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JOURNAL OF CLINICAL AND TRANSLATIONAL HEPATOLOGY

CONTENTS 2020 8(1):1–107

Original Articles

A Real-world Prospective Study of Mother-to-child Transmission of HBV in China Using a Mobile Health Application (Shield 01) Xueru Yin, Guorong Han, Hua Zhang, Mei Wang,Wenjun Zhang, Yunfei Gao, Mei Zhong, Xiaolan Wang, Xiaozhu Zhong, Guojun Shen, Chuangguo Yang, Huiyuan Liu, Zhihong Liu, Po-Lin Chan, Marc Bulterys, Fuqiang Cui, Hui Zhuang, Zhihua Liu and Jinlin Hou ...... 1

The Reliability of Fibro-test in Staging Orthotopic Liver Transplant Recipients with Recurrent Hepatitis C Panagiotis Trilianos, Adamantios Tsangaris, Augustine Tawadros, Vrushak Deshpande and Nikolaos Pyrsopoulos ...... 9

Review Articles

Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease Jie Li and Jian-Gao Fan ...... 13

COVID-19 and Liver Dysfunction: Current Insights and Emergent Therapeutic Strategies Gong Feng, Kenneth I. Zheng, Qin-Qin Yan, Rafael S. Rios, Giovanni Targher, Christopher D. Byrne, Sven Van Poucke, Wen-Yue Liu and Ming-Hua Zheng ...... 18

Assessing the Effectiveness of Strategies in US Birth Cohort Screening for Hepatitis C Infection Cynthia J. Tsay and Joseph K. Lim ...... 25

Pathophysiology and Prevention of Paracentesis-induced Circulatory Dysfunction: A Concise Review Anand V Kulkarni, Pramod Kumar, Mithun Sharma, T R Sowmya, Rupjyoti Talukdar, Padaki Nagaraj Rao and D Nageshwar Reddy...... 42

Clinical Updates in Primary Biliary Cholangitis: Trends, Epidemiology, Diagnostics, and New Therapeutic Approaches Artin Galoosian, Courtney Hanlon, Julia Zhang, Edward W. Holt and Kidist K. Yimam ...... 49

Portal Cavernoma Cholangiopathy in Children and the Management Dilemmas Moinak Sen Sarma and Aathira Ravindranath ...... 61

Liver Transplantation Beyond Milan Criteria VivekALingiah,MumtazNiazi,RaquelOlivo,FlavioPaterno,JamesVGuarreraandNikolaosTPyrsopoulos.... 69

Non-alcoholic Fatty Liver Disease: Growing Burden, Adverse Outcomes and Associations Ramesh Kumar, Rajeev Nayan Priyadarshi and Utpal Anand ...... 76 Modulating the Intestinal Microbiota: Therapeutic Opportunities in Liver Disease Cyriac Abby Philips, Philip Augustine, Praveen Kumar Yerol, Ganesh Narayan Ramesh, Rizwan Ahamed, SasidharanRajesh,TomGeorgeandSandeepKumbar...... 87

Case Report

Repurposing Pirfenidone for Nonalcoholic Steatohepatitis-related Cirrhosis: A Case Series Cyriac Abby Philips, Guruprasad Padsalgi, Rizwan Ahamed, Rajaguru Paramaguru, Sasidharan Rajesh, Tom George, Pushpa Mahadevan and Philip Augustine ...... 100

Letter to the Editor

Complementary and Alternative Medicine-related Drug-induced Liver Injury in Iran Mehdi Pasalar, Babak Daneshfard and Kamran Bagheri Lankarani ...... 106 Original Article

A Real-world Prospective Study of Mother-to-child Transmission of HBV in China Using a Mobile Health Application (Shield 01)

Xueru Yin1, Guorong Han2, Hua Zhang3, Mei Wang4, Wenjun Zhang5, Yunfei Gao6, Mei Zhong6, Xiaolan Wang7, Xiaozhu Zhong8, Guojun Shen9, Chuangguo Yang10, Huiyuan Liu11, Zhihong Liu1, Po-Lin Chan12, Marc Bulterys13, Fuqiang Cui14, Hui Zhuang15, Zhihua Liu1* and Jinlin Hou1*

1Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China; 2Department of Gynecology and Obstetrics, The Second Affiliated Hospital of the Southeast University, Nanjing, Jiangsu, China; 3Department of Obstetrics and Gynecology, Beijing Youan Hospital, Capital Medical University, Beijing, China; 4Department of Obstetrics and Gynecology, The Fifth Medical Centre, Chinese People’s Liberation Army General Hospital, Beijing, China; 5Department of Obstetrics and Gynecology, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China; 6Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China; 7Department of Obstetrics, Jiujiang Maternal and Child Care Center, Jiujiang, Jiangxi, China; 8Department of Infectious Diseases, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China; 9Department of Infectious Diseases, Third People’s Hospital of Jiujiang, Jiujiang, Jiangxi, China; 10Department of Infectious Diseases, The Third Affiliated Hospital of Southern Medical university, Guangzhou, Guangdong, China; 11Department of Severe Liver Diseases, Guangzhou Eighth People’s Hospital, Guangzhou, Guangdong, China; 12World Health Organization/Western Pacific Regional Office (WPRO), Manila, Philippines; 13Global Hepatitis Programme, World Health Organization, Geneva, Switzerland; 14School of Public Health, Peking University, Beijing, China; 15Department of Microbiology and Center of Infectious Diseases, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

Abstract were 446 mothers who received antiviral therapy, including 72.3% of the mothers with HBV DNA level >2,000,000 IU/mL Background and Aims: The World Health Organization and 21.0% of the mothers with HBV DNA level <2,000,000 (WHO) Western Pacific Region set a target of eliminating IU/mL. Eight infants were infected with HBV. The overall rate mother-to-child transmission (MTCT) of hepatitis B virus of MTCT was 0.9%. Birth defects were rare (0.5% among in- (HBV) by 2030. To assess the feasibility of this target in China, fants with maternal antiviral exposure versus 0.7% among we carried out an epidemiological study to investigate the infants without exposure; p=1.00). Conclusions:The MTCT status quo of MTCT in the real-world setting. Methods: One rate was lower than the WHO Western Pacific Region elimina- thousand and eight hepatitis B surface antigen-positive preg- tion MTCT target in this real-world study, indicating that a nant women were enrolled at 10 hospitals. Immunoprophy- comprehensive management composed of immunoprophylaxis was administered to infants. In addition, mothers with laxis to infants and antiviral prophylaxis to mothers may be HBV DNA level >2,000,000 IU/mL were advised to initiate a feasible strategy to achieve the 2030 WHO elimination goal. antiviral therapy during late pregnancy. A health application Citation of this article: Yin X, Han G, Zhang H, Wang M, called SHIELD was used to manage the study. Results: Nine Zhang W, Gao Y, et al. A real-world prospective study of hundred and five of the enrolled mothers, with 924 infants, mother-to-child transmission of HBV in China using a mobile completed the follow-up. Birth-dose hepatitis B vaccine and health application (Shield 01). J Clin Transl Hepatol hepatitis B immunoglobulin were received by 99.7% and 2020;8(1):1–8. doi: 10.14218/JCTH.2019.00057. 99.7% of infants, respectively, within 24 h after birth. There

Keywords: Mother-to-child transmission; Hepatitis B virus; Antiviral therapy; Introduction Immunoprophylaxis; Shield Project. Abbreviations: HBeAg, hepatitis B e antigen; HBIG, hepatitis B immunoglobulin; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HepB, hepatitis B Mother-to-child transmission (MTCT) is the major mode of vaccine; LAM, lamivudine; LdT, telbivudine; MTCT, mother-to-child transmission; hepatitis B virus (HBV) exposure in areas with a high PVST, post-vaccination serologic testing; TDF, tenofovir disoproxil fumarate; prevalence of HBV infection.1 MTCT among children born to WHO, World Health Organization. hepatitis B surface antigen (HBsAg)-positive mothers will Received: 4 December 2019; Revised: 21 January 2020; Accepted: 15 February 2020 induce high rates of chronic HBV infection and is associated *Correspondence to: Zhihua Liu, Hepatology Unit, Nanfang Hospital, Southern with high rates of severe clinical outcomes, such as liver cir- Medical University, Guangzhou, Guangdong 510515, China. Tel: +86-20- rhosis, liver failure and hepatocellular carcinoma.2,3 62787432, Fax: +86-20-62786530, E-mail: [email protected]; Jinlin Hou, Combined immunoprophylaxis, including hepatitis B Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China. Tel: +86-20-61641941, Fax: +86-20-62786530, vaccine (HepB) birth dose and hepatitis B immunoglobulin E-mail: [email protected] (HBIG), administered within 24 h of birth to infants born to

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 1–81

HBsAg-positive mothers is the most effective measure for infants following the national Chinese vaccination schedule, preventing MTCT of HBV.4,5 However, despite immunoprophy- i.e. HBIG and first dose of hepatitis B vaccine within 12 h after laxis, MTCT cannot be completely prevented, and immuno- delivery and the other two doses of hepatitis B vaccine at 1 and prophylaxis fails in 2-5% of infants born to mothers with 6 months of age. In addition, mothers with HBV DNA level high levels of HBV DNA.6–8 >2,000,000 IU/mL were advised to initiate antiviral therapy In 2017, the Regional Framework for Eliminating MTCT of during late pregnancy. Enrolled mothers and infants were Human Immunodeficiency Virus, Hepatitis B and Syphilis in prospectively followed until infant post-vaccination serologic Asia and the Pacific, 2018-2030 (Triple elimination) proposed testing(PVST)wasperformedat7-12monthsofage. an integrated and coordinated approach towards elimination of these three infections.9 The framework adopts the target of Data collection and laboratory testing 0.1% HBsAg prevalence among children by 2030, as set by the Global Health Sector Strategy on Viral Hepatitis 2016- The information of the enrolled mothers, including their demo- 2021. It also suggests antiviral therapy for mothers with graphic data, antiviral treatment history, pregnancy and labor high viral load for the Regional Framework for Eliminating history, and comorbidities, were collected from prenatal medical MTCT of hepatitis B, building upon previous successful hepa- records. HBV serologic markers, including HBsAg, antibody to titis B vaccination programs. HBsAg, HBeAg, antibody to HBeAg and antibody to hepatitis B In China, the HBV infection rate has been significantly core antigen, HBV DNA levels, alanine aminotransferase, decreasing since the universal immunization of newborns was aspartate aminotransaminase, total bilirubin, direct bilirubin, initiated.10 The latest epidemiological survey (in 2014) and albumin were measured at baseline, at gestational week showed that the HBsAg prevalence was as low as 0.32% 28, and at delivery for all enrolled mothers. Additional tests among children under 5 years-old, as compared to 9.67% in included liver ultrasound at baseline and PVST for infants at 1992.11 China has a large population of HBsAg-positive preg- 7-12 months of age. The mode of delivery, neonatal character- nant women. It was reported that there were approximately istics (height, weight, head circumference, Apgar score, and one million infants born to HBsAg-positive mothers in 2015.12 birth defect) and breastfeeding were noted. The patient’sHBV It is a great challenge, however, to decrease the HBsAg prev- DNA level and HBV serological markers were measured with the alence further in children under 5 years-old in order to Roche COBAS TaqMan platform (with the lower limit of detec- achieve the World Health Organization (WHO) 2030 goal. tion of 20 IU/mL) and Roche Elecsys (Basel, Switzerland). In the past decade, antiviral therapy during late pregnancy Maternal HBV DNA levels at gestational weeks 24-28 and in mothers with high HBV DNA has been shown to be effective infant HBV serological markers at 7-12 months were measured – in preventing MTCT of HBV7,8,13 15 and is recommended by at the central laboratory set up by the research group. All other hepatitis B guidelines for the management of pregnant markers were measured at each participating center. women with chronic hepatitis B.1,16–18 In previous studies, no infant was infected with HBV in the group of hepatitis B e Use of the mobile health application antigen (HBeAg)-positive mothers receiving antiviral therapy 7,8 during late pregnancy, which indicates that elimination of A mobile health application called “SHIELD” was developed HBV MTCT is possible with routine immunoprophylaxis for all and used in this study to collect data and provide support for infants and additional antiviral therapy for HBsAg-positive communication between mothers and their doctors. All labo- mothers with high viral load. ratory test reports, questionnaires and other relevant infor- Since 2007, China established a rigorous strategy for mation was uploaded into SHIELD and made available to prevention of HBV MTCT that requires immunization (timely mothers and doctors. Mothers could consult with their doctors HepB birth dose and HBIG plus two additional doses of HepB), via SHIELD during the follow-up (Fig. 1). and in 2010, universal screening of HBV in pregnant women was started. Given the increasing evidence of the effectiveness Outcomes of maternal antiviral therapy in preventing MTCT, we initiated the Shield Project across China — aiming towards zero HBV TheprimaryoutcomewastherateofMTCTfollowingthe 19 transmission rate in infants born to HBsAg-positive mothers. administration of immunoprophylaxis and antiviral therapy. The secondary outcome was any potential adverse effect Methods noted among infants following antiviral exposure, including birth defect and infant growth. The rate of MTCT was defined Study design and population as the proportion of infants who were positive for HBsAg, while they had a detectable HBV DNA level when PVST was performed. This was a prospective, multicenter, observational cohort study to investigate the status quo of MTCT of HBV in the real-world Statistical analysis setting (ClinicalTrials.gov No. NCT03539016). The study was conducted between July 2015 and May 2018 in 10 hospitals in Continuous variables are presented as the means ± standard China. The subjects included pregnant women at gestational deviation, and the means are compared by Student’s t-test or weeks 4-32 and their infants. The main inclusion criteria were the nonparametric Mann-Whitney U test. Categorical varia- pregnant women with positive HBsAg test for a minimum of six bles are presented as proportions and Pearson’s chi-square months, good compliance, and they and their infants could be test or Fisher’s exact test were used to compare groups. followed-up as planned. Pregnant women were excluded if The association between covariates and the rate of MTCT they had a positive serologic test for human immunodeficiency were assessed with univariate analysis. All statistical tests virus or hepatitis C virus, any comorbidity that might reduce were two-sided. A p-value <0.05 was considered statistically compliance, or if they were unable or unwilling to use the mobile significant. All data were analyzed by SPSS 22.0 (IBM Corp., health application. Immunoprophylaxis was administered to Armonk, NY, USA).

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Fig. 1. Use of the mobile health application SHIELD to manage this study.

Ethics approval Up to 73.4% of HBeAg-positive mothers had an HBV DNA

level of more than 6 log10 IU/mL, compared to 6.5% of The study was conducted in accordance with Good Clinical HBeAg-negative mothers (p<0.001). Practice, following local regulations and ethical principles described in the Declaration of Helsinki. The study protocol Maternal antiviral therapy was approved by the ethics committee at each participating center. All authors had access to the study data and reviewed There were 49.3% of the mothers enrolled in the study and approved the final manuscript. receiving antiviral therapy during pregnancy with either telbivudine (LdT), tenofovir disoproxil fumarate (TDF), or Results lamivudine (LAM). Among the three nucleot(s)ide analogues, LdT (76.8%) was the most frequently used, followed by TDF Enrollment and follow-up (19.9%) and LAM (3.3%). Nearly half (46.4%) of the 442 mothers received nucleot(s)ide analogues before gestational One thousand and twenty HBsAg-positive pregnant women week 28, while 49.5% received between gestational weeks were screened. Among them, 12 pregnant women were 28 to 32, and 4.1% after gestational week 32. A higher excluded due to not meeting the eligibility criteria, 23 percentage of HBeAg-positive pregnant women received pregnant women withdrew informed consent, 11 lost antiviral therapy compared to HBeAg-negative pregnant fetuses before delivery, and 69 were lost to follow-up. There- women (71.5% vs. 13.0%). Up to 72.3% of 499 pregnant fore, a total of 905 mothers and 924 infants completed follow- women with an HBV DNA level of more than 2,000,000 IU/mL up (Supplementary Fig. 1). The main reasons for loss to received antiviral therapy, compared to 21.0% of those with follow-up were moving away and parental unwillingness to an HBV DNA level of less than 2,000,000 IU/mL (Supple- let their infants undergo blood sampling. mentary Fig. 2).

Characteristics of the mothers and infants Neonatal immunoprophylaxis

The characteristics of the mothers and infants are listed in Data on the immunoprophylaxis was obtained from 918 infants Table 1. The distribution of viral load in HBeAg-positive whose records were available and complete. The coverage of mothers and HBeAg-negative mothers is depicted in Fig. 2. HepB birth dose and HBIG was 99.8% and 99.9%,

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 1–83 Yin X. et al: A real-world study of MTCT of HBV

Table 1. Characteristics of the HBV-infected pregnant mothers and their were both HBsAg- and HBeAg-positive mothers, and 1.6% in infants enrolled in the Shield study, China, 2015-2018 499 mothers who had an HBV DNA level of more than Characteristics Total, n=905 2,000,000 IU/mL, respectively. The characteristics of the eight infected infants and their mothers are listed in Table 2. Maternal characteristics at baseline In the 499 mothers with an HBV DNA level of more than Age in year, mean 6 SD 28.264.2 2,000,000 IU/mL, 72.3% received antiviral therapy during late pregnancy. Four HBV-infected infants had maternal Alanine aminotransferase antiviral exposure and four were without maternal antiviral No. of mothers with data* 892 exposure. Thus, the rates of MTCT were 1.1% and 2.9% in Mean 6 SD, U/L 32.2642.5 361 mothers with antiviral therapy and 138 without antiviral exposure, respectively (p=0.15). Distribution, n (%) <13ULN, 40 U/L 732 (82.1) Risk factors for MTCT $13ULN to <23ULN 101 (11.3) Based on the data for the entire cohort, we analyzed the risk $23ULN to <53ULN 47 (5.3) factors of MTCT. The results of univariate analyses are listed in $53ULN 12 (1.4) Table 3. In the univariate analyses, maternal viral load and HBeAg HBV DNA status were found to be associated with MTCT. We did not find an † association between MTCT and mode of delivery, feeding practice, No. of mothers with data 904 history of threatened abortion and threatened preterm birth, and 6 6 Log10 (HBV DNA), mean SD 5.7 2.6 history of invasive procedures during pregnancy.

Distribution as log10 HBV DNA, n (%) Infant safety outcomes Undetectable 38 (4.2) Detectable to <6 345 (38.2) Among the liveborn infants, there were no significant differ- $6 to <8 311 (34.4) ences in the rate of birth defect between infants with maternal $8 210 (23.2) antiviral exposure during pregnancy and those without maternal antiviral exposure (0.5% vs. 0.7%, p=1.00). The gestational HBeAg age, mode of delivery, weight, height, head circumference and No. of mothers with data‡ 893 Apgar score at birth were similar in the two groups (Table 4). Distribution, n (%) Discussion Negative 332 (37.2) Positive 561 (62.8) A real world study enrolling 1008 HBsAg-positive mothers Infant characteristics at birth, mean 6 SD and their infants was prospectively conducted to investigate the status quo of MTCT in the real world. The results show Head circumference in cm 33.261.3 that the rate of MTCT among HBV-exposed infants was 0.9%, Length in cm 49.861.8 in the context of high coverage of birth dose immunization Birth weight in kg 3.260.5 and maternal antiviral intervention during pregnancy. For all infants, free three-dose HepB was provided by the Apgar score 9.760.6 Chinese government, guided by the National Immunization * This category excludes 13 mothers without ALT records. Schedule. In this cohort, the timely HepB birth dose and HBIG † This category excludes one mother without HBV DNA data. were 99.7% and 99.7%, respectively. In addition, antiviral ‡ This category excludes 12 mothers without HBeAg records. therapy was administered to 49.3% of mothers in this cohort. Abbreviations: HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; SD, standard In this study, LdT is the most frequently used NA, followed by deviation; ULN, upper limit of normal. TDF and LAM. LdT, LAM and TDF were recommended for maternal antiviral therapy in the previous Chinese chronic respectively. The coverage of timely HepB birth dose within 24 hepatitis B guideline.1 In June 2014, TDF was approved in h after birth was 99.7%, and 98.1% within 12 h after birth. The China. Before that, LdT and LAM were used in preventing coverage of timely HBIG within 24 h was 99.7%, and 99.1% MTCT.8,13,14 However, TDF and LdT are preferably recommen- within 12 h after birth. The immunization schedule of four out ded for preventing MTCT of HBV, guided by the latest Chinese of ten hospitals involved in this study included two doses of recommendation,20 while TDF is preferably recommended in HBIG 100 IU, administered to infants born to HBsAg mothers the American Association for the Study of Liver Diseases and at birth and 1 month of age. In this cohort, 46.6% of 916 European Association for the Study of the Liver guidelines.16,17 infants received two doses of HBIG 100 IU. Eight infants were found to be infected by HBV who were all born to HBeAg-positive mothers with an HBV DNA level of Rate of MTCT more than 2,000,000 IU/mL. Four infants were born to mothers who received antiviral therapy and HBV DNA level

In total, eight infants were found to be positive for HBsAg and was less than 5 log10 IU/mL in two of four mothers and was had detectable HBV DNA after PVST. All eight infants were more than 6 log10 IU/mL in one of four mothers at delivery. born to HBeAg-positive mothers with a high viral load. All of This result indicated that in the real world, even if HBeAg- them received timely HepB birth dose and HBIG, except for positive mothers with a high viral load received antiviral one who was a preterm infant. The rates of MTCT were 0.9% therapy and their infants received immunoprophylaxis, perina- in 905 HBsAg-positive mothers, 1.4% in 561 mothers who tal infection cannot be completely prevented. One explanation

4 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 1–8 Yin X. et al: A real-world study of MTCT of HBV

Fig. 2. Viral load distribution in hepatitis B e antigen-positive and hepatitis B e antigen-negative mothers. for this is that intrauterine infection occurred in the infants. In HepB birth dose and HBIG is low, antiviral therapy during fact, one of the four infants was found to be positive for HBV pregnancy might be a compensatory approach for preventing DNA at birth, which indicated an intrauterine HBV infection. MTCT. Currently, it is important to objectively evaluate the Another possible explanation is that these infants’ infections significance of maternal antiviral therapy in preventing were caused by vaccine-escape mutants with altered binding MTCT of HBV. Based on immunoprophylaxis, maternal antivi- 21,22 capacity for the antibody to HBsAg. Another four infected ral therapy could be used to complement current prophylaxis infants were born to mothers who had an HBV DNA level of in mothers with high viral load to prevent MTCT. On the one 100,000,000 IU/mL but without antiviral therapy during preg- hand, we should avoid overuse of maternal antiviral therapy nancy. One infant did not receive birth dose vaccine due to in those who are unlikely to transmit to their infant perina- prematurity. Delayed immunoprophylaxis for premature or tally. On the other hand, we should do our best to prevent low-birthweight (<2000 g) infants is most likely the cause of MTCT and its consequent outcomes in HBsAg-positive these infections. It is recommended that premature infants mothers with a high viral load and in their infants. 23,24 should also receive timely immunoprophylaxis. Univariate analysis showed that HBeAg positivity and high In this study, the rates of MTCT were 0.9% and 1.4% in viral load were strongly associated with MTCT. To identify HBsAg-positive mothers and in HBsAg- and HBeAg-positive mothers who have a high risk of MTCT, HBeAg and HBV DNA mothers, respectively, which was much lower than the rates are the most important factors. Ideally, all HBsAg-positive reported in previous studies.10,25 Besides that, the MTCT rates mothers should have a measurement of HBV DNA levels so were 1.1% and 2.9% in HBsAg-positive mothers with high viral that antiviral prophylaxis can be administered to those with a load with antiviral exposure and those without antiviral expo- high viral load. However, HBV DNA quantification is expensive sure, respectively. Although an apparent reduction in the rate and may not be available in remote or under-developed areas of MTCT was found, there was no significant difference in the due to its cost and technical requirements. In our study, 73.4% of rates of MTCT between the two groups. The reason may be the HBeAg-positive mothers had an HBV DNA level of more than low rate of MTCTand that the sample size is not large enough in 2,000,000 IU/mL, compared to 6.5% of HBeAg-negative this study. Similar to our study, a recent study in Thailand mothers. Thus, if HBeAg was utilized to screen high-risk demonstrated that the immunoprophylaxis failure rate was 2.0% in the group without maternal antiviral therapy.7 mothers for antiviral therapy, 6.5% of HBeAg-negative mothers The threshold HBV DNA level for maternal antiviral therapy with an HBV DNA level of more than 2,000,000 IU/mL will be varies among hepatitis B guidelines. An HBV DNA level of missed, whereas 26.6% of HBeAg-positive mothers with an HBV more than 2,000,000 IU/mL is recommended in the China DNA level of less than 2,000,000 IU/mL will be over-treated. hepatitis B guidelines as the threshold for antiviral therapy to Considering that few infants born to HBsAg-positive and HBeAg- prevent MTCT,1 whereas HBV DNA level of more than 200,000 negative mothers would be perinatally infected by HBV after 11,25 IU/mL has been recommended in the American Association timely immunoprophylaxis at birth, HBeAg screening might for the Study of Liver Diseases and European Association for be an acceptable alternative to HBV DNA quantification, espe- the Study of the Liver guidelines.16,17 In addition, in Australia cially in areas where HBV DNA testing is unavailable. and New Zealand, the threshold is set at 10,000,000 IU/mL.26 The SHIELD mobile application was innovatively developed in In this real-world study, all eight HBV-infected infants were this study. First, this new tool was used as an electronic data born to mothers with HBV DNA level of more than capture system to collect data including laboratory test reports, 100,000,000 IU/mL. This result implied that the threshold immunoprophylaxis and antiviral therapy information, etc. In for antiviral therapy may be higher than the current level in addition, SHIELD broke down barriers among hepatologists, the context of high coverage of timely HepB birth dose and gynecologists and HBV-infected mothers, providing a platform HBIG. On the contrary, in the area where coverage of timely for communicating without restrictions of time and space.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 1–85 6

Table 2. Characteristics of HBV-infected infants and their mothers in the Shield study, China, 2015-2018

Infants Mothers ora fCiia n rnltoa Hepatology Translational and Clinical of Journal HBV Time DNA HBV of baseline DNA at Threatened Time HepB Age in delivery Baseline abortion/ Weight of birth Feeding in HBeAg log10 in log10 Antiviral ALT in Gestational Type of preterm No. in kg HBIG dose practiceÃ year status IU/mL IU/mL agents U/L week delivery labor Amniocentesis

1 3.4 <1 h <1 h Breastfeeding 38 Positive 8 7 No 19.0 38 Cesarean Threatened No section preterm labor 2 2.7 <1 h <12 h Breastfeeding 40 Positive 8 NT No 15.0 40 Vaginal No No 3 2.1 <2h - Breastfeeding + 33 Positive 8 NT No 19.0 33 Vaginal No No Formula 4 3.2 <1 h <1 h Formula 38 Positive 8 NT No 10.3 38 Vaginal No No 5 2.4 <1 h <1 h Formula 38 Positive 8 4 TDF at 10.2 38 Cesarean No No gestational section week 27 6 3.6 <1 h <1 h Formula 39 Positive 8 4 LdT at 12.8 39 Cesarean No No gestational section

2020 week 27 i X. Yin 7 3.2 <1 h <1 h Formula 39 Positive 8 NT TDF at 14.1 39 Cesarean No No

o.8|1 | 8 vol. gestational section

week 28 al et 8 2.9 <1 h <1 h Formula 40 Positive 8 6 TDF at 18.0 40 Vaginal Threatened No elwrdsuyo TTo HBV of MTCT of study real-world A : gestational abortion

– week 29 8 * Feeding practice was observed till infant post-vaccination serologic testing was done. Abbreviations: ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HBIG, hepatitis B immunoglobulin; HBV, hepatitis B virus; HepB, hepatitis B vaccine; LdT, telbivudine; NT, not tested; TDF, tenofovir disoproxil fumarate. Yin X. et al: A real-world study of MTCT of HBV

Table 3. Univariate analyses on risk factors of mother-to-child transmission

Mother-to-child transmission

Variables Yes No p-value

Total 8 (0.9%) 897 (99.1%) HBeAg 0.03 Positive 8 (1.4%) 553 (98.6%) Negative 0 (0.0%) 332 (100.0%)

HBV DNA level at gestational week 28 in log10 IU/mL 0.002 <6 1 (0.2%) 449 (99.8%) $6 to <8 0 (0.0%) 183 (100.0%) $8 7 (2.9%) 237 (97.1%) Initiation time of antiviral therapy 1.00 <28 weeks 2 (1.0%) 203 (99.0%) $28 weeks to <32 weeks 2 (0.9%) 215 (99.1%) $32 weeks 0 (0.0%) 20 (100.0%) Mode of delivery 0.54 Vaginal 4 (0.7%) 552 (99.3%) Cesarean section 4 (1.2%) 355 (98.9%) Feeding practice 0.48 Breastfeeding 3 (0.6%) 499 (99.4%) Formula feeding 5 (1.3%) 394 (98.8%) History of threatened abortion/threatened preterm labor 0.28 Yes 2 (1.7%) 116 (98.3%) No 6 (0.8%) 781 (99.2%) History of invasive procedure 1.00 Yes 0 (0.0%) 7 (100.0%) No 8 (0.9%) 890 (99.1%)

Abbreviations: HBeAg, hepatitis B e antigen; HBV, hepatitis B virus.

SHIELD is exploring a new model of management for pre- of MTCT for Hepatitis B, Human Immunodeficiency Virus, and venting MTCT. Syphilis Initiative. In July 2015, we launched the Shield Project The WHO has set the goal to eliminate viral hepatitis as a with the aim of reducing or even eliminating MTCT of HBV. A major public health threat by 2030, which includes reducing collaborative network for preventing MTCT has been established the prevalence of HBsAg among 5 year-old children to 0.1%.27 The WHO Regional Office for the Western Pacific set Table 4. Characteristics and birth defects of the infants 2.0% as the target for eliminating MTCT of HBV. In this study, the rate of MTCT could achieve this target in the real world, in Maternal antiviral therapy the context of high coverage of birth dose immunization and over 70% maternal antiviral intervention in mothers with high Variable Yes, n=446 No, n=459 p-value viral load. The overall coverage of hepatitis B vaccination has Head circumference 33.161.3 33.261.3 0.14 increased steadily, having reached 95% in urban areas in in cm China by 2005.28 Besides that, the recommendation for maternal antiviral intervention has been widely accepted. Length in cm 49.961.8 49.861.9 0.05 Thanks to the government’s policy, the prices of antivirals Birth weight in kg 3.260.5 3.260.5 0.39 fell sharply in China, improving the affordability and availabil- Apgar score 9.760.5 9.760.7 0.10 ity of antivirals. We believe that the 2030 elimination target would be achievable nationwide in the future. Gestational age in 39.461.5 39.161.7 0.12 China has achieved major success in the prevention and week control of hepatitis B.29 To further reduce the rate of MTCT of No. of cesarean 181 179 0.49 HBV, actions have already been taken by both government section (40.4%) (38.2%) sectors and non-government organizations in China. In No. of birth defect 2 (2/440, 3 (3/447, 1.00 December 2017, the National Health Commission (Ministry 0.5%) 0.7%) of Health) officially launched the aptly-named Triple Elimination

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 1–87 Yin X. et al: A real-world study of MTCT of HBV and 29,420 HBsAg-positive mothers and 1630 doctors were [5] Chen DS. Hepatitis B vaccination: The key towards elimination and eradication enrolled in 124 hospitals nationwide by September 2019. The of hepatitis B. J Hepatol 2009;50:805–816. doi: 10.1016/j.jhep.2009.01.002. [6] Lee C, Gong Y, Brok J, Boxall EH, Gluud C. Hepatitis B immunisation for newborn Shield Project will provide high-level evidence for implementing infants of hepatitis B surface antigen-positive mothers. Cochrane Database Syst public health policies to prevent MTCT of HBV and will facilitate Rev 2006:CD004790. doi: 10.1002/14651858.CD004790.pub2. the process of eliminating MTCT of HBV in China and globally. [7] Jourdain G, Ngo-Giang-Huong N, Harrison L, Decker L, Khamduang W, Tierney C, The limitation of this study is that the percentage of HBeAg- et al. Tenofovir versus placebo to prevent perinatal transmission of hepatitis B. N Engl J Med 2018;378:911–923. doi: 10.1056/NEJMoa1708131. positive mothers in this study could not represent the natural [8] Zhang H, Pan CQ, Pang Q, Tian R, Yan M, Liu X. Telbivudine or lamivudine use distribution in pregnant women with chronic HBV infection due in late pregnancy safely reduces perinatal transmission of hepatitis B virus in to more HBeAg-positive mothers being willing to participate in real-life practice. Hepatology 2014;60:468–476. doi: 10.1002/hep.27034. our study compared to HBeAg-negative mothers. [9] World Health Organization. Eliminating mother to child transmission of HIV, hepatitis and syphilis. Available from: https://www.who.int/westernpacific/ac- In conclusion, The MTCT rate was lower than the WHO tivities/eliminating-mother-to-child-transmission-of-hiv-hepatitis-syphilis. Western Pacific Region elimination MTCT target in this real- [10] Wang F, Zhang G, Zheng H, Miao N, Shen L, Wang F, et al. Post-vaccination world study, indicating that a comprehensive management serologic testing of infants born to hepatitis B surface antigen positive composed of immunoprophylaxis to infants and antiviral mothers in 4 provinces of China. Vaccine 2017;35:4229–4235. doi: 10. 1016/j.vaccine.2017.06.019. prophylaxis to mothers may be a feasible strategy to [11] Lu Y, Liang XF, Wang FZ, Yan L, Li RC, Li YP, et al. Hepatitis B vaccine alone achieve the 2030 WHO elimination target. may be enough for preventing hepatitis B virus transmission in neonates of HBsAg (+)/HBeAg (-) mothers. Vaccine 2017;35:40–45. doi: 10.1016/j. vaccine.2016.11.061. Acknowledgments [12] Cui F, Woodring J, Chan P, Xu F. Considerations of antiviral treatment to interrupt mother-to-child transmission of hepatitis B virus in China. Int J We thank Dr. Lan Zhang for her professional advice on this Epidemiol 2018;47:1529–1537. doi: 10.1093/ije/dyy077. ’ [13] Pan CQ, Han GR, Jiang HX, Zhao W, Cao MK, Wang CM, et al. Telbivudine pre- study, when she worked in the World Health Organization s vents vertical transmission from HBeAg-positive women with chronic hepatitis B. China Office. We thank all the participants who contributed to Clin Gastroenterol Hepatol 2012;10:520–526. doi: 10.1016/j.cgh.2012.01.019. the study, with the commitment to elimination of HBV [14] Han GR, Cao MK, Zhao W, Jiang HX, Wang CM, Bai SF, et al. A prospective and mother-to-child transmission. open-label study for the efficacy and safety of telbivudine in pregnancy for the prevention of perinatal transmission of hepatitis B virus infection. J Funding Hepatol 2011;55:1215–1221. doi: 10.1016/j.jhep.2011.02.032. [15] Pan CQ, Duan Z, Dai E, Zhang S, Han G, Wang Y, et al. Tenofovir to prevent hepatitis B transmission in mothers with high viral load. N Engl J Med 2016; This work was funded by the China Foundation for Hepatitis 374:2324–2334. doi: 10.1056/NEJMoa1508660. Prevention and Control (CFHPC) and National Natural [16] EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus Science Foundation of China (Grant No. 81673243) and the infection. J Hepatol 2017;67:370–398. doi: 10.1016/j.jhep.2017.03.021. [17] Terrault NA, Lok ASF, McMahon BJ, Chang KM, Hwang JP, Jonas MM, et al. Chinese National Research Grant of the Thirteenth Five-Year Update on prevention, diagnosis, and treatment of chronic hepatitis B: Plan for the Key Projects in Infectious Diseases (Grant No. AASLD 2018 hepatitis B guidance. Hepatology 2018;67:1560–1599. doi: 2017ZX10201201). 10.1002/hep.29800. [18] Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. – Conflict of interest Hepatol Int 2016;10:1 98. doi: 10.1007/s12072-015-9675-4. [19] Fan R, Yin X, Liu Z, Liu Z, Lau G, Hou J. A hepatitis B-free generation in China: from dream to reality. Lancet Infect Dis 2016;16:1103–1105. doi: 10. JLH has received consulting fees from AbbVie, Arbutus, Bristol 1016/S1473-3099(16)30327-9. Myers Squibb, Gilead Sciences, Johnson & Johnson, and [20] Hou J, Cui F, Ding Y, Dou X, Duan Z, Han G, et al. Management algorithm for Roche and has received grants from Bristol Myers Squibb interrupting mother-to-child transmission of hepatitis B virus. Clin Gastro- enterol Hepatol 2019;17:1929–1936.e1. doi: 10.1016/j.cgh.2018.10.007. and Johnson & Johnson. The other authors have no conflict of [21] Carman WF, Zanetti AR, Karayiannis P, Waters J, Manzillo G, Tanzi E, et al. interests related to this publication. Vaccine-induced escape mutant of hepatitis B virus. Lancet 1990;336:325– 329. doi: 10.1016/0140-6736(90)91874-a. [22] Ma Q, Wang Y. Comprehensive analysis of the prevalence of hepatitis B virus Author contributions escape mutations in the major hydrophilic region of surface antigen. J Med Virol 2012;84:198–206. doi: 10.1002/jmv.23183. Study concept and design (JH and Zhihua Liu), acquisition of [23] Schillie S, Vellozzi C, Reingold A, Harris A, Haber P, Ward JW, et al. Prevention data (Zhihua Liu, Zhihong Liu, XY, JH, GH, HZ, MW, WZ, YG, MZ, of hepatitis B virus infection in the United States: Recommendations of the advisory committee on immunization practices. MMWR Recomm Rep 2018; XW, XZ, GS, CY, and HL), analysis and interpretation of data (JH, 67:1–31. doi: 10.15585/mmwr.rr6701a1. Zhihua Liu, and XY), drafting of the manuscript (JH, Zhihua Liu, [24] World Health Organization. Hepatitis B vaccines: WHO position paper, July and XY), critical revision of the manuscript for important 2017- Recommendations. Vaccine 2019;37:223–225. doi: 10.1016/j. intellectual content (MJ, MJT, SS), administrative, technical, or vaccine.2017.07.046. [25] Chen HL, Lin LH, Hu FC, Lee JT, Lin WT, Yang YJ, et al. Effects of maternal material support, study supervision (PC, MB, FC, and HZ). screening and universal immunization to prevent mother-to-infant transmission of HBV. Gastroenterology 2012;142:773–781.e2. doi: 10.1053/j. gastro.2011.12.035. References [26] Wiseman E, Fraser MA, Holden S, Glass A, Kidson BL, Heron LG, et al. Peri- natal transmission of hepatitis B virus: an Australian experience. Med J Aust [1] Hou J, Wang G, Wang F, Cheng J, Ren H, Zhuang H, et al. Guideline of pre- 2009;190:489–492. doi: 10.5694/j.1326-5377.2009.tb02524.x. vention and treatment for chronic hepatitis B (2015 Update). J Clin Transl [27] World Health Organization. Draft global health sector strategy on viral hep- Hepatol 2017;5:297–318. doi: 10.14218/JCTH.2016.00019. atitis, 2016-2021 – The first of its kind. Available from: http://apps.who. [2] Lamberth JR, Reddy SC, Pan JJ, Dasher KJ. Chronic hepatitis B infection in int/gb/ebwha/pdf_files/WHA69/A69_32-en.pdf?ua=1. pregnancy. World J Hepatol 2015;7:1233–1237. doi: 10.4254/wjh.v7.i9.1233. [28] Lu FM, Zhuang H. Prevention of hepatitis B in China: achievements and [3] Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and challenges. Chin Med J (Engl) 2009;122:2925–2927. doi: 10.3760/cma.j. current and emerging prevention and control measures. J Viral Hepat 2004; issn.0366-6999.2009.24.001. 11:97–107. doi: 10.1046/j.1365-2893.2003.00487.x. [29] Cui F, Luo H, Wang F, Zheng H, Gong X, Chen Y, et al. Evaluation of policies [4] Wiesen E, Diorditsa S, Li X. Progress towards hepatitis B prevention through and practices to prevent mother to child transmission of hepatitis B virus in vaccination in the Western Pacific, 1990-2014. Vaccine 2016;34:2855– China: results from China GAVI project final evaluation. Vaccine 2013;31 2862. doi: 10.1016/j.vaccine.2016.03.060. Suppl 9:J36–J42. doi: 10.1016/j.vaccine.2012.11.061.

8 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 1–8 Original Article

The Reliability of Fibro-test in Staging Orthotopic Liver Transplant Recipients with Recurrent Hepatitis C

Panagiotis Trilianos, Adamantios Tsangaris, Augustine Tawadros, Vrushak Deshpande and Nikolaos Pyrsopoulos*

Division of Gastroenterology & Hepatology, University Hospital, Rutgers - New Jersey Medical School, NJ, USA

Abstract be associated with an accelerated rate of fibrosis progression.1–3 The advent of direct-acting antivirals (DAAs) dramat- Background and Aims: Liver biopsy remains the gold stand- ically changed the landscape of HCV treatment, with safe and ard for staging of chronic liver disease following orthotopic liver effective regimens that have high cure rates leading to signifi- transplantation. Noninvasive assessment of fibrosis with Fibro- cantly improved outcomes in the post-OLT setting4,5 Still, test (FT) is well-studied in immunocompetent populations with treatment of recurrent hepatitis C (RHC) in liver transplant chronic hepatitis C virus infection. The aim of this study is to recipients may pose a challenge.6 As such, there remains the investigate the diagnostic value of FT in the assessment of necessity to periodically restage the relatively few patients hepatic fibrosis in the allografts of liver transplant recipients who either fail or are unable to tolerate DAA treatment. with evidence of recurrent hepatitis C. Methods: We retro- Liver biopsy (LB) is considered the gold standard for spectively compared liver biopsies and FT performed within a assessment of hepatic fibrosis, although imperfect. Sample median of 1 month of each other in orthotopic liver transplan- size and quality factor greatly into diagnostic accuracy,7 and tation recipients with recurrent hepatitis C. Results: The study there is considerable inter- and intra-observer variability in population comprised 22 patients, most of them male (19/22), the interpretation of histologic findings.8 As a procedure, it and with median age of 62 years. For all patients, there was at is infrequently associated with both major and minor compli- least a one-stage difference in fibrosis as assessed by liver cations but does carry some mortality risk.9,10 Protocol biop- biopsy compared to FT, while for the majority (16/22) there sies, traditionally employed in the post-transplant setting for was at least a two-stage difference. The absence of correlation surveillance of allograft integrity and function, are currently between the two modalities was statistically demonstrated largely abandoned.11 (Mann-Whitney U test, p = 0.01). In detecting significant fib- Noninvasive assessment of fibrosis through serum rosis (a METAVIR stage of F2 and above), an FT cut-off of 0.5 markers, various types of elastography, imaging or combina- showed moderate sensitivity (77%) and negative predictive tions thereof mitigates the procedural risks associated with value (80%), but suboptimal specificity (61%) and positive LB, making it a very attractive concept. Reliable noninvasive predictive value (58%). Conclusions: In post-transplant pa- testing is currently accessible to aid in the care of patients tients with recurrent hepatitis C, FT appears to be inaccurately with chronic HCV infection. Such is Fibro-test (FT), a propri- assessing the degree of allograft fibrosis, therefore limiting its etary algorithm comprising both direct and indirect markers reliability as a staging tool. of fibrosis, that has been well-studied and validated as a Citation of this article: Trilianos P, Tsangaris A, Tawadros A, diagnostic tool in immunocompetent patients with viral hep- Deshpande V, Pyrsopoulos N. The reliability of fibro-test in atitis among other common chronic liver diseases.12,13 staging orthotopic liver transplant recipients with recurrent This study was designed to evaluate FT in the assessment hepatitis C. J Clin Transl Hepatol 2020;8(1):9–12. doi: of hepatic fibrosis in liver transplant recipients with RHC. 10.14218/JCTH.2019.00038.

Methods Introduction Retrospective review of charts of adult OLT recipients followed Allograft re-infection by the hepatitis C virus (HCV) in in our institution between the years of 2003 and 2016 was treatment-naïve or unsuccessfully treated orthotopic liver conducted. Criteria for enrollment included patients trans- transplant (OLT) recipients is swift and universal and used to planted for end-stage liver disease due to chronic hepatitis C infection, with recurrence of viremia in the post-transplant Keywords: Fibro-test; Orthotopic liver transplantation; Fibrosis; Recurrent period associated with histologically typical hepatic necroin- hepatitis C; Hepatitis C virus. flammation on LB (obtained for staging purposes), in the Abbreviations: DAA, direct-acting antiviral; FT, Fibro-Test; HCV, hepatitis C virus; absence of a concurrent cause of liver injury, such as cellular LB, liver biopsy; OLT, orthotopic liver transplant; RHC, recurrent hepatitis C; ROC, receiver operating characteristic; VCTE, vibration-controlled transient elastography. rejection, hepato-vascular complications, biliary strictures, Received: 22 August 2019; Revised: 24 November 2019; Accepted: 24 December and de novo liver disease (e.g., nonalcoholic fatty liver 2019 disease or autoimmune hepatitis). Patients with moderate *Correspondence to: Nikolaos Pyrsopoulos, Division of Gastroenterology & Hep- hepatitis (alanine aminotransferase >200 IU/mL), significant atology, University Hospital, Rutgers - New Jersey Medical School, Medical Science Building, Room H-536, 185 S. Orange Ave, Newark, NJ 07103, USA. Tel: +1-973- cholestasis (alkaline phosphatase or gamma-glutamyl trans- 972-5252, Fax: +1-973-972-3144, E-mail: [email protected] ferase >1.5x the upper limit of normal), decompensated liver

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 9–12 9

METAVIR classification system. The size of the histologic speci- Sensitivity 0.4 men was documented. Fibro Test was acquired through the commercially available assay (FibroSURE; LabCorp, Burlington, NC,USA),whichcomprisesa2-macroglobulin, haptoglobin, apo-lipoprotein A1, total bilirubin, and gamma-glutamyl trans- 0.2 ferase, while the addition of alanine aminotransferase offers a surrogate marker of hepatic necroinflammation (Acti-Test).14 Non-parametric testing (Mann-Whitney U test) was implemented to compare LB to FT results. The sensitivity, specificity, 0.0 and positive and negative predictive values of FT for any or 0.0 0.2 0.4 0.6 0.8 1.0 significant fibrosis (as defined by a METAVIR score of F2 and 1 - Specificity above) were calculated. The diagnostic value of FT for the Fig. 1. Diagnostic accuracy of FT as determined by area under the curve detection of significant fibrosis was further assessed by plotting (AUROC = 0.647). the receiver operating curve (ROC). All statistical analyses were performed with SPSS version 24 (IBM Corp, Armonk, NY, USA). within 1 year and cirrhosis developing in up to 40% of patients – Results within 5 years.1 3 There is no denying that the use of DAAs in liver recipients with RHC or even kidney recipients with de A total of 22 patients met the study criteria, including 19 men novo HCV infection has largely mitigated this problem, ena- and 3 women, mostly Caucasian (54%), with median age of bling timely, safe and cost-effective viral eradication.4,5,15 62 years. Infection with HCV genotype 1 was most prevalent However, data on the utilization of DAAs post-transplant are (18/22, 81.8%). The most frequently used immunosuppres- limited when compared to the abundance of large and small, sive agents were mycophenolate mofetil (82%) and tacroli- prospective and retrospective studies in immunocompetent mus (77%), often in combination (64%). Median time patients. The introduction of all-oral, interferon-free DAAs elapsed from OLT to LB was 45 months, while median into routine clinical practice is relatively recent (sofosbuvir/ chronological distance of FT from LB was 1 month. Median ledipasvir attained Federal Drug Administration approval in length of biopsy specimens acquired was 15 mm. More than October of 2014, with others following suit), and as such one-third of the study population had no histologic evidence there is paucity of robust data on the SVR rates in DAA- and of fibrosis (9/22, 41%). Mild fibrosis was seen in 8 patients more specifically the NS5A-experienced OLT recipients with (18%), moderate fibrosis in 8 (36%), and 1 patient had RHC. This is reflected in large clinical trials, where participants advanced fibrosis (5%). No patient was cirrhotic per LB. were either treatment-naïve or had received interferon- or There was at least a one-stage difference in fibrosis stage sofosbuvir-based regimens.16–20 The majority were also gen- as assessed by LB versus that of FT in all patients; a two-stage otype-1 infected.15–17,20 The issues of drug-drug interactions, difference was noted in 16 patients (73%), and in 4 patients LB varying degrees of concomitant renal insufficiency, and and FT were grossly discrepant. Mann-Whitney U test further rarely acute rejection also come into consideration in the confirmed the discrepancy between modalities (p = 0.01). In post-transplant setting, making the treatment of RHC more detecting any fibrosis (METAVIR F1 and above), FT was 100% challenging.6,17,21 What is more, sustained virological response sensitive. In detecting significant fibrosis (METAVIR F2 and rates appear to be lower in simultaneous liver-kidney trans- above), an FT cut-off of 0.5 showed a sensitivity of 77%, a plant recipients.17 As such, until a universally accepted, specificity of 61%, a positive predictive value of 58% and neg- streamlined, 100% effective and caveat-free regimen is found ative predictive value of 80%. A ROC curve was plotted, to treat RHC in liver recipients, there will be a need to monitor reflecting the overall performance (Fig. 1). As we only had disease progression in those few who fail or do not tolerate our one patient with biopsy-proven advanced fibrosis and none current options. with cirrhosis, we deferred measuring the respective values Liver biopsy is still accepted as the definitive method to for detecting advanced stages of liver disease. stage liver disease in the pre- and post-OLT setting, despite its well-documented shortcomings.7,9,10 Protocol biopsies are no Discussion longer the norm, although they do have proponents.11 Non- invasive assessment of fibrosis through (direct or indirect) Historical evidence demonstrates that the course of hepatitis serum markers or elastographic techniques (with or without C tends to be more accelerated following liver transplantation, concurrent imaging) has been gaining traction over the last with histologic hepatitis found in the majority of allografts 15 years. Simple, point-of-care clinical tools have been

10 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 9–12 Trilianos P. et al: Fibro-test in HCV post-liver transplant extensively studied and validated in viral hepatitis.22,23 grossly overestimated its degree. We applied strict selection Among proprietary tests, the Fibro Test (BioPredictive Paris, criteria to reasonably exclude patients with co-existing non- France) is now widely used in clinical practice as part of the HCV-related liver injury or severe hepatic necroinflammation, pretreatment evaluation of HCV infection.12,13,24,25 Moreover, either of which would confound the interpretation of FT. By the FT is well-validated in immunocompetent HCV-infected same token, the risk of selection bias is acknowledged. The patients,13,26 but the data in the post-transplant population mean chronological distance between FT and biopsy was 1.5 are decidedly mixed. months, making their acquisition practically concurrent and The largest study to research the utility of FT in the post- therefore eliminating the possibility of any meaningful fibrosis transplant setting was the one by Beckebaum et al.,27 com- progression in the time elapsed from one to the other. Fibro prising a population of 157 OLT recipients, a third of whom Test was also obtained either prior to or well after antiviral had RHC. In the HCV group, FT performed best, yielding an treatment, as improvement of biochemical parameters during area under the ROC curve of 0.79 for the diagnosis of treatment could also potentially affect its reliability. The small advanced fibrosis and 0.81 for cirrhosis, and it was less accu- number of patients is our foremost limitation. Among them, rate in the non-HCV group, with area under the ROC curve only one had biopsy-proven advanced fibrosis and none had values of 0.70 and 0.75 respectively. Of note, the diagnostic cirrhosis. Therefore, the utility of FT in detecting advanced value of FT in detecting moderate necroinflammation (grade stages of liver disease in post-transplant patients with RHC A2 and above) in patients with RHC appeared to be very could not be assessed in the present study. limited, with an area under the ROC curve of only 0.60. In contrast, vibration-controlled transient elastography (VCTE) Conclusions was found to be the most accurate noninvasive test in establishing advanced fibrosis and cirrhosis in both HCV and non- Contrary to immunocompetent HCV-infected patients, FT HCV OLT recipients. appears to significantly over-stage OLT recipients with RHC Earlier, the Bologna Liver Transplantation Group28 had also and therefore its diagnostic value as a non-invasive test for the evaluated the utility serologic markers (among them, FT) and assessment of fibrosis is questionable. This may be explained VCTE in detecting significant fibrosis (defined as a METAVIR by the reduction of apo-lipoprotein A1 levels, a component of score of F2 and above) in patients with RHC following liver FT, by calcineurin inhibitors.32,33 Similar to the findings transplantation. FT performed extremely poorly in this in immunocompetent patients, elastography appears to be – setting, with a cut-off of 0.8 yielding a sensitivity of 56% superior,28 30,34,35 though a universally accepted noninvasive and a specificity of 61%, and an overall area under the ROC strategy to detect hepatic fibrosis in the post-transplant setting curve of 0.56. Elastography performed excellently and was has yet to be established. found to be superior to other noninvasive testing procedures (area under the ROC curve of 0.94). Notably, parameters to Funding calculate the FT score were available for only a subgroup of the total population (36 out of 56 patients) and only a minor- None to declare. ity of those had advanced fibrosis or cirrhosis (n = 5); therefore, conclusions on the diagnostic value of FT in detecting late stages of fibrosis cannot be drawn from this study. Conflict of interest The accuracy of FT has also been investigated in the 29 setting of renal transplantation. In a 2009 French study Nikolaos Pyrsopoulos is a recipient of research grants from including 26 HCV-infected kidney recipients, FT correctly clas- Gilead, Abbvie, Merck and Roche. The other authors have no sified 80% of patients with no or mild fibrosis (F0-F1), per- conflict of interests related to this publication. forming similarly to transient elastography. However, the sensitivity and accuracy of FT in detecting advanced fibrosis or cirrhosis were rather dismal (sensitivity of ;31%, area Author contributions under the ROC curve of 0.55). A 2010 meta-analysis by Cholongitas et al.30 found VCTE to Study concept and design (NP, VD), data collection (AdT, be superior to serologic markers, including FT, in the assess- AuT), analysis and interpretation of data (PT, AdT), drafting of ment of significant fibrosis and cirrhosis in OLT recipients with manuscript (PT), critical revision, study supervision (NP). RHC; although, it should be noted that only one study28 evaluating FT was included. Most recently, the ability of FT to dis- References criminate between the presence and absence of significant fibrosis (as defined by an Ishak score of >3) was investigated [1] Gane E. The natural history and outcome of liver transplantation in hepatitis in an Italian study including 51 liver recipients with RHC;31 aFT C virus-infected recipients. Liver Transpl 2003;9:S28–S34. doi: 10. cut-off value of 0.716 had a positive predictive value of 58% 1053/jlts.2003.50248. [2] Forman LM, Lewis JD, Berlin JA, Feldman HI, Lucey MR. The association but a negative predictive value of almost 94% for significant between hepatitis C infection and survival after orthotopic liver transplanta- fibrosis, while the overall accuracy of FT (area under the ROC tion. Gastroenterology 2002;122:889–896. doi: 10.1053/gast.2002.32418. curve of 0.848) was second only to acoustic radiation forced [3] Berenguer M, Prieto M, Rayón JM, Mora J, Pastor M, Ortiz V, et al. Natural imaging and superior to all other chemistry-based indices. history of clinically compensated hepatitis C virus-related graft cirrhosis after liver transplantation. Hepatology 2000;32:852–858. doi: 10.1053/jhep. Combination of both acoustic radiation forced imaging and FT 2000.17924. appeared to offer no additional benefit. [4] Cotter TG, Paul S, Sandıkçı B, Couri T, Bodzin AS, Little EC, et al. Improved Our investigation reached a negative conclusion regarding graft survival after liver transplantation for recipients with hepatitis C virus in – the utility of FT in liver recipients with RHC. In a cohort of the direct-acting antiviral era. Liver Transpl 2019;25:598 609. doi: 10. 1002/lt.25424. 22 OLT recipients with RHC, FT was able to detect the [5] Belli LS, Perricone G, Adam R, Cortesi PA, Strazzabosco M, Facchetti R, et al. presence of any fibrosis but consistently and oftentimes Impact of DAAs on liver transplantation: Major effects on the evolution of

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 9–12 11 Trilianos P. et al: Fibro-test in HCV post-liver transplant

indications and results. An ELITA study based on the ELTR registry. J Hepatol [21] Smolders EJ, Jansen AME, Ter Horst PGJ, Rockstroh J, Back DJ, Burger DM. 2018;69:810–817. doi: 10.1016/j.jhep.2018.06.010. Viral hepatitis C therapy: Pharmacokinetic and pharmacodynamic consider- [6] Saab S, Rheem J, Jimenez M, Bau S, Choi G, Durazo F, et al. Curing hepatitis C ations: A 2019 update. Clin Pharmacokinet 2019;58:1237–1263. doi: 10. in liver transplant recipients is associated with changes in immunosuppressant 1007/s40262-019-00774-0. use. J Clin Transl Hepatol 2016;4:32–38. doi: 10.14218/JCTH.2016.00001. [22] Chou R, Wasson N. Blood tests to diagnose fibrosis or cirrhosis in patients [7] Bedossa P, Dargère D, Paradis V. Sampling variability of liver fibrosis in with chronic hepatitis C virus infection: a systematic review. Ann Intern Med chronic hepatitis C. Hepatology 2003;38:1449–1457. doi: 10.1016/j.hep. 2013;158:807–820. doi: 10.7326/0003-4819-158-11-201306040-00005. 2003.09.022. [23] Kim BK, Kim DY, Park JY, Ahn SH, Chon CY, Kim JK, et al. Validation of FIB-4 [8] Poynard T, Munteanu M, Imbert-Bismut F, Charlotte F, Thabut D, Le Calvez S, and comparison with other simple noninvasive indices for predicting liver et al. Prospective analysis of discordant results between biochemical markers fibrosis and cirrhosis in hepatitis B virus-infected patients. Liver Int 2010; and biopsy in patients with chronic hepatitis C. Clin Chem 2004;50:1344– 30:546–553. doi: 10.1111/j.1478-3231.2009.02192.x. 1355. doi: 10.1373/clinchem.2004.032227. [24] Poynard T, Imbert-Bismut F, Munteanu M, Messous D, Myers RP, Thabut D, [9] Piccinino F, Sagnelli E, Pasquale G, Giusti G. Complications following percuta- et al. Overview of the diagnostic value of biochemical markers of liver fibrosis neous liver biopsy. A multicentre retrospective study on 68,276 biopsies. J (FibroTest, HCV FibroSure) and necrosis (ActiTest) in patients with chronic Hepatol 1986;2:165–173. doi: 10.1016/s0168-8278(86)80075-7. hepatitis C. Comp Hepatol 2004;3:8. doi: 10.1186/1476-5926-3-8. [10] Seeff LB, Everson GT, Morgan TR, Curto TM, Lee WM, Ghany MG, et al. Com- [25] Munteanu M, Tiniakos D, Anstee Q, Charlotte F, Marchesini G, Bugianesi E, plication rate of percutaneous liver biopsies among persons with advanced et al. Diagnostic performance of FibroTest, SteatoTest and ActiTest in chronic liver disease in the HALT-C trial. Clin Gastroenterol Hepatol 2010;8: patients with NAFLD using the SAF score as histological reference. Aliment – 877–883. doi: 10.1016/j.cgh.2010.03.025. Pharmacol Ther 2016;44:877 889. doi: 10.1111/apt.13770. [11] Mells G, Mann C, Hubscher S, Neuberger J. Late protocol liver biopsies in the [26] Zarski JP, Sturm N, Guechot J, Paris A, Zafrani ES, Asselah T, et al. Compar- liver allograft: a neglected investigation? Liver Transpl 2009;15:931–938. ison of nine blood tests and transient elastography for liver fibrosis in chronic – doi: 10.1002/lt.21781. hepatitis C: the ANRS HCEP-23 study. J Hepatol 2012;56:55 62. doi: 10. [12] Poynard T, Morra R, Halfon P, Castera L, Ratziu V, Imbert-Bismut F, et al. 1016/j.jhep.2011.05.024. [27] Beckebaum S, Iacob S, Klein CG, Dechêne A, Varghese J, Baba HA, et al. Meta-analyses of FibroTest diagnostic value in chronic liver disease. BMC Assessment of allograft fibrosis by transient elastography and noninvasive Gastroenterol 2007;7:40. doi: 10.1186/1471-230X-7-40. biomarker scoring systems in liver transplant patients. Transplantation [13] Poynard T, Munteanu M, Deckmyn O, Ngo Y, Drane F, Castille JM, et al.Val- 2010;89:983–993. doi: 10.1097/TP.0b013e3181cc66ca. idation of liver fibrosis biomarker (FibroTest) for assessing liver fibrosis pro- [28] Corradi F, Piscaglia F, Flori S, D’Errico-Grigioni A, Vasuri F, Tamé MR, et al. gression: proof of concept and first application in a large population. J Assessment of liver fibrosis in transplant recipients with recurrent HCV infec- Hepatol 2012;57:541–548. doi: 10.1016/j.jhep.2012.04.025. tion: usefulness of transient elastography. Dig Liver Dis 2009;41:217–225. [14] Poynard T, Munteanu M, Ngo Y, Castera L, Halfon P, Ratziu V, et al. ActiTest doi: 10.1016/j.dld.2008.06.009. accuracy for the assessment of histological activity grades in patients with [29] Alric L, Kamar N, Bonnet D, Danjoux M, Abravanel F, Lauwers-Cances V, et al. chronic hepatitis C, an overview using Obuchowski measure. Gastroenterol Comparison of liver stiffness, fibrotest and liver biopsy for assessment of Clin Biol 2010;34:388–396. doi: 10.1016/j.gcb.2010.05.001. liver fibrosis in kidney-transplant patients with chronic viral hepatitis. [15] Axelrod DA, Schnitzler MA, Alhamad T, Gordon F, Bloom RD, Hess GP, et al. Transpl Int 2009;22:568–573. doi: 10.1111/j.1432-2277.2009.00834.x. The impact of direct-acting antiviral agents on liver and kidney transplant [30] Cholongitas E, Tsochatzis E, Goulis J, Burroughs AK. Noninvasive tests for costs and outcomes. Am J Transplant 2018;18:2473–2482. doi: 10. evaluation of fibrosis in HCV recurrence after liver transplantation: a system- 1111/ajt.14895. atic review. Transpl Int 2010;23:861–870. doi: 10.1111/j.1432-2277.2010. [16] Charlton M, Everson GT, Flamm SL, Kumar P, Landis C, Brown RS Jr, et al. 01142.x. Ledipasvir and sofosbuvir plus ribavirin for treatment of HCV infection in [31] Bignulin S, Falleti E, Cmet S, Cappello D, Cussigh A, Lenisa I, et al. Useful- – patients with advanced liver disease. Gastroenterology 2015;149:649 ness of acoustic radiation force impulse and fibrotest in liver fibrosis assess- 659. doi: 10.1053/j.gastro.2015.05.010. ment after liver transplant. Ann Hepatol 2016;15:200–206. doi: 10. [17] Saxena V, Khungar V, Verna EC, Levitsky J, Brown RS Jr, Hassan MA, et al. 5604/16652681.1193710. Safety and efficacy of current direct-acting antiviral regimens in kidney and [32] Chakkera HA, Mandarino LJ. Calcineurin inhibition and new-onset diabetes liver transplant recipients with hepatitis C: Results from the HCV-TARGET mellitus after transplantation. Transplantation 2013;95:647–652. doi: 10. study. Hepatology 2017;66:1090–1101. doi: 10.1002/hep.29258. 1097/TP.0b013e31826e592e. [18] Coilly A, Fougerou-Leurent C, de Ledinghen V, Houssel-Debry P, Duvoux C, Di [33] Chakkera HA, Kudva Y, Kaplan B. Calcineurin inhibitors: Pharmacologic Martino V, et al. Multicentre experience using daclatasvir and sofosbuvir to mechanisms impacting both insulin resistance and insulin secretion leading treat hepatitis C recurrence - The ANRS CUPILT study. J Hepatol 2016;65: to glucose dysregulation and diabetes mellitus. Clin Pharmacol Ther 2017; 711–718. doi: 10.1016/j.jhep.2016.05.039. 101:114–120. doi: 10.1002/cpt.546. [19] Reau N, Kwo PY, Rhee S, Brown RS Jr, Agarwal K, Angus P, et al. Glecapre- [34] Bhat M, Tazari M, Sebastiani G. Performance of transient elastography and vir/pibrentasvir treatment in liver or kidney transplant patients with hepatitis serum fibrosis biomarkers for non-invasive evaluation of recurrent fibrosis C virus infection. Hepatology 2018;68:1298–1307. doi: 10.1002/hep. after liver transplantation: A meta-analysis. PLoS One 2017;12:e0185192. 30046. doi: 10.1371/journal.pone.0185192. [20] Poordad F, Schiff ER, Vierling JM, Landis C, Fontana RJ, Yang R, et al. Dacla- [35] Carrión JA, Navasa M, Bosch J, Bruguera M, Gilabert R, Forns X. Transient tasvir with sofosbuvir and ribavirin for hepatitis C virus infection with elastography for diagnosis of advanced fibrosis and portal hypertension in advanced cirrhosis or post-liver transplantation recurrence. Hepatology patients with hepatitis C recurrence after liver transplantation. Liver Transpl 2016;63:1493–1505. doi: 10.1002/hep.28446. 2006;12:1791–1798. doi: 10.1002/lt.20857.

