485877 DOI: 10.1007/BF00687056]

14

Daum S, Ullrich R, Heise W, Dederke B, Foss HD, Stein H, Thiel E, Zeitz M, Riecken EO. Intestinal non-Hodgkin's lymphoma: a multicenter prospective clinical study from the German Study Group on Intestinal non-Hodgkin's Lymphoma. J Clin Oncol 2003; 21: 2740-2746 [PMID: 12860953 DOI: 10.1200/JCO.2003.06.026]

15

Egan LJ, Walsh SV, Stevens FM, Connolly CE, Egan EL, McCarthy CF. Celiac-associated lymphoma. A single institution experience of 30 cases in the combination chemotherapy era. J Clin Gastroenterol 1995; 21: 123-129 [PMID: 8583077 DOI: 10.1097/00004836-199509000-00012]

16

Gale J, Simmonds PD, Mead GM, Sweetenham JW, Wright DH. Enteropathy-type intestinal T-cell lymphoma: clinical features and treatment of 31 patients in a single center. J Clin Oncol 2000; 18: 795-803 [PMID: 10673521 DOI: 10.1200/JCO.2000.18.4.795]

17

Hertzberg M, Matthews JP, Stone JM, Dubosq MC, Grigg A, Ellis D, Benson W, Browett P, Horvath N, Januszewicz H, Abdi E, Green M, Bonaventura A, Marlton P, Cannell P, Wolf M; ALLG. A phase III randomized trial of high-dose CEOP + filgrastim vs standard-dose CEOP in patients with non-Hodgkin lymphoma: 10-year follow-up data: Australasian Leukaemia and Lymphoma Group (ALLG) NHL07 trial. Am J Hematol 2014; 89: 536-541 [PMID: 24481640 DOI: 10.1002/ajh.23684]

18

Cai MC, Cheng S, Wang X, Hu JD, Song YP, Huang YH, Yan ZX, Jiang YJ, Fang XS, Zheng XY, Dong LH, Ji MM, Wang L, Xu PP, Zhao WL. CEOP/IVE/GDP alternating regimen compared with CEOP as the first-line therapy for newly diagnosed patients with peripheral T cell lymphoma: results from a phase 2, multicenter, randomized, controlled clinical trial. Genome Med 2020; 12: 41 [PMID: 32349779 DOI: 10.1186/s13073-020-00739-0]

19


https://dx.doi.org/10.1007/s11899-018-0459-5




https://dx.doi.org/10.1212/01.wnl.0000066052.84194.6e


https://dx.doi.org/10.1136/gut.40.6.801


https://dx.doi.org/10.1136/jnnp.72.4.527


https://dx.doi.org/10.1097/00004836-199806000-00026


https://dx.doi.org/10.4103/2152-7806.103887





https://dx.doi.org/10.5009/gnl14457






https://dx.doi.org/10.1080/00855920410014605


https://dx.doi.org/10.1007/BF00687056




https://dx.doi.org/10.1097/00004836-199509000-00012


https://dx.doi.org/10.1200/JCO.2000.18.4.795


https://dx.doi.org/10.1002/ajh.23684


https://dx.doi.org/10.1186/s13073-020-00739-0



Sieniawski M, Angamuthu N, Boyd K, Chasty R, Davies J, Forsyth P, Jack F, Lyons S, Mounter P, Revell P, Proctor SJ, Lennard AL. Evaluation of enteropathy-associated T-cell lymphoma comparing standard therapies with a novel regimen including autologous stem cell transplantation. Blood 2010; 115: 3664-3670 [PMID: 20197551 DOI: 10.1182/blood-2009-07-231324]

20

Sibon D, Malamut G, Verkarre V, Derrieux C, Radford I, Meresse B, Macintyre E, Cellier C, Cerf-Bensussan N, Brousse N, Hermine O. Enteropathy-Associated T-Cell Lymphoma Type I, But Not Refractory Celiac Disease, Strongly Expresses CD30 and Might Benefit From Brentuximab Vedotin. Blood 2013; 122: 4252-4252 [DOI: 10.1182/blood.V122.21.4252.4252]

21

Khalaf WF, Caldwell ME, Reddy N. Brentuximab in the treatment of CD30-positive enteropathy-associated T-cell lymphoma. J Natl Compr Canc Netw 2013; 11: 137-40; quiz 140 [PMID: 23411380 DOI: 10.6004/jnccn.2013.0021]

22



https://dx.doi.org/10.1182/blood.V122.21.4252.4252


https://dx.doi.org/10.6004/jnccn.2013.0021

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47 - World Journal of Gastroenterology

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