12 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 9–12 Review Article

Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease

Jie Li1 and Jian-Gao Fan*2,3

1Department of Infectious Disease, Shandong Provincial Hospital Affiliated to Shandong University, Ji’nan, Shandong, China; 2Department of Gastroenterology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; 3Shanghai Key Lab of Pediatric Gastroenterology and Nutrition, Shanghai, China

Abstract The global outbreak of this disease is significantly bigger than the prior pandemic of SARS and Middle East respiratory An outbreak of severe acute respiratory syndrome coronavi- syndrome (MERS). Within 3 months, SARS-CoV-2 spread rus 2 (SARS-CoV-2) infection (2019 coronavirus disease, rapidly from Wuhan city to the entire country of China, and COVID-19) since December 2019, from Wuhan, China, has then into more than 120 countries worldwide. Up to March 14, been posing a significant threat to global human health. The 2020, a total of 81,021 cases of COVID-19 with 3194 deaths clinical features and outcomes of Chinese patients with in China and 63,016 cases with 2212 deaths outside of China COVID-19 have been widely reported. Increasing evidence have been confirmed. Unfortunately, the global numbers of has witnessed the frequent incident liver injury in COVID-19 both infected patients and fatalities will continue to grow in patients, and it is often manifested as transient elevation of the future months because no specific effective antiviral serum aminotransferases; however, the patients seldom have therapies nor vaccine have been identified.4 liver failure and obvious intrahepatic cholestasis, unless pre- With the number of cases increasing in China, abnormal existing advanced liver disease was present. The underlying liver function test results have been observed in some mechanisms of liver injury in cases of COVID-19 might patients with COVID-19, making this organ the most fre- include psychological stress, systemic inflammation re- quently damaged outside of the respiratory system.5–7 sponse, drug toxicity, and progression of pre-existing liver However, the clinical characteristics and outcomes of diseases. However, there is insufficient evidence for SARS- Chinese patients with COVID-19 might be changing with CoV-2 infected hepatocytes or virus-related liver injury in time, and the clinical-pathological manifestations and mech- COVID-19 at present. The clinical, pathological and labora- anism of the liver injury in COVID-19 remain largely unclear.8 tory characteristics as well as underlying pathophysiology and This review will summarize these important issues based etiology of liver injury in COVID-19 remain largely unclear. In on the recent developments in the field, for optimizing the this review, we highlight these important issues based on the management and treatment of COVID-19 patients with liver recent developments in the field, for optimizing the manage- injury. ment and treatment of liver injury in Chinese patients with COVID-19. Citation of this article: Li J, Fan JG. Characteristics and mech- General clinical characteristics and outcomes of anism of liver injury in 2019 coronavirus disease. J Clin Transl Chinese patients with COVID-19 Hepatol 2020;8(1):13–17. doi: 10.14218/JCTH.2020.00019. The clinical features of COVID-19 are similar to SARS and MERS, with typical manifestation of pneumonia and acute Introduction respiratory infection symptoms (Fig. 1). Most patients with SARS-CoV-2 infection usually have mild symptoms and In December 2019, a novel coronavirus (2019-nCoV), named good prognosis, with the exception of those involving patients 9 as severe acute respiratory syndrome coronavirus 2 (SARS- with older age and underlying health conditions. Guan et al. CoV-2), was discovered in Wuhan, Hubei Province, China.1–3 extracted data regarding 1099 patients with laboratory-con- The SARS-CoV-2 infection and related coronavirus disease firmed COVID-19 from 552 hospitals in mainland China 2019 (COVID-19), has aroused great concern worldwide. through January 29, 2020. They reported that the median age of the cases was 47 years, and 41.9% of the patient

Keywords: SARS-CoV-2; COVID-19; Liver injury; Clinical characteristics; population was female. The most common symptoms were Mechanism. fever (88.7%) and cough (67.8%). However, nausea or vom- Abbreviations: 2019-nCoV, 2019 novel coronavirus; ACE2, angiotensin convert- iting (5.0%) and diarrhea (3.8%) were present but uncom- ing enzyme 2; ALT, alanine aminotransferase; ARDS, acute respiratory distress mon. The median incubation period was 4 days, and 23.7% syndrome; AST, aspartate aminotransferase; CFR, case-fatality rate; COVID-19, 2019 coronavirus disease; ICU, intensive care unit; MERS, Middle East respiratory cases had at least one coexisting comorbid condition. Some syndrome; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. patients rapidly developed severe pneumonia, pulmonary Received: 9 March 2020; Revised: 13 March 2020; Accepted: 15 March 2020 edema, acute respiratory distress syndrome (ARDS), acute *Correspondence to: Jian-Gao Fan, Department of Gastroenterology, Xin Hua respiratory failure, or multiple organ failure. The primary Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai Key Lab of Pediatric Gastroenterology and Nutrition, Shanghai 200092, China. Tel: composite end-point occurred in 67 cases (6.1%), including + 86-21-2507-7340, E-mail: [email protected] 5.0% patients admitted to the intensive care unit (ICU), 2.3%

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 13–17 13

ratio of <300. Severe hypoxemia and acute respiratory failure usually occurred in the critical cases. Furthermore, the severe or critical patients still had laboratory characteristic findings of septic shock and multiple organ dysfunction or failure, such as liver injury, renal injury, and heart injury (Fig. 2). Abnormal liver enzymes in patients with COVID-19 was first reported by Chen et al.2 Among 99 cases with COVID-19 from Wuhan, 43 cases (43.4%) had increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactic dehydrogenase. Most of them had mild elevation of aminotransferase, and only one case had very high levels of aminotransferases (ALT of 7590 U/L and AST of 1445 U/L). However, no case was reported with the features of obvious intrahepatic cholestasis or liver failure. Holshue et al.12 reported the first Chinese case of COVID-19 confirmed in the USA with detailed information of liver function tests. The patient was admitted to hospital on day 4 of illness and progressed to pneumonia on day 9 of illness, and his serum levels of ALT increased from68to203U/LandASTincreasedfrom37to89U/L.In another report, among 12 severe patients with COVID-19 from Fig. 1. Clinical symptoms of patients with 2019 coronavirus disease. Shenzhen, only 1 had abnormal liver enzymes (ALT of 107 U/L and AST of 62 U/L).13 In 62 patients with COVID-19 from Zhe- patients who underwent invasive mechanical ventilation, and jiang, 16.1% patients had elevation of AST ($40 U/L) and the 1.4% patients who died. mean ALT level was 22 U/L.14 Among 305 patients with COVID- Recently, the Chinese Center for Disease Control and 19 from Wuhan, 39.1% (119/304) cases had elevation of ALT, Prevention published the largest case series to date of AST, or bilirubin at admission. Of them, 24 cases had increased COVID-19 in mainland China. Among 72,314 case records ALT ($80 U/L) and 19 had increased AST ($80 U/L), while only (updated through February11, 2020), 44,672 (62%) were 6 had increased bilirubin level.15 classified as confirmed cases of COVID-19, 16,186 (22%) as Recently, increasing evidence has highlighted the close suspected cases, 10,567 (15%) as clinically-diagnosed relationship of abnormal liver biochemistries with severity of cases, and 889 (1%) as asymptomatic cases. In total, 87% COVID-19. In the cohort of 1099 patients with COVID-19 of the cases represented ages 30 to 79 years-old. Among from mainland China, 39.4% had AST >40 U/L and 28.1% 44,415 confirmed cases, 14% cases were classified as severe had ALT >40 U/L, and most of them occurred in severe and and 5% as critical. There were 1023 deaths (2.3%) among critical cases.9 The mean levels of serum ALT, AST, and bilir- the 44,672 confirmed cases, all having involved critical cases. ubin in severe or critical cases were significantly higher than The case-fatality rate (CFR) was 8.0% in those aged 70 to 79 those in control cases in another multicenter retrospective years and 14.8% in those aged 80 years and older. The CFR study including 32 patients.16 Serum levels of ALT and AST was elevated among those with underlying diseases (10.5% in severe to critical cases were significantly higher than for cardiovascular disease, 7.3% for mellitus diabetes, 6.3% those in mild to moderate cases among 265 patients with for chronic respiratory disease, 6.0% for hypertension, and 17 5.6% for cancer).10 However, the information regarding coex- COVID-19 from Shanghai. Similarly, patients admitted to the ICU were more likely to have high levels of serum ALT, isting liver disease and liver-related morbidity and mortality 18,19 were not available in either article. AST, and total bilirubin. Furthermore, in a systematic review of 3470 patients with COVID-19, 11.5% were admitted to ICU, while the overall CFR was 3.7%. Compared to patients admitted outside of Hubei, China, those from Hubei had a significantly higher ICU admission rate (21.9% vs. 2.5%). Also, CFR attributed to COVID-19 in Hubei was significantly higher than that of non- Hubei admissions (10.4% vs. 0.6%).11

Characteristics of liver injury in Chinese patients with COVID-19

Laboratory changes of liver function test in patients with COVID-19

According to the diagnostic criteria, all hospitalized cases of COVID-19 were laboratory confirmed as SARS-CoV-2 infection by nucleic acid testing of respiratory tract samples. On admission, most of the patients with COVID-19 had lymphocytopenia, leukopenia, and elevated levels of C-reactive protein. Severe patients usually had blood oxygen saturation of <93%, and Fig. 2. Complications and disease mechanisms in patients with 2019 co- partial pressure of arterial oxygen to fraction of inspired oxygen ronavirus infection.

14 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 13–17 Li J. et al: Liver injury in COVID-19

In 52 critically ill patients with multiple organ damage, 35 liver injury is closely associated with corona virus infection. (67%) had ARDS, 15 (29%) had acute kidney injury, 15 But it’s unclear whether the liver injury can be caused directly (29%) had liver injury, and 12 (23%) had cardiac injury.20 by the coronavirus itself. It is well known that SARS-CoV-2 is Among 298 patients with COVID-19 from Shenzhen, the per- closely related to SARS-CoV, and they share the same recep- centage of liver injury in the severe group was substantially tor, angiotensin converting enzyme 2 (ACE2), and lung is the higher than that in the non-severe group.21 However, signifi- main target organ of the corona virus infection. Previous RNA- cant differences of serum levels of ALT, AST and total bilirubin seq data in the human protein atlas database demonstrates between patients with and without ARDS were not found in a expression of ACE2 in the liver.28 However, only a low fre- small sample cohort study from Wuhan.22 In addition, abnor- quency of ACE2 expression is observed in cholangiocytes, mal liver function test in cases of COVID-19 is often transient but not in hepatocytes, Kupffer cells, and endothelial cells.29 and often simultaneously combined with increased enzymes Recently, the single-cell RNA-seq data from two independent from muscle and heart; these laboratory changes can return cohorts suggest cholangiocyte-specific expression of ACE2 in to normal without liver-related morbidity and mortality. human liver samples. Similarly, single cell sequencing and immunohistochemistry study showed that ACE2 was only Pathological changes of liver in Chinese patients who expressed in bile duct epithelial cells of normal liver tissues, died from COVID-19 and very minimally in hepatocytes. In a mouse model of acute liver injury with partial The reported point CFR of COVID-19 patients in mainland hepatectomy, ACE2 expression in the liver was down-regu- China has varied widely, mainly based on the geographic lated on the first day, but it was elevated up to twice of the locality and the observation time. To date over 3100 patients normal level on the third day and returned to normal level on have died from COVID-19 in mainland China, all of them seventh day when the liver recovered and hepatocyte pro- belonging to the critical cases. However, only several cases liferation stopped. The experimental results implied the up- have undergone autopsies or post-mortem tissue biopsies. regulation of ACE2 expression in the liver was caused by Xu et al.23 first reported the pathological characteristics of compensatory proliferation of hepatocytes derived from bile post-mortem biopsy specimens from a 50-year-old man duct epithelial cells during acute liver injury.30,31 Considering who died from critical COVID-19. The results showed moder- the COVID-19 patients with liver injury mainly manifested as ate microvascular steatosis and mild lobular and portal activ- elevation of serum aminotransferases but alkaline phospha- ity in the liver. Liu et al.24 performed several cases of tase, SARS-CoV-2 might or might not affect the cholangio- autopsies in Wuhan, China, and concluded that the histolog- cytes through ACE2 damage to the hepatocytes. Liver injury ical characteristics of COVID-19 focuses on the lungs and in COVID-19 may thus have alternative pathophysiology and without sufficient evidence for other organ obvious injuries. etiology. The autopsy results of the liver include hepatomegaly with dark red, hepatocyte degeneration accompanied by lobular Stress and systemic inflammation-related liver injury focal necrosis and neutrophil infiltration, infiltration of lym- in COVID-19 phocytes and monocytes in the portal area, and congestion of hepatic sinuses with microthrombosis. However, neither Under physiological conditions, the liver is the important histological features of liver failure nor bile duct injuries organ that meets and filters a large amount of alien material, have been observed in these deceased cases. and then maintains immune tolerance through gut-liver axis. However, immune tolerance is interrupted under psycholog- Pathogenesis and etiology of liver injury in patients ical stress conditions in patients with severe COVID-19. with COVID-19 Hyperactivated immune responses and cytokine storm- related systemic inflammation in SARS-CoV-2 infection can Systemic inflammatory responses and pulmonary injury associ- affect and damage many organs, including the gut and liver. ated with coronaviruses are triggered by the innate and adaptive Peripheral blood levels of Th17 and CD8 T cells, interleukin-2, immune system. Expression of SARS-CoV S protein in animals is interleukin-6, interleukin-7, interleukin-10, tumor necrosis associated with strong inflammatory reaction and enhanced factor-a, granulocyte-colony stimulating factor, interferon- hepatitis. During SARS-CoV infection, host factors trigger an inducible protein-10, monocyte chemotactic protein 1, mac- immune response, which can inhibit virus replication, promote rophage inflammatory protein 1 alpha in severe patients were virus clearance, and trigger a prolonged adaptive immune significantly higher than those in control patients.18,21,22,32,33 response against the viruses. In SARS-CoV-infected patients, In the same way, abnormal liver biochemistries have also CD4+ T cells activate B cells then promote the production of virus- occurred often in severe COVID-19 cases. Stress-induced specific antibodies. A large amount of CD8+ T cells can be found in liver injury might be associated with hypoxia-reoxygenation, the pulmonary interstitium, which play a vital role in clearing the over-activation of Kupffer cells and oxidative stress, intestinal coronaviruses in infected cells and inducing immune injury.25 endotoxemia, and activation of the sympathetic nervous and However, it is important to note that an immune response out adrenocortical system in COVID-19 patients. of control can induce immunopathogenesis, which may result in Sepsis is not uncommon in severe and critical COVID-19 lung tissue damage and functional impairment in COVID-19 cases, especially in patients with intestinal microflora imbal- patients. However, reports about the underlying immune mecha- ance and existing liver cirrhosis.34 Sepsis is a dysregulated nism of liver injury in COVID-19 are still limited. immune response to an infection that leads to psychological stress and multiple organ dysfunction.35 The pathophysiology Coronavirus-related liver injury in COVID-19 of sepsis-related liver injury includes hypoxic liver injury due to ischemia and shock, cholestasis due to altered bile metab- Abnormal liver biochemistries have also been reported in olism, hepatocellular injury due to drug toxicity or over- patients with SARS and MERS,26,27 implying that potential whelming inflammation.36 Hence, sepsis in COVID-19

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 13–17 15 Li J. et al: Liver injury in COVID-19 patients might be one of the etiologies of liver injury and sub- present. Therefore, the pathophysiology and implication of stantially impairs the prognosis of COVID-19. Furthermore, liver injury have not yet been fully determined in COVID-19, severe hypoxia and hypovolemia are the major cause of as reports about liver injury as well as underlying mecha- ischemic/hypoxic liver injury in COVID-19 cases with acute nisms are limited. lung failure and/or shock. Ischemic/hypoxic liver injury is Being that liver is the most frequently affected outside of associated with metabolic acidosis, calcium overloading, and the respiratory system in COVID-19, more intensive surveil- changes of mitochondrial membrane permeability, and has lance or individually tailored therapeutic approaches is thus far usually manifested as very high aminotransferase needed for severe cases, especially among those with pre- concentrations in serum.37 existing advanced liver disease. Mechanistic understanding of the relationship of SARS-CoV-2 infection with liver injury is Drug-induced liver injury or existing liver disease in important for the clinical practice of managing COVID-19 COVID-19 patients with hepatic injury. Further study should focus on the mechanism of incident liver injury in COVID-19 and the effect Moderate microvascular steatosis with mild hepatic inflam- of underlying liver disease on treatment and outcome of mation in a COVID-19 patient indicates the possibility of drug- COVID-19 in the future. induced liver injury.23 In clinical practice, a large number of patients had history of using antipyretic drugs for treatment Funding of fever.38 Most antipyretic drugs contain paracetamol, which is generally recognized as a common reason for liver injury.39 Dr. Jie Li wishes to acknowledge support from the National In addition, many patients with COVID-19 have history of Science and Technology Major Project of China (2018ZX simultaneous use of multiple antiviral drugs, i.e. oseltamivir, 10302206-001-006), National Natural Science Foundation abidol, and lopinavir/ritonavir.40,41 These antiviral drugs can of China (81970545), Shandong Province Natural Science induce liver injury as well. A clinical trial of lopinavir/ritonavir Foundation (ZR2017MH102), and Shandong Province Key or abidol for treatment of COVID-19 from Shanghai showed Research and Development Project (2019GSF108145). Dr. that two cases of each group occurred liver injury among two- Jian-Gao Fan wishes to acknowledge support from the hundred and sixty-two patients.42 Thus, if abnormality of liver National Key R&D Program (2017YFSF090203), National enzymes occurs after using a hepatotoxic drug, drug-induced Natural Science Foundation of China (81873565), Shanghai liver injury should first be confirmed or ruled out. In addition, Leading Talent Plan 2017, and Innovative Research Team of given the high burden of chronic liver disease in China, non- High-Level Local Universities in Shanghai. alcoholic fatty liver disease, chronic hepatitis B, and liver cirrhosis might be the major alternative causes of liver injury in Chinese patients with COVID-19.43 SARS-CoV-2 infection and Conflict of interest related immune changes might be regarded as a “second hit” to simple fatty liver, and can induce incident liver injury and The authors have no conflict of interests related to this steatohepatitis. Stress and sepsis are particularly problematic publication. in cirrhotic patients, as either can trigger acute-on-chronic liver failure. However, the information of pre-existing liver Author contributions diseases has not been outlined in most studies of COVID-19 and the interaction between existing liver disease and SARS- Writing the manuscript (JL), developing the idea for the article CoV-2 infection has not yet been studied.36 and critically revising it (JGF). All authors read and approved the final version of the manuscript. Summary and future perspective

In conclusion, the SARS-CoV-2 infection outbreak has References become a global threat to human health over the past 3 months. The COVID-19 disease itself can result in severe and [1] Wu Z, McGoogan JM. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a even fatal respiratory diseases, and in mainland China 2.3% Report of 72314 Cases From the Chinese Center for Disease Control and of CFR is attributed to ARDS and multiple organ failure. Prevention. JAMA 2020. doi: 10.1001/jama.2020.2648. Increasing evidence has demonstrated frequent incident [2] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and liver injury in COVID-19, especially in patients with multiple clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet 2020;395:507–513. doi: 10. organ injury. The liver injury itself has often manifested as 1016/S0140-6736(20)30211-7. transient elevation of serum aminotransferases. However, [3] Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early transmission dynam- acute liver failure and obvious intrahepatic cholestasis have ics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med been reported in the available studies, but seldomly preva- 2020. doi: 10.1056/NEJMoa2001316. [4] Sohrabi C, Alsafi Z, O’Neill N, Khan M, Kerwan A, Al-Jabir A, et al. World lence of abnormal aminotransferases is significantly higher in Health Organization declares Global Emergency: A review of the 2019 severe cases than in mild cases, according to many studies of Novel Coronavirus (COVID-19). Int J Surg 2020;76:71–76. doi: 10.1016/j. COVID-19. The underlying mechanisms of liver injury in cases ijsu.2020.02.034. of COVID-19 might include psychological stress, systemic [5] Hu LL, Wang WJ, Zhu QJ, Yang L. [Novel coronavirus pneumonia related liver injury: etiological analysis and treatment strategy]. Zhonghua Gan Zang Bing inflammation response, drug toxicity, and progression of pre- Za Zhi 2020;28:E001. doi: 10.3760/cma.j.issn.1007-3418.2020.02.001. existing liver diseases, i.e. from simple fatty liver to steato- [6] Zu ZY, Jiang MD, Xu PP, Chen W, Ni QQ, Lu GM, et al. Coronavirus disease hepatitis. Currently, there is insufficient evidence for SARS- 2019 (COVID-19): A perspective from China. Radiology 2020:200490. doi: CoV-2 infected hepatocytes or virus-related liver injury in 10.1148/radiol.2020200490. [7] Ren M, Jie L, Jun S, Subrata G, Liang-Ru Z, Hong Y, et al. Implications of COVID-19 patients, and it is difficult to summarize the role of COVID-19 for patients with pre-existing digestive diseases. Lancet Gastro- the immune system in causing liver damage by the virus at enterol Hepatol 2020. doi: 10.1016/S2468-1253(20)30076-5.

16 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 13–17 Li J. et al: Liver injury in COVID-19

[8] Lai CC, Shih TP, Ko WC, Tang HJ, Hsueh PR. Severe acute respiratory syn- [25] Li G, Fan Y, Lai Y, Han T, Li Z, Zhou P, et al. Coronavirus infections and drome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID- immune responses. J Med Virol 2020;92:424–432. doi: 10.1002/jmv. 19): The epidemic and the challenges. Int J Antimicrob Agents 2020;55: 25685. 105924. doi: 10.1016/j.ijantimicag.2020.105924. [26] Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and chronic liver [9] Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Clinical characteristics of injury. Gut 2005;54:1024–1033. doi: 10.1136/gut.2004.053850. coronavirus disease 2019 in China. N Engl J Med 2020. doi: 10. [27] Chau TN, Lee KC, Yao H, Tsang TY, Chow TC, Yeung YC, et al. SARS-associ- 1056/NEJMoa2002032. ated viral hepatitis caused by a novel coronavirus: report of three cases. [10] Novel Coronavirus Pneumonia Emergency Response Epidemiology Team. Hepatology 2004;39:302–310. doi: 10.1002/hep.20111. [The epidemiological characteristics of an outbreak of 2019 novel coronavi- [28] Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, rus diseases (COVID-19) in China]. Zhonghua Liu Xing Bing Xue Za Zhi et al. Proteomics. Tissue-based map of the human proteome. Science 2015; 2020;41:145-151. doi: 10.3760/cma.j.issn.0254-6450.2020.02.003. 347:1260419. doi: 10.1126/science.1260419. [11] Zhangfu F, Fang Y, Kang W, Kefang L, Xizhuo S, Nanshan Z, et al. Clinical [29] Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H. Tissue characteristics of coronavirus disease 2019 (COVID-19): an updated sys- distribution of ACE2 protein, the functional receptor for SARS coronavirus. tematic review. medRxiv 2020 (v2). doi: 10.1101/2020.03.07.20032573. – [12] Holshue ML, DeBolt C, Lindquist S, Lofy KH, Wiesman J, Bruce H, et al. First A first step in understanding SARS pathogenesis. J Pathol 2004;203:631 case of 2019 novel coronavirus in the United States. N Engl J Med 2020;382: 637. doi: 10.1002/path.1570. 929–936. doi: 10.1056/NEJMoa2001191. [30] Guan GW, Gao L, Wang JW, Wen XJ, Mao TH, Peng SW, et al. [Exploring the [13] Liu Y, Yang Y, Zhang C, Huang F, Wang F, Yuan J, et al. Clinical and biochem- mechanism of liver enzyme abnormalities in patients with novel coronavirus- ical indexes from 2019-nCoV infected patients linked to viral loads and lung infected pneumonia]. Zhonghua Gan Zang Bing Za Zhi 2020;28:E002. doi: injury. Sci China Life Sci 2020;63:364–374. doi: 10.1007/s11427-020- 10.3760/cma.j.issn.1007-3418.2020.02.002. 1643-8. [31] Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A, et al. Clinical characteristics of 2019 [14] Xu XW, Wu XX, Jiang XG, Xu KJ, Ying LJ, Ma CL, et al. Clinical findings in a novel coronavirus infection in China. medRxiv 2020 (v1). doi: 10. group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) 1101/2020.02.06.20020974. outside of Wuhan, China: retrospective case series. BMJ 2020;368:m606. [32] Wan S, Yi Q, Fan S, Lv J, Zhang X, Guo L, et al. Characteristics of lymphocyte doi: 10.1136/bmj.m606. subsets and cytokines in peripheral blood of 123 hospitalized patients with [15] Fang D, Ma J, Guan J, Wang M, Song Y, Tian D, et al. Manifestations of diges- 2019 novel coronavirus pneumonia (NCP). medRxiv 2020 (v1). doi: 10. tive system in hospitalized patients with novel coronavirus pneumonia in 1101/2020.02.10.20021832. Wuhan, China: a single-center, descriptive study. Chin J Dig 2020. doi: 10. [33] Diao B, Wang C, Tan Y, Chen X, Liu Y, Ning L, et al. Reduction and functional 3760/cma.j.issn.0254-1432.2020.0005. exhaustion of t cells in patients with coronavirus disease 2019 (COVID-19). [16] Liu C, Jiang ZC, Shao CX, Zhang HG, Yue HM, Chen ZH, et al. [Preliminary medRxiv 2020 (v1). doi: 10.1101/2020.02.18.20024364. study of the relationship between novel coronavirus pneumonia and liver [34] Joung JY, Cho JH, Kim YH, Choi SH, Son CG. A literature review for the function damage: a multicenter study]. Zhonghua Gan Zang Bing Za Zhi mechanisms of stress-induced liver injury. Brain Behav 2019;9:e01235. 2020;28:148–152. doi: 10.3760/cma.j.issn.1007-3418.2020.02.003. [17] Lu H, Ai J, Shen Y, Li Y, Li T, Zhou X, et al. A descriptive study of the impact of doi: 10.1002/brb3.1235. diseases control and prevention on the epidemics dynamics and clinical fea- [35] Lelubre C, Vincent JL. Mechanisms and treatment of organ failure in sepsis. – tures of SARS-CoV-2 outbreak in Shanghai, lessons learned for metropolis Nat Rev Nephrol 2018;14:417 427. doi: 10.1038/s41581-018-0005-7. epidemics prevention. medRxiv 2020 (v1). doi: 10.1101/2020.02.19. [36] Strnad P, Tacke F, Koch A, Trautwein C. Liver - guardian, modifier and target 20025031. of sepsis. Nat Rev Gastroenterol Hepatol 2017;14:55–66. doi: 10. [18] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients 1038/nrgastro.2016.168. infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395: [37] Li J, Li RJ, Lv GY, Liu HQ. The mechanisms and strategies to protect from 497–506. doi: 10.1016/S0140-6736(20)30183-5. hepatic ischemia-reperfusion injury. Eur Rev Med Pharmacol Sci 2015;19: [19] Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical characteristics of 138 2036–2047. hospitalized patients with 2019 novel coronavirus-infected pneumonia in [38] Deng SQ, Peng HJ. Characteristics of and public health responses to the Wuhan, China. JAMA 2020. doi: 10.1001/jama.2020.1585. coronavirus disease 2019 outbreak in China. J Clin Med 2020;9:575. doi: [20] Yang X, Yu Y, Xu J, Shu H, Xia J, Liu H, et al. Clinical course and outcomes of 10.3390/jcm9020575. critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single- [39] Sun C, Fan JG. [Strengthen clinical research on liver injury in patients with centered, retrospective, observational study. Lancet Respir Med 2020. doi: novel coronavirus pneumonia]. J Prac Hepatol 2020;23:153–155. doi: 10. 10.1016/S2213-2600(20)30079-5. 3969/j.issn.1672-5069.2020.02.001. [21] Cai Q, Huang D, Ou P, Yu H, Zhu Z, Xia Z, et al. COVID-19 in a designated [40] Zhang C, Shi L, Wang FS. Liver injury in COVID-19: management and chal- infectious diseases hospital outside Hubei Province, China. medRxiv 2020 lenges. Lancet Gastroenterol Hepatol 2020. pii: S2468-1253(20)30057-1. (v1). doi: 10.1101/2020.02.17.20024018. doi: 10.1016/S2468-1253(20)30057-1. [22] Liu Y, Sun W, Li J, Chen L, Wang Y, Zhang L, et al. Clinical features and [41] Jiang F, Deng L, Zhang L, Cai Y, Cheung CW, Xia Z. Review of the Clinical progression of acute respiratory distress syndrome in coronavirus disease Characteristics of Coronavirus Disease 2019 (COVID-19). J Gen Intern Med 2019. medRxiv 2020 (v3). doi: 10.1101/2020.02.17.20024166. [23] Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of 2020. doi: 10.1007/s11606-020-05762-w. COVID-19 associated with acute respiratory distress syndrome. Lancet [42] Chen J, Ling Y, Xi X, Liu P, Li F, Li T, et al. Efficacies of lopinavir/ritonavir and Respir Med 2020. pii: S2213-2600(20)30076-X. doi: 10.1016/S2213-2600 abidol in the treatment of novel coronavirus pneumonia. Chin J Infect Dis (20)30076-X. 2020;38:E008. doi: 10.3760/cma.j.cn311365-20200210-00050. [24] Liu Q, Wang R, Qu G, Wang Y, Liu P, Zhu Y, et al. General anatomy report of [43] Wang FS, Fan JG, Zhang Z, Gao B, Wang HY. The global burden of liver novel coronavirus pneumonia patients. Journal of Forensic Medicine 2020; disease: the major impact of China. Hepatology 2014;60:2099–2108. doi: 36:21–23. doi: 10.12116/j.issn.1004-5619.2020.01.005. 10.1002/hep.27406.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 13–17 17 Review Article

COVID-19 and Liver Dysfunction: Current Insights and Emergent Therapeutic Strategies

Gong Feng#1, Kenneth I. Zheng#2, Qin-Qin Yan1, Rafael S. Rios2, Giovanni Targher3, Christopher D. Byrne4, Sven Van Poucke5, Wen-Yue Liu6 and Ming-Hua Zheng*2,7,8

1Xi’an Medical University, Xi’an, China; 2NAFLD Research Center, Department of Hepatology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; 3Section of Endocrinology, Diabetes and Metabolism, Department of Medicine, University and Azienda Ospedaliera Universitaria Integrata of Verona, Verona, Italy; 4Southampton National Institute for Health Research Biomedical Research Center, University Hospital Southampton, Southampton General Hospital, Southampton, UK; 5Ziekenhuis Oost-Limburg, Department of Anesthesiology, Critical Care, Emergency Medicine and Pain Therapy, Genk, Belgium; 6Department of Endocrinology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; 7Institute of Hepatology, Wenzhou Medical University, Wenzhou, China; 8Key Laboratory of Diagnosis and Treatment for The Development of Chronic Liver Disease in Zhejiang Province, Wenzhou, China

Abstract Introduction

The outbreak of coronavirus disease 2019 (COVID-19), caused Severe acute respiratory syndrome coronavirus 2 (SARS- by the severe acute respiratory syndrome coronavirus 2 CoV-2) has become a serious threat to global public health.1,2 (SARS-CoV-2), has attracted increasing worldwide attention. Although the virus appears to be only partially similar to Cases of liver damage or dysfunction (mainly characterized by severe acute respiratory syndrome coronavirus and Middle moderately elevated serum aspartate aminotransferase lev- East respiratory syndrome coronavirus, all of these viral els) have been reported among patients with COVID-19. infections are responsible for severe and potentially lethal However, it is currently uncertain whether the COVID-19- acute respiratory syndromes in humans.3 Unfortunately, to related liver damage/dysfunction is due mainly to the viral date, there are no specific/targeted drugs, or vaccines, and infection per se or other coexisting conditions, such as the use the number of SARS-CoV-2-positive patients is growing in of potentially hepatotoxic drugs and the coexistence of sys- many parts of the world. The world and China map showing temic inflammatory response, respiratory distress syndrome- the geographical distribution of coronavirus disease 2019 induced hypoxia, and multiple organ dysfunction. Based on the (COVID-19) is shown in Figure 1; the cut-off date for this current evidence from case reports and case series, this review data extraction was March 5, 2020.4 article focuses on the demographic and clinical characteristics, Surprisingly, in addition to the acute respiratory symp- potential mechanisms, and treatment options for COVID-19- toms, patients with COVID-19 also have varying degrees of related liver dysfunction. This review also describes the geo- liver damage/dysfunction. For example, Chen et al.5 showed graphical and demographic distribution of COVID-19-related that more than a third of COVID-19 patients have some liver liver dysfunction, as well as possible underlying mechanisms function test abnormalities. Most of these infected patients linking COVID-19 to liver dysfunction, in order to facilitate had mild-to-moderate elevations of serum alanine amino- future drug development, prevention, and control measures transferase (ALT) or aspartate aminotransferase (AST) for COVID-19. levels; one patient had severely elevated serum aminotrans- Citation of this article: Feng G, Zheng KI, Yan QQ, Rios RS, ferases (ALTof 7590 U/L, ASTof 1445 U/L).5 Since COVID-19- Targher G, Byrne CD, et al. COVID-19 and liver dysfunc- related liver dysfunction is now attracting widespread attention: Current insights and emergent therapeutic strat- tion, this review discusses the epidemiological characteris- egies. J Clin Transl Hepatol 2020;8(1):18–24. doi: 10.14218/ tics, the potential mechanisms, and the treatment options JCTH.2020.00018. for liver dysfunction induced by COVID-19.

Epidemiological characteristics of COVID-19-related liver dysfunction

To better understand the distribution of COVID-19-related Keywords: COVID-19; Liver dysfunction; SARS-CoV-2. liver dysfunction in different regions of the world, we Abbreviations: ACE, angiotensin converting enzyme; ALT, alanine aminotrans- searched the available literature between December 11, ferase; AST, aspartate aminotransferase; COVID-19, coronavirus disease 2019; 2019 and February 20, 2020. The literature search focused SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TLR, toll-like receptor. on initial case reports and case series covering COVID-19 Received: 8 March 2020; Revised: 16 March 2020; Accepted: 17 March 2020 patients, with a clear description of liver function tests and #These authors contributed equally to this study. results. All case reports without any data on patients’ liver *Correspondence to: Ming-Hua Zheng, NAFLD Research Center, Department of function tests were excluded. Finally, we included 14 eligible Hepatology, the First Affiliated Hospital of Wenzhou Medical University, No. 2 Fuxue Lane, Wenzhou 325000, China. Tel: +86-577-55579622, Fax: +86-577- studies, including 5 case reports (Table 1) and 9 case series 5–18 55578522, E-mail: [email protected] (Table 2). By reviewing these 14 studies, we have

18 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 18–24

Fig. 1. Geographical distribution of COVID-19, using the cut-off date for data extraction of March 5, 2020.

described geographical and demographic characteristics of levels, respectively.11,16 Of the six studies performed in COVID-19-related liver dysfunction (as summarized below). Wuhan, China,5,13–15,17,18 only four included data on the proportion of patients with abnormal liver function test results. Geographical distribution of COVID-19-related liver Specifically, in these four studies, the proportion of infected dysfunction patients with increased serum AST levels ranged from 24.1% to 36.6% (Fig. 2).5,13,17,18 In a survey from Zhejiang Prov- In two Chinese multicenter surveys, one of which was led by ince, China, the proportion of patients with increased serum the First Affiliated Hospital of Guangzhou Medical University, AST levels was only 16.1%, whereas the proportion of those including 552 hospitals in 31 provinces/provincial municipal- with increased serum ALT levels was not specified.12 It seems ities through January 29th, 2020 and the other was led by the First Hospital of Lanzhou University including 7 hospitals, a likely that the proportion of infected patients with increased considerable number of patients with COVID-19 had some serum AST levels in Wuhan (i.e. the area in which the epi- abnormalities in liver function tests.11,16 In particular, 6.2% demic of COVID-19 started) is much greater than cases to 22.2% of patients had increased serum AST levels (Fig. 2) reported outside Wuhan. It is plausible to speculate that and 21.3% to 28.1% of patients had increased serum ALT there may have been a higher viral load of COVID-19 in

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 18–24 19 Feng G. et al: COVID-19 and liver dysfunction

exposed patients in Wuhan, where the infection began and was concentrated in a greater proportion of the population.

Sex distribution of COVID-19-related liver dysfunction NA Antifungal drugs A total of six case series reporting the percentage of abnormal liver function test results amongst COVID-19 patients suggested that the proportion of infected men with increased serum AST levels was higher than that observed in infected Paramivir NA NA NA Antiviral drugs NA women.5,11–13,16,17 In fact, in these case series, the proportions of infected men with increased serum AST levels were, respectively, 68.7%, 58.2%, 58.1%, 72.4%, 62.8% and 73.2%, whereas the proportions of infected women were, respectively, 31.3%, 41.8%, 41.9%, 27.6%, 37.2% and 26.8%. It is possible to hypothesize that infected men are more predisposed to develop COVID-19-associated liver dys- Ceftazidime Linezolid, Oseltamivir Antibiotic drugs

Drugs function than infected women, and we suggest that further research is required to better understand this sex-related difference.

Age distribution of COVID-19-related liver dysfunction Prior history of liver diseases

Of the five case reports, three were in children and two were in adults. The age of children ranged from 3 months to 7 years, whereas the age of adult patients ranged from 35 to 56 years. None of these children had abnormal serum liver Non-severe No NA NA NA Non-severe NA NA COVID-19 Disease severity enzymes and, therefore, it is possible to hypothesize that older age is associated with a higher likelihood of liver damage/dysfunction. However, further studies are needed to confirm this finding. We look forward to more case reports/ case series on COVID-19-related liver damage/dysfunction in different age groups in the future. * 2 124 (30.3) 29

Putative mechanisms of COVID-19-related liver dysfunction * 1 Angiotensin-converting enzyme (ACE)2-mediated liver dysfunction

Whether SARS-CoV-2 has direct adverse effects on liver

Male NA function is currently not known. Some studies have suggested that SARS-CoV-2 predominantly enters alveolar epithelial cells through the human ACE2 receptor.19,20 Therefore, the lung is considered the main target organ of SARS-CoV-2 infection. However, previous studies have found that bile duct 3 months Female1 Normal7 Normal Male35 Non-severe Normal Male No Male Normal 33 Azithromycin, 72 (16.4) Severe 17 NA Non-severe Meropenem, No Levofloxacin NA NA Age (years) Sex AST (IU/L) ALT (IU/L) epithelial cells may also express ACE2 receptor at a concentration 20 times higher than in hepatocytes and these findings suggest that SARS-CoV-2 infection might also cause bile duct epithelial cell damage.21,22 However, significant increases in circulating levels of serum alkaline phosphatase, bilirubin or gamma-glutamyltransferase (that may reflect bile duct

China (Haikou) China (Wuhan) China (Shanghai) United States Canada 56 injury) have been rarely reported in COVID-19 patients.9 Liver histopathologic features from COVID-19 patients also 6 did not show any significant damage in hepatocytes or bile

7 23 8 duct cells. For this reason, it is reasonable to assume that COVID-19-related liver dysfunction is more likely due to secondary liver damage than the use of hepatotoxic therapies or

9 10 the coexistence of systemic inflammatory response, respiratory distress syndrome-induced hypoxia, or multiple organ Holshue ML, 2020 Chen F, 2020 Cai JH, 2020 Silverstein WK, 2020 Zhang YH, 2020 Standard deviation of liver enzyme fluctuations during hospitalization Author, Year Country

Table 1. Demographic and liver function characteristics of patients with COVID-19 based on the first case reports in three countries AST: aspartate aminotransferase. ALT: alanineNormal aminotransferase. ranges Liver for* diseases: AST and any ALT: liver AST < disease 40 that IU/L can and cause ALT serum < liver 40 enzyme IU/L. changes, such as viral hepatitis, autoimmune hepatitis, etc. NA: not available. dysfunction.

20 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 18–24 egG. Feng tal et OI-9adlvrdysfunction liver and COVID-19 :

Table 2. Main characteristics related to liver disease in patients with COVID-19 infection in different Chinese regions based on a series of case reports

Disease severity Drugs ora fCiia n rnltoa Hepatology Translational and Clinical of Journal Non- Antiviral Author, Sample Abnormal Abnormal Male, Age Severe severe History of liver Antibiotic drugs Antifungal Year Country size (n) AST (%) AST (IU/L) ALT (%) ALT (IU/L) n(%) (years) (%) (%) diseases (%) drugs (%) (%) drugs (%)

Guan WJ China 1099 168 NA 158 NA 640 47.0 173 926 23 (2.1) 632 393 30 (2.7) 202011 (Multi- (22.2) (21.3) (58.2) (35.0– (15.7) (84.3) (57.5) (35.8) center) 58.0) Xu XW, China 62 10 26 (20– NA 22 (14– 36 41.0 NA NA 7 (11.3) 28 (45.2) 55 (88.7) NA 202012 (Zhejiang) (16.1) 32) 34) (58.1) (32.0– 52.0) Chen China 99 35 34 28 39 67 55.5 6 NA NA 11 (11.1) 70 (70.7) 75 (75.8) 15 (15.2) NS, (Wuhan) (35.3) (26–48) (28.2) (22–53) (68.7) 35.1 20205 Chen L, China 29 7 NA 5 NA 21 56.01*14 15 NA NA NA NA 202013 (Wuhan) (24.1) (17.2) (72.4) (48.3) (51.7) Wang China 138 NA 31 NA 24 75 56.0 36 102 4 (2.9) 89 (64.5) 124 NA DW, (Wuhan) (24–51) (16–40) (54.3) (42.0– (26.1) (73.9) (89.9) 202014 68.0) Pan F, China 21 NA 32 6 20 NA 42 6 31 6 40.0 6 0 (0.0) 21 NA NA NA NA 202015 (Wuhan) (15–95) (12– (28.6) 9.0 (100.0)

2020 107) Liu C, China 32 2 (6.2) 25 9 26 (17– 20 38.5 4 28 1 (3.13) NA NA NA 202016 (Multi- (19–32) (28.1) 46) (62.5) (26.3– (12.5) (87.5) o.8|18 | 8 vol. center) 45.8) Huang C China 41 15 34 NA 32 (21– 30 49.0 13 28 1 (2.4) 41 38 NA 202017 (Wuhan) (36.6) (26–48) 50) (73.2) (41.0– (31.7) (68.3) (100.0%) (92.7%) 58.0) Chen HJ China 93 24 3 16 (11– 0 (0) 28 (26– 0 (0.0) 9 NA 9 6 NA – 18 421 24 2020 (Wuhan) (33.3) (21– (33.3) 58) 34) (100.0) (100.0%) (66.7%) 119)

Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; NA, not available. Liver diseases: any liver disease that can cause liver enzyme changes, such as viral hepatitis, autoimmune hepatitis, etc. Normal range for AST and ALT: AST < 40 IU/L and ALT < 40 IU/L. * Median, no interquartile range Feng G. et al: COVID-19 and liver dysfunction

inflammatory response syndrome, and multiple organ failure) may also cause hepatic ischemia and hypoxia-reperfusion dysfunction. Experimental data showed that hepatic cell death and inflammatory cell infiltration caused by hypoxia can be seen both in vivo and in vitro models of hepatic ischemia and hypoxia.29 This suggests that oxygen reduction and lipid accumulation in hepatocytes during shock and hypoxic conditions may lead to cell death. The subsequent marked increase in reactive oxygen species and their peroxidation products can act as a second messenger, activating redox- sensitive transcription factors, and further amplifying the Fig. 2. Proportion of patients with liver dysfunction in Chinese regions: release of multiple proinflammatory factors, causing liver 30 Wuhan and outside Wuhan. damage. All the aforementioned findings suggest that pneumonia-associated hypoxia is one of the most important factors causing secondary liver injury in COVID-19 patients. Drugs In summary, the COVID-19-related liver dysfunction may be considered as the result of secondary liver damage caused During the course of the COVID-19 epidemic, many infected mainly by several factors, such as the use of potentially patients have been treated with antipyretic agents. Most of hepatotoxic drugs, systemic inflammatory response, respiratory these medications contain acetaminophen, which is a drug distress syndrome-induced hypoxia, and multiple organ failure. recognized as being able to cause significant liver damage or In addition, critically ill COVID-19 patients with severe liver 23 induce liver failure. Itisknownthatanacuteingestionof>7.5 dysfunction are also more likely to have a poorer prognosis. to 10 g of acetaminophen in adults or 150 to 200 mg/kg in children is likely to cause hepatotoxicity.24 Although the US Food and Drug Administration Advisory Committee has pro- Treatment options for COVID-19-related liver dysfunction posed a decrease in the maximum daily dosage of acetaminophen from 4 to 3 g, and the maximum individual dosage from 1 Presently, there is no specific treatment for COVID-19 infec- 31 to 0.65 g, (relegating 500-mg tablets to prescription status), tion. Therefore, the cornerstone of COVID-19 management these recommendations have not been implemented world- is patient isolation and supportive medical care where neces- wide.25 In addition, although there is currently no targeted anti- sary, including pulmonary ventilation and prevention of the 32 viral treatment for COVID-19, many infected patients have also underlying inflammatory “storm” as well. been treated with some antiviral drugs, such as oseltamivir, From the findings discussed above, however, we believe abidol or lopinavir, which may have some hepatotoxic effects. that it is also reasonable to explore novel treatments for COVID-19 targeting of the ACE2 receptor. The ACE2 cellular Systemic inflammatory response syndrome receptor is highly expressed in human lung tissues, gastrointestinal tract, liver, vascular endothelial cells, and arterial smooth muscle cells.33 In addition, skin, nasal cavity, and oral Although many COVID-19 patients were not too unwell, this 27 infection in some patients has resulted in sudden deterioration, mucosa basal cells also express the ACE2 receptor. All organs with high expression of the ACE2 receptor may be tar- ending in multiple organ failure. Most experts believe that the 34 occurrence of multiple organ failure is mainly related to the geted by SARS-CoV-2 infection. Activation of the ACE2/Ang sudden initiation of an inflammatory “storm” in the critically ill COVID-19 patients.26 The so-called inflammatory “storm”,or systemic inflammatory response syndrome, is strongly related to activation of both natural and cellular immunity that is triggered by COVID-19 infection.27 In fact, the virus is able to directly induce multiple proinflammatory signals via toll-like receptors (TLRs) and activation of killer T lymphocytes.28 The activated T lymphocytes then attack the infected body cells, leading to their apoptosis and necrosis, until T lymphocytes are depleted. Damage-related pattern molecules released by dead infected cells can further amplify some inflammatory signals, such as TLRs. At the same time, T-lymphocyte depletion cannot control viral and bacterial infections, thereby activating multiple inflammatory signaling pathways, which lead to macrophage activation and secondary inflammatory reactions. Subsequently, when more inflammatory cytokines are released, more cell damage and necrosis are observed (Fig. 3). Such a vicious cycle is capable of causing multiple injuries, not only to Fig. 3. Schematic diagram showing the systemic inflammatory response the lungs but also to the liver, heart, and kidneys. syndrome induced by SARS-CoV2. After the SARS-CoV-2 infection, pathogenic T cells are rapidly activated, producing granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6 and other proinflammatory factors. GM-CSF will Hypoxia-reperfusion dysfunction further activate CD14+CD16+ inflammatory monocytes, producing a larger amount of IL-6 and other proinflammatory factors, and thereby inducing an inflammatory “storm” that leads to immune damage to other organs, such as the Hypoxia and shock induced by COVID-19-related complica- lungs and the liver. Both IL-6 and GM-CSF are two key proinflammatory factors tions (such as respiratory distress syndrome, systemic that trigger the inflammatory “storm” in patients with COVID-19.

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(1-7)/Mas signaling pathway or inhibition of the ACE/Ang II/ manuscript (MHZ, GT, CDB, SVP). All authors reviewed and AT1R pathway could be potential pathways for the treatment of commented on the manuscript and approved the final COVID-19. For SARS-CoV-2-infected patients, both ACE-inhibi- version. tors and angiotensin-II-receptor antagonists might be used not only for treating high blood pressure but also for reducing systemic inflammatory response and improving patient mortality.35 References Recently, Chen et al.36 reported that glycyrrhizic acid deriv- [1] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from atives might also have antiviral activity against SARS-CoV-2. patients with pneumonia in China, 2019. N Engl J Med 2020;382:727–733. Glycyrrhizic acid is one of the first-line drugs for anti-inflam- doi: 10.1056/NEJMoa2001017. matory protection in liver disease, and it has been used in [2] Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early transmission dynam- clinical practice for many years.37 In particular, glycyrrhizic ics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med 2020. doi: 10.1056/NEJMoa2001316. acid is a triterpene glycoside isolated from the root of the lic- [3] Zhang C, Shi L, Wang FS. Liver injury in COVID-19: management and chal- orice plant. ACE2 is a cellular type I membrane protein that is lenges. Lancet Gastroenterol Hepatol 2020. doi: 10.1016/S2468-1253(20) mostly expressed in the lungs, heart, kidneys, and intestine. 30057-1. Full-length ACE2 consists of an N-terminal peptidase domain [4] WHO. Coronavirus disease 2019 (COVID-19) situation report-49. 2020. Available from: https://www.who.int/docs/default-source/coronaviruse/si- and a C-terminal collectrin-like domain that ends with a single tuation-reports/20200309-sitrep-49-covid-19.pdf?sfvrsn=70dabe61_4 trans-membrane helix and a ;40-residue intracellular [5] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and segment.38 Glycyrrhizin has the potential to bind to ACE2 clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in receptor with an estimated DG (kcal/mol) of -9, with the Wuhan, China: a descriptive study. Lancet 2020;395:507–513. doi: 10. 36 1016/S0140-6736(20)30211-7. binding sites of ARG-559, GLN-388, ARG-393, and ASP-30. [6] Zhang YH, Lin DJ, Xiao MF, Wang JC, Wei Y, Lei ZX, et al. [2019-novel coronavirus infection in a three-month-old baby]. Zhonghua Er Ke Za Zhi 2020; Conclusions 58:E006. doi: 10.3760/cma.j.issn.0578-1310.2020.0006. [7] Chen F, Liu ZS, Zhang FR, Xiong RH, Chen Y, Cheng XF, et al. [First case of Our review shows the following: (1) In highly epidemic areas of severe childhood novel coronavirus pneumonia in China]. Zhonghua Er Ke Za COVID-19 infection, such as Wuhan, China, the proportion of Zhi 2020;58:E005. doi: 10.3760/cma.j.issn.0578-1310.2020.0005. [8] Cai JH, Wang XS, Ge YL, Xia AM, Chang HL, Tian H, et al. [First case of 2019 infected patients with abnormal liver function test results novel coronavirus infection in children in Shanghai]. Zhonghua Er Ke Za Zhi (mainly elevated serum AST levels) is greater than that 2020;58:86–87. doi: 10.3760/cma.j.issn.0578-1310.2020.02.002. observed in regions where a smaller proportion of cases of [9] Holshue ML, DeBolt C, Lindquist S, Lofy KH, Wiesman J, Bruce H, et al. First COVID-19 infection in the population have occurred. (2) The case of 2019 novel coronavirus in the United States. N Engl J Med 2020;382: 929–936. doi: 10.1056/NEJMoa2001191. proportion of infected patients with elevated serum trans- [10] Silverstein WK, Stroud L, Cleghorn GE, Leis JA. First imported case of 2019 aminase levels is higher in adults than in children and in men novel coronavirus in Canada, presenting as mild pneumonia. Lancet 2020; than in women, respectively. However, we suggest that further 395:734. doi: 10.1016/S0140-6736(20)30370-6. studies are needed to confirm these preliminary observations. [11] Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med 2020. doi: 10. In the meantime, we believe that the front-line medical staff 1056/NEJMoa2002032. should pay attention to liver function tests in patients infected [12] Xu XW, Wu XX, Jiang XG, Xu KJ, Ying LJ, Ma CL, et al. Clinical findings in a with COVID-19. For those patients with a pre-existing history group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) of liver diseases (especially older patients), special attention outside of Wuhan, China: retrospective case series. BMJ 2020;368:m606. doi: 10.1136/bmj.m606. should be paid to monitoring hepatic changes caused by [13] Chen L, Liu HG, Liu W, Liu J, Liu K, Shang J, et al. [Analysis of clinical features COVID-19, whilst carefully identifying the cause of the liver of 29 patients with 2019 novel coronavirus pneumonia]. Zhonghua Jie He He dysfunction.39 We also recommend that front-line medical staff Hu Xi Za Zhi 2020;43:E005. doi: 10.3760/cma.j.issn.1001-0939.2020.0005. should assess the use of appropriate hepatoprotective thera- [14] Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in pies, especially in patients with pre-existing liver disease, in Wuhan, China. JAMA 2020. doi: 10.1001/jama.2020.1585. order to attenuate the potentially deleterious impact of [15] Pan F, Ye T, Sun P, Gui S, Liang B, Li L, et al. Time course of lung changes on COVID-19-related liver damage/dysfunction. chest CT during recovery from 2019 novel coronavirus (COVID-19) pneumonia. Radiology 2020;200370. doi: 10.1148/radiol.2020200370. Funding [16] Liu C, Jiang ZC, Shao CX, Zhang HG, Yue HM, Chen ZH, et al. [Preliminary study of the relationship between novel coronavirus pneumonia and liver function damage: a multicenter study]. Zhonghua Gan Zang Bing Za Zhi This work was supported by grants from the National Natural 2020;28:148–152. doi: 10.3760/cma.j.issn.1007-3418.2020.02.003. Science Foundation of China (81500665), High Level Creative [17] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients Talents from Department of Public Health in Zhejiang Province infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395: 497–506. doi: 10.1016/S0140-6736(20)30183-5. and Project of New Century 551 Talent Nurturing in Wenzhou. [18] Chen H, Guo J, Wang C, Luo F, Yu X, Zhang W, et al. Clinical characteristics GT is supported in part by grants from the University School of and intrauterine vertical transmission potential of COVID-19 infection in nine Medicine of Verona, Verona, Italy. CDB is supported in part by pregnant women: a retrospective review of medical records. Lancet 2020; grants from the Southampton National Institute for Health 395:809–815. doi: 10.1016/S0140-6736(20)30360-3. [19] Li R, Qiao S, Zhang G. Analysis of angiotensin-converting enzyme 2 (ACE2) Research Biomedical Research Centre (IS-BRC-20004), UK. from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-nCoV. J Infect 2020. doi: 10.1016/j.jinf.2020.02. 013. Conflict of interest [20] Xu H, Zhong L, Deng J, Peng J, Dan H, Zeng X, et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci The authors have no conflict of interests related to this article. 2020;12:8. doi: 10.1038/s41368-020-0074-x. [21] Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A, et al. Specific ACE2 expression in cholangiocytes may cause liver damage after 2019-nCoV infection. bioRxiv Author contributions 2020(v1). doi: 10.1101/2020.02.03.931766. [22] Guan GW, Gao L, Wang JW, Wen XJ, Mao TH, Peng SW, et al. [Exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus- Design and data interpretation (MHZ, GF, KIZ, QQY, WYL), infected pneumonia]. Zhonghua Gan Zang Bing Za Zhi 2020;28:E002. doi: manuscript writing (GF, KIZ, RSR), critical revision of the 10.3760/cma.j.issn.1007-3418.2020.02.002.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 18–24 23 Feng G. et al: COVID-19 and liver dysfunction

[23] Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of [31] Zhang Z, Li X, Zhang W, Shi ZL, Zheng Z, Wang T. Clinical features and COVID-19 associated with acute respiratory distress syndrome. Lancet treatment of 2019-nCov pneumonia patients in Wuhan: report of a couple Respir Med 2020. doi: 10.1016/s2213-2600(20)30076-x. cases. Virol Sin 2020. doi: 10.1007/s12250-020-00203-8. [24] Hodgman MJ, Garrard AR. A review of acetaminophen poisoning. Crit Care [32] Zhang L, Liu Y. Potential interventions for novel coronavirus in China: A sys- Clin 2012;28:499–516. doi: 10.1016/j.ccc.2012.07.006. tematic review. J Med Virol 2020;92:479–490. doi: 10.1002/jmv.25707. [25] Krenzelok EP. The FDA Acetaminophen Advisory Committee Meeting - what [33] Santos RAS, Sampaio WO, Alzamora AC, Motta-Santos D, Alenina N, Bader is the future of acetaminophen in the United States? The perspective of a M, et al. The ACE2/Angiotensin-(1-7)/MAS axis of the renin-angiotensin – committee member. Clin Toxicol (Phila) 2009;47:784–789. doi: 10. system: focus on angiotensin-(1-7). Physiol Rev 2018;98:505 553. doi: 10.1152/physrev.00023.2016. 1080/15563650903232345. [34] Ding Y, Wang H, Shen H, Li Z, Geng J, Han H, et al. The clinical pathology of [26] Prompetchara E, Ketloy C, Palaga T. Immune responses in COVID-19 and severe acute respiratory syndrome (SARS): a report from China. J Pathol potential vaccines: Lessons learned from SARS and MERS epidemic. Asian 2003;200:282–289. doi: 10.1002/path.1440. Pac J Allergy Immunol 2020;38:1–9. doi: 10.12932/ap-200220-0772. [35] Sun ML, Yang JM, Sun YP, Su GH. [Inhibitors of RAS Might Be a Good Choice [27] Klimstra WB, Ryman KD, Bernard KA, Nguyen KB, Biron CA, Johnston RE. for the Therapy of COVID-19 Pneumonia]. Zhonghua Jie He He Hu Xi Za Zhi Infection of neonatal mice with sindbis virus results in a systemic inflamma- 2020;43:E014. doi: 10.3760/cma.j.issn.1001-0939.2020.0014. tory response syndrome. J Virol 1999;73:10387–10398. [36] Chen H, Du Q. Potential natural compounds for preventing 2019-nCoV infection. [28] Tartey S, Takeuchi O. Pathogen recognition and Toll-like receptor targeted Preprints 2020;2020010358(v3). doi: 10.20944/preprints202001.0358.v3. – therapeutics in innate immune cells. Int Rev Immunol 2017;36:57 73. doi: [37] Li JY, Cao HY, Liu P, Cheng GH, Sun MY. Glycyrrhizic acid in the treatment of 10.1080/08830185.2016.1261318. liver diseases: literature review. Biomed Res Int 2014;2014:872139. doi: [29] Yang L, Wang W, Wang X, Zhao J, Xiao L, Gui W, et al. Creg in hepatocytes 10.1155/2014/872139. ameliorates liver ischemia/reperfusion injury in a TAK1-dependent manner [38] Yan R, Zhang Y, Li Y, Xia L, Zhou Q. Structure of dimeric full-length human in mice. Hepatology 2019;69:294–313. doi: 10.1002/hep.30203. ACE2 in complex with B0ATI. bioRxiv 2020(v1). doi: 10.1101/2020.02.17. [30] Zhang XJ, Cheng X, Yan ZZ, Fang J, Wang X, Wang W, et al. An ALOX12-12- 951848. HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion [39] Cheng ZJ, Shan J. 2019 novel coronavirus: where we are and what we know. injury. Nat Med 2018;24:73–83. doi: 10.1038/nm.4451. Infection 2020. doi: 10.1007/s15010-020-01401-y.

24 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 18–24 Review Article

Assessing the Effectiveness of Strategies in US Birth Cohort Screening for Hepatitis C Infection

Cynthia J. Tsay1 and Joseph K. Lim*2

1Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; 2Yale Liver Center, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA

Abstract Introduction

Chronic hepatitis C infection in the USA is a highly morbid Chronic hepatitis C virus (HCV) infection is one of the most condition and current guidelines recommend one-time common indications for liver transplantation and the leading screening among the birth cohort (1945-1965). Understand- cause of hepatocellular carcinoma in the USA, accounting for ing strategies to optimize screening can help inform future approximately 19,000 deaths annually and substantial hepatitis C virus (HCV) screening guidelines. A focused healthcare costs.1 An estimated 4.1 million individuals in literature search was performed using PubMed and manual the USA are HCV antibody (Ab)-positive, indicative of past abstract review from major hepatology conferences over the or current infection. Among those, approximately 2.4 million past 2 years. The search strategy involved using Medical individuals have ongoing chronic HCV infection with positive Subject Headings terms for hepatitis C, screening, birth HCV RNA.2 The baby boomer “birth cohort” of individuals born cohort, baby boomers, and 1945-1965. The review was 1945-1965 comprise approximately 75% of HCV infections in limited to data from the USA. A total of 327 articles were the USA despite representing only 27% of the general popu- identified and 36 abstracts were included, with studies lation.3 One study in 2012 retrospectively applied birth cohort published between 2012-2019. Strategies including clinician to previously risk-based HCV screening using the National education, electronic medical record alerts, reflex HCV RNA Health and Nutrition Examination Survey (NHANES) database testing, point-of-care testing, multisite (outpatient, inpatient, and determined that optimal application of risk-based guide- emergency department, endoscopy suite) initiatives, direct lines would identify 82% of chronic HCV cases (number patient solicitation, and utilization of non-physician providers needed to screen 14.6) compared to birth-cohort screening, have increased HCV screening rates. However, broad imple- which would identify 76% of chronic HCV cases (number mentation remains less than optimal. Barriers include lack of needed to screen 28.7).4 patient acceptance to screening and engagement in the HCV The Center for Disease Control (CDC) and the USA care cascade. The Veterans Affairs Healthcare System has Preventative Services Task Force (USPSTF) issued recom- achieved higher birth cohort screening rates through an mendations in 20123,5 and 20136,7 recommending one-time integrated approach requiring high-level engagement by universal screening in all individuals born between 1945- leadership and institutional commitment. Multiple strategies 1965, irrespective of symptoms or risk factors. Cost effective- for increasing birth cohort screening have been successful, ness studies have confirmed birth cohort screening to be cost but overall rates of HCV screening remain low. These strat- saving for billions in test and treat models.5 However, recent egies can inform public health efforts to implement emerging studies indicate that HCV testing in the birth cohort minimally national recommendations for expansion of HCV screening to increased from 12.3% to 17.3% between 2013 and 2017.8 all U.S. adults age 18 or older. One unique population, veterans being cared for at the Citation of this article: Tsay CJ, Lim JK. Assessing the ef- Veteran Affairs Healthcare System, achieved more successful fectiveness of strategies in US birth cohort screening for hep- screening through automated clinical reminders through the atitis C infection. J Clin Transl Hepatol 2020;8(1):25–41. doi: electronic medical record (EMR) and also increased aware- 10.14218/JCTH.2019.00059. ness among primary care providers through national direc- tives. One study reported 79.5% of veterans born between 1945-1965 had been tested for the HCV Ab.9 Given the high cost and mortality associated with HCV, Keywords: Hepatitis C; Epidemiology; HCV antibody; Screening; Birth cohort; coupled with the presence of effective treatment options with Baby boomers; Care cascade; Electronic medical records. Abbreviations: Ab, antibody; BPAs, best practice alerts; CDC, Center for Disease direct-acting antivirals (DAAs), identification of patients with Control; DAAs, direct acting antivirals; ED, emergency department; EMR, elec- HCV is of paramount importance to public health efforts to tronic medical record; HCV, hepatitis C virus; NHANES, National Health and Nutri- achieve HCV elimination. The success of the World Health tion Examination Survey; POCT, point-of-care testing; RDT, rapid detecting test; Organization campaign for global elimination of HCV infection USPSTF, USA Preventative Services Task Force; VHA, Veterans Health Administra- 10,11 tion. by 2030 will depend on optimization of the HCV care Received: 18 December 2019; Revised: 4 February 2020; Accepted: 25 February cascade from screening/identification to linkage to treat- 2020 ment,12 and the associated funding required to support hep- *Correspondence to: Joseph K. Lim, Yale Liver Center, Section of Digestive Dis- atitis programs.13 eases, Yale University School of Medicine, 333 Cedar Street, LMP 1080, New Haven, CT 06520-8019, USA. Tel: +1- 203-737-6063, Fax: +1-203-785-7273, This review summarizes existing strategies for increasing E-mail: [email protected] the screening or testing of HCV infection within the birth

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 25

Copyright: © 2020 Authors. This article has been published under the terms of Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0), which permits noncommercial unrestricted use, distribution, and reproduction in any medium, provided that the following statement is provided. “This article has been published in Journal of Clinical and Translational Hepatology at DOI: 10.14218/JCTH.2019.00059 and can also be viewed on the Journal’s website at http://www.jcthnet.com”. Tsay C.J. et al: Birth cohort screening for HCV cohort (1945-1965). Although there have been substantial Care and Treatment of Viral Hepatitis.16 Subsequent studies efforts on HCV microelimination in several global regions, this confirmed the cost-effectiveness of birth cohort testing based article focuses primarily on evidence from the USA. Under- on health economic models, which projected that an addi- standing existing strategies for optimization of 1945-1965 tional 808,580 cases of chronic HCV infection cases would birth cohort screening may help inform future guidelines for be identified at a cost of $2874 per case, and that even with public health interventions focused on HCV screening, partic- a test-and-treat strategy using historical DAA plus pegylated ularly in the context of the burgeoning opioid epidemic. interferon/ribavirin, quality adjusted life-years increased by $532,000, with an incremental cost-effectiveness ratio of Methods $35,700 per quality adjusted life-years saved.17 Despite implementation of these recommendations in A focused literature search was performed using PubMed, 2012-2013, a recent review suggested that there was only with a combination of search terms, including screen, test, a modest increase in HCV screening within the birth cohort in hepatitis C virus, HCV, hepatitis C, birth cohort, 1945-1965, the USA, estimated to have risen from 12.3% in 2013 to and baby boomer, in August 2019. Manual abstract review 17.3% in 2017.8 Further analysis of this dataset identified the was performed (C.T.) for major hepatology conferences, primary care clinic as the setting in which prioritization of including The Liver Meeting 2017-2018 of the American testing should occur, permitting unscreened or untested eli- Association for the Study of Liver Diseases, Digestive Dis- gible patients access to follow-up care within the outpatient eases Week 2017-2019, and The International Liver Congress clinic.18 This information underlines the need for more inno- of the European Association for the Study of the Liver 2017- vative screening strategies to be implemented which involve 2019. The review was limited to USA data, with the exception a combination of patient and provider education, harnessing of reports from six countries (Argentina, Chile, Finland, the electronic health record automated alerts in the inpatient, France, Greece, and Japan)14 with similar recommendations outpatient, emergency departments, endoscopy, and colono- for birth cohort screening or one-time screening for all ages. scopy suites, as well as a patient-centered medical home There was no time restriction imposed on the search strategy approach19 utilizing non-physician providers to assist in of original manuscripts. improving screening, reflex testing, and linkage of care to the HCV care cascade. Results Innovative strategies for screening A total of 327 full text papers were identified by the database search and an additional 38 abstracts per manual review from EMR alerts major conferences. After individual assessment of abstracts, 36 were included in the review. Study characteristics and The advent of the EMR or electronic health record has created outcomes are summarized below (Table 1). All papers were a platform in which screening can become more streamlined published 2012-2019 but one study from 2012 was excluded with patient care in both the outpatient and inpatient settings. due to assessment prior to implementation of CDC birth Additionally, several states, including New York, Connecticut, 15 cohort recommendations. Massachusetts, California, and Colorado, have established laws mandating that baby boomers be offered screening with Current state of hepatitis C screening in the USA a one-time HCV Ab test. In New York state, laboratory data confirmed a 51% increase in the number of specimens The current recommendations of the CDC and USPTF regard- collected for HCV testing among birth cohort individuals ing HCV screening are summarized as follows: between 2013 and 2014, and New York Medicaid data revealed a 52% increase in average monthly testing for HCV CDC (August 17, 2012): In addition to testing Ab from 2012 to 201420 despite exclusion of HCV testing adults of all ages at risk for HCV infection, the CDC requirement in the emergency department (ED) setting.21 recommends: Furthermore, while EMR appears to be a cornerstone for All adults born during 1945–1965 receive one-time automated alerts, given the simplicity of identifying screening testing for the HCV. candidates based solely on date of birth, additional strategies Testing should begin with anti-HCV. If the anti-HCV test to augment EMR-based testing have resulted in significant is positive, or reactive, then a nucleic acid test should increases in screening rates, including one study employing follow. targeted education of resident physicians which found an All persons identified with current HCV infection should increase from 62% at baseline to 81% at 6 months post- 22 receive a brief alcohol screening and intervention, as intervention. Similar increases in screening rates employing clinically indicated, followed by referral to appropriate education plus EMR reminders in a resident physician context 23–24 care and treatment services.5 resulted in up to a 3-fold increase in HCV testing rates.

USPSTF (June 25, 2013) recommends: Outpatient Screening for HCV infection in persons at high risk for infection. Primary care and outpatient specialty clinics are the corner- One-time screening for HCV infection in adults born stone of population-based screening and the focus of most between 1945 and 1965. EMR alert-based interventions. Multiple studies have reported increases in HCV screening rates after implementation of EMR These recommendations were in line with recommenda- alerts25–31 in multiple quality improvement interventions, tions of the Institute of Medicine and the USA Department of including those incorporating formal Plan, Do, Study, Act Health and Human Services Action Plan for the Prevention, cycles; although, the magnitude of increase has ranged

26 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 Tsay C.J. et al: Birth cohort screening for HCV

Table 1. Characteristics and outcomes from HCV screening strategies among birth cohort individuals

Study Type Design Population Setting Sample size Outcomes

EMR alerts Outpatient Jones (2017) Abstract Retrospective Patients born Baylor Scott & n>30,000 Statistically chart review between 1945- White in Central significant increase 1965 not previously Texas Primary Care in baby-boomer screened for HCV Clinic from 2/ screening for 2014-2/2015 hepatitis C from 1.87% prior to EMR reminder to 14.14% after initiation of the reminder Kahn (2018) Abstract Retrospective Patients born Northshore n=99892 HCV tested: chart review between 1945- University Health 13.8% (13,804/ 1965 not previously System; 99,892) Highly screened for HCV Implemented 7/ varied adherence 2017 to screening guidelines by PCPs Konerman Abstract Retrospective Patients born Primary care n=52,5660 HCV screening (2017) cohort study between 1945- clinics increased 10-fold 1965 without prior from 7.6% for diagnosis of HCV patients with PCP infection, no prior visit in 6 months documented anti- prior to BPA HCV testing implementation to 72% over a 1-year period after implementation Soo (2017) Abstract Retrospective Patients born Automatic health n=29,987 HCV screening rate chart review between 1945- maintenance alert increased from 1965. Excluded in an integrated baseline 13.3% to patients with HCV medical group, 6/ 15.6% after 1 on problem list or if 2015-6/2016 month and to they had their one- 40.2% after 12 time HCV screen months HCV Ab- positive: 2.3% (684/29987) Soo (2018) Abstract Prospective Patients born Automatic health n=76288 HCV Ab-positive: cohort between 1945- maintenance alert 4.6% (3507/ 1965. Excluded module at primary 76,288); HCV patients with HCV care practices in screening rate on problem list the Providence increased 31.9% based on ICD9/10 Health and from 23.0% to code or positive Services and rates 54.9% anti-HCV or HCV assessed monthly RNA in five regions in the Western USA, 1/2017 - 12/2018

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Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Teply (2018) Abstract Retrospective All patients born 35 primary care n=29,703 Prealert HCV chart review between 1945- clinics within a (pre), 29,913 tested: 1.62% 1965 seen at a regional (post) (482/29.703) HCV primary care clinic healthcare system Ab-positive: 4.2% within a regional in Midwest USA (20/482) Postalert healthcare system (prealert), 6/1/ HCV tested: in Midwest USA 2016-11/30/ 19.0% (5685/ 2016; (postalert) 29,913) HCV Ab- 12/1/2016-5/31/ positive: 1.9% 2017 (107/5685) 10- fold increase in HCV screening Al-Hihi (2017) Full- Prospective All patients born Multiphysician Not reported Baseline screening text cohort- 2 PDSA between 1945- practice in the rate cycles 1965 seen in a Midwest USA preintervention: primary care clinic representing 84 30% (1674/5541) faculty physicians Screening rate at 3 and residents, 6/ mo: 45% 2016-3/2017, with Screening rate 3 BPA and health mo after maintenance alerts concurrent in the EMR and education session: education to 55% primary care providers via single educational sessions with a hepatologist Federman Full- Randomized Patients born 10 community and n=25, 620 Testing rates (2017) text control trial during birth cohort hospital-based study-eligible greater among period were primary care visits Birth Cohort pts in subjects. However, practices that intervention sites attending implemented BPA (20.2% v 1.8%, physicians and for HCV testing p<0.0001) EHR- medical residents among birth cohort embedded BPA were participants in adults, 4/2013-3/ markedly the study to see 2014 increased HCV how BPA affected screening, but the HCV testing and majority of eligible incidence of HCV pts did not receive Ab-positive tests testing indicating a need for more effective methods to promote uptake Nitsche Full Text Case control Patients born 7 primary care n=73,685; Screening rates at (2018) between 1945- sites in Virginia cases 37,783; the following times 1965 Mason Healthcare controls (case vs. control), System (greater 35,902 p<0.001 at all time Seattle area, WA), points Baseline: 8/1/2014-9/14/ 6.1% vs. 4.6% 2015 with 3 sites Time 1: 18.1% vs. given additional 10.4% Time 2: education 20.3% vs. 12.5% interventions Time 3: 22.2% vs. (case) not 13.7% Time 4: provided to the 23.4% vs. 14.7% remaining 4 Time 5: 24.2% vs. (control) 15.3% Time 6: 17.5% vs. 10.4%

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Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Shahnazarian Full- Case control Patients born Methodist Hospital Not reported PreEMR alert HCV (2015) text between 1945- in Brooklyn, NY screen: 47.2% 1965 prelegislative PostEMR alert HCV mandate (12/ screen: 87.9% 2013) and postmandate and postEMR intervention, 5/ 2014-2/2015 Yeboah- Full- Case control Patients born Northshore n=10,089 PreEMR alert HCV Korang (2018) text between 1945- University Health (pre); 45,188 screen: 0.68% 1965 in the System, 1/2010 to (post) (69/10,089) outpatient setting 12/2015, with PostEMR alert HCV retrospective chart screen: 10.76% review back to (5451/45,188) 2003 to identify 15.8-fold increase overall HCV testing in HCV testing rates (case) and rates then during 7/ 2015; BPA alert implemented 7/ 2017-11/2017 Inpatient Mehta (2017) Abstract Retrospective Adult admitted to 9/2014-9/2016 n=1128 HCV Ab-positive: cohort inpatient medicine 9.6% (108/1128) service born HCV-positive: 52% between 1945- (56/108) HCV RNA 1965 PCR-positive : 21% HCV RNA PCR-negative: 25% HCV RNA PCR not performed during hospitalization: 54% Only 18% of seropositive had outpatient gastrointestinal follow-up Shen (2018) Abstract Retrospective Patients born Patients admitted n=51657 Overall and initial cohort between 1945- to New York screening 1965 categorized Presbyterian improved pre- and by 3 timeframes Hospital- Weill postmandate from (premandate, Cornell; data 8% to 39% and postmandate but collected in 3 times 53% to 84% prereflex RNA, frames: 1. (p<0.01); this did postreflex RNA) Premandate (1/ not translate into and stratified by 2013-12/2013) 2. improved linkage screened vs not Postmandate but to care Follow-up screened prereflex (1/2014- care and initiation 8/2015) 3. of treatment Postreflex RNA (9/ decreased from 2015-12/2016) 31% to 20% and 9% to 5%, (p<0.01)

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Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 29 Tsay C.J. et al: Birth cohort screening for HCV

Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Turner (2015) Full- Prospective Patients born Safety-net hospital n=6140 HCV tested: 51% text cohort between 1945- in South Texas (3168/6140) HCV 1965 admitted to from 1/2012-1/ Ab-positive: 7.6% hospital 2014 with follow- (240/3168) HCV up through 12/ RNA-positive: 63% 2014 (134/214), 4.2% overall chronic HCV: 96.3% (129/ 134) were counseled and 80.6% (108/134) received primary care follow-up and 38.8% (52/134) received hepatology follow- up with 5 initiating anti-HCV treatment Direct patient solicitation- phone call, mailing Trowell (2018) Abstract Prospective Patients born Two-pronged n=15,583 BPA screened cohort between 1945- approach: 1. BPA 8786/15,583 1965 chosen from a created in EMR to Letters screened population in a prompt PCP to 3645/15,583 Baltimore city order tests for Screened via hospital patients; 2. Letters hospital or other mailed with affiliated locations educational 3152/15,583 HCV material, blood Ab-positive: 2.7% test request forms (426/15,583) HCV for pts without RNA-positive: prior HCV testing 1.3% (204/ 15,583) HCV positivity rates highest in 1951- 1960 birth cohort Kruger (2017) Full Text Prospective Project managers Three sites Not reported BPA was the cohort of each of the three implemented preferred sites implementing interventions to intervention at all HCV screening per increase birth- three sites, but CDC cohort testing site-specific recommendation through challenges (BEST-C sites). participation in the prevented success Filled out Birth-cohort of the solution in standardized Evaluation to two out of three questionnaires Advance Screening sites Despite about their and Testing for challenges in start- implementation Hepatitis C from up of the screening experiences and 12/2012-3/2014 in PCP settings, it qualitative analysis was deemed feasible and likely successful given dedicated resources, buy-in, and support from hospital administration

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30 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 Tsay C.J. et al: Birth cohort screening for HCV

Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Yartel (2018) Full- Randomized Patients born Patients randomly n=8992 Repeated mailing- text control trial between 1945- assigned to receive (mailing trial) intervention was 8 1965 not previously one of three n=14,475 times as likely to screened or independent (BPA trial) identify anti-HCV- diagnosed implementation n=8873 positive (adjusted strategies (patient relative risk: 8.0, (repeated mailing solicitation 95% confidence outreach, BPA, trial) interval: 2.8-23.0; direct patient adjusted solicitation), 12/ probabilities: 2012-3/2014 intervention 0.27%, control 0.03%) BPA trial was 2.6 times as likely to identify anti-HCV-positive (adjusted relative risk 2.6, 95% confidence interval: 1.1-6.4; adjusted probabilities: intervention 0.29%, control 0.11%) Patient- solicitation trial was 5 times as likely to identify anti-HCV-positive (adjusted relative risk 5.3, 95% confidence interval: 2.3-12.3) Colonoscopy Abu-Heija Abstract Retrospective Dominantly African Urban open access n=444 HCV tested: 140/ (2018) chart review American adults colonoscopy suite, 444 HCV Ab- undergoing 2014 positive: 43% (60/ colonoscopy born 140) HCV RNA between 1945- PCR-positive: 94% 1952, subgroup (56/60) university analysis with physician group university physician vs. non- university group or outsider physician group provider tested: 48% vs. 15% (p<0.05) Lost to follow-up after first visit: 47% Abu-Heija Abstract Retrospective Dominantly African Urban open access n=988 HCV tested: (2019) chart review American adults colonoscopy suite 40.3% (2017) vs. undergoing in 2014 or 2017 31.5% (2014) colonoscopy born (p=0.005); no between 1945- difference based 1958 on race or gender HCV Ab-positive: 31.5% (2017) vs. 42.9% (2014) HCV RNA PCR-positive: 97% (2017) vs. 96.5%

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Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Matin (2018) Abstract Prospective Veterans Veterans Affairs n=208 (38 did HCV tested: 145/ cohort undergoing facility, 7/2017- not show for 170 (85%) colonoscopy, 10/2017 appointments) registered nurse screen day prior and if no prior screen for HCV and born between 1945-1965, verbal consent obtained over the phone, HCV tested when intravenous line placed Sears (2013) Full- Prospective Adults aged 50-65 3 month period n=500 HCV tested: 72% text cohort years-old who (376/500) HCV received a Ab-positive: 4/376 colonoscopy HCV RNA PCR- answered positive: 1/4 questions in a survey and blood samples were collected for hepatitis B virus and HCV Endoscopy Hirode (2018) Abstract Prospective Adults undergoing Urban safety-net n=1752 Acceptance of test: cohort outpatient hospital, 7/2015- BC-RF+ > BC+RF- endoscopy 7/2017 > BC+RF+ Overall categorized into: 1) HCV Ab-positive: BC and at least one 3.4% BC+ RF+: RF 2) BC and no RF 12.5% BC- RF+: 3) non BC with one 4.9% BC+ RF-: RF 1.3% -higher in US born patients Hirode (2019) Abstract Observational Outpatient Urban safety-net n=3624 Eligible for HCV endoscopy-based hospital, 7/2015- screening (69.2%) patient navigator 9/2018 based on: BC: model for adults 89.8% At least 1 undergoing HCV RF: 30.4% endoscopy Eligible patients tested increased from 50.8% to 77.9% Wong (2017) Abstract Prospective Adults undergoing Underserved n=1125 Trend towards cohort outpatient safety-net lower HCV test endoscopy hospital, 7/2015- acceptance among 6/2016 BC (odds ratio 0.39, 95% confidence interval: 0.13- 1.14) High risk (including BC): 66.5% HCV test accepted: 85.4%

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32 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 Tsay C.J. et al: Birth cohort screening for HCV

Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

ED Hyun (2017) Abstract Prospective Adults born ED of community n=12,617 HCV tested: 40.2% cohort between 1945- hospital, 2/2016- (5069/12617) HCV 1965 presenting to 1/2017 Ab-positive: ED using 3.99% (202/5069) streamlined EHR HCV RNA PCR- ordering with positive: 1.32% patient navigators (67/5069) Linkage contacting of care rate 37.3% individuals with in 6 month period confirmed infection patient navigation; by automated awareness of certified letters and infection in phone calls for chronically infected linkage to care but not engaged in care: 38.8% (26/ 67) Minhas (2019) Abstract Retrospective Adults born between ED of urban n=1525 HCV Ab-positive: cohort 1945-1965 hospital, 2/2017- 15.5% (237/1525) presenting to ED 1/2018 HCV RNA PCR- with testing positive: 67.9% conducted on an (161/237) Referral opt-out basis (2/ to hepatology: 2017-11/2017) 75% (121/161) then when notification was no longer required on all-comers (11/ 2017-1/2018) with referral to affiliated hepatology clinic Allison (2016) Full- Cross sectional Adults born between ED of a large urban n=915 Structured text 1945-1965 academic hospital interview: 46.7% presenting to ED (Bellevue, WA) in a (427/915) HCV were provided study state where birth tested: 90.0% information sheet cohort is mandated (383/427) HCV and CDC by law in all non- Ab-positive: 7.4% information sheet in ED healthcare HCV testing in baby settings, 10/2014- boomers, then 7/2015 participated in researcher- administered questionnaire, those with positive HCV Ab were referred to clinic, non- attendance resulted in telephone call

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Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Cornett Full- Retrospective Adults born ED of small urban/ n=3046 HCV tested: (2018) text cohort between 1945- suburban area 96.1% (2928/ 1965 presenting to tertiary care 3046) HCV Ab- ED with an opt-out academic hospital, positive: 6.6% test order 6/2016-12/2016 (192/2928) HCV generated by the RNA PCR-positive: EHR seen between 43% (71/167) 11am-7pm and given handout explaining rationale with plan for contacting patients with results Galbraith Full- Cross sectional Adults born ED of a large n=3170 Unaware of HCV (2015) text between 1945- academic urban status: 73.2% 1965 presenting to hospital (UAB) in a (2323/3170) ED with an opt-out socioeconomically Opted out: 12.7% as part of standard disadvantaged (289/2323) clinical care, population, 9/ Automated test therefore no 2013-11/2013 order: 87.3% informed consent (1988/2323) HCV was required with tested: 76.9% ED nurses (1529/1988) HCV screening using Ab-positive: questionnaire 11.1% (170/1529) embedded in the HCV RNA tested: EHR with 88.2% (150/170) informational HCV RNA PCR- packet given to positive: 68.0% HCV-positive (102/150) individuals and linkage to care specialist information with coordinator arrange follow-up and phone call follow- up Hsieh (2016) Full- Retrospective Adults aged >17 ED of a large n=4713 HCV Ab-positive: text cohort identity years-old academic urban 13.8% (652/4713) unlinked presenting to a hospital (JHU) in a Undocumented seroprevalence large academic socioeconomically HCV infection: urban hospital ED disadvantaged 31.3% (204/652) with excess blood population, 6/ Diagnosed by BC: specimen 2013-8/2013 48.5% (99/204) Diagnosed by RF: 26.5% (54/204)

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Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Lyons (2016) Full- Cross sectional Adults between the ED of urban n=1034 HCV tested: 89% text seroprevalence ages of 18-64 academic hospital, (924/1034) HCV presenting to the 1/2008-12/2009 Ab-positive: 14% ED were consented (128/924) HCV to a “study of RNA PCR-positive: disease of public 81% (103/128) health importance” Birth cohort only and given testing would have compensation, risk missed 28% (36/ factors assessed 128) HCV Ab- via health positive, 25% (26/ questionnaires, 105) HCV RNA- deidentified data positive Awareness of prior diagnosis: 32% (41/128) Schechter- Full- Descriptive Individuals >13 ED of urban n=3936 HCV tested: 3808 Perkins (2018) text years-old of age academic hospital HCV Ab-positive: presenting to the (BMC), 11/2016- 13.2% (504/3808) ED undergoing 1/2017 HCV RNA PCR- phlebotomy for positive: 59.2% clinical purposes, (292/493) Outside non-targeted, opt- BC with active out screening with infection: 54% a best practice (115) Linkage to advisory alert with care: 76.4% (223) navigators to Appointments facilitate linkage to scheduled: 38% care for those with (102) Attended positive RNA LTC visit: 22.5% (66) White (2016) Full- Retrospective Adults born ED of Highland n=26639 HCV tested: 9.7% text cohort between 1945- Hospital-Alameda (2581/26,639) 1965 or reporting Health System, HCV Ab-positive: any use of injection single-center 10.3% (267/2581) drugs who were not urban ED, 4/2014- Screening Ab test: known to be HCV- 10/2014 79% (2028/2581) positive to triage Diagnostic Ab test: nursing, EMR, with 21% (553/2581) opt-out testing Screening HCV Ab- requiring consent positive: 9.1% with physicians (185/2028) able to choose Diagnostic HCV testing at clinical Ab-positive: discretion 14.8% (82/553) (diagnostic) with informational packets sent to HCV-positive patients with referral to primary care which could then be canceled if RNA test was negative

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Table 1. (continued ) Study Type Design Population Setting Sample size Outcomes

Non-physician providers Shelgrove Abstract Prospective Patients born Ampla Health (a n=5481 Detected HCV Ab (2018) cohort between 1945- Federally Qualified seropositivity in 1965 in Yuba, Health Center) 7.5% (410/5481). Sutter, Colusa offering medical, 45% (183/410) Counties. Also dental, mental RNA-positive. included patients health, specialty Overall, 3.3% ages 18-64 years- healthcare RNA-positive old in Butte, Glenn, services in which averages to Tehama Counties. Northern 20 HCV diagnosed Patients with high California, patients/month risk factors. screening from 8/ HCV Ab-positive: Followed HCV Ab 2017-4/2018 7.5% (410/5481) testing with reflex HCV RNA-positive: HCV RNA testing by 45% (183/410) PCR HCV RNA-positive overall: 3.3% (183/5481), average of 20 HCV diagnosed patients /month RNA- positive HCV reflex testing lead to timely diagnosis and LTC Patients attending follow- up appointment: 92% (168/183) Travis (2018) Abstract Retrospective Patients born Emory Midtown n=10,803 HCV screening chart review between 1945- University Primary rates increased 1965 Care Clinic, 12/1/ after intervention. 2015-5/1/2018. Before Implemented intervention was “HCV screen” on 5% (232/4336). patient intake form After intervention on 12/1/2016. screening rates went to 18% (765/ 3498) in 2016- 2017 and 23% (880/2969) in 2017-2018. Dong (2017) Full- Prospective Patients in Community n=83 HCV-Ab rapid POC- text cohort California in BBBC, pharmacy-based positive: 1.2% (1/ high risk patients HCV-Ab POC 83) with hx of IVDU, screening program crack cocaine or in California in methamphetamine collaboration with use. the local public health department. 3 month pilot, 6 community pharmacists.

Abbreviations: Ab, antibody; BBBC, baby boomer birth cohort; BC, birth cohort; BPAs, best practice alerts; BMC, Boston Medical Center; CDC, Centers for Disease Control; ED, emergency department; EHR, electronic health record; EMR, electronic medical record; HCV, hepatitis C virus; IVDU, intravenous drug use; JHU, Johns Hopkins University; LTC, linkage to care; PCP, Primary care physician; PDSA, plan, do, study, act; POC, point of care; RF, risk factor; UAB, University of Alabama-Birmingham. widely from increases of 1.8- to 15.8-fold.32,33 Two studies at 3 months after a single education session.28,34 While EMR examined the impact of educational intervention for outpa- alerts represent an easily adapted, systems-based interven- tient primary care providers versus EMR alerts alone, and tion to increase birth cohort screening, current studies have found an increase in screening rates as high as 45% to 55% been limited to largely small single-center interventions in

36 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 Tsay C.J. et al: Birth cohort screening for HCV unique clinical settings, with limited information on down- times control rates, respectively, suggesting that direct stream outcomes within the care cascade, including RNA con- patient contact may represent a valuable, complementary firmation, linkage to care, and HCV treatment. tool to augment birth cohort screening efforts. One of the few multicenter studies examining outpatient screening interventions was reported by Turner et al.35 who Colonoscopy evaluated the feasibility and impact of the Reach, Effective- ness, Adoption, Implementation, Maintenance model in a Patients undergoing routine colonoscopy for colorectal cancer state-funded program to implement birth cohort testing in screening overlap with patients born 1945-1965 (age 54-74 five federally qualified healthcare centers and one family years as of 2019). This concordance of age makes point-of- medicine residency program. Within a cohort of 27, 700 care testing (POCT) in the colonoscopy suite a unique setting baby boomers born 1945-1965, 13,334 (48.1%) successfully for HCV screening. Abu-Heija et al.42,43 reported the results of underwent HCV Ab testing, 695 were HCV Ab-positive a retrospective cohort study of a predominantly African Amer- (5.2%), 349 were HCV RNA-positive (2.6%), 82 initiated ican population undergoing colonoscopy in an urban open- DAA therapy, 74 completed DAA therapy, and 70 achieved a access colonoscopy suite, and identified an increase in birth sustained virologic response (SVR). In this grant funded cohort HCV testing from 31% to 40.3% between 2014-2017, intervention study involving multilevel practice engagement high rates of HCV Ab positivity (31.5 to 42.9%), and higher strategies, patient navigation, standardized HCV Ab and likelihood of screening among patients referred from univer- reflex HCV RNA testing, and access to free DAA therapy via sity-affiliated clinicians. Another study performed in USA vet- prescription assistance programs, HCV birth cohort screening erans examined the impact of a same-day HCV testing during increased from 0.8% to 48.1% between 2014 and 2018. This colonoscopy which involved registered nurse pre-screening study additionally identified multiple barriers and challenges and phone consent for HCV Ab testing in birth cohort patients to birth screening interventions, significant site-level variabil- 1 day prior to scheduled colonoscopy, followed by laboratory ity in performance, and the central importance of best prac- draw at the time of intravenous line placement for colono- tice alerts (BPAs), reflex RNA testing, access to specialty scopy; this intervention resulted in an 85% HCV Ab testing consultation via telemedicine, and role of local champions. rate in this setting.44 Other studies have reported slightly lower rates for HCV Ab testing among patients undergoing Inpatient colonoscopy targeted on the basis of risk factors identified by patient survey.45,46 The inpatient setting provides a unique place for birth cohort screening. Multiple studies have demonstrated that inpatient Endoscopy screening is effective in identifying new cases of HCV but may not translate to effective linkage to care or treatment, and is Outside the specific context of colorectal cancer screening, limited by factors such as socioeconomic status, racial dis- the endoscopy suite broadly provides an opportunity for HCV parities, and lack of insurance.36,37 Two studies examined the screening by gastroenterologists who are uniquely positioned effect of the 2014 New York state HCV screening law within to offer real-time Ab testing, results reporting, patient edu- large academic hospitals and showed that despite a signifi- cation, and linkage to care including DAA treatment. Few cant increase in screening rates, 8 to 39% and 53 to 84%, studies have examined prospective interventions in the respectively, very few patients were linked to care or received endoscopy suite incorporating both birth cohort- and risk DAA treatment, with an overall decrease in treatment initia- factor-based testing. One urban safety-net hospital in Cal- tion during the period of observation.38 The challenges of ifornia conducted a series of studies47,48 to examine HCV inpatient screening may be driven by variable access to screening practices in the endoscopy suite setting, and iden- reflex RNA testing, provider-led results reporting and educa- tified lower test acceptance among birth cohort versus non- tion, and outpatient follow-up after hospital discharge, and birth cohort populations (odds ratio: 0.39, 95% confidence underscores the need for additional research to clarify evi- interval: 0.13-1.14, p=0.09)48 and 82.5% versus 93.9% dence-based strategies to augment linkage to care. (p=0.004).47 Integration of a pre-screening tool and a patient navigator tool in the outpatient endoscopy suite Direct patient solicitation resulted in an overall increase in eligible patients who were tested (50.8% to 77.9%); although, the authors identified a Multiple strategies targeted at direct patient contact have persistent gap in HCV testing in positive risk factor groups, as been explored to increase screening rates, including direct well as significant challenges in achieving patient acceptance mail campaigns, with letters containing educational material and engagement.49 and blood test request forms distributed to birth cohort patients identified via EMR.39,40 One study compared live- ED person recruitment through either phone call, direct mail, or electronic health record prompt at three study sites and found Historic studies of HCV screening in ED populations prior to that all three methods were significantly limited by require- implementation of birth cohort screening guidelines revealed ment for substantial administrative and staffing resources a higher prevalence than in the general population, with rates beyond existing clinic infrastructure. One randomized con- ranging from 4% in Michigan to 18% in Baltimore.50–55 trolled trial assigned patients to one of three independent Although our review focused specifically on patients within implementation strategies: repeated mailings, BPA through the 1945-1965 year-range, some studies have revealed that electronic health record, or direct phone solicitation.41 Com- testing restricted to the birth cohort alone may miss identifi- pared to controls, all three methods were associated with cation of up to 28% of HCV Ab-positive patients seen in the ED increased screening, with repeat mailings, BPA, and direct setting.56 Recent reports have additionally identified high phone solicitation resulting in 8 times, 2.6 times, and 5 levels of patient acceptance of HCV testing in the ED setting

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(90% of those completed a structured interview), but sober- Birth cohort screening in USA veterans ing low rates of linkage to care following testing among Ab positive patients.54,57 A large CDC-funded study conducted The USA Veterans Health Administration (VHA) has repre- in an urban academic ED center in Alabama demonstrated sented a national leader in efforts to screen and manage HCV similarly high patient acceptance of HCV testing (88%), using systems-based approaches. One early retrospective high HCV Ab positivity (11%), and high HCV RNA testing of cohort study from 2011 in the Atlanta Veterans Affairs Medical Ab positive patients (88%).58 However, among patients con- Center described that over half of birth cohort veterans in care firmed with chronic HCV infection (68% RNA positive among had been tested for HCV, and those born in the birth cohort Ab-positive group), only 54% attended a hepatology clinic were 6 times more likely to have a positive HCV Ab test and 3 appointment following discharge despite automated phone times more likely to have chronic HCV compared to non-birth cohort veterans.66 A 2016 report examined HCV testing prac- calls and a care coordinator focused on linkage to care. tices within the national VHA system among 4.2 million birth Another study examining HCV testing of birth cohort versus cohort veterans receiving care between 2000-2013, and con- all adults in an urban ED in California revealed higher HCV Ab firmed that 51% had undergone HCV testing; significant local positivity in birth cohort versus all adults (13.7% vs. 10.3%), and regional variabilities in testing practices (7-83%) were limited HCV RNA confirmation testing (67%), similar propor- identified, and up to 20% of birth cohort veterans with FIB- tion of HCV RNA-positive patients confirmed with chronic 4 scores suggestive of advanced fibrosis or cirrhosis had not infection (70%), and poor linkage to care, with only 24% of received HCV testing.67 RNA positive individuals attending a postdischarge clinic In the context of national efforts by Veterans Affairs’ 59 appointment. A recent study in a large urban academic ED leadership to adopt birth cohort HCV screening recommen- in Boston similarly revealed high HCV Ab positivity (13.2%) dations, HCV Ab testing in birth cohort veterans had increased and poor attendance at postdischarge clinic appointment to nearly 70% as of 2017.67–69 The VHA has employed a mul- (22.5%) among HCV RNA-positive patients.60 These findings tifaceted, integrated approach, including the formation of underscore the high yield of targeted HCV screening in the ED regional HCV innovation teams focused on increasing birth context, as well as the need for innovative strategies to cohort testing, adoption of a national HCV testing electronic augment linkage to care to promote more effective patient clinical reminder, establishing HCV testing as a regional and navigation. national VA quality performance metric, and publishing quar- terly reporting of HCV birth cohort testing rates at site, regional, and national levels to enhance transparency and Non-physician providers accountability.68 Many of these strategies may potentially be applicable to large non-federal health systems but requires Non-physician clinicians including nurses, care coordinators, high-level engagement by leadership and institutional com- patient navigators, pharmacists, and advanced practice pro- mitment to achieving similar levels of success. viders, such as physician assistants, nurse practitioners, and advance practice registered nurses, play a central role in all aspects of HCV screening, care navigation, and treatment, POCT and rapid detecting tests (RDTs) and have featured prominently in nearly all interventional studies targeting birth cohort screening. However, few studies The CDC and USPSTF guidelines recommend that HCV Ab have examined the specific roles of non-physician providers. testing should be performed as a screening test of choice, and Shelgrove et al.61 highlighted the importance of care coordi- positive results should be reflexed to HCV RNA test by PCR or nators and clinic managers in the context of a federally quali- nucleic acid testing.70,71 The gold standard test for anti-HCV fied healthcare centers in California, with a focus on identification has been the enzyme immunoassay, which augmenting linkage to care. Among a cohort of patients takes several days to weeks to process a result, and has sig- screened for HCV, 7.5% were HCV Ab-positive (410/5481), nificant laboratory requirements, including high-cost equip- of whom 45% were HCV RNA-positive with reflex testing ment, trained technicians, continuous supply of electricity (183/410), and 92% (164/183) successfully attended an and high facility cost. In contrast, newer rapid point-of-care appointment with either a primary care physician or specialist immunoassays can provide results in as little as 20-40 m. The following diagnosis of chronic HCV infection; these findings OraQuick test represents the most widely used POCT in the USA, approved by the Federal Drug Administration in 2010, contrast sharply with the comparatively low rates of linkage and can detect HCV Ab in saliva or blood.72 One meta-analysis to care (22.5-37.3%) reported in other studies, such as the reported a pooled sensitivity of 98% (95% confidence inter- CDC Hepatitis Testing and Linkage to Care initiative.60,62,63 val: 98-100%) and specificity of 100% (95% confidence Utilizing medical assistants to identify eligible patients for interval: 100-110%) for HCV Ab rapid diagnostic test com- HCV screening by means of printed patient intake forms pared to the enzyme immunoassay reference standard. within an outpatient primary care clinic resulted in increased 64 Another pooled analysis of eight studies demonstrated that testing rates, from 5% up to 23%. Another research group OraQuick ADVANCE was associated with a sensitivity of reported the novel use of clinical pharmacists to offer point- 98% compared to 88% with other oral assays.73 RDTs are of-care HCV Ab testing to high risk and birth cohort patients often faster than POCTs but require specialized equipment 65 seen at a community-based pharmacy. It is likely that a and specially trained personnel, are limited to HCV Ab detec- combination of interventions using physician and non-physi- tion (unable to detect HCV RNA), and associated with lower cian providers across practice settings will be necessary to diagnostic performance than POCTs.74 Further studies to meaningfully expand HCV screening in both birth cohort and define optimal use of POCTs and RDTs may be helpful in iden- high-risk populations. tifying their appropriate role in HCV screening efforts.

38 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 Tsay C.J. et al: Birth cohort screening for HCV

Future directions screening strategy in populations with a HCV prevalence >0.07% among non-birth cohort adults.77 This emerging evi- Despite major advances in antiviral therapy for chronic HCV dence of increasing HCV incidence in younger adults78 and infection since 2014, persistent deficits in the HCV care resulting gap in identification of HCV-infected persons have cascade within the USA threaten to limit the capacity to prompted reconsideration of USA HCV screening guidelines. achieve meaningful changes in the burden of chronic infection In 2019, several organizations have publicly called for and downstream clinical outcomes, as well as ambitious expansion of HCV screening to a universal approach to offer targets for HCV elimination by the WHO and USA Department one-time HCV Ab testing to all USA adults age 18-79 years or of Health and Human Services. Although progress has been age 18 years and older, including draft statements by the 79 80 made within each step of the cascade, including screening USPSTF (August 2019) and CDC (October 2019), as well as (HCV Ab), confirmation (HCV RNA), linkage to care, and updated language within the HCV guidance document of the treatment, identification of uninfected patients represents Infectious Diseases Society of America and American Associa- 81 the critical rate-limiting step. Additional challenges to achiev- tion for the Study of Liver Diseases (November 2019). In the ing HCV elimination include ongoing late relapse and re- context of these emerging changes in national screening rec- infection among patients who achieve sustained virologic ommendations, significant additional research is needed to response. support evidence-based recommendations on population and Since implementation of new recommendations for one- system-level HCV screening strategies. Lessons learned from time HCV Ab testing in all USA adults born 1945-1965 by the early experiences with birth cohort screening studies may help CDC and USPSTF, birth cohort screening has provided a inform future research and public health efforts at implemen- central focus for health system, state, and national level tation of future universal screening initiatives. efforts to increase the diagnosis of chronic HCV. A multi- Funding faceted integrative approach to screening which integrates clinical education, clinical decision support, reflex HCV RNA None to declare. testing, and incorporation of non-physician providers have been proven to be successful in increasing HCV screening. However, multiple studies have confirmed that overall screen- Conflict of interest ing rates within the USA outside the VHA remain low, despite success within individual centers and health systems. Based The authors have no conflict of interests related to this on the available literature, no single strategy appears to be publication. easily applicable across clinical settings. Our review underscores the need for the following steps: 1) patient and provider education to address a persistent Author contributions deficit in knowledge, regarding both HCV risk factors as well Study concept and design, and critical revision of the manu- as national screening recommendations; 2) engagement of script for important intellectual content (JKL), acquisition of health systems to incorporate standardized tools, such as data (CJT), analysis and interpretation of data and drafting of reflex HCV RNA testing and EMR alerts to prompt clinicians to the manuscript (JKL and CJT). pursue HCV testing across the inpatient, emergency department, outpatient clinic, pharmacy, and endoscopy center settings; and 3) strategic use of novel diagnostic tests (e.g. References POCT/RDT) and emerging technologies (e.g. text alerts) in appropriate clinical settings. Significant resources may be [1] Ly KN, Hughes EM, Jiles RB, Holmberg SD. Rising mortality associated with required to fully support the multidisciplinary programs hepatitis C virus in the United States, 2003-2013. Clin Infect Dis 2016;62: 1287–1288. doi: 10.1093/cid/ciw111. required to meaningfully impact broader efforts for screening, [2] Hofmeister MG, Rosenthal EM, Barker LK, Rosenberg ES, Barranco MA, Hall EW, linkage to care, and treatment. et al. Estimating prevalence of hepatitis C virus infection in the United States, Furthermore, emerging data suggest that birth cohort 2013-2016. Hepatology 2019;69:1020–1031. doi: 10.1002/hep.30297. screening will be inadequate to identify the growing cohort [3] Smith BD, Morgan RL, Beckett GA, Falck-Ytter Y, Holtzman D, Ward JW. Hep- atitis C virus testing of persons born during 1945-1965: recommendations of non-birth cohort adults with chronic HCV, including a rising from the Centers for Disease Control and Prevention. Ann Intern Med 2012; population of young adults recently infected with HCV in the 157:817–822. doi: 10.7326/0003-4819-157-9-201211060-00529. context of substance use and the opiate epidemic. Recent [4] Tomaszewski KJ, Deniz B, Tomanovich P, Graham CS. Comparison of current NHANES reports suggest an overall decrease in chronic HCV US risk strategy to screen for hepatitis C virus with a hypothetical targeted birth cohort strategy. Am J Public Health 2012;102:e101–e106. doi: 10. prevalence from 1.32% to 0.80% between 1999-2004 and 2105/AJPH.2011.300488. 2011-2016; although, an estimated 1.90 million USA adults [5] Smith BD, Morgan RL, Beckett GA, Falck-Ytter Y, Holtzman D, Teo CG, et al. continued to have viremic HCV, of whom only 49.8% were Recommendations for the identification of chronic hepatitis C virus infection – reportedly aware of their infection.75 An updated report which among persons born during 1945-1965. MMWR Recomm Rep 2012;61:1 32. [6] Chou R, Cottrell EB, Wasson N, Rahman B, Guise JM. Screening for hepatitis C combined NHANES data with high-risk populations excluded virus infection in adults: a systematic review for the U.S. Preventive Services by NHANES (prisoners, unsheltered homeless persons, Task Force. Ann Intern Med 2013;158:101–108. doi: 10.7326/0003-4819- active-duty military personnel, nursing home residents) esti- 158-2-201301150-00574. mated a prevalence of 2.4 million HCV RNA-positive persons.2 [7] Moyer VA. Screening for hepatitis C virus infection in adults: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2013;159: Expansion from birth cohort to universal screening of all USA 349–357. doi: 10.7326/0003-4819-159-5-201309030-00672. adults may identify up to an estimated 28% of HCV-infected [8] Patel EU, Mehta SH, Boon D, Quinn TC, Thomas DL, Tobian AAR. Limited individuals who would be missed by birth cohort55–60 or risk coverage of hepatitis C virus testing in the United States, 2013-2017. Clin – factor-based screening strategies.76 In addition, economic Infect Dis 2019;68:1402 1405. doi: 10.1093/cid/ciy803. [9] Belperio PS, Chartier M, Ross DB, Alaigh P, Shulkin D. Curing hepatitis C virus models have demonstrated that a universal screening strat- infection: Best practices from the U.S. Department of Veterans Affairs. Ann egy in USA adults is more cost effective than a birth cohort Intern Med 2017;167:499–504. doi: 10.7326/M17-1073.

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[10] Gane E, Kershenobich D, Seguin-Devaux C, Kristian P, Aho I, Dalgard O, et al. [34] Al-Hihi E, Shankweiler C, Stricklen D, Gibson C, Dunn W. Electronic medical Strategies to manage hepatitis C virus (HCV) infection disease burden - record alert improves HCV testing for baby boomers in primary care setting: volume 2. J Viral Hepat 2015;22 Suppl 1:46–73. doi: 10.1111/jvh.12352. adults born during 1945-1965. BMJ Open Qual 2017;6:e000084. doi: 10. [11] World Health Organization. Global health sector strategies on viral hepatitis 1136/bmjoq-2017-000084. 2016-2021. Available from: https://www.who.int/hepatitis/strategy2016- [35] Turner BJ, Rochat A, Lill S, Bobadilla R, Hernandez L, Choi A, et al. Hepatitis C 2021/ghss-hep/en/. virus screening and care: Complexity of implementation in primary care [12] Saab S, Le L, Saggi S, Sundaram V, Tong MJ. Toward the elimination of practices serving disadvantaged populations. Ann Intern Med 2019. doi: hepatitis C in the United States. Hepatology 2018;67:2449–2459. doi: 10. 10.7326/M18-3573. 1002/hep.29685. [36] Turner BJ, Taylor BS, Hanson JT, Perez ME, Hernandez L, Villarreal R, et al. [13] Waheed Y, Siddiq M, Jamil Z, Najmi MH. Hepatitis elimination by 2030: Pro- Implementing hospital-based baby boomer hepatitis C virus screening and gress and challenges. World J Gastroenterol 2018;24:4959–4961. doi: 10. linkage to care: Strategies, results, and costs. J Hosp Med 2015;10:510– 3748/wjg.v24.i44.4959. 516. doi: 10.1002/jhm.2376. [14] Irvin R, Ward K, Agee T, Nelson NP, Vellozzi C, Thomas DL, et al. Comparison [37] Mehta A, Down C, Shen NT, Kumar S. Inpatient hepatitis C screening, health of hepatitis C virus testing recommendations in high-income countries. disparities, and inadequate linkage to outpatient care at a large academic World J Hepatol 2018;10:743–751. doi: 10.4254/wjh.v10.i10.743. medical center. Gastroenterology 2017;152:S1190. [15] Litwin AH, Smith BD, Drainoni ML, McKee D, Gifford AL, Koppelman E, et al. [38] Shen NT, Rosenblatt R, Chan K, Mehta A, Johnston C, Ma X, et al. Impact of Primary care-based interventions are associated with increases in hepatitis C state law mandated hepatitis C virus (HCV) screening and automated con- virus testing for patients at risk. Dig Liver Dis 2012;44:497–503. doi: 10. firmatory testing on screening disparities and delivery of healthcare across – 1016/j.dld.2011.12.014. medical and surgical departments. Hepatology 2018;68:279A 280A. [16] Satoskar R, Reau N. Potential consequences of healthcare recommenda- [39] Trowell J, Lowe G, Thuluvath PJ. Successful HCV screening among baby tions: a focus on the U.S. Preventive Services Task Force. Hepatology boomers using EMR pop-up and targeted mailing. Hepatology 2018;68: – 2013;58:422–427. doi: 10.1002/hep.26349. 896A 897A. [17] Rein DB, Smith BD, Wittenborn JS, Lesesne SB, Wagner LD, Roblin DW, et al. [40] Kruger DL, Rein DB, Kil N, Jordan C, Brown KA, Yartel A, et al. Implementa- The cost-effectiveness of birth-cohort screening for hepatitis C antibody in U. tion of birth-cohort testing for hepatitis C virus. Health Promot Pract 2017; – S. primary care settings. Ann Intern Med 2012;156:263–270. doi: 10. 18:283 289. doi: 10.1177/1524839916661495. 7326/0003-4819-156-4-201202210-00378. [41] Yartel AK, Rein DB, Brown KA, Krauskopf K, Massoud OI, Jordan C, et al. [18] MacDonald BR, Chu TC, Stewart RA, Ojha RP. Setting-based prioritization for Hepatitis C virus testing for case identification in persons born during birth cohort hepatitis C virus testing in the United States. Clin Infect Dis 1945-1965: Results from three randomized controlled trials. Hepatology – 2020;70:543–544. doi: 10.1093/cid/ciz440. 2018;67:524 533. doi: 10.1002/hep.29548. [19] Defining the PCMH. Available from: https://pcmh.ahrq.gov/page/defining- [42] Abu-Heija A, Tama M, Kathi P, Naylor PH, Ehrinpreis MN, Mutchnick MG. High rate of HCV positive patients in an urban open access colonoscopy suite: pcmh. potential point of care screening of a predominantly African American pop- [20] Flanigan CA, Leung SJ, Rowe KA, Levey WK, King A, Sommer JN, et al. Eval- ulation. Hepatology 2018;68:894A. uation of the impact of mandating health care providers to offer hepatitis C [43] Abu-Heija A, Mohamad B, Tama M, Kathi PR, Nayeem MM, Khalid M, et al. virus screening to all persons born during 1945-1965- New York, 2014. Hepatitis C screening in the colonoscopy suite: patients are there, why don’t MMWR Morb Mortal Wkly Rep 2017;66:1023–1026.doi: 10.15585/mmwr. we screen them? Gastroenterology 2019;156:S1338. mm6638a3. [44] Matin T, Shoreibah M, Williams W, Callaway J, Burksi C. Increasing hepatitis C [21] NYS Hepatitis C Testing Law. Available from: https://www.health.ny.gov/di- screening rates in veteran population outside of the primary care setting. seases/communicable/hepatitis/hepatitis_c/providers/testing_law.htm. Gastroenterology 2018;154:S1201. [22] Wong K, Abdelqader A, Camire L, Farshidpour M, Singh S, Abuwalla Z, et al.A [45] Sears DM, Cohen DC, Ackerman K, Ma JE, Song J. Birth cohort screening for resident initiative improves hepatitis C screening rates in primary care chronic hepatitis during colonoscopy appointments. Am J Gastroenterol clinics. J Grad Med Educ 2017;9:768–770. doi: 10.4300/JGME-D-17- 2013;108:981–989. doi: 10.1038/ajg.2013.50. 00199.1. [46] Final recommendation statement: Hepatitis B virus infection: Screening, [23] Aamar A, Madhani K, Singh P, Chia D. Hepatitis C screening: quality improve- 2014. Avaliable from: https://www.uspreventiveservicestaskforce.org/Pa- ment in a resident continuity clinic. Gastroenterology 2017;152:S1168. doi: ge/Document/RecommendationStatementFinal/hepatitis-b-virus-infection- 10.1016/S0016-5085(17)33906-9. screening-2014. [24] Madhani K. The integration of resident physicians in the outpatient manage- [47] Hirode G, Liu B, Bhuket T, Wong R. Among 1945-1965 birth cohort patients ment of chronic hepatitis C: Bridging the gap with primary care. Hepatology with at least one additional hepatitis C virus risk factor, one in eight were 2018;68:299A–300A. positive for HCV antibody: an underserved safety-net population experience. [25] Jones H, Patel P, Sears D. Epic babyboomer reminders increase screening by J Hepatol 2018;68:S316. 10 fold. Hepatology 2017;66:326A. [48] Wong R, Campbell B, Liu B, Baden R, Bhuket T. Low rates of hepatitis C virus [26] Kahn MQ, Majmudar K, Belopolsky Y, Beig MI, Yeboah-Korang A, Sonnenberg A, (HCV) testing and HCV awareness among individuals at high risk for chronic et al. HCV age cohort screening follows a power-law distribution. Hepatology HCV infection among an underserved safety-net population. J Hepatol 2017; – 2018;68:280A 281A. 66:S704–S705. [27] Soo S, Croghan A, Dale C, Spinelli T, Cardona Gonzalez MG, Hart ME, et al. [49] Hirode G, Liu B, Bhuket T, Wong R. An outpatient endoscopy-based patient Use of an automated health maintanence alert is associated with increased navigator model improves hepatitis C virus screening among high risk hepatitis C screening rates in an integrated medical group. Gastroenterology patients: A 3-year prospective observational study among a safety-net 2017;152:S1171. health system. J Hepatol 2019;70:e497. [28] Federman AD, Kil N, Kannry J, Andreopolous E, Toribio W, Lyons J, et al.An [50] Brillman JC, Crandall CS, Florence CS, Jacobs JL. Prevalence and risk factors electronic health record-based intervention to promote hepatitis C virus associated with hepatitis C in ED patients. Am J Emerg Med 2002;20:476– testing among adults born between 1945 and 1965: A cluster-randomized 480. doi: 10.1053/ajem.2002.32642. – trial. Med Care 2017;55:590 597. doi: 10.1097/MLR.0000000000000715. [51] Hall MR, Ray D, Payne JA. Prevalence of hepatitis C, hepatitis B, and human [29] Nitsche B, Miller SC, Giorgio M, Berry CA, Muir A. Improving hepatitis C immunodeficiency virus in a Grand Rapids, Michigan emergency department. identification: technology alone is not the answer. Health Promot Pract J Emerg Med 2010;38:401–405. doi: 10.1016/j.jemermed.2008.03.036. 2018;19:506–512. doi: 10.1177/1524839917725501. [52] Kelen GD, Green GB, Purcell RH, Chan DW, Qaqish BF, Sivertson KT, et al. [30] Konerman MA, Thomson M, Gray K, Moore M, Choxi H, Seif E, et al. Impact of Hepatitis B and hepatitis C in emergency department patients. N Engl J Med an electronic health record alert in primary care on increasing hepatitis c 1992;326:1399–1404. doi: 10.1056/NEJM199205213262105. screening and curative treatment for baby boomers. Hepatology 2017;66: [53] Minhas U, Matrachisia J, Picano JD, Martinez A. Hepatitis C screening in a high 1805–1813. doi: 10.1002/hep.29362. risk urban emergency department demonstrates high prevalence rates [31] Teply R, Mukherjee S, Goodman M, Guck T. Impact of a hepatitis C virus among birth cohort patients. Gastroenterology 2019;156:S-1340. electronic medical record screening alert for baby boomers. J Hepatol [54] Cornett JK, Bodiwala V, Razuk V, Shukla D, Narayanan N. Results of a hep- 2018;68:S327–S328. atitis C virus screening program of the 1945-1965 birth cohort in a large [32] Yeboah-Korang A, Beig MI, Khan MQ, Goldstein JL, Macapinlac DM, Maurer D, emergency department in New Jersey. Open Forum Infect Dis 2018;5: et al. Hepatitis C screening in commercially insured U.S. birth-cohort ofy065. doi: 10.1093/ofid/ofy065. patients: Factors associated with testing and effect of an EMR-based screen- [55] Hsieh YH, Rothman RE, Laeyendecker OB, Kelen GD, Avornu A, Patel EU, ing alert. J Transl Int Med 2018;6:82–89. doi: 10.2478/jtim-2018-0012. et al. Evaluation of the Centers for Disease Control and Prevention recom- [33] Soo S, Mukhtar NA, Senussi N, Baxter L, Kowdley KV. Implementation of a mendations for hepatitis C virus testing in an urban emergency department. health maintenance alert successfully increased rates of HCV screening Clin Infect Dis 2016;62:1059–1065. doi: 10.1093/cid/ciw074. across a large health care system serving the western United States. Hep- [56] Lyons MS, Kunnathur VA, Rouster SD, Hart KW, Sperling MI, Fichtenbaum CJ, atology 2018;68:306A. et al. Prevalence of diagnosed and undiagnosed hepatitis C in a midwestern

40 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 Tsay C.J. et al: Birth cohort screening for HCV

urban emergency department. Clin Infect Dis 2016;62:1066–1071. doi: 10. [70] Testing for HCV. infection: an update of guidance for clinicians and labora- 1093/cid/ciw073. torians. MMWR Morb Mortal Wkly Rep 2013;62:362–365. [57] Allison WE, Chiang W, Rubin A, O’Donnell L, Saldivar MA, Maurantonio M, [71] Alter MJ, Kuhnert WL, Finelli L. Guidelines for laboratory testing and result et al. Hepatitis C virus infection in the 1945-1965 birth cohort (baby reporting of antibody to hepatitis C virus. Centers for Disease Control and boomers) in a large urban ED. Am J Emerg Med 2016;34:697–701. doi: Prevention. MMWR Recomm Rep 2003;52:1-13, 15; quiz CE1-4. 10.1016/j.ajem.2015.12.072. [72] Smith BD, Drobeniuc J, Jewett A, Branson BM, Garfein RS, Teshale E, et al. [58] Galbraith JW, Franco RA, Donnelly JP, Rodgers JB, Morgan JM, Viles AF, et al. Evaluation of three rapid screening assays for detection of antibodies to hep- Unrecognized chronic hepatitis C virus infection among baby boomers in the atitis C virus. J Infect Dis 2011;204:825–831. doi: 10.1093/infdis/jir422. emergency department. Hepatology 2015;61:776–782. doi: 10.1002/hep. [73] Tang W, Chen W, Amini A, Boeras D, Falconer J, Kelly H, et al. Diagnostic 27410. accuracy of tests to detect Hepatitis C antibody: a meta-analysis and [59] White DA, Anderson ES, Pfeil SK, Trivedi TK, Alter HJ. Results of a rapid review of the literature. BMC Infect Dis 2017;17:695. doi: 10.1186/s12879- hepatitis C virus screening and diagnostic testing program in an urban emer- 017-2773-2. – gency department. Ann Emerg Med 2016;67:119 128. doi: 10.1016/j. [74] Shivkumar S, Peeling R, Jafari Y, Joseph L, Pant Pai N. Accuracy of rapid and annemergmed.2015.06.023. point-of-care screening tests for hepatitis C: a systematic review and meta- [60] Schechter-Perkins EM, Miller NS, Hall J, Hartman JJ, Dorfman DH, Andry C, analysis. Ann Intern Med 2012;157:558–566. doi: 10.7326/0003-4819- et al. Implementation and preliminary results of an emergency department 157-8-201210160-00006. nontargeted, opt-out hepatitis C virus screening program. Acad Emerg Med [75] Zou B, Yeo YH, Le MH, Henry L, Chang ET, Lok AS, et al. Prevalence of viremic 2018;25:1216–1226. doi: 10.1111/acem.13484. hepatitis C virus infection by age, race/ethnicity, and birthplace and disease [61] Snelgrove CA. A quality improvement initiative: making hepatitis C screen- awareness among viremic persons in the United States, 1999-2016. J Infect ing a standard of care. Hepatology 2018;68:317A. Dis 2020;221:408–418. doi: 10.1093/infdis/jiz479. [62] Hyun C, Hwang C, Lee S. HCV screening and linkage to care of baby boomers [76] Udompap P, Mannalithara A, Kwong AJ, Kim D, Kim WR. An alternative at community hospital emergency department using streamlined EHR order- screening strategy for hepatitis C virus (HCV) infection among Americans ing. Hepatology 2017;66:547A. not belonging in the baby boomer birth cohort. Gastroenterology 2017; [63] Patel RC, Vellozzi C, Smith BD. Results of hepatitis C birth-cohort testing and 152:S1078. linkage to care in selected U.S. sites, 2012-2014. Public Health Rep 2016; [77] Eckman MH, Ward JW, Sherman KE. Cost effectiveness of universal screening 131 Suppl 2:12–19. doi: 10.1177/00333549161310S203. for hepatitis C virus infection in the era of direct-acting, pangenotypic treat- [64] Travis N, Fluker S, Miller L. Medical assistant driven improvement in hepatitis – C screening rates in the primary care setting. Hepatology 2018;68:308A. ment regimens. Clin Gastroenterol Hepatol 2019;17:930 939.e9. doi: 10. [65] Dong BJ, Lopez M, Cocohoba J. Pharmacists performing hepatitis C antibody 1016/j.cgh.2018.08.080. point-of-care screening in a community pharmacy: A pilot project. J Am Pharm [78] Bian J, Schreiner AD. Population-based screening of hepatitis C virus in the – Assoc (2003) 2017;57:510–515.e2. doi: 10.1016/j.japh.2017.04.463. United States. Curr Opin Gastroenterol 2019;35:177 182. doi: 10. [66] Cartwright EJ, Rentsch C, Rimland D. Hepatitis C virus screening practices 1097/MOG.0000000000000520. and seropositivity among US veterans born during 1945-1965. BMC Res [79] Draft recommendation statement: Hepatitis C virus infection in adolescents Notes 2014;7:449. doi: 10.1186/1756-0500-7-449. and adults: Screening. Available from: https://www.uspreventiveservices- [67] Sarkar S, Esserman DA, Skanderson M, Levin FL, Justice AC, Lim JK. Dispar- taskforce.org/Page/Document/draft-recommendation-statement/hepatitis- ities in hepatitis C testing in U.S. veterans born 1945-1965. J Hepatol 2016; c-screening1. 65:259–265. doi: 10.1016/j.jhep.2016.04.012. [80] Center for Disease Control and Prevention. Recommendations for hepatitis C [68] Ross DB, Belperio PS, Chartier M, Backus LI. Hepatitis C testing in U.S. vet- screening among adults-2019; Request for comment. Available from: https: erans born 1945-1965: An update. J Hepatol 2017;66:237–238. doi: 10. //www.federalregister.gov/documents/2019/10/28/2019-23521/recom- 1016/j.jhep.2016.09.018. mendations-for-hepatitis-c-screening-among-adults-2019-request-for- [69] Justice AC, Esserman D, Sarkar S, Levin FL, Skanderson M, Lim JK. Reply to: comment. “Hepatitis C testing in U.S. veterans born 1945-1965: An update”. J Hepatol [81] AASLD-IDSA HCV guidance: Recommendations for testing, managing, and 2017;66:239. doi: 10.1016/j.jhep.2016.09.019. treating hepatitis C. Available from: https://www.hcvguidelines.org/.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 25–41 41 Review Article

Pathophysiology and Prevention of Paracentesis-induced Circulatory Dysfunction: A Concise Review

Anand V Kulkarni*1, Pramod Kumar1, Mithun Sharma1, T R Sowmya1, Rupjyoti Talukdar2, Padaki Nagaraj Rao1 and D Nageshwar Reddy2

1Department of Hepatology, Asian Institute of Gastroenterology, Hyderabad, India; 2Department of Gastroenterology, Asian Institute of Gastroenterology, Hyderabad, India

Abstract make it a less desirable option. Therapeutic paracentesis in patients with cirrhosis and tense ascites leads to circulatory Annually, 10% of cirrhotic patients with ascites develop dysfunction, which is termed paracentesis-induced circulatory refractory ascites for which large-volume paracentesis (LVP) dysfunction or post paracentesis circulatory dysfunction.6,7 is a frequently used therapeutic procedure. LVP, although a PICD usually occurs following LVP (>5–6 L) and results in safe method, is associated with circulatory dysfunction in a faster reaccumulation of ascites, hyponatremia, renal impair- significant percentage of patients, which is termed para- ment, and shorter survival.7 centesis-induced circulatory dysfunction (PICD). PICD results in faster reaccumulation of ascites, hyponatremia, renal Epidemiology impairment, and shorter survival. PICD is diagnosed through laboratory results, with increases of >50% of baseline plasma The incidence of PICD is estimated to be 80% among patients renin activity to a value $4 ng/mL/h on the fifth to sixth day not receiving any plasma volume expander, 33% to 38% in after paracentesis. In this review, we discuss the pathophysi- patients receiving polygeline/saline solution/dextran 70, and ology and prevention of PICD. 11% to 20% among patients receiving albumin.8,9 Citation of this article: Kulkarni AV, Kumar P, Sharma M, Talukdar R, Rao PN, Reddy DN. Pathophysiology and preven- The definition of PICD tion of paracentesis-induced circulatory dysfunction: A con- – cise review. J Clin Transl Hepatol 2020;8(1):42 48. doi: Gines et al.8 first defined PICD as an increase in plasma renin 10.14218/JCTH.2019.00048. activity (PRA) on the sixth day after paracentesis of more than 50% of the pretreatment value to a level > 4 ng/ml/h. The value of 4 ng/mL/h was determined based on 36 healthy volunteers who were on a 50 mmol/day sodium diet for 7 days. Introduction The mean value in healthy volunteers was 1.35 ± 0.94 ng/ mL/h (range, 0.1–4.0). Whether this value of 4 ng/mL/h Ascites is a major complication of liver cirrhosis, with an annual holds for patients with cirrhosis and refractory ascites who – 1 incidence of 5 10% in compensated cirrhosis. Dietary sodium have a hyperactive renin-angiotensin-aldosterone system is restriction and oral diuretics are the first treatment choices for still unclear. In the same study, 280 cirrhotic patients with 2,3 uncomplicated ascites. Annually, 10% of cirrhotic patients ascites had documented PRA value at baseline. The baseline with ascites develop refractory ascites, which is associated value in those who developed PICD (n = 85) was 8.0 ± 7.3 with a poor prognosis that reduces transplant-free survival to ng/ml/h, which was similar to those who did not (n = 195), 4 50% per year. In refractory ascites, transjugular intrahepatic i.e., 9.3 ± 11.5 ng/ml/h. The landmark study by Wilkinson portosystemic shunt (TIPS), increased doses of diuretics, or et al.10 showed that cirrhosis with refractory ascites would large-volume paracentesis (LVP) are the few effective meas- invariably have high PRA due to reduced renal blood flow ures apart from liver transplant in routine practice. Of these, with the highest value being 21.77 nmol/L/h and mean LVP is the preferred treatment of choice as it is faster, more value of 9.5 nmol/L/h, while those with ascites would have 5 4 effective, and associated with fewer side effects. TIPS, a value double that of the healthy controls (3.99 nmol/L/h although effective in a selected group of patients, may be asso- and 1.82 nmol/L/h, respectively). This indicates that the ciated with ascites recurrence and hepatic encephalopathy. In PRA needs to rise by >50% on day 6 in patients with cirrhosis addition, its high cost and lack of availability at some places and refractory ascites to be called PICD. The diagnosis of PICD is primarily based on the laboratory value of PRA and Keywords: Refractory ascites; Plasma renin activity; Large-volume paracentesis; can be diagnosed only after 5 to 6 days of paracentesis. Cirrhosis; Portal hypertension. Although it is a valid objective measure, the difficulties of Abbreviations: LVP, large-volume paracentesis; MAP, mean arterial pressure; PICD, paracentesis-induced circulatory dysfunction; PRA, plasma renin activity; measuring PRA due to the technical requirements (blood TIPS, transjugular intrahepatic portosystemic shunt. sample should be taken after overnight fasting and at least Received: 29 September 2019; Revised: 16 February 2020; Accepted: 23 February 1 h of bed rest), cost, and questionable benefit in patients 2020 with refractory ascites who undergo repeated paracentesis, *Correspondence to: Anand V Kulkarni, Department of Hepatology, Asian Insti- tute of Gastroenterology, Hyderabad, India. E-mail: [email protected], makes it a less favored option for clinicians. The PRA is 11,12 [email protected] dependent on age (PRA and plasma aldosterone levels

42 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48

Fig. 1. Mechanism of PICD and complications.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48 43 Kulkarni A.V. et al: Paracentesis-induced circulatory dysfunction colloid oncotic pressure and has a half-life of 15–21 days. albumin group (p = 0.03). When <6 L was tapped, the inci- With a molecular weight of 67 kDa and 607 amino acids, dence of PICD was similar in both groups (6.7% in saline vs. albumin is an asset for physicians. The free cysteine (Cys- 5.6% in the albumin group; p = 0.9). MAP, hemoglobin, and 34) residue accounts for the single free redox-active thiol potassium levels at baseline were predictors of PICD. The (-SH) moiety, which accounts for ;80% of extracellular anti- type of plasma expander was found to have a significant oxidant properties through nitrosylation, oxidation, and thio- preventive effect on PICD by both univariate and multivari- lation. Patients with ascites usually have hypoalbuminemia. ate analysis. Albumin was a better alternative when >6 L The causes of hypoalbuminemia are i) reduced hepatic syn- was tapped. thesis and secretion, ii) dilution by increased intravascular A pilot study by Richard Moreau et al.,31 conducted in and interstitial volume, and iii) loss in the ascitic fluid. The 2000 (published in 2006) comparing synthetic colloid combination of hypoalbuminemia and impaired albumin func- (3.5% polygeline) with albumin, concluded that albumin tion leads to marked disturbance in the transport, metabo- was more effective in preventing liver-related complications. lism, and excretion of many endogenous and exogenous The study was prematurely discontinued because of safety substances. Infusion of exogenous albumin in ascites patients concerns about bovine-derived products. A total of 68 may serve the dual purpose of replenishing the levels of cir- patients were enrolled: 30 in the albumin group and 38 in culating albumin and the functional activity of the albumin the polygeline group. One unit of the study colloid (blinded) pool. These are effects specific to albumin not shared by provided either 20 g albumin or 17.5 g polygeline. The other plasma volume expanders and may contribute to supe- amount transfused was fixed as 1 U if below 4 L, 2U if rior effectiveness. Albumin is available in preparations of 5%, between 4 L and below 6 L, 3U if between 6 L and below 8 20%, and 25% solutions, with a sodium content equivalent to L, and 4 U if 8 L or more. The patients were followed up for 6 that of serum. The studies on PICD have involved only 20% months, and the number of taps and complications due to albumin in either 8 g/L of ascitic fluid tapped or 4 g/L of ascitic liver disease was assessed. The polygeline group had a 1.6- fluid removed. fold higher risk of developing liver-related complications The first study was conducted by Pere Gines,29 who eval- than the albumin group. uated 105 patients with daily paracentesis of 4–6 L until the An open-label randomized study comparing half the dose disappearance of ascites. Fifty-two patients received of albumin (4 g/L of ascites tapped) with the conventional albumin (40 g with each tap), and the other group received dose (8 g/L ascites) concluded that half the dose was no volume expander. The number of complications (acute effective and economical for preventing the complications kidney injury [AKI] and hyponatremia) was significantly (PICD) of LVP.32 higher in patients who had no albumin infusion. However, Terlipressin33 Terlipressin (N-a-triglycyl-8-L-lysine-vas- the authors reported no mortality benefit with albumin infu- opressin) (formula: C52H74N16O15S2) is a vasopressin 1 (V1) sion. This landmark study paved the path for further studies receptor agonist with a molecular weight of 1227.4. It is a on albumin. cyclic dodecapeptide that exists as a white freeze-dried fluffy In 1996, Gines et al. compared albumin with dextran 70 powder, with a distribution half-life of ;8 m. Terlipressin and polygeline for the prevention of PICD.8 A total of 289 plasma concentrations decline exponentially following intra- patients undergoing paracentesis were enrolled, and it was venous IV administration, with an elimination half-life of reported that albumin decreased the incidence of PICD to approximately 1 h and plasma clearance of approximately 18.5% compared to 34.4% in the dextran 70 group and 9 ml/kg/min. Terlipressin, a prodrug, is cleaved by endopep- 37.8% in the polygeline group. The mean PRA was 9.2 ± tidases, resulting in slow release of lysine vasopressin, the 10.3 ng/ml/h in the albumin arm, 9.5 ± 11.7 in dextran active metabolite. Measurable concentrations of vasopressin 70, and 8.6 ± 9.8 in the polygeline group, indicating that appear in the plasma from about 20–30 m post-dose, the upper limit (i.e., two standard deviations) was about 30 ng/ peaking at 1–2 h post-dose. Although terlipressin is used in ml/h in some patients. PICD was associated with a shorter time to PICD, its short half-life requires it to be given for prolonged readmission and shorter survival. This was the first randomized periods to achieve a sustained MAP rise. Terlipressin study to demonstrate the superiority of albumin. increases systemic vascular resistance, reduces the portal A randomized study comparing saline versus albumin in pressure, and has been used for variceal bleed,34 hepatore- 2003 also showed significant benefits with albumin.30 nal syndrome (HRS),35 septic shock,36,37 and prevention of Seventy-two patients were enrolled (35 in the saline group PICD.38 and 37 in the albumin group). The baseline heart rate and A pilot study comparing the efficacy of terlipressin versus MAP in the saline group were 83.1 ± 8.6/m and 81.5 ± 12.2 albumin showed that terlipressin is as effective as albumin in mmHg and 82.4 ± 10.6/m and 83.1 ± 11.7 mmHg in the maintaining the arterial volume post paracentesis.38 The albumin group, respectively. The serum albumin was also authors randomly allotted 11 patients to terlipressin and 13 comparable with 2.67 ± 0.48 g/dL in the saline group and to the albumin arm. A total of 3 mg terlipressin was given, 2.56 ± 0.65 g/dL in the albumin group. The baseline PRA 1 mg at the onset of paracentesis, and then 1 mg at 8 h and value in the saline group was 5.4 ± 5.4, and in the albumin 16 h. Albumin was given at a dose of 8 g/L ascites removed. group was 5.2 ± 4.5 ng/mL/h. The volume of ascites Fifty percent within 2 h and 50% after 6 h of paracentesis. removed was comparable in both groups (>6 L) over a After completion, only 10 in each arm were included for anal- mean duration of ;90 min. The amount of albumin given ysis. The volume of ascites removed was similar in both was 260 ± 115.1 mL (20% albumin at 8 g/L ascites), and groups. They measured the plasma renin concentrations saline was 1075 ± 365 mL (170 mL of 3.5% saline solution (not the PRA). The number of patients (three in each arm) per L ascites removed at 999 mL/h). There were 10 patients who had decreased arterial blood volume (based on >50% with complications in the saline group compared to only 6 in decrease in plasma renin concentrations on day 6) did not the albumin group. The incidence of PICD (based on PRA differ between the two groups. Only one patient in each rise) was 33.3% in the saline group and only 11.4% in the group developed hyponatremia. There were no adverse

44 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48 Kulkarni A.V. et al: Paracentesis-induced circulatory dysfunction events noted in either group. The authors concluded that ter- midodrine in a fixed short-term dose is ineffective compared lipressin is as efficacious in maintaining the arterial blood to albumin. A total of 24 patients with 13 in the albumin arm volume post paracentesis by splanchnic vasoconstriction. and 11 in the midodrine arm were included. Midodrine was Another pilot study of 40 patients comparing terlipressin given at a dose of 12.5 mg three times a day for 2 days post with albumin showed that terlipressin is as efficacious as paracentesis and albumin at a dose of 8 g per L ascites 39 albumin in the prevention of PICD. The baseline PRA was removed. The causes of liver cirrhosis in the study were similar in both the treatment group with 19.15 ± 12.17 ng/ alcohol, hepatitis (the cause of hepatitis was not mentioned mL/h in the albumin group vs. 20.11 ± 10.60 ng/mL/h in the in the article), and idiopathic. The median volume of ascites terlipressin group. There were no side effects reported in the removed was 6.3 L. A total of 4 patients (2 in each arm) study; neither there was any difference in the occurrence of recruited were in Child’s class B, and 20 were Child C cir- PICD. There was an insignificant decline in sodium levels 6 rhotic (11 in the albumin and 9 in the midodrine groups). days after paracentesis, which was also similar in both PICD developed in six patients in the midodrine group groups. The authors concluded that terlipressin is equivalent (60%) and four patients (31%) in the albumin group. None to albumin. of the patients in the albumin group developed AKI com- Midodrine40 Midodrine is an a1-agonist whose active pared to two in the midodrine arm. Hyponatremia was metabolite is desglymidodrine. It acts on the alpha-adrener- noted in seven patients in the albumin group compared to gic receptors of the arteriolar and venous vasculature, pro- four in the midodrine group. Plasma renin concentration ducing an increase in vascular tone and elevation of blood increased from 677.5 to 1337.5 mU/mL in the midodrine pressure. It is rapidly absorbed orally. The bioavailability of midodrine is similar, whether given intravenously or orally. arm, whereas in the albumin arm, it increased from 385 to m Plasma levels of the prodrug peak after about 0.5 h and 402 U/mL on day 6. There were no side effects, even with decline with a half-life of approximately 25 m. Blood concen- 37.5 mg midodrine/day. 45 trations of desglymidodrine peak at 1 to 2 h after a dose of Noradrenaline Noradrenaline is the principal neuro- midodrine and has a half-life of about 3 to 4 h. The absolute transmitter of the sympathetic nervous system produced by oral bioavailability is 93% and is unaffected by food. Neither the adrenal medulla. Noradrenaline is preferred over adrena- midodrine nor desglymidodrine is bound significantly to line in shock as it causes global vasoconstriction, while adre- plasma proteins, and 80% of the drug is eliminated via the naline leads to venoconstriction and promotes venous return renal route by active secretion. An increase in PRA after short- to the heart. Noradrenaline is a potent alpha agonist. term noradrenaline infusion (also an alpha-adrenergic Although noradrenaline is an economical choice, its benefits agonist) has been demonstrated in patients with hepatorenal in the prevention of PICD are not well known. As the duration syndrome, suggesting a vasoconstrictive effect on the renal of action is only 1–2 m, it needs to be given by slow infusion. artery.41 It is unknown whether midodrine also exerts similar Noradrenaline infusion can cause severe peripheral and vis- effects. Midodrine has been used for refractory ascites,42 ceral vasoconstriction leading to a decrease in renal blood HRS,35 and prevention of PICD.43 flow by 20% and cerebral blood flow by 10% without affecting Two small pilot studies done on midodrine had conflicting the skeletal muscle and liver blood flow.46 Post paracentesis, results. One pilot study with 40 patients concluded that cirrhotics develop hypovolemia, and the systemic effects can 43 midodrine is as effective as albumin. Midodrine was be worsened by noradrenaline infusion. Moreover, cirrhotics administered at a dose of 5–10 mg (mean 8.62 ± 1.51mg) with ascites have higher levels of norepinephrine, but the three times per day to maintain a MAP at 10mmhg higher receptors are desensitized; hence, norepinephrine infusion than baseline. Albumin was given at a dose of 8 g/L ascitic is less effective in cirrhotics.47 Norepinephrine has been fluid removed (mean 48.4 ± 12.1 g). PRA at baseline and 6 used in shock,37 HRS,48 and PICD.49 days post paracentesis did not differ among the two groups. A small pilot study comparing noradrenaline with albumin Alcohol was the most common etiology of liver disease in reported that noradrenaline is an economical and efficacious both groups. Other causes of liver disease were hepatitis B choice over albumin.49 Noradrenaline was started in a contin- virus, hepatitis C virus, autoimmune, and idiopathic. Most uous infusion at an initial dose of 0.5 mg/h with a maximum interestingly, even with such a high Child’s score (;10), dose given up to 3 mg/h. The infusion rate was titrated in patients had preserved serum albumin levels of 3.2 ± 0.54 steps of 0.125 mg/h to maintain the MAP at 10 mmHg g/dL in the albumin group and 3.17 ± 0.49 g/dL in the mido- above the baseline. The MAP was maintained at that level drine group. Two patients had an increase in PRA by >50% for the next 72 h with close monitoring. Effective arterial from baseline in the albumin group, but none in the mido- blood volume, as indicated by PRA before and 6 days after drine group had significant PRA elevation. None developed ; AKI or hyponatremia, although there was a significant paracentesis, did not differ in the two groups (mean 20 decrease in serum sodium levels on day 6 in both groups. ng/mL/h). All patients underwent LVP. Two patients in the A significant increase in 24-h urine volume and urine sodium noradrenaline group and one in the albumin group had a excretion was observed in the midodrine group. One patient rise in PRA by >50% on the sixth day. None of the patients in the albumin group developed a febrile reaction, and one developed renal impairment or hyponatremia. Only one patient in the midodrine group developed headaches and patient receiving noradrenaline complained of restlessness. pruritis. Only three patients (two in the albumin group and Further, one patient in each arm developed spontaneous bac- one in the midodrine group) required repeat paracentesis terial peritonitis (SBP) on follow-up. within 3 months of the treatment. This was a randomized Contraindications to LVP: LVP leads to an accentuation of trial with a follow-up of 3 months and also reported the arteriolar vasodilation, which is already present in cir- adverse events. rhotic patients with ascites. Therefore, LVP should be deferred A pilot study by Appenrodt et al.44 evaluating the role of in patients with spontaneous bacterial peritonitis (SBP), midodrine in prevention PICD after LVP42 concluded that hepatorenal syndrome, and variceal bleed.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48 45 Kulkarni A.V. et al: Paracentesis-induced circulatory dysfunction

Combination therapies to no treatment, albumin reduced the odds of PICD by 93%, 61% when compared to another volume expander, and 30% A large trial of 150 patients (presented as an abstract at a when compared to a vasoconstrictor. Compared to untreated major conference) on combination therapy of vasoconstric- control patients in whom 16.5% developed hyponatremia, tor and albumin showed a significant decrease in the number only ;4% developed hyponatremia in the albumin group. of complications in patients receiving midodrine with In total, albumin reduced the odds of developing PICD by albumin.50 The benefit was lacking when terlipressin was 66%, hyponatremia by 42%, and death by 36%. used with albumin. However, terlipressin was given for only Another meta-analysis51 that included 16 trials on 1 day, whereas midodrine was given for 3 days. Complica- tions such as pain abdomen, headache, and bradycardia albumin concluded that albumin use significantly reduced were common in the terlipressin arm and whereas patients the risk of paracentesis-induced circulatory dysfunction, receiving midodrine experienced urinary retention, head- but there was a nonsignificant difference in complications ache, hypertension, and anxiety. The combination therapy and mortality. The authors also noted that there was a sig- should be further explored for the prevention and treatment nificant reduction in mortality and renal impairment when of PICD. albumin was used in cirrhosis with infection. A recent Cochrane review,52 which included 27 random- Meta-analysis ized trials, concluded that there was neither any benefit nor any adverse effect in using any plasma expander, including A meta-analysis by Bernardi et al. in 2012, which compared albumin, polygeline, dextrans, hydroxyethyl starch, intra- albumin with other therapies such as colloids or vasocon- venous infusion of ascitic fluid, and crystalloids in patients strictors, showed that albumin remains the best choice for undergoing paracentesis. The data on albumin/volume 9 patients undergoing LVP. Seventeen randomized controlled expanders originated from a few, small, short-term trials trials, reported from 1988 to 2010 with a total of 1,225 that were at high risk of systemic errors, selection bias, patients, met all selection criteria and were included and random errors. They also suggested that more exten- (Table 1). The mean age of the study population ranged sive trials with adherence to CONSORT and SPIRIT guide- from 46.9 to 61.4 years. Seventy-four percent were males lines are required to assess the role of plasma expanders, (range, 60.0-90.0%) with mean baseline serum albumin ranging from 26 to 29 g/L, and the mean Child-Pugh score including albumin, in paracentesis. The review did not con- ranged from 8.8 to 11.0. The mean volume of ascitic fluid sider trials comparing vasoconstrictors with volume removed ranged from 5.5 to 15.9 L. The dose of albumin expanders. The advantages and disadvantages of the varied from 5–10 g/L of fluid tapped. The vasoconstrictors drugs that have been used for PICD prevention are shown were terlipressin, midodrine, and noradrenaline. Compared in Table 2.

Table 1. Meta-analysis on the role of albumin in patients undergoing paracentesis

No. of trials Author included Characteristics of trials included Conclusions

Bernardi et al.9, 17 (n = 225 Nine trials comparing albumin with other Albumin remains superior to other 2012 participants) volume expanders formed the leading treatment modalities, and further added category, i.e. 74% (903 of 1,225) of all that the combination of vasoconstrictor patients in the meta-analysis, compared with albumin would further decrease the with 13% each for trials with no incidence of PICD, which has not been treatment or vasoconstrictor as the investigated to date. control regimen. Kwok et al.51, 16 (n = 1518 Four studies with no active comparator, Albumin use significantly reduced risk of 2013 participants) and eight studies comparing plasma paracentesis-induced circulatory expander to albumin, and four other dysfunction, but there was a studies of cirrhotic patients with infection nonsignificant difference in complications compared the use of antibiotics with and and mortality. without albumin Simonetti 27 (n = 1592 Five of the trials assessed plasma There was neither any benefit nor any et al.52, 2019 participants) expanders (albumin in four trials and adverse effect in using any plasma ascitic fluid in one trial) versus no plasma expanders including albumin, polygeline, expander. The remaining 22 trials dextrans, hydroxyethyl starch, assessed one type of plasma expander, i. intravenous infusion of ascitic fluid, and e. dextran, hydroxyethyl starch, crystalloids in patients undergoing polygeline, intravenous infusion of paracentesis. ascitic fluid, crystalloids, or mannitol versus another type of plasma expander, i.e. albumin in 20 of these trials and polygeline in one trial.

46 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48 Kulkarni A.V. et al: Paracentesis-induced circulatory dysfunction

Table 2. Drugs used in the prevention of PICD Way forward Drug Advantages Disadvantages More extensive randomized trials involving a larger sample Albumin Evidence from a Cost size on the prevention of PICD with vasoconstrictors (with or higher number of Need for IV without volume expanders) with clinical endpoints other than trials infusion laboratory-based are suggested. Terlipressin Efficacy similar to Evidence from Funding albumin small pilot studies None to declare. Noradrenaline Efficacy similar to Adverse events albumin Economical are common Conflict of interest Need for IV infusion/ The authors have no conflict of interests related to this admission publication. Midodrine Good oral Small pilot bioavailability studies Author contributions Maintains MAP Less studied effectively Efficacy still Study concept and design (AVK, MS, STR), compilation and Can be given in controversial critical revision (AVK, MS, RT, PK, PNR, and DNR). All daycare/outpatient members approved the final draft.

Abbreviations: IV, intravenous; MAP, mean arterial pressure. References

Table 3. Differential diagnosis of PICD complication [1] Ginés P, Quintero E, Arroyo V, Terés J, Bruguera M, Rimola A, et al. Compen- sated cirrhosis: natural history and prognostic factors. Hepatology 1987;7: Complication of PICD Common differentials in practice 122–128. doi: 10.1002/hep.1840070124. [2] Bernardi M, Laffi G, Salvagnini M, Azzena G, Bonato S, Marra F, et al. Efficacy Hyponatremia Diuretic induced/dilutional and safety of the stepped care medical treatment of ascites in liver cirrhosis: Encephalopathy Type B/C encephalopathy a randomized controlled clinical trial comparing two diets with different sodium content. Liver 1993;13:156–162. doi: 10.1111/j.1600-0676.1993. Dyselectrolytemia tb00624.x. Uremia [3] Angeli P, Dalla Pria M, De Bei E, Albino G, Caregaro L, Merkel C, et al. Randomized clinical study of the efficacy of amiloride and potassium canre- Acute kidney injury Diuretic-induced noate in nonazotemic cirrhotic patients with ascites. Hepatology 1994;19: 72–79. doi: 10.1002/hep.1840190113. [4] Bureau C, Thabut D, Oberti F, Dharancy S, Carbonell N, Bouvier A, et al. Transjugular intrahepatic portosystemic shunts with covered stents increase Differential diagnosis transplant-free survival of patients with cirrhosis and recurrent ascites. Gas- troenterology 2017;152:157–163. doi: 10.1053/j.gastro.2016.09.016. [5] Ginés P, Arroyo V, Quintero E, Planas R, Bory F, Cabrera J, et al. Comparison of The complications of PICD are usually attributed to cirrhosis paracentesis and diuretics in the treatment of cirrhotics with tense ascites. of the liver and are thought to be a complication of the disease Results of a randomized study. Gastroenterology 1987;93:234–241. doi: 10. (cirrhosis). The usual differentials, which are considered in 1016/0016-5085(87)91007-9. practice while missing the history of LVP, are shown in Table 3. [6] Runyon BA. Introduction to the revised American Association for the Study of Liver Diseases Practice Guideline management of adult patients with ascites due to cirrhosis 2012. Hepatology 2013;57:1651–1653. doi: 10.1002/hep. Treatment of PICD 26359. [7] EASL clinical practice guidelines on the management of ascites, spontaneous Volume expansion with albumin infusion is the mainstay of bacterial peritonitis, and hepatorenal syndrome in cirrhosis. J. Hepatol 2010; 53:397–417. doi: 10.1016/j.jhep.2010.05.004. treatment. The use of vasoconstrictors can also help in [8] Ginès A, Fernández-Esparrach G, Monescillo A, Vila C, Domènech E, Abecasis treatment but needs to be evaluated. However, avoiding R, et al. Randomized trial comparing albumin, dextran 70, and polygeline in paracentesis and withholding diuretics/beta-blockers during cirrhotic patients with ascites treated by paracentesis. Gastroenterology – PICD prevents the worsening of hemodynamics and 1996;111:1002 1010. doi: 10.1016/s0016-5085(96)70068-9. [9] Bernardi M, Caraceni P, Navickis RJ, Wilkes MM. Albumin infusion in patients encephalopathy. undergoing large-volume paracentesis: a meta-analysis of randomized trials. Hepatology 2012;55:1172–1181. doi: 10.1002/hep.24786. Conclusions [10] Wilkinson SP, Smith IK, Williams R. Changes in plasma renin activity in cirrhosis: a reappraisal based on studies in 67 patients and “low-renin” cirrhosis. Hypertension 1979;1:125–129. doi: 10.1161/01.hyp.1.2.125. PICD is a dreaded complication of paracentesis and is [11] Bauer JH. Age-related changes in the renin-aldosterone system. Physiolog- associated with a high incidence of morbidity and mortality. ical effects and clinical implications. Drugs Aging 1993;3:238–245. doi: 10. It is often overlooked and is a commonly missed diagnosis. 2165/00002512-199303030-00005. The differentials in any patient presenting with worsening of [12] James GD, Sealey JE, Müller F, Alderman M, Madhavan S, Laragh JH. Renin relationship to sex, race and age in a normotensive population. J Hypertens complications of cirrhosis should include PICD. The diagnosis Suppl 1986;4:S387–S389. is clinical, and the measurement of PRA may or may not aid in [13] Sharma AM, Schorr U, Oelkers W, Distler A. Effects of sodium salts on plasma diagnosis. PICD should be redefined as any complication renin activity and norepinephrine response to orthostasis in salt-sensitive – (kidney injury, hyponatremia, encephalopathy, variceal normotensive subjects. Am J Hypertens 1993;6:780 785. doi: 10. 1093/ajh/6.9.780. bleed) developing after paracentesis with or without a con- [14] Stern N, Sowers JR, McGinty D, Beahm E, Littner M, Catania R, et al. Circa- current rise in PRA. dian rhythm of plasma renin activity in older normal and essential

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48 47 Kulkarni A.V. et al: Paracentesis-induced circulatory dysfunction

hypertensive men: relation with inactive renin, aldosterone, cortisol and REM troesophageal variceal hemorrhage. Hepatology 2014;60:954–963. doi: 10. sleep. J Hypertens 1986;4:543–550. doi: 10.1097/00004872-198610000- 1002/hep.27006. 00005. [35] Cavallin M, Kamath PS, Merli M, Fasolato S, Toniutto P, Salerno F, et al. Terli- [15] Cohen EL, Conn JW, Rovner DR. Postural augmentation of plasma renin activ- pressin plus albumin versus midodrine and octreotide plus albumin in the ity and aldosterone excretion in normal people. J Clin Invest 1967;46:418– treatment of hepatorenal syndrome: A randomized trial. Hepatology 2015; 428. doi: 10.1172/JCI105543. 62:567–574. doi: 10.1002/hep.27709. [16] Lijnen P, Fagard R, Staessen J, Amery A. Effect of chronic diuretic treatment [36] Morelli A, Ertmer C, Rehberg S, Lange M, Orecchioni A, Cecchini V, et al. on the plasma renin-angiotensin-aldosterone system in essential hyperten- Continuous terlipressin versus vasopressin infusion in septic shock (TERLI- sion. Br J Clin Pharmacol 1981;12:387–392. doi: 10.1111/j.1365-2125. VAP): a randomized, controlled pilot study. Crit Care 2009;13:R130. doi: 10. 1981.tb01231.x. 1186/cc7990. [17] Seifarth C, Trenkel S, Schobel H, Hahn EG, Hensen J. Influence of antihyper- [37] Choudhury A, Kedarisetty CK, Vashishtha C, Saini D, Kumar S, Maiwall R, et tensive medication on aldosterone and renin concentration in the differential al. A randomized trial comparing terlipressin and noradrenaline in patients diagnosis of essential hypertension and primary aldosteronism. Clin Endo- with cirrhosis and septic shock. Liver Int 2017;37:552–561. doi: 10. crinol (Oxf) 2002;57:457–465. doi: 10.1046/j.1365-2265.2002.01613.x. 1111/liv.13252. [18] Cabrera J, Falcón L, Gorriz E, Pardo MD, Granados R, Quinones A, et al. [38] Singh V, Kumar R, Nain CK, Singh B, Sharma AK. Terlipressin versus albumin Abdominal decompression plays a major role in early postparacentesis hae- in paracentesis-induced circulatory dysfunction in cirrhosis: a randomized modynamic changes in cirrhotic patients with tense ascites. Gut 2001;48: study. J Gastroenterol Hepatol 2006;21:303–307. doi: 10.1111/j.1440- – 384 389. doi: 10.1136/gut.48.3.384. 1746.2006.04182.x. [19] Campbell PJ, Skorecki KL, Logan AG, Wong PY, Leung WM, Greig P, et al. [39] Moreau R, Asselah T, Condat B, de Kerguenec C, Pessione F, Bernard B, et al. Acute effects of peritoneovenous shunting on plasma atrial natriuretic Comparison of the effect of terlipressin and albumin on arterial blood volume peptide in cirrhotic patients with massive refractory ascites. Am J Med in patients with cirrhosis and tense ascites treated by paracentesis: a rand- – 1988;84:112 119. doi: 10.1016/0002-9343(88)90017-4. omised pilot study. Gut 2002;50:90–94. doi: 10.1136/gut.50.1.90. [20] Shaw-Stiffel T, Campbell PJ, Sole MJ, Greig P, Wong PY, Blendis LM. Renal [40] McTavish D, Goa KL. Midodrine. A review of its pharmacological properties prostaglandin E2 and other vasoactive modulators in refractory hepatic and therapeutic use in orthostatic hypotension and secondary hypotensive ascites: response to peritoneovenous shunting. Gastroenterology 1988; disorders. Drugs 1989;38:757–777. doi: 10.2165/00003495-198938050- – 95:1332 1338. doi: 10.1016/0016-5085(88)90369-1. 00004. [21] Greig PD, Langer B, Blendis LM, Taylor BR, Glynn MF. Complications after [41] Saló J, Ginès A, Quer JC, Fernández-Esparrach G, Guevara M, Ginès P, et al. – peritoneovenous shunting for ascites. Am J Surg 1980;139:125 131. doi: Renal and neurohormonal changes following simultaneous administration of 10.1016/0002-9610(80)90241-x. systemic vasoconstrictors and dopamine or prostacyclin in cirrhotic patients [22] Peltekian KM, Wong F, Liu PP, Logan AG, Sherman M, Blendis LM. Cardiovas- with hepatorenal syndrome. J Hepatol 1996;25:916–923. doi: 10. cular, renal, and neurohumoral responses to single large-volume paracent- 1016/s0168-8278(96)80297-2. esis in patients with cirrhosis and diuretic-resistant ascites. Am J [42] Singh V, Singh A, Singh B, Vijayvergiya R, Sharma N, Ghai A, et al. Midodrine Gastroenterol 1997;92:394–399. and clonidine in patients with cirrhosis and refractory or recurrent ascites: a [23] Ruiz-del-Arbol L, Monescillo A, Jimenéz W, Garcia-Plaza A, Arroyo V, Rodés J. randomized pilot study. Am J Gastroenterol 2013;108:560–567. doi: 10. Paracentesis-induced circulatory dysfunction: mechanism and effect on 1038/ajg.2013.9. hepatic hemodynamics in cirrhosis. Gastroenterology 1997;113:579–586. [43] Singh V, Dheerendra PC, Singh B, Nain CK, Chawla D, Sharma N, et al. Mido- doi: 10.1053/gast.1997.v113.pm9247479. drine versus albumin in the prevention of paracentesis-induced circulatory [24] Elsabaawy MM, Abdelhamid SR, Alsebaey A, Abdelsamee E, Obada MA, dysfunction in cirrhotics: a randomized pilot study. Am J Gastroenterol 2008; Salman TA, et al. The impact of paracentesis flow rate in patients with liver 103:1399–1405. doi: 10.1111/j.1572-0241.2008.01787.x. cirrhosis on the development of paracentesis induced circulatory dysfunc- [44] Appenrodt B, Wolf A, Grünhage F, Trebicka J, Schepke M, Rabe C, Lammert F, tion. Clin Mol Hepatol 2015;21:365–371. doi: 10.3350/cmh.2015.21.4.365. et al. Prevention of paracentesis-induced circulatory dysfunction: midodrine [25] Simon DM, McCain JR, Bonkovsky HL, Wells JO, Hartle DK, Galambos JT. vs albumin. A randomized pilot study. Liver Int 2008;28:1019–1025. doi: Effects of therapeutic paracentesis on systemic and hepatic hemodynamics 10.1111/j.1478-3231.2008.01734.x. and on renal and hormonal function. Hepatology 1987;7:423–429. doi: 10. [45] Hardman JG, Limbird LE, Gilman AG. Goodman and Gilman’s The Pharmaco- 1002/hep.1840070302. logical Basis of Therapeutics. 11th edition. New York, NY: McGraw-Hill 2006, [26] Caironi P, Gattinoni L. The clinical use of albumin: the point of view of a p. 248 specialist in intensive care. Blood Transfus 2009;7:259–267. doi: 10. [46] Swan HJ. Noradrenaline, adrenaline, and the human circulation. Br Med J 2450/2009.0002-09. – [27] Caraceni P, Tufoni M, Bonavita ME. Clinical use of albumin. Blood Transfus 1952;1:1003 1006. doi: 10.1136/bmj.1.4766.1003. 2013;11 Suppl 4:s18–25. doi: 10.2450/2013.005s [47] Nicholls KM, Shapiro MD, Van Putten VJ, Kluge R, Chung HM, Bichet DG, et al. [28] Garcia-Martinez R, Caraceni P, Bernardi M, Gines P, Arroyo V, Jalan R. Elevated plasma norepinephrine concentrations in decompensated cirrhosis. Albumin: pathophysiologic basis of its role in the treatment of cirrhosis and Association with increased secretion rates, normal clearance rates, and sup- – its complications. Hepatology 2013;58:1836–1846. doi: 10.1002/hep. pressibility by central blood volume expansion. Circ Res 1985;56:457 461. 26338. doi: 10.1161/01.res.56.3.457. [29] Ginès P, Titó L, Arroyo V, Planas R, Panés J, Viver J, et al. Randomized com- [48] Saif RU, Dar HA, Sofi SM, Andrabi MS, Javid G, Zargar SA. Noradrenaline parative study of therapeutic paracentesis with and without intravenous versus terlipressin in the management of type 1 hepatorenal syndrome: A – albumin in cirrhosis. Gastroenterology 1988;94:1493–1502. doi: 10. randomized controlled study. Indian J Gastroenterol 2018;37:424 429. doi: 1016/0016-5085(88)90691-9. 10.1007/s12664-018-0876-3. [30] Sola-Vera J, Miñana J, Ricart E, Planella M, González B, Torras X, et al. [49] Singh V, Kumar B, Nain CK, Singh B, Sharma N, Bhalla A, et al. Noradrenaline Randomized trial comparing albumin and saline in the prevention of para- and albumin in paracentesis-induced circulatory dysfunction in cirrhosis: a centesis-induced circulatory dysfunction in cirrhotic patients with ascites. randomized pilot study. J Intern Med 2006;260:62–68. doi: 10.1111/j.1365- Hepatology 2003;37:1147–1153. doi: 10.1053/jhep.2003.50169. 2796.2006.01654.x. [31] Moreau R, Valla DC, Durand-Zaleski I, Bronowicki JP, Durand F, Chaput JC, et [50] Kulkarni AV, Sukriti S, Maiwall R, Jindal A, Ali R, Thomas S. Midodrine with al. Comparison of outcome in patients with cirrhosis and ascites following albumin is safe and effective in preventing complications post-large volume treatment with albumin or a synthetic colloid: a randomised controlled pilot paracentesis-an open labelled randomised controlled trial (NCT-03144713). trail. Liver Int 2006;26:46–54. doi: 10.1111/j.1478-3231.2005.01188.x. Hepatology 2018;68:72A–73A. [32] Alessandria C, Elia C, Mezzabotta L, Risso A, Andrealli A, Spandre M, et al. [51] Kwok CS, Krupa L, Mahtani A, Kaye D, Rushbrook SM, Phillips MG, et al. Prevention of paracentesis-induced circulatory dysfunction in cirrhosis: Albumin reduces paracentesis-induced circulatory dysfunction and reduces standard vs half albumin doses. A prospective, randomized, unblinded pilot death and renal impairment among patients with cirrhosis and infection: a study. Dig Liver Dis 2011;43:881–886. doi: 10.1016/j.dld.2011.06.001. systematic review and meta-analysis. Biomed Res Int 2013;2013:295153. [33] Pesaturo AB, Jennings HR, Voils SA. Terlipressin: vasopressin analog and doi: 10.1155/2013/295153. novel drug for septic shock. Ann Pharmacother 2006;40:2170–2177. doi: [52] Simonetti RG, Perricone G, Nikolova D, Bjelakovic G, Gluud C. Plasma 10.1345/aph.1H373. expanders for people with cirrhosis and large ascites treated with abdominal [34] Seo YS, Park SY, Kim MY, Kim JH, Park JY, Yim HJ, et al. Lack of difference paracentesis. Cochrane Database Syst Rev 2019;6:CD004039. doi: 10. among terlipressin, somatostatin, and octreotide in the control of acute gas- 1002/14651858.CD004039.pub2.

48 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 42–48 Review Article

Clinical Updates in Primary Biliary Cholangitis: Trends, Epidemiology, Diagnostics, and New Therapeutic Approaches

Artin Galoosian*1, Courtney Hanlon2, Julia Zhang1, Edward W. Holt3 and Kidist K. Yimam4

1Department of Medicine, California Pacific Medical Center, San Francisco, CA, USA; 2Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA, USA; 3Department of Transplant, Division of Hepatology, California Pacific Medical Center, San Francisco, CA, USA; 4Director of the Autoimmune Liver Disease Program, Department of Transplant, Division of Hepatology, California Pacific Medical Center, San Francisco, CA, USA

Abstract with symptoms of pruritus, fatigue, other autoimmune diseases (including Sjogren’s syndrome, Hashimoto’s thyroiditis Primary biliary cholangitis, formerly known as primary biliary and/or rheumatoid arthritis), decreased bone mineral density, cirrhosis, is a chronic, autoimmune, and cholestatic disease hyperlipidemia, and xanthelasma.1,2 Without a prompt diag- ameliorating the biliary epithelial system causing fibrosis and nosis, treatment may be delayed; as such, the chronic inflam- end-stage liver disease, over time. Patients range from an mation and destruction of intrahepatic ducts contributes asymptomatic phase early in the disease course, to symp- significantly to disease-related mortality and morbidity. toms of decompensated cirrhosis later in its course. This The terminological and paradigm shift from “primary review focuses on the current consensus on the epidemiology, biliary cirrhosis” to “primary biliary cholangitis” reflects a diagnosis, and management of patients with primary biliary variety of changes within the PBC landscape in recent years, cholangitis. We also discuss established medical manage- including understanding of its pathogenesis and development ment as well as novel and investigational therapeutics in the of novel therapeutics. Since the majority of patients with the pipeline for management of PBC. diagnosis do not progress to cirrhosis, this change in the Citation of this article: Galoosian A, Hanlon C, Zhang J, Holt name was made to more accurately represent the histologic EW, Yimam KK. Clinical updates in primary biliary cholangitis: hallmarks of cholestasis and cholangitis.2 Trends, epidemiology, diagnostics, and new therapeutic ap- Despite ongoing efforts to improve treatment options for proaches. J Clin Transl Hepatol 2020;8(1):49–60. doi: patients with PBC, disease progression to cirrhosis and liver- 10.14218/JCTH.2019.00049. related complications contribute to recurrent hospitalizations, increased healthcare resource utilization, and increased morbidity and mortality overall.3 Improvement in the diagnosis, detection of early-stage disease, and understanding different Introduction treatment modalities are essential to reducing disease burden and complications. Thus, the aim of this paper is to elucidate Primary biliary cholangitis (PBC), formerly known as primary current trends in the epidemiology and diagnostic approaches biliary cirrhosis, is an autoimmune, T-cell-mediated condition of PBC by clarifying the current understanding of serologic and affecting the biliary epithelial cells. The granulomatous histologic factors associated with PBC. Additionally, we discuss destruction and subsequent necrosis of cholangiocytes the management of PBC, with use of both approved and inves- creates an accumulation of inflammatory infiltrates, which tigational agents, and discuss how to identify at-risk patients to eventually leads to cholestasis and fibrosis. Because of its reduce late-stage complications, morbidity, and mortality. cumulative nature, PBC can present as a spectrum of disease severity – from no symptoms to cholestasis to biliary cirrhosis resulting in end-stage liver disease. Patients may present Epidemiology

The overall prevalence and incidence of PBC remains low Keywords: Primary biliary cholangitis (PBC); Epidemiology; Treatment; UDCA; Obeticholic acid. compared to other liver disorders. PBC cases represented Abbreviations: AASLD, American Association for the Study of Liver Diseases; only 165 of the 8,250 liver transplantations done in 2018, ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMA, antimitochondrial according to the Organ Procurement and Transplantation antibody; APRI, aminotransferase to platelets ratio index; ASBT, apical sodium- Network.4,5 Despite not being a leading indication for liver dependent bile acid transporter; AST, aspartate aminotransferase; CI, confidence interval; FDA, Food and Drug Administration; FGF, fibroblast growth factor; FXR, transplantation, the overall global rate of PBC incidence con- 4,6–8 farnesoid X receptor; HCC, hepatocellular carcinoma; NASH, nonalcoholic steato- tinues to rise, with a marked increase since the 1980s. hepatitis; norUDCA, norursodeoxycholic acid; NOX, NADPH oxidase; OCA, obeti- Originally, PBC was thought to be a very rare disease, largely cholic acid; PBC, primary biliary cholangitis; PDC-E2, pyruvate dehydrogenase because of small sample sizes and lack of large longitudinal complex-dihydrolipoyltransacetylase; POISE, Perioperative Ischemic Evaluation Study; PPAR, peroxisome proliferator-activated receptor; PSC, primary sclerosing studies; moreover, reported prevalence and incidence data cholangitis; UDCA, ursodeoxycholic acid; ULN, upper limit of normal. have often been dramatically different between different Received: 7 October 2019; Revised: 21 December 2019; Accepted: 1 January studies and regions across the world (and even within differ- 2020 ent states in the USA).4 *Correspondence to: Artin Galoosian, Department of Medicine, California Pacific Medical Center, 1101 Van Ness Avenue, Suite 1120, San Francisco, CA 94109, In the USA, there are currently no longitudinal studies on USA. Tel: +1-310-560-2155, E-mail: [email protected] the epidemiology of PBC. In 2018, the Fibrotic Liver Disease

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Consortium reported a 12-year point prevalence of PBC of been accounted for by genetics.16,25–29 PBC frequently devel- 23.9 per 100,000 persons, but even this number varied ops around the ages of 40–60 years, with women more com- significantly by geographic region and patient demographics monly affected than men (at a ratio of 9:1).4,11,30 This female- within the USA.9 Their study found that prevalence increased to-male ratio also persists in other countries, such as Taipei by over 72% among women (33.5 to 57.8 per 100,000 (9:1), Japan (9.1:1), and mainland China (6.82:1).31,32 persons) and by over 114% among men (7.7 to 15.4 per Although there is a female predominance, men who develop 100,000 persons) during the 12-year period. Studies from PBC tend to have a more severe and aggressive disease Europe and Asia have also generally reported a steadily process with more severe lobular inflammation.33 Men are increasing prevalence of PBC within as early as the past also more likely to be unresponsive to the conventional first- decade.10,11 line treatment, ursodeoxycholic acid (UDCA).33

Geographic clusters Etiopathophysiology

It is estimated that the incidence of PBC ranges from 0.33 to Significant progress has been made in understanding the 5.8 per 100,000 persons, with the reported point prevalence immune responses involved in the pathophysiology of PBC. ranging from 1.91 to 33.8 per 100,000 persons, with large However, many aspects of the etiology and pathogenesis of differences seen in geographic region.12–15 Data on specific the destruction of biliary epithelial cells remains incompletely epidemiological trends and global incidence is scarce, again explained.4,23,34 It is known that PBC involves a predomi- largely due to the lack of a large longitudinal population- nantly T-cell mediated destruction of intrahepatic bile ducts, based studies and the marked geographic heterogeneity likely initiated when a genetically susceptible individual within different regions.4,8,16 comes into contact with an antigen in the environment such Both genetic and environmental factors contribute widely as an infectious pathogen, medication or other compound, to the pathogenesis of PBC, with epidemiologic data indicat- triggering an autoimmune event.2 ing variable prevalence rates of disease into distinct and Regardless of the autoimmune trigger, the disease begins different geographical areas, or clusters.17 For example, the with specific AMA targeting of the lipoic acid on the 2-oxo acid incidence of PBC developing in a well-defined population from dehydrogenases, which are located on the inner mitochondrial Rochester, Minnesota (USA) was 4.5 per 100,000 person- membrane. This enzyme then targets the E2 subunit of the years for women, and 0.7 per 100,000 person-years for pyruvate dehydrogenase complex, which eventually leads to men, with age and gender-adjusted prevalence for women biliary epithelial cell destruction. In addition, peptides from being 65.4 per 100,000 persons and 12.1 per 100,000 viruses or bacteria that are structurally similar to pyruvate persons for men.18 Another study aimed to highlight the epi- dehydrogenase complex-dihydrolipoyltransacetylase (referred demiology of PBC in Hong Kong; this study found that the to as PDC-E2) epitopes can trigger an autoimmune response – average age/sex adjusted annual incidence rate increased through molecular mimicry.2,34 36 An increase in PDC-E2 may from 6.7 to 8.1 per million person-years and the age/sex lead to a loss of humoral tolerance and increases the differ- adjusted prevalence increased from 31.1 to 82.3 per million entiation of T-cells in the liver, resulting in damage to the intra- between 2000 and 2015.14 hepatic biliary epithelial cells, scarring, fibrosis, and ductal – Europe and North America have the highest reported destruction.2,34 36 The exposure of extraductular liver paren- prevalence of PBC worldwide, as reflected in the increasing chyma to bile acids contributes to hepatocyte injury, fibrosis, rate of PBC-related hospitalizations over the last 30 and eventually cirrhosis. years.7,16,19 Recently, a large population-based analysis of both inpatient and outpatient registries in Sweden found Clinical manifestations of PBC that the prevalence of PBC increased steadily from 5.0 to 20 34.6 per 100,000 persons from 1987 to 2014, respectively. Signs, symptoms, and associated autoimmune Data collected from an Italian cohort between 2014 to 2015 conditions reported that the point-prevalence of PBC was 27.9 per 21 100,000 people in Italy. The clinical presentation of PBC can vary greatly. Up to 50– In addition to the scarcity of large population-based 60% of patients with PBC are asymptomatic at the time of studies, misdiagnoses and underreporting of patients may diagnosis, and only present with abnormal liver function also contribute to the incidence and prevalence of PBC, as tests.37 Fatigue is one of the most common symptoms, which 5–10% of patients may be antimitochondrial antibody (AMA)- is present in almost 80% of patients with PBC; however, there 16,22,23 negative. Despite these limitations, data support the is no correlation between fatigue and disease severity or dura- observation that global rates of PBC are rising, as many coun- tion.2,19,22 It is reported that 20% of these patients will have tries continue to improve diagnostic accuracy, reporting concomitant thyroid disorders, making it important for patients 4,16,19,22,23 measures, and surveillance of PBC patients. with PBC to get yearly thyroid function testing.2 Pruritus is a common symptom of PBC, which tends to be worse at night, in Heterogeneity the heat, and during pregnancy, and can affect up to 20–70% of patients.22,38 Dermatologic findings including hyperpigmen- Heterogeneity in prevalence can in part be explained by tation, jaundice, xanthomas, xanthelasmas, xerosis, and der- regional variation in diagnostic awareness or access to care; matographism are all common among patients with PBC.23,39 however, several studies have suggested that environmental Patients with PBC often have other rheumatologic or autoim- exposures may play a role in the development of specific mune diseases as well, most commonly Sjogren’ssyndrome regional clusters of PBC.8,16,24 Some studies support a high and autoimmune thyroid diseases, which can affect up to concordance among monozygotic twins and human leukocyte 40–65% of patients.23,39 About 5–10% of patients will also antigen alleles; yet, only about 15% of PBC variability has present with cutaneous scleroderma, and up to 10% of

50 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 Galoosian A. et al: Updates in primary biliary cholangitis patients may have concomitant rheumatoid arthritis.39 During Liver biochemistry and diagnostic algorithm the end stages of PBC, patients may develop symptoms of portal hypertension, which is typically seen in patients with Previous diagnostic guidelines focused on serologic, histo- at least bridging fibrosis.22,30,40 logic, and immunologic testing for a diagnosis of PBC. In 2018, the AASLD released updated practice guidelines, in Natural history which they outlined the currently used diagnostic criteria for PBC.23 Accordingly, a diagnosis of PBC today is established The natural history and prognosis of PBC has changed and based on the presence of two of three of the following: improved significantly over the last several decades. The 1. An elevated serum-based ALP level (>1.5 times the upper limit disease, without any treatment, has gone from a slow, of normal (ULN)) progressive disease resulting in liver fibrosis and cirrhosis to 2. Histologic evidence of chronic nonsuppurative biliary ductal de- eventual hepatic decompensation and death, to a disease struction (florid duct lesion) process with slower rates of progression and fibrosis, and 3. The presence of AMA at a titer of 1:40 or greater higher rates of clinical remission.41,42 This can be explained ALP levels are not only used for diagnosis but are associated partially by both the earlier diagnosis and earlier treatment of with both the severity of inflammation and ductopenia.49 ALP PBC. Without treatment, the intrahepatic bile ducts are levels also reflect markers of cholestasis, and higher levels of destroyed, causing bile acid to build up within the liver, ALP are associated with higher risk of liver transplantation and leading to cholestasis. Cholestasis contributes to chronic death.50,51 Similarly, aminotransferase and IgM levels reflect granulomatous inflammation, which eventually leads to fibro- inflammation and the severity of periportal and lobular sis and the subsequent development of cirrhosis and portal necrosis.49,52,53 Bilirubin has also been studied in PBC as a hypertension, along with its associated complications, includ- potential early marker for hepatic dysfunction; in conjunction ing a predisposition to hepatocellular carcinoma (HCC).43 with low albumin and low platelets, an elevated serum bilirubin With increasing awareness and understanding of the may precede the development of cirrhosis and portal hyper- natural history of the disease, as well as earlier diagnosis tension, and has been shown to be an important biomarker to and prompt treatment initiation, the progression to fibrosis assess disease severity.49,52,53 The discovery of more serologic and cirrhosis among PBC patients is becoming increasingly markers known to be associated with PBC will aid considerably rarer, as demonstrated by the decrease in the indications of in the diagnosis of patients with atypical clinical presentations. liver transplantation for PBC patients.19,41,44 The introduction Patients with symptoms typical for autoimmune-related biliary and use of UDCA for treating PBC has impacted the natural disease or patients with features of PBC-autoimmune hepatitis history of the disease. overlap syndrome may benefit from more accurate markers of disease. Patients may also present with dyslipidemia, particu- Diagnostic criteria larly with an elevated high-density lipoprotein level.

Serological features Role of liver biopsy Environmental triggers and autoimmunity may play crucial immunologic roles in the development of PBC. The current As stated above, the diagnosis of PBC can be made on the basis etiologic understanding suggests that the development of PBC of clinical features, liver biochemical and autoantibody testing, requires both a genetic predisposition and exposure to an and imaging to exclude extrahepatic biliary obstruction. Liver unknown environmental trigger, thereby initiating the immu- biopsy is not required in most cases but can be helpful in cases nologic cascade that destroys biliary cells.5,38 Autoantibodies without a certain diagnosis by evaluating for concomitant are commonly found in patients with PBC and are often used to conditions such as autoimmune hepatitis and nonalcoholic aid diagnosis. Approximately 90–95% of patients with PBC steatohepatitis (NASH). Liver biopsy also holds prognostic have detectable AMA, an autoantibody that targets lipoic acid value, based on disease activity and fibrosis stage. A classic present on the 2-oxo-acid dehydrogenase complexes within histologic characteristic of PBC is chronic nonsuppurative the inner mitochondrial membrane that has high disease spe- cholangitis affecting the interlobular and septal bile ducts. cificity.23,27,45 A large proportion of these patients also have Fibrosis in PBC can be staged using the METAVIR system, elevated levels of antinuclear antibodies, alkaline phosphatase which is a four-point scale, in which stage 0 represents normal (ALP), polyclonal IgM, and inflammatory cytokines, which liver, stage 1 represents portal inflammation with or without include TNF-alpha, IFN-gamma, IL-1, and IL-6.5,27,38,45–47 florid duct lesions, stage 2 represents periportal fibrosis, stage 3 represents bridging fibrosis, and stage 4 represents cirrho- Approximately 50% of patients are found to have antinuclear 54,55 antibody positivity, most commonly of nuclear-rim or nuclear- sis. The presence of cirrhosis is associated with a worse dot patterns, which are highly specific for PBC.38,48 prognosis and increased risk of developing complications. Between 5–10% of patients with PBC are AMA-negative, leading to potential misdiagnosis and under-treatment of the Radiographic features and noninvasive imaging actual disease.16,22,23 The American Association for the Study modalities of Liver Diseases (AASLD) recently updated its practice guidelines to include the antibodies against sp100 and gp210 as Magnetic resonance cholangiopancreatography and/or mag- serum markers, with the intent of helping to identify patients netic resonance imaging are a noninvasive method of imaging who are negative for AMA, via indirect immunofluorescence. the intrahepatic and extrahepatic biliary tree. These imaging These PBC-specific antinuclear antibodies are present in over modalities are not required but can aid in the exclusion of 30% of AMA-negative patients, allowing for more accurate patients with other cholestatic liver diseases, such as primary diagnosis in those who did not meet previous diagnostic sclerosing cholangitis (PSC), and to rule out concomitant guideline criteria. pancreatic or biliary masses causing biliary obstruction.2,56

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There are also newer imaging modalities, such as transient Table 1. Follow-up schedule for patients with PBC in the primary care elastography, that evaluate disease progression, prognosis, setting and the management of complications and treatment response. Transient elastography-derived 1. Liver function testing every 3 to 6 months (earlier if initiating treat- liver stiffness measurement has been used as a surrogate ment), including a complete metabolic panel, coagulation factors, and marker of liver fibrosis in PBC patients, allowing noninvasive complete blood count to assess platelet levels 54 2. Thyroid function studies every year monitoring over time. A prospective performance analysis 3. Bone mineral density, DEXA scans every 2–4 years was completed in 2012 which showed the benefit of noninva- 4. Fat soluble vitamin levels yearly, including vitamins A, D, and K sive testing using transient elastography in patients with PBC 5. Upper endoscopy every 1 to 3 years if patient has cirrhosis or if Mayo risk using UDCA. Using a generalized Cox linear regression model, score >4.1 (the 1-year risk of death was ;90% in patients with a Mayo risk score >6.0) the authors showed that, in general, patients had about an 6. Abdominal ultrasound and alpha fetoprotein in patients with known or 54 overall progression rate of 0.48 + 0.21 kPa/year (p = 0.02). suspected cirrhosis, including evidence of synthetic liver dysfunction in labs A cutoff value of 2.1 kPa/year was associated with an 8.4-fold 7. Screening for major depression and generalized anxiety disorder increased risk of liver decompensations, liver transplanta- 54 Abbreviations: DEXA, dual-energy X-ray absorptiometry; PBC, primary biliary tions, or deaths (p < 0.001). Accuracy is limited in patients cholangitis. with ascites obesity. Progression of liver stiffness over time is predictive of poorer outcomes, and patients with response to UDCA showed improvement in liver stiffness scores.54,56 The PBC. A randomized, double-blind, placebo-controlled study authors also purported that transient elastography is superior was conducted to evaluate the effects of modafinil with in determining liver fibrosis compared to using biochemical fatigue in patients with PBC, however no beneficial effects testing alone, which is important in the prognostication of on fatigue were found when compared with placebo.58 A clin- PBC patients over time. ical trial is currently in the enrollment phase to study the efficacy and impact of mindfulness-based interventions for the PBC complications treatment of moderate to severe fatigue in patients with PBC (ClinicalTrials.gov Identifier: NCT03684187). Patients with PBC are at an increased risk of developing HCC, though at a lower rate compared to those with other chronic Medical management liver diseases.37,43 In a multicenter study involving over 4,500 patients over a 40-year period, Trivedi et al.57 showed that the Without treatment, patients with PBC progress, on average, incidence of HCC among PBC patients was 3.4 cases per 1,000 one histologic stage within 2 years.59 Treatment of PBC is person-years. In particular, PBC patients with advanced age at aimed at reducing symptoms of cholestasis, preventing fibro- diagnosis, advanced disease, male sex, and suboptimal sis progression and avoiding complications of end-stage response to UDCA are at higher risk for development of disease.23 Previous data had shown the median survival of a HCC.23 However, biochemical nonresponse to treatment has patient with PBC not on treatment was dependent on symp- been shown to be a significant predictor of future risk for devel- toms, with median survival of symptomatic and asympto- oping HCC. According to one study, biochemical nonresponse matic patients of 7.5 years and 16 years, respectively.44,60 to treatment for PBC after one year had a hazard ratio of 3.44 Recent data suggest, however, that asymptomatic patients (p < 0.001) in developing HCC on multivariate analysis.57 with PBC often have less severe disease at diagnosis than Development of HCC in patients with PBC is associated those with symptomatic PBC; yet, the absence of symptoms with notably worse transplant-free and overall survival.43 alone is not associated with a better prognosis and does not Given this, PBC patients with known or suspected cirrhosis show a mortality benefit.61 Unlike other autoimmune dis- should receive screening ultrasounds for HCC with or eases, biologic and immune-based therapies have not been without alpha fetoprotein at regular 6 month intervals. Devel- shown to be effective in treating patients with PBC.62 Herein, opment of portal hypertension can also occur in these we describe approved treatments, off-label therapies, and patients with cirrhosis or in some patients before full-blown drugs in development for the treatment of PBC (see Fig. 1 cirrhosis, given granulomatous inflammation leading to pre- for a detailed schema of treatment options). sinusoidal portal hypertension. The main treatment paradigms of disease management for PBC patients are also at higher risk of osteopenia and patients with PBC include slowing the progression of the disease osteoporosis, mostly related to decreased bone formation and managing the symptoms and complications of chronic and concomitant vitamin D deficiency, which places them at cholestasis. The only two Food and Drug Administration (FDA) higher risk of fracture. Baseline bone mineral density scans medications that are approved for the treatment of PBC are should be done at diagnosis and then subsequent screening UDCA and obeticholic acid (OCA); however, over the last several should be continued on the basis of risk.23,43 General manage- years, there have been several therapies, currently under differ- ment includes calcium and vitamin D supplementation, ent phases of clinical trials, that have been shown to be effective encouraged weight-bearing exercises, and bisphosphonates as both primary and adjuvant medications for the treatment of to improve bone mineral density, though their effectiveness PBC. Herein, we will discuss the approved therapies for PBC, in PBC is not clear.23 Because of chronic cholestasis, hyperlipi- followed by the novel and investigational therapies that have demia is common but rarely of clinical significance; lipid-low- been under clinical investigations over the last several years. ering therapies should be considered in patients with other coexisting cardiovascular risk factors. Fat-soluble vitamin defi- Approved therapies: What is established ciencies are possible as well and can be treated with appropriate supplementation. Table 1 suggests a common follow-up UDCA schedule for patients with PBC in the primary care setting. In terms of managing symptom complications, no good UDCA is a naturally occurring, hydrophilic bile acid that was treatment exists for the treatment of fatigue in patients with originally used for gallstone dissolution. It was noted that

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Fig. 1. Treatment modalities for primary biliary cholangitis: What we know in 2019. patients treated with UDCA had associated decreases in their on the Rochester I, Barcelona, Paris I, Rotterdam, Toronto, ALP levels, leading to the eventual approval for its treatment Paris II, Rochester II, and Global treatment response crite- – of PBC. UDCA is incorporated into the bile acid pool, replacing ria.42,50,52,53,66 68 Although many models exist for determin- other more toxic bile acids and reducing inflammation, ing risk of disease progression, including the GLOBE and UK- cholestasis and cell lysis.63 Although UDCA has changed the PBC risk scoring systems, there is insufficient evidence to landscape of PBC treatment, only 40% to 60% of patients recommend one scoring system over another.50,69,70 Both with PBC respond adequately to UDCA.64 However, because the GLOBE and UK-PBC risk scoring systems were found to of its limited side effects (up to 2% report diarrhea and pru- be predictive of cirrhosis-related complications based on a 71 ritus), current guidelines recommend UDCA as the first-line recent multicenter Turkish study. Table 2 provides a treatment for PBC, at a dose of 13–15 mg/kg/day.63 Further, review of current prognostication models. higher doses have not been shown to be effective in PBC.51 Patients with incomplete biochemical response to UDCA To investigate the impact of UDCA on the incidence of and hence with risk of progressive liver disease and compli- cirrhosis-related complications and on overall survival in PBC, cations, should be evaluated for a second-line agent or be Harms et al.65 reviewed data from 16 liver centers in 10 coun- considered for available clinical trials. tries in Europe and North America in the Global PBC Study Group. This large study found that early diagnosis and early OCA treatment are independent protective factors in delaying PBC- related complications. Additionally, patients with higher aspar- OCA was first approved in May 2017 to be used in conjunction tate aminotransferase to platelets ratio index (commonly with UDCA for PBC patients with an inadequate response to known as the APRI) and patients who were UDCA nonrespond- UDCA, or as monotherapy for those who cannot tolerate ers were both more likely to experience disease complications; UDCA. OCA is a modified bile acid farnesoid X receptor patients who had both an elevated APRI score and proved to be (FXR) agonist which when activated, modulates various UDCA nonresponders experienced a 10-year complication rate steps in bile acid homeostasis, which culminates to a of 37.4% compared to 3.2% in those with a lower APRI score decrease in bile acid synthesis and an increase in its clear- and a response to treatment.2,65 ance. The FXR agonist also plays an important role in down- Guidelines continue to support the role of UDCA as first- regulating inflammatory signaling, which reduces line therapy for patients with PBC. After patients are placed on inflammation and cholestasis in the liver.72 Overall, OCA is an adequate dose of UDCA, treatment response should be well tolerated and has been shown to decrease ALP and measured after 1 year of treatment. Disease response is total bilirubin.65,73,74 In the landmark phase 3 Perioperative defined by biomarkers such as ALP and total bilirubin, based Ischemic Evaluation Study (POISE) trail, the primary

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Table 2. Assessment of biochemical response and prognostication after initiation of treatment, adapted from the 2018 Practice Guidelines from the AASLD23

Criterion Response criteria Response time

Mayo B Alkaline phosphatase <2 x ULN 6 months Barcelona B Alkaline phosphatase 40% from baseline or within normal limits 12 months Paris I B Alkaline phosphatase to < 3 x ULN, and decrease in aspartate aminotransferase 12 months <2 x ULN and normalized bilirubin Rotterdam Normalization of either bilirubin or albumin (one needed to be abnormal 12 months prior to treatment) Toronto Alkaline phosphatase <1.67 x ULN 24 months Paris II (early stage B Alkaline phosphatase to <1.5 x ULN or aspartate aminotransferase <1.5 x ULN 12 months PBC only) and normalized bilirubin Rochester II B Alkaline phosphatase <2 x ULN 12 months UK-PBC risk score Prognostic index; baseline albumin, platelet, bilirubin, alanine aminotransferase 12 months or aspartate aminotransferase, and alkaline phosphatase after 1 year on UDCA GLOBE score Prognostic index: baseline age, bilirubin, alkaline phosphatase, albumin, and 12 months platelet count after 1 year on UDCA

Abbreviations: AASLD, American Association for the Study of Liver Diseases; UDCA, ursodeoxycholic acid; ULN, upper limit of normal. endpoint of an ALP level of less than 1.67 times the ULN enrolled in the open-label extension and received OCA, but range, with a reduction of at least 15% from baseline and a only 60% of patients completed 5 years of the OCA treatment normal total bilirubin level met in an intention-to-treat anal- and 52 patients who had received OCA in the double-blind ysis. The primary endpoint occurred in more patients in the 5– phase of the trial completed 6 years of treatment after the 10 mg titration group (46%) and the 10 mg group (47%) than open-label extension was complete. The mean total bilirubin in the placebo group (10%; p < 0.001 for both compari- remained stable through 72 months of OCA treatment, and sons).74,75 Changes in noninvasive measures of liver fibrosis throughout the study, there was no significant worsening in did not differ significantly between either treatment group or hepatic stiffness as measured by transient elastography in a the placebo group at 12 months. However, the most common subset of patients. During the open-label extension, only adverse event of OCA is dose-dependent pruritus. Up to 56% eight patients (4%) discontinued treatment due to pruritus.76 of patients in the 5–10 mg titration group and 68% of patients Adverse events were consistent with the established safety in the 10 mg group discontinued OCA compared to 38% in the profile of OCA in PBC, with no new safety observations placebo arm.73,74 Pruritus returned back to baseline during during long-term treatment, out to 6 years. the open-lab extension phase of the study. Despite the Samur et al.80 conducted a simulation analysis of the clin- increase in pruritus, discontinuation rates remained low in ical impact and cost-effectiveness of OCA for the treatment of either arm of the study, with 0% of patients in the placebo PBC. This study found that over a 15-year period, UDCA + group versus 1% of patients discontinuing the drug in the OCA dual therapy could decrease the cumulative incidences 5–10 mg titration group and 10% of patients discontinuing of decompensated cirrhosis from 12.2% to 4.5%, of HCC the drug in the higher than 10 mg OCA dose group.76 In addi- from 9.1% to 4.0%, of liver-related mortality from 16.2% tion, OCA has also been studied in Child-Pugh Class A PBC to 5.7%, and of liver transplantations from 4.5% to 1.2%. patients in the POISE trial, with ongoing study in those This combination also increased transplant-free survival Child-Pugh Classes B and C with dose-reduction or caution from 61.1% to 72.9%.80 Despite these substantial improve- for usage in Child-Pugh Classes B and C. The data show that ments in long-term outcomes, the authors concluded that plasma exposure to OCA and its active conjugates and OCA is not currently cost-effective, as the lifetime cost of metabolites increases significantly in patients with moderate PBC treatment would increase from $63,000 to $902,000 (a to severe hepatic impairment.23 For example, compared to 1,330% increment) per patient.80 patients with normal liver function, Child-Pugh Class A patients have a 1.1-fold increase in OCA conjugates compared to a 4-fold increase in Child-Pugh B and 17-fold Emerging therapies: What is new and what is in the increase in Child-Pugh C patients.77 As such, it is recommen- pipeline ded for Child-Pugh B and C patients to start patients on 5 mg OCA weekly; then, doses can be increased to 10 mg twice Peroxisome proliferator-activated receptor-alpha weekly (3 days apart) after 3 months. and -gamma agonists Additionally, OCA has been showed to sustain improved liver biochemistry for up to 6 years in patients with PBC; the The peroxisome proliferator-activated receptor (PPAR) sub- results of the 5-year open-label extension of the phase III, families include a, b, d, and g, which regulate the transcription placebo-controlled POISE trial was reported most recently of various genes. PPAR-g is primarily found in the adipose during the AASLD 2019 Liver Meeting. POISE included a 12- tissue and the heart. An example of PPAR-g are the thiazoli- month double-blind phase with the 5-year open-label exten- dinediones. PPAR−d (also known as PPAR-b) is primarily sion.78,79 As many as 97.5% of patients (193 of 198 patients) found in the liver and in the peripheral tissues, and includes

54 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 Galoosian A. et al: Updates in primary biliary cholangitis drugs such as seladelpar (primarily PPAR-d agonist). PPAR-a with primary endpoint of ALP levels < 1.67 3 the ULN in (i.e. fibrates), which is found in the liver, muscle, and kidneys, patients with PBC. In a phase II study, 70 patients were is involved in the beta oxidation of fatty acids and regulation of screened at 29 sites in North America and Europe. During lipid metabolism. In addition, both PPAR-a and PPAR-d are the recruitment phase of the study, three of the seventy important regulators of bile acid homeostasis.72 Both PPAR-a patients developed a reversible and asymptomatic increase and PPAR-d work by acting on transcription factors which in their alanine aminotransferase (ALT) levels (one patient reduce inflammation (thus lowering IgM) and increase bile on the 50 mg dose, and two on the 200 mg dose), ranging acid excretion. The most common type of PPAR agonists are from just over 5- to 20-times the ULN. As such, the original the fibrates, which include fenofibrate (more PPAR-a selective) study with higher doses was terminated, and a newer low- and bezafibrate (pan-selective). dose phase II study (5 mg and 10 mg of seladelpar groups) A recent trial of fibrates in PBC enrolled 100 Korean patients was initiated and the 12-week interim results were first pub- with UDCA-refractory PBC, including 71 patients who received lished at the AASLD Liver Meeting in 2017,85 showing 45% UDCA monotherapy and 29 patients who received UDCA + and 82% of patients on the 5 mg group and 10 mg group, fibrate therapy (either 160 mg/day fenofibrate or 400 mg/day respectively, achieved the primary endpoint. Additionally, bezafibrate). At follow-up after 18 months, the UDCA + fibrate 12% of the 5 mg group and 45% of the 10 mg group had group showed a higher probability of normalizing ALP levels ALP less than the ULN at week 12. This 12-week analysis compared to the UDCA monotherapy group (hazard ratio = also revealed improvements in other biochemical parameters 5.00, 95% confidence interval (CI): 2.87–8.73, p <0.001).81 as well, including significant decreases in ALT and cholesterol Another randomized controlled trial in Europe included 100 levels.85 Given the promising results of the interim analysis, patients with PBC treated for 2 years with either UDCA + the study entered open enrollment extension phase to extend placebo or UDCA + bezafibrate (the BEZURSO trial).82 In this the duration, increase study participants, and add a 2 mg arm study, combination therapy was associated with improvement to assess the minimally effective dose. in ALP and 30% of patients saw normalization on all liver func- Additionally, a phase III multicenter and international tion tests. The combination group in this study also reported study (ENHANCE) was initiated at the end of 2018, and improvement in pruritus. Bezafibrate, however, is not currently enrolled 265 patients by mid-November 2019. However, on approved for use in the USA. November 25, 2019, the open-label extension phase of the More recently, during AASLD’s The Liver Meeting in 2019, study was placed on hold after a similar study which was it was published that bezafibrate was superior to placebo for evaluating the effectiveness of seladelpar in NASH patients the treatment of pruritus in cholestatic liver diseases, and found that patients receiving seladelpar had atypical histo- authors concluded that it should be the first-line treatment of logic findings characterized as interface hepatitis. As such, pruritus for patients with PBC and PSC, as determined from although the interim data was promising, further investiga- the Fibrates for Cholestatic ITCH Trial.83 The primary endpoint tions are placed on hold until investigators can try to identify a of the study was a 50% reduction in pruritus on a visual ana- mechanism of action and to understand if the observed logue scale, and 43.7% of patients in the bezafibrate treat- histological changes in patients taking seladelpar are NASH- ment arm achieved the primary endpoint compared to only specific or if this adverse event is universal among all liver 11% in the placebo arm (p = 0.003).42 disease etiologies. In addition, another pan-selective PPAR agonist, elafibra- nor, which targets both PPAR-a and PPAR-d, has been studied Fibroblast growth factor-19 analogues (antifibrotic in patients with PBC and results were discussed at the 2019 and anti-inflammatory) European Association for the Study of the Liver International Liver Congress Meeting. A phase 2 multi-center, double-blind, Fibroblast growth factor (FGF)-19 is an endocrine hormone randomized, placebo-controlled clinical trial was reported to that is induced by the activation of the FXR in ileal enter- evaluate the efficacy and safety of elafibranor after 12 weeks ocytes.86 After its activation, FGF-19 acts on hepatocytes to of treatment in patients with PBC with inadequate response to repress bile acid synthesis and gluconeogenesis, and to stim- UDCA. Forty-five patients were randomized into three arms: ulate hepatic glycogenesis and protein synthesis (Fig. 2).86 1) elafibranor at 80 mg, 2) elafibranor at 120 mg, or 3) FGF-19 is part of a signaling pathway that regulates the placebo. The primary endpoint of the study was a change in enterohepatic response of bile acid synthesis. In the normal serum ALP at the end of 12 weeks compared to baseline. Both liver, FGF-19 expression is absent; however, in cholestatic doses of elafibranor demonstrated a significant change in ALP, liver diseases, FGF-19 expression is increased in the liver, in a decrease of 48% from baseline in the 80 mg group and 41% response to both extrahepatic and intrahepatic cholestasis.87 decrease in the 120 mg group; as expected, there was a ;3% Recent evidence also suggests an antifibrotic activity of FGF- increase in ALP from baseline in the placebo group. A key 19 analogues.87 For example, FGF-19 is also involved in the secondary endpoint from this trial was the responder rate for regulation of proinflammatory cytokines in the cholangio- patients achieving the composite endpoint of serum ALP cytes.87 Serum levels of FGF-19 correlate with worse liver <1.67 3 the ULN, and ALP decrease >15%, and total bilirubin enzyme biochemistry levels; an elevated serum FGF-19 was

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 55 Galoosian A. et al: Updates in primary biliary cholangitis

Fig. 2. Steroidal and nonsteroidal FXR agonists and FGF-19 mechanism of action. FXR is a nuclear hormone receptor that is highly expressed in the gastrointestinal tract, adrenal glands, kidneys, and the liver.FXR is the primary regulator of BA homeostasis. By activating FXR, OCA is a steroidal FXR agonist and it is expected to do three things: 1) decrease fibrosis by decreasing fibrogenesis, stellate cell activation, and increase matrix degradation, 2) decrease inflammation, and 3) regulate BA homeostasis by decreasing its synthesis, uptake and absorption, and increasing its secretion. GS-9674 is a nonsteroidal FXR agonist in the intestine, which triggers release of FGF-19 from the intestinal mucosa. FGF-19, which is an endocrine hormone, then binds to hepatic receptor complex tyrosine kinase FGFR4 and its co-receptor b-klotho, which inhibits the gene transcription of the CYP7A1 BA synthesis gene (encoding cholesterol 7-alpha-hydroxylase), the SREBP1c lipogenic gene (encoding sterol regulatory element-binding protein 1C), and the G6PC gluconeogenic gene (encoding the glucose-6-phosphatase catalytic subunit), leading to decreases in BA synthesis, lipogenesis, and gluconeogenesis. Concurrently, FXR in the liver induces SHP, a negative nuclear receptor, which in turn also inhibits gene transcription of CYP7A1, SREBP1c, and G6PC. Cilofexor (GS-9674) has been shown to significantly reduce fibrosis and portal hypertension in a murine model of nonalcoholic steatohepatitis, to have anti-inflammatory property in vivo, and to increase eNOS. Abbreviations: BA, bile acid; eNOS, endothelial nitric oxide synthase; FGF, fibroblast growth factor; FXR, farnesoid X receptor; OCA, obeticholic acid; SHP, small heterodimer partner. patients who had inadequate response to UDCA.51 The without incomplete response to UDCA (ClinicalTrials.gov authors conclude that the administration of NGM282 (14 Identifier: NCT03394924). patients in the 0.3 mg group, 13 patients in the 3 mg group, In a recent phase II, randomized, double-blind, placebo and 15 patients in the placebo group) reduced ALP levels and controlled trial of 71 patients, the safety, efficacy, and toler- transaminase levels, as well as markers of cholestasis, hep- ability of cilofexor (GS-9674, which is a non-steroidal FXR atocellular injury and inflammation (IgM levels) in a follow-up agonist) evaluated in patients without advanced PBC. Fig. 2 of 28 days. The 0.3 mg group had an average of 15.9% shows the mechanism of action of this drug. This trial was decrease in ALP levels (95% CI: 3.9%–25.6%, p < 0.001) completed recently, and the results of the phase II trial were versus an average of 19.0% decrease in ALP in the 3 mg presented at AASLD’s The Liver Meeting in 2019. The authors group (95% CI: 6.7%–29.0%, p < 0.001). However, longer demonstrated that patients treated with cilofexor at 100 mg length studies are needed to assess overall effectiveness and had significant decreases (compared to placebo) in serum ALP, tolerability. gamma-glutamyltransferase, and ALT levels (ALP mean reduction of -13.8%, p = 0.005; gamma-glutamyltransferase mean reduction of 47.7%, p < 0.001; ALT mean reduction of Other FXR agonists (nonsteroidal) -17.8%, p =0.08).91 The study also showed a significant reduction in primary bile acids compared to placebo (reduction FXRs are nuclear hormone receptors that are integral in of −30.5%, p = 0.0008). At the same meeting, patient- bilirubin metabolism, primarily in the liver. The most reported outcomes in patients with PSC during treatment common FXR agonist studied and approved for treatment of with cilofexor were reported.92 Patient-reported outcomes PBC is OCA. Bile acids are the naturally occurring ligands of are important markers in clinical outcomes and contribute FXR. FXRs regulate the rate-limiting step in bile acid synthesis greatly to the efficacy of a drug. During long-term treatment (Fig. 2).89 FXR activation protects against cholestatic-induced with 100 mg cilofexor, patients experienced improvement in liver injury, and other studies have shown a protective effect some aspects of their patient-reported outcomes.92 of FXR activation in patients with nonalcoholic fatty liver disease.89,90 Additional FXR agonists are being investigated for PBC, including in a phase II dose-ranging, randomized, Antifibrotic agents double-blind, placebo-controlled trial, currently recruiting participants to evaluate the safety, tolerability, pharmacoki- Cholangiocyte injury and leakage of bile acid into the liver netics, and efficacy of EDP-305 in patients with PBC with or parenchyma are the key events in fibrosis progression in PBC

56 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 Galoosian A. et al: Updates in primary biliary cholangitis and other cholestatic liver diseases.93 Hepatic stellate cell but no significant decrease was observed in ALP levels.99 activation is ultimately the final common pathway in the for- Another phase II trial was completed in the UK to evaluate mation of fibrosis in chronic liver diseases. NADPH oxidase symptoms of fatigue in 71 patients with PBC.100 Using an (referred to as NOX) plays a role in stellate cell-mediated intention-to-treat analysis, this study found significant fibrogenesis. In vivo data has demonstrated that setanaxib decrease in fatigue at 3 months between the rituximab and (a dual inhibitor of NOX1 and NOX2) may be an effective anti- placebo arms (adjusted mean difference −0.9, 95% CI: fibrotic drug in PBC.93 Additional studies are needed, −4.6–3.1). Another open-label, active treatment study was however, to prove the efficacy of this and other compounds recently completed in 2018, which studied the efficacy and in human patients. safety of abatacept for the treatment of PBC in patients with incomplete response to UDCA. This study showed that, much Norursodeoxycholic acid and tauroursodeoxycholic like other biologic therapies, the treatment was ineffective in acid achieving biochemical responses associated with improved clinical outcomes.101 The molecular structure of norursodeoxycholic acid (norUDCA) In addition, another biologic therapy has failed to show is similar to UDCA, except that it lacks a methylene group in significant reductions in biochemical markers of cholesta- its side-chain. In theory, norUDCA can then allow for sis. A phase 2 open-label trial using ustekinumab, an anti- cholehepatic shunting of bile salts, instead of by enter- interleukin-12/23 monoclonal antibody, for PBC patients ohepatic circulation. norUDCA can also stimulate secretion with inadequate response to UDCA showed a modest of cholangiocytes found in bile ducts, which can help flush decrease in ALP after 28 weeks of therapy that was not the ducts of bile salts and reinforce the ductal wall epithe- significant, and overt proof-of-concept was not established lium. The use of norUDCA as treatment has been shown to be per the aprioriprimary endpoint’sproposedefficacy.45,102 beneficial in PSC, yet its usefulness in PBC has yet to be elucidated. Multiple animal studies have shown improve- Conclusions, remarks, and future directions ment in fibrosis and inflammation in mice with cholestatic liver diseases treated with norUDCA, including PBC.94,95 A PBC is a chronic and slowly progressive cholestatic liver multicenter, double-blind trial in China comparing taurourso- disease that is caused by the autoimmune and non-suppu- deoxycholic acid and UDCA was completed in 2013 and rative destruction of the bile ducts which can lead to fibrosis showed that tauroursodeoxycholic acid was non-inferior to and eventually cirrhosis. Mainstay treatments include UDCA UDCA in biochemical response and had a better profile in as first-line standard of care therapy, while OCA is used as an mitigating symptoms associated with PBC.96 Further adjuvant therapeutic agent for patients with incomplete studies are needed in the USA to account for external validity response to UDCA or as a first-line agent for patients who of this trial. cannot tolerate UDCA. Although UDCA continues to be the mainstay treatment, UDCA leaves over one-third of patients Ileal apical sodium-dependent bile acid transporter at risk for disease progression by using methods described in inhibitors Table 2. Other than UDCA and OCA, there are no other FDA- approved treatment agents for PBC at this time. As such, this The apical sodium-dependent bile acid transporter (ASBT) is review provides information regarding the promising thera- found in the brush-border membrane primarily expressed in peutic landscape for PBC, including many agents which are the distal ileum. It is responsible for the reuptake of bile acids currently under clinical trials. New anticholestatic pharmaco- and the maintenance of the enterohepatic circulation. ASBT is logic agents, such as PPAR agonists, choleretics, bile acid sup- up-regulated in PBC patients; therefore, the inhibition of pressants, antifibrotics and anti-inflammatories, provide ASBT may counteract the toxicity caused by bile acids. promising outlooks for treatment of PBC and possibly other Studies have shown that by inhibiting ASBT, both cholestatic diseases of cholestasis and NASH. In addition, early emerging injury and fibrosis improves by increasing bile acid excretion data indicates combination therapy may also contribute to the in fecal matter.97 There is currently a phase II trial underway landscape of PBC treatments with improved efficacy and that is comparing combination therapy of a selective ASBT potential benefits in symptoms such as pruritus and fatigue, inhibitor (lopixibat) with UDCA; the preliminary results, and in quality of life. however, show poor effects on ALP and pruritus, though there are reductions in transaminases.98 Funding

None to declare. Immunomodulating agents

Since PBC is an autoimmune disease, various immunological Conflict of interest hallmarks have been implicated in its pathogenesis, including humoral, cytotoxic and the innate immune response in The authors have no conflict of interests related to this destroying the small bile ducts. Rituximab is an anti-CD20 publication. monoclonal antibody that selectively decreases B cells and which has been used for years in other autoimmune diseases, such as rheumatoid arthritis, and other immune- Author contributions mediated disorders.99 Although this drug is used in other autoimmune disorders, a study evaluating 14 patients who Formulated manuscript, drafting of the manuscript, respon- did not respond to UDCA were given rituximab. Six months sible for organization of content, critical editing of manuscript after starting infusion therapy, there was a decrease in auto- (AG), writing of the manuscript, organization and formulation antibody production and a significant decrease in pruritus of content, major editing (CH), writing of the manuscript (JZ),

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 57 Galoosian A. et al: Updates in primary biliary cholangitis formulation and organization of drafts, major content revi- [23] Lindor KD, Bowlus CL, Boyer J, Levy C, Mayo M. Primary biliary cholangitis: sions and additions, expert review, study supervision (EWH), 2018 practice guidance from the American Association for the study of liver diseases. Hepatology 2019;69:394–419. doi: 10.1002/hep.30145. formulation and organization of drafts, major content revi- [24] Ala A, Stanca CM, Bu-Ghanim M, Ahmado I, Branch AD, Schiano TD, et al. sions and additions, expert review, study supervision (KKY). Increased prevalence of primary biliary cirrhosis near Superfund toxic waste sites. Hepatology 2006;43:525–531. doi: 10.1002/hep.21076. [25] Webb GJ, Siminovitch KA, Hirschfield GM. The immunogenetics of primary References biliary cirrhosis: A comprehensive review. J Autoimmun 2015;64:42–52. doi: 10.1016/j.jaut.2015.07.004. [1] Sylvestre PB, Batts KP, Burgart LJ, Poterucha JJ, Wiesner RH. Recurrence of [26] Cordell HJ, Han Y, Mells GF, Li Y, Hirschfield GM, Greene CS, et al. Interna- primary biliary cirrhosis after liver transplantation: Histologic estimate of tional genome-wide meta-analysis identifies new primary biliary cirrhosis incidence and natural history. Liver Transpl 2003;9:1086–1093. doi: 10. risk loci and targetable pathogenic pathways. Nat Commun 2015;6:8019. 1053/jlts.2003.50213. doi: 10.1038/ncomms9019. [2] Younossi ZM, Bernstein D, Shiffman ML, Kwo P, Kim WR, Kowdley KV, et al. [27] Hirschfield GM, Liu X, Xu C, Lu Y, Xie G, Lu Y, et al. Primary biliary cirrhosis Diagnosis and management of primary biliary cholangitis. Am J Gastroen- associated with HLA, IL12A, and IL12RB2 variants. N Engl J Med 2009;360: terol 2019;114:48–63. doi: 10.1038/s41395-018-0390-3. 2544–2555. doi: 10.1056/NEJMoa0810440. [3] Peery AF, Crockett SD, Murphy CC, Lund JL, Dellon ES, Williams JL, et al. [28] Nakamura M, Nishida N, Kawashima M, Aiba Y, Tanaka A, Yasunami M, et al. Burden and cost of gastrointestinal, liver, and pancreatic diseases in the Genome-wide association study identifies TNFSF15 and POU2AF1 as sus- United States: Update 2018. Gastroenterology 2019;156:254–272.e11. ceptibility loci for primary biliary cirrhosis in the Japanese population. Am J doi: 10.1053/j.gastro.2018.08.063. Hum Genet 2012;91:721–728. doi: 10.1016/j.ajhg.2012.08.010. [4] Podda M, Selmi C, Lleo A, Moroni L, Invernizzi P. The limitations and hidden [29] Liu JZ, Almarri MA, Gaffney DJ, Mells GF, Jostins L, Cordell HJ, et al. Dense gems of the epidemiology of primary biliary cirrhosis. J Autoimmun 2013; fine-mapping study identifies new susceptibility loci for primary biliary cir- 46:81–87. doi: 10.1016/j.jaut.2013.06.015. rhosis. Nat Genet 2012;44:1137–1141. doi: 10.1038/ng.2395. [5] Poordad F. Diagnosis, treatment, and monitoring of patients with primary [30] Flores A, Mayo MJ. Primary biliary cirrhosis in 2014. Curr Opin Gastroenterol biliary cholangitis. Gastroenterol Hepatol (N Y) 2016;12:561–564. 2014;30:245–252. doi: 10.1097/MOG.0000000000000058. [6] Hamlyn AN, Macklon AF, James O. Primary biliary cirrhosis: geographical [31] Chan CY, Lee SD, Huang YS, Wu JC, Tsai YT, Tsay SH, et al. Primary biliary clustering and symptomatic onset seasonality. Gut 1983;24:940–945. cirrhosis in Taiwan. J Gastroenterol Hepatol 1990;5:560–565. doi: 10. doi: 10.1136/gut.24.10.940. 1111/j.1440-1746.1990.tb01441.x. [7] Boonstra K, Kunst AE, Stadhouders PH, Tuynman HA, Poen AC, van Nieuwkerk [32] Sakauchi F, Mori M, Zeniya M, Toda G. A cross-sectional study of primary KM, et al. Rising incidence and prevalence of primary biliary cirrhosis: a large biliary cirrhosis in Japan: utilization of clinical data when patients applied to population-based study. Liver Int 2014;34:e31–e38. doi: 10.1111/liv.12434. receive public financial aid. J Epidemiol 2005;15:24–28. doi: 10.2188/jea. [8] McNally RJ, James PW, Ducker S, Norman PD, James OF. No rise in incidence 15.24. but geographical heterogeneity in the occurrence of primary biliary cirrhosis [33] Lohse AW, Weiler-Normann C. Not all PBC is the same! Gastroenterology – in North East England. Am J Epidemiol 2014;179:492 498. doi: 10. 2013;144:494–497. doi: 10.1053/j.gastro.2013.01.032. 1093/aje/kwt308. [34] Tanakaa A, Leung PS, Young HA, Gershwin ME. Toward solving the etiolog- [9] Lu M, Li J, Haller IV, Romanelli RJ, VanWormer JJ, Rodriguez CV, et al. Factors ical mystery of primary biliary cholangitis. Hepatol Commun 2017;1:275– associated with prevalence and treatment of primary biliary cholangitis in 287. doi: 10.1002/hep4.1044. – United States health systems. Clin Gastroenterol Hepatol 2018;16:1333 [35] Walden HR, Kirby JA, Yeaman SJ, Gray J, Jones DE, Palmer JM. Xenobiotic 1341.e6. doi: 10.1016/j.cgh.2017.10.018. incorporation into pyruvate dehydrogenase complex can occur via the exog- [10] Kim KA, Ki M, Choi HY, Kim BH, Jang ES, Jeong SH. Population-based epi- enous lipoylation pathway. Hepatology 2008;48:1874–1884. doi: 10. demiology of primary biliary cirrhosis in South Korea. Aliment Pharmacol 1002/hep.22540. Ther 2016;43:154–162. doi: 10.1111/apt.13448. [36] Yeaman SJ, Kirby JA, Jones DE. Autoreactive responses to pyruvate dehy- [11] Lleo A, Jepsen P, Morenghi E, Carbone M, Moroni L, Battezzati PM, et al. drogenase complex in the pathogenesis of primary biliary cirrhosis. Evolving trends in female to male incidence and male mortality of primary Immunol Rev 2000;174:238–249. doi: 10.1034/j.1600-0528.2002. biliary cholangitis. Sci Rep 2016;6:25906. doi: 10.1038/srep25906. 00021h.x. [12] Boonstra K, Beuers U, Ponsioen CY. Epidemiology of primary sclerosing [37] Onofrio FQ, Hirschfield GM, Gulamhusein AF. A practical review of primary cholangitis and primary biliary cirrhosis: a systematic review. J Hepatol biliary cholangitis for the gastroenterologist. Gastroenterol Hepatol (NY) 2012;56:1181–1188. doi: 10.1016/j.jhep.2011.10.025. 2019;15:145–154. [13] Chuang N, Gross RG, Odin JA. Update on the epidemiology of primary biliary [38] Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005;353: cirrhosis. Expert Rev Gastroenterol Hepatol 2011;5:583–590. doi: 10. 1261–1273. doi: 10.1056/NEJMra043898. 1586/egh.11.66. [39] Lindor KD, Gershwin ME, Poupon R, Kaplan M, Bergasa NV, Heathcote EJ. [14] Cheung KS, Seto WK, Fung J, Lai CL, Yuen MF. Epidemiology and natural Primary biliary cirrhosis. Hepatology 2009;50:291–308. doi: 10.1002/hep. history of primary biliary cholangitis in the Chinese: A territory-based study 22906. in Hong Kong between 2000 and 2015. Clin Transl Gastroenterol 2017;8: [40] Boberg KM, Chapman RW, Hirschfield GM, Lohse AW, Manns MP, Schrumpf e116. doi: 10.1038/ctg.2017.43. E. Overlap syndromes: the International Autoimmune Hepatitis Group [15] Rautiainen H, Salomaa V, Niemelå S, Karvonen AL, Nurmi H, Isoniemi H, (IAIHG) position statement on a controversial issue. J Hepatol 2011;54: et al. Prevalence and incidence of primary biliary cirrhosis are increasing in – Finland. Scand J Gastroenterol 2007;42:1347–1353. doi: 10. 374 385. doi: 10.1016/j.jhep.2010.09.002. 1080/00365520701396034. [41] Lee YM, Kaplan MM. The natural history of PBC: has it changed? Semin Liver – [16] Selmi C, Invernizzi P, Keeffe EB, Coppel RL, Podda M, Rossaro L, et al. Epi- Dis 2005;25:321 326. doi: 10.1055/s-2005-916323. demiology and pathogenesis of primary biliary cirrhosis. J Clin Gastroen- [42] Kuiper EM, Hansen BE, de Vries RA, den Ouden-Muller JW, van Ditzhuijsen terol 2004;38:264–271. doi: 10.1097/00004836-200403000-00013. TJ, Haagsma EB, et al. Improved prognosis of patients with primary biliary [17] Abu-Mouch S, Selmi C, Benson GD, Kenny TP, Invernizzi P, Zuin M, et al. cirrhosis that have a biochemical response to ursodeoxycholic acid. Gastro- – Geographic clusters of primary biliary cirrhosis. Clin Dev Immunol 2003;10: enterology 2009;136:1281 1287. doi: 10.1053/j.gastro.2009.01.003. 127–131. doi: 10.1080/10446670310001626526. [43] Janmohamed A, Trivedi PJ. Patterns of disease progression and incidence of [18] Kim WR, Lindor KD, Locke GR 3rd, Therneau TM, Homburger HA, Batts KP, complications in primary biliary cholangitis (PBC). Best Pract Res Clin Gas- et al. Epidemiology and natural history of primary biliary cirrhosis in a US troenterol 2018;34–35:71–83. doi: 10.1016/j.bpg.2018.06.002. community. Gastroenterology 2000;119:1631–1636. doi: 10.1053/gast. [44] Springer J, Cauch-Dudek K, O’Rourke K, Wanless IR, Heathcote EJ. Asymp- 2000.20197. tomatic primary biliary cirrhosis: a study of its natural history and progno- [19] Carey EJ, Ali AH, Lindor KD. Primary biliary cirrhosis. Lancet 2015;386: sis. Am J Gastroenterol 1999;94:47–53. doi: 10.1111/j.1572-0241.1999. 1565–1575. doi: 10.1016/S0140-6736(15)00154-3. 00770.x. [20] Marschall HU, Henriksson I, Lindberg S, Söderdahl F, Thuresson M, Wahlin [45] Hirschfield GM, Gershwin ME. The immunobiology and pathophysiology of S, et al. Incidence, prevalence, and outcome of primary biliary cholangitis in primary biliary cirrhosis. Annu Rev Pathol 2013;8:303–330. doi: 10. a nationwide Swedish population-based cohort. Sci Rep 2019;9:11525. doi: 1146/annurev-pathol-020712-164014. 10.1038/s41598-019-47890-2. [46] Cheung AC, LaRusso NF, Gores GJ, Lazaridis KN. Epigenetics in the primary [21] Marzioni M, Bassanelli C, Ripellino C, Urbinati D, Alvaro D. Epidemiology of biliary cholangitis and primary sclerosing cholangitis. Semin Liver Dis 2017; primary biliary cholangitis in Italy: Evidence from a real-world database. 37:159–174. doi: 10.1055/s-0037-1603324. Dig Liver Dis 2019;51:724–729. doi: 10.1016/j.dld.2018.11.008. [47] Xie YQ, Ma HD, Lian ZX. Epigenetics and primary biliary cirrhosis: a com- [22] Bowlus CL, Gershwin ME. The diagnosis of primary biliary cirrhosis. Auto- prehensive review and implications for autoimmunity. Clin Rev Allergy immun Rev 2014;13:441–444. doi: 10.1016/j.autrev.2014.01.041. Immunol 2016;50:390–403. doi: 10.1007/s12016-015-8502-y.

58 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 Galoosian A. et al: Updates in primary biliary cholangitis

[48] Worman HJ, Courvalin JC. Antinuclear antibodies specific for primary biliary acid therapy. Gastroenterology 2015;149:1804–1812.e4. doi: 10.1053/j. cirrhosis. Autoimmun Rev 2003;2:211–217. doi: 10.1016/s1568-9972(03) gastro.2015.07.061. 00013-2. [70] Carbone M, Sharp SJ, Flack S, Paximadas D, Spiess K, Adgey C, et al. The [49] Poupon R, Chazouillères O, Balkau B, Poupon RE. Clinical and biochemical UK-PBC risk scores: Derivation and validation of a scoring system for long- expression of the histopathological lesions of primary biliary cirrhosis. term prediction of end-stage liver disease in primary biliary cholangitis. UDCA-PBC Group. J Hepatol 1999;30:408–412. doi: 10.1016/s0168- Hepatology 2016;63:930–950. doi: 10.1002/hep.28017. 8278(99)80098-1. [71] Efe C, Tas¸çilar K, Henriksson I, Lytvyak E, Alalkim F, Trivedi H, et al. Vali- [50] Lammers WJ, van Buuren HR, Hirschfield GM, Janssen HL, Invernizzi P, dation of risk scoring systems in ursodeoxycholic acid-treated patients with Mason AL, et al. Levels of alkaline phosphatase and bilirubin are surrogate primary biliary cholangitis. Am J Gastroenterol 2019;114:1101–1108. doi: end points of outcomes of patients with primary biliary cirrhosis: an interna- 10.14309/ajg.0000000000000290. tional follow-up study. Gastroenterology 2014;147:1338–1349.e5; quiz [72] Santiago P, Scheinberg AR, Levy C. Cholestatic liver diseases: new targets, e15. doi: 10.1053/j.gastro.2014.08.029. new therapies. Therap Adv Gastroenterol 2018;11:1756284818787400. [51] Mayo MJ, Wigg AJ, Leggett BA, Arnold H, Thompson AJ, Weltman M, et al. doi: 10.1177/1756284818787400. NGM282 for treatment of patients with primary biliary cholangitis: A multi- [73] Erlinger S. Obeticholic acid in primary biliary cholangitis. Clin Res Hepatol center, randomized, double-blind, placebo-controlled trial. Hepatol Gastroenterol 2017;41:3–5. doi: 10.1016/j.clinre.2016.09.006. Commun 2018;2:1037–1050. doi: 10.1002/hep4.1209. [74] Nevens F, Andreone P, Mazzella G, Strasser SI, Bowlus C, Invernizzi P, et al. [52] Corpechot C, Abenavoli L, Rabahi N, Chrétien Y, Andréani T, Johanet C, et al. A placebo-controlled trial of obeticholic acid in primary biliary cholangitis. N – Biochemical response to ursodeoxycholic acid and long-term prognosis in Engl J Med 2016;375:631 643. doi: 10.1056/NEJMoa1509840. primary biliary cirrhosis. Hepatology 2008;48:871–877. doi: 10.1002/hep. [75] Hirschfield GM, Mason A, Luketic V, Lindor K, Gordon SC, Mayo M, et al. 22428. Efficacy of obeticholic acid in patients with primary biliary cirrhosis and [53] Corpechot C, Poujol-Robert A, Wendum D, Galotte M, Chrétien Y, Poupon inadequate response to ursodeoxycholic acid. Gastroenterology 2015; – RE, et al. Biochemical markers of liver fibrosis and lymphocytic piecemeal 148:751 761.e8. doi: 10.1053/j.gastro.2014.12.005. necrosis in UDCA-treated patients with primary biliary cirrhosis. Liver Int [76] Yimam KK, Bowlus CL. Obeticholic acid for the treatment of primary biliary – 2004;24:187–193. doi: 10.1111/j.1478-3231.2004.0918.x. cirrhosis. Expert Rev Clin Pharmacol 2014;2:1351 1358. doi: 10. [54] Corpechot C, Carrat F,Poujol-Robert A, Gaouar F,Wendum D, Chazouillères O, 1517/21678707.2014.964206. et al. Noninvasive elastography-based assessment of liver fibrosis progres- [77] Roda A, Aldini R, Camborata C, Spinozzi S, Franco P, Cont M, et al. Metabolic sion and prognosis in primary biliary cirrhosis. Hepatology 2012;56:198–208. profile of obeticholic acid and endogenous bile acids in rats with decompensated liver cirrhosis. Clin Transl Sci 2017;10:292–301. doi: 10.1111/cts. doi: 10.1002/hep.25599. 12468. [55] Goodman ZD. Grading and staging systems for inflammation and fibrosis in [78] Trauner MH, Nevens F, Shiffman ML, Drenth J, Bowlus CL, Vargas V, et al. chronic liver diseases. J Hepatol 2007;47:598–607. doi: 10.1016/j.jhep. Durable response in the markers of cholestasis through 36 months of open- 2007.07.006. label extension study of obeticholic acid in primary biliary cholangitis. Gas- [56] Kovac JD, Weber MA. Primary biliary cirrhosis and primary sclerosing chol- troenterology 2018;154:S-1211–S-1212. doi: 10.1016/S0016-5085(18) angitis: an update on MR imaging findings with recent developments. J 34002-2. Gastrointestin Liver Dis 2016;25:517–524. doi: 10.15403/jgld.2014. [79] Nevens F, Shiffman ML, Drent JPH, Bowlus CL, Vargas V, Andreone P, et al. 1121.254.vac. Durable response in the markers of cholestasis through 5 years of open- [57] Trivedi PJ, Lammers WJ, van Buuren HR, Parés A, Floreani A, Janssen HL, label extension study of obeticholic acid in primary biliary cholangitis. Hep- et al. Stratification of hepatocellular carcinoma risk in primary biliary cir- atology 2019;70:1481A–1482A. rhosis: a multicentre international study. Gut 2016;65:321–329. doi: 10. [80] Samur S, Klebanoff M, Banken R, Pratt DS, Chapman R, Ollendorf DA, et al. 1136/gutjnl-2014-308351. Long-term clinical impact and cost-effectiveness of obeticholic acid for the [58] Silveira MG, Gossard AA, Stahler AC, Jorgensen RA, Petz JL, Ali AH, et al.A treatment of primary biliary cholangitis. Hepatology 2017;65:920–928. randomized, placebo-controlled clinical trial of efficacy and safety: Modafinil doi: 10.1002/hep.28932. in the treatment of fatigue in patients with primary biliary cirrhosis. Am J [81] Chung SW, Lee JH, Kim MA, Leem G, Kim SW, Chang Y, et al. Additional Ther 2017;24:e167–e176. doi: 10.1097/MJT.0000000000000387. fibrate treatment in UDCA-refractory PBC patients. Liver Int 2019;39: [59] Locke GR 3rd, Therneau TM, Ludwig J, Dickson ER, Lindor KD. Time course 1776–1785. doi: 10.1111/liv.14165. of histological progression in primary biliary cirrhosis. Hepatology 1996;23: [82] Corpechot C, Chazouillères O, Rousseau A, Guyader D, Habersetzer F, 52–56. doi: 10.1002/hep.510230108. Mathurin P, et al. A 2-year multicenter, double-blind, randomized, [60] Mitchison HC, Lucey MR, Kelly PJ, Neuberger JM, Williams R, James OF. placebo-controlled study of bezafibrate for the treatment of primary Symptom development and prognosis in primary biliary cirrhosis: a study biliary cholangitis in patients with inadequate biochemical response to urso- – in two centers. Gastroenterology 1990;99:778 784. doi: 10.1016/0016- deoxycholic acid therapy (Bezurso). J Hepatol 2017;66:S89. doi: 10. 5085(90)90968-7. 1016/S0168-8278(17)30442-7. [61] Prince MI, Chetwynd A, Craig WL, Metcalf JV, James OF. Asymptomatic [83] de Vries E, Bolier R, Goet J, Pares A, Verbeek JPHM, Vree MD, et al. Bezafi- primary biliary cirrhosis: clinical features, prognosis, and symptom progres- brate is more effective than placebo in pruritus of chronic cholestasis: The – sion in a large population based cohort. Gut 2004;53:865 870. doi: 10. fitch trial. Hepatology 2019;70:9A. 1136/gut.2003.023937. [84] Jones D, Boudes PF, Swain MG, Bowlus CL, Galambos MR, Bacon BR, et al. [62] Bahar R, Wong KA, Liu CH, Bowlus CL. Update on new drugs and those in Seladelpar (MBX-8025), a selective PPAR-d agonist, in patients with primary development for the treatment of primary biliary cholangitis. Gastroenterol biliary cholangitis with an inadequate response to ursodeoxycholic acid: a – Hepatol (NY) 2018;14:154 163. double-blind, randomised, placebo-controlled, phase 2, proof-of-concept [63] Floreani A, Mangini C. Primary biliary cholangitis: Old and novel therapy. Eur study. Lancet Gastroenterol Hepatol 2017;2:716–726. doi: 10. – J Intern Med 2018;47:1 5. doi: 10.1016/j.ejim.2017.06.020. 1016/S2468-1253(17)30246-7. [64] Chascsa D, Carey EJ, Lindor KD. Old and new treatments for primary biliary [85] Hirschfield G, Boudes P, Bowlus C, Gitlin N, Michael G, Harrison S, et al. – cholangitis. Liver Int 2017;37:490 499. doi: 10.1111/liv.13294. Treatment efficacy and safety of seladelpar, a selective peroxisome prolif- [65] Harms MH, Lammers WJ, Thorburn D, Corpechot C, Invernizzi P, Janssen erator-activated receptor delta agonist, in primary biliary cholangitis HLA, et al. Major hepatic complications in ursodeoxycholic acid-treated patients: 12- and 26-week analysis from an ongoing international, random- patients with primary biliary cholangitis: Risk factors and time trends in ized, dose raging phase 2 study. J Hepatol 2018;68:S105–S106. doi: 10. incidence and outcome. Am J Gastroenterol 2018;113:254–264. doi: 10. 1016/S0168-8278(18)30429-X. 1038/ajg.2017.440. [86] Kliewer SA, Mangelsdorf DJ. Bile acids as hormones: The FXR-FGF15/19 [66] Angulo P, Lindor KD, Therneau TM, Jorgensen RA, Malinchoc M, Kamath PS, pathway. Dig Dis 2015;33:327–331. doi: 10.1159/000371670. et al. Utilization of the Mayo risk score in patients with primary biliary cir- [87] Li Z, Lin B, Lin G, Wu Y, Jie Y, Li X, et al. Circulating FGF19 closely correlates rhosis receiving ursodeoxycholic acid. Liver 1999;19:115–121. doi: 10. with bile acid synthesis and cholestasis in patients with primary biliary cir- 1111/j.1478-3231.1999.tb00020.x. rhosis. PLoS One 2017;12:e0178580. doi: 10.1371/journal.pone.0178580. [67] Parés A, Caballería L, Rodés J. Excellent long-term survival in patients with [88] Wunsch E, Milkiewicz M, Wasik U, Trottier J, Kempinska-Podhorodecka A, primary biliary cirrhosis and biochemical response to ursodeoxycholic Acid. Elias E, et al. Expression of hepatic Fibroblast Growth Factor 19 is enhanced Gastroenterology 2006;130:715–720. doi: 10.1053/j.gastro.2005.12.029. in Primary Biliary Cirrhosis and correlates with severity of the disease. Sci [68] Momah N, Silveira MG, Jorgensen R, Sinakos E, Lindor KD. Optimizing bio- Rep 2015;5:13462. doi: 10.1038/srep13462. chemical markers as endpoints for clinical trials in primary biliary cirrhosis. [89] Ali AH, Carey EJ, Lindor KD. Recent advances in the development of farne- Liver Int 2012;32:790–795. doi: 10.1111/j.1478-3231.2011.02678.x. soid X receptor agonists. Ann Transl Med 2015;3:5. doi: 10.3978/j.issn. [69] Lammers WJ, Hirschfield GM, Corpechot C, Nevens F, Lindor KD, Janssen 2305-5839.2014.12.06. HL, et al. Development and validation of a scoring system to predict out- [90] Liu X, Green RM. Beyond Farnesoid X receptor to target new therapies for comes of patients with primary biliary cirrhosis receiving ursodeoxycholic NAFLD. Hepatology 2017;66:1724–1726. doi: 10.1002/hep.29411.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 59 Galoosian A. et al: Updates in primary biliary cholangitis

[91] Kowdley KV, Minuk GY, Pagadala MR, Gulamhusein A, Swain MG, Neff GW, [100] Khanna A, Jopson L, Howel D, Bryant A, Blamire A, Newton JL, et al. Ritux- et al. The nonsteroidal farnesoid x receptor (FXR) agonist cilofexor imab for the treatment of fatigue in primary biliary cholangitis (formerly improves liver biochemistry in patients with primary biliary cholangitis primary biliary cirrhosis): a randomised controlled trial. Southampton (PBC): A phase 2, randomized, placebo-controlled trial. Hepatology 2019; (UK): NIHR Journals Library; 2018. doi: 10.3310/eme05020. 70:31A–32A. [101] Bowlus CL, Yang GX, Liu CH, Johnson CR, Dhaliwal SS, Frank D, et al. Ther- [92] Younossi ZM, Stepanova M, Nader F, Gulamhusein A, Hameed B, Thorburn D, apeutic trials of biologics in primary biliary cholangitis: An open label study et al. Patient-reported outcomes (PROS) in patients with primary sclerosing of abatacept and review of the literature. J Autoimmun 2019;101:26–34. cholangitis (PSC) during treatment with cilofexor (GS-9674). Hepatology doi: 10.1016/j.jaut.2019.04.005. 2019;70:805A–806A. [102] Hirschfield GM, Gershwin ME, Strauss R, Mayo MJ, Levy C, Zou B, et al. [93] Jiang JX, Venugopal S, Serizawa N, Chen X, Scott F, Li Y, et al. Reduced Ustekinumab for patients with primary biliary cholangitis who have an inad- nicotinamide adenine dinucleotide phosphate oxidase 2 plays a key role in equate response to ursodeoxycholic acid: A proof-of-concept study. Hepa- – stellate cell activation and liver fibrogenesis in vivo. Gastroenterology tology 2016;64:189 199. doi: 10.1002/hep.28359. 2010;139:1375–1384. doi: 10.1053/j.gastro.2010.05.074. [103] Webb GJ, Hirschfield GM. Work in progress: Drugs in development. Clin – [94] Beuers U, Trauner M, Jansen P, Poupon R. New paradigms in the treatment Liver Dis 2018;22:501 515. doi: 10.1016/j.cld.2018.03.004. [104] Dalekos G, Invernizzi P, Nevens F, Hans VV, Zigmond E, Andrade RJ, et al. of hepatic cholestasis: from UDCA to FXR, PXR and beyond. J Hepatol 2015; Efficacy of GKT831 in patients with primary biliary cholangitis and inad- 62:S25–S37. doi: 10.1016/j.jhep.2015.02.023. equate response to ursodeoxycholic acid: Interim efficacy results of a [95] Fickert P, Hirschfield GM, Denk G, Marschall HU, Altorjay I, Färkkilä M, et al. phase 2 clinical trial. J Hepatol 2019;70:e1–e2. doi: 10.1016/S0618- norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholan- 8278(19)30002-7. gitis. J Hepatol 2017;67:549–558. doi: 10.1016/j.jhep.2017.05.009. [105] Kaplan MM, Bonder A, Ruthazer R, Bonis PA. Methotrexate in patients with [96] Ma H, Zeng M, Han Y, Yan H, Tang H, Sheng J, et al. A multicenter, random- primary biliary cirrhosis who respond incompletely to treatment with urso- ized, double-blind trial comparing the efficacy and safety of TUDCA and deoxycholic acid. Dig Dis Sci 2010;55:3207–3217. doi: 10.1007/s10620- UDCA in Chinese patients with primary biliary cholangitis. Medicine (Balti- 010-1291-5. more) 2016;95:e5391. doi: 10.1097/MD.0000000000005391. [106] Ben AK, Dhahri R, Hana S, Fazaa A, Ouennich K, Kassab S, et al. Safety and [97] Baghdasaryan A, Fuchs CD, Österreicher CH, Lemberger UJ, Halilbasic E, efficacy of infliximab in primary biliary cirrhosis associated with rheumatoid Påhlman I, et al. Inhibition of intestinal bile acid absorption improves cho- arthritis. Intern Med 2017;7:250. doi: 10.4172/2165-8048.1000250. lestatic liver and bile duct injury in a mouse model of sclerosing cholangitis. [107] Dimopoulou D, Dimitroulas T, Akriviadis E, Garyfallos A. Infliximab as a – J Hepatol 2016;64:674 681. doi: 10.1016/j.jhep.2015.10.024. treatment option for patients with rheumatoid arthritis and primary biliary [98] Mayo MJ, Pockros P, Jones D, Bowlus C, Levy C, Patanwala I, et al.Clarity:A cirrhosis. Rheumatol Int 2015;35:1913–1916. doi: 10.1007/s00296-015- phase 2, randomized, double-blind, placebo-controlled study of lopixibat 3366-2. chloride (formerly lum001), a novel apical sodium-dependent bile acid trans- [108] Resorlu H, Kõlõc S, Isõk S, Gokmen F. Successful infliximab therapy in a porter inhibitor, in the treatment of primary biliary cirrhosis associated with patient with comorbid spondyloarthritis, primary biliary cirrhosis and gener- itching. J Hepatol 2016;64:S197. doi: 10.1016/S0168-8278(16)00146-X. alized morphea. Acta Clin Belg 2017;72:365–368. doi: 10.1080/17843286. [99] Myers RP, Swain MG, Lee SS, Shaheen AA, Burak KW. B-cell depletion with 2017.1290567. rituximab in patients with primary biliary cirrhosis refractory to ursodeox- [109] Hegade VS, Jones DE, Hirschfield GM. Apical sodium-dependent transporter ycholic acid. Am J Gastroenterol 2013;108:933–941. doi: 10.1038/ajg. inhibitors in primary biliary cholangitis and primary sclerosing cholangitis. 2013.51. Dig Dis 2017;35:267–274. doi: 10.1159/000450988.

60 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 49–60 Review Article

Portal Cavernoma Cholangiopathy in Children and the Management Dilemmas

Moinak Sen Sarma* and Aathira Ravindranath

Department of Pediatric Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Abstract statements are centered around adults with this disease; however, the problem is less highlighted in children due to Portal cavernoma cholangiopathy (PCC) is one of the most paucity of literature. In this review, we attempt to outline the harrowing complications of extrahepatic portal venous obstruc- epidemiology of PCC, pathogenesis, symptoms, yield of tion, as it determines the long-term hepatobiliary outcome. investigations and management in children. Although symptomatic PCC is rare in children, asymptomatic PCC is as common as that in adults. However, there are major Epidemiological burden of PCC gaps in the literature with regard to the best imaging strategy and management modality in children. Moreover, natural Though it has been more than 5 decades since the first history of PCC and effect of portosystemic shunt surgeries in description of biliary changes in EHPVO, it still remains as children are unclear. Neglected PCC would lead to difficult or one of the most dreaded complications of EHPVO, with a recalcitrant biliary strictures that will require endoscopic ther- direct impact on prognosis. PCC is the biliary complication apy or bilioenteric anastomosis, both of which are challenging which encompasses all abnormalities in the extrahepatic in the presence of extensive collaterals. There are limited biliary ducts, cystic duct, gall bladder and up to second studies on the effect of portosystemic shunt surgeries on the generation intrahepatic biliary ducts in patients with outcome of PCC in children compared to adults. In this review, EHPVO.1 Biliary changes are also described in patients with we aimed to collate all existing literature on PCC in childhood cirrhosis and noncirrhotic portal hypertension, but the major- and also compare with adult studies. We highlight the difficul- ity of cases have been described in EHPVO.2 In adults with ties of this disease to provide a comprehensive platform to EHPVO, PCC is present in 90–100%, of whom symptoms foster further research on PCC exclusively in children. related to PCC are variably seen in 5–38%.3,4 In contrast to Citation of this article: Sen Sarma M, Ravindranath A. Portal the West, EHPVO is the most common cause of portal hyper- cavernoma cholangiopathy in children and the management tension in Asian children (68–76%).5 The reported overall – dilemmas. J Clin Transl Hepatol 2020;8(1):61 68. doi: prevalence of PCC is 92% (85% asymptomatic; 7% sympto- 10.14218/JCTH.2019.00041. matic) in the authors’ study where 72 children were screened with multimodal investigations. Initial symptoms include cholestatic jaundice and recurrent cholangitis.6 In case of persis- Introduction tent obstruction to biliary drainage, secondary biliary cirrhosis can occur in the long run. PCC will affect long-term hepato- Extrahepatic portal venous obstruction (EHPVO) is a disorder biliary outcome independent of the other complications of that occurs predominantly in children and its manifestations portal hypertension in EHPVO. PCC is a silently progressive persist throughout childhood, adolescence and into adult- disease, seemingly innocuous yet deceptive in children. hood. Portal cavernoma cholangiopathy (PCC) is a distinct biliary complication of EHPVO. Cholangiography confirms Pathogenesis hepatobiliary structural changes, whereas vascular imaging delineates venous anatomy of these collaterals. Despite Deciphering the natural history of PCC and predicting EHPVO being a noncirrhotic disease with a favorable response to different types of surgical shunts require a outcome, cholangiopathy is identified only when the patient thorough understanding of pathogenesis, for which it is becomes symptomatic. Existing literature and consensus essential to simplify the complex biliary system venous drainage. The common bile duct (CBD) is essentially drained Keywords: Extra-hepatic portal vein obstruction; Portal cavernoma by tributaries of the portal vein (PV) and superior mesenteric cholangiopathy; Portosystemic shunt. vein (SMV). It is possible that in reality the SMV drains a Abbreviations: CBD, common bile duct; EHPVO, extrahepatic portal venous larger part of CBD than the PV.7 The reticulate epicholedochal obstruction; ERC, endoscopic retrograde cholangiogram; EUS, endoscopic ultra- and paracholedochal venous plexuses wrap around the CBD sound; MRC, magnetic resonance cholangiogram; MRPV, magnetic resonance portovenogram; PCC, portal cavernoma cholangiopathy; PV, portal vein; SMV, like a sieve, flush with the serosal surface. Perforator veins superior mesenteric vein. connect the two surface plexuses with the subepithelial veins Received: 2 September 2019; Revised: 8 December 2019; Accepted: 1 January and intramural plexuses. The surface plexuses form the mar- 2020 ginal veins at 3 o’clock and 9 o’clock positions. Inferiorly, the *Correspondence to: Moinak Sen Sarma, Department of Pediatric Gastroenter- ology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar veins communicate with the gastric veins, gastrocolic trunk Pradesh, India. Tel: +91- 522-2495379, E-mail: [email protected] and posterior superior pancreaticoduodenal veins. Superiorly,

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Copyright: © 2020 Authors. This article has been published under the terms of Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0), which permits noncommercial unrestricted use, distribution, and reproduction in any medium, provided that the following statement is provided. “This article has been published in Journal of Clinical and Translational Hepatology at DOI: 10.14218/JCTH.2019.00041 and can also be viewed on the Journal’s website at http://www.jcthnet.com”. Sen Sarma M. et al: Portal cavernoma cholangiopathy in children they drain into the hilar venous plexus and ultimately into the PV radicals.8 It is important to note that the posterior superior pancreaticoduodenal vein communicates with the posterior inferior pancreaticoduodenal vein and first jejunal vein, which are tributaries of SMV. Similarly, the gastrocolic trunk is also a tributary of SMV (Fig. 1). It is now possible to fathom the consequences of portal venous thrombosis. The paracholedochal and epicholedochal venous channels engorge when PV is blocked (Fig. 2). At this point, the congested bile duct drains through the SMV territory preferentially. Multiple collaterals reach out from the PV zone to the SMV, and a portal cavernoma is thus seen on imaging. However, the bypass drainage is insufficient and suboptimal, and consequently the enlarged paracholedochal veins will cause extrinsic compression on the bile ducts and the epicholedochal veins will cause fine irregularities of the bile duct contour. The subepithelial veins also Fig. 2. Enlargement of peribiliary collaterals and development of cholangiopathy after portal vein thrombosis. enlarge to form intracholedochal varices which protrude into the bile duct lumen.9 Compression by the enlarged venous plexus was ques- protective mechanism to avoid PCC. The authors observed tioned to be the sole mechanism behind PCC, as portosyste- that those with PCC had lower esophageal varices, smaller mic shunt surgeries failed to reverse all the biliary coronary vein diameter and acute angulation of CBD (due to abnormalities. Therefore, the ischemic hypothesis was pro- compression of pancreaticoduodenal veins). Those without posed, according to which ischemia to bile ducts could have PCC had dilated coronary veins due to decrease in hepatope- occurred at the time of PV thrombosis or due to extension of tal flow. In concomitant SMV block, collaterals may flow ret- 10 thrombosis into the draining veins or the arterial network. rograde through the right colic and gastroepiploic veins and With increasing duration of EHPVO, the periportal collaterals gastrocolic trunk. These drain into the anterior pancreatico- will develop surrounding fibrous connective tissue, causing a duodenal vein that communicates with the posterior pancrea- tumorous fibrous cavernoma encasing the CBD and leading to ticoduodenal vein.11 Hence, in a portomesenteric occlusion clinical consequences. It is already known that PCC will there is a preferential peribiliary flow rather than gastroeso- develop due to compression of the bile duct by the engorged phageal flow that worsens the PCC. peribiliary vessels and also due to ischemia. New issues in pathogenesis are related to the flow dynamics in the peribiliary vessels based on the extent of PV thrombosis and patency Implications of symptoms in children of SMV that can have long-term implications, even after portosystemic decompression. Concomitant thrombosis of the Though PCC has been documented in the majority of patients SMV occurs in as high as 64% of patients with EHPVO.6 This with EHPVO, most of the cases are asymptomatic in adults as results in a “double-choking” effect. Neither PV nor SMV have well as in children. Adults with PCC can present with symp- the scope of adequate drainage of bile duct. Thus, the PCC will toms in as high as 38%, but in children symptomatic PCC is be far more severe when there is an associated SMV block, as seen in only 7% cases.4,6 Studies in adults have demonstra- shown in the study by the authors6 (Fig. 3). ted that the probability of symptomatic PCC is related to the Walser et al.11 compared 19 patients with PCC and 41 duration of EHPVO and, hence, it is comprehensible that the patients without PCC in PV thrombosis. They postulated that majority of the children may not present with symptoms of in the event of PV thrombosis, there is a preferential decom- cholestasis as the total duration of EHPVO is shorter compression into the gastroesophageal varices rather than peri- pared to that in adults.12,13 pancreatic venous structures, possibly representing a natural

Fig. 3. Further enlargement of peribiliary collaterals when superior Fig. 1. Normal venous drainage of the biliary apparatus. mesenteric vein is also thrombosed.

62 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 61–68 Sen Sarma M. et al: Portal cavernoma cholangiopathy in children

In a study of adults with symptomatic PCC, the median age In the authors’ study, only 29% of EHPVO had dilated of presentation with symptoms of PCC was 41 years.14 The intrahepatic biliary radicals on ultrasound, while 92% actually mean interval between the first presentation with variceal had PCC on cholangiography.6 In another study that included bleed and jaundice was 7.4 years in another study of adults and children with EHPVO, ultrasound detected biliary adults.15 In a study of adults by Llop et al., it was shown abnormalities in 54% that included intrahepatic biliary radi- that the 5-year and 10-year actuarial probability of develop- cals dilatation, CBD narrowing, CBD wall thickening due to ing symptomatic PCC after diagnosis of chronic PV thrombosis mural varices, and choledocholithiasis.20 Despite the advan- was 9% and 13%, respectively.16 In the pediatric study by the tages of absence of irradiation and no requirement for seda- authors, the age at presentation and the duration of disease tion, ultrasonography is a poor screening tool for PCC. in asymptomatic PCC were 13.9 ± 2.3 and 6.9 ± 4.0 years, Computed tomography of the abdomen can clearly demon- respectively. This was significantly lower than the sympto- strate portal cavernoma, peribiliary varices, parabiliary matic group, where age and duration were 16.1 ± 0.9 and varices, extent of portal thrombosis, and other collaterals. 11.0 ± 1.4 years, respectively. Age at presentation and dura- Maximum information is provided by the portal venous tion of disease had significant linear correlation.6 phase. Computed tomography can reveal bile duct narrowing Zargar et al.17 followed 69 EHPVO children for 15 years, and and also show the corresponding compression by peribiliary 4% developed biliary obstruction. In one of the earlier studies varices.21 The use of computed tomography in children has involving children exclusively, cholestasis was found in the disadvantage of exposure to radiation, especially with 6.6%.18 Symptoms arise due to obstruction of bile flow and repeated imaging in the growing years. result in cholestatic jaundice, pruritus, cholangitis, and gall stones. The implication of finding symptomatic PCC in children Defining biliary and vascular changes by magnetic is grave. This would possibly mean tenacious strictures or resonance imaging stones that would entail multiple therapeutic endoscopies. A series of complications is anticipated. Endoscopic biliary inter- Combination of magnetic resonance cholangiogram (MRC) vention has technical limitations in younger children. Biliary and magnetic resonance portovenogram (MRPV) provides the drainage is associated with a risk of hemobilia from rupture best anatomical details. MRC with heavily T2-weighted of intracholedochal varices. Blood in the biliary tract may in images and maximum intensity projection can create excel- turn lead to biliary sepsis, cholangiolytic abscess, and biliary lent reconstruction of the biliary tree, similar to the images stones. Endoscopic intervention is easier for lower biliary stric- obtained by direct cholangiography.22 There are two major tures than higher strictures, more so in children. Refractory classification systems of biliary changes on cholangiography strictures may necessitate bilioenteric anastomosis. described in adult patients. The Chandra’s classification is Long-standing disease results in secondary biliary cirrho- based on the anatomy of the biliary changes (type I: involve- sis. Secondary biliary cirrhosis is an unfortunate consequence ment of extrahepatic bile duct; type II: involvement of intra- of a problem where a primary liver disease never existed in hepatic bile ducts only; type IIIa: involvement of extrahepatic the first place. It ultimately leads to end-stage liver disease. and unilateral intrahepatic bile ducts; type IIIb: involvement Considering the longevity of a child, quality of life in the of extrahepatic and bilateral intrahepatic bile ducts).2 growing years and gainful living, it is imperative to actively However, this classification does not reflect the severity. The search for asymptomatic biliary changes with serial imaging. Llop’s classification grades PCC as per the severity (grade I: “ Presentation as symptomatic PCC would be akin to missing irregularities and angulations of biliary tree; grade II: steno- ” the boat or practically end-stage disease. sis without dilatation; grade III: strictures with upstream dilatation) (Fig. 4). Biliary dilatation was defined as $4mm Investigative yield diameter of intrahepatic ducts and $7 mm diameter of extrahepatic duct.16 Only the patients with grade III PCC had Since symptomatic PCC is rare the diagnosis relies heavily on symptoms. However, these bile duct diameters cannot be investigations. In adult studies, it has been shown that 50% applied in children of all ages. of those with PCC had normal serum alkaline phosphatase In the pediatric study by Gauthier-Villars et al.,18 the intra- and normal bilirubin at the time of evaluation, despite having hepatic biliary diameter ranged from 2 to 15 mm and extra- jaundice in the past.15 Pediatric studies have also shown that hepatic biliary diameter from 3 to 11 mm. In the authors’ liver biochemical tests do not differentiate between those pediatric study, PCC was graded as per modified Llop’s clas- with and without PCC.6 Thus, normal liver biochemistry sification, by incorporating upper limits of biliary dilatation as does not rule out PCC. The definition of PCC itself involves reported by Zhang et al.6,23 Hence, in children, grade III stric- demonstration of biliary abnormalities by imaging. Pertinent tures with dilatation is defined as upstream dilatation of CBD to children, choosing the appropriate imaging modality and and common hepatic duct, with diameter >4.1 mm, and defining the biliary abnormalities are imprecise. Ultrasonog- central intrahepatic biliary diameter >2 mm. Thus, interpre- raphy is the initial screening tool and when combined with tation of MRC findings in children should be done with the color Doppler forms a handy investigative modality for diag- necessary modifications. Another classification used in nosing PCC. Color Doppler discerns periportal collaterals, tor- adults based on the compressive and ischemic changes on tuous collateral vessels in the gall bladder, and thickened bile biliary ducts divides PCC into varicoid, fibrotic and mixed duct walls due to mural varices (also known as pseudocholan- types. In the varicoid type, extrinsic compressions caused giocarcinoma sign).19 Though ultrasonography can detect by dilated peribiliary vessels produce irregular wavy contour stones, CBD dilatation and dilatation of intrahepatic biliary of the bile ducts and in the fibrotic type, ischemic changes will radicals, the exact number and levels of narrowing cannot produce bile duct narrowing with proximal dilatations. Most be distinguished. Moreover, visualization of CBD will be cases will have a mixture of both these varieties.24 None of more challenging in the presence of high level echoes at the the classifications include cholelithiasis and choledocholithia- porta and bowel gas obscuration, and is operator dependent. sis, which will have clinical implications. In the authors’ own

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Fig. 4. Magnetic resonance cholangiogram. A: Irregularities of the biliary tree (arrow). B: Indentations on the biliary tree (arrow). C: Strictures (arrow) in biliary tree with upstream dilatation. experience, in children, presence of biliary stones and intra- Role of pediatric endoscopic ultrasonography (EUS) hepatic strictures have a more convoluted outcome than simple extrahepatic biliary strictures in terms of therapeutic EUS is an attractive option for diagnosing PCC. Slimmer endoscopy. Hence, there is a dire need to reclassify PCC by diagnostic endosonoscopes are now available. EUS can taking into consideration the symptoms, anatomy, and grade provide information about the vascular changes in the peri- of biliary changes on MRC. biliary area and choledochal varices. Similar to EUS use in MRPV can delineate the extent of PV thrombosis, extent of adults, it can illustrate dilated choledochal perforators, intra- cavernoma, peribiliary and gall bladder collaterals, and dis- choledochal, and intramural gall bladder varices in children. tribution of all intra-abdominal collaterals. The relationship of Children have biliary diameters that are narrower than in biliary strictures with peribiliary collaterals can also be adults, more so due to compressive changes in PCC. Hence on illustrated. However, there are no standard grading systems EUS, the narrow bile duct surrounded by cavernoma is for density of peribiliary collaterals. Certain MRPV findings strenuously identified and appears as the “last log of wood have prognostic significance and aid in surgical planning. A engulfed in flames” (Fig. 5). In contrast to adults, the fine patent left branch of PV and the patency of the confluence differentiation of epi-, para- and peri-choledochal varices, in should be keenly looked for while planning the Meso-Rex reality, is difficult, as subepithelial and fibromuscular layers shunt. For alternate portosystemic shunts, patency of the splenic vein and SMV ought to be determined. As alluded to earlier, SMV forms an important drainage pathway for the peribiliary vessels. SMV block is associated with greater severity of PCC in children on MRC.6 Though MRC and MRPV provide crucial information, children have some unique issues, like requirement of sedation and poor breath-holding which will compromise the image quality. The authors believe that pre-emptive screening for PCC is warranted in children with EHPVO at the time of presentation.

Limitation of endoscopic retrograde cholangiogram (ERC)

The earliest of studies describing PCC used ERC for diagnosis. In the study by Dilawari et al.,3 abnormalities in the CBD were seen in 90%, left bile duct in 100% and right duct in 56%. The various findings described by Khuroo et al.4 included caliber irregularity, ectasias, external impressions, strictures, upstream dilatation, displacement of duct, angulations, and pruning of intrahepatic ducts. The role of diagnostic ERC in the current era is limited. In children with PCC, diagnostic ERC can produce iatrogenic cholangitis and expose to radiation. Moreover, it needs expertise, and size of outer diameter of scope is a major limiting factor for younger children. Hence, diagnostic ERC is not preferred over MRC. Therapeutic ERC is indicated in cases with cholangitis, choledocholithiasis and Fig. 5. Endoscopic ultrasound Doppler image showing dilated common biliary strictures which are symptomatic or persisting after bile duct engulfed by collaterals. portosystemic shunt surgeries.25 Abbreviations: GB, gall bladder; CBD, common bile duct; COLL, collaterals.

64 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 61–68 Sen Sarma M. et al: Portal cavernoma cholangiopathy in children are not adequately demonstrated in children. In the only left branch of PV is the part of the umbilical venous pathway pediatric study that incorporated EUS in PCC evaluation, it that remains patent despite PV block and creation of a conduit was found that there was a modest role in detecting biliary between SMV and recessus of Rex decompresses the portal calculi at the lower end of CBD that could be missed on MRC.6 system in the most physiological manner.27 In the study by Further studies are needed to assess the role of EUS in the Gauthier-Villars et al.,18 two of eight children with sympto- diagnosis and prognostication of PCC in children. EUS could matic PCC underwent Rex anastomosis and liver biochemistry hence play a vital role before ERC by providing information normalized after the shunt surgery. However, Meso-Rex shunt about the exact location of intracholedochal varices in avert- remains a utopian procedure for most children with EHPVO as ing hemobilia during therapeutic ERC. The major drawbacks the left branch of PV is not patent. Despite Meso-Rex bypass of endoscopic ultrasonography are lack of compatible sizes of being recommended in all children with EHPVO, it might not echoendoscopes for younger children and relatively poor be possible in those with non-favorable vascular anatomy. In delineation of higher biliary strictures. As such, this modality addition, in cases with long standing portal hypertension, may be used as an ancilliary investigation. issues related to large spleen predominate and the need for appropriate alternative portosystemic shunts then arises. Dilemmas in definitive management Total and selective portosystemic shunts have their own demerits of encephalopathy and liver ischemia. PCC determines the long-term hepatobiliary outcome in The effect of portosystemic shunt surgeries on PCC has children with EHPVO.26 In developing countries, late referral been shown in small series. In the study by Dhiman et al.10 of EHPVO patients is additive to the management complexity. which included five children, post-portosystemic shunt ERC Management of PCC is a situation of major dilemma for sur- showed that angulations and ectasias persisted and strictures geons and physicians. Choice of portosystemic shunt, reversed partially in all, except one. This study paved way for adequate decompression of biliary varices, appropriate time the ischemic hypothesis. In another study of adults with for bilioenteric anastomosis and prophylactic biliary dilatation EHPVO, among 19 patients with PCC, 10 underwent splenore- for strictures are well debated. Despite active screening for nal shunt surgery with jugular graft, after which symptoms PCC in all children, we must understand that symptoms arise resolved in 7 but there was no significant improvement in as a result of procrastination of treating asymptomatic PCC. liver function tests. Angiograms performed in five patients Symptomatic PCC definitely requires biliary drainage but after shunt surgery showed that portal cavernoma flow requirement of biliary decompression in asymptomatic PCC decreased in four patients, all of whom had patent SMV, and is a dilemma not only in children but also in adults. remained the same in one whose SMV was blocked.28 In a After detecting PCC, the logical step forward has to be recent study comprising 25 children with EHPVO who under- decompression of the portal hypertension with portosystemic went central end to side splenorenal shunt (where Meso-Rex shunt surgery. In those with symptomatic PCC, endoscopic shunt was not feasible), despite the patency of central end- therapy may be required before the shunt surgery. Presently to-side splenorenal shunt and decompression of the gastro- it is viewed that endoscopic therapy is akin to a tight rope esophageal bed, the PCC did not ameliorate at a median walk that has to balance between the risks involved and follow-up of 18 months. All those who had progressive PCC adequacy of biliary drainage. Hence, it is reserved for select had concomitant SMV block29 (Fig. 6) SMV block assumes cases of cholangitis and choledocholithiasis. Limitations such great importance, as it not only deems the case unfavorable as lack of appropriate sized endoscopes and biliary metallic for Meso-Rex bypass, it is also associated with greater stents (not approved yet) are unique issues in children. severity of PCC in children. It is possible that an early alternative pre-emptive portosystemic shunt surgery is warranted Problems of portosystemic shunt surgeries in this group.6 The choice of portosystemic shunt surgery and the Meso-Rex shunt is the most physiological shunt and the patency of SMV will play crucial roles in determining the procedure of choice in EHPVO. The recessus of Rex in the final outcome of PCC. Summary of all studies on PCC in

Fig. 6. Peribiliary collaterals. A: Decompressed after splenorenal shunt when SMV is patent. B: Increased after splenorenal shunt when SMV is blocked. Abbreviation: SMV, superior mesenteric vein.

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Table 1. Summary of the studies on portal cavernoma cholangiopathy in children

Author/Country/ Median Duration of Year/Sample age in disease in size Incidence years years Symptoms Treatment Outcome

Gauthier- 6.6% (8/121) 5 NA Jaundice Mesocaval Symptom Villars18/ (n =3) shunt (n =5) resolution France/2004/ Prutitus (n =3) Jejunocaval (n =8) n = 121 Hepatomegaly shunt (n =1) LFT (n =2) Meso-Rex normalization shunt (n =2) (n =8) Resolution of biliary dilatation (n =7) Zargar17/India/ 4.3% (3/69) NA NA NA Portosystemic NA 2004/n =69 shunt surgery (details NA) Sen Sarma6/ 92% (66/72) 13.9 6.8 Jaundice NA NA India/2018/ 82% - (n =3) n =72 asymptomatic, Cholangitis 7% - symptomatic (n =2) Ravindranath29/ NA 16 6.4 Cholangitis Central end-to- Symptom India/ (n =1) side resolution 2019/n =25 Jaundice splenorenal (n =2) (n =1) shunt (n = 24) Resolution Mesocaval of biliary shunt dilatation (n =1) (n =0)

Abbreviations: LFT, liver function test; NA, not available.

Fig. 7. Summary of management dilemmas of portal cavernoma cholangiopathy in children. Abbreviations: ERCP, endoscopic retrograde cholangiopancreatography; PCC, portal cavernoma cholangiopathy; SMV, superior mesenteric vein.

66 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 61–68 Sen Sarma M. et al: Portal cavernoma cholangiopathy in children children is provided in Table 1. Portosystemic shunt surgery is [3] Dilawari JB, Chawla YK. Pseudosclerosing cholangitis in extrahepatic portal not the final answer to PCC as it may not resolve in all. In venous obstruction. Gut 1992;33:272–276. doi: 10.1136/gut.33.2.272. [4] Khuroo MS, Yattoo GN, Zargar SA, Javid G, Dar MY, Khan BA, et al. Biliary those in whom biliary strictures persist, a second stage bil- abnormalities associated with extrahepatic portal venous obstruction. Hep- 30 ioenteric anastomoses may be contemplated. Bilioenteric atology 1993;17:807–813. doi: 10.1002/hep.1840170510. anastomosis is surgically daunting in naive EHPVO patients, [5] Yachha SK. Portal hypertension in children: an Indian perspective. J Gastro- due to dense hepatobiliary collaterals. The therapeutic strat- enterol Hepatol 2002;17 Suppl 3:S228–S231. doi: 10.1046/j.1440-1746. 17.s3.5.x. egy in asymptomatic PCC is more perplexing, as complete [6] Sen Sarma M, Yachha SK, Rai P, Neyaz Z, Srivastava A, Poddar U. Cholangi- reversal after portosystemic shunt surgery is still uncertain opathy in children with extrahepatic portal venous obstruction. J Hepatobili- (Fig. 7). Although symptomatic PCC occurs more often in ary Pancreat Sci 2018;25:440–447. doi: 10.1002/jhbp.582. adults, masterly inactivity in children with asymptomatic [7] Sen Sarma M, Yachha SK, Chaudhary A. Portal biliopathy. In: Barreto SG, Windsor JA. Surgical diseases of the pancreas and biliary tree. Singapore: PCC would increase the burden of complicated PCC and Springer; 2018:209–218. non-shuntable anatomy as they enter adulthood. Future [8] Ramesh Babu CS, Sharma M. Biliary tract anatomy and its relationship with studies are required to address the optimum management venous drainage. J Clin Exp Hepatol 2014;4:S18–S26. doi: 10.1016/j.jceh. modality in asymptomatic PCC. 2013.05.002. [9] Sharma M, Pathak A. Perforators of common bile duct wall in portal hypertensive biliopathy (with videos). Gastrointest Endosc 2009;70:1041–1043. Liver transplantation for PCC doi: 10.1016/j.gie.2009.03.040. [10] Dhiman RK, Puri P, Chawla Y, Minz M, Bapuraj JR, Gupta S, et al. Biliary After failure of endoscopic and surgical treatment for PCC the changes in extrahepatic portal venous obstruction: compression by collaterals or ischemic? Gastrointest Endosc 1999;50:646–652. doi: 10. only other feasible option is liver transplantation, though the 1016/s0016-5107(99)80013-3. available literature is scarce. The indications of liver trans- [11] Walser EM, Runyan BR, Heckman MG, Bridges MD, Willingham DL, Paz- plantation in this scenario would be recurrent cholangitis, Fumagalli R, et al. Extrahepatic portal biliopathy: proposed etiology on the secondary biliary cirrhosis, or intrahepatic cavernoma.31,32 basis of anatomic and clinical features. Radiology 2011;258:146–153. doi: 10.1148/radiol.10090923. The unique surgical challenge in those with PCC would be to [12] Peter L, Dadhich SK, Yachha SK. Clinical and laboratory differentiation of cir- achieve good portal inflow to the graft by using grafts from rhosis and extrahepatic portal venous obstruction in children. J Gastroenterol other veins or anastomosis with large recipient collater- Hepatol 2003;18:185–189. doi: 10.1046/j.1440-1746.2003.02943.x. als.33,34 Thus, deceased donor liver transplantation is pre- [13] Shukla A, Gupte A, Karvir V, Dhore P, Bhatia S. Long term outcomes of patients with significant biliary obstruction due to portal cavernoma cholangiopathy ferred over living donor due to the availability of suitable and extra-hepatic portal vein obstruction (EHPVO) with no shuntable veins. 35 graft PV length and also other venous conduits. J Clin Exp Hepatol 2017;7:328–333. doi: 10.1016/j.jceh.2017.04.003. [14] Oo YH, Olliff S, Haydon G, Thorburn D. Symptomatic portal biliopathy: a Conclusions single centre experience from the UK. Eur J Gastroenterol Hepatol 2009; 21:206–213. doi: 10.1097/MEG.0b013e3283060ee8. [15] Khare R, Sikora SS, Srikanth G, Choudhuri G, Saraswat VA, Kumar A, et al. PCC is an important complication of EHPVO that affects the Extrahepatic portal venous obstruction and obstructive jaundice: approach long-term prognosis in EHPVO. Optimum modality of imaging to management. J Gastroenterol Hepatol 2005;20:56–61. doi: 10.1111/j. and management of asymptomatic PCC in children still has 1440-1746.2004.03528.x. [16] Llop E, de Juan C, Seijo S, García-Criado A, Abraldes JG, Bosch J, et al. Portal many unanswered questions. Large scale studies are needed cholangiopathy: radiological classification and natural history. Gut 2011;60: to discern the natural history of PCC in children and the effect 853–860. doi: 10.1136/gut.2010.230201. of different types of portosystemic shunt surgeries on PCC [17] Zargar SA, Yattoo GN, Javid G, Khan BA, Shah AH, Shah NA, et al. Fifteen- with respect to SMV patency. year follow up of endoscopic injection sclerotherapy in children with extrahepatic portal venous obstruction. J Gastroenterol Hepatol 2004;19: 139–145. doi: 10.1111/j.1440-1746.2004.03224.x. Funding [18] Gauthier-Villars M, Franchi S, Gauthier F, Fabre M, Pariente D, Bernard O. Cholestasis in children with portal vein obstruction. J Pediatr 2005;146:568– None to declare. 573. doi: 10.1016/j.jpeds.2004.12.025. [19] Denys A, Hélénon O, Lafortune M, Corréas JM, Patriquin H, Moreau JF, et al. Thickening of the wall of the bile duct due to intramural collaterals in three patients with portal vein thrombosis. AJR Am J Roentgenol 1998;171:455– Conflict of interest 456. doi: 10.2214/ajr.171.2.9694474. [20] Kessler A, Graif M, Konikoff F, Mercer D, Oren R, Carmiel M, et al. Vascular and biliary abnormalities mimicking cholangiocarcinoma in patients with cav- The authors have no conflict of interests related to this ernous transformation of the portal vein: role of color Doppler sonography. J publication. Ultrasound Med 2007;26:1089–1095. doi: 10.7863/jum.2007.26.8.1089. [21] Aguirre DA, Farhadi FA, Rattansingh A, Jhaveri KS. Portal biliopathy: imaging manifestations on multidetector computed tomography and magnetic reso- – Author contributions nance imaging. Clin Imaging 2012;36:126 134. doi: 10.1016/j.clinimag. 2011.07.001. [22] Condat B, Vilgrain V, Asselah T, O’Toole D, Rufat P, Zappa M, et al. Portal Conceptualized the article and supervised manuscript writing cavernoma-associated cholangiopathy: a clinical and MR cholangiography (MSS), collected data and drafted the manuscript (AR), coupled with MR portography imaging study. Hepatology 2003;37:1302– approved the final manuscript as submitted and agree to be 1308. doi: 10.1053/jhep.2003.50232. [23] Zhang Y, Wang XL, Li SX, Bai YZ, Ren WD, Xie LM, et al.Ultrasonographic accountable for all aspects of the work (MSS, AR). dimensions of the common bile duct in Chinese children: results of 343 cases. J Pediatr Surg 2013;48:1892–1896. doi: 10.1016/j.jpedsurg.2012.11.047. [24] Shin SM, Kim S, Lee JW, Kim CW, Lee TH, Lee SH, et al. Biliary abnormalities References associated with portal biliopathy: evaluation on MR cholangiography. AJR Am J Roentgenol 2007;188:W341–W347. doi: 10.2214/AJR.05.1649. [1] Chawla Y, Agrawal S. Portal cavernoma cholangiopathy - history, definition [25] Bhatia V. Endoscopic retrograde cholangiography in portal cavernoma chol- and nomenclature. J Clin Exp Hepatol 2014;4:S15–S17. doi: 10.1016/j.jceh. angiopathy - results from different studies and proposal for uniform termi- 2013.04.001. nology. J Clin Exp Hepatol 2014;4:S37–S43. doi: 10.1016/j.jceh.2013.05. [2] Chandra R, Kapoor D, Tharakan A, Chaudhary A, Sarin SK. Portal biliopathy. J 013. Gastroenterol Hepatol 2001;16:1086–1092. doi: 10.1046/j.1440-1746. [26] Kumar M, Saraswat VA. Natural history of portal cavernoma cholangiopathy. 2001.02562.x. J Clin Exp Hepatol 2014;4:S62–S66. doi: 10.1016/j.jceh.2013.08.003.

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[27] Shneider BL, de Ville de Goyet J, Leung DH, Srivastava A, Ling SC, Duché M, [31] Dhiman RK, Saraswat VA, Valla DC, Chawla Y, Behera A, Varma V, et al. Portal et al. Primary prophylaxis of variceal bleeding in children and the role of cavernoma cholangiopathy: consensus statement of a working party of the MesoRex Bypass: Summary of the Baveno VI Pediatric Satellite Symposium. Indian national association for study of the liver. J Clin Exp Hepatol 2014;4: Hepatology 2016;63:1368–1380. doi: 10.1002/hep.28153. S2–S14. doi: 10.1016/j.jceh.2014.02.003. [28] Vibert E, Azoulay D, Aloia T, Pascal G, Veilhan LA, Adam R, et al. Therapeutic [32] Hajdu CH, Murakami T, Diflo T, Taouli B, Laser J, Teperman L, et al. Intra- strategies in symptomatic portal biliopathy. Ann Surg 2007;246:97–104. hepatic portal cavernoma as an indication for liver transplantation. Liver doi: 10.1097/SLA.0b013e318070cada. Transpl 2007;13:1312–1316. doi: 10.1002/lt.21243. [29] Ravindranath A, Sen Sarma M, Yachha SK, Lal R, Singh S, Srivastava A, et al. [33] Filipponi F, Urbani L, Catalano G, Iaria G, Biancofiore G, Cioni R, et al. Portal Outcome of portosystemic shunt surgery on pre-existing cholangiopathy in biliopathy treated by liver transplantation. Transplantation 2004;77:326– children with extrahepatic portal vein obstruction. J Hepatobiliary Pancreat 327. doi: 10.1097/01.TP.0000101795.29250.10. Sci 2019. doi: 10.1002/jhbp.692. [34] Zhang M, Guo C, Pu C, Ren Z, Li Y, Kang Q, et al. Adult to pediatric living [30] Chaudhary A, Dhar P, Sarin SK, Sachdev A, Agarwal AK, Vij JC, et al. Bile duct donor liver transplantation for portal cavernoma. Hepatol Res 2009;39:888– obstruction due to portal biliopathy in extrahepatic portal hypertension: sur- 897. doi: 10.1111/j.1872-034X.2009.00526.x. gical management. Br J Surg 1998;85:326–329. doi: 10.1046/j.1365-2168. [35] Gupta S, Taneja S. Liver transplantation for portal cavernoma cholangiop- 1998.00591.x. athy. J Clin Exp Hepatol 2014;4:S85–S87. doi: 10.1016/j.jceh.2014.01.001.

68 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 61–68 Review Article

Liver Transplantation Beyond Milan Criteria

Vivek A Lingiah*1, Mumtaz Niazi1, Raquel Olivo1, Flavio Paterno2, James V Guarrera2 and Nikolaos T Pyrsopoulos*1

1Department of Medicine, Division of Gastroenterology and Hepatology, Rutgers University, New Jersey Medical School, Newark, NJ, USA; 2Department of Surgery, Division of Liver Transplantation and Hepatobiliary Surgery, Rutgers University, New Jersey Medical School, Newark, NJ, USA

Abstract Introduction

Hepatocellular carcinoma (HCC) is one of the leading causes Hepatocellular carcinoma (HCC) is one of the leading causes of of cancer-related death worldwide, being the fifth most cancer-related death worldwide, being the fifth most common common cancer and the third most common cause of malignancy and the third most common cause of cancer- cancer-related mortality. The incidence of HCC has been related mortality.1,2 The incidence of HCC has been rising in rising in the USA over the last 20 years. Liver transplanta- the USA over the last 20 years and has been attributed to risk tion is an optimal treatment option, as it eliminates HCC as factors, such as chronic hepatitis C infection, hepatitis B infec- well as the underlying liver disease. The Milan criteria tion, heavy alcohol use, diabetes, and nonalcoholic fatty liver (1 lesion greater than or equal to 2 cm and less than or disease, among others.3 Recent data has shown that only 46% equal to 5 cm, or up to 3 lesions, each greater than or equal of HCC cases are diagnosed early on and most are unable to to 1 cm and less than or equal to 3 cm) have been adopted receive curative therapy.4 Currently, it is estimated that the by many transplant societies worldwide as the criteria to median survival for untreated HCC is less than 1 year.5 determine whether patients with HCC can move forward Surgical resection, orthotopic liver transplant (OLT), and with liver transplantation. However, many believe that the ablative techniques are potentially the only curative options Milan criteria may be too strict in regard to its size require- available for HCC. Liver transplant is an excellent therapeutic ments for lesions. This has led to a number of expanded option for long-term survival in patients with HCC, as it criteria for liver transplantation, concerning both overall eliminates both HCC and the underlying advanced liver size and number of lesions, as well as incorporation of other disease.2 Initially, poor patient selection (advanced tumor markers of tumor biology. Tumor markers, such as alpha- burden, unclear tumor etiology) resulted in high rates of fetoprotein, can also be used to follow treatment of HCC and tumor recurrence post-transplant.6 This changed in 1996 possibly exclude patients from transplant. HCC presenting when Mazzaferro and colleagues7 showed 75% 4-year sur- beyond Milan criteria can also be down-staged with locore- vival rate and 83% recurrence-free survival rate, utilizing gional therapy. Monitoring response to locoregional therapy their now well-known Milan criteria. These criteria (one and longer wait times after locoregional therapy prior to lesion greater than or equal to 2 cm and less than or equal transplant can serve as surrogate markers of tumor biology to 5 cm, or up to 3 lesions, each greater than or equal to 1 cm as well. and less than or equal to 3 cm, with no evidence of vascular Citation of this article: Lingiah VA, Niazi M, Olivo R, Pa- invasion or extra-hepatic metastases) showed comparable terno F, Guarrera JV, Pyrsopoulos NT. Liver transplantation beyond Milan criteria. J Clin Transl Hepatol 2020;8(1):69–75. survival outcomes compared to transplants performed on doi: 10.14218/JCTH.2019.00050. patients with cirrhosis but without HCC. The Milan criteria have seen widespread approval and have been incorporated into the United Network for Organ Sharing (UNOS) criteria since 2002, for listing patients with HCC for liver transplant. UNOS is the private, non-profit group that Keywords: Hepatocellular carcinoma; Liver transplantation; Cancer staging; handles the USA’s organ transplant system, under contract Tumor burden; Alpha-fetoprotein. Abbreviations: AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; with the US government. They manage the US transplant HCC, hepatocellular carcinoma; LRT, locoregional therapy; LT, liver transplanta- waiting list as well as the database that contains all the organ tion; MELD, Model for End-Stage Liver Disease; MMaT, Median MELD at Transplant; transplant data in the country.8 The Organ Procurement and mRECIST, modified response evaluation criteria in solid tumors; OLT, orthotopic Transplantation Network is the government workforce oper- liver transplantation; RETREAT, risk estimation of tumor recurrence after transplant; TACE, trans-arterial chemoembolization; TNM, tumor-node-metastasis; ating as a part of the Human Resources and Services Admin- TTV, total tumor volume; UCSF, University of California, San Francisco; UNOS, istration of the US Department of Health and Human United Network for Organ Sharing. Services.9 Currently in the United States, the model for end- Received: 7 October 2019; Revised: 4 February 2020; Accepted: 3 March 2020 *Correspondence to: Nikolaos T Pyrsopoulos, Gastroenterology and Hepatology, stage liver disease score exception points are granted to Rutgers University, New Jersey Medical School, MSB H-355, 185 S Orange Ave, patients with HCC within Milan criteria or who were initially Newark, NJ 07103, USA. Tel: +1-973-972-5252, Fax: +1-973-972-3144, E-mail: identified as outside the Milan criteria but successfully down- [email protected]; Vivek A Lingiah, Division of Gastroenterology and staged within the Milan. Hepatology, Rutgers University, New Jersey Medical School, MSB H-350, 185 S Orange Ave, Newark, NJ 07103, USA. Tel: +1-973-972-5252, Fax: +1-973-972- However, many believe that the Milan criteria may be 3144, E-mail: [email protected] too strict in regard to its size requirements for lesions.

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Publications from around the world support the idea that in 168 patients with pretransplant imaging and showed patients with HCC beyond Milan criteria can be transplanted 5-year recurrence-free survival of 81%, similar to Milan cri- with reasonable post-transplant outcomes (Table 1, Fig. 1). teria, while being able to offer OLT to an extra 5-20% of Additionally, the Milan criteria have been criticized as very patients with HCC not included by the Milan criteria.12 restrictive, as this system is based on tumor size and Duffy et al.13 from UCLA looked at liver transplants per- number, without taking tumor biology into account.10 This is formed for HCC between 1984 and 2006. In their cohort of where the concept of downstaging takes place, which is a 467 patients, 173 were within Milan criteria, 185 within UCSF process of applying locoregional therapy (LRT) to lesions cur- criteria, and 109 beyond UCSF. There were no significant dif- rently outside of the accepted transplant criteria, to reduce ferences in post-transplant survival rates between the Milan tumor burden, meet transplant criteria, and get transplanted. and UCSF criteria at 5 years, by pre-operative imaging (79% Many studies have reported acceptable long-term liver trans- vs. 64%, p = 0.061). Patients with tumors beyond UCSF cri- plant outcomes for patients with HCC beyond Milan criteria teria had 5-year post-transplant survival of less than 50%. who were successfully down-staged to within Milan criteria Further studies conducted more recently have validated the by applying LRT to reduce the tumor burden. UCSF criteria as having similar patient and tumor-free sur- The purpose of this article is to review the different staging vival compared to the Milan criteria.14 criteria present beyond the Milan criteria, types of LRT used in In 2000, the Pittsburgh criteria were conceived – these downstaging, and other factors used in evaluating post- criteria modified the tumor-node-metastasis (commonly transplant recurrence risk. known as TNM) classification and investigated such HCC characteristics as micro/macrovascular invasion, lobar distri- Other HCC transplant criteria used bution, tumor size, and lymph node involvement (all of which are independent predictors of tumor-free survival), as well as One of the most commonly used expanded HCC liver trans- evidence of metastatic disease. Tumor number was not found plant criteria was published by Yao et al.11 in 2001, known as to be a significant predictor. In this staging system, tumor- the University of California, San Francisco (UCSF) criteria. free survival is directly proportional to staging, and the These criteria are defined as a single tumor less than or likelihood of tumor recurrence in each stage is homoge- equal to 6.5 cm, or up to 3 lesions with the largest lesion nous.15 These criteria were able to increase the indications less than or equal to 4.5 cm, with a total tumor diameter no for transplant (being able to transplant larger-sized lesions greater than 8 cm. In that study, patients transplanted for that would not meet Milan criteria). HCC within these criteria had 1- and 5-year survival rates of Chen et al.16 showed in their study that the Pittsburgh 90% and 75.2% respectively, compared to a 1-year survival criteria better predicted outcomes and prognosis than the tra- rate of 50% in patients who exceeded these criteria.11 These ditional International Union against Cancer pTNM classifica- criteria were originally fashioned using explant HCC patholog- tion. In a later study, the Pittsburgh group applied Milan and ical data but were later validated in subsequent studies. UCSF criteria to a cohort of 393 patients who underwent OLT In 2007, the same group used the UCSF criteria prospectively for HCC. Of the 248 patients within Milan criteria, 5-year

Table 1. Various extended criteria for hepatocellular carcinoma and liver transplantation

Tissue/biopsy Criteria Description needed?

Milan criteria 1 lesion $2 cm and #5 cm OR up to 3 lesions, each $1 cm and #3cm No No evidence of vascular invasion or extra-hepatic metastases UCSF criteria 1 lesion #6.5 cm OR up to 3 lesions with the largest lesion #4.5 cm, with a total No tumor diameter #8cm Pittsburgh criteria Tumor number not a significant predictor Yes Modified TNM classification Micro/macrovascular invasion Lobar distribution Tumor size Lymph node involvement Evidence of metastatic disease Hangzhou criteria Total tumor diameter could be: Yes (1) #8cmOR (2) >8 cm, with histopathologic grade 1 or 2, and a preoperative alpha-fetoprotein value of #400 Up-to-seven 7 as total of the size of the largest lesion in cm and number of lesions Yes criteria No vascular invasion Toronto criteria No upper limit on size and number of lesions Yes No extra-hepatic metastases, evidence of venous or biliary tumor thrombus OR cancer-related symptoms All lesions beyond Milan criteria require a liver biopsy to evaluate for poor differentiation, which is exclusionary

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In 2016, the Toronto extended criteria were published. These criteria have no upper limit on size and number of lesions, but exclude patients with extrahepatic metastases, evidence of venous or biliary tumor thrombus, or cancer- related symptoms (weight loss >10 lbs or worsening performance status over 3 months).20 Most importantly, all patients with lesions beyond Milan criteria require a liver biopsy of the largest lesion to evaluate for poor differentiation, which would exclude patients as well. Prospective analysis was done comparing 138 patients meeting Milan criteria versus 105 patients beyond Milan criteria (72.4% of which were beyond UCSF criteria) but within Toronto criteria; the 5-year survival rates were not statistically different between Fig. 1. Comparison of 5-year overall and recurrence-free survival rates the two groups (78% vs. 68%). AFP was also reviewed after- between different transplant criteria. wards and values >500 at listing or at time of transplant were associated with worse outcomes, regardless of whether they were or were not within Milan criteria.20 These criteria are patient survival and recurrence-free survival rates were novel as they are more directly trying to measure tumor 67.3% and 95.7%. Of the 265 patients within UCSF criteria, biology. However, some may have issue with a lesional the 5-year patient survival and recurrence-free survival rates biopsy, as there is an increased risk of tumor seeding. were 67% and 94.4%.17 However, one of the main roadblocks to using these criteria is that information on vascular invasion and lymph node involvement is not easily obtained preoper- Biomarkers atively, limiting its use. In 2008, the Hangzhou criteria were established. These The previously mentioned extended criteria demonstrate that criteria dictated that the total tumor diameter could be either there is an increased desire for further information on tumor 1) less than or equal to 8 cm or 2) greater than 8 cm, with biology and surrogates of tumor biology when trying to histopathologic grade 1 or 2 (based on Edmonson criteria, determine transplant suitability. While histopathologic data which describe grade 1 as being well differentiated and grade 2 of lesions can be helpful in determining presence of micro- as being moderately differentiated) and a preoperative alpha- vascular invasion or poorly differentiated HCC, these characteristics are difficult to obtain in the pretransplant setting. fetoprotein (AFP) #400. Using these criteria, 5-year overall This limitation has led to an increased interest in identifying survival was 70.7% and recurrence-free survival was 62.4%, the best prognostic serologic biomarkers for HCC. similar to Milan criteria. However, with the Hangzhou criteria, AFP is the most common prognostic biomarker studied in an additional 37.5% of patients who would have been beyond relation to HCC. It has been utilized by numerous centers and Milan criteria were able to be transplanted, having favorable has been recently adopted by UNOS as a marker to exclude or long-term survival outcomes.18 These criteria, however, rely include patients from transplant listing (Table 2). Hameed on pretransplant lesional biopsies to estimate tumor differen- et al.21 investigated 211 patients who underwent OLT for tiation. As the American Association for the Study of Liver HCC and were within Milan criteria based on imaging. There Disease guidelines do not recommend routine biopsy of liver was a significant association between AFP levels and vascular lesions, this would be more difficult to implement. invasion, starting at AFP level greater than 300 ng/mL. AFP In 2009, Mazzafero and colleagues19 came up with the Up- greater than 1000 ng/mL was noted to be the strongest pre- to-Seven criteria. These criteria proposed that HCC with 7 as transplant variable predicting vascular invasion, which was the total of the size of the largest lesion in cm and number of the only significant predictor of tumor recurrence. The 5- lesions, without vascular invasion, could have survival out- year recurrence-free survival rate for patients with AFP comes as good as those within Milan criteria. Using a registry greater than 1000 ng/mL was 52.7% compared to 80.3% in of 1556 patients who underwent OLT for HCC in 36 centers, those with AFP less than 1000 ng/mL.21 Utilizing this as an 1112 had HCC beyond Milan criteria and 444 had HCC within exclusion in the trial would have excluded 4.7% of patients Milan criteria. A total of 454 of the 1112 patients had micro- and decreased the rate of HCC recurrence by 20%. Using a vascular invasion, and the 5-year survival of those outside lower cut-off of AFP greater than 400 ng/mL would have Milan criteria was 53.6% compared to the 73.3% 5-year sur- doubled the number of patients excluded (to around 9%) vival for those within Milan criteria. In the 283 patients without but with only an extra 6% HCC recurrence reduction.21 microvascular invasion but who were within the Up-to-Seven Duvoux et al.22 were able to demonstrate that increasing 19 criteria, 5-year overall survival was 71.2%. A limitation to AFP levels at time of listing were related to worse 5-year that study was that it utilized data from postoperative histology recurrence and survival rates. When patients were divided in making determinations on outcomes. This would not be into groups based on AFP less than 100 ng/mL, between available in the pretransplant setting. Other determinants of 100-1000 ng/mL, and greater than 1000 ng/mL, there were tumor biology (response to pretransplant treatment, AFP con- statistically significant different 5-year recurrence and overall centrations, etc.) were also not utilized.19 That being said, it survival rates (16.2% and 67.5% vs. 26.8% and 51.1% vs. was in this study that the ‘Metro Ticket Calculator’ was con- 53.0% and 39.1%, p<0.001).22 In particular, according to ceived, which can provide 3- and 5-year overall survival prob- their model, even subsets of patients exceeding Milan criteria abilities based on characteristics of the HCC lesion. The with one to three lesions and a largest lesion size of 6 cm or concept posits that the farther you travel (beyond Milan/estab- with four or more lesions with maximum tumor diameter of 3 lished criteria), the greater the price (higher post-transplant cm could be considered eligible for OLT if their AFP was less mortality and HCC recurrence rates).10 than 100 ng/mL. Conversely, the model identified patients

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Table 2. The 5-year recurrence, recurrence-free survival and overall survival rates based on pretransplant alpha-fetoprotein level

Study, First 5-year recurrence 5-year recurrence-free 5-year overall author Alpha-fetoprotein level rate, % survival rate, % survival rate, %

Hameed >1000 ng/mL - 52.7 - <1000 ng/mL - 80.3 - Duvoux <100 ng/mL 16.2 - 67.5 100-1000 ng/mL 26.8 - 51.1 >1000 ng/mL 53 - 39.1 Hangzhou #400 ng/mL - 62.4 70.7 Lai #400 ng/mL and/or tumor 4.9 74.4 - diameter #8cm Toso #400 ng/mL and TTV - - 74.6 (4-year) #115 cm3 within Milan criteria but with AFP greater than 1000 ng/mL Milan criteria HCC must have an AFP less than or equal to 1000 with high recurrence risk and significantly reduced survival.22 ng/mL. Candidates with AFP greater than 1000 ng/mL can However, the optimal AFP value for defining patients at undergo LRT, and if the AFP decreases to less than 500 ng/mL, higher risk for HCC recurrence has not been found. The then the candidate can be qualified for a standardized MELD Hangzhou criteria previously mentioned utilization of an AFP exception (as long as the AFP remains <500 ng/mL). Patients cut-off of up to 400 ng/mL and were able to obtain a 5-year with AFP greater than 500 ng/mL after LRT need to apply to the overall survival rate of 70.7% in patients meeting their national liver review board to receive MELD exception points.27 criteria.18 Lai et al.23 reviewed 158 patients undergoing OLT for HCC and noted that patients with the combination of AFP Downstaging up to 400 ng/mL and total tumor diameter no greater than 8 cm had a 5-year recurrence-free survival of 74.4%, similar to Besides using extended criteria to transplant patients with Milan and UCSF criteria, while this approach was able to HCC beyond Milan criteria, patients might undergo therapy increase the number of transplant candidates. Toso et al.24 until the tumor burden will be within the Milan criteria. The used similar parameters with their study, looking at total term, ‘downstaging’, refers to the use of LRT applied in order tumor volume (referred to here as TTV; being no greater to decrease the tumor burden until it meets suitable criteria than 115 cm3) and AFP no greater than 400 ng/mL. In their for OLT (usually Milan criteria).10 One of the advantages of prospective study of 166 patients transplanted for HCC, 134 this method is that tumor biology can be assessed based on were within Milan and 32 were beyond Milan but within TTV/ tumor behavior over a period of time. Good response to AFP criteria; the 4-year overall survival rates for the Milan downstaging has often been linked to the presence of histo- and TTV/AFP groups were similar at 78.7% vs. 74.6%. logic markers of good prognosis in the treated HCC (lack of Some studies have even recommended an AFP less than 25 microvascular invasion, low tumor grading, lack of satellite 100 ng/mL as an upper limit. Grat et al. performed a lesions), similar to patients being transplanted within Milan study evaluating 121 patients transplanted for HCC and dem- criteria at presentation.28 In the USA, UNOS policy now onstrated that increasing AFP had a nearly linear association requires a 6 month waiting period prior to granting HCC with increased risk of HCC recurrence in patients transplanted MELD exception points, so that tumor biology and response within UCSF and Up-to-Seven criteria. Patients transplanted to LRT can be assessed.27 under UCSF and within AFP limits had better 5-year recur- Yao et al.29 showed in 2015 that patients beyond Milan cri- rence-free survival than those within UCSF but not within teria undergoing downstaging to within Milan criteria for HCC the AFP limit (100% vs. 69%). This trend was also seen in prior to OLT had similar 5-year post-transplant survival and those meeting the Up-to-Seven criteria (100% vs. 71.9%). recurrence-free probabilities compared to patients within Recent publications have not only been investigating just a Milan criteria (T2 lesion) without downstaging (77.8% and static AFP prior to OLT but the trend of AFP in patients awaiting 90.8% vs. 81% and 88%). Even when assessing response OLT. This AFP ‘slope’ has been thought to be more reflective of via 5-year intention-to-treat survival rate, there was no differ- the dynamic nature of tumor biology than just a single value. ence between the downstaging and T2 groups (56.1% vs. 26 Giard et al. recently looked at 336 patients undergoing OLT for 63.3%). Similar post-transplant outcomes have been shown HCC who were within Milan criteria. Nearly all (98%) of the in other downstaging studies as well.5,30 Current UNOS policy patients had LRT at some time prior to transplant and the AFP includes a downstaging protocol to allow patients to obtain HCC slope was estimated by the AFP values during this pretransplant MELD exception points if specific criteria are met (Fig. 2).27 period. An AFP slope increase greater than 7.5 ng/mL/month The main options for LRT include ablative techniques despite LRT was significantly associated with post-transplant (radiofrequency ablation or microwave ablation), trans- HCC recurrence (3-time increasing risk compared to those arterial chemoembolization (TACE), or trans-arterial radio- with a decreasing AFP slope) and microvascular invasion.26 embolization. The decision as to what type of LRT should be The current UNOS HCC model for end-stage liver disease used depends on the degree of tumor burden, liver function, exception criteria take into account a candidate’s AFP. Candi- and location of the tumor.10 The most commonly used dates applying for standardized MELD exception for within staging system for HCC is the Barcelona Clinic Liver Cancer

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reduced tumor ablation in lesions larger than 3 cm.2 Microwave ablation uses electromagnetic energy to heat tissue and is less prone to the heat sink effect, allowing treatment of lesions near large blood vessels.28

Response to LRT and wait times

The response of HCC to different modalities of LRT is an important surrogate marker for survival, as well as a measure of tumor biology. The modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria were developed as a method for measuring treatment response based on tumor shrinkage. Fig. 2. Downstaging protocol according to UNOS policy. These criteria distinguish response into four categories – Candidates can be eligible for model for end-stage liver disease ex- complete response (disappearance of arterial enhancement in ception points if their pretreatment disease meets one of the criteria in tumor(s)), partial response (at least 30% reduction in sum of the left column. Once undergoing locoregional therapy, if the candi- diameters of viable tumor compared to baseline), stable date’s post-treatment disease meets T2 criteria (an option in the right disease (not meeting partial response or progressive disease column), they are eligible for a standardized model for end-stage liver criteria), and progressive disease (an increase of at least 20% disease exception. in sum of the diameters of viable tumor compared to baseline, or the appearance of new lesions).33 Gillmore et al.34 performed a study looking at 83 patients with HCC undergoing (commonly known as the BCLC) staging system. This system TACE with follow-up imaging done after a median of 64 days. evaluates tumor burden, liver function, as well as patient per- They found overall and target lesion response of 57% and 73% formance status when stratifying patients into different cate- based on mRECIST results, with a significant association gories. Given that patients being down-staged have advanced between survival and overall (complete or partial) mRECIST tumor burden, based on BCLC staging, TACE is usually utilized response in the form of a 42% risk reduction. Bargellini 35 most often, followed by trans-arterial radioembolization and et al. presented a study of 33 patients with HCC beyond ablative techniques.2 Milan criteria who underwent OLT after treatment with TACE. Since it has the best quality of evidence, TACE is the Tumor response was reviewed by CT scan at 1 month after recommended treatment option for large or multifocal HCC intervention based on mRECISTcriteria. The 5-year overall sur- without evidence of vascular invasion or extrahepatic meta- vival rate was significantly better in patients with complete stasis (BCLC stage B, intermediate).31 Conventional TACE response (94.4%) compared to partial response (45.4%) and involves arterial catheter delivery of chemotherapy (usually stable disease (50%). These significant differences were sim- doxorubicin or cisplatin) via lipiodol emulsion followed by vas- ilarly seen with 5-year recurrence-free rates (94.4% vs. 46.7% and 50%, respectively). Another study assessed patients cular embolization of tumor-feeding vessels. The combination undergoing OLT for HCC after TACE (both within and beyond of cytotoxic and ischemic injury tends to be significant as Milan criteria), with treatment response assessed using these lesions are usually fed entirely by arterial flow, as mRECIST. That study showed that the 5-year recurrence rate opposed to liver parenchyma that receives most of its blood was 5.3% in complete and partial responders compared to flow via the portal system.28 Patients with poor hepatic func- 17.6% in those with stable or progressive disease.36 tion (total bilirubin >2 mg/dL) or tumor burden greater than Along with response to LRT, the waiting time while listed for 50% of liver volume have higher risk for decompensation liver transplant can be used to further assess tumor biology after TACE.28 The advent of super-selective TACE, done to and identify aggressive HCC. Halazun et al.37 showed that minimize ischemic injury to nontumor liver tissue while still 32 patients with HCC receiving OLT in regions with shorter wait inducing tumor necrosis, can mitigate some of these risks. times (median of 1.6 months) had significantly higher post- Trans-arterial radioembolization is another treatment transplant mortality than patients transplanted in regions modality for HCC. With this modality, microspheres contain- with longer wait times (median 7.6 months), with 5-year sur- ing Yttrium-90 are infused into the hepatic artery and are vival rates of 67% versus 75%, respectively. The concept of preferentially delivered to the tumor area, where they emit ‘ablate and wait’ to assess tumor biology over time has been high-energy, low-penetration radiation to the tumor. Because shown to have merit in other studies, with the most current of the small size of the microspheres, trans-arterial radio- UNOS policy requiring a 6 month waiting period for patients 28 embolization can be used in portal vein thrombus. listed with HCC prior to receiving MELD exception points.27,38 Ablative techniques have also been used with downstaging, The policies in regard to UNOS MELD exceptions for HCC though these are usually used in concert with other forms of have been an evolving field. Up until recently, patients were LRT. These range from percutaneous ethanol injection to granted a MELD exception of 28 points after showing stable radiofrequency or microwave ablation. In radiofrequency abla- within-Milan criteria HCC for 6 months from listing (with an tion, cell death is attained by frictional heat using high AFP #1000 ng/mL). Escalating MELD exception points could frequency alternating current, this heat producing coagulation be accrued every 3 months, with a cap of 34. Since May 2019, necrosis.28 Location of the lesion is important when consider- however, new policies have been implemented, where ing radiofrequency ablation, as lesions near blood vessels may patients receive exception points equal to the median MELD not be treated appropriately due to a heat sink effect (impaired at transplant (MMaT) -3. The median MELD at transplant heating of tumor cells because of ‘cooler’ blood flow near the refers to the median of MELD scores at the moment of lesion). Lesions treated are usually no larger than 3 cm, as the transplant of all recipients 12 years and older transplanted ablative zone produced with radiofrequency ablation results in in hospitals within 250 nautical miles around the listing

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Table 3. RETREAT score components and scoring criteria breakdown

Characteristic Range of values RETREAT points

Alpha-fetoprotein at transplant in ng/mL 0-20 0 21-99 1 100-999 2 >1000 3 Microvascular invasion Absent 0 Present 2 Largest active tumor length (cm) + number of active tumors 00 1.1-4.9 1 5-9.9 2 $10 3 hospital in the last year.27 This change was made to make Conclusions sure that a candidate’s assigned score was more proportional to their needs and that candidates who relied on their native HCC is an ever-growing entity and is becoming a more MELD were not disadvantaged. This was also done to ensure common etiology to pursue orthotopic liver transplantation. that MELD exception points for HCC had a similar impact on Successful outcomes for transplantation due to HCC should transplantation in different parts of the country, each with have 5-year survival rates similar to those receiving liver 27,39 unique median MELDs at transplant. transplant for non-HCC related liver disease. While the Milan criteria have provided good, stable post-transplant survival Post-transplant recurrence and recurrence-free outcomes, there appear to be other expanded criteria that have been able to provide similar One of the major fears after liver transplant for HCC is tumor outcomes while opening the pathway of transplant to a recurrence, which has been noted to occur in 8-20% of larger cohort of patients. The majority of these criteria, transplanted patients. HCC recurrence usually occurs within however, require lesional tissue/histology to comment on 2 years after transplant, with a median survival of around 1 markers of tumor biology, like microvascular invasion or year after diagnosis.40 While many elements have been poor tumor differentiation. Downstaging of HCC to within linked to tumor recurrence post-transplantation, there are Milan criteria is the alternative option. Surrogates of tumor few standardized risk scores formed to ascertain a transplant biology can be assessed with this approach, such as response recipient’s chances of HCC recurrence. After successful OLT in to LRT and increased wait times after LRT before transplant. patients with HCC, further information can be obtained based AFP level or the AFP slope in producers can be another option on the explant pathology. to predict those with higher chances of recurrence and can be The Risk Estimation of Tumor REcurrence After Transplant used in patient selection for transplant. These methods can be (RETREAT) is a validated prognostic score to help predict considered to choose optimal transplant candidates and post-transplant HCC recurrence, detect patients who can be increase the number of patients who liver transplantation assisted by adjuvant therapy, and aid in defining post- can safely be offered to. transplant HCC surveillance schedules. When reviewing characteristics significant for HCC recurrence in 721 patients with Funding HCC within Milan criteria, three variables were noted to be independently associated with HCC recurrence. These None to declare. included 1) tumor burden of the explant liver (total of largest viable tumor diameter and the number of viable Conflict of interest tumors of explanted liver), 2) evidence of microvascular invasion, and 3) AFP at time of transplant. The RETREAT Nikolaos T Pyrsopoulos is a recipient of research grants from score was derived from these variables, with point values Gilead, Abbvie, Merck and Roche. The other authors have no being assigned to each of the criteria and with scores ranging conflict of interests related to this publication. from 0-8 (higher scores denoting higher risk of recurrence) (Table 3). A RETREAT score of 0 predicted 1- and 5-year HCC recurrence rates of 1% and 2.9%, whereas RETREAT scores of Author contributions 5 or more were associated with 1- and 5-year HCC recurrence rates of 39.3% and 75.2% (C-statistic of 0.77, 95% confi- Reviewed the literature, performed the majority of the dence interval of 0.71-0.82). This scoring system was vali- writing, and prepared the figures and tables (VAL), designed, dated in another cohort of 340 patients, with a C-statistic of co-authored and revised the manuscript (NTP), revised the 0.82 (95% confidence interval of 0.77-0.86).41 manuscript (MN, RO, FP, JVG).

74 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 69–75 Lingiah V.A. et al: Liver transplantation beyond Milan criteria

References [22] Duvoux C, Roudot-Thoraval F, Decaens T, Pessione F, Badran H, Piardi T, et al. Liver transplantation for hepatocellular carcinoma: a model including a-fetoprotein improves the performance of Milan criteria. Gastroenterology 2012; [1] Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. 143:986–994.e3. doi: 10.1053/j.gastro.2012.05.052. Cancer incidence and mortality worldwide: sources, methods and major pat- [23] Lai Q, Avolio AW, Manzia TM, Sorge R, Agnes S, Tisone G, Combination of terns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–E386. doi: 10. biological and morphological parameters for the selection of patients with 1002/ijc.29210. hepatocellular carcinoma waiting for liver transplantation. Clin Transplant [2] Marrero JA, Kulik LM, Sirlin CB, Zhu AX, Finn RS, Abecassis MM, et al. Diag- 2012;26:E125–E131. doi: 10.1111/j.1399-0012.2011.01572.x. nosis, staging, and management of hepatocellular carcinoma: 2018 practice [24] Toso C, Meeberg G, Hernandez-Alejandro R, Dufour JF, Marotta P, Majno P, guidance by the American association for the study of liver diseases. Hepa- et al. Total tumor volume and alpha-fetoprotein for selection of transplant tology 2018;68:723–750. doi: 10.1002/hep.29913. candidates with hepatocellular carcinoma: A prospective validation. Hepatol- [3] Kulik L, El-Serag HB. Epidemiology and management of hepatocellular carci- ogy 2015;62:158–165. doi: 10.1002/hep.27787. noma. Gastroenterology 2019;156:477–491.e1. doi: 10.1053/j.gastro. [25] Gra¸t M, Kornasiewicz O, Lewandowski Z, Ho1ówko W, Gra¸t K, KobrynK, et al. 2018.08.065. Combination of morphologic criteria and a-fetoprotein in selection of patients [4] Singal AG, El-Serag HB. Hepatocellular carcinoma from epidemiology to pre- with hepatocellular carcinoma for liver transplantation minimizes the vention: Translating knowledge into practice. Clin Gastroenterol Hepatol problem of posttransplant tumor recurrence. World J Surg 2014;38:2698– 2015;13:2140–2151. doi: 10.1016/j.cgh.2015.08.014. [5] Kim Y, Stahl CC, Makramalla A, Olowokure OO, Ristagno RL, Dhar VK, et al. 2707. doi: 10.1007/s00268-014-2647-3. [26] Giard JM, Mehta N, Dodge JL, Roberts JP, Yao FY. Alpha-fetoprotein slope Downstaging therapy followed by liver transplantation for hepatocellular carcinoma beyond Milan criteria. Surgery 2017;162:1250–1258. doi: 10. >7.5 ng/ml per month predicts microvascular invasion and tumor recurrence 1016/j.surg.2017.08.007. after liver transplantation for hepatocellular carcinoma. Transplantation – [6] Iwatsuki S, Gordon RD, Shaw BW Jr, Starzl TE. Role of liver transplantation in 2018;102:816 822. doi: 10.1097/TP.0000000000002094. cancer therapy. Ann Surg 1985;202:401–407. doi: 10.1097/00000658- [27] OPTN Policies. Available from: https://optn.transplant.hrsa.gov/me- 198510000-00001. dia/1200/optn_policies.pdf. [7] Mazzaferro V, Regalia E, Doci R, Andreola S, Pulvirenti A, Bozzetti F, et al. [28] EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. Liver transplantation for the treatment of small hepatocellular carcinomas in J. Hepatol 2018;69:182-236. doi: 10.1016/j.jhep.2018.03.019. patients with cirrhosis. N Engl J Med 1996;334:693–699. doi: 10. [29] Yao FY, Mehta N, Flemming J, Dodge J, Hameed B, Fix O, et al. Downstaging 1056/NEJM199603143341104. of hepatocellular cancer before liver transplant: long-term outcome com- – [8] What is UNOS? | About United Network for Organ Sharing. Available from: pared to tumors within Milan criteria. Hepatology 2015;61:1968 1977. https://unos.org/about/. doi: 10.1002/hep.27752. [9] Organ Procurement and Transplantation Network. About the OPTN. Available [30] Ravaioli M, Grazi GL, Piscaglia F, Trevisani F, Cescon M, Ercolani G, et al. Liver from: https://optn.transplant.hrsa.gov/governance/about-the-optn. transplantation for hepatocellular carcinoma: results of down-staging in [10] Rudnick SR, Russo MW. Liver transplantation beyond or downstaging within patients initially outside the Milan selection criteria. Am J Transplant 2008; – the Milan criteria for hepatocellular carcinoma. Expert Rev Gastroenterol 8:2547 2557. doi: 10.1111/j.1600-6143.2008.02409.x. Hepatol 2018;12:265–275. doi: 10.1080/17474124.2018.1417035. [31] Sieghart W, Hucke F, Peck-Radosavljevic M. Transarterial chemoemboliza- [11] Yao FY, Ferrell L, Bass NM, Watson JJ, Bacchetti P, Venook A, et al. Liver tion: modalities, indication, and patient selection. J Hepatol 2015;62: – transplantation for hepatocellular carcinoma: expansion of the tumor size 1187 1195. doi: 10.1016/j.jhep.2015.02.010. limits does not adversely impact survival. Hepatology 2001;33:1394– [32] Bryce K, Tsochatzis EA. Downstaging for hepatocellular cancer: harm or 1403. doi: 10.1053/jhep.2001.24563 benefit? Transl Gastroenterol Hepatol 2017;2:106. doi: 10.21037/tgh. [12] Yao FY, Xiao L, Bass NM, Kerlan R, Ascher NL, Roberts JP. Liver transplanta- 2017.11.18. tion for hepatocellular carcinoma: validation of the UCSF-expanded criteria [33] Lencioni R, Llovet JM. Modified RECIST (mRECIST) assessment for hepato- – based on preoperative imaging. Am J Transplant 2007;7:2587–2596. doi: cellular carcinoma. Semin Liver Dis 2010;30:52 60. doi: 10.1055/s-0030- 10.1111/j.1600-6143.2007.01965.x. 1247132. [13] Duffy JP, Vardanian A, Benjamin E, Watson M, Farmer DG, Ghobrial RM, et al. [34] Gillmore R, Stuart S, Kirkwood A, Hameeduddin A, Woodward N, Burroughs Liver transplantation criteria for hepatocellular carcinoma should be AK, et al. EASL and mRECIST responses are independent prognostic factors expanded: a 22-year experience with 467 patients at UCLA. Ann Surg for survival in hepatocellular cancer patients treated with transarterial embo- 2007;246:502–511. doi: 10.1097/SLA.0b013e318148c704. lization. J Hepatol 2011;55:1309–1316. doi: 10.1016/j.jhep.2011.03.007. [14] Abdelfattah MR, Elsiesy H, Al-Manea H, Broering DC. Liver transplantation for [35] Bargellini I, Vignali C, Cioni R, Petruzzi P, Cicorelli A, Campani D, et al. Hep- hepatocellular carcinoma within the Milan criteria versus the University of atocellular carcinoma: CT for tumor response after transarterial chemoem- California San Francisco criteria: a comparative study. Eur J Gastroenterol bolization in patients exceeding Milan criteria–selection parameter for liver Hepatol 2018;30:398–403. doi: 10.1097/MEG.0000000000001044. transplantation. Radiology 2010;255:289–300. doi: 10.1148/radiol. [15] Marsh JW, Dvorchik I, Bonham CA, Iwatsuki S. Is the pathologic TNM staging 09090927. system for patients with hepatoma predictive of outcome? Cancer 2000;88: [36] Kim DJ, Clark PJ, Heimbach J, Rosen C, Sanchez W, Watt K, et al. Recurrence 538–543. of hepatocellular carcinoma: importance of mRECIST response to chemo- [16] Chen J, Xu X, Ling Q, Wu J, Zheng SS. Role of Pittsburgh modified TNM embolization and tumor size. Am J Transplant 2014;14:1383–1390. doi: criteria in prognosis prediction of liver transplantation for hepatocellular car- 10.1111/ajt.12684. cinoma. Chin Med J (Engl) 2007;120:2200–2203. [37] Halazun KJ, Patzer RE, Rana AA, Verna EC, Griesemer AD, Parsons RF, et al. [17] Marsh JW, Dvorchik I. Liver organ allocation for hepatocellular carcinoma: Standing the test of time: outcomes of a decade of prioritizing patients with are we sure? Liver Transpl 2003;9:693–696. doi: 10.1053/jlts.2003.50086. hepatocellular carcinoma, results of the UNOS natural geographic experi- [18] Zheng SS, Xu X, Wu J, Chen J, Wang WL, Zhang M, et al. Liver transplantation ment. Hepatology 2014;60:1957–1962. doi: 10.1002/hep.27272. for hepatocellular carcinoma: Hangzhou experiences. Transplantation 2008; [38] Roberts JP, Venook A, Kerlan R, Yao F. Hepatocellular carcinoma: Ablate and 85:1726–1732. doi: 10.1097/TP.0b013e31816b67e4. wait versus rapid transplantation. Liver Transpl 2010;16:925–929. doi: 10. [19] Mazzaferro V, Llovet JM, Miceli R, Bhoori S, Schiavo M, Mariani L, et al. Pre- 1002/lt.22103. dicting survival after liver transplantation in patients with hepatocellular car- [39] National Liver Review Board (NLRB) Frequently asked questions for candi- cinoma beyond the Milan criteria: a retrospective, exploratory analysis. dates and caregivers. Available from: https://optn.transplant.hrsa.gov/me- Lancet Oncol 2009;10:35–43. doi: 10.1016/S1470-2045(08)70284-5. dia/2797/201901_nlrb_faq_patient.pdf. [20] Sapisochin G, Goldaracena N, Laurence JM, Dib M, Barbas A, Ghanekar A, [40] Clavien PA, Lesurtel M, Bossuyt PM, Gores GJ, Langer B, Perrier A. Recom- et al. The extended Toronto criteria for liver transplantation in patients with mendations for liver transplantation for hepatocellular carcinoma: an inter- hepatocellular carcinoma: A prospective validation study. Hepatology 2016; national consensus conference report. Lancet Oncol 2012;13:e11–e22. doi: 64:2077–2088. doi: 10.1002/hep.28643. 10.1016/S1470-2045(11)70175-9. [21] Hameed B, Mehta N, Sapisochin G, Roberts JP, Yao FY. Alpha-fetoprotein level [41] Mehta N, Heimbach J, Harnois DM, Sapisochin G, Dodge JL, Lee D, et al. >1000 ng/mL as an exclusion criterion for liver transplantation in patients Validation of a risk estimation of tumor recurrence after transplant with hepatocellular carcinoma meeting the Milan criteria. Liver Transpl 2014; (RETREAT) score for hepatocellular carcinoma recurrence after liver trans- 20:945–951. doi: 10.1002/lt.23904. plant. JAMA Oncol 2017;3:493–500. doi: 10.1001/jamaoncol.2016.5116.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 69–75 75 Review Article

Non-alcoholic Fatty Liver Disease: Growing Burden, Adverse Outcomes and Associations

Ramesh Kumar*1, Rajeev Nayan Priyadarshi2 and Utpal Anand3

1Department of Gastroenterology, All India Institute of Medical Sciences, Patna, India; 2Department of Radiodiagnosis, All India Institute of Medical Sciences, Patna, India; 3Department of Surgical Gastroenterology, All India Institute of Medical Sciences, Patna, India

Abstract Introduction

Nonalcoholic fatty liver disease (NAFLD) is a systemic disor- Nonalcoholic fatty liver disease (NAFLD) is characterized by der with a complex multifactorial pathogenesis and hetero- accumulation of fat in $5% of hepatocytes in the absence of genous clinical manifestations. NAFLD, once believed to be an significant alcohol consumption (<30 g/day for men and <20 innocuous condition, has now become the most common g/day for women) or secondary causes of hepatic steatosis.1 cause of chronic liver disease in many countries worldwide. Histologically, the spectrum of NAFLD ranges from simple NAFLD is already highly prevalent in the general population, steatosis (SS) that in some patients can progress to nonalco- and owing to a rising incidence of obesity and diabetes holic steatohepatitis (NASH), advanced fibrosis, cirrhosis, and mellitus, the incidence of NAFLD and its impact on global ultimately hepatocellular carcinoma (HCC), and liver failure. healthcare are expected to increase in the future. A subset of NAFLD, once believed to be an innocuous condition, has patients with NAFLD develops progressive liver disease lead- emerged as the leading cause of chronic liver disease in ing to cirrhosis, hepatocellular carcinoma, and liver failure. many countries worldwide.2,3 NAFLD is now pandemic world- NAFLD has emerged as one of the leading causes of cirrhosis wide and its prevalence has increased considerably over the and hepatocellular carcinoma in recent years. Moreover, HCC last two decades.4–7 can occur in NAFLD even in absence of cirrhosis. Compared The changing epidemiology of NAFLD in Asia during the with the general population, NAFLD increases the risk of liver- past two decades is well-documented.4,6,7 NAFLD is strongly related, cardiovascular and all-cause mortality. NAFLD is associated with metabolic syndrome (MetS), the components bidirectionally associated with metabolic syndrome. NAFLD of which include hypertension, hyperglycemia, abdominal increases the risk and contributes to aggravation of the obesity, and dyslipidemia.8 However, NAFLD is not merely a pathophysiology of atherosclerosis, cardiovascular diseases, hepatic manifestation of MetS but rather both a consequence diabetes mellitus, and chronic kidney disease. In addition, as well a predecessor of MetS. Compared with the general NAFLD is linked to colorectal polyps, polycystic ovarian population, NAFLD patients are at increased risk of liver- syndrome, osteoporosis, obstructive sleep apnea, stroke, related, cardiovascular and all-cause mortality. NAFLD has and various extrahepatic malignancies. Extended resection been associated with a large number of extrahepatic condi- of steatotic liver is associated with increased risk of liver tions, such as type-2 diabetes mellitus (T2DM), atherosclero- failure and mortality. There is an increasing trend of NAFLD- sis, cardiovascular disease (CVD), chronic kidney disease related cirrhosis requiring liver transplantation, and the (CKD), polycystic ovarian syndrome (PCOS), obstructive recurrence of NAFLD in such patients is almost universal. sleep apnea (OSA), extrahepatic malignancies, etc.9,10 This review discusses the growing burden of NAFLD, its Recent data suggest that NAFLD increases the susceptibility outcomes, and adverse associations with various diseases. and/or worsen outcome of acute pancreatitis (AP), cerebro- Citation of this article: Kumar R, Priyadarshi RN, Anand U. vascular accident (CVA), and osteoporosis.11–13 Non-alcoholic fatty liver disease: Growing burden, adverse out- There is growing trend of patients with NASH-related comes and associations. J Clin Transl Hepatol 2020;8(1):76–86. cirrhosis requiring liver transplantation (LT).14,15 The risk of doi: 10.14218/JCTH.2019.00051. developing progressive liver disease and associated extrahepatic diseases presents a challenge to the healthcare system to develop effective strategies in order to prevent an expo- Keywords: NAFLD; NASH; Metabolic; Outcome; Association. nential increase in morbidity and mortality related to it. This Abbreviations: AP, acute pancreatitis; CC, cryptogenic cirrhosis; CI, confidence review will focus on the growing burden of NAFLD, its out- interval; CKD, chronic kidney disease; CVA, cerebrovascular accident; CVD, car- comes and adverse associations with various extrahepatic diovascular disease; DM, diabetes mellitus; HCC, hepatocellular carcinoma; HR, hazard ratio; IR, insulin resistance; LT, liver transplantation; MetS, metabolic syn- diseases; of note, this review is not intended to discuss the drome; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; therapeutic aspects of NAFLD and its complications. NASH, nonalcoholic steatohepatitis; NHANES, National Health and Nutrition Examination Surveys; OSA, obstructive sleep apnea; OR, odds ratio; PCOS, polycystic ovarian syndrome; SS, simple steatosis; T2DM, type 2 diabetes mellitus. Global burden and rising prevalence Received: 10 October 2019; Revised: 3 December 2019; Accepted: 9 December 2019 According to current estimate, the global prevalence *Correspondence to: Ramesh Kumar, Department of Gastroenterology, All India Institute of Medical Sciences, 4th floor, OPD Block, Patna 801507, India. Tel: +91- of NAFLD among the general population may be as high 2 7765803112, E-mail: [email protected] as one billion. In a recent meta-analysis of 86 studies,

76 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86

Copyright: © 2020 Authors. This article has been published under the terms of Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0), which permits noncommercial unrestricted use, distribution, and reproduction in any medium, provided that the following statement is provided. “This article has been published in Journal of Clinical and Translational Hepatology at DOI: 10.14218/JCTH.2019.00051 and can also be viewed on the Journal’s website at http://www.jcthnet.com”. Kumar R. et al: NAFLD: a growing threat encompassing a sample size of 8,515,431 from 22 countries, sensitivity of diagnostic tools complicate diagnosis of NAFLD, the prevalence of NAFLD in the general population was often leading to an underestimate of the actual disease 25.24%, with the highest prevalence rates in the Middle burden. East and South America.5 That meta-analysis also demonstrated an increased prevalence of NAFLD, from 15% in Progression of NAFLD 2005 to 25% in 2010, and comparable prevalence rates between the West and the East. The natural course of liver disease progression in NAFLD is The National Health and Nutrition Examination Surveys still incompletely defined. A subset of such patients develops (NHANES) data collected from 1988 to 2008 show that the progressive liver disease leading to NASH, cirrhosis, HCC, and prevalence of NAFLD has doubled in the USA during that time liver failure (Fig. 1). Though early studies in 1990’s suggested period. From 1988 to 1994, NAFLD accounted for 46.8% of that SS does not progress to NASH or cirrhosis, subsequent chronic liver disease cases; from 1994 to 2004, its prevalence studies with paired liver biopsies have shown that SS is more further increased to 62.84%, and then to 75.1% from 2005 to 26–28 16 progressive than originally believed. Apart from compo- 2008. NAFLD is no longer a disease confined to the Western nents of MetS, genetic polymorphisms, such as PNPLA3 world, as studies from China, Korea, Taiwan, Japan and India I148M gene and transmembrane 6 superfamily member 2 have also found a high community prevalence of NAFLD, E167K gene variants, have a significant impact on NAFLD – 5,17 ranging from 15 49.8% (Table 1). Current USA projec- susceptibility and progression.25 Identification of such var- tions using a Markov model indicate a 21% increase in iants may help to identify NAFLD patients at higher risk for NAFLD from 2015 to 2030, leading to a 33.5% overall preva- liver disease progression and HCC. lence by 2030. The projected increase in the prevalence of NASH is 63%, which will cause a 168% increase in the Progression from SS and NASH number of patients with decompensated cirrhosis, and a 137% increase in the numbers of patients developing HCC 18 Prospective studies have revealed progression from SS to by 2030. Thus, with growing prevalence rates of NAFLD NASH in 23–44% of patients over a period of 26 months to affecting both adults and children, it is likely to emerge as 6.6 years.7,28 In a meta-analysis of 11 studies including 411 the leading cause of end-stage liver disease in the years to patients with paired liver biopsy performed at least 1 year come. apart, liver fibrosis progression occurred not only in patients Obesity and T2DM are important risk factors for NAFLD. with NASH but also in patients with nonalcoholic fatty liver – The prevalence of NAFLD is about 50 90% in obese sub- (NAFL), defined as SS alone or associated with mild inflam- 19 jects. The pooled prevalence of NAFLD in patients with mation. One stage of fibrosis progression occurred over 14.3 T2DM is 59.67% (95% confidence interval (CI): 54.31– years in patients with NAFL (95% CI: 9.1–50.0 years) and 7.1 20 64.92%) according to a meta-analysis of 24 studies. The years among patients with NASH (95% CI: 4.8–14.3 growing epidemic of obesity and T2DM have fueled an years).29 A very slow rate of progression of SS may partly 19–23 increasing prevalence of NAFLD worldwide. Moreover, account for the discrepancy between clinical and histological obesity and T2DM also increase the risk of NAFLD progression studies, as such patients may die from other causes before to NASH, cirrhosis, and HCC.22,23 In Asia, NAFLD can occur in developing advanced liver disease. In a systematic review of lean subjects with central obesity, which may be partly 10 studies, Argo et al. have found that 37.6% of 221 patients because of a higher metabolic activity of visceral fat and with NASH had progressive fibrosis over a mean follow-up genetic predisposition, such as the patatin-like phospholipase interval of 5.3 years.30 A recent meta-analysis also revealed domain-containing 3 (PNPLA3) polymorphism.24,25 It is worth occurrence of fibrosis progression in 41% of NASH patients, noting that a wide variation in clinical presentation and with 20% of them identified as being rapid progressors.5 Yet

Table 1. Recent Population-based prevalence studies on NAFLD based on ultrasonography

Study Country/region Year published Screened population, n NAFLD detected, n (%)

Kim et al.56 USA 2013 12317 4188 (34.00%) Caballería et al.111 Spain 2010 766 198 (25.80%) Suomela et al.112 Finland 2015 1621 246 (15.20%) van der Voort et al.95 Netherlands 2014 2292 779 (34.00%) Ruhl et al.113 USA 2013 12232 2446 (20.00%) Shen et al.114 Taiwan 2014 6511 1769 (27.17%) Younossi et al.115 USA 2013 6709 1448 (22.00%) Chang et al.116 South Korea 2013 43166 11652 (26.99%) Cai et al.117 China 2013 10605 3906 (36.83%) Dassanayake et al.118 Sri Lanka 2009 2985 974 (32.63%) Kim et al.65 South Korea 2012 4023 1617 (40.20%) Chalmers et al.17 India 2019 2158 1075 (49.8%)

Abbreviation: NAFLD, nonalcoholic fatty liver disease.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86 77 Kumar R. et al: NAFLD: a growing threat

Fig. 1. Progression of liver disease in patients with NAFLD. Data adapted from references: 9, 27, 28, 30 and 40. Abbreviations: HCC, hepatocellular carcinoma; NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis. another study has revealed a rapid fibrosis progression in of HCC in recent years. Multiple risk factors, such as compo- one-third of NASH patients who had any-stage of fibrosis pro- nents of MetS, ethnicity and hepatic siderosis, appear to have gression.31 To summarize, studies utilizing paired liver biop- an incremental effect on the risk of developing HCC among sies suggest that approximately 23–44% of patients with SS NAFLD patients. The cumulative incidence of HCC in patients progress to NASH and 37–41% of patients with NASH develop with NASH-related cirrhosis is quite high, ranging from 2.4 % progressive fibrosis. over 7 years to 12.8 % over 3 years.40 However, alarmingly, HCC can develop de novo in patients with NASH in the NASH-cryptogenic cirrhosis absence of cirrhosis.41,42 Kawada et al.,43 in a study of 1,168 HCC patients who underwent hepatic resection, found Clinical-histological study has revealed silent cirrhosis in 10% NASH as an etiology of HCC in 8 patients, 6 of who (75 %) had (8/80) of NAFLD patients with normal liver enzymes.32 noncirrhotic NASH. Similarly, Takuma et al.44 reported 7 out Around 9–25% of patients with NASH progress to cirrhosis of their studied 11 (65%) patients with NASH-related HCC over a period of 10–20 years. In a recent study, French inves- had noncirrhotic liver. In a review of 94 published cases of tigators identified 125,052 NAFLD/NASH patients from the NASH-related HCC, the patients were found to be predomi- French National Database on Hospital Care, of whom 1.2%, nantly elderly males, with 26% having noncirrhotic liver and 6.3%, and 0.9% were diagnosed with compensated cirrhosis, the majority (69%) having large (mean size 3.5 cm) and mul- decompensated cirrhosis, and HCC respectively. During 7 tifocal HCC.44 years of follow-up, 5.6% of the NAFLD/NASH patients pro- Several reports have confirmed the increasing burden of gressed to cirrhosis and 27.5% of the compensated cirrhosis NAFLD - related HCC worldwide. A recent large population - patients developed decompensation.33 Powell et al.34 have based Surveillance, Epidemiology and End Results (known as suggested that NASH should be recognized as a potential the SEER) study has demonstrated a 9% annual increase in cause of cryptogenic cirrhosis (CC). NAFLD-related HCC between 2004 and 2009.45 Dyson et al.46 Many of the patients with CC have features of MetS in noted a nearly 10 - fold increase in NAFLD - related HCC cases varying proportions.35,36 However, needle biopsy studies in the UK from 2000 to 2010. Another study from a hepatitis B have failed to demonstrate histological features of NASH in - endemic area in Korea has also demonstrated an increasing patients with CC. It seems that the features of NASH usually proportion of NAFLD - related HCC cases over time.47 More- regress concurrently with fibrosis progression.34,35 In con- over, patients with NAFLD-related HCC had a shorter survival trast to studies based on needle biopsy samples, explants time, more cardiovascular events, and more cancer-related from CC patients undergoing LT have revealed steatosis mortality than patients without NAFLD.48 In a study from (80%) and ballooning (70%) in a significant proportion.37 A Germany, where 1119 patients with HCC treated in an 11 study from India that assessed explants from patients with CC year period were retrospectively analyzed, the overall sur- revealed NASH to be the etiology in 63% patients.38 Another vival among the patients with NASH-related HCC (n = 45) study of explants from CC revealed NASH to be the most was lower compared to those with HCC of other etiologies.49 common etiology (33%).34 Moreover, the recurrence of stea- However, it appears that the worse natural history in such tosis in the allograft of CC patients is remarkably high (100% patients is not related to a more aggressive behavior of in 5 years).39 Therefore, NASH-cirrhosis cases appear to con- NAFLD - HCC, but mainly to detection at a later stage. stitute a significant proportion of patients previously labelled as CC. Long-term outcomes NAFLD and HCC Multiple studies have found that the overall mortality in HCC is the second leading cause of cancer - related death NAFLD patients is higher than that in matched individuals worldwide. NAFLD has emerged as one of the leading causes from a healthy population.50–55 A community-based cohort

78 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86 Kumar R. et al: NAFLD: a growing threat study with mean follow-up duration of 7.6 years found that of evidence to suggest that NAFLD is associated with many mortality in NAFLD patients was significantly higher than in of these diagnoses, independent of traditional risk factors, the general population (standardized mortality ratio of 1.34; such as components of MetS. Also, for some of the diseases, 95% CI: 1.003–1.76). Death was most commonly due to the association appears to be bidirectional. Therefore, malignancy and CVD.50 Using the third set of NHANES data, severity of NAFLD may influence the severity of associated Ong et al.51 found NAFLD to be associated with higher overall disease and vice versa. Similarly, management of one condi- (hazard ratio (HR): 1.038; 95% CI 1.036–1.041) and liver- tion may influence management of the associated one. related (HR: 9.32; 95% CI 9.21–9.43) mortality compared with the reference population. In both studies, liver disease T2DM was the third leading cause of death among NAFLD subjects. Two recent longitudinal studies have uniformly found that The association between NAFLD and T2DM is complex and stage of liver fibrosis irrespective of severity of hepatic necro- bidirectional. NAFLD is not only a consequence but also a inflammation is independently associated with overall and cause of T2DM. NAFLD is associated with increased risk of disease-specific mortality in patients with NAFLD.50,53 In one developing DM after adjustment for several metabolic con- study involving 619 NAFLD patients with median follow-up founders.54,58,59 Two large meta-analyses have confirmed period of 12.6 years, the risk of death or LT (n = 193) the association between NAFLD and incident T2DM.54 In a increased progressively with increasing stages of fibrosis prospective study of 129 biopsy-proven NAFLD patients, (HR for stage 1: 1.88, stage 2: 2.89, stage 3: 3.76, and 78% developed either T2DM (58%) or impaired glucose tol- stage 4: 10.9). Patients with fibrosis, regardless of NASH, erance (20%) during the 13.7 years follow-up.58 Moreover, had shorter survival times than patients without fibrosis.50 the risk of incident T2DM was threefold higher among patients Another longitudinal study with mean follow-up of 26.4 with NASH compared to those with SS. Moreover, T2DM years found that NAFLD patients (n = 229) had an increased increases the risk of NAFLD progression to NASH, cirrhosis, mortality compared with the reference population (HR: 1.29; 23 95% CI 1.04–1.59) and CVD constituted the most common and HCC. cause of death. Overall mortality was not increased in Because of systemic insulin resistance (IR), NAFLD patients with NASH and mild fibrosis, whereas patients with worsens the glycemic control in patients with T2DM. In diabetic subjects, NAFLD increases risk of all-cause mortality fibrosis stage >2, irrespective of NASH, had increased mortal- 60 ity (HR: 3.3; 95% CI 2.27–4.76, p < 0.001).53 by 2.2-fold compared with those without NAFLD. NAFLD and T2DM interact adversely to enhance the risk of atherosclero- In a meta-analysis of seven studies with follow-up ranging 23 from 7.3–24 years, liver -elated mortality was higher in sis, CKD, and retinopathy. Substantial evidence links NAFLD with an increased risk of developing CVD and arrhythmic patients with NASH compared to those with SS (OR: 5.71; 61 95% CI: 2.31–14.13).54 Kim et al.,56 in a large prospective complications in patients with DM. Thus, the coexistence cohort study of 11,154 USA adult participants from the of NAFLD and DM increases the risk of developing not only NHANES population found that NAFLD itself did not increase the more severe forms of NAFLD but also the vascular com- the risk of mortality. However, advanced fibrosis, as deter- plications of DM and all-cause mortality. mined by noninvasive fibrosis markers, significantly predicted the mortality, mainly from CVD causes, independent of other CVD known factors. In a systematic review and meta-analysis of five studies including 1,495 NAFLD patients with 17,452 Several studies have unequivocally demonstrated a strong 57 patient years of follow-up, Dulai et al. found an increased association between NAFLD and increased risk of CVD. NAFLD risk for all-cause mortality with increase in the stage of fibro- has been linked with increased biomarkers of endothelial sis, and such risk was more pronounced with regard to liver- dysfunction,62 increased carotid artery intima-media thick- related mortality. ness,63 increased arterial stiffness,64 coronary artery calcifi- It appears that higher stages of liver fibrosis are a strong cation,65 and impaired flow-mediated vasodilatation.66 A determinant of all-cause mortality in NAFLD, most likely recent meta-analysis of 27 studies supported the association because of the pronounced effect on liver-related mortality, of NAFLD with markers of subclinical atherosclerosis inde- whereas CVD accounts for an increased proportion of mortal- pendent of traditional CVD risk factors and MetS.67 Moreover, ity at lower stages of fibrosis. Thus, compared with the persistent and progression of NAFLD is associated with general population, NAFLD increases the risk of liver- increased risk and progression of subclinical carotid athero- related, cardiovascular and all-cause mortality, and the sclerosis, in a study comparing to subjects without NAFLD and impact of NAFLD on mortality appears to differ according to those with regression of NAFLD.68 its severity (Table 2). The disparity in estimates of risk across The widely prevalent dyslipidemia in NAFLD patients is studies might be attributed to variations in characteristics of highly atherogenic, being characterized by hypertriglyceride- study populations or follow-up. mia, high levels of low-density lipoprotein cholesterol, and low levels of high-density lipoprotein cholesterol—all of which Extrahepatic association of NAFLD are key risk factors for CVD.69 Studies in patients with NAFLD have shown abnormal left ventricular morphology and dia- NAFLD is closely associated with several extrahepatic dis- stolic dysfunction when compared with controls.70,71 Various eases, such as T2DM, CVD, malignancy, CKD, OSA, and PCOS factors have been implicated in the causation of left ventric- (Fig. 2). These associations do not truly represent extrahe- ular dysfunction in NAFLD, including hyperinsulinemia- patic manifestations of NAFLD. However, the implications of induced myocyte growth and interstitial fibrosis, alteration such association may influence clinical evaluation and treat- in myocardial metabolism of fatty acids, upregulation of ment decisions in NAFLD patients. Although these associa- angiotensin II (a neurohormone), decrease in myocardial tions may result from common risk factors, there are lines perfusion reserve, and increase in aortic stiffness.

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Table 2. Community-based longitudinal studies determining all-cause and cause-specific mortality in patients with NAFLD

Diagnostic Study, year Population Follow-up method Results

Adams et al.119, 420 community-based USA 7.6 years Histology and Patients with NAFLD had higher rates of 2005 NAFLD patients ultrasonography all-cause, CVD and liver-related mortality than the matched general population (standardized mortality ratio:ß 1.34; 95% CI: 1.003–1.76) Ekstedt et al.58, 129 Swedish biopsy-proven 13.7 years Histology Mortality was not increased in patients 2006 NAFLD patients with simple steatosis but patients with NASH had higher rates of all-cause (;2-fold), cardiovascular (;2-fold) and liver-related (;10-fold) mortality than the matched reference population Rafiq et al.121, 173 USA patients with 13 years Histology All-cause mortality did not differ 2009 biopsy-proven NAFLD between the NAFLD subtypes, but liver- related mortality was higher in patients with NASH The most common causes of mortality were CVD, malignancy and liver-related complications Söderberg 256 Swedish subjects with 28 years Histology 40% of the 118 NAFLD subjects died et al.52, 2010 raised liver enzymes, during follow-up. Compared with the including 118 biopsy-proven matched Swedish population, subjects NAFLD with NAFLD exhibited 69% increased mortality, more so with NASH (86%) Ekstedt et al.53, 229 Swedish patients with 26.4 6 5.6 Histology Patients with NAFLD have increased 2015 biopsy-proven NAFLD years all-cause mortality (HR: 1.29, 95% CI: 1.04–1.59), with a high risk of death from CVD and liver-related disease. The fibrosis stage rather than presence of NASH predicts the mortality Jepsen et al.38, 7,372 Danish patients with 6.2 years Ultrasound and Patients with NAFLD had higher rates of 2003 fatty liver, including 1,800 liver enzymes all-cause (2.6-fold), cardiovascular patients with NAFLD (2.1-fold) and liver-related (19.7-fold) mortality than the general population Haring et al.120, 4,160 community-based 7.3 years Ultrasound NAFLD was independently associated 2009 cohort of German adult with increased risk of all-cause and CVD subjects mortality in men (HR: 6.2, 95% CI: 1.2–31.6) Zhou et al.55, 3,543 community-based 4 years Ultrasound Patients with NAFLD had ;3-fold higher 2012 cohort study of Chinese rates of all-cause and CVD mortality adult subjects than those without NAFLD Kim et al.56, 11,154 USA adult 14.5 years Ultrasound and NAFLD with advanced fibrosis, not 2013 participants, including noninvasive NAFLD in general, is associated with 34.0% NAFLD, from the markers of liver increased mortality independent of Third NHANES-1988-94. fibrosis other known factors Zeb et al.122, 4,119 USA adult subjects, Median 7.6 Computed Overall 253 deaths reported, including 2016 including 728 NAFLD, years tomography 40 NAFLD subjects. NAFLD was without CVD at baseline independently associated with incident CVD and all-cause event (HR: 1.42, 95% CI: 1.00–2.03)

Abbreviations: CVD, cardiovascular disease; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

NAFLD is also associated with an increased risk of atrial population, and a prolonged QT interval increases the risk of fibrillation.72 NAFLD has also been reported as independently cardiac arrhythmias and sudden cardiac death.74 associated with QT prolongation.73 Notably, the duration of NAFLD contributes to the prothrombotic state by increas- the QT interval is a predictor for CVD death in the general ing plasma levels of plasminogen activator inhibitor 1, the

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Fig. 2. Association of NAFLD with cardiovascular diseases, extrahepatic malignancy, surgical complications, and various other diseases. Abbreviations: LT, liver transplantation; NAFLD, nonalcoholic fatty liver disease. elevated levels of which are related to increased risk of malignancy was the most common (28%) cause of death in myocardial infarction.75 In a recent prospective study NAFLD.54 In a recent longitudinal study comprising a com- among patients with clinical indication of coronary angiogram munity cohort of 4,722 NAFLD and 14,441 age- and sex- (n = 612), the presence of NAFLD was associated with matched control subjects, 2,224 incident cancers occurred severity of coronary artery stenosis and need for coronary during a median follow-up of 8 years and NAFLD was associ- intervention.76 In a large meta-analysis of 164,494 partici- ated with 90% higher risk of developing cancers.79 The pants from 21 cross-sectional and 13 cohort studies, NAFLD highest increase in the risk was noted for HCC, followed by was associated with an increased risk of incident (HR: 1.37; uterine, gastric, pancreatic and colonic cancer. Moreover, the 95% CI: 1.10–1.72) and prevalent (OR: 1.81; 95% CI: 1.23– association between obesity and cancer risk was small in the 2.66) CVD but not with CVD mortality.77 However, there was absence of NAFLD, suggesting that NAFLD may potentiate marked heterogeneity among studies and nonuniform defini- obesity-cancer relationship.79 tion of important variables, including DM, which could have In a cohort study of 129 biopsy-proven NAFLD patients, affected the results of mortality. Moreover, another meta- 5.6% of the patients with NASH died because of extrahepatic analysis of 40 studies assessing the natural history of malignancy during a mean follow-up of 13.7 years.58 Several NAFLD revealed that patients with NAFLD, irrespective of SS studies have found a higher prevalence of colorectal neo- or NASH, had a considerably greater risk of CVD mortality plasm in patients with NAFLD compared to patients without than the matched control population.54 NAFLD.80–82 A large cohort study (n = 5517) from Korea has Many prospective studies have demonstrated that CVD- found a two-fold increase in the occurrence of colorectal related death occurs in higher proportion than liver-related adenoma and a three-fold increase in the risk of colorectal death among patients with NAFLD.50,51 A meta-analysis of a cancer in patients with NAFLD compared to controls.80 total 16 observational studies with 34,043 adult individuals, Another study revealed that among NAFLD, patients with including 36.3% NAFLD and approximately 2,600 CVD out- NASH have a higher prevalence of adenomas (51.0% vs. comes over a median period of 6.9 years, revealed that 25.6%) and advanced neoplasms (34.7% vs. 14.0%) than NAFLD is significantly associated with an increased risk of those with SS. Moreover, NASH remained significantly asso- fatal and nonfatal cardiovascular events (OR: 1.64). ciated with a risk of adenomas and advanced neoplasms after However, the observational design of the studies included adjusting for demographic and metabolic factors.81 does not prove that NAFLD causes CVD.78 Thus, there In a retrospective cohort study on 1,522 subjects who seems to be little doubt that NAFLD is associated with underwent two consecutive colonoscopies between 2003 and increased incidence and prevalence of CVD, some controver- 2010, NAFLD was an independent risk factor (OR: 1.45; 95% sies surround as to whether NAFLD by itself is associated with CI: 1.07–1.98) for adenoma formation after a negative increased CVD mortality. Also, whether the association baseline colonoscopy.82 The adenoma group had a higher between NAFLD and CVD is because of the shared risk prevalence of NAFLD than the non-adenoma group (55.6% factors or NAFLD itself confers an additional risk is the vs. 38.8%; p < 0.05). NAFLD patients are more likely to subject of further extensive scrutiny. have multiple polyps localized more often in the right hemi- colon. In a case-control study, NAFLD was found to have a Extrahepatic malignancy significant association with breast cancer.83 NAFLD has also been found to be associated with malignancy of the esopha- NAFLD has been associated with several extrahepatic malig- gus, stomach, pancreas, kidney, and prostate.9,54,79 nancies. Malignancy is among the leading cause of death in However, concurrent presence of features of MetS and too NAFLD patients.50,51,54 In meta-analyses of seven longitudi- little available data limit drawing definite conclusions about nal studies with follow-up ranging from 7.3 to 24 years, a causal role of NAFLD in such association.

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CKD development and progression of NAFLD.92 Intermittent hypoxia activates hypoxia-inducible factors, which leads to The link between NAFLD and CKD has drawn considerable increased synthesis of hepatic fat, upregulated hepatic attention during recent times. Various studies have sug- inflammation, and fibrosis. The chronic intermittent hypoxia gested that NAFLD can accelerate the development and in morbidly obese subjects contributes to the severity of progression of CKD independent of traditional risk hepatic necroinflammation and fibrosis independent of factors.10,84,85 A recent meta-analysis, that included near adiposity.93 Furthermore, NAFLD in patients with OSA is asso- 64,000 subjects, found that NAFLD was associated with an ciated with higher CVD risks. Minville et al.94 have recently approximately 2-fold increase in risk of both prevalent (OR: demonstrated that in patients with OSA, hepatic steatosis was 2.12; 95% CI: 1.69–2.66) and incident CKD (HR: 1.79; 95% independently associated with endothelial dysfunction after CI: 1.65–1.95).84 Furthermore, histologically severe NAFLD adjustment for confounders. Therefore, among obese is more positively correlated with CKD. Both NASH and NAFLD patients with OSA, screening for the presence of underlying with advanced fibrosis are associated with a higher preva- NAFLD and subsequent monitoring for NAFLD progression lence (OR: 2.53 for NASH, OR: 5.20 for advanced fibrosis) should be considered. In patients with NAFLD, treatment of and incidence of CKD (HR: 2.12 for NASH, HR: 3.29 for OSA with continuous positive airway pressure may impact advanced fibrosis), as compared to those with SS.84 outcomes of future CVD. In a large longitudinal study, NAFLD was found to be associated with declining renal function in CKD patients Psoriasis independent of traditional risk factors, and the association was stronger in patients with advanced NAFLD.85 The patho- NAFLD is highly prevalent in patients with psoriasis. In a large physiologic basis of the linkage between the two appear to be prospective population-based cohort study of 2,292 subjects multifactorial. The pro-inflammatory milieu in NAFLD along with 118 (5.1%) patients with psoriasis, NAFLD prevalence with IR, dyslipidemia, oxidative stress, hypertension, and was higher among those with psoriasis (46.2% vs. 33.3%) the activated renin-angiotensin system may hasten the even after adjustment for important risk factors.95 The prev- development and progression of CKD. Compared to other eti- alence of NASH in patients with psoriasis is much higher ologies of cirrhosis, the risk of CKD and requirement of simul- (22%) than that in the general population (2–6%). Moreover, taneous liver-kidney transplantation are greater in patients data also suggest that the presence of NAFLD may increase with NASH-related cirrhosis.86 Furthermore, CKD may aggra- severity of psoriasis.96,97 Future studies are needed to assess vate NAFLD through uremic toxins, intestinal dysbiosis, whether there is a causal relationship between NAFLD and altered gut-barrier function, and alterations in glucocorticoid psoriasis. metabolism.87 Interestingly, there is evidence to suggest that in patients with NASH, improvement in liver histology by life- Osteoporosis style modification leads to improved kidney function.85 Several studies have demonstrated that NAFLD patients have PCOS lower bone mineral density compared to non-NAFLD subjects.98 In a study from China, NAFLD was independently PCOS is one of the most common endocrine disorders in associated with a ;2.5-fold increased odds of osteoporotic women during reproductive ages. It is associated with a fractures among men.99 A recent meta-analysis of six plethora of metabolic consequences, including glucose intol- studies has revealed that obese children with NAFLD are erance, dyslipidemia, and NAFLD. Several studies have con- more susceptible to osteoporosis than children with only sistently found that the prevalence and severity of NAFLD is obesity.13 The potential contribution of NAFLD to develop- markedly increased in women with PCOS, independent of ment of osteoporosis warrants further study. Chronic inflam- coexisting features of MetS.88 A recent systematic review and matory processes, vitamin D deficiency, disturbances of meta-analysis of 17 studies has revealed that PCOS patients growth hormone/insulin-like growth factor 1 axis are the pro- (n = 2,734) have increased prevalence of NAFLD (OR: 2.54, posed pathophysiological factors linking NAFLD with 95% CI 2.19–2.95) and the presence of NAFLD among them decreased bone mass. is associated with hyperandrogenism, in addition to IR and 89 adiposity. The prevalence of NAFLD among women with Sarcopenia PCOS is estimated to vary from 15% to 55%, whereas amongst the reproductive-aged women with NAFLD, the NAFLD has been recently associated with sarcopenia which is 90 prevalence of PCOS is as high as 71%. defined as a generalized, and progressive and loss of skeletal muscle mass, quality, and strength.100,101 Sarcopenia is OSA associated with increased risks and histological severity of NAFLD, independent of obesity and metabolic risk OSA is strongly associated with NAFLD independent of tradi- factors.100 In a longitudinal study, Kim et al.101 has demon- tional risk factors. In a meta-analysis of over 2,000 subjects strated that increases in relative skeletal muscle mass over from 18 studies, OSA was associated with an increased risk of time had significant beneficial association with incident NAFLD (OR: 2.99), NASH (OR: 2.37), and advanced fibrosis NAFLD (adjusted HR: 0.69; 95% CI: 0.59–0.82) and resolu- (OR: 2.30).91 This association is related to the degree of noc- tion of baseline NAFLD (adjusted HR: 4.17; 95% CI: 1.90– turnal hypoxemia caused by repetitive upper airway obstruc- 6.17). The pathophysiological mechanisms linking sarcopenia tion during sleep. Intermittent hypoxia can result in oxidative and NAFLD may include IR and chronic inflammation. IR pro- stress, IR, abnormal lipid metabolism, overactivation of the motes accumulation of triglycerides in muscles and exacer- sympathetic nervous system, inflammation, and mitochon- bates protein catabolism in association with the chronic drial dysfunction, each of which plays important roles in inflammatory milieu, leading to muscle depletion.

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CVA (5.1% vs. 1.8%) and worse patient (77% vs. 91 %) and graft (70% vs. 82%) survival at 2 years compared to patients NAFLD appears to be associated with an increased risk and receiving a nonsteatotic graft.108 NAFLD is an independent severity of stroke as well as with a worse functional outcome predictor of occurrence of post-LT MetS that increases the in stroke patients. In a prospective study including a total of risk of steatosis in the graft liver.109,110 Within a few months 25,800 subjects, Hadda et al.102 found an increased fre- of LT, steatosis developed in 60–100% of the patients and quency of CVA in NAFLD compared to the controls. A meta- NASH in 10–40% of the patients. Approximately 10% of the analysis of seven studies with a total of 6,183 subjects patients progressed to advanced fibrosis or cirrhosis in a revealed a significant association of NAFLD with elevated decade.110 risk of CVA.12 Also, there are data to suggest a role of In conclusion, NAFLD is highly prevalent in the general NAFLD in subclinical ischemic stroke and cognitive impair- population, and its prevalence is expected to increase in the ments.102 These observations are clinically relevant because coming years. It has emerged as one of the leading causes of strategies to prevent the progression of asymptomatic brain cirrhosis and HCC in recent years and is a growing indication lesions to overt CVA can be explored in patients with NAFLD. for LT. NAFLD is associated with various extrahepatic diseases Further studies are warranted to throw more light on these and increased risk of all-cause mortality. Increased aware- aspects. ness about consequences of NAFLD and development of strategies to change the course of this disease are needed AP to control its emerging global threat. The optimal treatment of NAFLD remains a clinical challenge, as there is no approved NAFLD increases the severity of AP. A recent study demon- pharmacotherapies. While traditional pharmacological thera- strated that the presence of NAFLD at admission portends a pies, such as insulin sensitizer (pioglitazone) and antioxida- higher risk of severe and moderately severe AP, as well as a tive agent (vitamin E), significantly improve steatosis and higher risk of organ failure.11 The mechanism by which NAFLD inflammation, they have no significant effect on liver fibrosis exacerbates pancreatitis remains unclear. The potential and have long-term safety issues. Currently, the therapeutic reasons could be imbalance of adipocytokines, increased options for NAFLD include diet and lifestyle modification and activity of hepatic Kupffer cells, and reduction of alpha-1-anti- pharmacological interventions targeting the components of trypsin levels. Pancreatic steatosis, which is frequently asso- MetS. Lifestyle changes, if sustained, can make significant ciated with NAFLD, may lead to a higher incidence of AP and difference in the trajectory of liver disease and overall out- may be an etiological factor in pancreatic cancer. comes. Finally, morbidly obese NASH patients can benefit from bariatric surgery, which may reduce liver fibrosis but Surgical implications of NAFLD carries a risk of decompensation in patients with cirrhosis.

Post-liver resection liver failure Funding

A normal liver has remarkable capacity to regenerate, which None to declare. makes it possible for surgeons to do a large liver resection without causing significant hepatic impairment. However, extended liver resection may lead to the development of Conflict of interest progressive liver failure in the postoperative period which is associated with very high mortality rate.103 Steatotic liver The authors have no conflict of interests related to this have poor ability to regenerate and reduced tolerance publication. against ischemic injury. Therefore, patients with NAFLD are at higher risk of post-liver resection liver failure. In a series of 135 patients who had undergone major hepatic resection at Author contributions the Mayo Clinic, acute liver failure occurred in 14% of patients with fatty liver versus 4% in those with normal liver.104 In a Conception of the study (RK, RNP, UA), design of the study cohort of 478 liver resection patients, Belghiti et al.105 dem- and drafting of the manuscript (RK), data collection (RK, RNP, onstrated that steatosis was an independent risk factor for UA), manuscript revision (RNP). postoperative complications. Another study on outcome after liver-resection for colorectal liver metastases in 406 patients has found that patients with steatohepatitis have a References significantly higher 90-day mortality than those without it (14.7 vs. 1.6%; OR: 10.5).106 In conclusion, major hepatic [1] Puri P, Sanyal AJ. Nonalcoholic fatty liver disease: Definitions, risk factors, – resection of steatotic liver, particularly in patients with NASH, and workup. Clin Liver Dis (Hoboken) 2012;1:99 103. doi: 10.1002/cld.81. [2] Loomba R, Sanyal AJ. The global NAFLD epidemic. Nat Rev Gastroenterol is associated with increased risk of liver failure and death. Hepatol 2013;10:686–690. doi: 10.1038/nrgastro.2013.171. [3] Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology Influence on LT and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther 2011;34:274–285. doi: 10.1111/j.1365-2036.2011.04724.x. Transplantation of steatotic grafts is associated with an [4] Farrell GC, Wong VW, Chitturi S. NAFLD in Asia–as common and important increased risk of primary nonfunction, early allograft dysfunc- as in the West. Nat Rev Gastroenterol Hepatol 2013;10:307–318. doi: 10. tion, and posttransplant vascular and biliary complications in 1038/nrgastro.2013.34. cadaveric as well as living-donor LT.107 A study involving large [5] Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment series of LT patients has demonstrated that patients receiving of prevalence, incidence, and outcomes. Hepatology 2016;64:73–84. doi: up to 30% of fatty liver had a higher rate of primary nonfunction 10.1002/hep.28431.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86 83 Kumar R. et al: NAFLD: a growing threat

[6] Li Z, Xue J, Chen P, Chen L, Yan S, Liu L. Prevalence of nonalcoholic fatty liver Gastroenterol Hepatol 2015;13:643–654.e9. doi: 10.1016/j.cgh.2014.04. disease in mainland of China: a meta-analysis of published studies. J Gas- 014. troenterol Hepatol 2014;29:42–51. doi: 10.1111/jgh.12428. [30] Argo CK, Northup PG, Al-Osaimi AM, Caldwell SH. Systematic review of risk [7] Kojima S, Watanabe N, Numata M, Ogawa T, Matsuzaki S. Increase in the factors for fibrosis progression in non-alcoholic steatohepatitis. J Hepatol prevalence of fatty liver in Japan over the past 12 years: analysis of clinical 2009;51:371–379. doi: 10.1016/j.jhep.2009.03.019. background. J Gastroenterol 2003;38:954–961. doi: 10.1007/s00535-003- [31] Harrison SA, Torgerson S, Hayashi PH. The natural history of nonalcoholic 1178-8. fatty liver disease: a clinical histopathological study. Am J Gastroenterol [8] Kim D, Touros A, Kim WR. Nonalcoholic fatty liver disease and metabolic 2003;98:2042–2047. doi: 10.1111/j.1572-0241.2003.07659.x. syndrome. Clin Liver Dis 2018;22:133–140. doi: 10.1016/j.cld.2017.08. [32] Sorrentino P, Tarantino G, Conca P, Perrella A, Terracciano ML, Vecchione R, 010. et al. Silent non-alcoholic fatty liver disease-a clinical-histological study. J [9] Kumar R. Hard clinical outcomes in patients with NAFLD. Hepatol Int 2013;7 Hepatol 2004;41:751–757. doi: 10.1016/j.jhep.2004.07.010. Suppl 2:790–799. doi: 10.1007/s12072-013-9455-y. [33] Boursier J, Fraysse J, Lafuma A, Fabron C, Sanatan S. THU-299-Increased [10] VanWagner LB, Rinella ME. Extrahepatic manifestations of nonalcoholic risk of mortality with liver disease progression in non-alcoholic fatty liver fatty liver disease. Curr Hepatol Rep 2016;15:75–85. doi: 10. disease/non-alcoholic steatohepatitis patients: An analysis of French 1007/s11901-016-0295-9. national hospital care. J Hepatol 2019;70:e291–e292. doi: 10. [11] Xu C, Qiao Z, Lu Y, Zhang D, Jia Z, Zhuang X, et al. Influence of fatty liver on 1016/S0618-8278(19)30562-6. the severity and clinical outcome in acute pancreatitis. PLoS One 2015;10: [34] Powell EE, Cooksley WG, Hanson R, Searle J, Halliday JW, Powell LW. The e0142278. doi: 10.1371/journal.pone.0142278. natural history of nonalcoholic steatohepatitis: a follow-up study of forty- [12] Hu J, Xu Y, He Z, Zhang H, Lian X, Zhu T, et al. Increased risk of cerebro- two patients for up to 21 years. Hepatology 1990;11:74–80. doi: 10. vascular accident related to non-alcoholic fatty liver disease: a meta-anal- 1002/hep.1840110114. ysis. Oncotarget 2017;9:2752–2760. doi: 10.18632/oncotarget.22755. [35] Ayata G, Gordon FD, Lewis WD, Pomfret E, Pomposelli JJ, Jenkins RL, et al. [13] Sun Y, Dai W, Liang Y, Yang P, Yang Q, Liang M, et al. Relationship between Cryptogenic cirrhosis: clinicopathologic findings at and after liver transplan- nonalcoholic fatty liver disease and bone mineral density in adolescents tation. Hum Pathol 2002;33:1098–1104. doi: 10.1053/hupa.2002.129419. with obesity: a meta-analysis. Diabetes Metab Syndr Obes 2019;12:199– [36] Nayak NC, Jain D, Vasdev N, Gulwani H, Saigal S, Soin A. Etiologic types of 207. doi: 10.2147/DMSO.S192256. end-stage chronic liver disease in adults: analysis of prevalence and their [14] Wong RJ, Cheung R, Ahmed A. Nonalcoholic steatohepatitis is the most temporal changes from a study on native liver explants. Eur J Gastroenterol rapidly growing indication for liver transplantation in patients with hepato- Hepatol 2012;24:1199–1208. doi: 10.1097/MEG.0b013e32835643f1. cellular carcinoma in the U.S. Hepatology 2014;59:2188–2195. doi: 10. [37] Bugianesi E, Leone N, Vanni E, Marchesini G, Brunello F, Carucci P, et al. 1002/hep.26986. Expanding the natural history of nonalcoholic steatohepatitis: from crypto- [15] Wong RJ, Aguilar M, Cheung R, Perumpail RB, Harrison SA, Younossi ZM, genic cirrhosis to hepatocellular carcinoma. Gastroenterology 2002;123: et al. Nonalcoholic steatohepatitis is the second leading etiology of liver 134–140. doi: 10.1053/gast.2002.34168. disease among adults awaiting liver transplantation in the United States. [38] Jepsen P, Vilstrup H, Mellemkjaer L, Thulstrup AM, Olsen JH, Baron JA, et al. Gastroenterology 2015;148:547–555. doi: 10.1053/j.gastro.2014.11.039. Prognosis of patients with a diagnosis of fatty liver—a registry-based cohort [16] Younossi ZM, Stepanova M, Afendy M, Fang Y, Younossi Y, Mir H, et al. study. Hepatogastroenterology 2003;50:2101–2104. Changes in the prevalence of the most common causes of chronic liver [39] Nayak NC, Vasdev N, Saigal S, Soin AS. End-stage nonalcoholic fatty liver diseases in the United States from 1988 to 2008. Clin Gastroenterol disease: evaluation of pathomorphologic features and relationship to cryp- Hepatol 2011;9:524–530.e1. doi: 10.1016/j.cgh.2011.03.020. togenic cirrhosis from study of explant livers in a living donor liver trans- [17] Chalmers J, Ban L, Leena KB, Edwards KL, Grove JL, Aithal GP, et al. Cohort plant program. Hum Pathol 2010;41:425–430. doi: 10.1016/j.humpath. profile: the Trivandrum non-alcoholic fatty liver disease (NAFLD) cohort. 2009.06.021. BMJ Open 2019;9:e027244. doi: 10.1136/bmjopen-2018-027244. [40] Said A, Ghufran A. Epidemic of non-alcoholic fatty liver disease and hepa- [18] Estes C, Razavi H, Loomba R, Younossi Z, Sanyal AJ. Modeling the epidemic tocellular carcinoma. World J Clin Oncol 2017;8:429–436. doi: 10. of nonalcoholic fatty liver disease demonstrates an exponential increase in 5306/wjco.v8.i6.429. burden of disease. Hepatology 2018;67:123–133. doi: 10.1002/hep. [41] Paradis V, Zalinski S, Chelbi E, Guedj N, Degos F, Vilgrain V, et al. Hepato- 29466. cellular carcinomas in patients with metabolic syndrome often develop [19] Divella R, Mazzocca A, Daniele A, Sabbà C, Paradiso A. Obesity, nonalcoholic without significant liver fibrosis: a pathological analysis. Hepatology fatty liver disease and adipocytokines network in promotion of cancer. Int J 2009;49:851–859. doi: 10.1002/hep.22734. Biol Sci 2019;15:610–616. doi: 10.7150/ijbs.29599. [42] Piscaglia F, Svegliati-Baroni G, Barchetti A, Pecorelli A, Marinelli S, Tiribelli [20] Dai W, Ye L, Liu A, Wen SW, Deng J, Wu X, et al. Prevalence of nonalcoholic C, et al. Clinical patterns of hepatocellular carcinoma in nonalcoholic fatty fatty liver disease in patients with type 2 diabetes mellitus: A meta-analy- liver disease: A multicenter prospective study. Hepatology 2016;63:827– sis. Medicine (Baltimore) 2017;96:e8179. doi: 10.1097/MD. 838. doi: 10.1002/hep.28368. 0000000000008179. [43] Kawada N, Imanaka K, Kawaguchi T, Tamai C, Ishihara R, Matsunaga T, [21] Li L, Liu DW, Yan HY, Wang ZY, Zhao SH, Wang B. Obesity is an independent et al. Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic stea- risk factor for non-alcoholic fatty liver disease: evidence from a meta-anal- tohepatitis. J Gastroenterol 2009;44:1190–1194. doi: 10.1007/s00535- ysis of 21 cohort studies. Obes Rev 2016;17:510–519. doi: 10.1111/obr. 009-0112-0. 12407. [44] Takuma Y, Nouso K. Nonalcoholic steatohepatitis-associated hepatocellular [22] Polyzos SA, Kountouras J, Mantzoros CS. Obesity and nonalcoholic fatty carcinoma: our case series and literature review. World J Gastroenterol liver disease: From pathophysiology to therapeutics. Metabolism 2019; 2010;16:1436–1441. doi: 10.3748/wjg.v16.i12.1436. 92:82–97. doi: 10.1016/j.metabol.2018.11.014. [45] Younossi ZM, Otgonsuren M, Henry L, Venkatesan C, Mishra A, Erario M, [23] Hazlehurst JM, Woods C, Marjot T, Cobbold JF, Tomlinson JW. Non-alcoholic et al. Association of nonalcoholic fatty liver disease (NAFLD) with hepato- fatty liver disease and diabetes. Metabolism 2016;65:1096–1108. doi: 10. cellular carcinoma (HCC) in the United States from 2004 to 2009. Hepatol- 1016/j.metabol.2016.01.001. ogy 2015;62:1723–1730. doi: 10.1002/hep.28123. [24] Kumar R, Mohan S. Non-alcoholic fatty liver disease in lean subjects: Char- [46] Dyson J, Jaques B, Chattopadyhay D, Lochan R, Graham J, Das D, et al. acteristics and implications. J Clin Transl Hepatol 2017;5:216–223. doi: 10. Hepatocellular cancer: the impact of obesity, type 2 diabetes and a multi- 14218/JCTH.2016.00068. disciplinary team. J Hepatol 2014;60:110–117. doi: 10.1016/j.jhep.2013. [25] Sookoian S, Pirola CJ. Genetic predisposition in nonalcoholic fatty liver 08.011. disease. Clin Mol Hepatol 2017;23:1–12. doi: 10.3350/cmh.2016.0109. [47] Cho EJ, Kwack MS, Jang ES, You SJ, Lee JH, Kim YJ, et al. Relative etiological [26] Teli MR, James OF, Burt AD, Bennett MK, Day CP. The natural history of role of prior hepatitis B virus infection and nonalcoholic fatty liver disease in the nonalcoholic fatty liver: a follow-up study. Hepatology 1995;22:1714– development of non-B non-C hepatocellular carcinoma in a hepatitis 1719. doi: 10.1002/hep.1840220616. B-endemic area. Digestion 2011;84 Suppl 1:17–22. doi: 10.1159/000333210. [27] Wong VW, Wong GL, Choi PC, Chan AW, Li MK, Chan HY, et al. Disease [48] Wong SW, Ting YW, Chan WK. Epidemiology of non-alcoholic fatty liver progression of non-alcoholic fatty liver disease: a prospective study with disease-related hepatocellular carcinoma and its implications. JGH Open paired liver biopsies at 3 years. Gut 2010;59:969–974. doi: 10.1136/gut. 2018;2:235–241. doi: 10.1002/jgh3.12070. 2009.205088. [49] Weinmann A, Alt Y, Koch S, Nelles C, Düber C, Lang H, et al. Treatment and [28] McPherson S, Hardy T, Henderson E, Burt AD, Day CP, Anstee QM. Evidence survival of non-alcoholic steatohepatitis associated hepatocellular carci- of NAFLD progression from steatosis to fibrosing-steatohepatitis using noma. BMC Cancer 2015;15:210. doi: 10.1186/s12885-015-1197-x. paired biopsies: implications for prognosis and clinical management. [50] Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatchar- J Hepatol 2015;62:1148–1155. doi: 10.1016/j.jhep.2014.11.034. oenwitthaya P, et al. Liver fibrosis, but no other histologic features, is asso- [29] Singh S, Allen AM, Wang Z, Prokop LJ, Murad MH, Loomba R. Fibrosis pro- ciated with long-term outcomes of patients with nonalcoholic fatty liver gression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a sys- disease. Gastroenterology 2015;149:389–397.e10. doi: 10.1053/j. tematic review and meta-analysis of paired-biopsy studies. Clin gastro.2015.04.043.

84 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86 Kumar R. et al: NAFLD: a growing threat

[51] Ong JP, Pitts A, Younossi ZM. Increased overall mortality and liver-related [73] Hung CS, Tseng PH, Tu CH, Chen CC, Liao WC, Lee YC, et al. Nonalcoholic mortality in non-alcoholic fatty liver disease. J Hepatol 2008;49:608–612. fatty liver disease is associated with QT prolongation in the general popu- doi: 10.1016/j.jhep.2008.06.018. lation. J Am Heart Assoc 2015;4:e001820. doi: 10.1161/JAHA.115. [52] Söderberg C, Stål P, Askling J, Glaumann H, Lindberg G, Marmur J, et al. 001820. Decreased survival of subjects with elevated liver function tests during a [74] Robbins J, Nelson JC, Rautaharju PM, Gottdiener JS. The association 28-year follow-up. Hepatology 2010;51:595–602. doi: 10.1002/hep. between the length of the QT interval and mortality in the Cardiovascular 23314. Health Study. Am J Med 2003;115:689–694. doi: 10.1016/j.amjmed.2003. [53] Ekstedt M, Hagström H, Nasr P, Fredrikson M, Stål P, Kechagias S, et al. 07.014. Fibrosis stage is the strongest predictor for disease-specific mortality in [75] Verrijken A, Francque S, Mertens I, Prawitt J, Caron S, Hubens G, et al. NAFLD after up to 33 years of follow-up. Hepatology 2015;61:1547– Prothrombotic factors in histologically proven nonalcoholic fatty liver 1554. doi: 10.1002/hep.27368. disease and nonalcoholic steatohepatitis. Hepatology 2014;59:121–129. [54] Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural history doi: 10.1002/hep.26510. of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non- [76] Wong VW, Wong GL, Yeung JC, Fung CY, Chan JK, Chang ZH, et al. Long- invasive tests for liver disease severity. Ann Med 2011;43:617–649. doi: term clinical outcomes after fatty liver screening in patients undergoing 10.3109/07853890.2010.518623. coronary angiogram: A prospective cohort study. Hepatology 2016;63: [55] Zhou YJ, Li YY, Nie YQ, Huang CM, Cao CY. Natural course of nonalcoholic 754–763. doi: 10.1002/hep.28253. fatty liver disease in southern China: a prospective cohort study. J Dig Dis [77] Wu S, Wu F, Ding Y, Hou J, Bi J, Zhang Z. Association of non-alcoholic fatty 2012;13:153–160. doi: 10.1111/j.1751-2980.2011.00571.x. liver disease with major adverse cardiovascular events: A systematic review [56] Kim D, Kim WR, Kim HJ, Therneau TM. Association between noninvasive and meta-analysis. Sci Rep 2016;6:33386. doi: 10.1038/srep33386. fibrosis markers and mortality among adults with nonalcoholic fatty liver [78] Targher G, Byrne CD, Lonardo A, Zoppini G, Barbui C. Non-alcoholic fatty disease in the United States. Hepatology 2013;57:1357–1365. doi: 10. liver disease and risk of incident cardiovascular disease: A meta-analysis. J 1002/hep.26156. Hepatol 2016;65:589–600. doi: 10.1016/j.jhep.2016.05.013. [57] Dulai PS, Singh S, Patel J, Soni M, Prokop LJ, Younossi Z, et al. Increased [79] Allen AM, Hicks SB, Mara KC, Larson JJ, Therneau TM. The risk of incident risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Sys- extrahepatic cancers is higher in non-alcoholic fatty liver disease than tematic review and meta-analysis. Hepatology 2017;65:1557–1565. doi: obesity - A longitudinal cohort study. J Hepatol 2019;71:1229–1236. doi: 10.1002/hep.29085. 10.1016/j.jhep.2019.08.018. [58] Ekstedt M, Franzén LE, Mathiesen UL, Thorelius L, Holmqvist M, Bodemar G, [80] Lee YI, Lim YS, Park HS. Colorectal neoplasms in relation to non-alcoholic et al. Long-term follow-up of patients with NAFLD and elevated liver fatty liver disease in Korean women: a retrospective cohort study. J Gastro- enzymes. Hepatology 2006;44:865–873. doi: 10.1002/hep.21327. enterol Hepatol 2012;27:91–95. doi: 10.1111/j.1440-1746.2011.06816.x. [59] Fraser A, Harris R, Sattar N, Ebrahim S, Davey Smith G, et al. Alanine [81] Wong VW, Wong GL, Tsang SW, Fan T, Chu WC, Woo J, et al. High prevalence aminotransferase, gamma-glutamyltransferase, and incident diabetes: of colorectal neoplasm in patients with non-alcoholic steatohepatitis. Gut the British Women’s Heart and Health Study and meta-analysis. Diabetes 2011;60:829–836. doi: 10.1136/gut.2011.237974. Care 2009;32:741–750. doi: 10.2337/dc08-1870. [82] Huang KW, Leu HB, Wang YJ, Luo JC, Lin HC, Lee FY, et al. Patients with [60] Younossi ZM, Stepanova M, Rafiq N, Makhlouf H, Younoszai Z, Agrawal R, nonalcoholic fatty liver disease have higher risk of colorectal adenoma after et al. Pathologic criteria for nonalcoholic steatohepatitis: interprotocol negative baseline colonoscopy. Colorectal Dis 2013;15:830–835. doi: 10. agreement and ability to predict liver-related mortality. Hepatology 2011; 1111/codi.12172. 53:1874–1882. doi: 10.1002/hep.24268. [83] Bilici A, Ozguroglu M, Mihmanli I, Turna H, Adaletli I. A case-control study of [61] Targher G, Lonardo A, Byrne CD. Nonalcoholic fatty liver disease and chronic non-alcoholic fatty liver disease in breast cancer. Med Oncol 2007;24:367– vascular complications of diabetes mellitus. Nat Rev Endocrinol 2018;14: 371. doi: 10.1007/s12032-007-0034-8. 99–114. doi: 10.1038/nrendo.2017.173. [84] Musso G, Gambino R, Tabibian JH, Ekstedt M, Kechagias S, Hamaguchi M, [62] Pugh CJ, Spring VS, Kemp GJ, Richardson P, Shojaee-Moradie F, Umpleby et al. Association of non-alcoholic fatty liver disease with chronic kidney AM, et al. Exercise training reverses endothelial dysfunction in nonalcoholic disease: a systematic review and meta-analysis. PLoS Med 2014;11: fatty liver disease. Am J Physiol Heart Circ Physiol 2014;307:H1298– e1001680. doi: 10.1371/journal.pmed.1001680. H1306. doi: 10.1152/ajpheart.00306.2014. [85] Targher G, Chonchol MB, Byrne CD. CKD and nonalcoholic fatty liver [63] Kim HC, Kim DJ, Huh KB. Association between nonalcoholic fatty liver disease. Am J Kidney Dis 2014;64:638–652. doi: 10.1053/j.ajkd.2014. disease and carotid intima-media thickness according to the presence of 05.019. metabolic syndrome. Atherosclerosis 2009;204:521–525. doi: 10.1016/j. [86] Singal AK, Salameh H, Kuo YF,Wiesner RH. Evolving frequency and outcomes atherosclerosis.2008.09.012. of simultaneous liver kidney transplants based on liver disease etiology. [64] Lee YJ, Shim JY, Moon BS, Shin YH, Jung DH, Lee JH, et al. The relationship Transplantation 2014;98:216–221. doi: 10.1097/TP.0000000000000048. between arterial stiffness and nonalcoholic fatty liver disease. Dig Dis Sci [87] Musso G, Cassader M, Cohney S, Pinach S, Saba F, Gambino R. Emerging 2012;57:196–203. doi: 10.1007/s10620-011-1819-3. liver-kidney interactions in nonalcoholic fatty liver disease. Trends Mol Med [65] Kim D, Choi SY, Park EH, Lee W, Kang JH, Kim W, et al. Nonalcoholic fatty 2015;21:645–662. doi: 10.1016/j.molmed.2015.08.005. liver disease is associated with coronary artery calcification. Hepatology [88] Targher G, Rossini M, Lonardo A. Evidence that non-alcoholic fatty liver 2012;56:605–613. doi: 10.1002/hep.25593. disease and polycystic ovary syndrome are associated by necessity rather [66] Sookoian S, Pirola CJ. Non-alcoholic fatty liver disease is strongly associ- than chance: a novel hepato-ovarian axis? Endocrine 2016;51:211–221. ated with carotid atherosclerosis: a systematic review. J Hepatol 2008;49: doi: 10.1007/s12020-015-0640-8. 600–607. doi: 10.1016/j.jhep.2008.06.012. [89] Rocha ALL, Faria LC, Guimarães TCM, Moreira GV, Cândido AL, Couto CA, [67] Oni ET, Agatston AS, Blaha MJ, Fialkow J, Cury R, Sposito A, et al. A system- et al. Non-alcoholic fatty liver disease in women with polycystic ovary syn- atic review: burden and severity of subclinical cardiovascular disease drome: systematic review and meta-analysis. J Endocrinol Invest 2017;40: among those with nonalcoholic fatty liver; should we care? Atherosclerosis 1279–1288. doi: 10.1007/s40618-017-0708-9. 2013;230:258–267. doi: 10.1016/j.atherosclerosis.2013.07.052. [90] Kelley CE, Brown AJ, Diehl AM, Setji TL. Review of nonalcoholic fatty liver [68] Sinn DH, Cho SJ, Gu S, Seong D, Kang D, Kim H, et al. Persistent nonalco- disease in women with polycystic ovary syndrome. World J Gastroenterol holic fatty liver disease increases risk for carotid atherosclerosis. Gastro- 2014;20:14172–14184. doi: 10.3748/wjg.v20.i39.14172. enterology 2016;151:481–488.e1. doi: 10.1053/j.gastro.2016.06.001. [91] Heinzer R, Vat S, Marques-Vidal P, Marti-Soler H, Andries D, Tobback N, [69] Sahebkar A, Chew GT, Watts GF. New peroxisome proliferator-activated et al. Prevalence of sleep-disordered breathing in the general population: receptor agonists: potential treatments for atherogenic dyslipidemia and the HypnoLaus study. Lancet Respir Med 2015;3:310–318. doi: 10. non-alcoholic fatty liver disease. Expert Opin Pharmacother 2014;15: 1016/S2213-2600(15)00043-0. 493–503. doi: 10.1517/14656566.2014.876992. [92] Mesarwi OA, Loomba R, Malhotra A. Obstructive sleep apnea, hypoxia, and [70] Karabay CY, Kocabay G, Kalayci A, Colak Y, Oduncu V, Akgun T, et al. nonalcoholic fatty liver disease. Am J Respir Crit Care Med 2019;199:830– Impaired left ventricular mechanics in nonalcoholic fatty liver disease: a 841. doi: 10.1164/rccm.201806-1109TR. speckle-tracking echocardiography study. Eur J Gastroenterol Hepatol [93] Aron-Wisnewsky J, Minville C, Tordjman J, Lévy P, Bouillot JL, Basdevant A, 2014;26:325–331. doi: 10.1097/MEG.0000000000000008. et al. Chronic intermittent hypoxia is a major trigger for non-alcoholic fatty [71] Fotbolcu H, Yakar T, Duman D, Karaahmet T, Tigen K, Cevik C, et al. Impair- liver disease in morbid obese. J Hepatol 2012;56:225–233. doi: 10.1016/j. ment of the left ventricular systolic and diastolic function in patients with jhep.2011.04.022. non-alcoholic fatty liver disease. Cardiol J 2010;17:457–463. doi: 10. [94] Minville C, Hilleret MN, Tamisier R, Aron-Wisnewsky J, Clement K, Trocme C, 1097/mbp.0b013e328339e2c8. et al. Nonalcoholic fatty liver disease, nocturnal hypoxia, and endothelial [72] Käräjämäki AJ, Pätsi OP, Savolainen M, Kesäniemi YA, Huikuri H, Ukkola O. function in patients with sleep apnea. Chest 2014;145:525–533. doi: 10. Non-alcoholic fatty liver disease as a predictor of atrial fibrillation in middle- 1378/chest.13-0938. aged population (OPERA study). PLoS One 2015;10:e0142937. doi: 10. [95] van der Voort EA, Koehler EM, Dowlatshahi EA, Hofman A, Stricker BH, 1371/journal.pone.0142937. Janssen HL, et al. Psoriasis is independently associated with nonalcoholic

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86 85 Kumar R. et al: NAFLD: a growing threat

fatty liver disease in patients 55 years old or older: Results from a popula- [110] Yalamanchili K, Saadeh S, Klintmalm GB, Jennings LW, Davis GL. Nonalco- tion-based study. J Am Acad Dermatol 2014;70:517–524. doi: 10.1016/j. holic fatty liver disease after liver transplantation for cryptogenic cirrhosis jaad.2013.10.044. or nonalcoholic fatty liver disease. Liver Transpl 2010;16:431–439. doi: 10. [96] Miele L, Vallone S, Cefalo C, La Torre G, Di Stasi C, Vecchio FM, et al. Prev- 1002/lt.22004. alence, characteristics and severity of non-alcoholic fatty liver disease in [111] Caballería L, Pera G, Auladell MA, Torán P, Muñoz L, Miranda D, et al. Prev- – patients with chronic plaque psoriasis. J Hepatol 2009;51:778 786. doi: alence and factors associated with the presence of nonalcoholic fatty liver 10.1016/j.jhep.2009.06.008. disease in an adult population in Spain. Eur J Gastroenterol Hepatol 2010; [97] Gisondi P, Targher G, Zoppini G, Girolomoni G. Non-alcoholic fatty liver 22:24–32. doi: 10.1097/MEG.0b013e32832fcdf0. disease in patients with chronic plaque psoriasis. J Hepatol 2009;51:758– [112] Suomela E, Oikonen M, Virtanen J, Parkkola R, Jokinen E, Laitinen T, et al. 764. doi: 10.1016/j.jhep.2009.04.020. Prevalence and determinants of fatty liver in normal-weight and overweight [98] Targher G, Lonardo A, Rossini M. Nonalcoholic fatty liver disease and decreased bone mineral density: is there a link? J Endocrinol Invest 2015; young adults. The Cardiovascular Risk in Young Finns Study. Ann Med 2015; – 38:817–825. doi: 10.1007/s40618-015-0315-6. 47:40 46. doi: 10.3109/07853890.2014.966752. [99] Li M, Xu Y, Xu M, Ma L, Wang T, Liu Y, et al. Association between nonalcoholic [113] Ruhl CE, Everhart JE. Relationship of non-alcoholic fatty liver disease with fatty liver disease (NAFLD) and osteoporotic fracture in middle-aged and cholecystectomy in the US population. Am J Gastroenterol 2013;108:952– elderly Chinese. J Clin Endocrinol Metab 2012;97:2033–2038. doi: 10. 958. doi: 10.1038/ajg.2013.70. 1210/jc.2011-3010. [114] Shen HC, Zhao ZH, Hu YC, Chen YF, Tung TH. Relationship between obesity, [100] Koo BK, Kim D, Joo SK, Kim JH, Chang MS, Kim BG, et al. Sarcopenia is an metabolic syndrome, and nonalcoholic fatty liver disease in the elderly agri- independent risk factor for non-alcoholic steatohepatitis and significant fib- cultural and fishing population of Taiwan. Clin Interv Aging 2014;9:501– rosis. J Hepatol 2017;66:123–131. doi: 10.1016/j.jhep.2016.08.019. 508. doi: 10.2147/CIA.S59538. [101] Kim G, Lee SE, Lee YB, Jun JE, Ahn J, Bae JC, et al. Relationship between [115] Younossi ZM, Otgonsuren M, Venkatesan C, Mishra A. In patients with non- relative skeletal muscle mass and nonalcoholic fatty liver disease: A 7-year alcoholic fatty liver disease, metabolically abnormal individuals are at a – longitudinal study. Hepatology 2018;68:1755 1768. doi: 10.1002/hep. higher risk for mortality while metabolically normal individuals are not. 30049. Metabolism 2013;62:352–360. doi: 10.1016/j.metabol.2012.08.005. [102] Mahfood Haddad T, Hamdeh S, Kanmanthareddy A, Alla VM. Nonalcoholic [116] Chang Y, Jung HS, Yun KE, Cho J, Cho YK, Ryu S. Cohort study of non- fatty liver disease and the risk of clinical cardiovascular events: A system- alcoholic fatty liver disease, NAFLD fibrosis score, and the risk of incident atic review and meta-analysis. Diabetes Metab Syndr 2017;11 Suppl 1: diabetes in a Korean population. Am J Gastroenterol 2013;108:1861– S209–S216. doi: 10.1016/j.dsx.2016.12.033. [103] Ray S, Mehta NN, Golhar A, Nundy S. Post hepatectomy liver failure - A 1868. doi: 10.1038/ajg.2013.349. comprehensive review of current concepts and controversies. Ann Med [117] Cai W, Wu X, Zhang B, Miao L, Sun YP, Zou Y, et al. Serum uric acid levels Surg (Lond) 2018;34:4–10. doi: 10.1016/j.amsu.2018.08.012. and non-alcoholic fatty liver disease in Uyghur and Han ethnic groups in [104] Behrns KE, Tsiotos GG, DeSouza NF, Krishna MK, Ludwig J, Nagorney DM. northwestern China. Arq Bras Endocrinol Metabol 2013;57:617–622. doi: Hepatic steatosis as a potential risk factor for major hepatic resection. J 10.1590/s0004-27302013000800006. Gastrointest Surg 1998;2:292–298. doi: 10.1016/s1091-255x(98) [118] Dassanayake AS, Kasturiratne A, Rajindrajith S, Kalubowila U, Chakrawarthi 80025-5. S, De Silva AP, et al. Prevalence and risk factors for non-alcoholic fatty liver [105] Belghiti J, Hiramatsu K, Benoist S, Massault P, Sauvanet A, Farges O. Seven disease among adults in an urban Sri Lankan population. J Gastroenterol hundred forty-seven hepatectomies in the 1990s: an update to evaluate Hepatol 2009;24:1284–1288. doi: 10.1111/j.1440-1746.2009.05831.x. the actual risk of liver resection. J Am Coll Surg 2000;191:38–46. doi: [119] Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, 10.1016/s1072-7515(00)00261-1. et al. The natural history of nonalcoholic fatty liver disease: a population- [106] Vauthey JN, Pawlik TM, Ribero D, Wu TT, Zorzi D, Hoff PM, et al. Chemo- based cohort study. Gastroenterology 2005;129:113–121. doi: 10.1053/j. therapy regimen predicts steatohepatitis and an increase in 90-day mortal- gastro.2005.04.014. ity after surgery for hepatic colorectal metastases. J Clin Oncol 2006;24: [120] Haring R, Wallaschofski H, Nauck M, Dörr M, Baumeister SE, Völzke H. 2065–2072. doi: 10.1200/JCO.2005.05.3074. Ultrasonographic hepatic steatosis increases prediction of mortality risk [107] Linares I, Hamar M, Selzner N, Selzner M. Steatosis in liver transplantation: from elevated serum gamma-glutamyl transpeptidase levels. Hepatology Current limitations and future strategies. Transplantation 2019;103:78– 2009;50:1403–1411. doi: 10.1002/hep.23135. 90. doi: 10.1097/TP.0000000000002466. [121] Rafiq N, Bai C, Fang Y, Srishord M, McCullough A, Gramlich T, et al. Long- [108] Marsman WA, Wiesner RH, Rodriguez L, Batts KP, Porayko MK, Hay JE, et al. Use of fatty donor liver is associated with diminished early patient and graft term follow-up of patients with nonalcoholic fatty liver. Clin Gastroenterol – survival. Transplantation 1996;62:1246–1251. doi: 10.1097/00007890- Hepatol 2009;7:234 238. doi: 10.1016/j.cgh.2008.11.005. 199611150-00011. [122] Zeb I, Li D, Budoff MJ, Katz R, Lloyd-Jones D, Agatston A, et al. Nonalcoholic [109] Malik SM, Devera ME, Fontes P, Shaikh O, Sasatomi E, Ahmad J. Recurrent fatty liver disease and incident cardiac events: The multi-ethnic study of disease following liver transplantation for nonalcoholic steatohepatitis cir- atherosclerosis. J Am Coll Cardiol 2016;67:1965–1966. doi: 10.1016/j. rhosis. Liver Transpl 2009;15:1843–1851. doi: 10.1002/lt.21943. jacc.2016.01.070.

86 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 76–86 Review Article

Modulating the Intestinal Microbiota: Therapeutic Opportunities in Liver Disease

Cyriac Abby Philips*1, Philip Augustine1, Praveen Kumar Yerol2, Ganesh Narayan Ramesh3, Rizwan Ahamed1, Sasidharan Rajesh1, Tom George1 and Sandeep Kumbar1

1The Liver Unit, Monarch Liver Lab and Division of Gastroenterology, Cochin Gastroenterology Group, Ernakulam Medical Centre, Kochi, Kerala, India; 2Department of Gastroenterology, State Government Medical College, Thrissur, Kerala, India; 3Department of Gastroenterology, Aster Medcity, Kochi, Kerala, India

Abstract culturomics). Apart from high-throughput culturing, the ‘continuous culturing’ utilizes an open system which is constantly Gut microbiota has been demonstrated to have a significant supplied at one end with fresh growth medium and overflow impact on the initiation, progression and development of allowed to drain from the vessel at the other end, diluting out complications associated with multiple liver diseases. Notably, toxic metabolites and dead cells and leading to a ‘steady- nonalcoholic fatty liver diseases, including nonalcoholic steato- state’ of microbial activity that can be further studied. hepatitis and cirrhosis, severe alcoholic hepatitis, primary scle- These techniques have been overwhelmed with the advent rosing cholangitis and hepatic encephalopathy, have strong links of sequence-based approaches, in which, without the need for to dysbiosis – or a pathobiological change in the microbiota. In growing microbes in the lab, complete detail of the species this review, we provide clear and concise discussions on the present in the sample can be attained within a short time. human gut microbiota, methods of identifying gut microbiota Marker gene survey is the most common sequence-based and its functionality, liver diseases that are affected by the gut approach used for microbial characterization. This method microbiota, including novel associations under research, and utilizes identification and comparison of the microbiome in provide current evidence on the modulation of gut microbiota the sample with universal marker genes, thereby identifying all and its effects on specific liver disease conditions. known species within the sample based on unique conserved Citation of this article: Philips CA, Augustine P, Yerol PK, DNA. The universal marker gene that is most widely employed Ramesh GN, Ahamed R, Rajesh S, et al. Modulating the intes- is the small subunit ribosomal RNA gene (16s rRNA gene for tinal microbiota: Therapeutic opportunities in liver disease. bacteria and archaea, 18s rRNA gene for eukaryotes). J Clin Transl Hepatol 2020;8(1):87–99. doi: 10.14218/ Using the standard PCR technique, en masse sequencing of JCTH.2019.00035. the extracted DNA is performed, and resulting data is clustered by comparative sequence similarity into Operational Taxo- nomic Units that are mapped against a comprehensive reference database to assign taxonomic classifications – thereby, Identification and study of the gut microbiota (GM) representing an approximation of species within the sample. Whole-genome sequencing employs sequencing of multiple The microbiota is defined as all of the microbes associated microbial genomes in a single run, by multiplexing samples with complex organisms such as humans, whereas the micro- through the addition of unique sequence tags. Shotgun- biome is the complete representation of these microbes and sequencing — in which extracted DNA is randomly fragmented their genes. Initial methods to identify and characterize before sequencing and then the resulting overlapping microbiota were predominantly based on culture techniques. sequence data combined bioinformatically by mapping onto High-throughput culturing, which combines controlled and an existing reference genome into continuous stretches — is automated cell-culturing systems to grow dozens of cultures the standard method of whole-genome sequencing. Whole- at once over long periods, has essentially improved charac- genome-sequencing typically requires that the organism be terization of novel microbes and strains (also known as grown in culture first before DNA extraction and sequencing. With metagenomics, direct sequencing is performed on DNA Keywords: Microbiome; Metagenomics; NGS; Cirrhosis; Illumina. extracted from a sample, followed by bioinformatical piecing Abbreviations: ACLF, acute-on-chronic liver failure; AH, alcoholic hepatitis; AIH, together of the sequenced data into continuous data, allowing autoimmune hepatitis; ALD, alcoholic liver disease; APAP, acetaminophen; FMT, fecal microbiota transplantation; FXR, farnesoid-X receptor; GM, gut microbiota; for study of the qualitative as well as functional aspect of the HCV, hepatitis C virus; HE, hepatic encephalopathy; IL, interleukin; LPS, lipopo- microbiota without culturing. Single-cell genomics utilizes iso- lysaccharide; MELD, model for end-stage liver disease; NAFLD, nonalcoholic fatty lation of individual microbes from the sample, after which whole liver disease; NASH, nonalcoholic steatohepatitis; PSC, primary sclerosing chol- genome amplification is performed. This powerful technique angitis; TGR5, G protein-coupled bile acid receptor 1 and membrane-type bile acid receptor; TNF, tumor necrosis factor. allows for recovery of genomic data of rare species and helps in Received: 11 August 2019; Revised: 11 October 2019; Accepted: 27 October the understanding of organisms that are capable of carrying out 2019 a particular metabolic function, even if such genomic and *Correspondence to: Cyriac Abby Philips, The Liver Unit, Monarch Liver Lab functional information is missing from comparative databases. and Division of Gastroenterology, Cochin Gastroenterology Group, Ernakulam Medical Centre, Kochi, Kerala 682028, India. Tel/Fax: +91-484-2907000, Metatranscriptomics, or RNA sequencing, in contrast to E-mail: [email protected] metagenomics, provides detailed information on the functional

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 87

Fig. 1. Various methods for identifying and studying the gut microbiota and microbiome. Culturomics help in phenotyping the microbial communities, and further whole genome sequencing improves identification of microbial diversity up to the species and functional levels. DNA-based genomic analysis through 16s rRNA sequencing curtails time to identification of microbial species, while RNA-based analysis helps in studying microbial function at the transcriptome, proteome and metabolite levels.

88 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 Philips C.A. et al: Intestinal microbiota reinstitution therapy produced in the gut include short-chain fatty acids, branched- and related compounds which promote systemic inflamma- chain fatty acids, branched-chain amino acids, biogenic tion through transcription of IL-6. amines, organic gases, and other secondary plant-derived Indole-3-propionate acts at the pregnane X receptor compounds, such as choline, bacterial cell wall components, (referred to as PXR) and down-regulates tumor necrosis polyamines, and volatile organic compounds. The plethora of factor (TNF)-alpha production in enterocytes by limiting metabolites thus formed are further absorbed, distributed or bacterial translocation and lipopolysaccharide (LPS) infiltra- excreted by multiple, highly dynamic processes and pathways tion into the circulation, thereby reducing metabolic endotox- 19–22 that significantly affect human health by regulating pro- and emia and host inflammation. Various microbes or groups anti-inflammatory processes, immunological landscape of microbes are associated with carrying out specific regula- (innate and adaptive immunity), and detoxification. For tory processes in the human gut, which is directly or indirectly maintaining homeostasis, the intestinal barrier limits expo- associated with liver health (Supplementary Fig. 3). sure of the host immune system to the microbiota through multifactorial and dynamic processes that operate at the Microbiota and the gut-liver-axis luminal and mucosal level.12,13 The GM caters to and provides multiple benefits to the Since the liver is an organ that has privilege in placement with host, such as nutrient metabolism and assimilation, protec- regards to maximal exposure to gut microbes and its metab- tion and control over pathogenic species, and maintenance of olites, studies on ‘healthy state’ and diseases associated with immune functions. For example, the colonic bacteria express the hepatobiliary system have been on the forefront in the carbohydrate-active enzymes, which empower them to current bench-to-bedside research. Changes associated with ferment complex carbohydrates, thereby generating metab- the GM are implicated in the pathogenesis of many liver olites which regulate cellular processes such as gene expres- diseases. This alteration in general is termed dysbiosis, in sion, chemotaxis, differentiation, proliferation, and apoptosis. which there is an imbalance between the symbionts and On the other hand, specific anaerobes produce acetate while pathobionts in the gut. The intestine and liver have a bidirec- propionate and butyrate are produced by different subsets of tional communication mediated through the biliary tract, gut bacteria through distinct molecular pathways. In the portal vein, and systemic circulation. The liver communicates human intestine, propionate is mainly produced by Bacter- with the gut through bile acids and other metabolic media- oidetes, while the production of butyrate is mainly by Firmi- tors. In the gut, the microbes metabolize endogenous and cutes. Starch fermentation by Eubacterium rectale or exogenous compounds, end-products of which translocate to Eubacterium hallii that belong to Firmicutes, significantly con- the liver through the portal vein, influencing the liver micro- tributes to butyrate production in the colon and Akkermensia environment and functions. The liver receives and filters large muciniphila has been found to majorly contribute to propio- amounts of nutrients, bacterial products, toxins and metab- nate production through mucin degradation, the latter which olites through the portal vein, with an efferent circulation via the biliary system. This ‘metabolic endotoxemia’, as described is primarily absorbed by the liver. Propionate has been shown 23 to reduce cancer cell proliferation and through its action on by Cani et al. in patients with diabetes and metabolic syndrome promotes a steady-state of low-grade inflammation beta-cell function, ameliorates reward-based eating behavior within the liver microenvironment, driven by unhealthy though striatal pathways. In addition, butyrate is known for changes in the GM. its anti-inflammatory activities in the liver microenvironment, Similarly, a ‘tip of the balance’ towards a more pathogenic acting by attenuating bacterial translocation and enhancing profile of microbiota leads to increased exposure of the liver gut barrier strength by improving tight-junction function. to pathogen and microbe-associated molecular patterns Similarly, the short-chain fatty acids produced by the colonic through an increase in bacterial translocation and leakiness GM regulate the immune system and inflammatory processes of the gut. This exposure results in proinflammatory cellular by influencing the production of interleukin (IL)-18, which is signaling within the hepatic environment driven by major involved in maintenance and repair of mucosal epithelial cytokines, such as IL-1, IL-6 and TNF-alpha. Continuous integrity as well as in modulation of appetite regulation and proinflammatory state, in the presence of persistence of energy utilization in the host, which are associated with met- factors that promote it (such as alcohol, drugs, obesity, abolic syndrome and obesity. diabetes) leads to production of reactive oxygen species Apart from carbohydrate metabolism, pertinent lipid which promote liver injury and fibrosis. metabolism in the host is also driven by the GM. For Liver, a highly active site of metabolism and immune example, the facultative and anaerobic bacteria of the colon homeostasis, handles and secretes multiple immunogenic produce secondary bile acids which enter the systemic molecules and metabolites into the gut, which affects the circulation to modulate hepatic and systemic lipid metabolism microbiota and vice-versa. Secretory immunoglobulin A, bile through nuclear or G protein-coupled receptors. Akkerman- acids and fatty acids processed by the liver activate various sia, Christensenellaceae, Tenericutes, Eggerthella, Pasteurel- nuclear receptors, such as the G-protein coupled receptor and laceae, and Butyricimonas are associated with body mass farnesoid-X receptor (FXR), that regulate glucose and lipid index in patients with metabolic syndrome as well as levels metabolism and homeostasis as well as conjugation and 14–18 of triglycerides and high-density lipoproteins. With detoxification of exogenous and endogenous toxins. Gut- regards to protein metabolism, the microbiota-derived derived hormones (for example, fibroblast growth factor, metabolites produced from aromatic amino acids (tyrosine, glucagon-like-peptide-1 and serotonin) also play an impor- tryptophan, and phenylalanine) affect host signaling path- tant role in maintaining homeostasis and energy assimilation ways interacting with host immunity. Bacteroides thetaiotao- and balance by affecting the steady metabolic state, via their micron, Proteus vulgaris, and Escherichia coli act through action on appetite and food intake. Dysbiosis has been shown tryptophanase activity, producing indole which is sulfated in to initiate, promote or cause progression of liver diseases, the liver and resulting in the production of 3-indoxyl sulfate such as alcoholic liver disease (ALD), alcoholic hepatitis (AH),

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 89 Philips C.A. et al: Intestinal microbiota reinstitution therapy nonalcoholic fatty liver disease (NAFLD) and nonalcoholic and progression of NAFLD-associated conditions.28,29 Lower steatohepatitis (NASH), drug-induced toxic liver injury, liver levels of Bacteroides are associated with obese patients with fibrosis, cirrhosis and its complications, hepatocellular carci- NASH, while the lower abundance of Firmicutes was demon- noma, and chronic cholestatic and autoimmune hepatobiliary strated in non-obese NASH patients. Reduction in Lachno- 24–27 disease (Fig. 2). spira, Ruminococcus and Lactobacillus was notable in lean patients with NASH. In adolescents, the abundance of Bacter- GM and diseases of the liver oides followed a ‘U’ pattern, based on the dietary pattern of fat intake. In those with high fat intake, low and high abun- NAFLD and NASH dances were noted, while in those with low fat intake, a moderate level of abundance was found. NAFLD encompasses steatosis, steatohepatitis, advanced Bilophila, Paraprevotella and Suturella are associated with fibrosis, cirrhosis and related hepatocellular carcinoma. higher hepatic fat content, in contrast to Oscillospira and Var- High-quality studies in humans with and without NAFLD/ ibaculum for which a negative association with steatosis has NASH have shown a strong correlation of GM in the initiation been demonstrated. Higher levels of Bilophila wadworthia

Fig. 2. The gut-liver axis and microbiota related cross-talk. In the presence of factors that disrupt microbial diversity and function (alcohol, metabolic diseases, drugs, environmental toxins, diet), pathobionts that promote disease causation or progression evolve in the dysbiotic milieu. This leads to gut barrier disruption, enhancement of local proinflammatory cascade, endotoxemia and ultimately systemic inflammation, leading to end organ adverse events.

90 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 Philips C.A. et al: Intestinal microbiota reinstitution therapy were found to be associated with T helper-1-mediated gut liver cancer in obese mouse models has been linked to the inflammation. Lower levels of Oscillospira were associated persistence of low-grade systemic inflammation that is ini- with an increase in the metabolite 2-butanone, which was tiated from dysbiotic microbiota. Thus, there is robust evi- related to the onset of NAFLD. Increased levels of Ruminococ- dence that GM plays a central role in steatosis, inflammation cus, Dorea, Robinsoniella and Roseburia were found to be and progression of fibrosis in NAFLD patients. associated with progression of inflammation and fibrosis in GM targeted therapies are upcoming strategies for treat- NASH. Patients with NASH fibrosis $2 were found to have ment of NAFLD, NASH and NASH-related HCC. Currently, the higher abundance of Bacteroides and Ruminococcus, and focus of such treatments has been solely on probiotics and lower levels of Prevotella; while, in those with advanced fib- prebiotic supplementation – most commonly utilizing Lacto- rosis and cirrhosis, Proteobacteria were relatively higher. At bacillus and Bifidobacterium. Administration of probiotics has the species level, it was shown that Eubacterium rectale and been shown to reduce liver enzymes, total cholesterol, TNF- Bacteroides vulgaris were relatively abundant in patients with alpha, serum endotoxin levels, liver fat and NASH activity mild to moderate NASH, while Bacteroides vulgatus and index in small-animal as well as human models. Probiotics E. coli were predominant among patients with NASH-related increase PPAR-alpha activity and reduce metalloproteinases advanced fibrosis and cirrhosis.30–32 2 and 9 and cyclooxygenase expression. Lactobacillus casei Changes in GM in NAFLD patients have been shown to strain Shirota reduced the development of NASH in methio- have positive correlation with small-intestinal bacterial over- nine- and choline-deficient diet mouse models by lowering growth, defined as total bacteria growth of more than 103 serum LPS concentration; whereas Bifidobacteria and Lacto- colony-forming-units of coliform bacteria per milliliter of prox- bacillus rhamnosus GG ameliorated liver steatosis by acting imal jejunal fluid, which is directly related to endotoxemia, on the sirtuin-1-mediated signaling pathway and reducing circulating bacterial DNA and leakiness of the gut, and asso- nuclear factor-kB inhibitor protein expression.36–38 Meta- ciated with worsening of steatosis and inflammation. Intesti- analysis of randomized controlled trials on probiotics and pre- nal dysbiosis results in lower levels of colonic junctional biotics have shown that supplementation led to reduction in adhesion molecule A expression, increase in intestinal perme- aminotransferase level, reduced body fat, and improved ability, leading to liver exposure to higher levels of bacterial glucose metabolism.34,39–41 LPS, and endogenous ethanol, acetone and butanoic acid, Fecal microbiota transplantation (FMT), generally called that leads to hepatic inflammation. The GM also modulates ‘stool transplant’, utilized the transfer/transplantation of fecal choline metabolism, in which dietary choline is converted to microbiota from a healthy individual into a patient with dimethylamine and trimethylamine — increased levels of dysbiosis, aiming to restore intestinal microbial diversity. which result in hepatic inflammation. Lower levels of dietary There have been strong notions challenging the current choline lead to changes in GM and its functionality, which pro- terminology of FMT. The term ‘fecal/stool’ as a treatment motes fatty liver disease which, however, can reverse with modality has negative implications within the scientific com- high choline diet in small animal models. Escherichia, which munity, pharmaceutical industry and funding sources as well increases production of endogenous ethanol utilizing the as among patients and their families with regards to accept- ‘mixed-acid-fermentation’ pathway was shown to be signifi- ance. Khoruts et al.42 proposed the term ‘intestinal micro- cantly associated with NASH and higher grades of fibrosis. biota transplantation’, while Bajaj et al.43 proposed that FMT The end metabolites that form out of ethanol degradation be renamed ‘microbiome restoration therapy’. However, both include acetate, which takes part in fatty acid synthesis, and these terms feature inadequacies. Even though considered by acetaldehyde, which promotes cytotoxic effects within the some as ‘an organ’, the fecal microbiota is not an organ and is liver microenvironment. Gut dysbiosis and small-intestinal a highly different, unexplored entity, which differs in accord- bacterial overgrowth in NASH patients have been shown to ance with sex and region, and even between individuals. be associated with higher levels of circulating serum TNF- Hence, the term transplantation is inaccurate. We are yet to alpha and IL-8, through signaling mediated by toll-like recep- define a ‘matching/healthy’ microbiota donor. Microbiome, as tors -9 and -4. discussed, is the genetic totality associated with the micro- Bile acids have significant effect on GM and have great biota, which remains unique to the person. Studies have impact on the metabolism of bile acids. Taurine- and glycine- mostly looked at bacterial communities, even though eukar- conjugated primary and secondary bile acids were found to be yotes, protozoa, viruses and phages are also part of the resto- higher in patients with NASH compared to healthy controls. ration, and which remain undefined. Hence, the term Bile acids, through regulatory effects on FXR and G protein- microbiome becomes vaguely general and does not hold coupled bile acid receptor 1 and membrane-type bile acid well as a replacement to current terminology of FMT. receptor (referred to as TGR5), affecting the natural history of The term Microbiota Restoration Therapy™ has been NAFLD. Agonists of FXR and TGR5 have been shown to reduce patented by Rebiotix Inc (Ferring, Saint-Prex, Switzerland; liver fat, improve NASH histology, and promote weight loss in www.rebiotix.com) to define their microbiota-based thera- a small animal model of NAFLD.33–35 pies. In this regard, an ideal, general, novel terminology for Intestinal bacteria have been shown to reduce expression FMT, for utilization in discussions and in trials, in the light of of fasting-induced adipocyte factor on the enterocytes, current studies, would be ‘intestinal microbiota reinstitution leading to induction of lipoprotein lipase activity and accu- therapy’, which can be modified accordingly, to further mulation of hepatic triglycerides. Gut bacteria also promote characterize different sites and specific components of the increased production of short-chain fatty acids, acetate, microbiota in future studies. We propose ‘reinstitution’ and propionate and butyrate, contributing to obesity, metabolic not ‘restoration’ because the latter means ‘to bring something disease and increase in liver steatosis. LPS production by back to the original form/former position or condition’, when specific microbiota groups has been shown to promote liver in reality, we are unaware of ‘original’ condition of microbiota carcinogenesis in mouse models, while in the germ-free mouse within the recipient prior to the disease state; moreover, FMT model, hepatocarcinogenesis was reduced. The occurrence of actually modifies the microbiota more towards the donor

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 91 Philips C.A. et al: Intestinal microbiota reinstitution therapy profile and is not technically a ‘restoration’. ‘Reinstitution’ Grander and colleagues50 showed that Akkermansia muci- means the act of establishing something again – and with niphila abundance reduced with increasing severity of ALD FMT, we aim to establish a healthy microbiota. Studies on FMT and was lowest in AH. Ciocan and colleagues51 showed that in animal models have demonstrated amelioration of steato- in patients with cirrhosis and AH, total plasma bile acid levels hepatitis in high-fat diet mice.44 Utilization of FMT for meta- were higher, while levels of total and secondary bile acids bolic syndrome in humans was first performed by Vrieze were lower compared to those without cirrhosis or AH. The et al.45 They found that FMT from lean donors temporarily relative abundance of Actinobacteria was higher and that of increased peripheral insulin sensitivity and reduced hepatic Bacteroidetes which was lower in alcoholic cirrhosis with AH. steatosis without statistical significance. Xue et al.46 pre- Puri et al.52 found that in alcohol-consuming patients, there sented preliminary data on the effects of FMT (200 mL/day was an enrichment of bacteria with genes related to methano- for 3 days) in human NAFLD. They showed that change in a genesis and denitrification. Both heavy drinking controls and ™ fat-attenuated parameter, as measured by FibroTouch , was patients with severe AH demonstrated activation of a type III significantly lower after treatment with FMT compared with secretion system associated with gram-negative bacterial vir- the control group. Clinical trials assessing the efficacy and ulence. In patients with AH compared to non-alcohol consum- safety of FMT for NASH (NCT02469272) and NASH-related ing controls, there was an increase in isoprenoid synthesis cirrhosis (NCT02721264) are ongoing. through upregulation of the mevalonate and anthranilate degradation, which are known modulators of gram-positive ALD bacterial growth and biofilm production, respectively. Bluemel et al.53 investigated the microbiota in the jejunum, Continuous or binge alcohol use over long periods results in ileum, cecum, feces and liver of mice subjected to chronic ALD, which comprises liver steatosis, AH, alcoholic cirrhosis ethanol feeding; they found that chronic ethanol administra- and acute-on-chronic liver failure (ACLF). Characteristic tion modified alpha diversity in the ileum and the liver, largely changes in intestinal microbiota have been shown to predis- driven by an increase in gram-negative phyla, resulting in pose to severe forms of ALD. Dysbiosis is associated with AH endotoxemia. Specifically, the gram-negative Prevotella in animal models, which were reversed with healthy donor increased in the mucus layer of the ileum and also in liver FMT. Similarly, progressive worsening of dysbiosis is associ- tissues, suggesting the central role of dysbiosis and bacterial ated with the progression of alcoholic cirrhosis and its translocation leading to liver injury with alcohol use. complications. Severe AH was associated with the higher In an open-label randomized controlled trial, probiotic-rich fecal abundance of Bifidobacteria, Streptococci and Entero- in Bifidobacterium bifidum and Lactobacillus plantarum,com- bacteria. Germ-free mice (C57BL/6) demonstrated higher pared to placebo in patients with AH, led to a reduction in susceptibility to alcohol-induced liver injury than conven- hepatic inflammation in the form of improvement in liver bio- tional mice.47 This impresses the fact that complete chemistry, while the addition of Lactobacillus casei Shirota 3 absence of intestinal microbiota as well as an imbalance in strain thrice daily for 30 days improved neutrophilic phagocytic 54 microbiota both predispose to alcohol-related liver injury; capacity in ALD patients, when compared with baseline. A hence, a ‘eubiosis state’ properly defines protection against placebo-controlled trial showed that supplementation with alcohol-induced liver injury. 1.5 g of Bacillus subtilis and Enterococcus feacium daily for 7 Defining the microbial communities that promote this days improved liver function and reduced systemic inflamma- 55 eubiosis is still a matter of research. Change in GM has also tion and endotoxemia in AH. The first pilot study of FMT in been implicated in alcohol-induced damage to the liver steroid ineligible severe AH demonstrated an improvement in through modulation of immune responses, expression of 1-year survival in FMT-treated patients compared to historical alcohol metabolism, oxidative stress, fat metabolism and controls (87.5% vs. 33.3%). The relative abundance of Pro- endotoxemia. Alcohol use was shown to be associated with teobacteria was high and that of Actinobacteria low in patients decreased levels of butyrate-producing Clostridiales and with severe AH at baseline. Post-FMT, this was significantly increased levels of pro-inflammatory Enterobacteriaceae reversed, along with coexistence of protective symbiotic and Proteobacteria. Lower abundance of Ruminococcus was donor and recipient species at 12 months. Reduction in relative associated with increased intestinal permeability and dysbio- abundance of pathogenic species [Klebsiella pneumonia (10% sis, which was reversed with abstinence. In AH, reduction in to <1%)], and increase in non-pathogenic species [Enterococ- the relative abundance of Clostridium leptum and Fecalibac- cus villorum (9% to 23%) and Megasphaera elsdenii (10% to terium prausnitzii has been demonstrated.46 Philips et al.48 60%)] was demonstrated. After FMT, reduction in methane demonstrated that patients with severe AH had a higher rel- metabolism, bacterial invasion of the epithelial cells, inflam- ative abundance of Enterobacter, Megaspaera, Dialister, Pre- matory and cytotoxic pathways, and aromatic amino acid gen- votella and Klebsiella, while in healthy controls, Akkermansia, eration was noted.48 Veillonella, Oscillopsira, Lachnospira, Bacteroides, Egger- In a retrospective observational study comparing FMT to thella, Coriobacterium and Bifidobacterium were higher. At other conventional modalities of treatment for AH, the pro- the functional level, LPS biosynthesis, glycosyl transferase portions of patients surviving at the end of 3 months in the and valine-leucine-isoleucine degradation pathways were steroids, nutrition, pentoxifylline, and FMT group were 38%, affected in patients with AH, while in healthy controls, 29%, 30%, and 75% (p =0.036).56 In patients with severe AH alanine-aspartate-glutamic acid metabolism and non-aro- and non-responders to steroids with ACLF grades 0, 1 and 2 + matic amino acid metabolism were significantly up-regulated. 3, the 90-day survival rates were 68.1%, 45.8% and 36.7%. The gut microbiota composition in healthy and ALD and its Philips and colleagues57 showed that, at the end of 548 days effect on intestinal permeability in ALD pathogenesis point follow up, the proportion of ACLF-AH patients surviving, after toward emerging evidence on GM modulation in ALD as a FMT, in the lower (ACLF 0 + 1) and higher grade (ACLF 2 + 3) mode of treatment from preliminary clinical and non-clinical groups were 72.7% and 58.3% respectively, which was higher studies.49 than what is seen with current medical therapies and

92 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 Philips C.A. et al: Intestinal microbiota reinstitution therapy comparable to liver transplantation. Future studies on FMT in reversed towards a beneficial pattern after FMT, leading to AH could identify better methods for fecal transfer, refine tar- stable liver disease severity scores. The same group studied the geted therapy, and utilize precision metabolomics to modulate utility of FMT capsules in patients with recurrent HE. In this the intestinal milieu to improve outcomes. phase 1 study, they found that oral capsule-based FMT treatment was safe and well-tolerated in patients with cirrhosis Role of GM in cirrhosis and its complications and recurrent HE and was associated with improved duodenal mucosal alpha diversity, reduced dysbiosis, antimicrobial In liver cirrhosis, the presence of portal hypertension results peptide expression, reduced LPS binding protein level and 62,63 in structural changes to the intestinal mucosa and vascula- improved EncephalApp performance. ture, leading to an increase in intestinal permeability that Even though circulating bacterial DNA, plasma endotoxin worsens with gut microbial changes and associated functional levels, and inflammatory and vasoactive markers in ascites metabolism. Altered microbiota has been found in the intes- and blood have been linked to infections in cirrhosis, espe- tinal mucosa, stool and saliva samples from patients with cially spontaneous bacterial peritonitis, no direct linkage to cirrhosis of variable etiologies. The dysbiotic microbiota in dysbiosis or specific patterns of bacterial community changes patients with cirrhosis reveal a reduction in Bacteroides and have been studied. Lachnospira and increase in Proteobacteria, Enterobacteria and Veillonella. The progressive increase in Enterobacteria GM in liver cancer correlates with complications of cirrhosis, especially hepatic encephalopathy (HE). The severity of cirrhosis with regards to High-quality studies concerning experimental animal models Child-Pugh class correlated positively with Streptococcus and supporting the role of GM changes and hepatocarcinogenesis negatively with Lachnospiraceae (Coprococcus, Pseudobutyr- are well known in the literature. In earlier studies, gut ivibrio, Roseburia). The decrease in autochthonous taxa such sterilization by antimicrobials in carcinoma animal models as Lachnospiraceae, Ruminococcaceae and Clostridiales XIV, was shown to reduce tumor incidence and growth. Helico- and the relative increase in Staphylococcaceae, Enterococca- bacter hepaticus co-administration in the AFB-1 model of hep- ceae and Enterobacteriaceae were found to be associated atocarcinogenesis revealed greater tumor number and size with progressive liver failure and endotoxemia. The cirrhosis compared to AFB-1 alone. Chronic administration of diethylni- to dysbiosis ratio, between indigenous and non-indigenous trosamine decreased the abundance of Lactobacillus, Entero- taxa, negatively correlated with endotoxemia, was highest coccus and Bifidobacterium species, leading to the promotion among healthy controls and lowest in patients with decompen- of tumor development and growth, which was then arrested by sated cirrhosis. A higher proportion of bacteria of buccal origin probiotic supplementation. LPS administration also resulted in (Streptococcus and Veillonella) within the gut microbiome of increased number and size of HCC in animal models, which patients with cirrhosis suggested that the oral microbiota was attenuated via antibody and antimicrobial use. High-fat invaded the gut, thereby contributing to the progression of diet-related increase in gut dysbiosis and subsequent increase the disease. Composition of the microbiota differed between in deoxycholic acid resulted in hepatic tumor formation, which patients with and without HE, only in mucosa but not in stool decreased with antibiotic treatment. Higher abundance of Ato- samples. Veillonella, Megasphaera, Bifidobacterium and Enter- pobium, Bacteroides vulgatus, Bacteroides acidifaciens, Bac- ococcus were prevalent in HE, whereas Roseburia was more teroides uniforms, Clostridium cocleatum, Clostridium abundant in the non-HE groups. Minimal HE was associated xylanolyticum and Desulfovibrio spp in a NASH mouse model with higher levels of Streptococcus salivarius, while in overt was found to be associated with HCC development, which was HE, fecal levels of Alcaligenaceae and Porphyromonadaceae again associated with a change in bile acid fractions in the liver were associated with poor cognition. Salivary dysbiosis was tissue and plasma. The number and size of tumors was ame- greater in patients with cirrhosis who developed 90-day hospi- liorated using cholestyramine in the small animal model of talizations. Stool Bacteroidaceaeae and Clostridiales XIV pre- NASH-related HCC. Prevotella and Oscilibacter,thatarepro- dicted 90-day hospitalizations independent of such clinical ducers of anti-inflammatory metabolites, were found to be predictors as Child-Pugh class and model for end-stage liver negatively associated with liver tumor formation. Studies disease (commonly known as MELD) score.58–60 linking dysbiosis and specific bacterial communities in human Bajaj et al.61 demonstrated distinct gut microbial profiles HCC is lacking, but targeting GM and its metabolites in patients associated with ACLF in hospitalized patients. The cirrhosis- with chronic liver disease is an exciting frontier for HCC man- 64,65 to-dysbiosis ratio was lower in those with ACLF and also those agement in the future. with renal failure. Enterobacteriaceae, Campylobacteriaceae, Pasteurellaceae, Enterococcaceae and Streptococcaceae Role of GM in other liver diseases – emerging were associated with the development of poor outcomes, indications while Lachnospiraceae and Clostridiales were associated with a reduction in poor outcomes. Changes in the microbiota Studies have shown that the intestinal microbiome could and dysbiosis had an independent and significant association affect the development of autoimmune hepatitis (AIH) in with extrahepatic organ failure, intensive unit admissions, predisposed individuals. A decrease in fecal Bifidobacterium ACLF, and death in cirrhosis patients in-hospital. and Lactobacillus abundance along with an increase in plasma In a randomized controlled trial, rectal enema-based FMT LPS was notable in patients with higher severity of AIH.66 In improved cognition among cirrhotic patients with recurrent HE, germ-free mice, protection against experimental AIH was significantly higher than seen in the control group. The MELD notable, in the presence of lower levels of leukocyte infiltra- score transiently worsened post-antibiotics but reverted to tion and inflammatory cytokines and absence of hepatocyte baseline after FMT. Antibiotic therapy in the control group apoptosis due to the deficiency in activation of natural killer T reduced beneficial taxa and decreased microbial diversity cells in the liver microenvironment, that predisposes to auto- concurrent with Proteobacteria expansion, which was again antibody-mediated liver injury. Humans studies on the

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Fig. 3. Healthy donor microbiota restitution therapy through upper gastrointestinal endoscopy. Given to a patient with primary sclerosing cholangitis (A). The bacterial communities at the family level in the donor, the patient and the patient after 4 weeks (B). The modification of gut bacterial communities is evident, associated with improved clinical outcomes. microbiome in AIH are lacking. Wei et al.67 showed that the gut Roseburia and Bacteroides were associated with PSC and microbiome of steroid treatment-naïve AIH had lower alpha- inflammatory bowel disease.68 Even though a single-case diversity with distinct overall microbial composition when com- study, done longitudinally over 12 months, Philips et al. 69 pared with healthy controls. Reduction in obligate anaerobes demonstrated that endoscopic FMT (250 mL, distal duodenum) and increase in pathobionts such as Veillonella was associated repeated weekly for 4-weeks improved symptoms, liver bio- with AIH – of which, Veillonella dispar was the most signifi- chemistry, bile acid fractions and survival in tandem with ben- cantly disease-associated taxa with positive correlation with eficial changes in bacterial communities and functional the elevation of aspartate aminotransferase. Thus, micro- metabolites in a patient with advanced PSC and recurrent- biota-based biomarkers could help identify AIH disease cholangitis listed for liver transplantation (Fig. 3). Allegretti severity as well as being potential therapeutic targets. et al.70 performed the first pilot study on FMT in 10 patients GM-driven pathophysiological progression of primary scle- with PSC, of whom 9 had ulcerative colitis, and 1 had Crohn’s rosing cholangitis (PSC) and primary biliary cholangitis are colitis. The mean baseline alkaline phosphatase level was 489 well documented in animal models. Bile acid metabolism is U/L. Overall, 30% experienced a $50% decrease in alkaline heavily handled by the GM and is central for the pathogenesis phosphatase levels post-FMT. The bacterial diversity increased of PSC and primary biliary cholangitis. Functional gut micro- in all patients post-FMT, in the first week itself and abundance bial activities associated with bile acid metabolisms, such as of engrafted microbial communities after FMT also correlated dehydrogenation, conjugation and deconjugation and degra- with the decrease in alkaline phosphatase levels. dation of primary and secondary bile acids, and subsequent Functional and compositional changes of GM have been metabolite and toxin generation play an important role in demonstrated in patients with chronic hepatitis B virus autoimmune-mediated cholestatic inflammatory disease. infection-related cirrhosis, in the form of reduced abundance Germ-free mice were found to have severe PSC features, in of Bifidobacteria and Lactobacillus, and high levels of Enter- comparison to conventional mice, as the complete lack of ococcus. In another study, it was shown that, in hepatitis B microbiota resulted in alteration of needful bile acid metab- virus-related cirrhosis reduction in Bacteroidetes and olism that is associated with worsening fibrosis and liver increased levels of Proteobacteria compared to the healthy injury. Experimental animal studies have demonstrated that group was notable. Healthy donor FMT, in addition to standard enteric but not colonic dysbiosis was associated with hepato- antivirals, was shown to be significantly more effective in biliary inflammation, and small bowel bacterial overgrowth in clearance of the hepatitis B e antigen when compared to anti- rats resulted in hepatobiliary inflammation resembling histo- viral therapy alone.71,72 logical and cholangiography features of PSC. In patients with hepatitis C virus (HCV)-related cirrhosis, Human studies have revealed that increased abundance of the microbial diversity was found to be lower when compared Escherichia and Megasphaera and lower levels of Prevotella, to healthy controls. HCV can alter the GM through

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Table 1. Summary of association of gut microbiota in various liver diseases

Disease Pertinent associated microbiota and metabolites Comments

NAFL Increase Blautia, Dorea, Streptococcus, Clostridium In animal models, reversing microbiota Butanoic acid, Propanoic acid, Phenylacetic acid, changes reversed hepatic steatosis in Isobutyric acid, Unconjugated cholic acid, Ethanol the absence of weight loss Decrease Oscillospira, Coprococcus, Fecalibacterium 2-butanone NASH Increase Escherichia, Blautia, Dorea, Lactobacillus, Clostridium, Allisonella, Bacteroides Chenodeoxycholic acid, Unconjugated cholate, Lithocholic acid, Ethanol, 4-Methyl-2-pentanone Decrease Oscillospira, Coprococcus, Fecalibacterium NASH-related Increase Blautia, Roseburia, Streptococcus, Lactobacillus, advanced Enterococcus, Bacteroides, Escherichia, Klebsiella fibrosis 3-Phenylpropanoate, 3–4-Hydroxyphenyl-lactate Decrease Ruminococcus, Akkermansia NAFLD-related Increase Enterococcus, Oscillospira, Bacteroides HCC Decrease Blautia, Bifidobacterium NASH in obese Increase Prevotella, Escherichia coli children Decrease Bifidobacterium, Alistipes, Blautia Alcoholic liver Increase Proteobacteria disease without Threonine, Glutamine, Guanidino-succinate, cirrhosis Propionate, Isobutyrate, Dimethyl disulfide, Dimethyl trisulfide, Urinary 3- hydroxytetradecanedioic acid, and so-citric acid Decrease Bacteroidetes, Ruminococcaceae Urinary sebacic acid Alcoholic Increase Enterobacteriaceae cirrhosis with Decrease Lachnospiraceae and Ruminococcaceae abstinence Alcoholic Increase Oral-origin microbiota and Lactobacillaceae cirrhosis with Decrease Citrate, Malate, Phosphate, Threonine, Ornithine, active drinking Serine, Ribosine, Orotic acid, Hexanoate Alcoholic Increase Enterobacteriaceae, Streptococcaceae, Higher total serum bilirubin in patients hepatitis Actinobacterium, Bifidobacterium, Fusobacteria with higher fecal abundance of Eicosapentaenoate, Docosapentaenoate, Benzoic Enterobacteria Lower total serum acid metabolites bilirubin in patients with higher fecal Decrease Akkermansia muciniphila abundance of Clostridiales Monoacylglycerols, Malate, Fumarate, Citrate, Akkermansia muciniphila abundance Glycodeoxycholate reduced with increasing severity of alcoholic liver disease; lowest in alcoholic hepatitis Cirrhosis (any Increase Proteobacteria, Fusobacteria, Clostridium Reduction in levels of Lachnospira and etiology) clusters XI increase in level of Streptococcus Enterobacteriaceae, Streptococcaceae, associated with higher Child-Pugh Leuconostocaceae, Lactobacillaceae, scores Alcaligenaceae Enterobacterium associated with Acidaminococcus, Enterococcus, Burkholderia, spontaneous bacterial peritonitis Ralstonia Bacteroidaceaeae and Clostridiales XIV Proteus predictors of 90-day hospitalization Decrease Bacteroidetes and higher Child-Pugh and MELD Lachnospiraceae, Ruminococcaceae scores Clostridium-Incertae sedis – XIV Dorea, Subdoligranumum

(continued)

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Table 1. (continued ) Disease Pertinent associated microbiota and metabolites Comments

Acute-on- Predictors Enterobacteriaceae, Campylobacteriaceae, chronic liver of poor Pasteurellaceae, Enterococcaceae, failure outcomes Streptococcaceae Reduction Lachnospiraceae, Clostridiales in poor outcomes Hepatic Increase Alcaligenaceae, Porphyromonadaceae encephalopathy Veillonella, Megasphaera, Bifidobacterium, Enterococcus, Streptococcus salivarius Decrease Roseburia HCC Increase Escherichia coli, Escherichia-Shigella, Enterococcus, Proteus, Veillonella, Actinobacterium, Gemmiger Decrease Fecalibacterium, Rumonococcus, Ruminoclostridium Pseudobutyrivibrio, Lachnoclostridium Phascolarctobacterium, Parabacteroides Chronic Increase Proteobacteria, Enterococcus hepatitis B Decrease Bifidobacterium, Lactobacillus virus-related cirrhosis Chronic Increase Prevotella, Fecalibacterium hepatitis C Decrease Acinetobacter, Veillonella, Phascolarctobacterium virus-related cirrhosis Autoimmune Increase Veillonella dispar V. dispar associated with elevation of hepatitis Decrease Bifidobacterium, Lactobacillus aspartate aminotransferase levels and severity of autoimmune hepatitis Primary Increase Barnesiellaceae, Lachnospiraceae Additionally, increased proportion of sclerosing Blautia, Escherichia, Ruminococcus, Megasphaera fungi Exophiala and a decreased cholangitis Decrease Uncultured Clostridiales II proportion of Saccharomyces Prevotella, Roseburia, Bacteroides cerevisiae notable in patients with primary sclerosing cholangitis and inflammatory bowel disease Drug-induced Increase in Mucispirillum, Turicibacter and Ruminococcus liver injury associated with higher risk of toxicity to acetaminophen Metabolite 1-phenyl-1,2-propanedione associated with diurnal variation of acetaminophen induced hepatotoxicity Post-liver Higher fecal levels of Klebsiella, Escherichia, Shigella in post- Fecal microbiome index consisting of transplantation transplantation period associated with infections Staphylococcus and Prevotella useful in identifying patients post-liver transplant who develop abnormal liver tests

* Pertinent metabolites associated with prominent bacterial communities in the given liver disease condition Abbreviations: HCC, hepatocellular carcinoma; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. immunoglobulin A produced by infected B-lymphocytes. In The intestinal microbiota influences drug and xenobiotic the GM of Egyptian patients with HCV, higher abundance of metabolisms, that can affect drug efficacy and toxicity. Prevotella and Faecalibacterium and lower levels of Acineto- Microbiota-related drug metabolism is important for activa- bacter, Veillonella and Phascolarctobacterium were notable. tion of some prodrugs.74 The GM also takes part in additional The role of Prevotella or Faecalibacterium to Bifidobacterium metabolic reactions associated with some drugs, such as ace- ratio has been demonstrated as a biomarker for fibrosis pro- tylation, decarboxylation, dihydroxylation and demethyla- gression in HCV-infected patients. However, in patients with tion. Microbiota-derived metabolites can indirectly affect HCV-related cirrhosis, gut dysbiosis can persist, regardless of xenobiotic metabolism pathways. It was demonstrated long-term sustained viral response.73 that the intestinal microbiota modulated susceptibility to

96 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 Philips C.A. et al: Intestinal microbiota reinstitution therapy acetaminophen (APAP)-induced acute liver injury. The rela- PA, SR, RA), critically reviewing and revising the manuscript tive abundance of Mucispirillum, Turicibacter and Ruminococ- (CAP, PA, PKY, GNR, RA, SR, TG, SK). cus before APAP dosing was found to be associated with increased hepatotoxicity.75 APAP-induced liver injury has diurnal variation. APAP causes more hepatotoxicity during consumption at night compared to References morning. It was demonstrated that the gut microbial metabo- [1] Gutleben J, Chaib De Mares M, van Elsas JD, Smidt H, Overmann J, Sipkema D. lite, 1-phenyl-1,2-propanedione was involved in the rhythmic The multi-omics promise in context: from sequence to microbial isolate. Crit hepatotoxicity induced by APAP, by depleting hepatic gluta- Rev Microbiol 2018;44:212–229. doi: 10.1080/1040841X.2017.1332003. thione levels. The anti-inflammatory drug, salicylazosulfapyr- [2] Zuñiga C, Zaramela L, Zengler K. Elucidation of complexity and prediction of – idine, underwent degradation in conventional rats and when interactions in microbial communities. Microb Biotechnol 2017;10:1500 1522. doi: 10.1111/1751-7915.12855. cultured with human gut bacteria but not in germ-free rats, [3] Fondi M, Liò P. Multi-omics and metabolic modelling pipelines: challenges and demonstrating the role of GM in drug transformations. Wang tools for systems microbiology. Microbiol Res 2015;171:52–64. doi: 10. et al.76 showed that healthy donor FMT prevented HE in rats 1016/j.micres.2015.01.003. with carbon tetrachloride-induced acute liver failure. [4] Abram F. Systems-based approaches to unravel multi-species microbial community functioning. Comput Struct Biotechnol J 2014;13:24–32. doi: 10. The intestinal microbiota has been shown to play a central 1016/j.csbj.2014.11.009. role in sensitization of sepsis-induced liver injury, and micro- [5] Cani PD. Human gut microbiome: hopes, threats and promises. Gut 2018; biota-associated granisetron production resulted in amelio- 67:1716–1725. doi: 10.1136/gutjnl-2018-316723. ration of liver injury during sepsis development in a mouse [6] Mohajeri MH, Brummer RJM, Rastall RA, Weersma RK, Harmsen HJM, Faas M, 77 et al. The role of the microbiome for human health: from basic science to model of cecal ligation puncture. clinical applications. Eur J Nutr 2018;57:1–14. doi: 10.1007/s00394-018- A recent case series showcased beneficial outcomes with 1703-4. FMT-related reconstitution of the gut microbiome on immune [7] Goodrich JK, Davenport ER, Beaumont M, Jackson MA, Knight R, Ober C, et al. Genetic determinants of the gut microbiome in UK twins. Cell Host checkpoint inhibitors in colitis associated with a relative – 78 Microbe 2016;19:731 743. doi: 10.1016/j.chom.2016.04.017. increase in regulatory T-cells within the colonic mucosa. [8] Liang D, Leung RK, Guan W, Au WW. Involvement of gut microbiome in 79 Lu et al. showed that there was a higher relative abun- human health and disease: brief overview, knowledge gaps and research dance of Klebsiella, Escherichia and Shigella and reduced opportunities. Gut Pathog 2018;10:3. doi: 10.1186/s13099-018-0230-4. levels of beneficial butyrate-producing bacteria among liver [9] Dieterich W, Schink M, Zopf Y. Microbiota in the gastrointestinal tract. Med Sci (Basel) 2018;6:116. doi: 10.3390/medsci6040116. transplant recipients. The authors established a fecal micro- [10] Dominguez-Bello MG, Godoy-Vitorino F, Knight R, Blaser MJ. Role of the biome index (specific alterations in Staphylococcus and Pre- microbiome in human development. Gut 2019;68:1108–1114. doi: 10. votella) that could be used for assessing liver recipients at risk 1136/gutjnl-2018-317503. of liver dysfunction. A summary of pertinent GM association [11] Gordo I. Evolutionary change in the human gut microbiome: From a static to a dynamic view. PLoS Biol 2019;17:e3000126. doi: 10.1371/journal.pbio. with various liver diseases is shown in Table 1. 3000126. [12] Thursby E, Juge N. Introduction to the human gut microbiota. Biochem J Conclusions 2017;474:1823–1836. doi: 10.1042/BCJ20160510. [13] Yang JY, Kweon MN. The gut microbiota: a key regulator of metabolic diseases. BMB Rep 2016;49:536–541. doi: 10.5483/bmbrep.2016.49.10.144. GM plays a central role in the initiation and progression of [14] Gomes AC, Hoffmann C, Mota JF. The human gut microbiota: Metabolism certain liver diseases and changes in GM drive the patho- and perspective in obesity. Gut Microbes 2018;9:308–325. doi: 10. physiology of select liver diseases. There are robust data on 1080/19490976.2018.1465157. the role of GM in the development of NAFLD and NASH, severe [15] Lamichhane S, Sen P, Dickens AM, Oresic M, Bertram HC. Gut metabolome meets microbiome: A methodological perspective to understand the rela- AH, complications of cirrhosis and especially HE and PSC. tionship between host and microbe. Methods 2018;149:3–12. doi: 10. High-quality studies have also shown specific roles played by 1016/j.ymeth.2018.04.029. the GM in the progression of diseases such as drug-induced [16] Nagpal R, Kumar M, Yadav AK, Hemalatha R, Yadav H, Marotta F, et al. Gut liver injury, AIH and chronic viral hepatitis. The benefits of microbiota in health and disease: an overview focused on metabolic inflammation. Benef Microbes 2016;7:181–194. doi: 10.3920/bm2015.0062. therapeutic modulation of GM in liver diseases such as AH, HE [17] Boulangé CL, Neves AL, Chilloux J, Nicholson JK, Dumas ME. Impact of the and PSC are well documented but high-quality randomized gut microbiota on inflammation, obesity, and metabolic disease. Genome trials are lacking. The future of liver disease management Med 2016;8:42. doi: 10.1186/s13073-016-0303-2. could well include microbiota modulation based on the high- [18] Janssen AW, Kersten S. Potential mediators linking gut bacteria to metabolic health: a critical view. J Physiol 2017;595:477–487. doi: 10.1113/JP272476. quality bench-to-bedside research in the coming years. [19] Ghazalpour A, Cespedes I, Bennett BJ, Allayee H. Expanding role of gut microbiota in lipid metabolism. Curr Opin Lipidol 2016;27:141–147. doi: Funding 10.1097/MOL.0000000000000278. [20] Diether NE, Willing BP. Microbial fermentation of dietary protein: An impor- None to declare. tant factor in diet-microbe-host interaction. Microorganisms 2019;7:19. doi: 10.3390/microorganisms7010019. [21] Bik EM, Ugalde JA, Cousins J, Goddard AD, Richman J, Apte ZS. Microbial biotransformations in the human distal gut. Br J Pharmacol 2018;175:4404– Conflict of interest 4414. doi: 10.1111/bph.14085. [22] Madsen L, Myrmel LS, Fjære E, Liaset B, Kristiansen K. Links between dietary protein sources, the gut microbiota, and obesity. Front Physiol 2017;8:1047. CAP receives advisory fees and research grant support from doi: 10.3389/fphys.2017.01047. ® ® Cipla and Samarth Lifesciences . The other authors have no [23] Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Metabolic conflict of interests related to this publication. endotoxemia initiates obesity and insulin resistance. Diabetes 2007;56: 1761–1772. doi: 10.2337/db06-1491. [24] Giannelli V, Di Gregorio V, Iebba V, Giusto M, Schippa S, Merli M, et al. Micro- Author contributions biota and the gut-liver axis: bacterial translocation, inflammation and infection in cirrhosis. World J Gastroenterol 2014;20:16795–16810. doi: 10. 3748/wjg.v20.i45.16795. Designing the research study (CAP, PA, PKY, GNR), collecting [25] Fukui H. Gut-liver axis in liver cirrhosis: How to manage leaky gut and endo- the data (CAP, RA, SR, TG, SK), writing the manuscript (CAP, toxemia. World J Hepatol 2015;7:425–442. doi: 10.4254/wjh.v7.i3.425.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 87–99 97 Philips C.A. et al: Intestinal microbiota reinstitution therapy

[26] Usami M, Miyoshi M, Yamashita H. Gut microbiota and host metabolism in alcoholic liver disease. Gut 2018;67:891–901. doi: 10.1136/gutjnl-2016- liver cirrhosis. World J Gastroenterol 2015;21:11597–11608. doi: 10. 313432. 3748/wjg.v21.i41.11597. [51] Ciocan D, Voican CS, Wrzosek L, Hugot C, Rainteau D, Humbert L, et al. Bile [27] Patel VC, White H, Støy S, Bajaj JS, Shawcross DL. Clinical science work- acid homeostasis and intestinal dysbiosis in alcoholic hepatitis. Aliment Phar- shop: targeting the gut-liver-brain axis. Metab Brain Dis 2016;31:1327– macol Ther 2018;48:961–974. doi: 10.1111/apt.14949. 1337. doi: 10.1007/s11011-015-9743-4. [52] Puri P, Liangpunsakul S, Christensen JE, Shah VH, Kamath PS, Gores GJ, [28] Wiest R, Albillos A, Trauner M, Bajaj JS, Jalan R. Targeting the gut-liver axis in et al. The circulating microbiome signature and inferred functional metage- liver disease. J Hepatol 2017;67:1084–1103. doi: 10.1016/j.jhep.2017.05. nomics in alcoholic hepatitis. Hepatology 2018;67:1284–1302. doi: 10. 007. 1002/hep.29623. [29] Le Roy T, Llopis M, Lepage P, Bruneau A, Rabot S, Bevilacqua C, et al. Intes- [53] Bluemel S, Wang L, Kuelbs C, Moncera K, Torralba M, Singh H, et al. Intes- tinal microbiota determines development of non-alcoholic fatty liver disease tinal and hepatic microbiota changes associated with chronic ethanol admin- in mice. Gut 2013;62:1787–1794. doi: 10.1136/gutjnl-2012-303816. istration in mice. Gut Microbes 2019. doi: 10.1080/19490976.2019. [30] Tripathi A, Debelius J, Brenner DA, Karin M, Loomba R, Schnabl B, et al. The 1595300. gut-liver axis and the intersection with the microbiome. Nat Rev Gastroen- [54] Kirpich IA, Solovieva NV, Leikhter SN, Shidakova NA, Lebedeva OV, Sidorov PI, terol Hepatol 2018;15:397–411. doi: 10.1038/s41575-018-0011-z. et al. Probiotics restore bowel flora and improve liver enzymes in human [31] de Faria Ghetti F, Oliveira DG, de Oliveira JM, de Castro Ferreira LEVV, Cesar alcohol-induced liver injury: a pilot study. Alcohol 2008;42:675–682. doi: DE, Moreira APB. Influence of gut microbiota on the development and pro- 10.1016/j.alcohol.2008.08.006. gression of nonalcoholic steatohepatitis. Eur J Nutr 2018;57:861–876. doi: [55] Han SH, Suk KT, Kim DJ, Kim MY, Baik SK, Kim YD, et al. Effects of probiotics 10.1007/s00394-017-1524-x. (cultured Lactobacillus subtilis/Streptococcus faecium) in the treatment [32] de Groot PF, Frissen MN, de Clercq NC, Nieuwdorp M. Fecal microbiota trans- of alcoholic hepatitis: randomized-controlled multicenter study. Eur J Gastro- – plantation in metabolic syndrome: History, present and future. Gut Microbes enterol Hepatol 2015;27:1300 1306. doi: 10.1097/MEG.0000000000000458. 2017;8:253–267. doi: 10.1080/19490976.2017.1293224. [56] Philips CA, Pande A, Shasthry SM, Jamwal KD, Khillan V, Chandel SS, et al. [33] Behrouz V, Jazayeri S, Aryaeian N, Zahedi MJ, Hosseini F. Effects of probiotic Healthy donor fecal microbiota transplantation in steroid-ineligible severe – and prebiotic supplementation on leptin, adiponectin, and glycemic param- alcoholic hepatitis: A pilot study. Clin Gastroenterol Hepatol 2017;15:600 eters in non-alcoholic fatty liver disease: A randomized clinical trial. Middle 602. doi: 10.1016/j.cgh.2016.10.029. East J Dig Dis 2017;9:150–157. doi: 10.15171/mejdd.2017.66. [57] Philips CA, Phadke N, Ganesan K, Ranade S, Augustine P. Corticosteroids, [34] Kolodziejczyk AA, Zheng D, Shibolet O, Elinav E. The role of the microbiome nutrition, pentoxifylline, or fecal microbiota transplantation for severe alco- – in NAFLD and NASH. EMBO Mol Med 2019;11:e9302. doi: 10.15252/emmm. holic hepatitis. Indian J Gastroenterol 2018;37:215 225. doi: 10. 1007/s12664-018-0859-4. 201809302. [58] Fukui H. Gut microbiome-based therapeutics in liver cirrhosis: Basic consid- [35] Lynch SV, Pedersen O. The human intestinal microbiome in health and eration for the next step. J Clin Transl Hepatol 2017;5:249–260. doi: 10. disease. N Engl J Med 2016;375:2369–2379. doi: 10.1056/NEJMra1600266. 14218/JCTH.2017.00008. [36] Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, et al. [59] Hartmann P, Chu H, Duan Y, Schnabl B. Gut microbiota in liver disease: too The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell much is harmful, nothing at all is not helpful either. Am J Physiol Gastrointest homeostasis. Science 2013;341:569–573. doi: 10.1126/science.1241165. Liver Physiol 2019;316:G563–G573. doi: 10.1152/ajpgi.00370.2018. [37] Cammarota G, Ianiro G, Tilg H, Rajilic-Stojanovi c M, Kump P, Satokari R, [60] Bajaj JS. The role of microbiota in hepatic encephalopathy. Gut Microbes et al. European consensus conference on faecal microbiota transplantation 2014;5:397–403. doi: 10.4161/gmic.28684. in clinical practice. Gut 2017;66:569–580. doi: 10.1136/gutjnl-2016- [61] Bajaj JS, Vargas HE, Reddy KR, Lai JC, O’Leary JG, Tandon P, et al. Associa- 313017. tion between intestinal microbiota collected at hospital admission and [38] Dong TS, Jacobs JP. Nonalcoholic fatty liver disease and the gut microbiome: outcomes of patients with cirrhosis. Clin Gastroenterol Hepatol 2019;17: Are bacteria responsible for fatty liver? Exp Biol Med (Maywood) 2019;244: 756–765.e3. doi: 10.1016/j.cgh.2018.07.022. 408–418. doi: 10.1177/1535370219836739. [62] Bajaj JS, Kassam Z, Fagan A, Gavis EA, Liu E, Cox IJ, et al. Fecal microbiota [39] Sharpton SR, Ajmera V, Loomba R. Emerging role of the gut microbiome in transplant from a rational stool donor improves hepatic encephalopathy: A nonalcoholic fatty liver disease: From composition to function. Clin Gastro- randomized clinical trial. Hepatology 2017;66:1727–1738. doi: 10. enterol Hepatol 2019;17:296–306. doi: 10.1016/j.cgh.2018.08.065. 1002/hep.29306. [40] Sáez-Lara MJ, Robles-Sanchez C, Ruiz-Ojeda FJ, Plaza-Diaz J, Gil A. Effects [63] Bajaj JS, Salzman NH, Acharya C, Sterling RK, White MB, Gavis EA, et al. of probiotics and synbiotics on obesity, insulin resistance syndrome, type 2 Fecal microbial transplant capsules are safe in hepatic encephalopathy: A diabetes and non-alcoholic fatty liver disease: A review of human clinical phase 1, randomized, placebo-controlled trial. Hepatology 2019;70:1690– trials. Int J Mol Sci 2016;17:928. doi: 10.3390/ijms17060928. 1703. doi: 10.1002/hep.30690. [41] Ma YY, Li L, Yu CH, Shen Z, Chen LH, Li YM. Effects of probiotics on non- [64] Sanduzzi Zamparelli M, Rocco A, Compare D, Nardone G. The gut micro- alcoholic fatty liver disease: a meta-analysis. World J Gastroenterol 2013; biota: A new potential driving force in liver cirrhosis and hepatocellular – 19:6911 6918. doi: 10.3748/wjg.v19.i40.6911. carcinoma. United European Gastroenterol J 2017;5:944–953. doi: 10. [42] Khoruts A, Brandt LJ. Fecal microbiota transplant: A rose by any other name. 1177/2050640617705576. Am J Gastroenterol 2019;114:1176. doi: 10.14309/ajg.0000000000000286. [65] Wang L, Wan YJY. The role of gut microbiota in liver disease development and [43] Bajaj JS, Hays RA. Manipulation of the gut-liver axis using microbiome resto- treatment. Liver Research 2019;3:3–18. doi: 10.1016/j.livres.2019.02.001. ration therapy in primary sclerosing cholangitis. Am J Gastroenterol 2019; [66] Czaja AJ. Factoring the intestinal microbiome into the pathogenesis of auto- – 114:1027 1029. doi: 10.14309/ajg.0000000000000191. immune hepatitis. World J Gastroenterol 2016;22:9257–9278. doi: 10. [44] Zhou D, Pan Q, Shen F, Cao HX, Ding WJ, Chen YW, et al. Total fecal micro- 3748/wjg.v22.i42.9257. biota transplantation alleviates high-fat diet-induced steatohepatitis in mice [67] Wei Y, Li Y, Yan L, Sun C, Miao Q, Wang Q, et al. Alterations of gut microbiome via beneficial regulation of gut microbiota. Sci Rep 2017;7:1529. doi: 10. in autoimmune hepatitis. Gut 2019. doi: 10.1136/gutjnl-2018-317836. 1038/s41598-017-01751-y. [68] Hov JR, Kummen M. Intestinal microbiota in primary sclerosing cholangitis. Curr [45] Vrieze A, Van Nood E, Holleman F, Salojärvi J, Kootte RS, Bartelsman JF, et al. Opin Gastroenterol 2017;33:85–92. doi: 10.1097/MOG.0000000000000334. Transfer of intestinal microbiota from lean donors increases insulin sensitiv- [69] Philips CA, Augustine P, Phadke N. Healthy donor fecal microbiota transplan- ity in individuals with metabolic syndrome. Gastroenterology 2012;143: tation for recurrent bacterial cholangitis in primary sclerosing cholangitis - A 913–916.e7. doi: 10.1053/j.gastro.2012.06.031. single case report. J Clin Transl Hepatol 2018;6:438–441. doi: 10. [46] Xue LF, Luo WH, Wu LH, He XX, Xia HHX, Chen Y. Fecal microbiota trans- 14218/JCTH.2018.00033. plantation for the treatment of nonalcoholic fatty liver disease. Explor Res [70] Allegretti JR, Kassam Z, Carrellas M, Mullish BH, Marchesi JR, Pechlivanis A, Hypothesis Med 2019;4:12. doi: 10.14218/ERHM.2018.00025. et al. Fecal microbiota transplantation in patients with primary sclerosing [47] Llopis M, Cassard AM, Wrzosek L, Boschat L, Bruneau A, Ferrere G, et al. cholangitis: A pilot clinical trial. Am J Gastroenterol 2019;114:1071–1079. Intestinal microbiota contributes to individual susceptibility to alcoholic doi: 10.14309/ajg.0000000000000115. liver disease. Gut 2016;65:830–839. doi: 10.1136/gutjnl-2015-310585. [71] Kang Y, Cai Y. Gut microbiota and hepatitis-B-virus-induced chronic liver [48] Philips CA, Pande A, Shasthry SM, Jamwal KD, Khillan V, Chandel SS, et al. disease: implications for faecal microbiota transplantation therapy. J Hosp Healthy donor fecal microbiota transplantation in steroid-ineligible severe Infect 2017;96:342–348. doi: 10.1016/j.jhin.2017.04.007. alcoholic hepatitis: A pilot study. Clin Gastroenterol Hepatol 2017;15:600– [72] Ren YD, Ye ZS, Yang LZ, Jin LX, Wei WJ, Deng YY, et al. Fecal microbiota 602. doi: 10.1016/j.cgh.2016.10.029. transplantation induces hepatitis B virus e-antigen (HBeAg) clearance in [49] Scarpellini E, Forlino M, Lupo M, Rasetti C, Fava G, Abenavoli L, et al. Gut patients with positive HBeAg after long-term antiviral therapy. Hepatology microbiota and alcoholic liver disease. Rev Recent Clin Trials 2016;11:213– 2017;65:1765–1768. doi: 10.1002/hep.29008. 219. doi: 10.2174/1574887111666160810100538. [73] Aly AM, Adel A, El-Gendy AO, Essam TM, Aziz RK. Gut microbiome alterations [50] Grander C, Adolph TE, Wieser V, Lowe P, Wrzosek L, Gyongyosi B, et al. in patients with stage 4 hepatitis C. Gut Pathog 2016;8:42. doi: 10. Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates 1186/s13099-016-0124-2.

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[74] Wilson ID, Nicholson JK. Gut microbiome interactions with drug metabolism, [77] Gong S, Yan Z, Liu Z, Niu M, Fang H, Li N, et al. Intestinal microbiota mediates efficacy, and toxicity. Transl Res 2017;179:204–222. doi: 10.1016/j.trsl. the susceptibility to polymicrobial sepsis-induced liver injury by granisetron gen- 2016.08.002. eration in mice. Hepatology 2019;69:1751–1767. doi: 10.1002/hep.30361. [75] Gong S, Lan T, Zeng L, Luo H, Yang X, Li N, et al. Gut microbiota mediates [78] Wang Y, Wiesnoski DH, Helmink BA, Gopalakrishnan V, Choi K, DuPont HL, diurnal variation of acetaminophen induced acute liver injury in mice. J et al. Fecal microbiota transplantation for refractory immune checkpoint Hepatol 2018;69:51–59. doi: 10.1016/j.jhep.2018.02.024. inhibitor-associated colitis. Nat Med 2018;24:1804–1808. doi: 10.1038/s41591- [76] Wang WW, Zhang Y, Huang XB, You N, Zheng L, Li J. Fecal microbiota trans- 018-0238-9. plantation prevents hepatic encephalopathy in rats with carbon tetrachlor- [79] Lu HF, Ren ZG, Li A, Zhang H, Xu SY, Jiang JW, et al. Fecal microbiome data ide-induced acute hepatic dysfunction. World J Gastroenterol 2017;23: distinguish liver recipients with normal and abnormal liver function from healthy 6983–6994. doi: 10.3748/wjg.v23.i38.6983. controls. Front Microbiol 2019;10:1518. doi: 10.3389/fmicb.2019.01518.

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Repurposing Pirfenidone for Nonalcoholic Steatohepatitis-related Cirrhosis: A Case Series

Cyriac Abby Philips*1, Guruprasad Padsalgi2, Rizwan Ahamed2, Rajaguru Paramaguru3, Sasidharan Rajesh4, Tom George4, Pushpa Mahadevan5 and Philip Augustine2

1The Liver Unit and Monarch Liver Lab, Cochin Gastroenterology Group, Ernakulam Medical Centre, Kochi, Kerala, India; 2Department of Gastroenterology, Cochin Gastroenterology Group, Ernakulam Medical Centre, Kochi, Kerala, India; 3Department of Pathology, Al Salam International Hospital, Bneid Al Qar, Kuwait, India; 4Department of Diagnostic and Interventional Radiology, Department of Gastroenterology and The Liver Unit, Cochin Gastroenterology Group, Ernakulam Medical Centre, Kochi, Kerala, India; 5Department of Clinical Pathology, VPS Lakeshore Hospital, Kochi, Kerala, India

Abstract female with hypothyroidism and family history of diabetes mellitus, fatty liver disease and dyslipidemia. Patient-3 is a We repurposed the antifibrotic drug pirfenidone—which is 58 year-old obese female with dyslipidemia, diabetes melli- approved for treatment of idiopathic lung fibrosis—in a series tus, hypothyroidism and systemic hypertension, with family of patients with nonalcoholic steatohepatitis-related cirrhosis. history of fatty liver disease and coronary artery disease. All Our report demonstrates the observed improvements in three patients had biopsy-proven NASH cirrhosis (percuta- necroinflammation and regression of cirrhosis with pirfeni- neous, right lobe only, 16-gauge cutting needle, minimum of done use for 12-weeks, associated with classical hepatic five un-fragmented cores, each with minimum length of repair complex features on follow-up liver biopsies. This novel 20mm and with at least 10 identifiable portal tracts) in the work could help stimulate further randomized trials of pirfe- absence of portal hypertension. Other causes for chronic liver nidone in patients with nonalcoholic steatohepatitis-related disease, including chronic viral hepatitis, alcoholic liver liver fibrosis or cirrhosis, for whom no recommended drug disease, Wilson’s disease, autoimmune hepatitis, chronic treatments exists currently. cholestatic liver disease and hemochromatosis, were explic- Citation of this article: Philips CA, Padsalgi G, Ahamed R, itly ruled out as per standard diagnostic recommendations. Paramaguru R, Rajesh S, George T, et al. Repurposing pirfe- All three patients were started on PFD (Pirfenex®;Cipla, nidone for nonalcoholic steatohepatitis-related cirrhosis: A India) at 200mg thrice daily for 12 weeks. All underwent case series. J Clin Transl Hepatol 2020;8(1):100–105. doi: Shearwave elastography (LOGIQ E9; GE-Healthcare, USA) as 10.14218/JCTH.2019.00056. per manufacturer’s guideline recommendation, with at least 4 h of fasting prior to the procedure at baseline and after Introduction 12 weeks post-therapy. The right lobe of the liver was chosen for stiffness measurement in all patients, with 10 read- Regression of cirrhosis has been demonstrated with antiviral ings taken by the same radiologist; the final result was gener- therapy in patients with chronic hepatitis B and hepatitis C ated by the system software as an average (kPa). Liver virus infections and with immunosuppressants in autoim- function tests were performed once weekly for the first mune hepatitis.1 Pharmacological therapies leading to regres- 4 weeks and once monthly thereafter. Patients were monitored sion of nonalcoholic steatohepatitis (NASH)-related cirrhosis for adverse events through monthly telephonic interview. have not been demonstrated. We present here proof-of- Percutaneous liver biopsy was performed in all three concept in regression of NASH-cirrhosis, through serial liver patients at the end of 12 weeks (in view of liver stiffness biopsies, achieved by repurposing the antifibrotic drug pirfe- measurement changes noted at this arbitrary time point) and nidone (PFD) in three patients. compared to baseline. Two trained liver pathologists who were blinded to the treatment but not to the patients assessed the liver biopsies, working independently. Informed Case report consent was obtained from all patients included in the study, prior to the start of PFD therapy and before baseline and Patient-1 is a 30 year-old overweight male, with occasional follow-up liver biopsy procedures. The study was approved by alcohol intake, abstinent for 18 months after diagnosis of the Institutional Ethics and Review Committee and all proto- chronic liver disease, and with a family history of diabetes cols conformed to the ethical guidelines as laid out by the mellitus and cirrhosis. Patient-2 is a 49 year-old overweight Declaration of Helsinki and its latest amendments. The complete patient details at baseline and end of 12 weeks Keywords: NASH; Fibrosis; Antifibrotic; Hepatitis; Pirfenidone. are shown in Table 1. Abbreviations: NASH, nonalcoholic steatohepatitis; PFD, pirfenidone. Received: 28 November 2019; Revised: 4 January 2020; Accepted: 17 January 2020 Results *Correspondence to: Cyriac Abby Philips, Philips Augustine Associates, The Liver Unit and Monarch Liver Lab, Cochin Gastroenterology Group, Erna- kulam Medical Centre, Palarivattom, Cochin 682025, Kerala, India. E-mail: Post-treatment, at the end of 12 weeks, in the absence of [email protected] intentional weight loss (exercise regimen- and dietary

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Table 1. Baseline and follow-up parameters of all three patients with nonalcoholic fatty liver disease-related cirrhosis on pirfenidone treatment

Parameters Patient-1 Patient-2 Patient-3

Age in years 30 49 58 Sex Male Female Female Height in cm 168 154 152 Weight in kg, at baseline / 12 weeks 79 / 78 72 / 73 81 / 79 Body mass index in kg/m2, at baseline / 28 / 27.6 30.4 / 30.8 35.1 / 34.2 12 weeks Associated chronic diseases None Hypothyroidism Dyslipidemia, diabetes mellitus, hypothyroidism, systemic hypertension Associated drug intake, dose (duration) None Thyroxine 75 Rosuvastatin 10 mg (10 years), metformin mcg once daily 1000 mg (10 years), glimepiride 1 mg (14 years) (4 years), thyroxine 50 mcg (8 years), metoprolol 50 mg (3 years) Hemoglobin in g/L, at baseline / 12 weeks 12.2 / 11.8 13.4 / 13 11.8 / 12.1 Total leucocyte count as 3109/L, at baseline / 5.6 / 6.4 7.2 / 6.7 6.2 / 7.0 12 weeks Platelet count as 3103/mL, at baseline / 220 / 230 210 / 194 260 / 272 12 weeks Total bilirubin in mg/dL, at baseline / 0.8 / 1.1 1.2 / 0.9 1.2 / 0.9 12 weeks Direct bilirubin in mg/dL, at baseline / 0.4 / 0.6 0.8 / 0.4 0.3 / 0.4 12 weeks Aspartate transaminase in IU/L, at baseline / 54 / 48 62 / 60 78 / 56 12 weeks Alanine transaminase in IU/L, at baseline / 112 / 74 98 / 78 102 / 44 12 weeks Alkaline phosphatase in IU/L, at baseline / 98 / 88 84 / 92 78 / 92 12 weeks g-Glutamyl transpeptidase in IU/L, at 42 / 54 38 / 72 62 / 44 baseline / 12 weeks Serum albumin in g/dL, at baseline / 4.2 / 4.1 4.4 / 4.1 4.2 / 3.9 12 weeks Serum creatinine in mg/dL, at baseline / 0.8 / 1.1 0.9 / 0.6 1.0 / 0.9 12 weeks Serum sodium in mmol/L, at baseline / 141 / 140 139 / 140 138 / 136 12 weeks Serum potassium in mmol/L, at baseline / 4.2 / 3.8 4.1 / 4.0 4.5 / 4.8 12 weeks International normalized ratio, at baseline / 1.1 / 0.9 1 / 1.2 1.2 / 1.2 12 weeks HbA1c, at baseline / 12 weeks 5.6 / 6.1 6.1 / 5.9 7.4 / 7.8 APRI score, at baseline / 12 weeks1*0.61/0.78 / 0.77 0.75 / 0.51 0.52 FIB4 score, at baseline / 12 weeks2* 0.7 / 0.73 1.46 / 1.72 1.72 / 1.8 Shearwave elastography in kPA, at baseline / 22.4 / 7.8 18.6 / 9.8 9.4 / 10.2 12 weeks NAFLD activity score (known as NAS) 5 / 2 5 / 3 5 / 4 Steatosis-Activity-Fibrosis (known as SAF) S2A2F4 / S2A2F4 / S1A4F4 / S0A2F3 score, at baseline / 12 weeks S0A0F3 S0A1F4

* Noninvasive scores like the aspartate aminotransferase-to-platelet ratio index (APRI) and the Fibrosis-4 score (FIB4) have been studied and fully validated in patients with chronic hepatitis C and in patients with hepatitis C and human immunodeficiency virus-related coinfection and not in patients with NASH-cirrhosis, and have low diagnostic accuracy when used alone.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 100–105 101 Philips C.A. et al: Pirfenidone for NASH fibrosis restriction-based), substantial reduction in liver elastography studies on the anti-inflammatory and antifibrotic effects of values were notable in two patients and a reduction in alanine PFD in small animal models of bleomycin-induced lung fibrosis, transaminase was notable in all three patients. The liver cardiac, renal and hepatic fibrosis, and allergen-induced biopsy evaluation revealed amelioration in steatosis as well airway-remodeling have been published.2 PFD exerts multifac- as inflammation, associated with features of cirrhosis regres- eted actions on inflammatory and fibrosis mediators to sion, such as thinning and perforation of fibrous septae, improve inhibitory effects on multiple pathways, which ulti- presence of isolated thick collagen fibers, delicate periportal mately lead to liver fibrosis (Fig. 2). spikes, clusters of hepatocytes within portal tracts, splitting of PFD was shown to reduce hepatocyte apoptosis and tumor septa by hepatocytes and loss of distinction of hepatic necrosis factor-a-related fibrogenesis and to markedly nodules; components of the ‘hepatic repair complex’ were attenuate liver fibrosis in a rodent model of human NASH.3 1 observed, in varying degrees, in all three patients (Fig. 1). It was also recently demonstrated that prolonged release PFD The complete representational images of pre- and post-treat- in an animal model of NASH led to increase in the peroxisome ment liver biopsies of all three patients are shown in the Sup- proliferator-activated receptors-g and -a and liver - X receptor- – plementary Figs. 1 6. No adverse events were reported. a-related metabolic transcriptional factors, causing a reduc- However, Patient-1 and Patient-3 reported unintentional tion in steatosis, and down-regulation of transforming growth weight loss (of approximately 2.2 kg at 12 weeks) in the factor-b1, nuclear factor-kB and interluekins-1, -6 and -17A absence of dietary restrictions, anorexia and exercise eliciting antifibrotic effects.4 In a pilot trial, patients with regimen. chronic hepatitis C virus-related advanced liver fibrosis were given 1200mg/day of PFD for 1 year. The authors found that Discussion there was marked reduction in necrosis, inflammation and steatosis on follow-up biopsy, along with liver cell regenera- PFD is an orally bioavailable pyridone derivative, approved for tion (measured by antiproliferating cell nuclear antigen the treatment of idiopathic pulmonary fibrosis. Oral PFD is immunostaining). Fibrosis was reduced in 30% of patients rapidly absorbed, reaching maximal concentration at 30 min at the end of 1 year and mRNAs coding for profibrogenic mol- in fasted, older adults, having a terminal half-life of 2.5 h. PFD ecules, such as collagen type I a-1, transforming growth is primarily metabolized through the cytochrome P450 enzyme factor-b1 and tissue inhibitors of metalloproteinases, were CYP1A2 and excreted through urine. Several high-quality markedly down-regulated by the end of treatment.5

Fig. 1. Percutaneous liver biopsy features showing various components of the hepatic repair complex in patients with nonalcoholic steatohepatitis-related cirrhosis after a 12-week course of pirfenidone. (A) Loss of distinct nodularity and absence of steatosis (black arrows; H&E stain, 40x). (B) Thinning of fibrous septa with delicate periportal spikes (arrows; MTS, 100x). (C) Splitting of thinned out fibrous septa by clusters of hepatocytes (black arrow; MTS, 100x), with isolated dense bands of collagen (yellow arrow). (D) Pale staining of edematous resorptive septa, with loss of fibrous appearance (red arrow; MTS, 100x). (D, E) Thinned and perforated fibrous septae (black arrows; MTS, 100x). (E, F) Clusters of hepatocytes and hepatocyte buds within areas of fibrosis regression (E, yellow arrow and F, black arrows; MTS, 100x). H&E, hematoxylin and eosin stain; MTS, Masson’s trichrome stain.

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Fig. 2. Multiple anti-inflammatory and antifibrotic effects of pirfenidone on advanced liver disease confirmed in studies based on cell culture and small animal models. Abbreviations: Cyt-C, cytochrome C; ICAM, intercellular adhesion molecule 1; IL, interleukin; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PPAR, peroxisome proliferator-activated receptors; TNF, tumor necrosis factor; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases.

Angulo et al.6 studied the utility of PFD (2400 mg/day for 12 related advanced liver disease. However, even though not months) in patients with primary sclerosing cholangitis. They very conclusive, our findings are strong enough to stimulate found no significant changes in liver biochemistries, Mayo risk further studies on PFD in advanced NASH-fibrosis/cirrhosis. It score, degree of inflammation, fibrosis nor histologic changes may be argued that the reduction in fibrosis observed in our in the treated patients at end of 1 year but found adverse study could be due to sampling error, but other features of the events (gastrointestinal symptoms and skin rash) in 83% hepatic repair complex were strikingly evident in all our (which disappeared shortly after stopping PFD) of treated treated patients, being highly suggestive of PFD-related patients. This study, however, used a very high dose of PFD regression of cirrhosis independent of weight loss or dietary and overlooked cholangitis episodes and other factors that restrictions. Drug-induced weight loss is a known event with promoted worsening of primary sclerosing cholangitis. A crit- PFD use, and this in itself could have caused reversal features ical review on effects of PFD in animal models of liver fibrosis of cirrhosis, independent of a direct drug effect.9 and clinical trials in humans is shown in Table 2. Verma et al.10 demonstrated acute liver failure due to PFD To date, there are no drug treatments that promote regres- in an elderly male, aged 77 years, with idiopathic pulmonary sion of NASH-cirrhosis. Glass et al.7 has shown reversal of fibrosis and multiple comorbidities, including Parkinson’s advanced NASH fibrosis in patients who lost $10% total body disease. This single report, however, did not consider drug- weight through bariatric and nonsurgical methods. However, drug interactions of PFD with other medications ingested by achieving such targets through rigorous exercise (the ideal the patient that could have potentiated the liver injury, as was dose yet to be defined) or through surgery may not be univer- shown in a report by Benesic et al.11 (also in an elderly male, sally possible or acceptable. Of the 47 drugs that are currently in aged 75 years) which featured the concomitant use of eso- various phases of clinical trials for the treatment of NASH, none meprazole. In our patient, simultaneous use of other medica- are postulated or demonstrated to target reversal of cirrhosis. tions was well documented and adverse events associated Considering the fact that a reasonable number of these potential with PFD was not seen at 3 months. Ideally, Sirius red and therapeutic agents are in very early testing phases, it is safe to smooth muscle actin staining for quantification of collagen assume that a long wait is required before the next generation and the fibrotic area needs to be performed to demonstrate of NASH drugs are available for clinical use. In this regard, drug fibrosis regression; unfortunately, the technology and image repurposing becomes the need of the hour.8 analysis software for such was lacking at our facility. Our report is of a small case series and may not be Future studies assessing fibrosis regression with off-label adequately powered to identify efficacy of PFD in NASH- or experimental drug use for NASH should ideally include

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Table 2. Small animal model and human subject clinical study on efficacy of pirfenidone in liver fibrosis

Author / Year Study Findings Comments

Animal models Tada et al. / Dimethyl nitrosamine rat 40% decrease in fibrotic area 2001 model of fibrosis Significant reduction in liver 500 mg/kg, oral gavage hydroxyproline content Significant reduction in expression of collagen 1 mRNA Garcia et al. Carbon-tetrachloride mouse Pirfenidone use resulted in 70% reduction Increased survival when / 2002 model of cirrhosis in fibrosis staining from baseline when compared to placebo due One group received used after stopping carbon-tetrachloride to less liver-related pirfenidone after Pirfenidone use resulted in 40% reduction events discontinuation of carbon- in fibrosis staining from baseline when tetrachloride used along with carbon-tetrachloride Second group received Total liver hydroxyproline levels reduced pirfenidone along with by 40% carbon-tetrachloride Number of hepatic stellate cells reduced Additional group of bile duct significantly ligation rat model of cirrhosis Di Sario et al. Dimethyl nitrosamine rat 70% reduction in fibrosis staining area / 2004 model of fibrosis/cirrhosis Significant reduction in alanine Pirfenidone (0.5% of liquid transaminase, necroinflammatory score, diet) dosed at weeks 3 to 5 in hepatic stellate cell accumulation a 5-week model Transforming growth factor-beta expression decreased significantly Procollagen-1 mRNA expression decreased Salazar- Carbon-tetrachloride rat Liver fibrosis decreased by 40% Markers of liver injury: Montes et al. model of fibrosis Collagen 1 mRNA expression decreased transaminases and total / 2008 Bile duct ligation rat model of Nitrite and malondialdehyde, superoxide bilirubin decreased cirrhosis dismutase and catalase mRNA levels significantly compared to 200 mg/kg (markers of oxidative stress) reduced in controls Control group – placebo the liver Human studies Angulo et al. Primary sclerosing cholangitis No improvements in necroinflammation, Very high dose of / 2002 n =24 fibrosis, Mayo risk score, bile duct pirfenidone utilized 12-month study duration changes on imaging Ideal dose finding not 2400 mg/day 75% gastrointestinal adverse events, performed 46% severe fatigue, 42% photosensitive Pathophysiology of rash primary sclerosing Discontinuation of pirfenidone in 50% cholangitis not wholly patients targeted Cholangitis episodes, dominant strictures and other clinical events that could worsen primary sclerosing cholangitis not looked into in depth Armendariz- Hepatitis C virus-related Reduction in necroinflammatory scores, Uncontrolled study Borunda advanced fibrosis liver steatosis, fibrosis Associated factors that et al. / 2006 n =15 Collagen 1-A1 protein expression, affected improvement in 12-month study duration transforming growth factor-beta liver fibrosis and 1200 mg/day expression and tumor necrosis factor-a necroinflammation not levels were reduced significantly at end of looked into 1 year Gastrointestinal and photosensitivity in 15% Complete resolution of adverse events after 2 to 3 months of therapy

104 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 100–105 Philips C.A. et al: Pirfenidone for NASH fibrosis quantification analyses with methods/technologies that have PA, PM, RP), drafting of the manuscript (CAP, GP), critical more subjective acceptance and better reproducibility. Other revision of the manuscript for important intellectual content biomarker combinations, such as those obtained by the (CAP, GP, RA, RP, SR, TG, PM, PA). Fibrotest® or Enhanced Liver Fibrosis™ test and ProC3, were also not available at our hospital; regardless, they are not yet validated in Indian patients with advanced NASH-fibrosis. However, the presence of serial liver biopsies (gold References standard) along with the findings from the validated method [1] Hytiroglou P, Theise ND. Regression of human cirrhosis: an update, 18 years for liver stiffness assessment in our patients add to the after the pioneering article by Wanless et al. Virchows Arch 2018;473:15–22. improvement in the objective findings of our study. doi: 10.1007/s00428-018-2340-2. [2] Macías-Barragán J, Sandoval-Rodríguez A, Navarro-Partida J, Armendáriz- Conclusions Borunda J. The multifaceted role of pirfenidone and its novel targets. Fibro- genesis Tissue Repair 2010;3:16. doi: 10.1186/1755-1536-3-16. [3] Komiya C, Tanaka M, Tsuchiya K, Shimazu N, Mori K, Furuke S, et al. Even though our case series of three patients with conclusions Antifibrotic effect of pirfenidone in a mouse model of human nonalcoholic that are based on raw observations require future, prospec- steatohepatitis. Sci Rep 2017;7:44754. doi: 10.1038/srep44754. tive, randomized placebo-controlled trials to confirm our [4] Armendariz-Borunda J, Rodriguez-Echevarria R, Macias-Barragan J, Mendivil- findings related to regression of cirrhosis with use of PFD, Rangel E, Vera-Cruz J, Garcia-Banuelos J. Prolonged-release pirfenidone is a our study has demonstrated a proof-of-concept in regression dual activator for PPARalpha and PPARgamma and improves NASH features induced by high fat/carbohydrate diet. J Hepatol 2017;66:S605. of NASH-cirrhosis with 12-week use of low-dose PFD. This [5] Armendáriz-Borunda J, Islas-Carbajal MC, Meza-García E, Rincón AR, Lucano could stimulate initiation of clinical trials to evaluate the S, Sandoval AS, et al. A pilot study in patients with established advanced liver repurposing of low- or high-dose of PFD given in the inter- fibrosis using pirfenidone. Gut 2006;55:1663–1665. doi: 10.1136/gut.2006. mediate or long term for early and advanced NASH fibrosis, 107136. which could eventually become an important component in [6] Angulo P, MacCarty RL, Sylvestre PB, Jorgensen RA, Wiesner RH, LaRusso NA, et al. Pirfenidone in the treatment of primary sclerosing cholangitis. Dig the armamentarium of treatments against NASH. Dis Sci 2002;47:157–161. doi: 10.1023/a:1013240225965. [7] Glass LM, Dickson RC, Anderson JC, Suriawinata AA, Putra J, Berk BS, et al. Funding Total body weight loss of $ 10 % is associated with improved hepatic fibrosis in patients with nonalcoholic steatohepatitis. Dig Dis Sci 2015;60:1024– None to declare. 1030. doi: 10.1007/s10620-014-3380-3. [8] Sookoian S, Pirola CJ. Repurposing drugs to target nonalcoholic steatohepatitis. World J Gastroenterol 2019;25:1783–1796. doi: 10.3748/wjg.v25.i15. Conflict of interest 1783. [9] Cottin V, Maher T. Long-term clinical and real-world experience with pirfenidone in the treatment of idiopathic pulmonary fibrosis. Eur Respir Rev 2015; The authors have no conflict of interests related to this 24:58–64. doi: 10.1183/09059180.00011514. publication. [10] Verma N, Kumar P, Mitra S, Taneja S, Dhooria S, Das A, et al. Drug idiosyn- crasy due to pirfenidone presenting as acute liver failure: Case report and – Author contributions mini-review of the literature. Hepatol Commun 2017;2:142 147. doi: 10. 1002/hep4.1133. [11] Benesic A, Jalal K, Gerbes AL. Acute liver failure during pirfenidone treatment Study concept and design (CAP, GP), acquisition of data (RA, triggered by co-medication with esomeprazole. Hepatology 2019;70:1869– RP, PM, SR, TG), analysis and interpretation of data (CAP, GP, 1871. doi: 10.1002/hep.30684.

Journal of Clinical and Translational Hepatology 2020 vol. 8 | 100–105 105 Letter to the Editor

Complementary and Alternative Medicine-related Drug-induced Liver Injury in Iran

Mehdi Pasalar1,2, Babak Daneshfard*3 and Kamran Bagheri Lankarani4

1Research Center for Traditional Medicine and History of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; 2Essence of Parsiyan Wisdom Institute, Phytopharmaceutical Technology and Traditional Medicine Incubator, Shiraz University of Medical Sciences, Shiraz, Iran; 3Traditional Medicine Clinical Trial Research Center, Shahed University, Tehran, Iran; 4Health Policy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Citation of this article: Pasalar M, Daneshfard B, Lankarani As mentioned correctly by Philips et al.,1 one of the major KB. Complementary and alternative medicine-related drug- limitations in identifying these side effects is absence of label- induced liver injury in Iran. J Clin Transl Hepatol 2020;8(1): ing that mentions the ingredient and component details of the 106–107. doi: 10.14218/JCTH.2020.00008. remedies. A national survey in Iran revealed that a considerable portion of medicinal plants available in the market are Dear Editor, faced with ambiguous identification, contamination, and adul- We read the interesting review article by Philips et al.1 teration.7 These problems occur mainly in traditional herbal regarding the complementary and alternative medicine shops (Attari), which are also the most common source of (CAM)-related drug-induced liver injury (DILI) in Asian coun- medicinal herb delivery and often present their products in a tries. They have addressed a critical issue in CAM practice disordered and messy set-up (Fig. 1). which has posed a serious challenge to healthcare providers In the past decade, Iran has started a new model of all around the world. promoting evidence-based traditional medicine. Many The authors have vigorously reviewed the evidence from research centers are working on providing evidence for the India and many East Asian countries, however, they did not effectiveness of these remedies.8 Realizing the personal nature comment on the situation in West Asia. Traditional Persian of disease in the philosophy of TPM, the target is to find which medicine (TPM) is one of the oldest comprehensive schools of drug is effective for which patient. Knowing the temperaments medicine, with a history of more than 7000 years which is of the patients is one of the mainstays in this approach. practiced in the Middle East and India widely today. It is a The Ministry of Health and Medical Education in Iran has Hikmat (philosophy)-based holistic medical school based on established a specialized office to promote the use of TPM humoral medicine, including the theory of mizadj (tempera- while supervising the practice and products. The main ment) as well.2 Introducing the genius sages, such as Avi- mission is to provide the remedies with good manufacturing cenna (980–1037 AD)3 and Rhazes (865–925 AD),4 TPM practice and under strict control within the industry (Fig. 2). has played an important role in the progression of medical sciences across the world. There is an observed increasing trend of CAM usage in Asian countries, including West Asian countries and in the Middle East, for various acute and chronic ailments.5,6 This increase in use of traditional medicines, along with widely accepted belief that herbal therapy is totally harmless, has resulted in increasingly greater occurrence of liver injuries caused by these remedies. For instance, in our center, we have faced few hepatotoxicity cases due to inappropriate consumption of borage (Echium amoenum) resulting in hepatic failure and hospital admission. It is worthy of mention that based on the temperamental viewpoint in TPM, many of the hepatotoxic herbs, such as E. amoenum, possess hot and dry nature, which can increase the liver enzymes – even causing liver failure.

Abbreviations: CAM, complementary and alternative medicine; DILI, drug- induced liver injury; TPM, traditional Persian medicine. Received: 29 January 2020; Accepted: 23 March 2020 *Correspondence to: Babak Daneshfard, Traditional Medicine Clinical Trial Research Center, Shahed University, Enghelab Sq., North Kargar St., No. 1471, Tehran 1417953836, Iran. Tel/Fax: +98-21-66464322, E-mail: babakdanesh- Fig. 1. Medicinal herb delivery in a representative Iranian traditional [email protected] herbal shop (Attari).

106 Journal of Clinical and Translational Hepatology 2020 vol. 8 | 106–107

New legislation and regulatory processes have been issued by the health authorities in recent years and active supervision is currently running in Iran. Hence, there is still a long way to go until we reach a desirable state in the healthcare delivery of CAM.

Funding

None to declare.

Conflict of interest

The authors have no conflict of interests related to this publication.

References

[1] Philips CA, Augustine P, Rajesh S, Y PK, Madhu D. Complementary and alternative medicine-related drug-induced liver injury in Asia. J Clin Transl Hepatol 2019;7:263–274. doi: 10.14218/JCTH.2019.00024. [2] Atarzadeh F, Daneshfard B, Dastgheib L, Jaladat AM, Amin G. Early description of diet-induced blistering skin diseases in Medieval Persia: Avicenna’s point of view. Skinmed 2016;14:367–370. [3] Nimrouzi M, Tafazoli V, Daneshfard B, Zare M. Optimal fluid intake in daily diet: Avicenna’s view. J Integr Med 2016;14:241–244. doi: 10.1016/S2095-4964 (16)60262-5. [4] Daneshfard B, Dalfardi B. Medieval roots of modern knowledge regarding carotid sinus syncope. Int J Cardiol 2014;173:342–343. doi: 10.1016/j. ijcard.2014.03.030. [5] Bordbar M, Pasalar M, Safaei S, Kamfiroozi R, Zareifar S, Zekavat O, et al. Complementary and alternative medicine use in thalassemia patients in Shiraz, southern Iran: A cross-sectional study. J Tradit Complement Med 2017;8:141–146. doi: 10.1016/j.jtcme.2017.05.002. [6] Hashempur MH, Heydari M, Mosavat SH, Heydari ST, Shams M. Comple- mentary and alternative medicine use in Iranian patients with diabetes mellitus. J Integr Med 2015;13:319–325. doi: 10.1016/S2095-4964(15) 60196-0. [7] Joharchi MR, Amiri MS. Taxonomic evaluation of misidentification of crude herbal drugs marketed in Iran. Avicenna J Phytomed 2012;2:105–112. [8] Pasalar M, Vardanjani HM. Trend of clinical trials in Persian medicine: a decade – Fig. 2. Herbal medicine delivery in a representative university-affiliated of endeavours. Integr Med Res 2020;9:10 11. doi: 10.1016/j.imr.2019.11. health center in Iran. 010.

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